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THE MICROBIOLOGY OF 
STARCH AND SUGARS 



5 



Uniform with this Volume 

THE MICROBIOLOGY OP CELLULOSE, HEMECELLtH 
PECTIN, AND GUMS. By A. C. THATSBN and H. J. IB 
Demy 8vo. Cloth. Pp. viii + 363, with illustrations in the t< 
9 plates. 2 

OXFORD UNIVERSITY PRESS 



THE MICROBIOLOGY OF 
STARCH AND SUGARS 



BY A. C. THAYSEN AND 
L. D. GALLOWAY 





OXFORD UNIVERSITY PRESS 

LONDON : HUMPHREY MILFORD 

1930 



OXFOBD UNIVERSITY PB.BSS 

AMEN HOUSH, B 0. 4 

tONEON HDINBURQH GLASGOW 

IHIPZIG NHWYOBK TORONTO 

MHEBOITBNB CAPETOWN BOMBAY 

CALCUTTA MADHAS 8IIANGHAI. 

HUMPKRET MILFOED 

PUBLB3HBB TO THB 

TOTVHRSITT ' 






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PHIWEHD IN QBHAX BBETAIN AT IHH TOlVaiiaCTY PRH33, OXFORD 
BY JOHN JOHNSON, PBINTJ1IR TO TUB UMIVHRSITY 



PREFACE ' x 

, '< - 

THB treatise embodied in the following pages has been com- 
piled as a -complementary volume to the -Microbiology of 
Cellulose, HemicelMoses, Pectin, and Gums published by one 
of the writers (A. C. Thaysen) in collaboration with Mr. H. J. 
Bunker in 1927. ' 

Taken togetheif the two volumes endeavour to review the 
microbiology* of the carbohydrates, a subject, it will be 
realized^ whicB covers a very wide field, particularly the 
section which is now placed in the hands of the publishers 
under the Mtle of tne Microbiology^ of Starch and Sugars. 
Considerably more than three thousand original publications 
bearing on the subject have had to be examined in detail, 
and though it is hoped that the bulk of the relevant literature 
has been considered, the writers are anxious to acknowledge 
that some papers, even perhaps some quite important papers, 
may have escaped their notice, particularly any such which 
have been published in languages other than English, French, 
German, Dutch, Danish, Swedish, and Norwegian. 

The volume has been written from the point of view of the 
research worker, and in addition to compiling existing know- 
ledge it endeavours to point out paths which might be 
followed by workers who desire to extend their knowledge 
of the action of micro-organisms on starch and sugar. 

As in the treatise on the microbiology of cellulose, the term 
'micro-organisms' has been adopted to designate all micro- 
scopic organisms, whether belonging to the animal or to 
the vegetable kingdom. The word 'bacteria' has been used 
colloquially for all rod-shaped schizomycetes belonging to 
the JSubacteriales. Where a distinction in the Latin nomen- 



vi PREFACE 

clature has been necessary among the "bacteria, the system 
followed by Lehmann and Neumann and by Bergey has been 
adopted, a spore producing rod being termed 'BacttlMs' and 
a non-sporing rod 'Bacterium'. 

The writers are anxious to place on record the valuable 
help rendered them by members of the staff of the Depart- 
ment of Scientific Research of the Admiralty and of the 
Department of Scientific and Industrial Research. 

They also greatly appreciate the permission granted them 
by the Lords Commissioners of the Admiralty to publish 
their treatise. 

Last but not least they are most sincerely indebted to the 
publishers," the Oxford University Press, for their never 
failing patience, which must have been taxed to the utmost 
by the long-delayed delivery of a promised manuscript. The 
writers can only plead in their defence, that the task under- 
taken by them was found to be much greater than had been 
originally anticipated. 

HOLTOST HEATH, 
September 1929. 




CONTENTS 
PREFAOB ...... 

PABT ONE 

I. Outline of the Constitution and the Bio- 
chemical Properties of Starch, Glycogen, and 

Tnnlin ....... 3 

n. Microbiological Hydrolysis of Starch, Glyco- 

gen, and Inulin ...... 16 

HI. The Hydrolysis of Tetra-, Tri-, and Disac- 

charides ....... 40 

IV. Hydrolysis of Gluoosides .... 65 

PART TWO 
V. The Fermentation of Monoses ... 77 

VI. The Dehydrogenation of Hexoses resulting in 
the Production of Gluconic, Saccharic, Suc- 
cinio, Fumario, Oxalic, and Citric Acids . 92 

VET. The Aerobic Dehydrogenation of Hexoses, 
resulting in the Formation of Acetic and 
Formic Acids, Acetylmethylcarbinol and 2:3 
Butyleneglycol 106 

VIII. The Facultative Anaerobic Dehydrogenation 
of Hexoses resulting in the Production of 
Acetic Acid, Formic Acid, Ethyl Alcohol, 
Acetylmethylcarbinol and 2:3 Butyleneglyool 111 

IX. The Facultative Anaerobic Dehydrogenation 
of Hexoses involving the Formation of Lactic 
Acid as an essential Decomposition Product 
/. The groups of Bact. coli commune and 
Bact. lactis aerogenes . . . .116 

X. The Facultative Anaerobic Dehydrogenation 
of Hexoses involving the Formation of Lactic 
Acid as an essential Decomposition Product 
//. The group of the lactic acid bacteria . 125 



viii CONTENTS 

PAGE 

XI. The Facultative Anaerobic Dehydrogenation 
of Hexoses, involving the Formation of Lactic 
Acid as an essential Decomposition Product 
///. Propionic acid bacteria . . .136 

XII. The Obligatory Anaerobic Dehydrogenation of 
Hexoses 
The production of n-butyric acid and n-buiyl 

alcohol 142 

TTTT. The Fermentation of Pentoses . . .162 

PART THREE 

XIV. The Synthetic Activities of Micro-organisms . 173 
XV. The Mucus Fermentations . . . .181 

PART FOUR 

XVI. The Microbiology of Cereals and Cereal Pro- 
ducts 191 

XVII. The Microbiology of Grain and its Milling 

Products, Bran and Flour . . . .196 

XVIII. The Microbiology of Starch-containing Sizing 

Materials and Adhesives . . . .210 

XIX. The Microbiology of Baking . . .219 
XX. Diseases of Bread 246 

PART FIVE 

XXI. The Microbiology of Sugar-Cane, Sugar-Beet, 

and their Raw Juices . . . . .261 

XXII. The Microbiology of Cane Juice and Beet Juice 

in Manufacture ...... 278 

XXTTT. The Microbiological Deterioration of Sugar in 

Storage 289 

INDUS OE AUTHOBS 306 

SUBJECT INDEX 324 



PART ONE 



CHAPTER I 

OUTLESTE OF THE CONSTITUTION AND THE BIO- 
CHEMICAL PROPERTIES OF STARCH, 
GLYCOGEN, AND INUUN 

Starch. The constitution of starch, its depolymerisation 
and its conversion through hydrolysis into simpler carbo- 
hydrates have long been favourite subjects for investigation. 
But though recent years have witnessed a certain simplifica- 
tion of the conception of these problems, much work has still 
to be done before the chemistry of starch and the constitution 
of this polysaccharide are fully understood. 

The bulk of the existing literature on starch and starch 
hydrolysis is now of little more than historical interest and 
cannot be dealt with here. It must suffice briefly to refer to 
the few publications which have had a direct bearing on more 
recent fundamental researches. Among these must be men- 
tioned Kirohoff's 1 observation in 1812 that boiling with dilute 
mineral acids converts starch into a sugar; de Saussure's 2 
record in 1819 of the spontaneous decomposition of starch 
with the formation of sugars; and Dubrunfaut's 3 study of 
the action of malt extracts on starch, which led to Payen and 
Persoz's 4 isolation of diastase (malt amylase) in 1833. In 
1847 Dubrunfaut 6 showed that the sugar, termed maltose by 
him, which he obtained from starch by the action of malt 
amylase, and which in the course of time had come to be 
regarded as identical with glucose, was in fact different from 
this sugar, yielded by starch on hydrolysis with dilute acids. 
Dubrunfaut's researches were considerably extended by 
O'Sullivan 6 from 1872 to 1876. O'Sullivan found that the 
maltose obtained from starch by the action of malt amylase 
was a disacoharide with about double the rotatory power of 
glucose, but with a lesser reducing action towards Folding's 
solution. 

During the eighty years following the discovery of malt 

B2 



4 CONSTITUTION AND BIOCHEMICAL PROPERTIES OF 
amylase, the study ol the chemistry of starch became largely 
an investigation of the action of this enzyme mixture and of 
the nature of the dextrins, intermediate decomposition pro- 
ducts of starch. Comparatively few of the publications dating 
from this period have proved of direct value to recent re- 
search. Of interest is Lintner's 7 observation in 1891 that a 
new sugar, isomaltose, is formed as a decomposition product 
of starch when the polysacoharide is subjected to the action 
of malt amylase. Lintner's statement was not accepted at 
the time, but subsequent investigations (Ling and Nanji 8 and 
Kuhn 9 ) supported his view. 

Another fruitful observation was reported by Maquenne 
and Eoux 10 in 1906. These authors succeeded in decomposing 
the starch granule into two distinct substances, which they 
termed amylose and amylopeotin respectively. The amylose 
was found to constitute from 80 to 85 per cent, of the starch 
granule, while amylopeotin represented the bulk of the re- 
maining 16 to 20 per cent. Amylose showed the characteristic 
blue iodine reaction of starch to an intensified degree, while 
the amylopeotin gave no blue coloration with iodine. In this 
detail the French authors differ from Ling and Nanji, who 
find that their amylopectin, when dissolved, gives a blue- 
black colour and precipitate with iodine. Pringsheim, 11 on the 
other hand, records a brownish red colour for amylopeotin, 
which he regards as identical with glyoogen. 

Amylose was shown by Maquenne and Roux to be soluble 
in boiling water. On treatment with malt amylase it was 
readily and completely converted into maltose. Amylopeotm, 
on the other hand, had to be boiled to become dispersed in 
water and then yielded a viscous liquid. Amylopeotin is thus 
responsible for the gelatinization of starch suspensions pre- 
pared with hot water. Treated with malt amylase, amylo- 
peotin was found to saccharify slowly, yielding dextrins. 
Nevertheless it was taken for granted by Maquenne and 
Roux 13 that amylopectin consisted entirely of maltose resi- 
dues, since it was converted completely into maltose (using 
potato starch paste) when treated with malt amylase at a 



STARCH, GLTCOGEN, AND DfDUN 6 

favourable hydrogen ion concentration. It should be added 
here that Ling 13 denies that amylopeotin can be converted 
into maltose by amylase, which merely, he says, dephospha- 
tises it and depolymerises it into a hexa-amylose. Maquenne 
and Eoux's work, which demonstrated the presence of at 
least two substances in the starch granule, in many ways 
confirmed the views of the earliest investigators, for instance 
that of Nageli, 14 who referred to two compounds in the starch 
granule, an interior 'granulose', the mother substance of 
maltose, and an exterior compound, 'starch-cellulose '. Meyer 16 
shared the view of the presence of two substances in the 
starch granule, terming them a- and /?-amylose, corresponding 
to Nageli's granulose and starch cellulose respectively. But 
he regarded them as one and the same substance dehydrated 
to different degrees. 

Gatin-Gruzewska's 16 researches demonstrated experiment- 
ally that the water-soluble amylose is identifiable with 
starch granulose and thus constitutes the interior substance 
of the starch grain, while the amylopectin, asmentionedabove, 
represents the starch cellulose of earlier workers. It separates 
as empty shells of the original starch granule when a starch 
paste is subjected to the treatment required for the separa- 
tion of its two composing substances. By the treatment 
evolved by Gatin-Gruzewska, as much as 40 per cent, of 
amylopectin can be obtained from potato starch. Tanret 17 
examined a variety of other starch types and came to the 
conclusion that the yield of amylopectin may be much 
higher. In chestnut starch he found as much as 67 per cent, 
and in banana starch 79-5 per cent, amylopectin. Of other 
workers who have determined quantitatively the amount of 
amylopectin present in starch may be mentioned Ling and 
Nanji, 8 whose yields correspond to those of Maquenne and 
Roux; Pringsheim and Wolfsohn, 18 who report having ob- 
tained as much as 66 per cent, from potato starch, using the 
method recommended by Ling and Nanji ; and Samec, 19 who 
reached a yield of 77 per cent, when carrying out the separa- 
tion in a manner different from that used by Gatin-Gruzewska. 



6 CONSTITUTION AND BIOCHEMICAL PROPERTIES OF 

Other fruitful researches which should be mentioned are 
those of Fouard 20 and of Sameo and his collaborators 21 on the 
phosphorus content of starch. Sameo 's investigations particu- 
larly have thrown light on the function of the phosphorus 
found associated with starch in the starch granule. It is now 
generally accepted that amylopeotin represents the phos- 
phoric ester of a polysaccharide, while amylose is free from 
chemically bound phosphorus. 

Of interest are Olayson and Schryver's 22 and Schryver and 
Thomas's 23 observations on the presence of hemicelluloses in 
certain types of starch, in sago and maize starch, for instance, 
where these substances occur in quantities up to 3-8 per cent. 
These hemicelluloses are not acted upon by amylase, at least 
not by the enzyme present in taka-diastase. Potato starch 
does not appear to contain any hemicelluloses. 

Additional work on the amylohemicelluloses has been 
carried out by Ling and his collaborators, 13 who find the 
percentages present considerably greater, reaching figures of 
16 to 17 per cent, in potato tubers and rice respectively. It 
should be added also that Taylor and Lehrmann 24 have ob- 
served the presence of small quantities of fatty acids in maize 
starch, substances which they claim are organically combined 
with the amylopeotin. 

Special interest attaches to Schardinger's 86 observations, 
dating from 1909 to 1911, on the formation of crystalline 
decomposition products of starch by tha action of Sac. 
mac&rans. In the hands of Pringsheim and his collaborators 
these observations have led to the elaboration of a possible 
interpretation of the chemical structure of amylose and of 
amylopectin. Prom the liquefied starch paste which Bac. 
macerans had acted upon for some days Schardinger obtained 
two crystalline substances which he termed a- and yff-amylose 
respectively. In most of their physico-chemical properties 
these bodies resembled dextrins, substances to which reference 
will be made later. They were soluble in water but insoluble 
in alcohol, ether and choroform, and they did not reduce 
Fehling's solution. A solution of a-amylose gave a yellowish- 



STARCH, GLYCOGEN, AND INUUN 7 

green and of /?-amylose a reddish-brown colour with iodine. 
Pringsheim and Langhans, 26 who repeated Schardinger's 
experiments, were able to confirm his results. They found 
that the a-amylose was composed of four groups of (C e H 10 6 ), 
a hypothetical hexosan to which they applied the term 
'amylose'. The -amylose of Schardinger was found to con- 
tain six of the hypothetical 'amylose' groups. Pringsheim 
and Langhans succeeded in depolymerising the tetra-amylose 
(Schardinger's a-amylose) into a diamylose, while the hexa- 
amylose (Sohardinger's /?-amylose) on depolymerisation gave 
a triamylose. The relationship between these polyamyloses 
(tetra- and hexa-amylose) and the amylose and amylopectin 
of Maquenne and Roux was studied by Pringsheim and 
Wolfsohn. 18 They found that Maquenne's amylose could be 
converted into the same diamylose which was obtained from 
Schardinger's a-amylose, while a triamylose, identical with 
that isolated from Schardinger's /?-amylose, could be obtained 
from amylopeotin either through acetylation or through 
heating the polysaccharides in glycerine at 200 C., a method 
used by Pictet and Jahn 27 for the depolymerisation of starch. 
Experimental evidence had thus been brought forward in 
support of Pringsheim's already expressed view 28 that the 
basic unit of Maquenne's amylose is a diamylose and that of 
amylopectin a triamylose. Pringsheim and Langhans also 
observed that diamylose and its polymers gave iodine addi- 
tion products of metallic green needles, while triamylose and 
its polymers gave reddish-brown iodine compounds. 

On treating the polyamyloses formed by Bac. mac&rans 
with cold concentrated hydrochloric acid, Pringsheim and 
Leibowitz 29 obtained a disaccharide CjJ3. 2Z O w , which they 
termed amylobiose. It reduced Fehling's solution. The same 
substances could be obtained on treatment of amylose (Ma- 
quenne and Roux) with this reagent, while amylopectin yielded 
a reducing trisaccharide under these conditions, a substance to 
which Pringsheim and Leibowitz gave the name amylotriose. 

Both amylobiose and amylotriose, when acted upon by 
malt diastase, were converted quantitatively into maltose. 



8 CONSTITUTION AND BIOCHEMICAL PROPERTIES OF 

Karrer and Nageli, 80 from their study of the behaviour of 
Pringsheim's polyamyloses and of starch itself towards acetyl 
bromide, came to the conclusion that Pringsheim's diamylose 
as well as starch are maltose anhydrides, the starch having 
been polymerised to such an extent as to contain no less 
than eight amylose groups. 31 Karrer and his collaborators 
regard maltose anhydride as the fundamental unit of the 
whole of the starch molecule both of the amylose of Maquenne 
and Roux and of amylopeotin. 

Ling and Nanji, 8 on the other hand, regard both amylose 
and amylopeotin as built up of hexosans as basic units, the 
amylose hexosan being of a-glucosidio nature, while the 
amylopectin hexosan shows both a- and ^-glucoside Unkings 
in addition to one phosphoric acid group in ester formation 
for every three hexose groups. By adopting this formula for 
amylopectin, Ling and Nanji claim to have explained the 
formation of isomaltose as a decomposition product of amylo- 
pectin. 

Finally Irvine and Macdonald 32 have come to the conclusion 
from their study of methylated starch and starch derivatives 
that the ultimate unit of the starch molecule is a trihexosan, 
thus supporting, in some measure at least, ling and Nanji's 
contention. Their researches do not indicate, however, 
whether starch is to be considered as two different constitu- 
ents embodied in the amylose and amylopectin respectively 
of Maquenne and Boux, or whether it is to be assumed that 
starch is a polymer of maltose anhydride only. 

In the following pages the subject of the hydrolysis of 
starch will be dealt with on the assumption that it consists of 
two definite components, amylose and amylopectin, as de- 
fined by Maquenne and Roux. 

One of the most characteristic physical properties of starch 
is its swelling to form a viscous opalescent paste when heated 
in water above a certain temperature. This property is 
attributed to the amylopectin, while another property of the 
carbohydrate, the formation of a deep blue compound with 
iodine, is ascribed to the amylose. If a very dilute solution 



STARCH, GLYCOGEN, AND INTJLIN 

of a staroh-hydrolysing enzyme, an amylase, is allowed to 
act on a starch paste under favourable conditions, the viscous 
nature of the paste diminishes and disappears without the 
resulting solution losing its property of staining blue with 
iodine. Sameo 21 observed this and pointed out that, as a 
result of the enzyme action, the amylopectin, the bearer of 
the viscous properties of the paste, must have undergone 
changes either through saponification of its ester Unkings or 
through depolymerisation into non-viscous soluble products. 
That the latter has actually occurred is clear from the fact 
that the carbohydrates of the paste still contain their phos- 
phorus chemically bound. The first stage in the action of 
enzymes upon starch is therefore a depolymerisation of at 
least one constituent of the starch granule. For this reaction 
to take place, a reaction which does not lead to the formation 
of FehUng-reduoing substances (since the elements of water 
do not enter the polyamylose molecule), Pringsheim assumes 
the presence of specific depolymerising enzymes in amylase. 

This view is not accepted by Nishimura, 88 who regards 
amylase as being composed of one enzyme only, having the 
ability to perform both the saooharification and the de- 
polymerisation, the latter notably in the presence of an 
activator, perhaps identical with Pringsheim and Beiser's 34 
co-enzyme. 

Earlier investigators had interpreted the conversion of the 
viscous opalescent starch paste into a clear solution in a 
different manner. The resolution was connected by them 
with a partial hydrolysis of the starch granule through an 
enzyme, sometimes termed dextrinase (Pottevin 313 ), leading 
to the formation of a series of dextrins ranging from the 
amylodextrins, which showed comparatively small Fehling- 
reducing powers, and retained the faculty of giving a blue 
reaction product with iodine, to the achroodextrins, or simply 
dextrins (Czapek 86 ), where the Fehling-reducing properties 
had increased considerably and the iodine coloration had 
been lost. Between these extremes were found other dex- 
trins, sometimes termed erythrodextrins, which showed an 



10 CONSTITUTION AND BIOCHEMICAL PROPERTIES OF 
intermediate Fehling reduction and stained reddish-brown 
with iodine. How far it is justifiable to assume with Prings- 
heim that amylase contains both a depolymerising and a 
dextrinising enzyme in addition to saccharifying enzymes 
remains an open question. The assumption does not appear 
essential, since the opening of the depolymerised amylose 
and amylopectin through the introduction of the elements of 
water might well be due to a saccharifying enzyme if present 
in the amylase. That thin saccharifying enzyme IB different 
from the liquefying enzyme is clear from the work of Olsson, 87 
who found that addition of certain poisons reduced its action 
without interfering with the activity of the liquefying enzyme. 

The saooharification of the depolymerised amylose and 
amylopectin does not proceed at the same rate. This was 
noted very early in the history of starch sacoharification. 
The amylose always becomes smoothly and rapidly converted 
into the theoretical amount of maltose, a disaccharide which 
under normal conditions represents the final stage of starch 
hydrolysis by many types of amylase. The saocharification 
of the amylopectin, on the other hand, is slower, and usually 
ceases when about 78 per cent, of the theoretical amount of 
maltose has been formed. The residue, a 'residual dextrin', 
was found by Pringsheim and Beiser 34 to be a trisaccharide 
identical with trihexosan. But even this, according to Prings- 
heim and Beiser, may be converted into maltose by the 
saccharifying enzyme when a co-enzyme or complement, in 
the form of yeast extract, is added. The formation of this 
residual dextrin and its saccharification to maltose in the 
presence of a complement lend considerable weight to Prings- 
heim's suggestion that amylopectin is composed of a triglu- 
cose as basic unit. The nature of the complement has been 
studied by Pringsheim, Bondi, and Thilo 38 . It would appear 
to be a trypsin decomposition product of albumin, and it is 
present in yeast owing to autolysis of the yeast cells. 

Pringsheim 11 visualizes the conversion of a triglucosan into 
maltose by assuming that the active enzyme splits off one 
molecule of maltose, leaving the remaining glucose radical to 



STARCH, GLYCOGEN, AND ETOUN 11 

condense with another glucose radical liberated from another 
molecule of the trigluoosan. In support of this explanation 
he quotes Hess, Weltzien, and Messmer's 39 researches on the 
auto-condensation of glucose anhydride to form cellobiose, 
a view which is not accepted by Kuhn. 40 That the incomplete 
conversion of amylopectin into maltose may sometimes be 
due to the absence of optimal hydrogen-ion concentrations 
was shown by Maquenne and Roux. 41 

Interesting light has subsequently been thrown on the 
nature of the various existing amylases by the researches of 
Kuhn. 42 He bases his investigations on the observations of 
Brown and Heron 43 that the hydrolysis of starch by malt 
amylase at 50 0. proceeds at the same rate both as regards 
increase in copper-reducing properties and polarimetric 
changes, and on Brown and Morris's 44 conclusions that this 
is not the case when the hydrolysis takes place at a low tem- 
perature. Kuhn brings forward evidence to show that these 
divergences are due to the fact that malt amylase liberates 
maltose in the /J-glucosidic form. At low temperatures and 
at the optimal hydrogen-ion concentration of malt amylase 
this j8-maltose is converted comparatively slowly into the 
stable a-j8-maltose mixture. The establishment of a polari- 
metric equilibrium in the hydrolysed starch paste will there- 
fore lag behind the changes in copper-reducing powers in 
this case. 

Where pancreas amylase or taka-diastase is used to hydro- 
lyse starch, Kuhn finds that the maltose is initially liberated 
in the a-glucosidio form. This shows that two distinct types 
of amylase exist, an a-amylase originating from animal 
sources and from taka-diastase, and a j6-amylase from vege- 
table sources other than taka-diastase and present in emulsin 
and in malt amylase. 

In a later paper Kuhn 42 remarks that in view of its behaviour 
towards animal and vegetable amylases the starch granule 
probably consists of carbohydrates in which a- and jS-linkings 
occur in regular sequence between the glucose residues com- 
posing them. This, ap will be recollected, had been already 



12 CONSTITUTION AND BIOCHEMICAL PROPERTIES OF 
suggested by Ling and Nanji in the case of amylopectin, 
Kuhn does not accept the -view that Pringsheim's polyamy- 
loses or hexosans are the basic units of the starch molecule, 
since each of them only shows the characteristics of starch 
to a limited extent. 

A deduction which is of interest from the point of view of 
the miorobiologist was made by Pringsheim and Leibowitz 46 
from Kuhn's earlier work. They considered that if both a- 
and jS-gluoosidic Hnkings occur in the carbohydrates of starch 
it should be possible to obtain glucose direct from starch 
if a mixture of a- and /?-amylase is made to act on a starch 
paste. That this was possible they demonstrated experi- 
mentally, thus showing that it is not essential to assume, as 
had been done by earlier investigators (Beijerinck 46 ), that a 
special maltase must be present in an amylase which sac- 
charifies starch with the production of both maltose and 
glucose. 

Before leaving the subject of the chemistry of starch, a few 
words must be devoted to the observations of Wolff and 
Fernbach 47 that unripe cereals and leaves contain an enzyme 
which precipitates soluble starch from its solutions. This 
enzyme they term amylocoagulase. Beyond the observa- 
tions by Joszt 48 that amylocoagulase is destroyed on heating 
to 63 0. for six minutes, and that it differs markedly from 
the saccharifying enzyme, little is known of this interesting 
substance, which in the writers' view probably plays a con- 
siderable role in the action of micro-organisms on starch, 
since it frequently occurs that a starch paste inoculated with 
starch hydrolysing micro-organisms has its colloidal properties 
destroyed long before the production of aoidic fermentation 
products can have accumulated sufficiently to cause this 
change. 

Glycogen. Glycogen, the reserve carbohydrate of animals 
and of many lower saprophytio and parasitic plants, re- 
sembles starch in some of its properties. It forms a white 
amorphous powder which yields opalescent colloidal sus- 
pensions in water. With iodine it forms a reddish-brown 



STAECH, GLYCOGEN, AND rNULIN 13 

compound resembling that of the erythrodextrins. When 
acted upon by amylase it undergoes decomposition processes 
similar to those of starch (Musculus and Mering), 49 and yields 
dextrins and maltose. These changes, according to Cremer, 60 
proceed slower than in the case of starch. According to Har- 
den and Young 61 the rate seems to vary with the type of 
glycogen used. The glycogen-hydrolysing enzyme, glyco- 
genase, was found by Pick 62 in various animal tissues, and was 
observed as an endo-enzyme in yeast by Buchner and Rapp. 68 
Norris 5 * found that the hydrolysis proceeded much faster 
during the early stages and seldom converted the carbo- 
hydrate quantitatively. The presence or absence of electro- 
lytes in the enzyme was shown by him to have a considerable 
effect on the rate of hydrolysis, dialysed enzymes having 
practically no action at all. An interesting account of the 
constitution of glycogen has been given by Pringsheim and 
Beiser, 34 who suggest that, apart perhaps from its content of 
electrolytes, it is identical with amylopectin. They arrive at 
this conclusion from their observations that both glycogen 
and amylopeotin yield the same depolymerisation products 
on heating with glycerin and by treatment with hydrochloric 
acid. 

Inulin. This, the third important polysaccharide which 
will be touched upon in subsequent pages, forms white sphere- 
crystals and occurs as a reserve carbohydrate in the roots and 
tubers of certain plants. Czapek 86 records its presence in 
certain algae. On hydrolysis it is converted quantitatively 
into fructose. The determinations of the molecular weight of 
its acetyl ester led Pringsheim. and Aronowsky 66 to conclude 
that its molecule contains nine fructose residues. Karrer 66 
assumes that these are present without internal polymerisa- 
tion, but Pringsheim and Aronowsky see in its molecule an 
aggregation of three groups of an anhydro-tri-fructosan, a 
view which appears to have been accepted by Irvine. 57 A 
characteristic of inulin is its rapid hydrolysis to fructose 
without the formation of detectable intermediate decom- 
position products. This would appear to indicate that the 



14 CONSTITUTION AND BIOCHEMICAL PROPERTIES OF 
individual fructose radical occurs in a particularly active 
form, as a so-called y-sugar (Irvine 58 ). Intermediate con- 
densation products between fructose and inulin have been 
shown by Tanret 59 to occur in tissues of certain plants. 

UTEEATUEE 

1. S. R. Kirchoff, Account by Nasse, J.f. Chem. and Physik, vol. 4, p. Ill, 

1812. 

2. Th. de Saussure, Ann. Chim. Phys., vol. 11, p. 379, 1819. 

3. P. Dubrunfaut, Jour.filr technische und okonomische Chemie (Erdmann), 

vol. 4, p. 156, 1830. 

4. A. Payen and J. Persoz, Ann. Chtm. Phys., vol. 63, p. 73, 1833. 

5. P. Dubnmfaut, Ann. Ohim. Phys., vol. 3, p. 178, 1847. 

6. C. O'Sullivan, J. Ohem. Soc. Trans., vol. 25, p. 570, 1872 ; vol. 29, p. 479, 

1876; vol. 30, p. 125, 1876. 

7. C. J. Lintner, Zeitech.f. d. gesamt. Brauwesen, vol. 16, p. 284, 1891. 

8. A. R. Ling and D. R. Nanji, J. Chem. Soc. Trana., vol. 123, p. 2666, 

1923. 

9. R. Kuhn, Berichte, vol. 57, p. 1965, 1924. 

10. L. Maquenne and E. Roux, Ann. CJiim. Phys., vol. 0, p. 179, 1906. 

11. H. Pringsheim, BericJtte, vol. 57, p. 1581, 1924. 

12. L. Maquenne and E. Rom, Comptea rend., vol. 142, p. 1069, 1906. 

13. A. R. Ling, J. Soc. Chem. Ind., vol. 46, p. 279 T, 1927. 

14. W. Nageli, Die StdrJeekorner, Zurich, 1868. 

15. A. Meyer, Untersuchungen -uber die St&rkekbrner, Jena, 1895. 

16. Z. Gatin-Gruzewska, Comytes rend. soc. liol., vol. 64, p. 178, 1908; 

Comptesrend., vol. 146, p. 640, 1908. 

17. C. Tanret, Comptea rend., vol. 168, p. 1353, 1914; vol. 159, p. 630, 1914. 

18. H. Pringaheim and K. Wolfsohn, Benchte, vol. 67, p. 887, 1024. 

19. M. Sameo, EoUoiddiern. Betiiefie, vol. 4, p. 23, 1914. 

20. E. Fouard, Comptes rend., vol. 144, p. 146, 1907-8; Intern. Cong. Appl. 

Chem. London, vol. 7, VI. B. Fermentation, p. 129, 1909. 

{M. Sameo and F. v. Hoefift, EoUoidchem. Beihefte, vol. 4, 132, 1912; 
vol. 6, p. 141, 1913 ; vol. 6, p. 201, 1014. 
M. Sameo and H. Haerdtl, Kottoidchem. Beihefte, vol. 12, p. 281, 1920. 
M. Sameo and A. Meyer, gottoidchem. Beihefte, vol. 16, p. 89, 1922. 
M. Samec, Kottoidchem. Zeitachr., voL 47, p. 81, 1929. 

22. D. H. F. Clayaon and S. B. Scnryver, Biochem. J., vol. 17, p. 403, 1923. 

23. S. B. Sohryver and E. M. Thomas, Biochem. J., vol. 17, p. 497, 1923. 

24. F. C. Taylor and L. Lehrmann, J. Am. Chem. Soc., vol. 48, p. 1739, 

1026. 

26. F. Sohardinger, ZentrU.f. Bokt., Abt. H, vol. 22, p. 08, 1909 ; LT, vol. 29, 
p. 188, 1911. 



STARCH, GLYCOGEN, AND INTILDSr 15 

_. fH. Pringsheim and A. Langhans, Berichte, vol. 45, p. 2533, 1912. 
\H. Pringsheim and F. Eiflsler, Benchte, vol. 46, p. 2959, 1913. 

27. A. Piotet and R. Jahn, Hdv. Chim. Acta, vol. 6, p. 640, 1922. 

28. H. Pringsheim, Berichte, vol. 57, p. 1581, 1924. 

29. H. Pringsheim and J. Leibowitz, Benchte, vol. 57, p. 884, 1924. 

30. P. Karrer and 0. Nfigeli, Hdv. Ohtm. Acta, vol. 4, pp. 169, 185, 263, 

1921. 

31. P. Karrer, C. Nageli, 0. Hurwitz and A. Walti, Hdv. Chim. Acta, vol. 4, 

p. 678, 1921. 

fj. C. Irvine, J. CJiem. Soc. Trans., vol. 123, p. 898, 1923. 
32X J. C. Irvine and J. Maodonald, J. CJiem. Soc. Trans , vol. 129, p. 1502, 
l_ 1926. 

33. S. Nishimura, Btochem. Ze.it , vol. 200, p. 81, 1928. 

34. H. Pringsheim and A. Beiser, Btochem. Zetts., vol. 148, p. 336, 1924. 

35. H. Pottevin, Ann. Inst. Pasteur, vol. 13, p. 665, 1899. 

36. F. Czapok, Dte Biochemie der Pflamen, Gustav Fischer, Jena, 1913-21. 

37. U. Olason, Z.physwl. Chem., vol. 126, p. 29, 1923. 

38. H. Pringsheim, J. Bondi, and E. Thilo, Biochem. Zeit., vol. 197, p. 143, 

1928. 

39. K. Hess, W. Weltzien, and E. Messner, Liebig's Ann., vol. 435, p. 1, 

1923. , 

40. B. Kuhn, Ann. d. Chem., vol. 443, p. 1, 1925. 

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42. R. Kuhn, Berichte, vol. 56, p. 857, 1923; vol. 57, p. 1965, 1924. 

43. H. T. Brown and J. Heron, J. Chem. Soc. Trans., vol. 35, p. 696, 1879. 

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1895. 

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47. J. Wolf and A. Fernbaoh, Comptes rend., vol. 137, p. 718, 1903. 

48. A. Joszt, Chem. Zentralbl, vol. 96, p. 1327, 1925. 

49. F. Musoulus and von Mering, Zeitschrift physiol. Chem., vol. 2, p. 413, 

1882. 
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1902. 

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63. E. Buchner and R. Rapp, Benchte, vol. 31, p. 214, 1898. 

64. R. U. Norris, Biochem. J., vol. 7, pp. 26 and 622, 1913. 

65. H. Pringsheim and A. Aronowsky, Benchte, vol. 64, p. 1281, 1921. 

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59. C. Tanret, Butt. Soc. Chim., Pans, vol. 9, p. 622, 1893. 



OHAPTEB II 

MICROBIOLOGICAL HYDROLYSIS OF STARCH, 
GLYCOGEN, AND INULIN 

IT was mentioned in Chapter I that de Saussure 1 had ob- 
served that a starch paste left exposed to the atmosphere 
became spontaneously decomposed. This is probably the 
first record of a hydrolysis of starch by micro-organisms. 
Six decades later, in 1877 and in 1878, Fitz 2 studied the 
schizomycete fermentation of starch and dextrin, while 
Wortmann 8 succeeded in isolating enzymes from a crude 
culture of Bact. i&rmo, a species which is now considered 
synonymous with the putrefying Bact. vulgare Lehmann 
and Neumann. These investigations were followed in rapid 
succession by a large number of publications, illustrating the 
widespread property among micro-organisms of hydrolysing 
starch. 

Following the procedure adopted by Thaysen and Bunker 4 
in their treatise on the microbiological decomposition of 
cellulose, the writers have grouped the existing information 
on microbiological starch hydrolysis under three headings, 
as caused by bacteria, by actinomyoetes, and by fungi. 

No reference will be made to the amylolytic properties of 
the myxomycetes and of the algae, a subject which is dealt 
with in several leading text-books on plant physiology and 
plant chemistry. Even among the three classes of micro- 
organisms selected it has been found necessary to restrict the 
discussion to the most important groups. The fact that most, 
if not all, existing fungi, a very large number of bacteria, and 
many actinomyoetes hydrolyse starch makes it quite im- 
possible within the scope of the present volume to give a 
detailed description of each individual type producing 



r. 



STABCH, GLYCOGEN, AND INTJLIN 17 

THE HTDBOLYSIS Off STARCH BY BAOTHEIA. 

Following the first more or less incidental observations of 
Fitz, 2 Marcano, 5 Wortmann, 3 Prillieux, 6 and Bitter, 7 Fermi 8 
undertook a more comprehensive study of the behaviour of 
a number of well-known bacteria towards starch. The list in 
Table I contains those types found by him to produce starch- 
hydrolysing enzymes as well as some additional forms which 
from time to time have been studied from this point of view. 
The list is not complete, and could not claim to be complete, 
since the whole subject of the action of individual species of 
bacteria on starch, glycogen, and inulin is one which has not 
yet been systematically explored. 

The nature of bacterial amylase has not been studied in 
detail. The isolation of the enzyme appears to have been 
carried out only in a few cases, for instance in those of Bac. 
mesent&rious (Effronf 4 ), and of Bact. t&rmo, now known as 
Bad. vulgare. Here Wortmann obtained an amylase by the 
addition of alcohol to a culture of the test organism. He 
found that the enzyme, when dissolved in water, acted both 
at neutral and at acid reactions, the latter being the more 
favourable. 

In most cases where bacterial amylase has been studied, 
the investigators have employed, not the isolated enzyme, 
but a bacterial culture, added to a starch paste or a solidified 
mixture of starch paste and gelatine (Beijerinck 32 ), often 
after the addition of an antiseptic such as toluene (Gottheil 16 ). 
A technique such as this cannot be regarded as altogether 
satisfactory, particularly where no care is taken to destroy 
the living cells producing the enzyme. And even with this 
ensured it cannot a priori be assumed that the cultures do 
not contain other enzymes, in addition to the amylase, which 
would seriously influence the nature of the products of hydro- 
lysis. This deficiency in technique is conceivably responsible 
for the great difference of opinion which prevails as to the 
changes suffered by starch through the action of bacterial 
amylase. Leaving out of account Fitz's assertion that only 
the amylose of the starch granule becomes affected by the 

o 



18 



MICROBIOLOGICAL HYDROLYSIS OF 



TABLE I 



Name of Organism. 


Hydrolysis 

of 
Starch. 


Reaction 
of medium 
at which 
hydrolysis 
occurred. 


Name of Investigator. 


Azotdbacter croococcum 


+ 


j 


Omehansky 9 


Bac. anthracts 


+ 


neutral 


Fermi 8 ; Maumus 10 


Bac. amylobacter species 


+++ 


pH 3-9-4-6 


Vffliers u ; Behrens 18 








Bredemann 13 ; Makn- 








nov 14 


Bac. amylozyma 


++ 


? 


Perdnx" 


Bac. asterosporua 


+++ 





Gottheil 19 


Bac. botulmus 


++ 





Holland" 


Bac. ffitztanus 


++ 




Fermi 8 


Bac. graveolens 


++ 





Gottheil 19 


Bac. macerana 
Bac. Megatherium 


+ + + + + 


pH 6-75-6 
very acid 


Sohardinger 18 
Fermi 8 


Bac. mesentericus ruber 


+++ 


acid 


Dupont 19 


Bac. mycoides Flugge 


+++ 


neutral 


Fermi 8 


Bac. oedematis maltgni 


++ 


acid 


Kerry and Frankd 20 


Bac petasites 


++ 





Gotbheil" 


Bac. polymyxa 


+4- 





Gruber 21 


Bac. rumtnatus 


++ 





Gottheil 19 


Bac. subtilis 


++ 


neutral 


Fermi 8 




When pep 








tone is 








present as 








a food 






Bac. tennis Duolaux (Ty- 


++ 


neutral 


Fernbach 22 


rothnx tennis) 








Bac. thermoamylolyticus 


+++ 


acid 


Coolhaas 28 


Bac. Welchii 
Bact. dysenteriae 


_+ + 


~ 


Fleming and Neill" 
Eijkman 26 


Bact. fluorescens lique- 


+ 


1 


Emmerhng and Rei- 


faciens 






ser 29 


Bact. induatnum (an 
acetic acid bacterium) 


+ 


acid 


Eenneberg 27 


Bact . lactis aerogenes 


+ 


? 


jaybourn 28 




in some 








strains 






Bact. pestis 
Bact. phosphorescens 


+++ 


very acid 


Sijkman 25 
Fermi 29 



STAKGH, GLTCOGEN, AND ETOLIN 



19 







Reaction 






Hydrolysis 


of medium 




Name of Organism. 


of 


at which 


Name of Investigator. 




Starch. 


hydrolysis 








occurred. 




Bact. pyogenes foetidum 


+ 


aoid 


Fermi 8 


Bact. typhosum 


traces 


neutral 


Fermi 8 


Bact. violaceae 


+ 


very aoid 


Fermi 8 


Bact. vulgare 


++ 


neutral to 


Sclavo and Goaio 30 






aoid 




Bact. Zopfii 


traces 


neutral 


Fermi 8 


Glycdbacter species 


+++ 


? 


Wollman 31 


Ghnnulobacter species 


++ 


aoid 


Beijennok 32 


Micrococcus gonorrhoeae 


+ 


? 


Vedder 33 


Micrococcus mastttidis 


+++ 


? 


Fermi 20 


gangraenosae 








Micrococcus tetragenus 


++ 


aoid 


Fermi 9 


Staphylococcus pyogenes 


+ 


neutral 


Fermi 8 


aureus 








F*&no cholerae 


+ + + 


aoid 


Bitter 7 ; Fermi 8 



enzyme, an assertion which is contradicted by all subsequent 
investigators, the available information indicates that the 
attacked starch may be decomposed in different ways. It 
may be depolymerised to lower hexosans (Schardinger 18 ), 
and these be converted into the final fermentation products 
with the formation of but a slight excess of reducing sugars 
(Euler and Svanberg 36 ). In other cases dextrins may be 
formed and no reducing sugars (Villiers 11 ), Kerry and Fran- 
kel 20 and Siedish 36 . Finally, in some cases both dextrins 
and reducing sugars may be produced, the latter composed 
either of maltose (Beijerinck 32 ) or of glucose (Bitter 7 and Du- 
pont 19 ). Further work is evidently required to elucidate the 
nature of the decomposition products, particularly as the 
same type of bacterium is sometimes recorded to have yielded 
one set of products and sometimes another. At the moment 
the changes caused by bacterial amylase are obscure. On one 
point only do the various investigators appear to agree, and 
that is in allotting two enzymes to the bacterial amylases, a 
depolymerising enzyme and a saccharifying enzyme. Whether 

02 



20 MICROBIOLOGICAL HYDROLYSIS OF 

the latter is of on a- or a j5-glucosidic nature or a mixture of 
both remains to be decided. The reported conversion of 
starch into glucose as the final product of hydrolysis (Du- 
pont 19 , and Sclavo and Gosio 30 ) might conceivably point to 
the latter being the case. 

Very little additional information is available on bacterial 
amylases. Efiront 37 records that they are readily extractable 
by maceration. The enzymes studied by Fermi were active 
between the temperatures of 4 and 50 C. Effront's work 
indicates that their optimum temperature lies at 40 C. 
Heating to 60 C., and in some cases to 70 C., was sufficient in 
Fermi's experiments to destroy them. They would neverthe- 
less appear to be more thermolabile than malt amylase. On 
the subject of the range of their hydrogen ion concentrations 
little is known beyond Fermi's observations recorded in 
Table I. It should be mentioned also that 6 per cent, of phenol, 
or saturation of their solutions with salicylic acid, or addition 
of 10 per cent sodium carbonate, were found by Fermi not to 
interfere with their action. The latter statement is interesting 
since it indicates that a hydroxyl ion concentration equal to 
a pH value of about pH 11-0 should be unable to affect the 
normal functioning of the enzyme. 

Of interest from the point of view of pathological bacteri- 
ology are Kodama and Takeda's 38 observations on the starch 
hydrolysing properties of Vibro cholerae and Vedder's 33 on 
those, of Micrococcus gonorrhoeae. In the case of Vibrio 
cholerae the observations have inspired the elaboration of a 
method for detecting this micro-organism in faecal matter, 
and in the case of Micrococcus gonorrhoeae the preparation of 
a suitable culture medium. 

Kodama and Takeda recommend the inoculation of cho- 
lera suspected excreta into peptone water containing starch, 
followed by incubation for 7 to 24 hours. If, after this time, 
the addition of iodine indicates the disappearance of the 
starch, through the absence of the typical blue reaction with 
iodine, the suspicion is justified that Vibrio cholerae was 
present in the faecal matter tested. The brief incubation 



STARCH, GLYCOGEN, AND INULIN 21 

period allowed is necessary to avoid interference by other 
starch hydrolysing micro-organisms, such as Vibrio Metchni- 
kofffoi instance, which is less active than Koch's vibrio. 

For the cultivation of Micrococcua gonorrJioeae Vedder 
recommends a beef infusion agar to which 1 per cent, of 
maize starch is added. He finds that the gonoooocus remains 
alive for two to three weeks on this medium as against two 
to three days on the usual salt-free veal agar. 

The utilization of bacterial enzymes for the removal of 
starchy matter from textile materials has been recommended 
by Boidin, 39 and by JBoidin and EfEront, 40 who use the amylases 
secreted by Bac. mesentericus and Bac. subtiUs for this 
purpose. 

Bac. mesent&ricua has been recommended also by Effront 84 
for the liquefaction of starch-containing mashes in the dis- 
tillery. He grows the organism on distillery residue, a medium 
which appears to be highly suitable, since 1 kg. of the residue 
on which Bac. mesentericus has been allowed to develop is 
stated to be equal in starch liquefying power to 20 kg. of 
malt. The optimum temperature of this amylase is given as 
40 C. It is very resistant to alkali but somewhat more sensi- 
tive to acid, a 1 per mille concentration of hydrochloric acid 
(pH value about 2-9) arresting its action completely. Like 
Fermi, Efiront isolates the enzyme from the culture of the 
organism by precipitation with alcohol. Ammonium sulphate 
also appears suitable for this purpose. The products of hydro- 
lysis are stated to consist of dextrins and maltose 41-2 per 
cent, of the latter. 

For the saccharincation of starch mashes in distilleries 
Jouola 41 advocates the use of Bac. burdigalense, a type related 
to Bac. tennis, Duclaux, Lehmann and Neumann, and recom- 
mended also by Bettinger 42 for this purpose. This organism 
is used either alone or in symbiosis with a Mucor species, 
Mucor eloeis, which is claimed to possess twice the sacchari- 
fying power of Mucor Rouxii. Though Bac. burdigalense is 
capable of developing even at 60 C. the mash must be cooled 
to 36 C., when a mixed culture of the rod and Mucor eloeis is 



22 MICROBIOLOGICAL HYDROLYSIS OF 

employed, since the fungus is unable to develop at the higher 
temperature. 

Other thermophilio or thennotolerant amylase producing 
bacilli have been isolated by Coolhaas 28 from mud. The most 
active form, Bac. tTiermoamylolyticiis, penetrates the starch 
granules attacked by it, gradually dissolves them, and con- 
verts them into maltose. In the course of 9 days this organ- 
ism was capable of converting 80 per cent, of the starch into 
maltose, a conversion equal in magnitude, though not in 
speed, to that of malt amylase. The maltose is subsequently 
converted into various fermentation products the nature of 
which indicates that Sac. th&rmoamylolyticus belongs to the 
group of butyric acid bacilli. 

THE HYDROLYSIS OF STAJROH BY AOTINOMYOETBS 

Beyond the observation that a great many Actinomycetes 
hydrolyse starch, little is known on the subject. Of interest 
is Fermi's 8 observation that such important species as Coryne- 
bacterium diphtheriae and Mycobacterium tuberculosis actively 
hydrolyse starch. The hydrolysing properties of the latter 
are regarded by Fermi 43 as an additional proof for its relation- 
ship to the Actinomycetes. Of other species producing 
amylase may be mentioned Actinomyces boms &-Q& Actinomyces 
dicLstaiicus. In their studies on the classification of the Acti- 
nomycetes both Waksman and Curtis 47 and Krainsky 45 have 
paid attention to the amylolytio properties of the various 
species examined. It should be recorded also that Rullmann 46 
found that the presence of starch in cultures of Actinomyces 
odorifera gave rise to the emanation of an intensified odour 
of damp earth. 

HYDEOLYSIS OF STAKOH BY FUNGI 

Commensurate with its greater industrial importance starch 
hydrolysis by fungi has received more attention in the past 
than saccharification through bacteria or through actino- 
mycetes. In the Far East certain fungi have been used for 
many centuries (Kozai 47 ) for purposes where in the West 



STABCH, GLYCOGEN, AND INHLIN 23 

malt is employed. The preparation of alcoholic beverages 
such as sake" , and of the aromatic sauce soya, is based on the 
saocharification of starch by species of lower fungi. The value 
of these products, taking Japan alone, was stated by Taka- 
mine 48 to amount to 40,000,000 in 1914. 

Both in the case of sake 1 and of soya manufacture, the first 
operation consists in the preparation of a 'koji', or culture 
of the micro-organisms active in the hydrolysis of the starch. 
The following description of this process is taken from Atkin- 
son's 49 classic account in 1881, though Korsohelt 60 can lay 
claim to a still earlier description : 

After removing the husks and the inner skin the cleaned 
grain, usually rice, is soaked in water overnight and heated 
on the following morning in a current of steam until the 
grains have become elastic to the touch. This operation 
takes from four to five hours. The soft grain is then placed 
on mats and cooled with stirring till its temperature has fallen 
to 28-30 C. To every 75 kgs. of the cooked grain 'three 
salt spoons full' of spores of the active fungus, usually Asper- 
gillus oryzae, are added. The spores are first mixed with a 
small proportion of the steamed grain, and this mixture then 
scattered over the rest. The inoculated rice is gathered in 
baskets and carried to the coolest part of the growing cham- 
ber, which normally is built underground. The grain is thrown 
in a heap on the floor, covered with mats and left overnight. 
After about 20 hours it is removed and sprinkled with water. 
The temperature of the grain thereby falls to about 23 C. 
Where the koji is to be used for sake" brewing this sprinkling 
process is omitted. In this case the preparation is termed 
raw koji. In the afternoon of the second day the sprinkled 
grain is spread thinly on trays, placed in the growing chamber 
underneath other trays containing nearly finished koji. The 
temperature of the grain rises during the night, and by the 
following morning has reached a figure of about 25 C. It 
is now gathered in small heaps on the trays and allowed to 
remain undisturbed for about three to four hours. By then 
its appearance has changed. Through the development of 



24 MICROBIOLOGICAL HYDROLYSIS OF 

mycelium it has become woolly. The temperature of the 
heaps has reached 40-41C. In order to cool the grain 
the heaps are broken up, aerated, and again built up, to be 
left for another four to five hours until the temperature has 
risen to 40 C. The grain is now finally scattered in thin layers 
on trays and the fungus allowed to spread its silky threads 
of mycelium throughout the mass. The koji is left on the 
trays for about 20 hours until it has set to a solid cake, the 
ionn in which it is disposed of for industrial purposes. 

For other types of koji, bean flour, millet or roasted barley 
are used, and other species of fungi, among them Asp&rgillua 
batotoe, which yields a black koji, coloured by the dark pig- 
mented conidiospores of the fungus (Saito 61 ). 

In India, according to Hutchinson and Ramayyar, 52 an 
enzyme similar to koji is prepared from rice, powdered roots, 
and other parts of certain plants. It is known under the name 
of Bakhar, and is used in the manufacture of rice beers. It 
contains a great variety of fungi, the most active being Asper- 
gillus oryzae. Its saccharifying powers are markedly less than 
that of koji, and its alcohol-producing properties equally 
deficient owing to the inferior fermenting power of the yeast 
types present. 

In Formosa a species of Rhizopus is used for a similar pur- 
pose in the preparation of an alcoholic beverage, 'Biityn' 
(Takeda 53 ), and in the Malay States the Chinese inhabitants 
prepare a distilled beverage, using Mucor Rouxii (Bishop and 
Feik 54 ) as the fungus for the hydrolysis of the raw material, 
rice. 

From the fact that various Asiatic countries have acquired 
what amounts to a monopoly in the industrial application of 
fungi to the hydrolysis of starch, it is not to be deduced that 
the fungi met with in Asia are exceptionally vigorous amylase 
producers. Nill, 56 who undertook to ascertain whether this 
might be the case, found that a number of Rhizopus species 
could be isolated from German cereals which produced amylase 
to the same extent as the well-known Asiatic species, Rhizopus 
tonkinevisis, and Asp&rgiUus oryzae. 



STAECH, GLTCOGEN, AND INULBT 26 

In soya koji, Saito 61 found a number of other fungi besides 
Aap&rgillua oryzae ; among them EMzopus ja/panicus, Olado- 
sporium herbarum, and Penicittium glaucum. The active type 
was considered to be Aspergillus oryzae. Nishiwaki 68 reports 
the preparation of, a soya koji, using Monilia sitopMla as 
active fungus. 

A description of the preparation of soya is given by Saito. 
The previously prepared koji, for which bean flour and wheat 
flour are frequently used in the proportions of 60 parts to 46. 
parts, is mixed with a steam-boiled solution of 2-8 per cent, 
sodium chloride in the proportions of equal parts of koji and 
salt- water, and the mixture stirred from time to time during 
the whole process of fermentation, which takes from one to 
one-and-a-quarter years to complete. The mash gradually 
acquires a deep reddish-brown colour. When the fermenta- 
tion is finished the mash is pressed in bags, previously soaked 
in the tannin containing juice of unripe date plums (Diospyros 
Kaki). On standing the filtrate separates into an oily and a 
watery layer. The latter, the soya, is left standing for a few 
days after separation from the oil and, when clear, is bottled 
and heated to 50 C. for the purpose of sterilization. 

In the preparation of taka-koji, a substance which has 
become widely known in the form of an extract termed taka- 
diastase, Takamine 48 uses wheat bran instead of grain. The 
bran is mixed in a rotatory drum with an antiseptic and a 
requisite amount of warm water, and is gelatinized by live 
steam. After cooling, the mass is inoculated with spores of 
Asp&rgillus oryzae, acclimatized to the antiseptic used, and 
cool air is passed through the drum to prevent excessive 
heating. After 48 hours' incubation the koji is finished. 
Takamine reports that Ortved found 4 per cent, of this koji 
capable of hydrolysing a starch paste in 15 to 20 minutes. 

The types of antiseptics suitable for the suppression of in- 
fections in preparations of taka-diastase have been investi- 
gated by Oshima, 67 who recommends cresol in concentrations 
of 0-15 to 0-4 per cent., calculated on the solution of taka- 
diastase, lysol 0-5 to 2-0 per cent., phenol 0-4 to 1-5 per cent., 



26 MICROBIOLOGICAL HYDROLYSIS OF 

thymol 0-06 to 0-2 per cent., and mixtures of phenol and 

cresol. 

Chloral hydrate, chloroform, clove oil, formalin, potassium 
cyanide, mercuric chloride, sodium fluoride, sodium benzoate, 
salicylic acid, toluene, and xylol were all found unsuitable. 

By extracting the koji with water and precipitating the 
extract with alcohol, for which purpose Sherman and Tan- 
berg 58 recommended a strength of 60 to 65 per cent, by 
volume, the active enzyme is obtained in dry form. This 
substance has been extensively investigated during the last 
decade. A review of the more important literature is given 
in the following lines. 

Takamine states that taka-diastase is a mixture of several 
enzymes, the amylase being considerably more resistant to 
acids than malt diastase and more stable on storage. A fuller 
account of the various enzymes present in taka-diastase is 
given by Wohlgemuth, 69 who identified amylase, maltase, 
trypsin, rennet, erepsin, lipase, and haemolysin all the 
enzymes of the pancreatic juice except peptolytic enzymes. 
He found that one gram of taka-diastase contained as much 
trypsin as 10 cc. of active pancreatic juice. In addition to 
the above enzymes Nishimura 60 records the presence in taka- 
diastase of sacoharase, protease, lactase, oatalase, inulase, 
sulphatase, and amidase. The experimental data of a number 
of investigations support the view that the amylase of taka- 
diastase is a mixture of depolymerising and saccharifying 
enzymes. Thus Sherman and Tanberg 58 record that the 
amyloclastic enzyme is considerably more active than that of 
the best malt amylase, while the saccharifying enzyme is less 
active than that of malt amylase. Expressed in figures, Waks- 
man 61 gives the ratio of amyloclastic to saccharifying enzymes 
as 1 :1, or even 1-5 :1, as against 1 :4 or 1 .5 for malt amylase. 

A comparative study of the hydrogen ion concentration 
ranges of taka-diastase or rather of the amylase of Asper- 
gittus oryzae malt amylase, and pancreas amylase was under- 
taken by Sherman, Thomas, and Baldwin. 62 They found that 
Asp&rgillus amylase was active between the pH values of 



STAECH, GLYCOGEN, AND INUUN 27 

2-6 and 8-0, with an optimum at 4-8. Malt amylase was active 
between pH values 2-5 and 9-0, with an optimum between 
4-4 and 4-5. These ranges, however, should not be regarded 
as fixed and typical for this particular enzyme. According to 
Chrzaszcz, Bid /in ski, and Krause, 63 they will vary with the 
prevailing temperature, with the protective action of the 
starch, and with the presence or absence of substances acting 
as buffers. Conditions, these writers found, which are un- 
favourable to the action of amylase activity shifted the 
hydrogen ion concentration towards the alkaline side of the 
neutral point, while favourable conditions moved jit towards 
the acid side. 

The resistance of taka-diastase to acids is emphasized by 
Okada, 64 who states that the enzyme regains its former activity 
on neutralization after having been kept for some time at a 
pH value not exceeding that which usually obtains in the 
gastric juice of man (0-9-1-6). 

The temperature range for 'polyzime ', another preparation 
of the enzymes of Aspergillus oryzae, was investigated by 
Takamine jun. and Oshima. 65 They record a maximum 
activity at 40 C. Kept at 50 C. for three hours the prepara- 
tion lost 55 per cent, of its diastatic powers. At 40 C. and 
less it could be kept for six months in a closed vessel without 
any loss of activity. 

The action of electrolytes on the enzymes of Aspergillus 
oryzae and on their preparations has been studied by a num- 
ber of investigators. Effront 66 finds that phosphates and 
aluminium salts have an accelerating action, and he has noted 
a similar action by asparagin. 

Sodium chloride and potassium chloride had no action on 
the enzymes in Sherman and Tanberg's experiments, while 
acid phosphates accelerated, and alkaline phosphates re- 
tarded, the activity of taka-diastase. For the protection of 
the enzymes on storage Kita 67 recommended concentrated 
solutions of sodium chloride. Calcium salts in Kita and 
Kyoto's 68 experiments accelerated the amyloclastic enzyme 
but sometimes retarded the saccharifying enzyme. 



28 MICBOBIOLOGICAL HYDEOLYSIS OF 

Under optimum conditions taka-diastase converts from 
70 to 80 per cent, of the available starch into the reducing 
sugars maltose and glucose, and attains a degree of conversion 
therefore Himi1a.r to that of malt diastase. Taken in large 
excess, however, Nishimura 69 found that an extract from 
A&pergillus oryzae, and therefore presumably also taka- 
diastase, was able to hydrolyse 95 per cent, of the available 
starch, irrespective of whether a complement (Pringsheim 
and Otto 70 ) was present or not. 

The incomplete conversion of the starch by taka-diastase 
under ordinary conditions is therefore perhaps not caused 
by the absence of a complement, but may be due to the 
accumulation of reducing sugars and even of saccharose 
substances which various "investigators, Grezes, 71 Katz, 72 
Funke, 73 and Yamagishi, 74 have found to inhibit the progress 
of the hydrolysis in proportion to their concentration. 

For the quantitative determination of the diastatio power 
of taka-diastase a number of methods have been recom- 
mended. Those recommended by Lintner 75 or by Wohlge- 
muth 76 for malt diastase might of course be used. A more 
satisfactory method appears to be that evolved by Waksman 61 . 
It is based on the resolution of the starch of a potato starch 
paste by the enzyme to be tested without considering the 
question of how much of the starch is converted into poly- 
amyloses, how much into dextrins, and how much into re- 
ducing sugars. The potato starch used is first stained red 
with neutral red and then made into a 2 per cent, starch 
paste. The paste is filled into test tubes, 100 o.cms. in each, and 
these placed in an incubator at 40 C. When the paste has 
reached this temperature sufficient enzyme is added to the 
tube to cause a resolution of the starch in not less than one 
minute, and in not more than fifteen minutes. Tubes liquefied 
outside these limits are rejected. The resolution of the starch 
is easily recognized by the liquid becoming clear and reddish 
coloured. The various tubes must be shaken from time to 
time during digestion to ensure thorough mixing. As each 
tube clears the time taken is recorded. 



STARCH, GLYCOGEN, AND INULJN 29 

The enzymatic power is deduced from the f ollo^w&g f ormulay ' 

F = v v 

ExT " - - v- 

where F represents the enzymatic power at 40 C. ; D the 
dilution of the enzyme; t the time taken for the standard 
unit of enzyme to resolve the starch (t 30 min.) ; E the quan- 
tity of diluted enzyme ; and T the time taken by the actual 
amount of enzyme added to liquefy the starch. Thus if the 
enzyme has been diluted ten times and 0-2 o.cms. of this dilu- 
tion has been found to liquefy 10 o.oms. of the starch paste in 
six minutes the enzymatic power expressed as units would be 

-30 min. 10X30 

, AO rt = = 350 units. 

.40C. o-2x6 

Attempts to increase the diastatic properties of taka- 
diastase through precipitation were made by Sherman and 
Tanberg. 68 They found that extraction of the commercial 
product with water, precipitation of the solution with am- 
monium sulphate, subsequent removal of the salt by dialysis, 
and final precipitation of the resulting solution of the enzyme 
by alcohol yielded an amylase which in some cases showed 
an increased activity of nearly thirty times that of the com- 
mercial product. 

In addition to Aspergillus oryzae, a number of other Asper- 
gillus and Penicillium species have been studied from the 
point of view of their amylolytic enzymes. Among them may 
be mentioned Aspergillus niger, investigated by Gayon 77 
and by Fernbach, 78 Aspergillus batatae and Aspergillus 
pseudoflavus by Saito, 51 Aspergillus atibus, Aspergillus candidus, 
and Aspergillus Okazakii by Okazaki, 79 Aspergillus terricola 
by Scales, 80 and Aspergillus glaucus and Penicillium glaucum 
by Duolaux. 81 

Though all of these species hydrolyse starch and many of 
them are used in the East for the manufacture of alcoholic 
beverages, they do not convert carbohydrates into alcohol 
at least not to any appreciable extent. Sanguinetti 82 reports 
a yield of 4 per cent, on the carbohydrates converted in the 



2205 



30 MICROBIOLOGICAL HYDEOLYSIS OP 

case of Aspergill/us oryzae. Far more active in this respect 
are a number of Mucor species used in the East for fermenta- 
tion purposes. The first Mucor species reported capable of 
hydrolysing starch and dextrins and converting the resulting 
saccharides into alcohol were Mucor circindloides and Mucor 
dltemans, studied by Gayon and Dubourg 83 in 1886 and 1887. 
The utilization of Muoor species for industrial purposes was 
not seriously considered, however, until after Calmette's 84 
publication on 'Chinese yeast' in 1892. This 'yeast' was 
found by Calmette to be a Mucor species, which he named 
Amytomyces Rouxii, and which is now usually referred to as 
Mucor JRouxii. It produces a very active amylase when 
grown aerobioally. On elimination of the oxygen supply it 
utilizes the available carbohydrates as hydrogen acceptors* 
in the place of oxygen and converts them to ethyl alcohol, as 
much as 35 per cent, of alcohol being formed from the sugar 
fermented. 

Calmette succeeded in isolating the amylase produced by 
Mucor JRouxii, using Duclaux's method a method to which 
a few lines must be devoted here, since it is often advocated 
as suitable for the isolation of fungus enzymes. Duclaux 86 
inoculates a culture medium containing inorganic and organic 
food substances Ranlin 's medium, for instance with spores 
of the fungus from which he desires to obtain an enzyme, and 
allows growth to proceed to maturity. This is usually reached 
after 96 hours' incubation at the optimum temperature. At 
this stage the fungus covers the culture medium with a densely 
matted layer of mycelial growth containing numerous spo- 
rangia. The accumulation of enzyme in the medium is now 
particularly rapid, and the spent culture medium, with its 
salts and decomposition products, is therefore replaced by 
distilled water, into which the enzyme continues to diffuse. 
An aqueous solution of the enzyme is thus obtained and can 
be concentrated by evaporation in vacuo. Or the enzyme 
may be separated from the aqueous solution by precipitation 

* See Chapter V. 



STARCH, GLYCOGEN, AND INULIN 31 

with alcohol. Duclaux in his account of the secretion of 
enzymes emphasizes the need for the presence in the culture 
medium of the substance specifically decomposed by the 
enzyme to be collected. This, however, does not appear to be 
essential in all cases. Thus taka-diastase has been found to 
contain both rennet and haemolysin, though its mode of 
preparation precludes the presence of either milk or red blood 
corpuscles during its secretion by Aapergittus oryzae. Katz 72 
in his studies on the amylolytic properties of Asp&rgillus niger, 
Penicillwm gkwcum, and Sac. Megath&riwm found that 
amylase was secreted by these types even where starch was 
absent from the culture medium. The secretion of the enzyme 
could, however, be influenced by the composition of the 
medium. If for example the medium contained 5 per cent, 
of saccharose or 2 per cent, of glucose, amylase secretion 
became completely inhibited in the case of P&nicillium 
glaucum and Bac. Megatherium, while even 30 per cent, 
of saccharose was insufficient completely to check the secre- 
tion of amylase in the case of AspergiUus niger. This obser- 
vation confirms the experimental evidence discussed in 
Chapter XXm on the action of species of Aspergilli on 
sugar stored in bulk. 

Maltose was found to be less inhibitory to the secretion of 
amylase than glucose or saccharose, and erythrodextrin even 
less. Peptone was found to accelerate amylase production in 
the case of all three types of micro-organisms with which 
Katz experimented. The addition of tannin, which removed 
by precipitation the enzyme secreted, was also found to in- 
crease its production by the test organism. 

In discussing the question of the influence of the medium 
on the secretion of enzymes it is interesting to recall that 
Duclaux 81 found that a typical starch hydrolysing fungus, 
such as Aspergill^ niger, did not germinate from its spore in 
a medium consisting exclusively of ungelatinized starch. To 
achieve growth it was necessary to add traces of water soluble 
carbohydrates, such as maltose or saccharose. In starch 
pastes, however, germination took place within the normal 



32 MICROBIOLOGICAL HYDROLYSIS OF 

time, even when no traces of copper-reducing substances 

could be detected in the paste. 

The available information on the physical and chemical 
properties of the amylase isolated by Calmette from Mucor 
Souxii is not very definite. Calmette himself states that the 
optimum activity of the enzyme is reached between 35 C. and 
38 C., and that heating to 72 C. destroys it. The addition of 
calcium carbonate is claimed by Calmette to impede its 
activity. An indication of its acidity range is supplied by 
Bellinger and Delaval. 88 They found that optimal conditions 
prevailed when Mucor Rouxii acted in a mash containing 
6 litres of concentrated hydrochloric acid per 1,000 kg. of 
grain. Assuming their mash to have been of 10 per cent, 
strength, the acid added would be sufficient to establish a 
hydrogen ion concentration equal to a pH value of about 
4-0 in the mash. 

Subsequent to Calmette's publication a number of de- 
scriptions of other amylolytio Mucoraceae have been given. 
Among these may be mentioned Mucor Cambodia (Chrzaszcz 87 ), 
Mucor doeis (Joucla 88 ), and Mucor batatas (Nakazawa 89 ). 
The amylase in Mucor stolonifer was studied by Durandard, 90 
who records a temperature range for_this enzyme from 10 C. 
to 65 C., with an optimum at 46 C. 

Though this is not the place to enter into a discussion of 
the alcohol-producing properties of the various Mucor species, 
a brief account has been included of the methods adopted for 
the utilization of muoor species in distilleries, in the processes 
frequently described under the names of the Amylo process 
and the Boulard process. Most of the literature dealing with 
this subject is difficult of access owing to patent restrictions, 
and Galle's 91 account in 1923 therefore fills a long-felt want. 
The data supplied here are taken from Galle's paper, which is 
based on practical experience with the working of the Amylo 
process. For the laboratory cultivation of the fungus a mash 
containing 16 to 17 per cent, of starch is filled in test tubes to 
5 o.cms. and in litre flasks to 250 c.oms. and sterilized for 20 min. 
at 1-5 atm. After 24 hours' standing the media are sterilized 



STARCH, GLYCOGEN, AND INDLIN 33 

again. A further medium used consists of rice (20 gr.) mois- 
tened with 1 o.om. of water and contained in sufficiently large 
containers to allow abundant air-space. This medium, like 
the liquid mash, is sterilized in the autoclave on two con- 
secutive days at 1-5 atm. for 20 min. 

When required for starting large-scale fermentations the 
sterile tubes containing liquid mash are inoculated with a 
pure culture of Mucor Rouxii, or any other Mucor strain 
which may have been chosen, and incubated at 38 C. After 
four to five days' incubation these cultures are fully developed 
and the surface of the liquid is covered with a mass of black 
sporangia. The ripe cultures are placed in a dark, cool place 
when not required for immediate use. Before the inoculation 
of the sterilized rice grains the contents of a tube are carefully 
shaken until the spores contained in the sporangia have been 
distributed throughout the liquid in the tube. The rice is 
then sprinkled with this liquid and incubated at 38 C. for 
eight days. After satisfactory development it is shaken with 
260 o.cms. of sterile mash and left at 38 C. for 24 hours for 
the spores to germinate. If free from infecting bacteria this 
material is used to inoculate a seed tank holding a quantity 
of mash equal to 10 per cent, of that finally to be fermented. 
The mash in the seed tank is sterilized by live steam for one 
hour at 110 C. (0-9 atm.) and cooled to 39 C. before inocula- 
tion. After 16 hours' incubation the first hyphae become 
visible. For the satisfactory development of the fungus it is 
essential that the reaction of the mash, as well as its tempera- 
ture, should be maintained at the optimum. The acidity of 
the mash must not be allowed to increase beyond a concen- 
tration which requires 2 to 3 c.cms. of NaOH for neutrali- 
zation of 20 c.oms. of the mash. After IShours in the seed tank 
the mash is used as inoculant for the mash in the fermenting 
vat. Here the acidity must be checked every six hours and 

not allowed to increase beyond 5 c.cms. of NaOH per 
20 o.cms. of the mash. Twenty-four hours after inoculation the 



34 ' MICROBIOLOGICAL HYDROLYSIS OF 

mash in the fermenting vat is cooled to 32 C. and inoculated 
with ayeast mixture. Thereupon the mash is aerated for about 
six hours. After 4=8 hours the fermentation is completed. The 
whole process in the factory therefore takes three days. At 
the end of the fermentation the acidity may have risen to 

7 c.cms. aoid per 20 c.oms. of mash. 

In many factories where the Amylo and the Boulard pro- 
cess are worked it is customary to assist the liquefaction of 
the grain by soaking it at 70 C. with the requisite amount of 
water to which 0-8 per cent, of sulphuric aoid have been 
added, calculated on the weight of the grain taken. During 
the subsequent cooking the mash becomes more liquid than 
if no aoid had been used. In consequence higher concentra- 
tions of grain than would normally be possible can be used. 
Bellinger and Delaval in their publication already referred to 
mention that the addition of acid is more favourable for the 
subsequent activity of the amylase than the use of a small 
amount of malt added for the purpose of liquefaction of the 
mash. 

The production of amylase by higher fungi cannot be dealt 
with in these pages. It has already been mentioned that very 
few species belonging to the order of the Eumycetes lack the 
property of amylase secretion. Its presence in the mycelium 
of the higher fungi has frequently been recorded, for instance 
by Zellner. 92 The importance of starch hydrolysis in the 
microbiological destruction of wood was referred to in Thay- 
sen and Bunker's 4 treatise on the microbiology of cellulose. 

It remains to draw attention to the connexion which must 
exist between the amylase-producing properties of many 
fungi and the discoloration and destruction of starch in 
storage. In this destruction fungi participate and may per- 
haps even be solely responsible. It would seem that they are 
capable not only of spreading throughout the mass of the 
starch but actually of penetrating the individual starch 
grains. Lindner's 93 observations on mouldy potatoes at least 
indicate that this is what may occur. 



STARCH, GLYCOGEN, AND INULIN' 35 

Before leaving the subject of starch hydrolysis reference 
must be made to the interesting observation of Gramenitzki 94 
on the regeneration of amyloclastic enzymes after exposure 
to high temperatures. It has already been mentioned that 
temperatures of from 70 C.-80 C. destroy the characteristic 
properties of starch hydrolysing enzymes. Gramenitzki finds 
that this destruction in many cases is an inactivation rather 
than a destruction an inaotivation from which the enzymes 
may recover. Thus a solution of taka-diastase heated for a 
short time to 80-85 C. will show an appreciable regeneration 
of its amyloclastic properties when left at room temperature 
for one or two days, When left at 40 C. the recovery of its 
specific properties is more rapid, though less complete, and 
is maintained for only four to sis hours. Kept at 45 C. there 
is only a slight regeneration, which is lost again after two to 
three hours. Kept at 50 C. no regeneration at all takes place. 
The degree of regeneration therefore depends on the tempera- 
ture at which the enzyme is kept after exposure. But in 
addition it is governed also by the degree to which the enzyme 
has been heated, a short exposure to 115 C. being the maxi- 
mum beyond which no regeneration occurs. The more active 
an enzyme the more readily will it become regenerated, and as 
a general rule dilute solutions of taka-diastase are more easily 
regenerated than concentrated solutions. Whether the enzyme 
solution has been previously dialysed or not does not appear to 
affect its regenerative powers, which therefore must be regarded 
as independent of the conductivity of the enzyme solution. 
It is interesting to note that Gramenitzki found the sac- 
charifying enzyme of taka-diastase less resistant to heat than 
the amyloclastic enzyme, and that it lost its regenerative 
power at a lower temperature (100 C.) than the amyloclastic 
enzyme. This observation offers additional proof for the 
contention that the two enzymes are different. 

Glycogen. The hydrolysis of glycogen by micro-organ- 
isms has been investigated in the past almost exclusively 
from the point of view of the Saccharomycetea, in the cell of 
which it occurs as a reserve carbohydrate. Occasionally a 

D2 



36 MICROBIOLOGICAL HYDROLYSIS OF 

reference is met with in the literature, such as that of Eeinze 91 
or that of Pringsheim and Lichtenstein, 96 which deals in some 
detail with the action on glycogen of specific micro-organisme 
other than yeast. In such cases the conclusion is invariably 
arrived at that this polysacoharide is hydrolysed by those 
micro-organisms which produce amylolytic enzymes. Realiz- 
ing the close relationship, bordering on identity, which exists 
between glycogen and amylopectin this result is not surprising 

Inulin . Even more unsatisfactory is the existing knowledge 
of the microbiological decomposition of inulin. No sys- 
tematic investigation like that of Fermi's on the amylolytio 
micro-organisms has so far been carried out, and the occa- 
sional reference to inulin decomposition leaves no impression 
as to the extent to which this polysaccharide is utilized for 
instance by bacteria and actdnomycetes. Among fungi the 
secretion of an inulase appears to be fairly widespread, and 
inulin decomposition has been observed both among higher 
and lower types. Thus Bourquelot 97 found inulase in Asper- 
giUus niger, Griiss 98 in Ustilago Maydis, Dean 99 in Aspergillus 
niger and a Penicillium species, Iron 100 in Morchetta, Weh- 
mer 101 in some Mucar species, Hanzawa 102 in EUzopus Dele- 
mar, introduced by Boidin into the Amylo process, and 
Castellani and Taylor 108 in Manilla macedoniensis. 

The physical constants of the inulase of Aspergittus niger 
were studied by Boselli, 104 who determined the optimum 
temperature as 51 C. and the optimum acidity at this tem- 
perature as H 2 S04, equal to a hydrogen ion concentration 

of the pH value 2-3. A 'slight* alkalinity was found by him 
to arrest the action of the enzyme. In Dean's experiments 
the optimum temperature of the enzyme is given as 56 C. 
and the optimum hydrogen ion concentration at a pH value 
of 4-0. Dean also determined the alkalinity capable of arrest- 
ing the action of the enzyme. It was equal to a pH value of 
10-14. In its sensitiveness to alkalies the microbiological 
inulase greatly resembles the inulase isolated by Green 106 
from Jerusalem artichokes. 



STARCH, GLYCOGEN, AND INUUN 37 

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38 MICROBIOLOGICAL HYDROLYSIS OF 

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62 H. C. Sherman, A. W. Thomas, and M. E. Baldwin, J. Am Chem. Soc , 
vol. 41, p. 231, 1919. 

63. T. Chrzoszcz, Z. Bidzinaki and A. Kranse, Biochem. Z., vol. 160, p 166, 

1925. 

64. S. Okada, Biochem. J., vol. 10, p. 130, 1916. 

65. J. Takamine Jr. and K. Oshima, J. Am. Ghem. Soc., vol. 42, p. 1261, 

1920. 
60. J. Effront, Comptes rend., vol. 116, p. 1324, 1892. 

67. G. Kita, J. Ind. Eng. Ghem., vol. 5, p. 220, 1913. 

68. G. Kita and E. Kyoto, Mem. Cdl. Imp. University, Tokyo, vol. 2, p. 1, 

1918. 

69. S. Nishimura, Chem. Absts., vol 22, p 2233, 1928. 

70. H. Pringsheim and G. Otto, Btochem. Z , vol. 173, p 399, 1926. 

71. G. Grazes, Ann. Inst. Pasteur, vol 26, p. 656, 1912. 

72. J. Katz, Jahrb. Wtss. Botantk, vol. 31, p. 599, 1898. 

73. G. L. Funke, Rec. trav. lot. Neerland., vol. 23, p. 200, 1926. 

74. H. Yamagishi, Japan J. Biol., vol. 4, p. 61, 1928. 

76. C. J. Lintner, Z gesamm. Brauwesen, vol. 8, p. 281, 1886. 

76. J. Wohlgemuth, Biochem. Z , vol. 9, p. 1, 1908. 

77. U. Gayon, Comptes rend., vol. 86, p. 52, 1878. 

78. A Fernbach, Ann. d. brasserie et disttllerie, vol. 2, p 409,1889. 

79. K. Okazaki, Zentrbl f. Bakt., Abt II, vol. 42, p. 225, 1914. 



STAECH, GLYCOGBN, AND INULm 39 

80. P. M. Scales, J. Sid. Ohem., vol. 19, p. 469, 1914. 

81. E. Duolaux, Ann. Inst. Pasteur, vol. 3, p. 97, 1889 ; vol. 6, p. 97, 1889. 

82. J. Sanguinetti, Ann. Inst. Pasteur, vol. 11, p. 264, 1897. 

83. U. Gayon and E. Dubourg, Oomptes rend., vol. 103, p. 885, 1886. Ann. 

Inst. Pasteur, vol. 1, p. 632, 1887. 

84. A. Oalmette, Ann. Inst. Pasteur, vol, 6, p. 604, 1892. 

86. E. Duolaux, Fremy's Enoydopedie Chimique, vol. 9, part I, p. 191, 1883. 
Dunot, Paris. 

86. P. Bellinger and H. Delaval, Butt, oasoc. chim. sucr. et diet., vol. 37, 

p. 264, 1920. 

87. T. Ohrzaszcz, Zentrbl.f. Bakt., Abt. IE, vol. 7, p. 326, 1901. 

88. H. Joucla, Fr. Pat., 474948, 1914. 

89. R. Nakazawa, Zentrbl.f. Bakt., Abt. IE, vol. 24, p. 482, 1909. 

90. M. Durandard, Comptes rend., vol. 167, p. 471, 1913. 

91. E. Galle, Z. angew. Ohem., vol. 30, p. 231, 1909. 

92. J. Zellner, Monatshefte d. Ohem., vol. 30, p. 231, 1909. 

93. P. Lindner, Z.f. 8piritusmd., vol. 43, p. 213, 1920. 

94. M. J. Gramenitzki, Z f. pjiysiol. Ohem., voL 69, p. 286, 1910. 
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96. H. Pringaneim and S. Liohtenstein, Benchte, vol. 49, p. 364, 1916. 

97. E. Bourqnelot, Comptes rend., vol. 116, p. 1143, 1893. 

98. J. Gruss, Berichte d. deutsch. botan. OeseUsch., vol. 20, p. 213, 1902. 

99. A. L. Dean, Botanical Gazette, vol. 36, p. 24, 1903. 

100. G. Frou, Oomptes rend., voL 140, p. 1187, 1906. 

101. C. Wehmer, Lafar's Handbuch der techn. Mykdlogte, vol. 4, p. 627, 

1905-7. Gustav Fischer, Jena. 

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105. J. R. Green, Annals of Botany, vol. 1, p. 223, 1888. 



CHAPTER in 

THE HYDROLYSIS OF TETRA-, TRI-, AND 
DISACCHAEIDES 

IT seems obvious that before starch, glycogen, and inulin 
can be assimilated by micro-organisms they must undergo 
a depolymerisation and hydrolysis to soluble carbohydrates. 
This has not been seriously disputed. But in the case of 
soluble carbohydrates among those saccharides which Prings- 
heim 1 terms polysacoharides of the first order, the concep- 
tion of a preliminary hydrolysis before assimilation has never 
been generally accepted although a vast amount of evidence 
has been accumulated demonstrating the frequent occurrence 
of hydrolysis of such saccharides. Abderhalden's 2 theory, 
therefore, that a food substance must be decomposed to its 
simplest units before it can be assimilated has not always 
been accepted for the tetra-, tri-, and disaccharides. For 
instance it has occasionally been maintained that a di- 
saccharide might readily be assimilated by micro-organisms 
but its component monosacoharides be of little or no value as 
a food substance. Thus Rose 8 found that Endomyces Magnusii 
could assimilate maltose, while glucose, though fermented, 
did not promote growth. Similar observations were recorded 
by Lindner and Saito 4 for certain types of yeasts. Kluyver, 6 
who reinvestigated this question and confirmed that certain 
yeasts showed better development in maltose than in glucose 
solutions, was able to trace this anomaly to the presence of 
small amounts of nitrogenous impurities in the pure maltose 
used. On removal of these, maltose was found to behave 
exactly like glucose, thus showing that the readier assimilation 
of maltose was due not to the greater suitability of the carbo- 
hydrate, but to the presence of nitrogen which, when added to 
glucose, rendered the latter as readily assimilable as the 
disaccharide. 



THE HYDBOLYSIS OF TETRA-, TRI-, AND DISACCHAEIDES 41 
A direct assimilation of saccharose is stated by Pringsheim 
and Zemplen 8 to be possible in the case of certain fungi. The 
experimental evidence given in support of this statement is 
not altogether convincing, and is based solely on the failure 
of the investigators to demonstrate the presence of a hydro- 
lysing enzyme in the press juice and in the dried mycelium 
of some of the fungi studied which were capable of hydro- 
lysing saccharose in vitro. It is conceivable, as Kluyver 
points out in the paper referred to above, that the hydrolysis 
in this case in some way had been connected with the life 
function of the fungi and therefore evaded detection. But it 
might be also that the technique adopted by the investigators 
for the preparation of the mycelial extracts was unsuitable 
and had destroyed the inverting enzyme responsible for the 
hydrolysis. Whatever the explanation, Pringsheim and 
Zemplen's investigations do not suffice to dispose of the 
theory that a soluble polysaccharide must be decomposed to 
its basic units before it can be assimilated by micro-organ- 
isms. No more convincing is Violle's 7 observation on a certain 
lactic acid-producing Str&ptococcits, the behaviour of which 
has been quoted in support of the theory that polysaccharides 
may be assimilated without preliminary hydrolysis. Violle 
finds that his organism develops exceptionally well on media 
containing saccharose, and reports that it is unable to utilize 
either glucose or fructose. He emphasizes, however, that 
an inverting enzyme is contained in such cultures, and thus 
indirectly admits that the surprising results of his experi- 
ments with glucose and fructose must have been due to faulty 
technique and not to an abnormal behaviour of the micro- 
organisms studied. More important evidence in favour of a 
direct assimilation of saccharose was brought forward by 
Gayon and Dubourg, 8 who observed that certain mannitol- 
producing bacteria could ferment saccharose readily without 
producing mannitol, but yielded considerable quantities of 
this alcohol when acting on fructose. But even here it re- 
mains to be shown that fructose as liberated from saccharose 
cannot act differently in statu nascendi from the fructose 



42 THE HYDROLYSIS OF TETRA-, TRI-, AND DISACGHAErDES 
from which the strain of mii.Tmit.nl bacteria studied by Gayon 
and Dubourg yielded mn.Tmit.nT. 

More recently Willstatter and Lowry 9 have recorded ob- 
servations on the behaviour of a saccharose fermenting yeast 
which, according to these writers, support the view that a 
direct fermentation of disaccharides is possible without 
preliminary hydrolysis. Willstatter and Lowry found that 
a certain saccharose fermenting yeast would retain from 
70-80 per cent, of its fermenting properties unimpaired under 
conditions which reduced the action of its saccharase by 
95 per cent. It is not shown, however, that this remaining 
5 per cent, of saccharase is insufficient to maintain the some- 
what reduced fermentative activity of the yeast in question, 
or that sufficient enzymes, as suggested by von Euler, 10 may 
not be produced during the actual fermentation to ensure this 
reduced rate of fermentation. This view is held by Cohn, 11 
who points out that the performance of an enzyme is not a 
simple function of its quantity, and that a retardation of the 
fermentation of saccharose occurs on the partial inaotivation 
of the saccharase of the yeast employed for its fermentation. 
There are thus at present no grounds, as Cohn rightly 
asserts, for assuming a direct fermentation of saccharose. 

In recording the available information on the action of 
micro-organisms on the natural tetra-, tri-, and disaccharides 
there is at present little justification for not inquiring in the 
first instance into the hydrolysing actions taking place before 
proceeding to discuss the chemical changes to which monoses 
are subjected by micro-organisms for the liberation of the 
energy accumulated in these substances. 

HYDROLYSIS OF TETEASAOOHABIDES 

Stachyose, a tetrasaccharide present in a number of plants, 
was~fiB3t~ discovered by von Planta and Schulze 12 in the tubers 
of Stachys tub&rif&ra. On partial hydrolysis it yields one 
molecule of fructose and one molecule of mannotriose, the 
latter yielding glucose and galactose on decomposition. 



THE HYDROLYSIS OF TETEA-, TEI-, AND DKACOHAEIDES 43 

Theinformationavailable as to the action of mioro-organisms 
on stachyose, as well as on other tetra-, tri-, and disaocharides, 
will show that microbiological action runs parallel if it is not 
identical with the action of other hydrolysing agents. To 
demonstrate this is the chief justification for including in the 
present pages the often very fragmentary information avail- 
able on the subject. 

The hydrolysis of stachyose by Aspergillus nig&r was 
studied by Tanret, 13 who found that the tetrasacoharide was 
first broken down to fructose and mannotriose, the latter 
subsequently being slowly decomposed. He was unable to 
decide whether the action on mannotriose was due to the 
saccharase produced by the fungus or to some other enzyme. 
Vintilesco, 14 who reinvestigated the action otAspergittus nig&r 
on stachyose, came to the conclusion that saccharase had no 
action on mannotriose, but that in the presence of saccharase 
and emulsin-oontaining j8-glucosidio enzymes stachyose 
breaks down into its component monoses, fructose, glucose, 
and galactose. More recently Neuberg and Lachmann 16 have 
studied the action of the enzyme mixture of kephir on 
stachyose and have found that it liberates fructose. They 
ascribe this action to the lactase contained in the enzyme 
mixture and mention that yeast maltase contained in the 
enzyme mixture has a similar action on stachyose. The 
latter statement undoubtedly requires verification, since mal- 
tase hydrolyses a-glucosides only, while lactase decomposes 
/J-glucosidio carbohydrates. 

It is noteworthy that lactase should have been unable to 
attack mannotriose in Neuberg and Lachm arm's investiga- 
tions since emulsin, according to them, has a slight hydro- 
lysing action on the trisaccharides. 

HYDBOLTSIS OF TKESAOOHAJRIDBS 

Gentianose. Meyer 18 gave the first description of gentianose, 
which he obtained from the rhizomes of Gentiana litiea. The 
carbohydrate was shown by Bourquelot and He'rissey 17 to be 
a trisaccharide. 



44 THE HYDROLYSIS OP TETEA-, TEI-, AND DISACCHARIDES 

On subjecting gentianose to the action of the enzyme 
mixture produced by AspergUhts niger, Bourquelot 18 ob- 
served a complete hydrolysis of the trisaccharide into its 
component monoses, fructose and glucose. That this action 
was due to more than one enzyme he concluded from the fact 
that sacoharase obtained from yeast liberated fructose but 
left unattacked a disaccharide, gentiobiose, composed of two 
gluoose molecules, and regarded by Berlin 19 as identical with 
Fischer's isomaltose. In a later paper Bourquelot and 
H&issey 20 state that the complete hydrolysis of gentianose 
by AspergiUus niger occurs in two stages, the liberation of 
fructose by the saccharase of the fungus and the subsequent 
hydrolysis of the gentiobiose by an enzyme either identical 
with, or contained in, emulsin. 

Gentianose has a limited practical use in mountainous 
countries where Gentiana species occur in abundance. Here 
the roots are used for the preparation of an alcoholic beverage, 
the making of which was investigated by Guyot. 21 

Raffinose or Melitriose occurs as a normal constituent of 
the carbohydrates in sugar beet. It is therefore of consider- 
able practical interest. It was first isolated by Loiseau 22 from 
beet molasses and haa shown to yield fructose, galactose, and 
glucose on hydrolysis. 

Neuberg 28 maintains, probably rightly, that the hydrolysis 
of raffinose by bacteria and fungi follows the lines of the 
decomposition of this carbohydrate by yeast. For Asper- 
gillus niger Bourquelot 24 has shown this to be the case, since 
the hydrolysis proceeds in two stages, resulting first in the 
liberation of fructose and the formation of melibiose, and 
subsequently in the hydrolysis of melibiose to glucose and 
galactose. Gillot 25 confirms the formation of the three monoses 
by raffinose as the result of the action of Asp&rgittus niger on 
the polysaccharide. Whether in the case of bacteria and 
fungi an action is possible such as that observed by Neuberg 
in the case of emulsin, that is, with the liberation of galactose 
and saccharose, was not definitely established by Neuberg and 
remains to be determined. 



THE HYDROLYSIS OF TETRA-, TRI-, AND DISACCHARIDES 46 

A fermentation of raffinose by JSact. lactiarabinosum was 
observed by Fred, Peterson, and Anderson, 26 by Sact. Fried- 
lander by Erankland, Stanley, and Frew, 27 and by Monilia 
sitophila by Went. 28 

Hie enzyme responsible for the hydrolysis of raffinose, 
according to Willstatter and Kuhn, 29 is not to be regarded as 
identical with sacoharase. 

Melezitose. Melezitose was first isolated by Villiers 80 
from the manna of Alhagi camelorum, and has been referrei 
to by von Schrenk 31 as contained in the droplets of liquid 
which exude from the sporophores of various Polyporaceae, 
types of wood destroying higher fungi. The trisaccharide 
was shown by Alekhine 82 to be decomposable by dilute acids 
into glucose and a disacoharide turanose, which in turn, and 
under the same influence, yielded one further molecule of 
glucose and one molecule of fructose. It might be thought 
therefore that melezitose was a dehydration product of one 
molecule of glucose and one molecule of saccharose. This, 
however, does not appear to be so since the saccharase pro- 
duced by yeast is unable to decompose it (Alekhine). At- 
tempts have been made by Kuhn and von Grundherr 33 to 
explain the mode of combination of saccharose with glucose 
in melezitose, but it must be concluded with Bridel and 
Aagaard 34 that there is at present no experimental evidence 
to show how this combination has been brought about. 

The information available on the microbiological decom- 
position of melezitose is very scanty indeed. That melezitose 
is unfermentable, presumably by yeast, was the conclusion 
arrived at by Alekhine. 

Bourquelot and H&rissey 86 found that the enzyme of Asp&r- 
gittus niger hydrolyses melezitose to glucose and turanose, 
while Kayser 36 showed that a lactic acid producing bacterium 
isolated by him from 'sauerkraut' was capable of fermenting 
and, presumably, of hydrolysing it. Perhaps this type of 
lactic acid bacterium was related to the Bact. lactiarabinosum 
of Fred, Peterson, and Anderson, 26 which is claimed to be a 
vigorous melezitose fermenting type. 



48 THE HYDROLYSIS OF TETRA-, TBI-, AND DISAOCHAEIDES 
HYDROLYSIS OF DISAOOHABIDHS 

Trehalose. Trehalose, a digluoose not reducing Fehling, 
was first isolated by Bourquelot. 87 He found it to be widely 
distributed among fungi, where it is produced in the sporo- 
phores on commencement of spore formation. During the 
ripening of the spores the carbohydrate disappears, a specific 
enzyme, trehalase, being responsible for its conversion into 
glucose. This enzyme was found by Bourquelot to be different 
from sacoharase, amylase, and emulsin. Ivanov 38 draws 
attention to the presence of trehalose in Myxomycetes, organ- 
isms which at certain stages of their development produce 
also the corresponding hydrolytio enzyme trehalase. 

Where the enzyme is absent, for instance in the stipe of 
the sporophore of many higher fungi, trehalose accumulates 
in such quantities that these tissues may be used as a raw 
material for the isolation of the carbohydrate. 

Among the lower fungi Bourquelot found the enzyme 
trehalase present in the mycelium of Aspergillus niger. He 
showed that it was different from the maltase produced by 
the same fungus, since the two enzymes withstood high 
temperature to a varying extent. 

Fischer 89 found that yeast is capable of hydrolysing tre- 
halose, and that an aqueous suspension of dried pure yeast 
converted about 20 per cent, of any trehalose present into 
reducing sugars. An aqueous.extraot of living yeast, however; 
had no action on the carbohydrate, Bourquelot 87 had pre- 
viously shown that yeast sacoharase had no action on tre- 
halose. 

According to Went, 28 trehalase is produced by Monilia 
sitophila when trehalose is added to the culture medium in 
which the fungus is grown. 

An attempt to utilise trehalose for diagnostic purposes was 
made by Koser, 40 who found that Bact. paratyphosum, Bact. 
jSchottmull&ri, and Bact. enteritidis fermented it with the 
evolution of gas and production of acid, while Bact. suipestifer 
was unable to hydrolyse it. Kayser 36 in his investigations on 
lactic acid bacteria found one of his strains capable of fer,' 



THE HYDROLYSIS OF TETRA-, TEI-, AND MSACCHARIDES 47 
menting trehalose. Trehalose, according to Frouin and 
GuiHaumie, 41 would appear also to be a carbohydrate suitable 
for maintaining the growth of the tubercle bacterium. 

Saccharose. The observation that yeast produces alcohbl 
from saccharose long ago caused chemists and biologists to 
take an interest in the reactions involved in this conversion. 
Quevenne 42 reports that before 1832, and independently of 
each other, Baudrimont and Dubrunfaut had established 
that saccharose becomes converted into non-crystallizable 
sugars when left in contact with yeast. Berthelot 48 records 
that Pasteur endeavoured to correlate this reaction with the 
presence of a 'soluble ferment' in the yeast cell but failed to 
do so, and therefore concluded that the appearance of the 
copper-reducing sugars was the result of a side reaction during 
fermentation, due to the action on saccharose of the succinic 
acid formed by yeast during fermentation. In 1860 Berthelot 
undertook to test this hypothesis experimentally. For the 
purpose he allowed sucoinio acid to act on a saccharose solu- 
tion under conditions which in the presence of yeast would 
have resulted in the hydrolysis of the disaccharide. The 
experiment showed that no inversion of the saccharose took 
place. He obtained further proof that the hydrolysis was 
independent of the presence of sucoinic acid by fermenting 
saccharose at alkaline reactions under conditions which 
excluded succinic acid from exercising a hydrolytio action. 
As reducing sugars nevertheless were formed under these 
conditions, Berthelot concluded that the inversion must be 
more intimately connected with the activity of the yeast 
than Pasteur had assumed. He therefore attempted to isolate 
the substances responsible for the conversion of saccharose 
into reducing sugars. He succeeded in showing that an 
aqueous extract of yeast was capable of hydrolysing saccha- 
rose and that the reactive substance could be precipitated 
from the extract by addition of alcohol. This convinced 
Berthelot that the living yeast cell was not itself the ferment, 
but that it produced one, capable of acting independently of 
the life functions of the cell. 



48 THE HYDROLYSIS OF TETEA-, TRI-, AND DISACCHARDDES 

More than twenty years later Gayon 44 confirmed Berthelot's 
experiments, and again showed that succinic acid has no 
hydrolysing action on saccharose at" ordinary temperatures. 

The substance saccharase which Berthelot isolated from 
yeast is frequently described under the name invertase. The 
enzyme is widely distributed among micro-organisms, particu- 
larly among fungi, where most of the higher fungi as well as 
many lower fungi, particularly the Aspergilli and the Peni- 
cittia, hydrolyse cane sugar. Among the Mucoraceae the 
enzyme saccharase is less widely distributed, and only one 
species, Mucor rac&mosus, has so far been shown to produce 
the enzyme. Even within this one species Kostytschew and 
Eliasberg 46 have found that only the minus strain is able to 
hydrolyse the carbohydrate. 

Among bacteria saccharase is undoubtedly less frequently 
found. Nevertheless the few saccharose hydrolysing types 
mentioned by Fermi 48 and Fermi and Montesano 47 by no 
means exhaust the number of saccharose hydrolysing bac- 
teria. 

Certain types of colif orm bacteria studied by Bum 48 and by 
Thaysen 49 are of particular interest. On first being isolated 
from their natural habitat these types do not decompose 
saccharose, but apparently acquire the property of doing so 
on being cultivated on media containing cane sugar. Types 
acting similarly towards lactose had been investigated 
previously by Neisser, 50 Massini, 51 Burri, 48 and others. 

In the case of bacteria saccharase does not appear to have 
been isolated in substance. Fermi and his collaborators used 
cultures of the test organisms grown in broth for about four- 
teen days as solutions of the enzyme, a method which seems 
to have been adopted by all subsequent investigators. 

A similar procedure of utilizing a solution of the enzyme 
secreted into the culture medium or into water has usually 
been adopted, except in the case of yeast saocharase and taka- 
diastase saccharase. Some authors have preferred to work 
with the juice pressed or triturated from the mycelium of the 
fungus. This was noticeably the case in Fischer and Lind- 



THE HYDROLYSIS OF TETEA-, TRI-, AND DISACOHARIDES 49 
ner's 62 experiments, carried out to ascertain the stage in the 
life-cycle of M onilia Candida at which sacoharase was being 
formed by the fungus. Kscher and Lindner disintegrated 
fresh cells of Monilia Candida in a mortar after mixing them 
with glass powder and found that the resultant paste hydro- 
lysed saccharose as effectively as did cells of the fungus, thus 
showing that saccharase, as had been assumed, was not pro- 
duced only on the drying out of the cells. In exceptional 
oases only has saccharase been separated from such solutions. 

The information available on the nature and properties of 
sacoharase has in most cases been derived from a study of 
the enzyme obtainable from yeast. The review of the pro- 
perties of sacoharase given in the following pages refers 
this enzyme therefore where not otherwise stated. 

Before proceeding to discuss these properties it should be 
mentioned that in none of the experiments of Fermi and his 
collaborators were aotinomycetes found capable of hydro- 
lysing saccharose. Subsequent work by Waksman 68 has 
shown that some actinomycetes are able to do so, and to 
develop moderately well on media containing saccharose as 
sole source of carbon. Generally speaking, however, saccha- 
rose cannot be regarded as a carbohydrate favouring the 
growth of actinomycetes. 

Though available as a solid, the substance known until 
recently as saccharase was a very heterogeneous compound 
containing a large percentage of mineral matter, of gum 
(hemicelluloses), and pectin. Great efforts were made to 
establish the connexion of these substances with the enzyme 
and the part played by them in the action of saccharase on 
cane sugar. The existing literature contains a large number 
of publications dealing with this question, which now has no 
more than historical interest, von Euler 64 and his collabora- 
tors and Willfltatter 55 in their publications have been able 
not only to increase the efficiency of their saccharase solution 
by elaborate methods of purification, but the latter investi- 
gator has shown that the purified saccharase prepared by 
him contains no mineral matter, carbohydrates, or protein. 



50 THE HYDROLYSIS OF TETRA-, TBI-, AND DISACCHAEIDES 

It is not within the scope of this volume to give a detailed 
description of the modern methods by which saccharase of 
high purity can be prepared, but a broad outline of the 
principles adopted may perhaps be included. 

The yeast used can be extracted while still living or after 
destruction through drying, "disintegration, or autolysis. In 
the former case, and when very cold water is used, a less active 
enzyme solution is obtained (Salkowski 66 ) than when the 
extraction is carried out at temperatures between 30 and 
40 C. When dried or autolysed yeast is used, the enzyme 
solution contains a large percentage of impurities. 

The most efficient way of removing these impurities is at 
present that adopted by Willstatter and his collaborators. 
This consists in treatment of the crude enzyme with kaolin 
followed by precipitation of the purified sacoharase with 
tannin at 0. In this way Willstatter, Schneider, and 
Wenzel 57 have prepared an enzyme of even greater purity 
than their earlier preparations, which had been entirely free 
from gum and proteins. 

The existing very extensive literature dealing with the 
physical and chemical properties of saccharase has been 
largely based on data collected with less highly purified 
enzyme preparations than Willstatter's. This can hardly 
have failed to affect the results obtained. In interpreting 
the available information on the chemical and physical 
properties of saccharase, it is advisable to bear this in mind. 
In his studies on saccharase von Euler 10 expresses the view 
that yeast does not secrete the enzyme but synthesizes it 
during the actual fermentation with energy derived from the 
breakdown of carbohydrates. This statement postulates 
that yeast grown on media in the absence of carbohydrates 
should contain no saccharase. How far this is the case does 
not appear to have been confirmed experimentally. Kertesz, 68 
however, maintains that Penicillium glaucum when grown on 
a medium containing 6 per cent, of glycerol or of invert sugar 
as the only source of carbon produces no saccharase. 
On the other hand the presence of carbohydrates un- 



THE HYDROLYSIS OF TETRA-, TRI-, AND DISACCEARIDES 51 
doubtedly favours the production of saocharase . Thus von Euler 
and Cramer 69 increased the saccharase content of yeast by 20per 
cent, by adding mannose to the culture medium. Willstatter, 
Lowry, and Schneider 60 found a similar though slighter effect 
when using maltose, but nolle where lactose or glycerin was 
added to the medium. In the case of Aspergillus niger, 
Grezes 61 increased the saccharase content of the fungus by 
cultivating it in a medium containing cane sugar. A suitable 
incubation temperature also appears to favour saccharase 
production. Thus Svanberg 62 records that yeast cultivated 
between 25 C. and 28 C. showed a higher sacoharase con- 
tent than cultures grown outside this optimum. At the 
optimum temperature the production of saccharase by a non- 
multiplying culture of yeast was found by von Euler and 
Svanberg 63 to remain constant throughout the period of 
investigation. The optimum coincided with the optimum 
hydrogen ion concentration of the enzyme (pH values 4-0 to 
6-0 in von Euler and Laurin's 64 experiments). Willstattor, 
Lowry, and Schneider 60 record a somewhat wider optimum 
range, between pH values of 4-5 and 7-0. 

In the case of the mycelium of Penicittium glaucum grown 
at or below 16 C. von Euler 96 found that the largest amount 
of saccharase was produced on the fourth day after inocula- 
tion. It amounted to only one-tenth of that produced under 
optimum conditions by a bottom yeast or one-sixth of that 
of a top yeast. The conidia of the Penicittium species studied 
contained but one-third of the saccharase found in the 
mycelium. In a species of Fusarium investigated in 1887 by 
Wasserzug 66 copper-reducing carbohydrates were not observed 
in a saccharose solution acted upon by the fungus until the 
fifth day after inoculation. Here, therefore, the production 
of saccharase had been even slower than in the case of Peni- 
cittium glaucum. 

A reduction in saccharase production was observed by 
Fermi and Montesano in the case of their test-organisms 
when grown beyond their optimum temperatures. This may 
have been due to a general lowering of the vitality of the 

E 2 



52 THE HYDROLYSIS OF TETRA-, TRI-, AND DISACCHARIDES 
organisms since the recovery of enzyme production did not 
take place for a number of generations. At 35 C. von Euler 
and Svanberg 67 were unable to detect sacoharase production 
by their yeast strain. 

The action of sacoharase was thought by Brown 68 to 
lead in the first instance to a compound being formed 
between the enzyme and the hydrolyte, cane sugar. This 
view became discredited, but has recently been accepted as 
correct by Colin and Chaudun. 69 It is also supported by 
Fermi and Montesano's observation that sacoharase shows 
greater resistance to high temperatures in the presence of 
cane sugar than in the absence of the carbohydrate. Accord- 
ing to H. E. and E. F. Armstrong 70 sacoharase extends its 
influence over the whole of the saccharose molecule, and 
differs in this respect from other disaccharide hydrolysing 
enzymes such as maltase, which affects only one part of the 
hydrolyte. 

From. O'Sullivan and Thompson's 71 investigations in 1890 
to IngersolTs 72 observations in 1926, the subject of the rate 
of hydrolysis of saccharose by its specific enzyme has received 
considerable attention. Under favourable conditions, and in 
solutions containing up to 5 per cent, of saccharose, the rate 
will increase proportionally to the amount of hydrolyte 
present. Beyond this the rate remains stationary until a 
20 per cent, concentration of saccharose is reached (von 
Euler and Myrback 73 ), when a decrease in rate sets in. The 
reason for this decrease has not been satisfactorily explained 
so far. Colin and Chaudun 74 associate it with the greater 
viscosity of high concentrations, pointing out that glycerin, 
as was first observed by Michaelis and Pechstein 76 and by 
Bourquelot, 78 produces a similar inhibition. But increased 
viscosity apparently (Ingersoll 72 ) is not an important factor. 
Perhaps the saccharose itself, as Ingersoll suggests, may be 
the factor governing the retardation. 

Another type of inhibition in the rate of hydrolysis is that 
caused by the two products of reaction, glucose and fructose, 
even at favourable concentrations of saccharose. It is usually 



THE HYDROLYSIS OF TBTEA-, TRI-, AND DISACCHARIDES 53 
observed that saccharase from yeast is iiihibited by fructose 
and that Aspergittus saocharase is similarly affected by the 
addition of glucose. For this reason the former enzyme has 
been regarded as different from the latter, possessing a 
specific affinity for the fructose half of the saccharose mole- 
cule as against the specificity of the Aspergillus saccharase 
for the glucose half of saccharose. The terms fruoto- and 
gluco-saocharase respectively have sometimes been applied 
to the two enzymes (Kuhn and Munch 77 ). The study of the 
action of glucose, fructose, and methylglucoside on the rate 
of hydrolysis of saccharose is at present being actively pursued. 
Contributions to this study have been made by Nelson and 
Anderson, 78 Nelson and Post, 79 Josephson, 80 von Euler and 
Josephson, 81 and by Weidenhagen, 82 so far, however, without 
any definite conclusions being reached as to the significance 
of the various observations made. 

An accelerating action of various acids on the activity of 
saccharase was observed by Bertrand and Rosenblatt. 83 
They found that the saccharase of Asp&rgillua niger usually 
was less markedly affeoted in its action by acids than yeast 
saocharase, though the effect differed with the nature of the 
acid. In the case of propionic acid the concentration inducing 
the most rapid acceleration of the reaction was found to be 
the same for both enzymes. Formic, phosphoric, and nitric 
acids, on the other hand, acted at lower concentrations on 
Aspergillus saccharase than on yeast saccharase. The effect 
of the acid on saccharase therefore was dependent both on 
its kations and anions. An increased action of saccharase 
was noticed by H. E. and E. F. Armstrong 70 on adding 
glyoine to the reacting enzyme solution. 

Unlike maltose, sacoharase, according to Michaelis and 
Rona, 84 remains soluble at all hydrogen ion concentrations. 

Unexplained so far is the inhibition caused by sodium 
chloride on the action of koji saccharase (Kellner, Moi, and 
Nagaoka 85 ). Fales and Nelson 86 studied this action in greater 
detail, and showed it to depend on the prevailing hydrogen 
ion concentration of the saccharose solution. They do not 



64 TEE HYDROLYSIS OP TETBA-, TRI-, AND DISACCEARIDES 
explain, however, why the action of sodium chloride should 
be negligible at the optimum hydrogen ion concentration of 
saccharase and increase with increasing deviations from this 
optimum. 

The effect of magnesia on the action of saccharase was 
studied by Tribot, 87 who found that less cane sugar is 
hydrolysed by saccharase in a given time when magnesia 
is present, provided the time taken is sufficiently short. 
Giving a longer time for the reaction the effect apparently 
is the opposite, the magnesia inducing the enzyme to greater 
activity except at temperatures above 40 C., when the 
activity of the enzyme becomes independent of the presence 
of magnesia. 

The action of silver nitrate and mercury bichloride was 
studied by von Euler and Svanberg. 88 Both salts were found 
to have a very marked effect on the enzyme, the former in 
proportion to its concentration. The action depends also on 
the concentration of the enzyme solution and on its reaction. 
It is interesting to note that the elimination of the metal 
from the poisoned enzyme resulted in regeneration of the 
enzyme. 

The question of the regeneration of sacoharase acquired 
renewed interest some time ago by the investigations of 
Durieux, 89 who found that, after being heated to boiling 
point, the enzyme would recover as much as 10 per cent, of 
its former hydrolysing power. If heated only to 70 C. to 
80 C. it was completely inactivated. Durieux's explanation 
of this behaviour of saccharase is not entirely convincing. 
He assumes that the proteins present in the saccharase solu- 
tion become coagulated on being heated to 70 C. to 80 C. 
and that on precipitating they carried with them the saccha- 
rase held in suspension, thus removing the enzyme from the 
solution. A further increase of the temperature, Durieux 
suggests, causes the saccharase to become water soluble, aiid 
therefore to return into solution. Only when the temperature 
rises considerably above the boiling point of water is the 
enzyme finally destroyed. A reinvestigation of Durieux's 



THE HYDROLYSIS OF TETRA-, TRI-, AND DISACCHAEIDES 65 
observation with the saccharose preparation of WiUstatter 
would be of great interest, since this preparation contains no 
protein, at least no traces of coagulable protein. 

Durieux's actual observations on the action of heat on 
saccharase have been confirmed by Bertrand and Rosenblatt. 90 
These writers remark that the property of regeneration is not 
possessed by the maltase of yeast. In this fact they see 
additional support for the view that the two enzymes are 
different. 

Sacoharase, according to Bourquelot and Bridel, 91 also has 
a hydrolysing action on the three carbohydrates raffinose, 
gentianose, and stachyose at a rate decreasing in the order 
named. In each case fructose is liberated, showing that 
saccharose constitutes a part of the molecule of each of these 
sugars. Bourquelot and Bridel find that the remainder of the 
hydrolysed carbohydrate exercises an inhibitory action not 
only on the inversion of these sugars but also, as already 
mentioned, on that of saccharose. 

The question of the reversibility of the action of saccharase, 
and consequently of the biological synthesis of saccharose, is 
still open. Visser, 92 who published some very interesting data 
on this subject, came to the conclusion that a reversibility 
does exist, while both Blagowestschenski 98 and Hudson and 
Paine 94 deny that this is the case. Hudson and Paine attribute 
the decline in rotatory power of a solution of fructose and 
glucose on addition of sacoharase a decline which Visser 
interpreted as support for his contention to the action of 
the hydrogen ions present on the rotation of fructose. In 
future investigations on this subject Bourquelot and Bridel's 95 
researches on the activity of saccharase in high concentra- 
tions of ethyl alcohol should not be overlooked. They found 
that the hydrolysing properties of saccharase remained 
practically unaffected by dissolving the enzyme in ethyl 
alcohol even of 90 per cent, strength, that is, under conditions 
which should facilitate the elimination of any cane sugar 
synthesized from glucose and fructose owing to its low 
solubility in this strength of alcohol. 



66 THE HYDROLYSIS OP TETRA-, TR1-, AND DISACCFARIDES 

Maltose. The production by micro-organisms of an 
enzyme capable of hydrolysing maltose was first suggested by 
Bourquelot 96 in 1886. As he was unable to trace the presence 
of glucose in the maltose-containing medium in which yeast 
developed, and since apparently he did not feel justified in 
assuming that the maltose was being assimilated directly, he 
concluded that a specific maltose hydrolysing enzyme would 
be necessary and suggested that it was being produced by 
the yeast cell within the cell wall. 

The conception of maltase as a specific enzyme was not 
shared by all investigators, and as late as 1894 Emil Ksoher 97 
suggested that the maltose hydrolysing enzyme might be 
identical with saccharase. The observation made by Roh- 
mann 98 that commercial saccharase does not act on maltose 
was explained by Fischer on the assumption that the treat- 
ment with alcohol to which commercial saccharase is sub- 
jected rendered it incapable of acting on maltose. Subsequent 
research has proved Eischer's views to be incorrect. In 
addition to Rohmann's discovery that maltase is destroyed 
by alcohol, a chemical to which saccharase is very resistant, 
Lintner and KrOber" have brought forward evidence to show 
that the optimum temperatures of the two enzymes differ 
materially, that of maltase being 40 C. and that of saccharase 
52-53 0. 

The optimum hydrogen ion concentration of maltase was 
shown by Mohaelis and Rona 100 to be covered by the pH 
values of 6-1 to 6-8, a range considerably less acid than the 
optimum usually attributed to saccharases. At a pH value 
of 4-5 maltase was found to be quite inactive. Michaelis and 
Rona point out that maltase is active only as an anion, while 
the uncharged molecule of saccharase is capable of hydro- 
lysing cane sugar. They also found that, while maltase is 
precipitated at its isoelectri point, saccharase remains in 
solution at all hydrogen ion concentrations. A further 
difference between the two enzymes noted by them was that 
maltase is absorbed by kaolin, while sacoharase is not. 
Nevertheless a certain similarity between the two enzymes 



THE HYDROLYSIS OF TETRA-, TRI-, AND DISACCHARTDES 57 
appears to exist. When discussing the action of aoids on 
sacoharase it was mentioned that this action depended as 
much on the anion of the acid as on its kation. According to 
Kopaczewsky 101 this is the case also with maltase where the' 
optimum hydrogen ion concentration, when trichloraoetic 
acid is used to establish it, is equal to a pH value of 2-8, but 
using acetic acid it amounts to a pH value of 6-97. 

It has been suggested that maltase might be capable of 
hydrolysing a-gluoosides, seeing that maltase itself is an a- 
glucoside. This, however, does not appear to be the case. 
Aubry 102 , for instance, found that the enzyme mixture ex- 
tracted from Aapergittus niger, though hydrolysing maltose, 
had. no action on a-methylglucoside. Among yeasts Aubry 
found some types which did not produce a-methyl glucosidase 
though all of them produced maltase. 

Maltase is produced by a large number of micro-organisms 
other than yeast. In fact, judging from the observation of 
Bokorny 103 that maltose is a highly suitable carbohydrate 
for a wide range of organisms, it is to be concluded that the 
production of maltase is common among micro-organisms. 

In his study of the production of maltase by Monilia 
sitophila Went made several interesting observations. He 
found that not only maltose but several other carbohydrates 
were capable of inducing the secretion of maltase, among 
them raffinose, trehalose, starch, cellulose, and xylose. That 
this action cannot have been in the nature of a trigger action, 
as observed in the case of Microspira agarliguefaciens by 
Gray and Chalmers 104 , is clear from the fact that maltase 
production in the case of Monilia sitoyihila increased pro- 
portionally with the increase in carbohydrate concentration 
until a maximum had been reached. Exceeding this point an 
inhibition set in which according to Went was not caused by 
the increase in osmotic pressure. 

Since Bourquelot's investigations it has generally been 
accepted that the diffusion of maltase through the cell wall 
of yeast cells is impossible. Some years ago, however, Will- 
statter, Oppenheimer, and Steibelt 105 came to the conclusion 



58 THE HYDROLYSIS OF TETEA-, TBI-, AND DISACCHAEIDES 
that a diffusion did occur but that the maltase as it left the 
cell was destroyed by the acid produced during fermentation. 

Gentiobiose. Bourquelot and He"rissey 106 obtained this 
disaccharide from gentianose and showed that the enzyme 
mixture of Asp&rgiUua niger was capable of hydrolysing it. 
A later investigation 107 indicated that yeast enzymes were 
unable to attack it. 

Gellobiose. This disaccharide contains two glucose resi- 
dues. It can be obtained by hydrolysis of cellulose acetate 
or by the action of micro-organisms on cellulose. 

The property of hydrolysing cellobiose is probably wide- 
spread among micro-organisms, but so far only two groups of 
bacteria, Bad. coli commune and Bad. lactis aerogenes, appear 
to have been tested for their fermentative action on cellobiose. 
Both Jones and Wise 108 and Koser 109 have found that cello- 
biose is fermented by Bad. ladis aerogenes while the typical 
Bad. coli commune does not do so. They see in this fact a 
means of differentiating the two types. Possessing a j8- 
glucosidic constitution emulsin has been regarded as the 
enzyme responsible for the hydrolysis of cellobiose (Fischer 
and Zemple'n 110 ). Bertrand and Compton 111 have shown, 
however, that this is not the case and that a specific cellobiose 
or cellase occurs in nature. The cellobiase isolated from 
A&pergillus niger was found by them to be most active at a 
pH value of about 6-0, while the optimum activity of emulsin 
was found to be nearer the alkaline side of the neutral point. 
That the sugar resulting from the hydrolysis of cellobiose 
by Aspergilli is glucose was shown by Bertrand and Hol- 
derer. 112 

Lactose. The association of lactose hydrolysing enzymes 
with the activity of lactose fermenting micro-organisms was 
first suggested by Naegeli (see Oppenheimer 113 ). Seven years 
later, in 1889, Beijerinck 114 turned his attention to lactose 
fermenting yeasts and came to the conclusion that their 
fermentative power was intimately connected with the pro- 
duction by them of a lactose hydrolysing enzyme which he 
termed lactase. Subsequently Emil Fischer 116 obtained a 



THE HYDROLYSIS OF TETRA-, TRI-, AND DISACOEARIDES 69 
laotaae from kephir granules, a material from which he found 
the enzyme easier to extract than from yeast. Fischer also 
suggested that the lactase of yeast might be different from 
that of kephir since the latter is more resistant towards 
alcohol than the former. 

The presence of laotase in fungi other than yeast has 
occasionally been reported, both in the case of higher fungi 
(Polypoms suJ/pfwreus by Bourquelot and H6rissey u6 ) and in 
that of lower types such as Asp&rgillus nig&r (Pottevin 117 ; 
Bierry and Coupin 118 ), certain Rhizopua species (Nakazawa 119 ; 
Hauzawa 120 ), and Mucor Eouxii (Wehmer 121 ). 

Its presence in actinomycetes, organisms which are some- 
times reported (Bergey 122 ) to produce acid in milk, has not 
been demonstrated. 

In many types of bacteria which ferment lactose it is to be 
assumed that lactase is present. But except for those types 
which with yeast constitute the kephir granules, the presence 
of the enzyme has not been demonstrated. And even here 
the lactase may have been derived from the yeast and not 
from the bacteria. 

The isolation of lactase in substance has never progressed 
beyond Beijerinok's 114 attempts at precipitating it from a 
yeast culture with 86 per cent, alcohol, and Barendrecht's 123 
method of adsorbing it on kieselguhr added to a culture of 
kephir yeast grown in whey for 48 hours. 

Information on the nature and the properties of lactase is 
limited to a few indirect observations on its temperature 
range and hydrogen ion concentration. 

From Wehmer's 121 observation that-3/^wcor R&uxii ferments 
lactose more readily at 80 C. than at 15 C. it may be con- 
cluded that the optimum temperature for the activity of 
lactase is to be found nearer the former than the latter tem- 
perature. And from Bokorny's 124 demonstration that lactase 
is highly resistant even to high concentrations of lactic acid it 
follows that its optimum temperature is to be sought well on 
the acid side of the neutral point. 

Though Beijerinck and Barendrecht obtained their lactase 



60 THE HYDROLYSIS OF TETRA-, TRI-, ND DISACCHARIDES 
preparations from the medium in which kephir yeast had 
developed without first destroying and disintegrating the 
cells, the opinion is frequently expressed (Pottevin 117 ; Bierry 
and Coupin 118 ; and Coupia 126 ) that lactase is an endoenzyme. 
This view is supported by Fischer's statement that it is 
necessary to disintegrate the cells before lactase can be ex- 
tracted from kephir yeast. 

Before leaving the subject of lactase reference must be 
made to certain bacteria of the coH-paratyphosum group, 
first observed by Neisser and subsequently studied by Mas- 
sini 51 , Burri 48 , and others. These types possess the faculty of 
acquiring fermentative powers towards lactose or saccharose 
when grown for some days in a medium containing lactose 
and saccharose respectively. 

When first observed, the acquisition by these bacteria of 
fermentative power towards lactose and saccharose appeared 
to be spontaneous, occurring in a few cells of a colony or a 
culture. The acquisition was for that reason described as a 
mutation. This interpretation of the change, however, was 
shown by Burri 48 and by Pringsheim 126 to be misleading and 
has now been abandoned in favour of an explanation which 
allocates to each cell of the culture a latent power of ferment- 
ing lactose or saccharose, when either of these sugars is 
provided in the absence of other suitable carbohydrates. 
The nature of the enzymes thus activated has not been studied. 

Melibiose is a product of hydrolysis of raffinose or meli- 
triose. It was first observed and isolated by von Lippman 127 
from the stalks of certain Malvaceae. Very little is known of 
the extent to which micro-organisms are capable of hydro- 
lysing melibiose. That many types of yeast, notably bottom 
yeast, are capable of doing so was noted by Fischer and 
Lindner. 68 



THE HYDROLYSIS OF TETRA-, TBI-, AND DISACCHABIDES 6] 

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1914. 

95. E. Bourquelot and M. Bridel, J. Pharm. et Chim. (7), vol. 9, p. 321, 1914. 

96. E. Bourquelot, J. de I'Anatomie etdela Physiologic, vol. 22, p. 162, 1886. 

97. E. Fischer, Berichte, vol. 27, p. 3479, 1894. 

98. F. Rehmann, Berichte, vol. 27, p. 3251, 1894. 

99. C. J. Lmtner and E. KrOber, Berichte, vol. 28, p. 1050, 1895. 

100. L. Miohaelis and P. Rona, Biochem. Zeits., vol. 67, p. 70, 1913. 

101. W. Kopaczewsky, Comptea rend., vol. 168, p. 640, 1914. 

102. A. Aubry, J. Pharm. et Chim. (7), vol. 10, p. 23, 1914. 

103. Th. Bokorny, Zentrbl.f. Bakt., Abt. H, vol. 47, p. 191, 1917. 

104. P. H. H. Gray and C. H. Chalmers, Ann. App. Biol, vol. 11, p. 324, 

1924. 



64 THE HYDROLYSIS OF TETEA-, TBI-, AND DISAOCHAEIDBS 

105. R. Willst&tter, T. Oppenheimer, and W. Steibelt, Z. phyaiol. Chem., 

vol. 110, p. 232, 1920. 

106. E. Bonrciuelot and H. Herissey, J. Pharm. et Ghim. (6), vol. 7, p. 369, 

1898. 

107. E. Bourquelot and H. Herissey, Comptes rend., vol. 132, p. 671, 1901. 

108. H. N. Jones and L. E. Wise, J. Bacterial., vol. 11, p. 359, 1926. 

109. A. Koser, J. Infect. Dis., vol. 38, p. 606, 1926. 

110. E. Fischer and G. Zemple'n, Ann. der Chem., vol. 366, p. 1, 1909. 

111. G. Bertrand and A. Compton, Butt. Soc. (Mm. (4), vol. 7, p. 996, 1910. 

112. G. Bertrand and M. Holderer, Com/pies rend., voL 149, p. 1385, 1909. 

* 13. C. Oppenheimer, Die Fermente, 5th edition, vol. 1, p. 627, 1924 (Guatav 

Fischer, Jena). 
14. M. W. Beijerinok, Z&ntrblf. Bakt., vol. 6, p. 44, 1889. 

16. E. Fischer, Benchte, vol. 27, p. 3479, 1894. 

L6. E. Bourquelot and H. Henssey, Comptes rend., vol. 137, p. 56, 1903. 

17. H. Pottevin, Ann. Inst. Pasteur, vol. 17, p. 31, 1903. 

118. H. Bierry and F. Coupin, Comptea rend., vol. 157, p. 246, 1913. 

119. R. Nakazawa, Zentrbl f. Bakt., Abt. n, vol. 24, p. 482, 1909. 

120. J. Hanzawa, Mykologisehes Zembrbl., vol. 1, p. 76, 1912. 

121. C. Wehmer, ZentrU. f. Bakt., Aht. n, vol. 6, p. 353, 1900. 

122. D. H. Bergey, Manual of Determinative Bacteriology, 2nd edition, 

1928 (Bailliere, Tindal & Cox, London). 

123. H. P. Barendrecht, Z&iischr . /. physikal. Ohem., vol. 54, p. 357, 1906. 

124. Th. Bokorny, Ohem. Zentrbl, vol. 74, p. 1334, 1903. 

125. F. Coupin, J. de Physiol. Path, gen., vol. 16, p. 419, 1914. 

126. H. Pringsheim, Die Variabflitdt niederer Organismen, 1910 (Julius 

Springer, Berlin). 

127. E. O. von Lippmann, Benchte, vol. 53, p. 2069, 1920. 



CHAPTER IV 
HYDROLYSIS OF GLUCOSIDES 

IKT spite of the great interest which attaches to the micro- 
biological decomposition of glucosides, both from a pharma- 
cological and from a physiological point of view, little has been 
done to study the problem even in the case of such important 
gluoosides as digitalin, digitoxin, and strophanthin. Judging 
from the available literature, interest appears to have been 
limited almost entirely to ascertaining whether micro-organ- 
isms do or do not hydrolyse glucosides. Only in the case of 
the industrially important glucoside indican and the equally 
important glucosidio substance tannin has an attempt been 
made to investigate in greater detail the hydrolysis and the 
subsequent decomposition. 

In general it may be claimed that many bacteria and fungi 
possess the property of hydrolysing glucosides. Among 
aotinomycetes, however, the property is rare. 

Bourquelot 1 found that amygdalin, sambucin, coniferin, 
aesculin, and salioin were hydrolysed by many wood-destroy- 
ing fungi. Kohnstamm 2 added to the list of glucosides arbutin 
and helicin. Among the lower fungi Bourquelot and Herissey 3 
found that the enzyme mixture of AapergiUus nig&r hydro- 
lysed amygdalin, salicin, ooniferin, arbutin, aesculin, helicin, 
populin, and phloridzin. Penicillium glaucum was tested by 
Gerard 4 on amygdalin and on salioin and was found to hydro- 
lyse both. 

The readiness with which a fungus hydrolyses a glucoside 
was studied by Brunstein 5 and was found by him to vary 
considerably. Amygdalin and coniferin were most easily 
decomposed. Arbutin, helicin, and salicin were less so, owing, 
he suggests, to the inhibitory action exercised on the growth 
of the fungus by the products of hydrolysis. Nevertheless, 
Castellani's 6 investigations on certain bacteria indicate that 
salicin is far more frequently hydrolysed than amygdalin. 



66 HYDROLYSIS OF GLUCOSIDES 

As a general rule Brunstein found that a well-nourished 
mycelium acted more quickly and more completely on 
glucosides than did a starved mycelium. For that reason he 
recommended the use of a starved fungus for the study of the 
progress of the hydrolysis, since the slower rate of conversion 
of the glucoside by such a mycelium made it possible to 
follow the intermediate stages of the breakdown and to show 
that the carbohydrate liberated during hydrolysis becomes 
subsequently assimilated by the fungus. 

This liberation of a carbohydrate indicates that glucosides 
are capable of serving as a food-substance for the organisms 
hydrolysing them, a view which is supported by Puriewitsoh's 7 
observation that spores of Asp&rgiUus niger, Aspergillus 
glaucus, and Penicillium glaucwm germinate and form a 
mycelium in an aqueous solution of helicon. 

That glucoside hydrolysing enzymes are present in the 
mycelium of a fungus from the time of the germination of its 
spore was shown by Javillier and Tschernoroutzky. 8 In their 
experiments Javillier and Tschernoroutzky also observed 
that the presence of a zinc salt in the culture medium was 
favourable to the secretion of glucosidase. Studying Asper- 
gillus and Penicillium species these workers established that 
less enzyme diffuses into the surrounding medium from 
young cultures than from older ones, and that the rate of 
diffusion may vary according to the nature of the enzyme. 
The optimum hydrogen ion concentration for the activity 
of amygdalase by the species mentioned was determined by 
Javillier and Tschernoroutzky to be equal to a pH value of 
about 3-8 to 4-0. 

Among bacteria the glucoside hydrolysing properties of 
the coli-typhosum group have been studied by Twort, after 
Inghilleri 10 had shown that Sact. coli commune decomposes 
amygdalin while Bact. typhosum does not. Twort examined 
the behaviour of a large number of strains of the ooli-typhosum 
group towards no less than forty-nine different gluoosides, 
with a view to discovering differences in the behaviour of 
these strains, applicable to their classification. He found 



HYDROLYSIS OF GLUCOSIDES 67 

that as a rule the strains which hydrolysed lactose attacked 
gluoosides fairly readily. But he was unable to base any 
system of classification on their behaviour towards glucosides 
since two types of one sub-group might differ in this respect 
while two members of different sub-groups might show an 
identical behaviour towards gluoosides. 

A year before Twort's publication van der Leck u had 
emphasized the value of the use of glucosides as a means of 
differentiating bacteria and had shown that Bact. coli commune 
and Bact. lactis aerogenes are both capable of hydrolysing 
indican and aesculin. Van der Leek based his technique on 
the method introduced by Molisch, 12 who tested bacteria for 
glucoside hydrolysing properties by cultivating them on an 
agar prepared by the addition of the required percentage of 
the gel to an extract of Indigof&ra leaves. Molisch found that 
types of bacteria capable of hydrolysing indioan produced a 
blue growth on this medium. 

The action of bacteria on gluoosides was also studied by 
Fermi and Montesano, 13 who showed that amygdalin is hydro- 
lysed by M icrococcus pyogenes tenuis and usually by Vibrio 
Metchnikoff, at least when freshly isolated strains are em- 
ployed. JBact. coli commune, on the other hand, sometimes 
gave negative results, an observation which is not altogether 
surprising considering the wide range of variation represented. 
The findings of Fermi and Montesano were confirmed some 
years later by Twort's observations referred to above. 

In Fermi and Montesano's experiments Bac. Megatherium, 
Sarcina aurantiaca, and Corynebacterium dipMieriae were 
sometimes found capable of hydrolysing amygdalin. This is 
of considerable interest, particularly as regards the last-named 
type, since none of the typical aotinomycetes tested by 
Fermi and Montesano were found capable of hydrolysing 
glucosides. 

On the subject of the physical properties of glucosidases 
secreted by bacteria and fungi, information is limited to the 
observations of Bourquelot and Aubrey 14 and of Fischer 16 
that these enzymes are highly sensitive to acids. In Bourque- 

F2 



68 HYDROLYSIS OF GLUCOSIDES 

lot and Aubrey's experiments even 'very small' quantities 

of acetic acid destroyed the j8-glucosidase of fungi. 

The hydrolysis of glucosides by micro-organisms has 
acquired practical importance in two directions, in the pro- 
duction of natural indigo and in the fermentation of tannin. 
In both cases the hydrolysis of a glucoside, or at least of a 
compound related to the gluoosides, is an essential part of the 
industrial process involved. 

MAJTDTAOTUBB OE INDIGO 

Like retting, the preparation of natural indigo is an agri- 
cultural process traceable to remote antiquity. And even 
more than retting has indigo preparation been allowed to 
proceed on old established lines without any serious attempt 
to apply the result of scientific investigation, despite the 
pressure of competition brought about by the introduction 
of synthetic indigo. 

Indigo can be prepared from plants which produce the 
glucoside indican. The process, in brief outline, consists in 
an extraction of the indican-containing tissues, usually the 
leaves, with eight to ten parts of water at ordinary tempera- 
tures, followed by an oxidation of the extract in a process 
termed the beating process, during which the indoxyl in 
the extract is converted into indigotin. The extraction, 
usually known as the fermentation, is allowed to proceed 
as a rule for 12 to 14 hours, and is the stage during 
which microbiological activity predominates. Though in- 
vestigators agree that a varied and vigorous microflora exists 
in the extraction vats during fermentation, the part it takes 
in the hydrolysis of indican to indoxyl and glucose has been 
very differently estimated. Some investigators, notably 
Br^audat 16 and Beijerinck, 17 have expressed the view that 
the conversion of the indican to indoxyl is due solely to the 
action of a specific enzyme secreted by the plant tissues. 
Others, Molisch 12 for instance, while not denying that micro- 
organisms are capable of converting indican to indigotin and 
admitting (Bergtheil 18 ) that the microflora may exercise 



HYDROLYSIS OF GLUCOSIDES 69 

some slight action, still see the chief hydrolysing agency in a 
specific enzyme produced by the plant tissues, or regard the 
conversion as a purely chemical reaction (Molisoh 12 ). Alvarez, 19 
and more recently Davis 20 , maintain that the mioroflora 
present in the steeping vats is the chief hydrolysing agent and 
regard the action of any indican hydrolysing enzyme pro- 
duced by the plant tissues as of secondary importance if not 
actually detrimental. As early as 1887 Alvarez isolated a 
bacterium in pure culture from the content of steeping vats, 
a bacterium which he found capable of hydrolysing indican 
and therefore termed Bact. indigogenum. He described it as 
a short rod with rounded ends, often occurring in pairs or 
short chains. 

Davis 20 accepts Alvarez's claim that micro-organisms 
constitute the normal agency for the conversion of indican 
into indoyl, and supplies data supporting this, showing also 
that in actual practice the yield of indigotin depends on the 
presence of an adequate number of suitable bacteria in the 
tanks used for extraction, a number which may be supplied 
naturally with the steeping water or introduced artificially 
as pure cultures. 

Using a medium consisting of an indigo leaf extract and 
the requisite percentage of agar, Hutchinson (see Davis 20 ) 
was able to sub-divide these indican hydrolysing micro- 
organisms into two groups, an active group producing in- 
tensely blue colonies and a poor type giving pale blue or 
colourless colonies. Where the former type predominated 
good yields of indoxyl could always be relied upon, while a 
large number of faintly staining types gave rise to destructive 
changes with resulting loss in yield of quality of indigo. 

Though he gives but few details on the fermentation 
process, it is clear from Davis's papers that considerable 
quantities of carbon dioxide are produced during the de- 
composition of the glucose liberated through the hydrolysis 
of indican. In fact this carbon dioxide is claimed to be almost 
entirely responsible for the very marked acidity which, 
if not checked, may cause considerable loss in yield and 



70 HYDROLYSIS 0! GLUCOSIDES 

quality of the resulting dye. On the other hand, a certain 
acidity of the water used for extraction is stated to be essen- 
tial, since the indican cannot diffuse out into the water 
surrounding the leaves under neutral or alkaline conditions. 
Davis observed that the extraction of the glucoside and the 
development of acidity proceeded almost concurrently. Both 
increased during the earlier stages in direct proportion to 
time. An accumulation of indioan in the extract did not 
occur, the glucoside being hydrolysed into indoxyl and 
glucose as rapidly as it passed into solution. 

The liquor from the steeping or fermentation vats is usually 
of a pale yellow colour with a greenish fluorescence. Im- 
mediately on being drawn from the vat it is subjected to an 
oxidation through which the comparatively unstable indoxyl 
is converted into indigotin. The efficiency of this conversion 
was found by Davis to depend on several factors, principally 
on the acidity developed during steeping. Where the fer- 
mentation had given rise to a high acidity, owing to the 
accumulation of acid forming micro-organisms, the efficiency 
of the oxidation was low. Neutralizing the acidity resulted 
in improved yields. Davis regards the conversion of indoxyl 
into indigo brown, a substance which does not oxidize to 
indigotin under normal conditions, as due to an excessive 
initial acidity in the steeping liquor. He sees a further sign 
of the serious effects of a high acidity in the solution of plant 
proteins and their subsequent precipitation during beating. 
These are the proteins which are described in indigo tech- 
nology as indigo gluten. Not infrequently this indigo gluten 
represents as much as 20 per cent, of an ordinary cake of 
indigo. 

The varied microflora which must be introduced into the 
steeping vats with fresh leaves no doubt contains many types 
capable of producing volatile and non-volatile acids from the 
glucose liberated during the hydrolysis of the indican. It is 
noteworthy that this flora should be unable to form these 
acids under the conditions prevailing in the steeping vats. 
Davis specifically mentions that volatile acids such as formic, 



HYDROLYSIS OF GLUCOSIDES 71 

acetio, and butyric acids are not met with, in appreciable 
quantities during normal fermentations, and that lactic, 
citric, and tartaric acids usually were absent. This apparent 
anomaly renders a further study of the physiology of the true 
indican hydrolysing bacteria most desirable. 

The oxidation process completed, the indigotin is allowed 
to settle, and, together with admixed indigo brown, indigo 
gluten, and other impurities, is collected in paste form. The 
paste is usually heated to a temperature sufficient to prevent 
further microbiological activity and is then allowed to dry 
under neutral conditions. 

THE PBODUOTION" OF OAUUKJ AOTD THROUGH 
THE FERMENTATION" OF TANNIN" 

The conversion of tannin into gallic acid must have been one 
of the earliest biological reactions to be seriously investigated. 
As early as 1785 Scheele 21 expressed the view that gallic acid 
exists in gall apples but cannot be extracted directly owing to 
its being protected by other substances which only a decay 
of the apples can remove. This view was not accepted by 
subsequent investigators. A clear conception of the formation 
of gallic acid was not obtained until nearly a century later 
when van Tieghem 22 showed that gallic acid is formed by the 
decomposition of tannin present in gall apples. Since then 
interest has become centred on the question of the agent 
responsible for the conversion of tannin. Various fungi 
developing on gall apples during their decay, i.e. their fer- 
mentation, were usually thought to be associated with the 
conversion, though Robiquet 23 maintained that a plant 
enzyme, a pectase, was the responsible agent. Van Tieghem 
ascribed the conversion to the activity of an enzyme secreted 
by the fungi present, but failed to prove this. When Duclaux 24 
succeeded in doing so a fairly accurate impression was ob- 
tained of the reaction taking place. 

Duclaux gives the following description of the preparation 
of gallic acid from gall apples. Gall apples which normally 
contain from 40 to 60 per cent, of tannin are made damp and 



72 HYDROLYSIS OF GLUCOSIDES 

incubated at 25 C. to 30 0. for a month under moist con- 
ditions. The apples swell and, through the action of the fungi 
developing on tfiem, undergo a 'fermentation' in which no 
appreciable evolution of gas occurs except where yeast is 
added to remove the glucose liberated by the hydrolysis of 
the tannin. The progress of the fermentation is followed by 
determining the decrease in glucose present. When com- 
plete, a yield of 30 to 60 per cent, of gallic acid should have 
accumulated. In practice, however, the yield is often as low 
as 20 per cent. The reason for this was shown by Knudson 26 
to be due to various causes, primarily to the absence of suit- 
able fungi, and secondly to lack of a certain percentage of an 
additional food other than tannin. In his experiments Peni- 
cittium rugulosum and AspergiHus niger were the only fungi 
studied which developed normally at tannin concentrations 
higher than 10 per cent. This growth was found most rapid 
hi concentrations of about 16 per cent, of tannin and under 
aerobic conditions. But to ensure an economic conversion of 
the tannin into gallic acid he found it necessary to add 10 per 
cent, of cane sugar. This prevented the fungi from breaking 
down the liberated gallic acid. In his investigations 1 mg. of 
mycelium of Aap&rgillus nig&r, the more active of the two 
fungi, was found sufficient to hydrolyse 2-70 mg. of tannin 
in 10 days. 



LITERATURE 

1. E. Bourquelot, Comptes rend., vol. 117, p. 383, 1893. 

2. Ph. Kohnstamm, Beihefte botan. Zentrbl, vol. 10, p 90, 1901. 

3. E. Bourquelot and H. IKrisaey, Oomptea rend., vol. 121, p. 693, 1895. 

4. E. Geraxd, Comptes rend. Soc. biol., vol. 5 (9), p. 651, 1893. 
6. A. Brnnstein, Beihefte botan. Zentrbl, vol. 10, p. 1, 1901. 

6. A. Castellani, Zentrbl. f. Bakt., Abt. I, vol. 62, p. 262, 1912. 

7. K. Puriewitsch, Ber. deut. botan. Oesell, vol. 16, p. 368, 1898. 

8. M. Javillier and H. TBohernoroutzky, Ann. Inet. Pasteur, vol. 27, Bull. 

Sd. Pharm., vol. 20, p. 132, 1913. 

9. B 1 . W. Twort, Proc. Eoyal Soc., Series B, vol. 79, p. 329, 1907. 

10. . Inghilleri, Zentrbl. f. Bakt., vol. 16, p. 821, 1894. 

11. G. van derLeok, Zentrbl. f. JBakt., Abt. n, vol. 17, p. 366, 1906. 



HYDROLYSIS OF GLUOOSIDES 73 

12. H. Mohsoh, Srtzber. Akad. Wiss. Wien, Math.-NatunotM. Kl., vol. 

107, p. 758, 1898. 

13. C. Fermi and G. Montesano, Zentrbl.f. BaTct., vol. 15, p. 722, 1894. 

14. E. Bourquelot and A. Aubrey, Oomptes rend., vol. 116, p. 742, 1915. 

15. E. Fischer, Z.physiol. Ohem., vol. 107, p. 176, 1919. 

16. L. Breaudat, Comptes rend., vol. 127, p. 769, 1898. 

17. M. W. Beijerinok, Proc. Eon. Akad. van Wetenschap, Amsterdam, 

Section Science, vol. 3, p. 101, 1900-1. 

18. 0. Bergtheil, Trans. Ch&m. 8oc., vol. 86, p. 870, 1904. 

19. E. Alvarez, Oomptes rend., vol. 105, p. 286, 1887. 

20. W. A. Davis, Publication of the Agricultural Research Institute, Pusa. 

Indigo publication, No. 9, 1921 ; No. 11, 1922. 

21. K. W. Soheele, Opunoula, voL 2, p. 229, 1786. 

22. Ph. van Tieghem, Arch. d. Sciences naturelks bot. (5), vol. 8, p. 240, 

1867. ' 

23. E. Robiquet, Ann. Chim. Phys. (3), voL 39, p. 453, 1853. 

24. E. Duolaux, Fr&ny'a Encydoptdie chimique, vol. 9, p. 1, p. 226, 1883. 

(Durod, Paris.) 

25. L. Knudson, J. Biol Chem., vol. 14, pp. 169 and 285, 1913. 



PART TWO 



CHAPTER V 
THE FERMENTATION OF MONOSES 

THE hydrolytic processes by which polysaccharides are 
converted by micro-organisms into their component monoses 
results in the liberation of very little energy, far from 
sufficient to cover the requirements of the cells responsible 
for the hydrolysis. To satisfy these requirements the liberated 
monoses must undergo decomposition. 

The fact that numerous products result from this de- 
composition has been regarded as an indication that micro- 
organisms possess a large number of enzymes, each of them 
responsible for a specific change of the monose molecule. 
Recent investigations, however, indicate that these changes 
are the result of comparatively few and simple reactions, 
catalysed in some manner hitherto not definitely ascer- 
tained. To explain these reactions Oppenheimer and Neu- 
berg 1 suggest the functioning of a single group of enzymes 
which they term 'desmolases'. 

Quastel 2 favours the assumption of the presence of powerful 
electrical fields on the surface of the bacterial cell membrane. 
But it may well be that some other explanation will in time 
be found to be a more suitable working theory. 

Until about the second decade of the present century it 
was customary to speak of the carbohydrate decomposing 
activities of micro-organisms as oxidation processes by which 
food substances such as monoses became decomposed, either 
in the presence of atmospheric oxygen (aerobic respiration), 
or of oxygen contained as an integral part of the mole- 
cule of one of the substances which were being decomposed 
(anaerobic respiration, fermentation). 

With the accumulation of experimental evidence in support 
of Wieland's 8 theory on the chemistry of respiration this view 
had to be abandoned in favour of the assumption that hydro- 
gen atoms, contained in the decomposing molecule, became 



78 THE FERMENTATION OF MONOSES 

'activated* before oxygen entered the field of reaction. 
Through their activation the hydrogen atoms were rendered 
capable of combining with oxygen or with any other sub- 
stances capable of combining with activated hydrogen. The 
molecule containing the activated hydrogen atoms is termed 
the hydrogen donator, the oxygen, or any other hydrogen 
absorbing molecule or molecule radical, the hydrogen ac- 
ceptor. Such substances as methylene blue, litmus, and 
nitrates can function as hydrogen acceptors. 

Very interesting work on the activating properties of 
certain facultative anaerobic and obligatory aerobic micro- 
organisms has been carried out by Quastel and his collabo- 
rators ;* work which has demonstrated experimentally that 
these organisms activate the molecules of the substances to be 
dehydrogenated (oxidized) before a fermentation takes place. 
This activation involves in some cases the hydrogen donator 
only, in others both the hydrogen donator and the hydrogen 
acceptor. It is clear from Quastel's work that the activation 
of the hydrogen donator is essential in all cases, but that 
certain hydrogen acceptors, methylene blue for instance, 
are able to function without preliminary activation. 

Atmospheric oxygen possibly belongs to those hydrogen 
acceptors which function without preliminary activation. 
Some authorities, however, among them Hopkins, 6 Rapkine 
and Wurmster, 6 Oppenheimer, 7 von Scent-Gybrgyi, 8 and 
Kluyver and Donker, 9 favour the view that an activation is 
essential before oxygen becomes capable of combining with 
activated hydrogen. 

As a result of the transfer of hydrogen to the acceptor, 
the energy necessary for growth is liberated and substances 
are formed which, according to Quastel and Stephenson, 10 
serve as the basis for the synthetic processes of the micro- 
organisms responsible for the activation. 

Thus the essential feature of respiration and of fermenta- 
tion as viewed in the light of Wieland's theory, is an action 
of the living plasma, or of enzymes produced by the plasma, 
or of electrical fields functioning on the surface of the cell 



THE FERMENTATION OF MONOSES 79 

membrane (on the actual agency opinions still differ), on 
certain molecules of the medium in which the plasma is 
suspended. A transfer of hydrogen thereby becomes possible. 
The function of oxygen has been reduced to that of an ac- 
ceptor for hydrogen, a function which it shares with a good 
many other substances, which in the case of anaerobic 
respiration at least are more suitable than oxygen itself. 

The principle of this more recent conception of respiration 
and fermentation is an interaction between hydrogen donator 
and hydrogen acceptor, through what Oppenheimer 11 de- 
scribes as oxidation-reductions. 

Why oxygen should be of less value than other acceptors 
is not yet clearly explained. It is conceivably connected with 
the inability of anaerobic organisms to produce catalase, an 
enzyme responsible for the destruction of the peroxide of 
hydrogen formed as the final product of reaction between 
activated hydrogen and oxygen. 

In these and subsequent pages the above conception has 
been adopted to explain the manifold and apparently uncon- 
nected data which in the course of time have been collected 
on the subject of the action of micro-organisms on monoses. 
This conception is an indispensable part of the only existing 
working theory which makes it possible to reduce the various 
known data on bacterial fermentations to something like 
order, and which makes it feasible to co-ordinate this subject 
into a comprehensible system a system which already shows 
signs of being capable of withstanding criticism based on 
experimental evidence. 

In order to appreciate the system adopted for the grouping 
of the various types of bacterial fermentation an outline must 
be given of the working theory on which it is based. 

It is generally accepted that a monose must be esterified 
into monosephosphoric esters before it can be decomposed by 
yeast, or by any other living cell or cell extract, into com- 
pounds containing chains of less than six carbon atoms. The 
exact nature of these esters has not yet been clearly defined. 
They may be identical with the hexosediphosphoric ester 



80 THE FERMENTATION OF MONOSES 

first isolated by Harden and Young j 12 or with the hexose- 
monophosphorio ester of Robison ; u or they may perhaps be 
represented by specially labile esters which on stabilization 
yield either hexosediphosphorio or hexosemonophosphorio 
esters (Kluyver and Struyk 14 ). 

Following the formation of these phosphoric esters it is 
possible to conceive the conversion of monoses into any or all 
of the known fermentation products on the assumption of 
the occurrence of progressive oxidation-reduction reactions, 
activated by the responsible micro-organisms. Instead of a 
multitude of specific enzymes the working theory adopted 
in these pages requires the presence of two catalytic agencies 
only, one responsible for the esterification of a monose into 
monose phosphoric esters and another activating the hydro- 
gen transfer from a hydrogen donator to a hydrogen acceptor. 
The diversity of the final fermentation products is conceived 
to be the result, of variations in the functions of the activating 
agency, in the faculty of a specific organism to utilize certain 
substances as hydrogen acceptor, and conceivably also in the 
possession by the organism of protective powers against the 
accumulation of certain substances hydrogen peroxide for 
instance which in higher concentrations might destroy the 
cell. 

As early as 1907 Meyer 16 suspected that methylglyoxal, 

CH 3 CO C TT, might be an intermediate fermentation 



product of glucose when acted upon by yeast. This view was 
further developed by Neuberg and Kerb, 16 who assume that 
glucose when fermented by yeast is first converted into two 
molecules of methylglyoxal, each of which is further dis- 
integrated until alcohol and carbon dioxide results. In the 
course of time numerous attempts have been made to ascer- 
tain the behaviour of a large number of micro-organisms 
towards methylglyoxal, sometimes with positive results 
showing that this substance is decomposable into the normal 
fermentation products of the test organism, and some- 
times with negative results. Through these investigations it 



THE FERMENTATION OF MONOSES 81 

oame to be widely accepted that methylglyoxal is the first 
intermediate decomposition product in all those fermenta- 
tions in which a rupture of the monose chain occurs. Recently, 
however, work by Lambie 17 and by Kermaok, Lambie, and 
Slater 18 on the function of methylglyoxal in insulin hypo- 
glycaemia has thrown doubt on the soundness of this view. 
It would appear that methylglyoxal cannot replace glucose 
in the alleviation of insulin hypoglycaemia, while dihydroxy- 
acetone, CH a OH.CO.CH a OH, another compound possessing 
a three carbon chain, is even better suited to do so than 
glucose itself. Methylglyoxal, therefore, cannot be a physio- 
logical intermediary of glucose, at least not in the form 
in which it was tested by Lambie. If a substance is to 
function as an antidote against insulin hypoglycaemia, it 
must be affected by insulin in the same way as glucose is 
affected and for that reason must be either glucose itself or a 
physiological derivative of glucose. 

It may be, therefore, that dihydroxyacetone and not 
methylglyoxal is the first detectable intermediate decom- 
position product of hexose. This was suspected many years 
ago by Bertrand 19 and by Boysen-Jensen. 20 

However this may be, there is considerable experimental 
evidence in support of the assumption that on saponification 
of a monosephosphorio ester by micro-organisms, the liberated 
hexose is converted into two molecules possessing three 
carbon chains. 

Kluyver and Donker, 9 who are the authors of the working 
theory adopted in these pages for the purpose of grouping 
the various microbiological fermentations, explain the con- 
version of glucosephosphoric esters into methylglyoxal by 
the following scheme: 

(1) CH 2 OH.CH.CH*OH.CHOH.OHOH.CHO-> 



PO a R 2 



82 TKE I'ERMENTATION OF MONOSES 

(2) CH 2 OH.CH.CHO + CE 2 OH.CHOH.CEO-* 



POgR 2 glyceraldehyde 

(3) CH a OH.CH.CHO -f- E 2 -> CH 2 OH.CHOH.CHO 
| +P0 4 R 2 H 

P0 8 R 2 

glyceraldehyde phosphorio glyceraldehyde -f- phosphoric 

ester eater 



(4) CE 2 .CH*OE.C<r -*CE 3 COH. 

^0-^ H O'Qg- 

glyceraldehyde anhydride 

OH /BE r\ 

CH 3 C<(-C-OH-* CH 3 .CO.</ 

C\~Cf \ \TT 

UL \OH M 

methylglyozal 

The activating properties of the organism attacking the 
monose are assumed to affect, in the first instance, the hydro- 
gen atom bound to the fourth carbon atom. This is indicated 
above by an asterisk placed against this atom. Through the 
activation, the third carbon atom becomes capable of func- 
tioning as a hydrogen acceptor, with the result that the 
activated hydrogen wanders to this carbon atom. Thereby 
the six carbon chain is broken up into one glyceraldehyde 
molecule and one glyceraldehyde phosphoric ester molecule. 
The latter becomes hydrolysed, yielding one additional mole- 
cule of glyceraldehyde and one molecule of phosphorio acid, 
the latter combining with a fresh monose molecule so long as 
the esterifying agency continues to function. 

Under the influence of the fermenting organism the hydro- 
gen of the second carbon atom in the glyceraldehyde anhy- 
dride molecule is activated and transferred to the third. The 



THE FERMENTATION OF MONOSES 83 

introduction of a molecule of water gives rise to the formation 
of methylglyoxal. 

The assumption of the occurrence of dihydroxyacetone as the 
first detectable intermediate product of fermentationpostulates 
a slightly different conception of this initial stage of fermenta- 
tion. In this case the activated hydrogen of the second carbon 
atom would be transferred to the first carbon atom thus : 

/OH / OH: 

(1) CH a .CHOH.C< -> CH a .COE.C-H 

\0^ H V \H 

glyoeraldehyde anhydride 

On introduction of one molecule of water dihydroxyacetone 
would result 

,OH 

(2) CH 2 .COH.CH 2 OH -> CH 2 OH.<-CE 2 OH-> 



H OH 

CH 2 OH.CO.CH a OH+H 2 

dihydroxyacetone 

From methylglyoxal perhaps it would be more correct 
to have assumed from dihydroxyacetone Kluyver and 
Donker conceive the formation of all known fermentation 
products through straightforward oxidation reductions, 
occurring : (i) within the molecule itself Intramolecular oxida- 
tion reductions', (ii) between two molecules of intermediate 
products Int&rmolecvJar oxidation reductions ; or (iii) through 
Condensations, that is, through oxidation reductions resulting 
in the coupling of the hydrogen donator with the hydrogen 
acceptor. 

- As instances of intramolecular oxidation reductions of 
methylglyoxal the following reactions may be mentioned : 



, 
CH 8 .CO.C-OH -> CH 3 .CHOH.COOH 

Na 

methylglyoxal hydrate laotio aoid 

G2 



84 THE FERMENTATION OF MONOSES 



CH 3 .CO.C-OH -> CH 3 .C<f +H.COOH 

\H H 

methylglyoxal hydrate acetaldehyde formio aoid 

/ H / 

CH 3 .CO.C-OH -> CHgCO.O^ + 2H 

methylglyoxal hydrate pyruvio aoid 

H.COOH V CO a +2H 

formio acid 

Instances of intermolecular oxidation reductions would 
comprise the following reactions: 
CH 8 .CHO+2H -> CH 3 CH 2 OH 

aoetaldehyde ethyl alcohol 

CH 3 .CO.CHOH.CH 3 +2H -* CH 3 CHOH.CHOH.CH 3 
aoetyhnethyloarbinol 2 : 3 butyleneglycol 

CH 3 CH a CH a COOH+4H - OH 3 CH 2 CH a CH a OH+H a O 

n-butyrio acid n-butyl alcohol 

C a H 12 6 -f2H -* C 6 H 14 6 

fructose mannitol 

CH 3 .CH(OH) a -> CH 3 .COOH+2H 

acetaldehyde hydrate acetic acid 

Condensations would be represented by the following re- 
actions: 

/OH 

CH 3 .C-OH + >C.CH 3 -> CH 3 .CO.CHOH.CH 3 

\c\y 
H u 

aoetaldehyde hydrate aoetaldehyde acetylmethyloarbhioi 

/o H \ / OH / OH 

CH 3 .C^ +H-C-C-OH->CH 3 .CHOH.CH a .C-OH-> 

H H/ ^H \H 

aoetaldehyde acetaldehyde /J-hydroxybutaldehyde 

hydrate 

CH 3 .CH a .CH a .COOH+H 2 

n-butyrio aoid 



THE FERMENTATION OF MONOSES 85 

X> H \ 
CH 3 Of +H-C-COOH -> CH 3 .CO.OH 2 .COOH - 

\OTT / 



aoetio acid acetic acid aoetoacetio acid 



acetone 

To illustrate Kluyver and Donker's conception by a concrete 
example the behaviour of Bact. coli commune may be chosen. 
The fermenting properties of this bacterium have frequently 
been investigated. It has been established by various in- 
vestigators that it produces from glucose lactic, formic, and 
acetic acids as well as ethyl alcohol, carbon dioxide, and 
hydrogen. All of these substances can be visualized as de- 
rived from glucose through oxidation reductions with glucose 
phosphoric esters, glyceraldehyde, and methylglyoxal (or 
perhaps dihydroxyacetone) as intermediate fermentation 
products. 

2C 6 H 12 6 -* 2C 6 H 10 6 .R 2 P0 4 -* 

glucose glucose phosphoric ester 

-4CH 8 .CO.C/ 

ul 
4C 3 H 6 3 me thylgl y oxal 

Xr 4CH a OH.CO.CH 2 OH-> 

glyceraldehyde dihydroxyaoetone 



.C/ 



^ 
2CH3.CHOH.COOH+2CH 3 .C< + 2H.COOH 

/\ X H /\\ 

lactic acid acetaldehyde formic acid 

\ / i \ 

CH 3 .CH 2 OH 2H 2C0 2 H a 

acetic acid ethyl alcohol activated hydrogen 

However, in the case of the fermentation of glucose by Bact. 
coli commune there are not sufficiently definite data available 
on the actual yields of the various fermentation products 



86 



THE FERMENTATION OF MONOSES 



from which support could he derived for the assumption that 
the conversion of glucose to the final fermentation products 
had actually followed the lines suggested above. Nor have 
all of the fermentation products of this bacterium been 
definitely established. 

A carefully determined list of all the fermentation products, 
other than those synthesised into plasma and plasma content 
is available, however, for another organism, Bac. acetoeihylicus, 
which Donker 21 studied for this purpose* 

In columns 1 and 2 of Table n a list is given of these 
fermentation products and of the percentage of each of them 
obtained from glucose when fermented by Bac. acetoethylicus. 

TABLE n 

Bac. acetoethylicus grown in yeast-water containing 2 per cent, 
of glucose and 1 per cent, of calcium carbonate. 



Fermentation 
product. 


Percentage 
of fermenta- 
tion prodiict 
calculated 
on glucose 
fermented. 


Number of gramme molecules produced 
per 50 gramme molecules of glucose 
fermented. 


Carbon 
dioxide. 


Hydrogen. 


Acetaldehyde. 


Carbon dioxide 
Hydrogen 
Fonnio aoid 
Acetic acid 
Ethyl alcohol 
Acetone 
Aoetymethyl- 
carbinol 
2:3,Butylene- 
glyool 


52-6 
152 
2-6 
5-2 
31-2 
9-1 
traces 

06 


+107-6 
+ 5-1 

14-1 


+ 684 
+ 5-1 
7-8 
+ 61-0 
28-2 

+ 0-6 


+ 7-8 
+61-0 
+28-2 

+ 1-2 


Total 




08-6 


99-1 


982 



Assuming the fermentation of the glucose to have pro- 
ceeded in this case via methylglyoxal to acetaldehyde and 
formic aoid, and the formic acid to have been partly converted 
into carbon dioxide and hydrogen, and postulating also that 



THE FERMENTATION OF MONOSES 87 

the whole of the various fermentation products, as recorded 
in columns 1 and 2 of Table n, were derived by oxidation 
reductions from this acetaldehyde and formic acid, it is clear 
not only that equimolecular quantities of acetaldehyde and 
formic acid or of hydrogen and carbon dioxide must have 
been produced from the glucose, but that the sum total of all 
the fermentation products, when expressed as gramme mole- 
cules of acetaldehyde and formic acid (carbon dioxide and 
hydrogen respectively), must have been exactly double that 
of the number of gramme molecules of glucose fermented. 

The following equation of the fermentation of glucose 
by Bac. acetoethylicus will illustrate the necessity for this 
assumption. 

^ y 

C 6 H 12 6 -^2CH 8 .CO.cf -*2CH a Cf +2H.COOE-* 
N H X H 

glucose methylglyoxal acetaldehyde formic acid 

^ 
2CHg.Of +2H 2 +2CO a 

X H 

aoetaldehyde hydrogen carbon dioxide 

The actual yield therefore of each of the final fermentation 
products as recorded in column 2 of Table II must have been 
a definite fraction of this total number of gramme molecules 
of acetaldehyde and formic acid (or carbon dioxide and hydro- 
gen), and their total when expressed as gramme molecules of 
aoetaldehyde, hydrogen, and carbon dioxide must have been 
double that of the gramme molecules of glucose fermented. 

That this is actually the case is clear from Table II. In the 
last three columns of this table the numbers of gramme 
molecules of each of the fermentation products obtained from 
50 gr. molecules of glucose fermented have been entered. 
These figures were arrived at on the basis of the reasoning 
given below. It will be seen from these figures that the total 
number of gramme molecules of acetaldehyde, hydrogen, and 
carbon dioxide are very nearly double that of the gramme 
molecules of glucose fermented by Bac. acetoethyhcus. 



68 THE FERMENTATION OF MONOSES 

These figures were arrived at in the following manner. 
Ethyl alcohol was produced by the organism to the extent 
of 31-2 per cent, calculated on the glucose fermented. This 
amount is equal to 61-04 gr. molecules of ethyl alcohol per 
50 gr. molecules of glucose since 

* 60x180x31-2 _ 
46XZ= - ; JS:=61-04. 

This alcohol was assumed to have been formed from 
acetaldehyde by hydrogenation on the lines shown below : 

+2H->CH 8 CH a OH. 



It is to be concluded, therefore, that there must have been 
available 61-04 gr. molecules of acetaldehyde and 61-04 gr. 
molecules of hydrogen for this purpose. Both of these quanti- 
ties are entered in their respective columns of Table IT, pre- 
ceded by a sign. 

In the case of the acetic acid produced by Bac. acetoethy- 
licus 5-2 per cent, was obtained from the sugar fermented. 
This amount is equivalent to 7-8 gr. molecules per 50 gr. 
molecules of glucose, since 



100 

The acetic acid was derived from aoetaldehyde by de- 
hydrogenation according to the following formula : 



+2H 

aoetaldehyde aoetio acid. 

hydrate 

The 7-8 gr. molecules of acetic acid therefore must have 
required the presence of 7-8 gr. molecules of acetaldehyde 
* indicates molecular weight. 



THE FERMENTATION OE MONOSES 89 

hydrate less 7-8 gr. molecules of hydrogen. These amounts 
have been entered in their respective columns of Table n as 
7-8 gr. molecules of acetaldehyde and 7-8 gr. molecules 
of hydrogen. 

For the purpose of calculation the yields of the remaining 
fermentation products have been similarly converted. 
Together they give the totals recorded in Table n. 

Similar evidence in support of Kluyver and Donker's 
theory of fermentation has been obtained by the last-named 
writer in the case of a number of other glucose-fermenting 
bacteria. It is to be hoped that the scope of this work will be 
extended to embody all other types of fermentation in order 
that it may be ascertained to what extent Kluyver and 
Donker's efforts to co-ordinate and to simplify the conception 
of bacterial fermentations are justifiable experimentally. 

The conception that the fermentative activity of micro- 
organisms is a function of their hydrogen activating properties 
admits of a sub-division of these activities into aerobic, 
facultative anaerobic, and obligatory anaerobic fermenta- 
tions, depending on the faculty of a given organism to utilize 
oxygen as a hydrogen acceptor. But a grouping of this order 
would be wholly inadequate to account for the great diversity 
of ways in which carbohydrates are decomposed by micro- 
organisms. 

A further though still incomplete grouping is undoubtedly 
possible on the above assumption and has been attempted by 
Kluyver and Donker. 9 These writers reduced the fermenta- 
tive activities of micro-organisms to seven types as occurring : 
I. among the organisms producing gluoonic acid ; 
II. among the aerobic spore forming bacteria ; 
HE. in yeast fermentations ; 

IV. in the group of Bact. coli commune Bact. typhosum ; 
V. among the true lactic acid bacteria ; 
VI. among the propionic acid bacteria ; and 
VII. among the butyric acid butyl alcohol bacilli. 
In broad outline Kluyver and Donker's subdivision has 
been adhered to in the following pages, but attention has been 



90 THE FEEMENTATION OF MONOSES 

paid also to the property of micro-organisms of utilising 

oxygen as hydrogen acceptor. 

The information available on the fermentation of pentoses 
has been included in a separate chapter Chapter XTTT 
not because this type of fermentation shows a marked 
difference from that of hexoses, but because of lack of in- 
formation on this type of fermentation. A separate chapter 
has been devoted also to the mucus fermentations in which a 
synthesis of hexoses to hexosans occurs. 

Among the obligatory aerobic micro-organisms two dis- 
tinct modes of hexose fermentation are clearly discernible. 
In the first group the carbohydrate is subjected to direct 
dehydrogenation, frequently without the rupture of the six 
carbon chain. This group comprises the acetic acid bacteria 
and certain fungi. It is dealt with in Chapter VI. 

In the second group of aerobic fermentations a preliminary 
disintegration of the six carbon chain occurs. To this group 
belong the obligatory aerobic soil bacilli, dealt with in 
Chapter VII. 

Among the facultative anaerobic micro-organisms two 
main types of fermentations are possible, both of them in- 
volving a preliminary cleavage of the hexose chain. 

The first, dealt with in Chapter Vlil, does not give rise to 
lactic acid formation under normal conditions. In this group 
must be placed such types as Bact. fluorescens liquefaciens, 
JSact. prodigiosum, JBac. ethaceticus and Sac. acetoethylicus. 

The second involves the production of lactic acid in smaller 
or larger quantities. This mode of fermentation must be 
divided up into two sub-groups, the first comprising micro- 
organisms which, in addition to lactic acid, produce a number 
of other important fermentation products ; the second com- 
posed of the true lactic acid bacteria which convert the bulk 
of the glucose into lactic acid. These facultative anaerobic, 
lactic acid producing types are discussed in detail in Chapters 
IX and X. 

Among the facultative anaerobic micro-organisms with 
decided preference for anaerobiosis and among the obligatory 



THE FERMENTATION OF MONOSES 91 

anaerobes lactic acid production is not an important final 
fermentation product. It may be produced as an inter- 
mediate, however, as in the case of the propionic acid bacteria 
dealt with in Chapter XI and by the butyric acid and butyl 
alcohol bacteria discussed in Chapter XII. 

LITERATURE 

1. C. Oppenheimer and C. Neuberg, Biochem. Zeits., vol. 166, p. 260, 1025. 

2. J. H. Quastel, Biochem. J., vol. 20, p. 166, 1926. 

3. C. Wieland, Oppenheimer 1 a Handbuch der Biochemie, 2nd edition, vol. 2, 

p. 252. Qustav Fischer, Jena, 1923. 

4. J. H. Quastel, M. Stephenson, and M. D. Whetham, Biochem. J., vol. 19 

pp. 304, 520, 645, 652, 1925. 

6. E. Hopkins, Opening address to the International Congress of Physio- 
logists in StocWiolm, 1926. 

6. L. Rapkine and R. Wurmster, Proo. Roy. Soc., vol. 102 (B), p. 128, 

1927. 

7. C. Oppenheimer, Ohem^Ztg., vol. 62, p. 709, 1928. 

8. A. von Soent-GyGrgyi, Biochem. Zeits., vol. 150, p. 195, 1924. 

9. A. J. Kluyvei and H. J. L. Donker, Zeits. d, ZeUe und Gewebe, vol. 13, 

p. 134, 1926. 

10. J. H. Quastel and M. Stephenson, Biochem. J., vol. 19, p. 660, 1925. 

11. C. Oppenheimer, Die Fermente und ihre Wirkungen t 6th edition, G. 

Thieme, Leipzig, 1924-6. 

12. A. Harden and W. J. Young, Proc. Boy. Soc., B., vol. 82, p. 321, 1910. 

13. R. Robison, Biochem. J., vol. 16, p. 809, 1922. 

14. A. J. Kluyver and A. P. Struyk, Proc. Kon. ATcad. -van Wetenschap, 

Amsterdam, vol. 30, p. 871, 1927 ; vol. 31, p. 882, 1928. 
16. P. Mayer, Biochem. Zeits., vol. 2, 435, 1907. 

16. C. Neuberg and J. Kerb, Biochem. Zeits., vol. 58, p. 158, 1913. 

17. C. G. Lambie, J. Soc. Chem. Ind., vol. 46, p. 300 T., 1927. 

18. W. 0. Kermack, C. G. Lambie, and R. H. Slater, Biochem. J., vol. 21, 

p. 40, 1927. 

19. G. Bertrand, Ann. Chvm. Phys. (8), vol. 3, p. 181, 1904. 

20. P. Boysen-Jensen, Biochem. Zeits., vol. 58, p. 451, 1914. 

21. H. J. L. Donker, Bijdrage tot de Kennis der Boterzuur-Butyl alcoholen 

Acetonegishngen. Dissertation, W. D. Meinema, Delft, 1926. 



CHAPTER VI 

THE DEHYDROGENATION OF HEXOSES RESULT- 
ING IN THE PRODUCTION OF GLUCONIC, SAC- 
CHARIC, SUCCINIC, FUMARIC, OXALIC, AND 
CITRIC ACIDS. 

THE grouping of the substances dealt with in this chapter as 
products of one single type of fermentation indicates the 
existence of an intimate connexion between their various 
modes of formation by micro-organisms. This can hardly be 
substantiated experimentally at present, since the observa- 
tions made on the production of gluconio, saccharic, succinic, 
fumaric, citric, and oxalic acids by micro-organisms have 
done Httlj^ beyond ftfltabKahirig *heir actual ocourrence_ajB 
definite hexose fermentation products. A relationship be- 
tween these substances is indicated, nevertheless, by the fact 
that one and the same type of organism, or group of organ- 
isms, can produce one or more, or sometimes all of the com- 
pounds mentioned, being governed in this respect by external 
conditions such as the reaction of the culture medium and 
the presence or absence of certain food-substances and 
powerful hydrogen acceptors. 

A discussion of the available, somewhat extensive literature 
will indicate the present position of knowledge on the subject 
and will show how far the suggested grouping is justifiable. 

PBODUXmON OB 1 GLUOONIO AOTO 

One of the most interesting observations which has been 
made was that recorded recently by Muller, 1 - a that a press 
juice can be prepared from the mycelium of species of 
Aspergillus and Penicillium which, when acting on glucose, 
converts the carbohydrate into an acid which can be identi- 
fied as gluoonic acid. The conversion takes place only in the 
presence of an abundance of oxygen, within a temperature 



PRODUCTION OF GLUCONIC AND OTHER ACIDS 93 
range of C. to 30 C. and at a definite hydrogen ion con- 
centration equal to a pH value of 6-5 to 6-5. Particularly 
noteworthy is Muller's observation that phosphates fail to 
accelerate the action of the press juice. This can only be 
interpreted as indicating that the conversion of the glucose 
to gluconic acid proceeds without the intervention of phos- 
phoric esters and supports Kluyver and Donker's 3 contention 
that the glucose molecule as such is dehydrogenated without 
the preliminary formation of intermediates containing 
shorter carbon chains. The conversion of glucose to gluconio 
acid therefore may be visualized as a dehydrogenation of the 
enolic form of glucose in the presence of a powerful hydrogen 
acceptor such as oxygen or, according to Hoyer, 4 methylene 
blue, litmus or indigotin. 

/ H 

CHOH.CHOH.CHOE.CHOH.CHOH.C-OH-* 



^ 
CH 2 OH.CHOH.CHOH.CHOH.CHOH.Cf +2H. 

The formation of gluconic acid by micro-organisms was 
first observed by Boutroux 5 in 1878, who at the time was 
under the impression that the acid which he had obtained 
was lactic acid. At Pasteur's suggestion he had undertaken 
to investigate the behaviour of an organism, closely related 
to Pasteur's Mycoderma aceti (Bact. Past&wrianum) towards 
glucose. He came to the conclusion that his organism was 
identical with Pasteur's lactic acid 'ferment' since it pro- 
duced a colourless, tasteless, viscous, and non-crystalline 
acid from glucose. Subsequently Boutroux 6 corrected this 
view and showed that the acid which he had obtained and in 
1880 7 had termed zymogluconic acid was identical with the 
gluconio acid obtainable from glucose by oxidation with 
bromine. The formation of gluoomc acid by Bact. Pasteuri- 
anum was confirmed by Brown 8 in 1886. Since then several 
writers, notably Henneberg, 9 have reported on the production 



94 DEHYDROGENATION OP EEXOSES RESULTING IN 
of gluconic acid by acetic acid bacteria. From these observa- 
tions it may be concluded that the direct dehydrogenation of 
glucose to gluconio acid is a special feature of the activity of 
the acetic acid bacteria. 

Acids have been observed to be produced by acetic acid 
bacteria also from fructose and from various di- and tri- 
saccharides such as saccharose, maltose, lactose, and raffinose. 
The nature of these acids, however, has but rarely been 
ascertained. In Hoyer's* experiments the action of Sact. 
xylinum on saccharose was definitely shown to result in the 
production of gluconic acid, but Hermann's 10 observations 
that Bact. gluconicwm produces acetic acid from maltose and 
from fructose leaves it an open question whether the types 
studied by Henneberg 11 and by Zeidler 12 produced gluconic 
or acetic acid from carbohydrates other than glucose. It is 
felt by the writers of these pages that a renewed investigation 
of the action of acetic acid bacteria on carbohydrates would 
be of considerable theoretical interest, particularly in view of 
the observations made by Neuberg and Simon 13 that Bact. 
ascendens, when allowed to act on glucose under anaerobic 
conditions, produces ethyl alcohol in considerable quantities. 
The acetic acid bacteria apparently have at their command, 
in addition to their direct dehydrogenating powers, other 
means of attacking hexoses, resembling those of yeasts and 
certain alcohol producing bacteria. 

The action of acetic acid bacteria on saccharose is utilized 
in China for the preparation of a drink known as ' Kombucha '. 
According to Hermann 10 this drink is prepared by allowing 
acetic acid bacteria, in the form of a mucous covering, to 
develop on sweetened tea, thereby converting any saccharose 
present into gluconic acid. This is the only case known to the 
writers in which practical use has been made of the acidic pro- 
perties of gluconic acid. There are many reasons, notably an 
increased nutritive value over acetic acid, why gluconic acid, 
as suggested by Herzfeld and Lanart, 14 should be more exten- 
sively used in the preparation of foods if a cheap and efficient 
method for its production could be devised. The utilization 



PRODUCTION OP GLUCONIO AND OTHER ACIDS 96 
of micro-organisms for this purpose might be worthy of 
consideration. 

The direct dehydrogenating properties of acetic acid 
bacteria are evident in their behaviour towards glycerol, 
which is converted by them into dihydroxyaoetone when 
conditions are favourable, that is, when a powerful hydrogen 
acceptor is available. The reaction may be visualized by the 
following formula : 

(l)CH a OH.CHOH.CH 2 OH->CH 2 OH.CO.CH a OH+2H 

glyoerol dihydroxyaoetone 

where the available CHOH group, the only group in the 
glycerol molecule which, according to Bertrand, 16 can be 
oxidized (dehydrogenated), has had its H atoms activated. 

The production of dihydroxyacetone by micro-organisms 
was first observed by Bertrand in his study of an organism 
which he described as the sorbose bacterium but which has 
since been identified with Bact. xyUnum. The sorbose bac- 
terium was given its name by Bertrand because it was found 
to be able to convert sorbitol, present in the juice of mountain 
ash berries, into the corresponding carbohydrate sorbose. 

Virtanen and Barlund 16 have studied the conditions favour- 
ing the formation of dihydroxyacetone and find that a suit- 
able hydrogen ion concentration is essential for the conversion. 
The reaction, according to them, proceeds best at an acidity 
in the culture medium equal to a pH value of 5-0. The 
method has been considerably elaborated by the German 
chemical industries, who have secured patents 17 protecting 
the conversion of glycerol to dihydroxyacetone in the presence 
of food material such as hay infusions and distillery waste 
extracts. To this mixture of food solutions and glycerol 
the requisite bacteria are added. Bact. xylinum and Bact. 
ffuboxidans would appear to be particularly suitable for the 
purpose. 

A reference was made above to work by Muller on the 
extraction of an enzyme from species of Aspergillus and 
Penicillium, which converted glucose into gluconic acid. 



96 DEHYDROGENATION OJ HEXOSES RESULTING IN 
Production of gluconic acid by fungi was first recorded in 
1922 by Molliard. 18 Subsequent work by Falck and Kapur, 19 
Falok and van Beyma thoe Enigma, 20 Butkewitsch, 21 Weh- 
mer, 22 May, Herrick, Thorn and Church, 28 Herrick and May, 24 
and notably by Bernhauer, 26 has demonstrated that the pro- 
duction of this aoid by species of Aspergittiis and by some 
P&nicittia is extremely common, at least when certain physio- 
logical conditions prevail. It is clear from the publications 
quoted that the presence of an abundant supply of oxygen 
is essential for the smooth conversion of glucose to gluconio 
acid. It has been established also that the production pro- 
ceeds best at a reaction approaching neutrality, that is, in 
the presence of calcium carbonate, and in the absence of 
excessive amounts of nitrogen. An optimum temperature 
for the conversion would appear to exist, 30 C. to 36 C. 
being quoted by Bernhauer 26 and by MoUiard, 26 22 C. by 
Falck and Kapur, presumably depending on the particular 
strain of fungus used. 

Under the most favourable conditions May and his col- 
laborators succeeded in converting 80 per cent, of the avail- 
able glucose into gluconic acid in the course of 12 days, and 
under such conditions gluconio acid was found to be the only 
dehydrogenation product formed, at least when Penicittium 
lut&wm purpurogenum was used as the activating organism. 

Both in the case of fungi and in that of bacteria the action 
on the carbohydrate may take a different course and various 
other dehydrogenation products may be formed either in 
addition to gluconio acid or in its place. 

Of such Boutroux 87 recorded the production from glucose 
and gluconic acid of oxygluconic acid, CH 2 OH.CO.CHOH 

^ 
.CHOH.CHOH.Of by a coccus isolated by him and 

X OH 

claimed to be m'milar to a previously isolated gluoonic aoid 
producing type. 

Griiss 28 isolated a species of yeast Amphiernia rubra which 
carried the dehydrogenation of glucose still farther, producing 



PRODUCTION OF GLTJCONIC AND OTHER ACIDS 97 
saccharic acid, COOH.CHOH.CHOH.CHOH.CHOH.COOH, 
an acid which was detected also by Challenger, Subramanian 
and Walker 29 as a dehydrogenation product when glucose was 
acted upon by Asp&rgillus niger. There are at present no 
observations available to indicate the conditions required 
for the production of these higher oxidation products of 
glucose, but seeing that their formation does not involve a 
disruption of the glucose molecule, it may be permitted per- 
haps to assume that the agency and the conditions governing 
the production of gluoonio acid may suffice possibly if 
further intensified to convert glucose to oxygluconic and 
saccharic acid. 

Far more difficult is the interpretation of the formation of 
the various other substances which have been obtained as 
dehydrogenation products of glucose when acted upon by 
micro-organisms discussed here, notably by acetic acid bac- 
teria and various species of Asp&rgillus and, Penicillium. 
These substances comprise succinic acid, fumario acid, oxalic 
acid, and citric acid. 

The first two of these acids possess a four carbon chain, 
the oxalic acid a two carbon chain, while citric acid has six 
carbon atoms in its molecule. As these six carbon atoms do 
not form a straight chain, it must be assumed that citric acid, 
like the other acids referred to, has been derived from the 
glucose molecule after preliminary disruption of the six 
carbon chain of the carbohydrate. There is at present nothing 
to indicate how this disruption can have taken place. Pre- 
liminary conversion of the glucose into glucosephosphoric 
esters does not appear to occur, since the addition of phos- 
phoric acid, according to Bernhauer and Wolf, 30 does not 
increase the rate of production of citric acid any more than 
other acids capable of establishing a suitable hydrogen ion 
concentration. It is noticeable also that Bernhauer and 
Schon, 31 who detected the presence of acetaldehyde in 
saccharose-containing cultures of Asp&rgillus niger, emphasize 
that acetaldehyde is in no way connected with the production 
of citric acid by this fungus. 



98 DEHYDROGENATION OF HEXOSES RESULTING IN 

Raistriok and dark's 88 suggestion that the glucose molecule 
is first split into a two and a four carbon molecule, and that 
the production of citric acid results from the union of one 
molecule of acetic acid derived from the two carbon mole- 
cule and one molecule of oxalacetio acid resulting from 
the four carbon molecule is purely speculative, and is based 
solely on the observation of these workers that Asp&rgillus 
niger, which produces citric acid, develops well on four 
carbon dibasic acids such as suooinic and fumario which, as 
already mentioned, are produced from glucose by this and 
other fungi. 

The only other proposal which has been advanced to throw 
light on the theoretical aspect of the conversion of glucose 
into organic acids is that of Bernhauer, 34 who suggests that 
the formation of citric acid and perhaps also of the other 
acids involved is preceded by the disruption of the six 
carbon chains into two three-carbon chains. This, however, 
is equally unfounded experimentally. As indications of the 
changes taking place, there remain the empirically established 
facts that one and the same type of fungus or two systemati- 
cally closely related fungi can produce from glucose and from 
certain other hexoses, gluponio acid, sucoinio acid, fumaric 
acid, citric acid, and oxalic aoidjjtooording to the conditions 
prevailing during the action of the organisms on the carbo- 
hydrate. 

It is not to be understood that any one of these acids can 
be normally produced to the complete exclusion of all the 
others. Mogfcjgritgra^^^thejubject agree that gluoonic acid 
is produced first and under aUjxmditions, and that this acid 
is present therefore at one stageor anotEer of the conversion 
of glucose by fungi. 

Succinio and fumaric acids mixed with gluconio acid 
(Wehmer 33 ) are formed, for instance, by Aspergittusfumaricus, 
while the most typical citric acid producing types are capable 
of producing gluconio acid (Bernhauer 34 ), and probably do so 
during the early stages of their action on glucose. This might 
appear to indicate that gluconic acid is the first intermediate 



PRODUCTION OF GLUCONIC AND OTHER ACIDS . &9 
roduct in the conversion of glucose to the various acidic 
>mpoundfl dealt with, a view which has been widely held,- 
it which is strongly disputed by Bernhauer. 35 The fact 
imains nevertheless that gluconic acid can be converted 
to citric acid by various fungi such as Aap&rgittus fumaricus 
ichreyer 38 ). There is another significant observation which 
LTOWS light on the important part played by gluoonic acid 
the action of fungi and glucose. Both Wehmer 37 and 
jhreyer 88 have observed that the typical fumario acid pro- 
icing fungus Asp&rgillus fumaricus loses the power of 
educing its characteristic acid when cultivated on artificial 
edia for a prolonged time and then produces chiefly gluconio 
id. 

Oxalic acid has been shown to be formed both by acetic 
id bacteria and by fungi not only from glucose but from 
[iconic acid, succinic acid, and fumario acid (Raistrick and 
ark 32 ), and from citric acid (Banning 39 ). It might be thought 
eref ore that this acid represents a further dehydrogenation 
oduot of the various acids mentioned, and that succinic, 
mario, and citric acids are intermediaries between gluconic 
d oxalic acid. This may be the case, but it has not yet been 
nfirmed experimentally. In accepting this view it should 
noted, however, that recent work by Kostytschev and 
chesnokov 40 and by Chrzaszoz and Tinkow 41 indicates that 
e formation of oxalic acid is dependent on outside influences, 
rticularly on the presence of a sufficient alkalinity in the 
Iture medium, and that it is not an essential stage in the 
hydrogenation of glucose by the micro-organism dealt 
th in this chapter. 

PRODUCTION OF SUOOINIO ACTED 

ie conversion of glucose to suocinic acid COOH.CH 2 . 
I 2 .COOH has been demonstrated only in the case of two 
scies of Mucoraceae, Mucar Eouxii and Mucor stolonifer. 
ling the former as inoculant, Goupil 42 obtained succinic acid 
the main fermentation product when the fungus developed 

H2 



100 DEHYDROGENATION OF HBXOSES RESULTING IN 
on glucose containing media under aerobic conditions. In 
Butkewitsch and Fedoroff's 43 experiments Mucor stolonifer 
yielded mainly fumaric acid COOH.CHrCH.COOH, but 10 
per cent, of the sugar converted could be identified as 
suocinio acid. 

Presence of alkali, or at least of calcium carbonate, and 
oxygen would appear to be essential for the conversion. No 
work has been done to ascertain the effect of varying the 
concentration of the hydrogen acceptor present on the pro- 
portional yields of fumario and succinic acids obtained. 
As fumaric acid must be a dehydrogenation product of suc- 
cinio acid, it is to be expected that a restricted supply of 
hydrogen acceptors oxygen for instance must affect the 
conversion of glucose by Mucor stolonifer in favour of the 
formation of increased amounts of suocinic acid. 



PEODTJOTION OF FUMABIO AOID 

Fumario acid has been shown to be formed both by species of 
Mucor [Mucor stolonifer] and of Asp&rgillus [Aspergillus 
fumaricus]. Ehrlich 44 first recorded its formation by the 
former, Wehmer 46 by the latter. Shortage of nitrogen in the 
culture medium, a hydrogen ion concentration approaching 
neutrality, restricted supply of hydrogen acceptors (oxygen), 
and a temperature between 21 and 23 C. are conditions 
favouring the production of fumaric acid by Aspergillus 
fumaricus. But even with these conditions assured the 
organism loses its power of forming fumaric acid on prolonged 
artificial cultivation (Schreyer 38 ) and then reverts to a strain 
giving essentially gluconio and citric acids (Wehmer 37 ). 

The subject of fumario acid production by fungi is at 
present of theoretical interest only. The proposal has been 
advanced by Bailey and Potter, 46 however, that fumaric acid 
might be used for the synthesis of indigo. Should this method 
be found industrially important the microbiological conver- 
sion of sugars to fumaric acid might become of practical 
interest. 



102 DEHTDBOGENATION OF HBXOSES RESULTING IN 

In Aapergillus the sterigmata are formed simultaneously 
in smaller or larger numbers over the whole surface of the 
vesicle, which has been formed previously at the top of the 
conidiophore. In recent text-books, notably in that of Thorn 
and Church, 51 this classification of Citromyces as a distinct 
genus has been abandoned, since it has been possible to show 
experimentally that all species of Aspergilli and some Pencillia 
are capable of forming citric acid, and that the characteristics 
of Citromyces as denned by Sartory and Bainier are insufficient 
to distinguish it from the Aspergilli. 

Apart from the Asp&rgilli, various other fungi, such as 
Penicillium glaucum (Butkewitsch 52 ), Penicillium luteum 
(Wehmer 58 ), PeniciUium citrinum and Penicillium divaricatum 
(May and collaborators 28 ), as well as Mucor pwiformis (Weh- 
mer 63 ), have been shown to produce citric acid from hexoses. 

The Aspergilli giving the highest yields of citric acid 
over 25 per cent, calculated on the carbohydrates decomposed 
were claimed by Falck and van Beyma thoe Kingma 20 to 
be those producing bJteck^JiiQjen,_pr jawn, pigments. The 
moderately active jtypes, giving from 10 to 20 per cent, of 
acid, were those producing a yeUpjPvish-greenpigment ; the 
inferior types, with less than 10 per cent, yields, were those 
with slight pigmentation or none at all. 

After the first unsuccessful attempt by Wehmer the indus- 
trial application of fungi for the production of citric acid was 
allowed to lapse. Recently, and noticeably since Currie's 54 
investigations of the reaction, renewed interest has been 
taken in the matter and patents have been secured for the 
protection of what are claimed to be reliable and economic 
methods for the production of citric acid (Fernbach and 
Yuill 65 ). 

The outstanding feature of Currie's investigations is his 
observation that the formation of citric acid proceeds far 
more readily at distinctly acid reactions than at the hydrogen 
ion concentrations prevailing when calcium carbonate is 
added to the food solution. Currie recommends an initial 
hydrogen ion concentration of the pH value 3-4 to 3-5. He 



PRODUCTION OF GLUCONIC AND OTHER ACIDS 103 
secures this by adding hydrochloric acid to the food solution, 
which is composed of the following substances : 
Ammonium nitrate . . .0-2 to 0-25 per cent. 
Potassium dihydrogen phosphate 0-076 to 0-1 
Magnesium sulphate (crystalline) 0-02 to 0-025 
Saccharose .... 12-5 to 15 
Tap_watej to a total of . . 100 

During the first two or three days after inoculation little 
production of citric acid occurs as during this period the 
fungus spreads and covers the surface of the liquid. When 
this has been effected, after the third to the eighth day, the 
rise in acidity is very rapid, corresponding to a daily pro- 
duction of 2 per cent, of citric acid. 

When the hydrogen ion concentration of the solution has 
reached a pH value of 1-4, fungus development has become 
practically impossible, and the solution therefore is effectively 
protected against the growth of infecting fungi. This is impor- 
tant, since Wehmer's early attempts failed partly owing to the 
mashes becoming infected by undesirable types of fungi. It will 
be noticed that Currie recommends the use of saccharose and 
not of glucose as carbohydrate. In this respect he is following 
the usage of earlier investigators, and even of Wehmer himself, 
who found saccharose as suitable as glucose for the production 
of citric acid ; this incidentally confirms the view already ex- 
pressed in this chapter that the dehydrogenation of the hexose 
molecule by acetic acid bacteria and by the fungi discussed in 
these pages proceeds via the enolic form of the carbohydrate. 
Citric acid production has been shown by Butkewitsch 66 
and by Bernhauer 57 to be favoured by the presence of an 
abundance of nitrogen in the food solution. The optimum 
temperature for the conversion would appear (Bernhauer 57 ) 
to depend to some extent on the prevailing nitrogen con- 
centration, 30 C. being most favourable in the presence of 
abundant nitrogen, 35 C. at lower nitrogen concentrations. 
A restriction of the oxygen supply available in the culture 
medium favours citric acid production (Buchner and Wiisten- 
f eld, 58 and Bernhauer 57 ) . 



DEHYDROGENATION OF HEXOSES RESULTING BT 

LITERATURE 

. D. Muller, Biochem. Zeits., vol. 199, p. 136, 1928. 
i. D. Muller, Biochem. Zeits., vol. 206, p. Ill, 1929. 

3. A. J. Kluyver and H. J. L. Donker, Zeits. f. Chem. d. Zelle und Qewebe, 

vol. 13, p. 134, 1926. 

4. D. P. Hoyer, Die deutsche EssigindvMrie, vol. 3, p. 1, 1899. 
6. L. Boutroux, Oomptes rend., vol. 86, p. 605, 1878. 

6. L. Boutroux, Comptes rend., vol. 104, p. 369, 1887. 

7. L. Boutroux, Comptes rend., vol. 91, p. 236, 1880. 

8. A. J. Bro-wn, J. Chem. Soc., vol. 49, p. 172, 1886. 

9. W. Henneberg, Die detutsche Essigtndustrie, vol. 2, p. 146, 1898. 

10. T. S. Hermann, Biochem. Zeits., vol. 192, pp. 176 and 188, 1928. 

11. W. Henneberg, Zentrbl.f. Bakt., Abt. IE, voL 4, p. 14, 1898. 

12. A. Zeidler, Zentrbl.f. Bakt., Abt. IE, vol. 2, p. 729, 1896. 

13. C. Neuberg and E. Simon, Biochem. Zeds., vol. 197, p. 259, 1928. 

14. A. Herzfeld and G. Lanart, Zeits. Verein d. deutsch. Zuckerindustrie, 

Teoh. Teil, vol. 69, p. 122, 1919. 

15. G. Bertrand, Comptes rend., vol. 122, p. 900, 1896 ; vol. 127, p. 842, 

1898. .47171. Chim. Phys. (8), vol. 3, p. 181, 1904. 

16. A. L Virtanen and B. Barlund, Biochem. Zeits., vol. 169, p. 169, 1926. 

17. I. G. Farbenindustne, B. Pat., 269, 950, 1928. 

18. M. Molliard, Comptes rend., vol. 174, p. 881, 1922. 

19. R. Falck and S. N". Kapur, Benchte, vol. 57, p. 920, 1924. 

20. R. Falck and van Beyma thoe Kingma, Benchte, vol. 57, p. 915, 1924. 

21. W. Butkewitsoh, Biochem. Zeits., vol. 164, p. 177, 1924. 

22. C. Wehmer, Benchte, vol. 58, p. 2616, 1925. 

23. 0. E. May, H. T. Herriok, C. Thorn and M. B. Church, J. BioL Chem , 

vol. 75, p. 417, 1928. 

24. H. T. Herriok and 0. E. May, J. Biol. Ohem., vol. 77, p. 185, 1928. 

25. i. K. Bernhauer, Biochem. Zeits., vol. 153, p. 517, 1924. 
ii. K. Bernhauer, Biochem. Zeits., voL 172, p. 313, 1926. 

iii. K. Bernhauer, Z. physiol. Chem., vol. 177, p. 86, 1928. 

26. M. Molliard, Oomptes rend., vol. 178, p. 161, 1924. 

27. L. Boutroux, Comptes rend., vol. 127, p. 1224, 1898. 

28. J. Gruss, Jahrb.f. wiss. Bot., vol. 66, p. 109, 1926. 

29. F. Challenger, V. Subramanian and T. K. Walker, Nature, vol. 119, 

p. 674, 1927. 

30. K. Bernhauer and H. Wolf, Z. physiol. Chem., vol. 177, p. 270, 1928. 

31. K. Bernhauer andK. SohOn, Biochem. Zeits., vol. 202, p. 164, 1928. 

32. H. Raistriokand A. B. Clark, Biochem. J., vol. 13, p. 329, 1919. 

33. C. Wehmer, Biochem. Zeits., vol. 197, p. 418, 1912. 

34. K. Bernhauer, BiocJiem. Zeits., vol. 197, p. 278, 1928. 

35. K. Bernhauer, Biochem. Zeits , vol. 197, p. 1164, 1928. 

36. R. Sohreyer, Berichte, vol. 68, p. 2647, 1926. 

37. C. Wehmer, Biochem. Zeits., vol. 197, p. 418, 1928. 



PRODUCTION OF GLUCONIO AND OTHER ACIDS 105 

38. R. Sohreyer, Biochem. Zeits., vol. 202, p. 131, 1028. 

39. F. Banning, ZentrU.f. Bakt., Abt. n, vol. 8, p. 395, 1902. 

40. S. Kostytsohev and V. Tsohesnokov, Botan. OentrU., vol. 164, p. 158, 

1928. 

41. T. Chrzaszcz and D. Tinkow, Biochem. Zeits , vol. 204, p. 106, 1929. 

42. R. Groupil, Commies rend., vol. 153, p. 1172, 1912. 

43. W. S. Butkewitsch and M. W. Fedoroff, Biochem. Zeits., vol. 206, p. 440, 

1929. 

44. F. Ehrlioh, Berichte, vol. 44, p. 3737, 1911. 

45. C. Wehmer, Berichte, vol. 51, p. 1663, 1918. 

46. G. 0. Barley and R. S. Potter, J. Amer. Ohem. Soc., vol. 44, p. 215, 1922. 

47. W. Zopf, Ber. d. deutsch. bot. Gesell., vol. 18, p. 32, 1900. 

48. 0. Wehmer, Botan. Zeitg., vol. 9, p. 163, 1891. Ber. d. deutsch. lotan. 

GeseU., vol. 11, p. 333, 1893. 

49. C. Wehmer, F. Lafar'a Handbuch d. technischen MyJeologie, vol. 4, p. 234, 

1906/7. Gustav Fischer, Jena. 

50. A. Sartory and S. Bainier, Comptes rend. Soc. Btol., vol. 70, p. 873, 1911. 

51. C. Thorn and M. B. Church, The Asperg&U, Williams & Wilkins, 

Baltimore, 1926. 

52. W. Butkewitsoh, Biochem. Zeits., vol. 129, p. 464, 1922. 

63. C. Wehmer, Chem. Zeitg., vol. 21, p. 1022, 1897. 

64. J. N. Currie, J. Biol. Chem., vol. 31, p. 15, 1917. 

65. A. Fernbaoh, J. L. Yuill and Messrs. Rowntree & Co., B. Pot., No. 

266414, 1925, 

56. W. Butkewitsch, Biochem,. Zeits., vol. 131, p. 338, 1922. 

57. K. Bernhauer, Biochem. Zeits., vol 172, p. 324, 1926. 

68. E. Buohner and H. Wustenfeld, Biochem. Zeits., vol. 17, p. 397, 1909. 



CHAPTER VII 

THE AEROBIC DEHYDROGENATION OF HEXOSES, 
RESULTING IN THE FORMATION OF ACETIC 
AND FORMIC ACIDS, ACETYLMETHYLCARBINOL 
AND 2:3 BUTYLENEGLYCOL 

A PATKLY clear conception of this mode of hexose dehydro- 
genation has been gained in recent years by the study of 
certain obligatory aerobic bacilli. 

The earliest efforts to establish the action of these micro- 
organisms on hexoses were made by Pere^ 1 who reported 

^ 

finding glyceraldehyde CH 2 OH.CHOH.Cf and formalde- 

X H 
hyde H.CHO in his glucose containing cultures. 

Subsequent investigations by Desmots 2 on the behaviour 
of Bac. mesentericus vulgatus, Bac. mesent&ricus niger, and 
Bac. mesent&ricus ruber ; by Fernbaoh 3 on Bac. tenuis (Tyro- 
thrix tenuis), and by Lemoigne 4 on certain types claimed by 
him to be Bac. subtdia and Bac. mesent&ricus >, showed that 
the substance characterized by P6r6 as glyceraldehyde was 
acetylmethylcarbinol CH 3 .CO.CHOH.CH 3 . In addition to 
this product Desmots found acetic and valeric acids as well 
as traces of ethyl alcohol. 

Perm's observation on the formation of formaldehyde was 
confirmed by Fernbaoh 3 in the case of Bac. tenuis. Fernbach 
also reported the presence in cultures of this organism on 

/ 

glycerine of methylglyoxal CH 3 .CO.C\ , dihydroxyacetone 

X H 

CH 2 OH.CO.CH 2 OH, and acetic acid. In addition Aubel 5 
detected pyruvio acid CH 3 .CO.COOH in glycerine cultures 
of Bac. subtihs, while 2:3 butyleneglycol CH 3 .CHOH.CHOH. 
CH 3 was detected by Lemoigne 4 as a decomposition product 
of glucose by the strains described by hi as Bac. subtilis 
and Bac. mesentericus strains which produced considerable 



FORMATION OF ACETIC AND FORMIC ACIDS, ETC. 107 
quantities of carbon dioxide from the carbohydrate. With 
macerations of an organism related to Bac. Megatherium 
Lemoigne 6 observed the formation of ^hydroxybutyrio acid, 
CH 3 .CHOH.CH 2 .COOH. 

The absence of lactic acid as a product of decomposition 
of glucose in these cases is noteworthy. That it is not likely 
to have been overlooked is supported both by theoretical 
consideration and by experimental evidence. In their study 
on facultative anaerobiosis Quastel and his collaborators 7 
found that only those organisms which were capable of 
activating nitrates to function as hydrogen acceptors could 
be expected to utilize lactic acid for growth. The aerobic 
bacilli do not belong to the nitrate reducing types. This 
was shown by Quastel in the case of Bac. subtilis. It had 
been demonstrated previously by Lemoigne 8 that the growth 
of his strain of Bac. subtilis, which is claimed to produce 
carbon dioxide, was extremely slow in a medium containing 
calcium lactate as sole source of carbon. 

The various observations referred to above indicate that a 
direct dehydrogenation of glucose to gluconic acid does not 
occur. The first fermentation product is invariably a sub- 
stance with less than six carbon atoms. It is probable there- 
fore that the first stage in the action of the aerobic bacilli on 
glucose is the formation of hexose phosphoric esters. 

A relationship to the group of the acetic acid bacteria is 
discernible nevertheless in the property of the aerobic bacilli 
of dehydrogenating glycerine to dihydroxyacetone, a reaction 
which, it may be recalled, was shown by Bertrand 9 to occur 
when Bact. xylinum acted on glycerine. 

Dehydrogenation of methylglyoxal would result in the 
formation of pyruvio acid, the presence of which was reported 
by Aubel 5 as an intermediate. The functions of pyruvic acid 
may be assumed with Cambier and Aubel 10 and with Quas- 
tel 11 to be connected, partly at least, with the synthetic 
processes of the cell plasma. Through intramolecular hydro- 
gen transfer methylglyoxal might give rise to acetaldehyde 
and formic acid (Kluyver and Donker 12 ), of which the latter 



108 AEROBIC DEHYDROGENATION OF HEXOSES 

was detected by Per6 as a fermentation product of some of 

the micro-organisms discussed in this chapter. 

Acetaldehyde has not to the writers' knowledge been 
discovered as an intermediate fermentation product of the 
aerobic bacilli. There is every reason to assume, however, 
that acetaldehyde is produced, since ethyl alcohol, acetyl- 
methyloarbinol, 2:3 butyleneglycol, and j8-hydroxybutyric 
acid have been found. It is true that Lemoigne 4 suggests a 
different origin for acetylmethylcarbinol and butyleneglycol. 
He states that the glucose molecule becomes dislocated 
through the action of the bacteria and that 2 :3 butyleneglycol 
is thereby produced with evolution of carbon dioxide. The 
acetylmethylcarbinol is regarded by him as formed from 2 :3 
butyleneglycol by subsequent oxidation. But this is hardly 
a correct interpretation. The work of Neuberg and Rein- 
furth, 13 and of Kluyver, Donker, and Visser't Hooft 14 on the 
formation of acetylmethyloarbinol by yeast, shows that 
acetaldehyde is the mother substance of this compound and 
that it is produced from acetaldehyde in the presence of 
hydrogen acceptors, notably of oxygen. 

The formation of the ^-hydroxybutyric acid, detected by 
Lemoigne, might result from the coupling of one molecule of 
acetaldehyde with one molecule of acetic acid, 

O -^\ O 

CH 8 .C/ + H-C-C/ -> CH 8 .CHOH.CH a .C/ 
X X \ 



V H H / X OH 

acetaldehyde acetio acid j8-hydroxybutyrio acid. 

The origin of the valeric acid observed by Per6 has not 
been traced by subsequent investigators; in fact it seems 
problematic whether this substance was really derived from 
carbohydrates. 

The presence of formic acid as a fermentation product of 
the aerobic bacteria has not been reported though formalde- 
hyde, as already mentioned, was found by PerS 1 and by 
Fernbach. 8 

Reviewing the above observations, it is to be assumed that 



FORMATION OF ACETIC AND FORMIC ACIDS, ETC. 109 
the hexose molecule in the mode of fermentation dealt with 
in this chapter is converted first into hexosephosphoric 
esters. On saponification of these glyceraldehyde results, 
followed by methylglyoxal, pyruvio acid, acetaldehyde, and 
formic acid, with side reactions leading to 2 :3 butyleneglyool, 
acetylmethylcarbinol, acetic acid, and/8-hydroxybutyrio acid. 

In the ordinary bacteriological methods of diagnosis these 
changes would be indicated only by the production of acid 
in litmus glucose broth, coupled perhaps with a reduction of 
litmus. Where the test organism produced lactose hydro- 
lysing enzymes, acid production, and possibly discoloration 
or coagulation, would occur in litmus milk. 

The formation of acetylmethyloarbinol in the presence of 
peptone would give rise to a positive Voges and Proskauer 15 
reaction a fluorescent discoloration of the upper part of the 
culture, similar to that of a dilute alcoholic solution of eosin, 
on addition of potash to the glucose broth culture and 24 
hours' standing of the culture at room temperature. 

Harden 16 showed that this reaction was due to the presence 
of acetylmethylcarbinol. The reaction is now no longer used 
for the identification of acetylmethylcarbinol, but has been 
replaced by that devised by Lemoigne 17 or its modification . 
recommended by Kluyver, Donker, and Visser't Hooft, 14 both J\ 
of which allows of a quantative estimation of both acetyl- 
methylcarbinol and 2:3 butyleneglycol. 

Some very early investigations of the action of Bac. subtilis 
on glucose, carried out by Vandevelde 18 in 1884, differ materi- 
ally from the view set out above. Vandevelde obtained from 
glucose lactic acid, butyric acid, caproio acid, and two 
alcohols, one of which boiled below 100 C. 3 and the other 
above. It is probably justifiable to assume with Lemoigne 4 
that the organism studied by Vandevelde was not Bac. 
subtilis, particularly as it is stated to have produced a gas, 
consisting of carbon dioxide, hydrogen, nitrogen, and an 
undetermined residue. 

It is much to be regretted that a list of the various micro- 
organisms acting on glucose in a manner similar to that of the 



110 AEROBIC DEHYDROGENATION OF HEXOSES 
aerobic bacilli cannot be compiled. Here, as in the case of 
most other fermentations, the study of the action of the 
organisms on carbohydrates has been far too superficial to 
make such compilations possible or even desirable at present. 

LITERATURE 

1. A. Pere", Ann. Inat. Pasteur, vol. 10, p. 417, 1896. 

2. H. Desmots, Comptes rend., vol. 138, p. 581, 1904. 

3. A. Fernbaoh, Gomptes rend., vol. 151, p. 1004, 1910. 

4. M. Lemoigne, Comptes rend., vol. 162, p. 1873, 1911 ; vol. 155, p. 792, 

1912. Ann. Inat. Pasteur, vol. 27, p. 856, 1913. 

5. E. Aubel, Comptes rend. Soc. Siol, vol. 84, p. 574, 1921, 

6. M. Lemoigne, Comptes rend., vol. 176, p. 1761, 1923. 

7. J. H. Quastel, M. Stephenson and M. D. Whetham, Biochem. J., vol. 19, 

p. 304, 1925. 

8. M. Lemoigne, Comptes rend., vol. 177, p. 652, 1923. 

9. G. Bertrand, Gomptea rend., vol. 122, p. 900, 1895. 

10. R. Cambier and E. Aubel, Com/ptes rend., vol. 175, p. 71, 1922. 

11. J. H. Quastel, Biochem. J., vol. 19, p. 641, 1925. 

12. A. J. Kluyver and H. J. L. Donker, Zeits. f. Chem. d. ZeUe und Geivebe, 

vol. 13, p. 134, 1926. 

13. C. Neuberg and E. Reinfurth, Biochem. Zeits., vol. 143, p. 653, 1923. 

14. A. J. Kluyver, H. J. L. Donker, and F. Visser't Hooft, Biochem. Zetts., 

vol. 161, p. 361, 1925. 
16. 0. Voges and B. Proakauer, Z. f. Hygiene, vol. 28, p. 20, 1898. 

16. A. Harden, Proo. Eoy. Soc., Series B, vol. 77, p. 424, 1926. 

17. M. Lemoigne, Comptes rend., vol. 170, p. 131, 1920. 

18. G. Vandevelde, Z. physiol. Chem., vol. 8, p. 367, 1884. 



CHAPTER 

THE FACULTATIVE ANAEROBIC DEHYDROGENA- 
TION OF HEXOSES RESTJLTESTG IN THE PRO- 
DUCTION OF ACETIC AGED, FORMIC ACID, 
ETHYL ALCOHOL, ACETYLMETHYLCARBINOL 
AND 2:3 BUTYLENEGLYCOL 

THERE appear to be three methods by which hexoses can 
be fermented by facultative anaerobic micro-organisms 
without the formation of lactic acid. These three modes of 
fermentation are dealt with in the present chapter as one 
group. 

The first is represented by the action of Bad. vulgare, the 
second by that of Bad. fluorescent liquefaciens and Bact. 
prodigiosum, and the third by that of Bact. pneumoniae and 
Bac. acetoethylicus. 

The action of Bact. vulgare on glucose was studied by 
Smith 1 from the point of view of gas production. He obtained 
a mixture of gaseous products consisting of 33 per cent, of 
carbon dioxide and 66 per cent, of hydrogen. Lemoigne 2 
showed that Bact. vulgare produced both acetylmethyl- 
oarbinol CH 8 .CO.CHOH.CH 3 and 2:3 butyleneglycol, 
CH 3 .CHOH.CHOH.CH a . Quastel and Wooldridge 3 found 
that Bact. vulgare activated no other hexoses than glucose 
and fructose. A slight activation of fumarates and malates 
was also noted. Nitrates were the only markedly activated 
hydrogen acceptors. 

The formation of aoetylmethylcarbinol by Bact. vulgare 
must be taken as an indication that acetaldehyde and acetic 
acid are formed as intermediate decomposition products, if 
it is assumed with Kluyver and Donker 4 that the fermentation 
of hexoses proceeds along lines of comparatively simple 
hydrogenations, dehydrogenations, and condensations. On 
the same assumption the formation of acetaldehyde would 
indicate the occurrence of methylglyoxal CH 3 .CO.CHO, or 



112 ANAEROBIC DEHYDROGENATION OF HEXOSES 
perhaps of dihydroxyacetone GE 2 .OH.CO.CH a OH, glycer- 
aldehyde (JE a OH.CflIOH.CHO, and hexosephosphorio esten* 
as precursors. The formation of these intermediaries hw* 
still to be proved experimentally, however. 

On being converted into aoetaldehyde, methylglyoxal 
would yield one molecule of formic acid for every molecule- 
of acetaldehyde. The formic acid on being dehydrogenated 
would yield carbon dioxide and hydrogen in equal propor- 
tions, part of the latter being used for the hydrogenation of 
acetylmethyloarbinol to 2:3 butyleneglycol. Smith's yields 
referred to above do not agree with the quantities to IK* 
expected under the above scheme. 

The fermentation of hexoses by Bact. fluorescens lique- 
faciens, Bact. pyocyaneum, and Bact. prodigiosum has been 
studied by Lemoigne, 5 Aubel, 6 Quastel and Whetham, 7 and 
Quastel and Wooldridge. 8 According to Quastel and bin 
collaborators the activating properties of both species aro 
much more marked than those of Bact. vulgare. 

Lemoigne 5 found that a species which he terms Bact. 
prodigiosum, but which is more likely to have been Bact. 
ruber indicum, a type capable of fermenting saccharose 
(HefFeran 9 ), produced traces of acetaldehyde and ethyl 
alcohol, acetylmethylcarbinol, and 2 : 3 butyleneglycol. Evolu- 
tion of carbon dioxide by Bact. prodigiosum was observed by 
Sohottelius 10 and by Hefferan.* 

When acting on glucose Bact. pyocyaneum was found by 
Aubel 6 to yield formic acid and acetic acid in the proportions 
of 1 :2. Ethyl alcohol was also detected by him. An evolution 
of carbon dioxide or of hydrogen from hexoses by Bact. 
pyocyaneum or Bact. fluorescens liquefaciens has not been 
reported, nor have acetylmethyloarbinol and 2:3 butylene- 
glycol been found. 

In spite of these differences, which on further investigation 
may be shown to be less marked than appears to be the case, 
a similarity in the action of Bact. fluorescens liquefaciens, 
Bact. pyocyaneum, and Bact. prodigiosum has undoubtedly 
been established. It is noteworthy, for instance, that in none 



PRODUCTION OF ACETIC ACID, FORMIC ACID, ETC. 113 
of the cases does the breakdown of glucose lead to the forma- 
tion of lactic acid. Gas evolution is restricted in both cases 
and never leads to the evolution of molecular hydrogen. And 
ethyl alcohol is formed as a fermentation product by both 
types. 

It is probable that the fermentation proceeds in both cases 
via hexosephosphoric esters, methylglyoxal or dihydroxy- 
acetone to pyruvic acid, CH 8 .CO.COOH, with side reactions 
to acetaldehyde and formic acid, acetic acid, ethyl alcohol, 
acetyhnethyloarbinol, and 2 :3 butyleneglycol. The scheme of 
fermentation suggested by Aubel 6 supports this assumption. 

The third method of facultative anaerobic glucose fermenta- 
tion not involving the production of lactic acid is represented 
by the action of Bact. pneumoniae, Bac. ethaceticus, and Bac. 
acetoethylicus. 

The most exhaustive investigation of this mode of fermenta- 
tion was carried out by Frankland and his collaborators 11 in 
the case of Bact. pneumoniae and Bac. etTtaceticus, and by 
Northrop, Ashe and Senior, 13 and Donker 13 in the case of Bac. 
acetoethylicus. 

The principal glucose fermentation products of Bact. 
pneumoniae and Bac. ethaceticus were shown by Frankland 
to be acetic acid, formic acid, and ethyl alcohol, in equal 
molecular proportions, as well as traces of succinio acid. 
Hydrogen and carbon dioxide were given off under anaerobic 
conditions in equal proportions. Calcium lactate was fer- 
mented, yielding the same fermentation products as glucose 
but in Different molecular proportions, four molecules of 
acetic acid being formed to one molecule of ethyl alcohol. 
The observation of Grimbert 14 that Mactio acid is also pro- 
duced has not been confirmed. 

As fermentation products of Bac. acetoethylicus, Northrop, 
Ashe and Senior detected formic and acetic acids, ethyl 
alcohol, acetone, carbon dioxide, and hydrogen. Traces of 
i propyl and butyl alcohols were also reported present by them. 

! Neither acetyhnethylcarbinol nor 2:3 butyleneglycol was 



114 ANAEROBIC DEHYDROGENATION OF HEXOSES 

found. Details of the experimental data obtained by Donker 
in his study of the fermentation of glucose by this organism 
were given in Chapter V, page 86. Donker does not confirm 
the presence of propyl or butyl alcohol, but records finding 
traces of ethylmethylcarbinol and measurable quantities of 
2:3 butyleneglycol. 

Of particular interest is the appearance of suocinic acid as 
a fermentation product of Bac. pneumoniae and Bac. ethace- 
ticus and of acetone in the case of Bac. acetoethylicus. 

Succmio acid, COOH.CH 2 .CH 2 .COOH, was found by Grey 16 
to be a fermentation product of Bact. coli commune. Grey 
concluded from his observations that the precursor of succinic 
acid in the case of Bact. coli commune was identical with that 
of acetic acid and ethyl alcohol that is, with methylglyoxal 
or dihydroxyaoetone. If this view must be taken as correct, 
the production of succinic acid would follow a different course 
to that taken in the formation of succinic acid by the micro- 
organisms dealt with in Chapter VI. 

Experimental evidence on the formation of acetone from 
acetic acid by microbiological activity was brought forward 
by Desborough 16 in the case of an obligatory anaerobic ba- 
cillus, Bac. acetonigenus (Clostridium acetonigenum), Donker. 13 
Its formation from the same intermediate in the case of Bac. 
acetoethylicus has not been proved, but if likely to occur 
would result in two molecules of acetic acid combining to 
form one molecule of acetone and one molecule of carbon 
dioxide. 

In the case of Bac. efhaceticus and Bac. acetoethylicus, the 
final fermentation products again indicate the preliminary 
conversion of the hexose molecule into phosphoric esters, 
followed by the production of glyceraldehyde, methylglyoxal 
(or perhaps dihydroxyacetone), acetaldehyde, and formic 
acid, with various side reactions giving rise to the other 
fermentation products which have been identified. Of the 
chief intermediate fermentation products of Bac. acetoethylicus 
acetaldehyde has actuaUy been isolated by Peterson and 
Fred. 17 



PRODUCTION 0$ ACETIC ACID, FORMIC ACID, ETC. 116 

UTERATUBE 

1. Th. Smith (see K. B. Lehmann and R. 0. Neumann, Bakterid. Diagnos- 

tile, 5th edition). Lehmann, Munohen, 1912. 

2. M. Lemoigne, Comptes rend. Soc. bid., vol. 88, p. 498, 1923. 

3. J. H. Quastel and W. R. Wooldridge, Biochem. J., vol. 19, p. 652, 1925. 

4. A. J. KHuyver and H. J. L. Donker, Z.f. Chem. d. Zette und Gewebe, vol. 

13, p. 134, 1926. 

5. M. Lemoigne, Gomptes rend. Soc. bid., vol. 82, p. 234, 1919. 

6. E. Aubel, Comptes rend., vol. 173, p. 1493, 1921. 

7. J. H. Quastel and M. D. Whetham, Biochem. J., vol. 18, p. 519, 1924. 

8. J. H. Quastel and W. R. WooMridge, Btochem. J., vol. 19, p. 662, 1925. 
fl. M. Hefferan, ZentrbLf. Bate., Abt. n, vol. 11, p. 311, 1904. 

10. M. Schottehus, ZentrbLf. Bakt., vol. 2, p. 439, 1887. 

f P. F. Frankland, A. Stanley and W. Frew, J. Chem. Soc., vol. 59, p. 253, 

1891. 
P. F. Frankland, G. Frankland and J. Fox, Chem. News, vol. 60, p. 181, 

1889. 

P. F. Frankland and J. S. Lumsden, J. Chem. Soc., vol. 61, p. 432, 
. 1892. 

12. J. H. Northrop, L. H. Ashe and J. K. Senior, J. Biol. tihem., vol. 39, 

p. 1, 1919. 

13. H. J. L. Donker, Bijdrage tot de JZennis der Bolerzuur-Butyl akoholen 

Acetonegtsttngen, W. D. Meinema, Delft, 1926. 

14. L. Grimbert, Comptes rend., vol. 132, p. 706, 1901. 

16. E. C. Grey, Proc. JBoy. Soc., Sec. B, vol. 90, p. 76, 1917. 

16. A. P. H. Desborough, Eng. Pat., 128403, 1918. 

17. W. H. Peterson and E. B. Fred, J. Bid. Chem., vol. 44, p. 29, 1929. 




12 



CHAPTER IX 

THE FACULTATIVE ANAEROBIC DEHYDROGENA- 
TION OF HEXOSES INVOLVING THE FORMA- 
TION OF LACTIC ACID AS AN ESSENTIAL DE- 
COMPOSITION PRODUCT 

/. TEE GROUPS OF BAGT. COLI COMMUNE AND BAOT. 
LACTIS AEROGENE8 

TEH mode of fermentation represented by this group is 
undoubtedly extremely common among micro-organisms. 
It was investigated in considerable detail by Harden 1 in the 
case of Bact. typhosum and Bact. coli commune, organisms 
which he found capable of converting approximately 50 per 
cent, of any available hexoses into lactic acid, the remainder 
yielding ethyl alcohol and acetic acid in approximately equal 
molecular proportions, with formic acid, carbon dioxide, and 
hydrogen in lesser proportions. Succinic acid COOH.CH 2 . 
CH a .COOH was also detected by Harden as a fermentation 
product of Bact. coli commune. Observations made by Tn'm 
in collaboration with Penfold 2 on specially treated strains of 
Bact. coli commune convinced Harden that the fermentation 
of hexoses by this organism is performed by three independent 
enzymes, one leading to the production of lactic acid, one to 
ethyl alcohol, acetic acid and formic acid, and one to carbon 
dioxide and hydrogen. The view that three independent 
stages occur has been confirmed and elaborated by subse- 
quent investigators, notably by Grey, 3 Aubel, 4 and Aubel and 
Salabartan. 5 Formic acid production and evolution of carbon 
dioxide and hydrogen are regarded by them as the result of 
the activity of one enzyme, the formation of acetic acid, ethyl 
alcohol, and succinic acid as that of another, and lactic acid 
production as the result of the function of a third and entirely 
independent enzyme. 

It is questionable whether this conception is correct. 
Recent work by Quastel and collaborators 6 indicates that the 



FOBMATION OP LACTIC ACID 117 

governing factor in these reactions is more likely to be found 
in the hydrogen activating properties of the organisms than 
in the existence of several specific enzymes. 

Formation of formic acid, carbon dioxide, hydrogen, acetic 
acid, ethyl alcohol, and suooinic acid at least are closely con- 
nected with these properties. This is particularly noticeable 
in the case of the conversion of formic acid to carbon dioxide 
and hydrogen, a reaction which is readily performed under 
normal conditions by Bact. coli commune when once formic 
acid has been activated to function as hydrogen donator. 

Grey, who realized the connexion between production of 
formic acid and the evolution of carbon dioxide and hydrogen, 
pointed to the close relationship of these reactions with the 
formation of ethyl alcohol, and suggested that the rate at 
which ethyl alcohol, acetic acid, and succinic acid are pro- 
duced depends upon the availability of hydrogen liberated 
by the breakdown of formic acid. Where an excess of 
hydrogen was available Grey observed an increase in alcohol 
production from the acetaldehyde which Neuberg and Nbrd 7 
detected as an intermediate fermentation product of Bact. 
coli commune, while a scarcity of active hydrogen resulted in 
an increased production of succmic acid. To obtain activated 
hydrogen Grey added formates to a glucose containing culture 
of Bact. coli commune. 

This observation that formates break down with the libera- 
tion of nascent hydrogen under the influence of micro-organ- 
isms is of very great importance, and, as Grey himself suggests, 
should have an important bearing on the study of other 
fermentation processes in which the production of activated 
hydrogen is aimed at. Grey's experiments lead him to con- 
clude that the formation of lactic acid is independent of the 
other reactions performed by Bact. coli commune. 

It is clear from the work of Grey, as well as from that of 
Stephenson and Whetham, 8 that the formation of lactic acid 
by Bact. coli commune and presumably by all other micro- 
organisms producing this acid is intimately connected with 
the growth of the cells. Grey found that practically no lactic 



118 ANAEROBIC DEHYDROGENATION OF EEXOSES 
acid was formed by Bact. coli commune during the period 
characterized by rapid death of the cells the latter part of 
the fermentation while 70 per cent, of the hexoses con- 
sumed were converted into lactic acid during the period 
following immediately upon the rapid multiplication of the 
cells the early part of fermentation. 

The period of destruction of the cells was characterized by 
the transformation of hexoses into alcohol, acetic acid, formic 
acid, and suocinic acid. 

The limitation of lactic acid production -to the period of 
growth of the cells justifies Stephenson and Whetham's 
assumption that its formation is intimately connected with 
the liberation of the energy required for the synthetic activi- 
ties of the cells. 

The question of the precursors of lactic acid has engaged 
the attention of Neuberg and his pupils, 9 of Aubel, and of 
several other writers. That the precursor is likely to be 
methylglyoxal, CH 8 .CO.CHO, had been suggested by Nef. 10 
Dakin and Dudley 11 have since proved that lactic acid is 
produced by animal cells and tissues from this intermediate. 
Neuberg, Aubel, and de Graaf and le F&vre 12 found that Bact. 
coli commune decomposed methylglyoxal, and, according to 
Neuberg and Gorr, 9 with a theoretical yield of lactic acid. 
Aubel accepts methylglyoxal as the precursor of lactic acid. 
As mother substance of methylglyoxal, Kluyver and Donker 13 
suggest glyceraldehyde, CH 2 OH.CHOH.CHO, which de Graaf 
and le F&vre found readily decomposed by Bact. coli com- 
mune and its related types, though not with the production 
of methylglyoxal or lactic acid, but with formation of carbon 
dioxide, acetic acid, and traces of ethyl alcohol. 

Neither acetylmethylcarbinol, CHg.CO.CHOH.CH 3 ,nor 2:3 
butyleneglyool, CHg.CHOH.CHOH.CHg, appears to be a 
normal fermentation product of Bact . typhosum, Bact. paraty- 
phosum or Bact. coli commune, though de Graaf and le Fe"vre 
report the presence of traces of the former in cultures of 
Bact. coli commune containing glyceraldehyde and dihy- 
droxyacetone, CH 2 OH.CO CH 2 OH. 



FORMATION OF LACTIC ACID 119 

As a further decomposition product of glucose Aubel 14 
detected pyruvic acid, CH 8 .CO.COOH, which both QuasteP 
and Cambier and Aubel 16 regard as the basic substance used 
in the synthetic processes of this and other carbohydrate 
decomposing micro-organisms. Kluyver and Donker consider 
that the pyruvio acid is derived from methylglyoxal, and this 
in turn from glyceraldehyde. Aubel 4 does not agree that 
methylglyoxal can be the precursor of pyruvic acid, since he 
found Bact. coli commune unable to decompose methylglyoxal. 
He must have been unaware of de Graaf and le Fdvre's 
observations, however, that certain strains of Bact. coli 
commune do so, and must have been studying types of the 
organism incapable of activating methylglyoxal to function 
as hydrogen donator. That such strains exist is emphasized 
by de Graaf and le Fevre. The reactions giving rise to the 
formation of succinic acid are not yet clear. Its formation 
from glutamic acid by yeast was reported by Ehrlioh ; 17 from 
ketoglutamic acid by Neuberg and Ringer. 18 Virtanen 19 
regards it as the product of a side reaction of carbohydrate 
decomposition, during which no phosphoric esters are formed 
that is presumably a production similar to that occurring 
among the species of Asp&rgillus and Mucor discussed in 
Chapter VI. 

Summarizing the observations discussed in the preceding 
pages, the following scheme for the fermentation of hexoses 
by Bact. coli commune may be suggested. 

-> f lactio acid 

Hexose->glyceraldehyde->-methylglyoxalr->- J pyruvio acid 
(or perhaps dihydroxyacetone) j 

-> [_aoetaldehyde-(-fonmo acid 

I I 

ethyl alcohol carbon dioxide 

I + 

acetic acid hydrogen 

This scheme differs somewhat from that suggested by Neu- 
berg and Nord, 20 who assume that the pyruvic acid is 
converted into acetaldehyde and carbon dioxide, and the 
aoetaldehyde to ethyl alcohol and acetic acid by a Cannizzaro 



120 ANAEROBIC DEHYDROGENATION OF EEXOSES 
reaction. In the scheme advanced by these workers, the 
molecular hydrogen is thought to have been derived from the 
conversion of glucose to pyruvic acid. 

In the mode of fermentation outlined above lactic acid is 
regarded as a final fermentation product. It should not be 
concluded, however, that this acid cannot be further de- 
composed by the organism. Both Quastel and Aubel have 
shown that lactic acid can enter the fermentation when 
suitable hydrogen acceptors for instance nitrates are 
available. It is then utilized for the production of pyruvio 
acid. 

The fermentation of hexoses by Bact. typkosum would 
follow lines similar to those of Bact. coli commune. But since 
Bact. typhosum does not possess activating powers towards 
formic acid, carbon dioxide and hydrogen are not evolved by 
this organism. 

The fermentation of hexoses by Bact. paratyphomm and 
by its numerous related types occurring in the soil, on plants 
and in water, follows a middle course between those of the 
two previous types, formic acid being activated in some cases, 
with a resulting evolution of carbon dioxide and hydrogen. 

Parallel with its analytic functions, or perhaps preceding 
them, Grey 21 thought he observed a marked synthetic activity 
in Bact. coli commune. This action is stated to be particularly 
noticeable during the early stages of the fermentation of 
glucose and is claimed to result in the production of starch. 
Further work would appear to be desirable before the observa- 
tions of Grey can be accepted as correct. 

The fermentation of hexoses by Bact. lactis aerogenes differs 
markedly in certain respects from that by the Bact. typhosum- 
Bact. coli group, though in this case also the formation of 
lactic acid is an important stage in the reaction. Harden and 
Walpole, 22 who subjected the fermentative activity of Bact. 
lactis aerogenes to exhaustive investigation, state that ethyl 
alcohol, acetic acid, succinic acid, formic acid, carbon dioxide 
and lactic acid are produced. The evolution of gas was found 
to be greater than in the case of Bact. coli commune. The 



FORMATION OF LACTIC ACID 121 

proportion of carbon dioxide in the mixture was consider- 
ably greater than that of hydrogen in Harden and Walpole's 
experiments, indicating, they suggest, that the former is 
derived from a source additional to formic acid. It has still 
to be shown, however, that the excess of carbon dioxide 
over hydrogen cannot have been due to a consumption of 
part of the hydrogen derived from formic acid in inter- 
mediate reactions. 

In this connexion it is perhaps worth recording that the 
presence of methane 7 per cent, of the gas mixture of Bact. 
lactis aerogenes was recorded by Baginsky. 23 

Of the fermentation products mentioned above ethyl 
alcohol accounted for 17-1 to 18-2 per cent, of the hexoses 
fermented, as against 12-85 per cent, in the case of Bact. coli 
commune; acetic acid represented 4-2 to 8-6 per cent, as 
against 18-84 per cent, in the case of Bact. coli commune; 
suocinic acid 2-4 to 4-5 per cent, as against 5-2 per cent. ; 
lactic acid 4-7 to 9-1 per cent., as against 31-9 per cent. ; 
formic acid 0-75 to 1-7 per cent. ; carbon dioxide 35-2 to 38 
per cent. 

Altogether these products represented no more than 66 per 
cent, of the carbon of the glucose fermented. A search for the 
remaining 33 per cent, revealed the presence in the cultures of 
acetylmethylcarbinol and 2-3 butyleneglycol. The former of 
these substances was shown by Harden 24 to be responsible 
for the Voges-Proskauer reaction. 26 

Harden and Walpole suggest that both the acetylmethyl- 
carbinol and the 2 :3 butyleneglycol are formed at the expense 
of those intermediate fermentation products which, in the 
case of Bact. coli commune, lead to acetic acid and lactic acid, 
and Walpole 26 subsequently asserted that the 2:3 butylene- 
glycol was the mother substance of the acetylmethylcarbinol, 
the latter being formed from the former through oxidation. 
The work of Neuberg and Reinfurth 27 and of Kluyver, Donker 
and Visser't Hooft 28 on the formation of acetylmethylcarbinol 
and 2.3 butyleneglycol by yeasts, indicates a different origin, 
however. Both Neuberg and Kluyver found that the addition 



122 ANAEROBIC DEHYDBOGENATION OF HEXOSES 
of acetaldehyde to a culture of a fermenting yeast led to the 
production of acetylmethylcaibinol, and Kluyver and his 
collaborators showed that in the absence of sufficient oxygen 
the acetylmethylcarbinol could be made to function as hydro- 
gen acceptor, thereby becoming reduced to 2:3 butylene- 
glycol. Moreover, where methylene blue or sulphur was 
added to a culture of yeast containing glucose, added 
acetaldehyde could be recovered by Kluyver, not as acetyl- 
methylcarbinol, but as 2:3 butyleneglycol. 

A similar course is probably followed in the case of Bact. 
lactis aerogenes, where acetylmethylcarbinol and 2 :3 butylene- 
glycol are produced, the mother substance of both, acetalde- 
hyde, being first converted into acetyhnethylcarbinol, and 
this subsequently reduced to 2 :3 butyleneglycol. 

That lactic acid, as suggested by Harden and Walpole, 
should be an intermediate of acetyhnethylcarbinol and its 
product of hydrogenation does not appear likely in view of 
the fact that lactic acid when it is fermented by Bact. coli 
commune yields pyruvic acid. 

Though the intermediate stages in the fermentation of 
hexoses by Bact. lactis aerogenes have by no means been fully 
explored, it does not appear unreasonable to suggest the 
following scheme for the reactions taking place during this 
fermentation : 

Hexofle->-hexoBepho8phorie esters 

rlaotio acid 



< pyruvio acid 

Laoetaldehyde + formio acid 



acetio acid ethyl alcohol aoetylmcthylcarbinol 

I 
2 : 3 butyleneglyool 

carbon dioxide hydrogen 

Of these reactions the conversion of methylglyoxal to lactic 
acid by Bact. lactis aerogenes has been experimentally con- 
firmed by Neuberg and Gorr. 29 





FORMATION OP LACTIC ACID 123 

An entirely different conception of the fermentation of 
hexoses by Bact. lactis aerogenes has been advanced by 
Virtanen 19 and by Myrback and von Euler. 80 Studying 
various aspects of the functions of enzymes Virtanen came 
to the conclusion that Bact. lactis aerogenes carried out the 
fermentation of glucose without the aid of a co-enzyme, and 
that consequently the initial stages of the fermentation 
differed from that of yeast and of lactic acid bacteria, which 
produce co-enzymes and form hexose phosphoric esters as 
preliminary fermentation products. 

The assumed absence of these esters in the breakdown of 
glucose by Bact. lactis aerogenes suggested to Virtanen the 
possibility that the glucose molecule might in the first instance 
be converted into a two carbon and a four carbon inter- 
mediate. In other words, that the fermentation might proceed 
on lines similar to those suggested by Raistrick and Clark 81 
for Asp&rgillus niger, giving rise in the first instance to the 
production of succinic acid and acetaldehyde. 

O 

C fl H 12 6 ^COOH.CH 2 .CH 2 .COOH+CH 3 .Cf +H 2 

\ 



Buooinio acid acetaldehyde. 

Myrback and von Euler see in acetaldehyde the mother 
substance of the acetic acid and formic acid which have been 
detected as additional fermentation products of Bact. lactis 
aerogenes. 

In referring to this conception of the fermentation of 
glucose by Bact. lactis aerogenes, the writers desire to empha- 
size that they cannot accept it without additional and more 
convincing evidence than has so far been supplied. 



124 ANAEROBIC DEHYDROGENATION OF HEXOSES 

LITERATURE 

1. A. Harden, J. Chem. 8oc., vol. 79, p. 612, 1901. 

2. A. Harden and W. J. Penfold, Proc. Boy. Soo., Ser. B, vol. 86, B, p. 416, 

1912. 

3. E. 0. Grey, Proc. Eoy. Soc. t Ser. B, vol. 87, p. 461, 1914; Ser. B, vol. 90, 

p. 76, 1917 ; Ser. B, vol. 90, p. 92, 1917 ; Ser. B, vol. 91, p. 294, 1920. 

4. E. Aubel, Comptes rend., vol. 181, p. 671, 1926 ; vol. 183, p. 572, 1926. 
6. E. Aubel and J. Salabartan, Comptes rend., vol. 180, p. 1183, 1926; 

vol. 180, p. 1784, 1926. 
C J. H. Quastel, M. Stephenson and M. D. Whetham, Biochem. J., vol. 

19, p. 304, 1926. 
J. H. Quastel and H. D. Whetham, Biochem. J., vol. 19, p. 620, 1926. 



6. 



J. H. Quastel and M. D. Whetham, Biochem. J., vol. 19, p. 645, 1925. 



J. H. Quastel and W. E. Wooldridge, Biochem. J., vol. 19, p. 626, 1925. 
I J. H. Quastel and M. Stephenson, Biochem. J., vol. 19, p. 660, 1925. 

7. C. Neuberg and P. T. Nord, Biochem. Zeits., vol. 96, p. 133, 1919. 

8. M. Stephenson and M. D. Whetham, Biochem. J., vol. 18, p. 498, 1924. 

9. C. Neuberg and G. Gorr, Biochem. Zeits., vol. 162, p. 490, 1925. 

10. J. U. Kef, Annolen, vol. 335, p. 279, 1904. 

11. H. D. DaMn and H. W. Dudley, J. Btol. Chem., vol. 14, p. 155, 1913. 

12. W. 0. de Graaf and A. J. le Fevre, Biochem. Zeits., vol. 155, p. 313, 1925. 

13. A. J. Kluyver and H. J. L. Donker, Zeits. f. Chem. d. Zette und Qewbe, 

vol. 13, p. 134, 1926. 

14. E. Aubel, Comptes rend., vol. 181, p. 571, 1926. 
16. J. H. Quastel, Biochem. J., voL 19, p. 641, 1926. 

16. E. Cambier and E. Aubel, Comptes rend., vol. 176, p. 71, 1922. 

17. F. Ehrlioh, Biochem. Zeits., vol. 18, p. 391, 1909. 

18. 0. Neuberg andM. Einger, Biochem. Zeits., vol. 71, p. 226, 1915. 

19. A. I. Virtanen, Ann. Acod. 8ci. Fennicoe, vol. 29, p. 16, 1927. 

20. C. Neuberg and F. T. Nord, Biochem. Zeits., vol. 96, p. 130, 1919. 

21. E. C. Grey, Biochem. J., vol. 18, p. 712, 1924. 

22. A. Harden and G. S. Walpole, Proc. Soy. Soc., Ser. B, vol. 77, p. 399, 

1906. 

23. A. Baginsky, Z.f. phyaiol. Chem., vol. 12, p. 434, 1888. 

24. A. Harden, Proc. Hoy. Soc., Ser. B, vol. 77, p. 424, 1906. 

26. 0. Voges and B. Proskauer, Z.f. Hygiene, vol. 28, p. 20, 1898. 

26. G. S. Walpole, Proc. Roy. 3oc. t Ser. B, vol 83, p. 272, 1911. 

27. C. Neuberg and E. Reinfurth, Biochem. Zeits., vol. 143, p. 663, 1923. 

28. A. J. Kluyver, H. J. L. Donker and F. Viaser't Hooft, Biochem. Zeits., 

vol 161, p. 361, 1926. 

29. C. Neuberg and G. Gorr, Biochem. Zeits., vol. 166, p. 482, 1925. 

30. K Myrbaok and H. von Euler, Berichte, Ser. B, vol. 57, p. 1073, 1924. 

31. H. Eaifltriok and A. B. Clark, Biochem. J., vol. 13, p. 329, 1919. 



CHAPTER X 

THE FACULTATIVE ANAEROBIC DEHYDROGENA- 
TION OF HEXOSES INVOLVING THE FORMA- 
TION OF LACTIC ACID AS AN ESSENTIAL DECOM- 
POSITION PRODUCT 

II. THE GROUP OF THE LACTIC AOID BACTERIA 

THE earliest investigations on the action of lactic acid bacteria 
on sugars indicated that other fermentation products than 
lactic acid, notably acetic acid, were frequently formed. 

Subsequently, and owing primarily to the researches of 
Kayser, 1 it became customary to subdivide the lactic acid 
bacteria into two main groups, one which in addition to lactic 
acid produced volatile acids in appreciable, though varying, 
quantities, and another, the true lactic acid bacteria, which 
gave 95 per cent, or more of fixed acids. 

Even the true lactic acid bacteria, however, were shown to 
yield volatile acids in measurable quantities. Thus Bertrand 
and Weissweiler 2 obtained 1-5 per cent, of acetic acid and 
traces of formic acid from cultures of Bact. bulgqricum, a type 
isolated by Grigoroff 3 from yoghurt. 

For this reason differentiation of the lactic acid bacteria 
on the basis of their fermentative activity might seem un- 
satisfactory, particularly as several investigators, Kayser 1 
and Barthel, 4 for instance, have found that the percentage of 
volatile acid produced increases in the presence of oxygen. 
It is not to be overlooked, however, that those members of 
the group which produce appreciable quantities of volatile 
acids convert fructose into its corresponding alcohol, mannitol, 
a reaction which is unknown among the true lactic acid 
bacteria. 

A fairly extensive literature is already in existence dealing 
with the mannitol producing lactic acid bacteria, and some 



126 ANAEROBIC DEHYDROGENATION OF HEXOSES 
writers have attempted to establish the various intermediary 
and final fermentation products formed by them. Others, 
notably Gayon and Dubourg 5 6 and Muller-Thurgau and 
Osterwalder, 7 have studied these bacteria from the point of 
view of their activities as infections in wine. 

Kruis and Raymann 8 isolated from malt a type which 
produced from saccharose formic and acetic acids, traces of 
ethyl alcohol, mannitol, and lactic acid. Tate 9 studied a type 
resembling Streptococcus mesenteroides (Leuconostoc) which 
gave 2 molecules of ethyl alcohol, 7 to 8 molecules of lactic 
acid, 1 molecule of succinic acid, and smaller quantities of 
acetic acid and formic acid, from 9 molecules of glucose. 
Peterson and Fred 10 investigated other types isolated from 
manure, silage, and soil. The last-named writers suggest that 
the mannitol is formed as a coincident product of fermentation 
through the reduction of fructose by nascent hydrogen 
and that it becomes subsequently broken down to lactic and 
acetio acids. Previously Gayon and Dubourg 6 and Muller- 
Thurgau and Osterwalder 7 had failed to secure growth of 
their mannitol bacteria on media containing mannitol as the 
sole source of carbohydrate. The organism studied by Tate 
fermented mannitol with the formation of ethyl alcohol, 
acetio acid, formic acid, and lactic acid. 

A more exhaustive study of the mannitol producing lactic 
acid bacteria was carried out by Smit 11 in 1916 and by van 
Steenberge in 1920. 12 The figures recorded in Table HE, 
showing the quantitative yields of fermentation products of 
a typical mannitol producing lactic acid bacterium, are taken 
from Smit's paper. 

The organism in question, Bact. lactofermentum (Lacto- 
bacterium fermewtum), had been described previously by 
Beijerinok. 13 

It will be seen that qualitatively, and in some respects 
quantitatively, the fermentation of glucose by this bacterium 
recalls the mode of action of Bact. coli commune, except that 
hydrogen is absent, that the yield of acetic acid is lower, and 
that glycerol has appeared as an important product of fer- 



FORMATION OF LACTIC ACID 127 

mentation. The proportions of carbon dioxide, ethyl alcohol, 
lactic acid, and of formic acid are of the same order. 

How far this can be taken as indicating a similarity in the 
mode of action of the two types of micro-organisms has not 
yet been shown. 

According to van Steenberge the mannitol producing 
lactic acid bacteria possess marked reducing powers towards 
selenium and tellurium salts, and are capable of activating 

TABLE HE 



Fermentation products. 


Glucose, 


Fructose. 


Carbon dioxide - 
Ethyl alcohol - 
Lactic aoid 


14-1 pei 
16-9 
47-1 


oen 


t. 


1-6 per. cent. 
12-3 


Acetic aoid 


3-7 






12-9 


Jt 


Formic acid 


0-1 






0-2 





Suooinio aoid ... 


1-2 






1-4 


if 


Mannitol .... 


m 






60-1 




Glyoerol - 
Bacterial substance 


6-3 
35 






nil 



these salts to function as acceptors for activated hydrogen. 
This observation supports the view that the formation of 
mannitol is attributable to the same cause, and that inter- 
mediately formed formic acid, therefore, produced at one 
stage or another in the fermentation of hexoses by these 
organisms, is decomposed with liberation of atomic hydrogen. 

Among the true lactic acid bacteria the activating powers 
are much less marked. According to van Steenberge they 
are unable to reduce selenium and tellurium salts, and fructose 
is converted into lactic aoid by them. 

They still retain the property of reducing methylene blue 
to its leuco-compound (Jensen 14 ), a reaction which does not 
involve a preliminary activation of the molecule of the 
hydrogen acceptor. 

Catalase is not produced by the true lactic acid bacteria, 
an observation which Beijerinck 16 made twenty-five years 



128 ANAEROBIC DEKSDROGENATION OF HEXOSES 
ago, and which Jensen 14 subsequently confirmed. This must 
be taken to indicate that hydrogen peroxide is unlikely to be 
formed as a result of the fermentation of carbohydrates by 
the true lactic acid bacteria, and indicates no doubt an in- 
creased sensitiveness towards oxygen by these organisms. 
The absence of catalase in the cells of the true lactic acid 
bacteria was utilized by Beijerinck 15 for detecting their 
colonies in artificial cultures. He found that a droplet of 
dilute hydrogen peroxide solution, when placed on these 
colonies, remained undeoomposed for a considerable time. 

Though the true lactic acid bacteria are undoubtedly more 
anaerobic than the mannitol producing types, they still 
develop though sparingly in the presence of oxygen. 

A further characteristic of these types is their inability 
readily to convert methylglyoxal into acetaldehyde and 
formic acid (Kluyver and Donker 16 ). Their action on this 
important intermediate is restricted almost entirely to a 
conversion into lactic acid, a reaction which, according to 
Neuberg and Gorr, 17 gives rise to almost theoretical yields. 

Several other observations have been made during recent 
years which facilitate the interpretation of the course of 
carbohydrate decomposition among the true lactic acid 
bacteria. Thus Myrback and von Euler 18 have shown that 
these micro-organisms produce a co-enzyme, and that, as in 
the case of yeast, a fermentation of glucose by these bacteria 
is impossible in the absence of the co-enzyme. It can be 
concluded, therefore, that the first stage in the fermentation 
of monoses is the formation of hexose phosphoric esters. 
That these esters are subsequently converted into methyl- 
glyoxal is supported by Neuberg and Gorr's researches 
already referred to. 

A certain percentage of the methylglyoxal must be con- 
verted into acetaldehyde and formic acid, since Bertrand and 
Weissweiler 2 observed the formation of the latter substance 
in cultures of Baet. bulgcuricum. 

Formation of pyruvic acid by the true lactic acid bacteria 
has recently been reported by Kostytchev and Soldatenkov. 19 



FORMATION OF LACTIC ACID 129 

In broad outline the fermentation of glucose by the true 

lactic acid bacteria may probably be schematized as follows : 

glucose > glucose phosphoric estera-> glyoeraldehyde-- 

methylgloxal 

4 \ 

acetaldehyde+ formic acid lactic acid 

I 
acetic aoid 

\ 
ethyl alcohol ( ?) 

The optical properties of the lactic acid produced by 
bacteria has engaged the attention of many workers. In the 
early investigations fermentation lactic acid was generally 
assumed to be inactive (Lewkowitsch 20 ). 

Reports did not fail to appear, however, containing de- 
scriptions of lactic acid bacteria producing either dextro-aoid 
only (Nencki and Sieber; 21 Gunther and Thierfelder 22 ), or 
laevo-lactic acid only (Tate 9 and Leiohmann 28 ). Again, other 
types were stated to produce a non-compensated mixture of 
dextro- and laevo-aoids (Bertrand and Weissweiller 2 and 
Harden 24 ), the proportions of the two acids depending in 
many cases (Pe're' 25 ) on the conditions of the growth of the 
test organisms, notably on the presence in the cultures of a 
suitable source of nitrogen, or on the temperature prevailing 
(Kozai 26 ). A comprehensive review of the investigations on 
this subject up to 1907 is given by Heinemann. 27 Some order 
was brought into the general confusion by MoKenzie, 28 who 
showed that the reason for the divergent views could be 
ascribed to the methods adopted for the analysis of the acids 
obtained, it being customary to examine the zinc salts of the 
lactic aoid formed, usually after one or more recrystalliza- 
fcions to ensure purity. By these purifications, however, the 
-feme proportions of the lactic acid formed became entirely 
obscured owing to differences in solubility of the zinc salts, 
-that of the inactive (racemic) lactic acid being less soluble 
fchan that of the two active components. McKenzie expressed 
-fche view that all fermentation lactic acid consists of an equal 
mixture of dextro- and laevo-acid. 



130 ANAEROBIC DEHTDROGENATrON OF HEXOSES 

In this view McKenzie is supported by Harden's 24 observa- 
tion on the optical nature of the lactic acid formed by Bad. 
coli commune and Sact. typhosum. Fully aware of the com- 
plications due to differences in the solubilities of the zinc 
salts of the inactive and the active acids, Harden conducted 
his investigation in such, a way as to avoid these difficulties, 
and found that the lactic acid produced by his two test 
organisms consisted of a mixture of inactive acid and laevo- 
laotio acid. 

Nevertheless, the question of the optical properties of 
fermentation lactic acid is still obscure. In investigations by 
Neuberg and Gorr 29 on the action of Sact. coli commune on 
methylglyoxal it is claimed that inactive acid is produced, 
and as late as 1926 Pederson, Peterson and Fred 30 asserted 
that the optical nature of the acid produced by a lactic acid 
bacterium studied by them was influenced by the presence 
in the culture of an infection form unable to produce lactic 
acid. 

THE BOOSTOMIG IMPORTANCE OF THE LAOTIO 
AOU> BACTERIA 

The very considerable and growing economic importance of 
the lactic acid bacteria necessitates a short reference to this 
subject. 

The question of the application of these bacteria in the 
distillery for the checking of undesirable bacterial infections, 
of their introduction into milk for the preparation of various 
types of sour milk and all types of cheese, is most conveniently 
dealt with in an account specially devoted to these subjects 
and will not be referred to here. The occurrence and activity 
of the lactic acid bacteria in flour and doughs will be discussed 
subsequently. 

There remains to be reviewed, therefore, the manufacture 
of lactic acid for medicinal and technical purposes. The first, 
and at that time universally adopted method, was described 
by Boutron and Freiny, 81 who worked under the conception 
that casein was the agency responsible for the conversion of 



FORMATION OP LACTIC ACED 131 

lactose into lactic acid. Subsequently von Blticher 32 observed 
that saccharose could be similarly converted in the presence 
of casein, and recommended this method as superior to that 
of Boutron and Fremy. Gobley, 83 like Boutron and Fremy, 
added lactose to millr to increase the carbohydrate con- 
centration, but allowed the conversion to proceed spon- 
taneously in the presence of chalk. Bensch, 34 like von Bliicher, 
utilized saccharose as the source of carbohydrate, and added 
cheese as a source of casein. In order, as he thought, to 
facilitate the inversion of the saccharose, he added small 
amounts of tartaric acid to his saccharose solution. The 
improvement obtained by the introduction of this acid must 
have been due to other causes, however probably to the 
checking of the development of wild yeast and butyric acid 
bacteria, two sources of infection which invariably affected 
the yields of acid obtained in the early methods of lactic 
acid production. 

With the realization of the function of bacteria consider- 
able further improvements were introduced. One of the 
pioneers in the manufacture of lactic acid was Avery, 36 who 
took out a number of patents during 1881 to 1885 for the 
protection of his method, in which the danger of undesirable 
side-reactions, notably of butyric acid and alcoholic fermenta- 
tions, was guarded against though by no means excluded. 
A very detailed account of this method was given by Claflin 36 
in 1897, an account which appears to have served as a model 
for the industry until quite recently, when genuine pure 
culture work was introduced. 

Avery's method, as improved by Claflin, conforms on the 
whole with the requirements of modern technique and may 
serve as a basis for the description of lactic acid manufacture 
to be given in these pages. 

Though most lactic acid bacteria develop well in milk and 
whey it is doubtful whether these liquids now serve as the 
raw materials for the Deduction of lactic acid. The difficul- 
ties involved in the recovery of the acid from milk and whey 
militates against their use in practice. The carbohydrates 

K2 



132 ANAEROBIC DEHYDROGENATION OF HEXOSES 
commonly used are saccharose, in the form of molasses, 
glucose, and maltose, obtained by the hydrolysis of starch. 

The necessary nitrogen, which for preference should be 
present as organic nitrogen (Kayser 1 ), is obtained from bran 
or from the grain in which the starch is contained. The total 
nitrogen of the finished mash should amount to 2 per cent, 
of the available carbohydrates though obviously it must 
be lower in the case of the procedure proposed by von Sait- 
cew, 37 who utilizes molasses without the addition of a nitrogen 
source. 

The concentration of carbohydrates in the mash should 
not be below 7-6 per cent., preferably about 10 per cent. 

If the mash is left slightly acid, at a pH value of about 3-7, 
its sterilization can be carried out without the application of 
high pressures, an important point, since pressure treatment is 
inclined to caramelize the dissolved carbohydrates. In many 
cases boiling at atmospheric pressure should be sufficient to 
ensure complete sterility under acid conditions. 

The sterilized mash is cooled to 50-55 C. with aseptic 
precautions, and is then run into a fermentation vat, where it 
is inoculated with a suitable type of true lactic acid bacteria. 
In earlier days Streptococcus (Bacterium) lactis acidi, Gunther 
and Thiefelder, was generally used. To-day a type of Bact. 
acidificans longissimum, Lafar, 38 is usually chosen, since it 
withstands higher temperatures. Bact. Ddbrucki is recom- 
mended by von Saitcew. 37 Calcium carbonate, or lime, in 
sterile suspension is added to the fermenting mash to main- 
tain its reaction at a pH value of 3-7 to 4-5, which, according 
to Bachraoh and Cardot, 39 represent the optimum hydrogen ion 
concentrations of most lactic acid bacteria, though Virtanen, 
Wichmann and LrndstrGm 40 place it at a pH value of 6-2. 

When, after 3 to 6 days, the conversion of the carbohy- 
drates is" completed, the lactic acid obtained should amount 
to 98 per cent, of the theoretical yield. The mash is now 
filtered and immediately concentrated so as to supply, on 
decomposition of the calcium lactate with sulphuric acid, an 
acid containing 50 per cent, lactic acid. 



FORMATION OF LACTIC ACID 133 

The use of zinc oxide or magnesium oxide instead of lime 
or chalk has been recommended by some workers. According 
to Mayer, 41 however, these oxides are less favourable than 
calcium salts. 

The crude lactic acid may be purified in various ways, but 
can be used in most industrial undertakings without further 
preparation. It finds a growing market in the textile indus- 
tries, notably in the woollen industries, for mordanting with 
potassium dichromate. In the leather industry an appreciable 
quantity is consumed in the deliming of the raw hides before 
tanning. 

The technical production of mannitol was discussed by 
Strecker 42 in 1854. He obtained it in exceptionally large 
yields up to 10 per cent, of the saccharose taken when 
fermenting a saccharose solution at room temperature under 
the conditions recommended by Bensch 34 for the production 
of lactic acid. Draggendorf 43 obtained 5 per cent, under 
somewhat similar conditions. Dox and Plaisance 44 recom- 
mended isolating mannitol from silage, in which they regularly 
found it present. 

The production of mannitol in appreciable quantities at 
comparatively low temperatures agrees with the observations 
that the mannitol producing lactic acid bacteria have a lower 
optimum temperature for growth than the true lactic acid 
bacteria. 

Before leaving the subject of lactic acid a reference must be 
made to the many attempts which have been made to intro- 
duce systems of classification for the numerous types which 
have been described at various times. It is questionable 
whether any of these systems, including those of Beijerinck, 15 
Lohnis, 46 Jensen, 46 and van Steenberge, 12 will be found of 
permanent value. The safest subdivision of the lactic acid 
bacteria remains at present the division based partly on their 
morphological characters and partly on their mode of action 
on fructose, whether they are capable of producing mannitol 
or not. 

Finally, it should be mentioned that the property of fer- 



134 ANAEROBIC DEB^DROGENATION OF HEXOSES 
menting carbohydrates with, the formation of small quantities 
of lactic aoid is also met with among certain fungi. This was 
observed by Eijkman 47 in the case of Mucor Eouxii and con- 
firmed for this fungus by Chrzaszcz 48 and by Saito 49 for 
Mucar chinensis. 

LITERATURE 

1. E. KayBer, Ann. Inst. Pasteur, vol. 8, p. 736, 1894. 

2. G. Bertrand and G. Weissweiler, Ann. Inst. Pasteur, vol. 20, p. 977 

1906. 

3. S. Grigoroff, Rev. mid. de la Suisse Eomande, vol. 26, p. 714, 1905. 

4. Chr. Barthel, Zentrbl.f. Bakt., Abt. H, vol. 6, p. 417, 1900. 

6. U. Gayon and E. Dub.ourg, Ann. Inst. Pasteur, vol. 8, p. 108, 1894. 

6. U. Gayon and E. Dubourg, Ann. Inst. Pasteur, vol. 15, p. 527, 1901. 

7. H. MtUler-Thurgau and A. Osterwalder, Zentrbl. f. Bakt., Abt. II, vol. 

36, p. 129, 1912. 

8. K. Kruia and B. Rayman, Lafcw*s Tech. Mykologie, vol. 5, p. 295, 1906. 

Gustav Fischer, Jena. 

9. G. Tate, J. Chem. Soc., vol. 63, p. 1263, 1893. 

10. W. H. Peterson and E. B. Fred, J. Biol. Chem., vol. 42, p. 273, 1920. 

11. J. Smit, Zeits.f. Qarungsphysiol., vol. 6, p. 273, 1916. 

12. P. van Steenberge, Ann. Inst. Pasteur, vol. 34, p. 803, 1920. 

13. M. W. Beijerinok, Arch, n&erland (2), vol. 6, p. 212, 1901. 

14. 0. Jensen, Zentrbl.f. Bakt., Abt. n, vol. 18, p. 211, 1907. 

16. M. J. Beijerinok, Zeits.f. Spiritusindustrie, vol. 26, p. 531, 1902 

16. A. J. Kluyver and H. J. L. Donker, Zeits. f. Ohemie d. ZeUe und Qewebe, 

vol. 13, p. 134, 1926. 

17. C. Nenberg and G. Gorr, Biochem. Zetts., vol. 173, p. 476, 1926. 

18. K. Myrbaok and H. von Euler, Berichte, Ser. B, vol. 57, p. 1073, 1924. 

19. S. Kostytchev and S. Soldatenkov, Z. physwl. Chem., vol. 168, p. 124, 

1927. 

20. J. Lewkowitsoh, Berichte, vol. 16, p. 2720, 1883. 

21. M. Nencki and N". Sieber, Monatsh. d. Chem., vol. 10, p. 632, 1889. 

22. C. Gtinther and H. Thierfelder, Arch.f. Hygiene, vol. 25, p. 164, 1895. 

23. G. Leichmann, Zentrbl.f. Bact., Abt. H, vol. 2, p. 777, 1896. 

24. A. Harden, Trans. Chem. Soc , vol. 76, p. 610, 1901. 

25. A. Pere, Ann. Inst. Pasteur, vol. 7, p. 737, 1893. 

26. T. Kozai, Zeits.f. Hygiene, vol. 31, p. 337, 1899 

27. P. G. Heinemann, J. Biol. Chem., vol. 2, p. 603, 1907. 

28. A. McKenzie, J. Chem. Soc., vol. 87, p. 1373, 1905. 

29. C. Neuberg and G. Gorr, Biochem. Zeits., vol 162, p. 490, 1925. 

30. C. S Pederaon, W. H. Peterson and E. B. Fred, J. Bwl. Chem., 

vol. 68, p. 181, 1926. 

31. F. Boutron and E. Fremy, Ann. Chem. Phys. (3), vol. 2, p. 257, 1841. 



FORMATION OF LACTIC ACID 135 

32. H. von BHicher, Ann. d. Physik und Ghem., vol. 63, p. 425, 1844. 

33. . Gobley, J. d. Pharm. et Ghem. [3], vol. 6, p. 64, 1844. 

34. A. Benaoh, Annalen, vol. 61, p. 174, 1847. 

35. C. E. Avery, Pharmaceut. J., vol. 13, Series m, p. 109, 1881-2. 

36. A. A. Claflia, J. Soc. Ghem. Ind., vol. 16, p. 516, 1897. 

37. J. von Saitoew, Zentrll.f. Bakt., Abt. n, vol. 72, p. 4, 1927. 

38. F. Lafor, Z&ntrU.f. Bakt., Abt. H, vol. 2, p. 194, 1896. 

39. E. Baohraoh and H. Cardot, Comrptea rend. Soc. liol, vol. 86, p. 1127, 

1922. 

40. A. I. Virtanen, E. Wiohmann and B. Linstrflm, Z. phystd. Chem., vol. 

166, p. 21, 1927. 

41. A. Mayer, Johresber. d. Tierdwm., vol. 22, p. 598, 1892. 

42. A. Strecker, Annalen, vol. 92, p. 80, 1854. 

43. G. Draggendorf, Arch.f. Pharm., Series HE, vol. 16, p. 47, 1879. 

44. A. W. Box, Q. P. Plaisanoe, J. Amer. Chem. Soc., vol. 39, p. 2078, 1917 

45. F. Lfihnifl, Zentrblf. Bakt., Abt. n, vol. 18, p. 96, 1907. 

46. 0. Jensen, ZentrU.f. Bakt., Abt. n, vol. 44, p. 144, 1915. 

47. C. Eijkman, Zentrblf. Bakt , vol. 18, p. 97, 1894. 

48. F. Chrzaszoz, Z&ntrU.f. Bakt., Abt. n, vol. 7, p. 326, 1901. 

49. K. Saito, ZentrU.f. Bakt., Abt. n, vol. 29, p. 289, 1911. 



CHAPTER XI 

THE FACULTATIVE ANAEROBIC DEEYDROGENA- 
TION OF HEXOSES, INVOLVING THE FORMA- 
TION OF LACTIC AdD AS AN ESSENTIAL DECOM- 
POSITION PRODUCT 

///. PROPIONIC ACID BACTERIA 

SiRHOKHR's 1 observations in 1854 on the formation of pro- 
pionic acid from calcium lactate by fermentation and the 
still earlier work referred to by him were reinvestigated and 
confirmed in 1879 to 1880 by Fitz, 2 who found that a solution 
of calcium lactate inoculated with bacteria contained in cows' 
dung broke down under the influence of these bacteria to a 
mixture of calcium propionate and calcium acetate. 

His analysis of the fermentation products indicated that 
the reaction had taken the following course : 

3 CH 3 .CHOH.COOCa = 2CH 3 .CH a .COOCa + 

oalo. .laotate calc. propionate. 

CH 3 .COOCa + C0 a + H a O 

oalo. acetate. 

The first pure cultures of propionic acid bacteria were 
isolated and described by von Freudenreich and Jensen. 3 
They obtained them from Emmenthaler cheese, in which 
these micro-organisms were shown to be responsible for the 
formation of 'holes' during the ripening process of the 
cheeses. 

Like the types studied by Strecker and by Fitz, von 
Freudenreich and Jensen's propionic acid bacteria, Bact. 
acidi propionici a and b and Bac. acidi propionici (a non- 
spore-forming rod but longer than the two other species), 
decomposed the lactic acid, previously formed from lactose 
in the young cheeses by true lactic acid bacteria. 

In addition to the decomposition of lactic acid, two of the 
types of Bact. acidi propionici isolated by von Freudenreich 



FORMATION OP LACTIC ACID 137 

and Jensen were shown to be capable of fermenting lactose 
with the production of carbon dioxide, propionic acid and 
acetic acid, the last named in somewhat larger proportions 
than those yielded where calcium lactate was fermented. 
Two further types resembling Bact. acidi propionici a and b 
were described by ThOni and AUemann 4 under the names of 
Bact. acidi propionic var. fuscum and Bact. acidi propionici 
var. rubrum, their varietal names referring to the pigmenta- 
tion of their colonies. A further type inclined to zoogloea 
production was isolated by Troili Peterson 6 from a certain 
type of Swedish cheese. 

More recently, Whittier, Sherman and Albus 6 have studied 
the properties of a related type, isolated by Sherman and 
Shaw 7 from Emmenthaler cheese. They found this type 
capable of fermenting both lactose and its component 
monoses as well as saccharose and maltose. 

Of these carbohydrates maltose was most readily fermented. 
Saccharose and maltose were found by van Niel 8 not to be 
fermentable by one of the types isolated by von ITreudenreich 
and Jensen. Glycerol appears to be fermentable by all known 
propionic acid bacteria (van Niel 8 ). 

The propionio acid bacteria are facultative anaerobes with 
a preference for anaerobiosis. Most of them are short, some- 
times cocooid, rods, von Freudenreich and Jensen's Bac. 
acidi propionici being longer. They produce yellowish-brown 
to red colonies under suitable conditions of growth. They do 
not produce endospores, are Gram positive, and in their 
morphology usually resemble Streptococcus lactia acidi very 
strikingly. Grown under conditions where oxygen can gain 
iccess, the cells become irregular, resembling those of Bact. 
"adicicola found in nodules of Leguminoseae. The propionic 
icid bacteria, according to Sherman 9 and to van Niel, 8 are 
,ypical catalase producers in spite of their reducing power 
/owards methylene blue, which Jensen 10 found to be three 
imes greater than that of the lactic acid bacteria. They all 
)roduce propionic and acetic acids from calcium lactate in 
he approximate proportions of 1-8:1 (van Niel 8 ). 



138 ANAEROBIC DEHYDEOGENATION OF HEXOSES 

In addition to the above types, which may be termed the true 
propionic acid bacteria, reference has been made in the litera- 
ture to types which are claimed to produce smaller amounts 
of propionic acid. Botkin 11 mentions one which fermented 
lactose with the production of butyric acid, butyl and ethyl 
alcohols, small amounts of acetic and formic acids, and traces 
of propiomc acid. Tissier and Gasching 13 describes another, 
Sac. T^ctopropylobutyricus non liquefaciens, which resembles 
Sac. perfringens, and which is stated to produce butyric and 
propionio acids in the proportions 2 :1, in addition to appreci- 
able quantities of lactic acid. It is highly probable, as sug- 
gested by van Mel, that the detection of propiomc acid in the 
fermentation products of these types is due to misinter- 
pretation of analytical data. 

The ability of the propionic acid bacteria to decompose 
lactic acid has naturally led to this acid being regarded 
as on important intermediate in the fermentation of carbo- 
hydrates by these micro-organisms. Virtanen 13 emphasizes 
this function of lactic acid, and considers that the hexose 
which, is finally converted into acetic and propionic acids is 
first dehydrogenated into lactic acid, and the latter subse- 
quently converted into a mixture of the two volatile acids. 

He suggests the following scheme for this mode of glucose 
fermentation. 

3C 6 H 12 O a ->6CH 3 .CHOH.COOH->4CH 8 .CH 2 .COOH+ 

glucose laotio acid propiomc acid 

2CH 8 .COOH-f-2CO a +2H 2 

acetic acid. 

The dehydrogenation of lactic acid to acetic acid and 
propionic acid has been studied in considerable detail by van 
Niel, 8 who concludes from his experimental data that the 
conversion occurs without the intermediate formation of 
acetaldehyde, since small quantities of this substance added 
to an active culture of propiomc acid bacteria remain essen- 
tially undecomposed, and are certainly not converted into 
acetic acid. In place of acetaldehyde van Niel suggests 



FORMATION" OF LAOTIO AOID 139 

pyruvic acid CH 8 .CO.COOH as the intermediate stage 
between lactic acid and propionic and acetic acids. Pyruvio 
acid was found both by Virtanen and by van Niel to be as 
readily converted into propionic and acetic acids as lactic 
acid, while acetaldehyde was hardly affected at all. Those 
traces of the aldehyde which were affected could be recovered 
as acetylmethylcarbinol, a substance which van Niel detected 
in small quantities in his culture of propionic acid bacteria. 
The conversion of pyruvio acid to propionio, acetic, and car- 
bonic acids can be secured only if the pyruvic acid is assumed 
to function as a powerful hydrogen acceptor, thus : 

2CHi.CO.GC +2H-^CH 3 .CH a .COOH+CH 8 .COOH+C0 2 . 
X)H 

That it does so is emphasized by van Niel, who comments 
also on the importance of this observation that a fermenta- 
tion of hexoses has here been found in which the production 
of aoetaldehyde is of quite insignificant importance. 

The following scheme probably represents the essential 
phases in the fermentation of glucose by the propionio acid 
bacteria : 

Grlucose-> glucose phosphoric esters >-glyoeraldehyde->-methylglyoxal->- 
laotio aoid-vpymvio acid->-propionio aoid+aoetio acid+CO a 

Compared with the lactic acid bacteria the propionic acid 
bacteria have received but slight attention in the past. 
Ihough present among the intestinal microflora of herbivo- 
'ous fl.niTYifl.1g and among that of silage, participation of the 
propionic acid bacteria in the essential reactions occurring in 
ihe intestine and in the silage heap is probably insignificant. 
Pheir economic importance is restricted at present to their 
ictivity during the ripening of cheese and to the use of 
Dropionic acid in the perfume industry. 

The use of propionic acid and its esters has been suggested 
is solvents for pyroxylin and as substitutes for cellulose 
icetate. To facilitate this application of propionio acid 
>acteria, Sherman 14 and his collaborators attempted to 



140 ANAEROBIC DEHYDROGENATION OF HEXOSES 
accelerate the fermentation of carbohydrates by them 'and to 
increase the yields of propionic acid. They found that the 
presence in the cultures of propionic acid bacteria of JSact. 
casei, or of Bact.alcaligenes or Bact.wdgare, greatly accelerated 
the formation of propionic acid and favoured the production 
of the acid at the cost of other fermentation products. Thus, 
while pure cultures of their test organism a (Bact. acidi pro- 
pionici d) fermented 66 per cent, of the available lactose within 
30 days and gave a yield of 1 part of acetic to 1-7 parts of 
propionio acid, the presence of one of the afore-mentioned 
micro-organisms not only changed these proportions to 1 part 
of acetic acid and 11-5 parts of propionic acid, but increased 
the rate of fermentation of the carbohydrates to an appreci- 
able extent. In a patent taken out by Sherman 14 this mode 
of propionio acid preparation is suggested as a suitable 
method for industrial purposes. 

In concluding this review of the propionic acid bacteria, 
it should be mentioned that Virtanen 13 has laid great stress 
on an observation of his that succinic acid is produced by 
propionic acid bacteria in considerable quantities, up to 
about 30 per cent, of the total fermentation products. Most 
other investigators have obtained suocinic acid as a fermenta- 
tion product of the propionio acid bacteria but in far smaller 
proportions. Virtanen explains the occurrence of succinic 
acid as due to the property of the propionic acid bacteria of 
decomposing hexoses without the intervention of hexose 
phosphoric esters, a reaction which takes place when a coen- 
zyme is absent or only present in insufficient quantities. By 
this abnormal fermentation process the glucose molecule is 
stated to be broken down into succinic acid and acetaldehyde, 
the latter giving rise to acetic acid formation. 

Myrback and von Euler 15 hold a similar view. The correct- 
ness of this conception, however, is warmly disputed by 
Maurer. 16 And it must be admitted that it is rendered highly 
questionable by van Niel's observation on the effect of pro- 
pionio acid bacteria on added aoetaldehyde. Van Niel ascribes 
the small amounts of succinic acid formed by the propionic 



FORMATION OF LACTIC ACID 141 

acid bacteria to the action of the organisms on protein sub- 
stances, on the lines suggested by Ehrlich, 17 Neuberg and 
Binger, 18 and Kostytchev and Erey 19 in the case of yeast, and 
by Kozai 20 in the case of bacteria. 

LITERATURE 

1. A. Strecker, Annalen, vol. 92, p. 80, 1854. 

2. A. Fitz, Benchte, vol. 13, p. 1309, 1880. 

3. E. von Freudenreich and 0. Jensen, Zentrbl. f. Bakt., Abt. IE, vol. 17, 

p. 629, 1907. 
4.- 1. Thfini and 0. Allemann, Zentrbl. f. Bakt., Abt. H, vol. 25, p. 8, 1910. 

5. G. Troili Peterson, Zentrbl. f. Bakt., Abt. H, vol. 24, p. 333, 1909. 

6. E. 0. Whittier, J. M. Sherman and W. R. Albus, Ind. and Eng. Ghem., 

vol. 16, p. 122, 1924. 

7. J. M. Sherman and R. H. Shaw, J. Bid. Ghem., vol. 56, p. 695, 1923. 

8. C. M. van Niel, Theproptomc acid bacteria, Dissertation, publ. by Messrs. 

Uitzeverszack Boissevain & Co., Haarlem, 1928. 

9. J. M. Sherman, J. Bact., vol. 11, p. 417, 1926. 

'0. O. Jensen, Zentrbl. f. Bakt., Abt. IE, vol. 18, p. 211, 1907. 

1. S. Botkin, Z.f. Hygiene, vol. 11, p. 421, 1891. 

2. H. Tissier and P. Gasohmg, Ann. Inat. Pasteur, vol. 17, p. 540, 1903. 

3. A. I. Virtanen, Berichte, vol. 58, p. 2441, 1926. 

4. J. M. Sherman, UJ3. Pat., No. 1469969, 1923. 

5. K. Myrbaok and H. von Euler, Benchte, Ser. B, vol. 67, p. 1073, 1927. 

6. K. Maurer, Biochem. Zeits., vol. 191, p. 83, 1927. 

7. F. Ehrlioh, Zeits. f. angew. Ghem., vol 27, p. 48, 1914. 

8. C. Neuberg and M. Ringer, Biochem. Zeits., vol. 91, p. 131, 1918. 

9. S. Kostytsohev and L. Frey, Zetts. physiol. Ghent., vol. 146, p. 276, 1925. 
0. Y. Kozai, Zeite.f. Hygiene, vol. 38, p. 386, 1901. 



CHAPTER XII 

THE OBLIGATORY ANAEROBIC DEHYDROGENA- 
TION OF HEXOSES 

THE PRODUCTION OF N-BUTYRIC ACID AND N-BUTYL 

ALCOHOL. 

THE type of hexose fermentation which results in the pro- 
duction of butyric acid as a chief fermentation product 
attracted the attention of investigators at an early date in 
the history of microbiology, not only because of the apparent 
unusual conversion of the hexose molecule, but also, and 
perhaps mainly, owing to the remarkable fact, first observed 
by Pelouze and Gelis, 1 that butyric acid may be produced 
by the fermentation of calcium laotate, or, according to Fitz, 2 
from glycerol, substances which contain three carbon atoms 
in their molecule as against the four found in the butyric 
acid molecule itself. 

Buohner and Meisenheimer 3 were the first to undertake 
systematic investigations with a view to throwing light on the 
reactions involved in the conversion of a three carbon chain 
substance like glycerol into butyric acid containing four 
carbon atoms in its molecule. To do so they compared the 
fermentation products of glucose and of glycerol when 
fermented by a typical butyric acid producing bacterium. 

The result of their quantitative analyses, given in Table IV 
below, is the first detailed record of the final products of a 
typical butyric acid fermentation : 

TABLE IV 
Final fermentation products in percentage 

















Carbon 




Material, 


n-Buty- 
ric acid. 


Acetic 
acid. 


Formic 
acid. 


Lactic 
acid. 


n-Butyl 
alcohol. 


Ethyl 
alcohol. 


di- 
oxide. 


Hydro- 
gen. 


100 gr. 


















glycerine 


0-7 


1-0 


4-0 


3-4 


19-6 


10-4 


42-1 


19 


100 gr. 


















glucose 


26-0 


7-5 


3-4 


10-0 


0-7 


2-8 


481 


1-6 



ANAEROBIC DEHYDROGENATION OF HEXOSES 143 
Buchner and Meisenheimer utilized for their investigations 
a culture of Sac. butylicus isolated by Fitz 4 from cows' dung 
and stated to be capable of producing butyric acid under 
both aerobic and anaerobic conditions. The fermentation 
products, which amounted to 83 and 85 per cent, respectively 
of the substances fermented, were found to be identical 
whether glycerol or glucose were taken, but differed quantita- 
tively. This identity was considered to indicate a common 
origin. As such Buchner and Meisenheimer suggested lactic 
acid, which Sohade 6 had previously contemplated as a possible 
mother substance of acetaldehyde and formic acid. Postu- 
lating the production of acetaldehyde from lactic acid, Buchner 
and Meisenheimer explained the formation of butyric acid 
by assuming that aoetaldehyde was first condensed to aldol, 

J> 

CH 3 .CHOH.CH a .C<^ , and this subsequently reduced to 

N H 

butyric acid. The n-butyl alcohol was thought to have 
been formed by the reduction of crotonic aldehyde, CH 8 . 



CH.CH.O^ , which in turn had been derived from aldol. 

NBC 

The ethyl alcohol was considered to have been formed from 
acetaldehyde on the lines known from the action of yeast on 
glucose. The origin of the acetic acid was not discussed by 
Buchner and Meisenheimer. 

That acetaldehyde occurs as an intermediate fermentation 
product in the butyric acid fermentation was first demon- 
strated by Neuberg and Arinstein, 6 who 'fixed' appreciable 
quantities of this substance in fermenting cultures of Sac. 
butylicus, Mtz. Though these writers emphasize that the 
fixing of acetaldehyde by means of sodium sulphite results 
in the disappearance of butyric acid and of butyl alcohol from 
the final fermentation products, they decline to accept aoet- 
Eildehyde as an intermediate of butyric acid. Instead they 
propose pyruvio acid aldol CH 8 .COH.COOH, a substance 

CEL.CO.COOH 



144 ANAEROBIC DEHYDROGENATION OF HEXOSES 
which they found could be decomposed to butyric acid by 
BOA. butylicus. This conception, as pointed out by Donker, 7 
is not likely to be correct, since pyruvic acid CH 3 .CO.COOH, 
when added to a culture of Bac. butylicus, does not become 
converted into butyric acid, but is transformed into acetic 
and formic acids. Neuberg and Arinstein had already ob- 
served this to be the case. 

Important observations were made on the reactions taking 
place in the fermentation of glucose by butyric acid bacteria 
during the investigations carried out between 1917 and 1919 
in the laboratory of one of the writers, and subsequently 
published by Beilly and collaborators 8 and by Thaysen. 9 
These investigations showed that the fermentation of glucose 
by butyric acid bacteria can be divided into two well-defined 
stages an early one, during which acids are produced and 
carbon dioxide and hydrogen evolved, and a later one, during 
which most of the acids are converted into neutral products. 

The acids formed were found to consist essentially of acetic 
and butyric acids in varying proportions, with smaller 
amounts of formic and of lactic acids. The neutral products 
comprised n-butyl alcohol, acetone, and ethyl alcohol. 

By the addition of an excess of calcium carbonate to a 
fermenting culture of the test organism, or by the adjustment 
of the reaction of the culture medium nearer to the alkaline 
side of the neutral point, the fermentation could be influenced 
in such a way that almost the whole of the available carbo- 
hydrates was converted into acids,- carbon dioxide, and 
hydrogen, the usual neutral fermentation products being 
practically eliminated. Similar observations were made by 
Speakman. 10 

These observations indicated that any attempt made to 
elucidate the intermediate stages of the butyric acid fermenta- 
tion had to concentrate in the first instance on the reactions 
leading to the formation of acids. Unfortunately Reilly and 
his collaborators did not pursue their investigations in this 
direction beyond ascertaining that the two volatile acids 
formed were produced in the proportions of 1 molecule of 



ANAEROBIC DEHYDKOGENATION OF HEXOSES 146 
acetic acid to 1-8 molecule of butyric acid. Speakman's 11 
publication on this subject added nothing of interest to the 
solution of the problem. He suggested that the acids originate 
direct from the carbohydrate, being formed in equimolecular 
proportions with simultaneous liberation of oxygen. Other 
publications have not. appeared. 

There is, therefore, at present no direct evidence to show- 
how hexoses are broken down into acetic acid and butyric 
acid by butyric acid bacteria. The experimental data of 
Neuberg and Arinstein on the intermediate formation of 
acetaldehyde and those of Donker 7 on the occurrence of 
formic acid and aoetylmethyloarbinol among the fermenta- 
tion products support the hypothesis, however, that the 
fermentation proceeds via methylglyoxal or perhaps di- 
hydroxyacetone to acetaldehyde and formic acid. 

The formic acid would be the mother substance of carbon 
dioxide and hydrogen evolved, and the acetaldehyde that of 
butyric acid through aldol condensation and reduction, of 
acetic acid, and of acetylmethyloarbinol CH 3 .CO.CHOH. 
CH 3 . Support for this hypothesis is found in the results of 
the quantitative analysis of the fermentation products of a 
typical butyric acid producing bacterium, Bac. Past&wrianus 
(Clostridium Pasteurianum), which was investigated by 
Donker. Table V shows that the fermentation products of 
Bac. Pasteurianus, when expressed as their equivalent 
quantities of acetaldehyde, hydrogen, and carbon dioxide, 
were equal in each case to double the number of gramme 
molecules of carbohydrates converted. The bearing of similar 
observations in the case of Bac. acetoethylicus on the inter- 
pretation of the course taken by a fermentation was discussed 
in detail in Chapter V, p. 86. 

The second stage in the fermentation of carbohydrates by 
butyric acid bacteria, when the acids formed are con- 
verted into neutral compounds, varies very greatly with 
the species. In some cases the active hydrogen resulting 
from the break-up of formic acid takes very little part in the 
subsequent reactions and is given off almost entirely as mole- 



146 ANAEROBIC DEHYDROGENATION OF HEXOSES 
cular hydrogen. Here the fermentation invariably comes to a 
standstill before the whole of the available carbohydrates 
has been consumed, unless precautions are taken to prevent 
an accumulation of free acids by the addition of a neutrn.lJ7.ing 
agent such as calcium carbonate. Typical representatives of 



TABLE V 

Bac. Pasteurianus grown in yeast water containing 2 per cent, 
of glucose and 1 per cent, of calcium carbonate 



Fermentation 
product. 


Percentage of 
fermentation 
product cal- 
culated on 
glucose fer- 
mented. 


Number of gramme molecules produced per 
50 gramme molecules of glucose fermented. 


Carbon 
dioxide. 


Hydrogen. 


Acetalde- 
hyde. 


Carbon dioxide 
Hydrogen 
Formic acid 
Acetio aoid 
Butyrio acid 
n-bntyl alcohol 
Acetone 
2:3 Butylene- 
glyool 


47-4 
152 

11-4 
126 
212 
1-1 


+97-0 
-1-7 


+68-4 
-17-1 

+51-6 
-34 


+ 17-1 
+26-8 
+61-6 
+ 3-4 


95-3 


995 


97-9 



such strains are Vibrion butyrigue, Pasteur j 12 Bac. butyricus, 
Prazmowski ; 13 Bac. Pasteurianus, Winogradski ; 14 Bac. 
saccharobutyricus (Qranulobacter saccharobutyricum), Beije- 
rinck; 15 and Grassberger and Schattenfroh's 16 non-motile 
butyric acid bacillus. The chief hexose fermentation products 
of these types are butyric and acetic acids, carbon dioxide, 
and hydrogen. 

The strains which activate butyric and acetic acids to 
function as hydrogen acceptors usually convert the whole of 
the available hexoses and produce appreciable quantities of 
neutral fermentation products, notably n-butyl alcohol, 



ANAEROBIC DEEYDROGENATIOlSr OF HEXOSES 147 
acetone and ethyl alcohol. The most characteristic repre- 
sentative of this group is the type introduced by Weizmann 17 
for industrial purposes and described variously in the literature 
as Bac. granulobacter pectinovorum, Speakman; 10 as Clostri- 
dium acetonigenum, Donker ; 7 or as Clostridivm acetobutylicum 
(Wilson, Peterson, Fred 18 ). Colloquially it has become known 
as the 'acetone bacillus'. In these pages it will be referred 
to as Bac. acetonigenus. 

The reactions involved in the production of neutral fer- 
mentation products by the strains activating acetic and 
butyric acids have engaged the attention of several workers 
since the industrial processes for the manufacture of n-butyl 
alcohol and acetone by fermentation were introduced. That 
a connexion existed between the disappearance of n-butyric 
acid and the formation of n-butyl alcohol was clear from the 
investigation of Reilly and collaborators, but with the ex- 
ception of some little known observations by Hall and Ran- 
dall 19 on the behaviour of Bac. Welchii, it appears to have 
escaped the notice of investigators studying the behaviour of 
pathogenic types. 

It was shown by Reilly that butyric acid added to a fer- 
menting culture of the test organism became converted into 
n-butyl alcohol through the action of available active hydro- 
gen. It is now generally accepted as correct that n-butyl 
alcohol is derived from the corresponding acid through 
hydrogenation. The figures for hydrogen evolution among 
various types of butyric acid bacteria which have been sup- 
plied by Donker confirm that a hydrogenation of butyric 
acid takes place during the period of the formation of n-butyl 
alcohol. 

The interpretation of the origin of acetone has been debated 
at great length, but can now be regarded as settled in favour 
of the view, first advanced by Desborough, 20 that it is formed 
from acetic acid. 

Desborough found that an addition of acetic acid to a fer- 
menting culture of Bac. acetonigenus resulted in an increase 
of acetone, amounting to 80 per cent, of wnat might be 

L2 



150 ANAEROBIC DEHYDROGENATION OB 1 EEXOSES 
classification of these types. The former may be termed the 
true butyric aoid bacteria, the latter the butyl alcohol 
bacteria. In both cases the fermentation proceeds best under 
anaerobic conditions, though some species, for instance Bac. 
butyticus, ITitz, are able to develop also in the presence of oxygen. 

Obligatory aerobic bacteria no doubt exist which produce 
small quantities of butyric acid in the presence of oxygen. 
Sohattenfroh and Grassberger 25 make reference to them in 
one of their well-known publications. One of the best known 
of these types is probably Bac. butylicua, Hiippe, 26 which is 
stated by Lehmann and Neumann 27 to be related to Bac. 
Megaih&rium and Bac. mesentericus. Another type, Bac. 
mes&ntericus rtib&r, is stated by Dupont 28 to produce not only 
butyric acid and acetic aoid in small quantities, but also traces 
of valeric acid. 

The progress of the hexose fermentation in these cases has 
not yet been investigated. 

A special reference must be made also to those butyric 
acid bacteria, first isolated by Omelianski, 29 which ferment 
cellulose. A detailed account of these types, notably of Bac. 
methanigenes and Bac. fossicularum, was given in the mono- 
graph of Thaysen and Bunker 30 on the microbiology of cellu- 
lose and allied substances. Though the activity of these 
types is restricted to cellulose, glucose probably occurs at one 
stage or another in the reaction. In other respects the inter- 
mediate stages of the fermentation remain entirely unex- 
plored. 

Both groups of starch and hexose fermenting anaerobic 
butyric acid bacteria have acquired considerable industrial 
importance and are used for the production of butyric acid, 
n-butyl alcohol and acetone. In the following rdswnA an 
account will be given of the processes by which butyric acid 
and n-butyl alcohol may be obtained by fermentation. 

THE BTJTYBIO AOID FERMENTATION 

It was mentioned above that Pelouze and Gelis 1 had found 
the mixture recommended by Boutron and Fr&ny 31 for 



ANAEROBIC DEHYDROGENATION OF HEXOSES 161 
the preparation of lactic acid to undergo a further decom* 
position when kept under suitable conditions. Pelouze and 
Gelis found that this secondary fermentation took place 
with the evolution of considerable quantities of hydrogen and 
with the formation of butyric acid, derived not from the 
hexose but from the lactic acid to which the hexoses present 
had been converted in the first instance. 

That this fermentation was due to the activity of micro- 
organisms was first demonstrated by Pasteur. 82 Stricht 88 was 
able to show that butyric acid can be produced direct from a 
carbohydrate. Later other investigators, among them Fitz, 84 
confirmed this, and showed that butyric acid can be produced 
also from glyoerol. The wide distribution in soil, milk, water, 
faecal matter and wounds, of bacteria producing butyric acid 
under anaerobic conditions was abundantly proved by 
subsequent investigators. Efforts at grouping and describing 
the numerous types proceeded actively with the isolation 
of new strains and are still proceeding. Various schemes have 
been suggested for this classification. The best known are 
perhaps those of Grassberger and Schattenfroh 16 and of Brede- 
mann. 85 The most recent and the most extensive from the 
point of the carbohydrate metabolism is that of Donker. 7 

In spite of the interest which has been taken in the butyric 
acid bacteria little progress has been made with the improve- 
ment of the methods for the preparation of butyric acid. It 
is questionable even whether pure cultures of the most 
suitable types are in use for the purpose. The comparatively 
recent method suggested by Lefranc and Co. 36 would appear 
to indicate this. It is impossible, therefore, to give more than 
a tentative description of the industrial production of butyric 
acid as it should be carried out under ideal conditions. 

The raw material chosen, usually starch, maltose, saccha- 
rose (molasses), or glucose, should be suspended or dissolved 
in the requisite amount of water and sterilized by heat. 

On the whole, starch in the form of maize or rice is prefer- 
able as raw material, since its use obviates the necessity for 
separate processes for the preparation of maltose or glucose. 



162 ANAEROBIC DEHYDROGENATION OF 
Where molasses are used it will invariably be f our*-* 
to supply nitrogenous food substances to ensure 
progress of fermentation. As such may be used 
grain, or the gluten remaining after the preparatio 
from rice or wheat (Legg 37 ). 

The temperatures required for sterilization wo" 1 
on the hydrogen ion concentrations maintained i* 
(Chick 38 ) and on the concentration of the mash. 

The sterile mash would have to be cooled to 37 
cooled mash fed continuously into a previously st 
containing a vigorously fermenting culture of t>3 
type of butyric acid bacterium, until the vat was 
filled. While the fermentation proceeded the hy 
concentration of the mash would have to be ma/i 
its optimum, presumably between pH values of 5 < 
as to prevent the fermentation from coming to a- 
standstill. On completion of the fermentation SL 
four days the mash, containing salts of the vo] 
produced, should be evaporated to dryness and ~t 
acid recovered by fractional distillation of the ca 
in the presence of sulphuric acid or oxalic acid. 

THE PRODUCTION OP BTJTYL ALCOHOL BY FBRMJB 

Not until 1911 was any serious interest taken i 
duction of butyl alcohol by fermentation. In that 
bach and Strange 39 patented a fermentation proc< 
purpose, based, it is understood, on the utHizati< 
butylicus, Mtz. 

Previously the butyl alcohol producing bacteria 
a subject of research only in connexion with the e 

i of the origin of the 'fusel oils' of crude alcohol. 

' raised this question in 1877 and Perdrix 40 had sul 

reverted to it. From his investigations on Bac a,n 
isolated from Paris tap water, Perdrix had come t 
elusion that the appearance of 'fusel oils' was un 
the result of contamination of worts with this or j 



ANAEROBIC DEHYDROGENATION OF EEXOSES 163 
teria. A bacterial origin of the 'fusel oils' was favoured also 
by Emmerling. 41 Pringsheim's 42 investigations of the problem 
threw doubt on this contention, however, and showed that 
'fusel oils' rarely, if ever, contain n-butyl alcohol, but are 
composed of amyl and propyl alcohols besides iso-butyl 
alcohol. It was noted also by Pringsheim that bacteria such 
as Bac. amylazymus are unable to develop in worts containing 
10 per cent, of ethyl alcohol, a concentration usually estab- 
lished in worts under industrial conditions before the pro- 
duction of 'fusel oils' becomes apparent. The question of 
the origin of 'fusel oils' was finally settled by Ehrlich, 48 who 
found that they resulted from the action of yeast on nitro- 
genous organic substances. 

Pernbach and Strange's efforts at producing butyl alcohol 
on an industrial scale do not appear to have met with marked 
success, and the industry remained of insignificant proportions 
until the outbreak of the war of 1914 to 1918, when an ab- 
normal demand for acetone arose. 

At this time attention was drawn by Weizmann 17 to the 
property of butyl alcohol bacteria of producing considerable 
quantities of acetone. Liberal financial support from public 
funds made it possible to institute a comprehensive inquiry 
into the most suitable methods by which the butyl alcohol 
bacteria could be utilized for the production of acetone. As 
a result factories were erected in which starch and maltose 
were fermented under aseptic conditions with a species of 
butyl alcohol bacterium, supplied by Weizmann, discussed 
in this treatise under the name of Bac. acetonigenus. 

The morphology, biology, and fermentative action of this 
strain have been the subject of fairly detailed investigations 
by Speakman, 10 Reilly and collaborators, 8 Thaysen, 9 and 
notably by Donker, 7 who came to the conclusion that it 
differed sufficiently from other similar organisms studied by 
him to justify him in regarding it as a new species. 

Its most characteristic properties are its marked liquefying 
action on gelatine and its formation of appreciable quantities 
of acetylmethylcarbinol. All other similar strains examined 



164 ANAEROBIC DEHYDROGENATION OF HEXOSES 
by Donker lacked proteolytio action and reduced any inter- 
mediately formed acetylmethyloarbinol to 2 :3 butyleneglycol. 

A striking feature of the fermentation of hexoses by this 
organism is the rise of the titratable acidity of the fermenting 
mash to a clearly denned maximum. Under the most favour- 
able conditions this maximum is reached within the first 
fifteen hours of fermentation, though it may be delayed 
for various reasons until the thirtieth hour. It is followed 
immediately by a fall to a Trm'TvimnTn which, apart from 
slight variations, remains stationary until the completion 
of the fermentation. The curve shown in Fig. 1 illustrates 
this. A similar rise and fall in the hydrogen ion concentra- 
tion of the fermenting mash can be observed, but the 
maximum concentration is not as clearly defined as in the 
case of the titratable acidity. Unpublished observations 
by the writers show that a hydrogen ion concentration 
equal to a pH value of 4-3 is established under normal con- 
ditions within the first ten hours of the fermentation, and that 
this concentration, after an intermediate rise which coincides 
with the attainment of the maximum titratable acidity, is again 
established towards the end of the fermentation (see Fig. 1). 

The evolution of gas, a varying mixture of hydrogen and 
carbon dioxide, does not coincide with the rise and fall in 
titratable acidity, but commences at a later stage as shown 
in Fig. 2. 

By checking the relationship between evolution of gas and 
the production of titratable acidity at frequent intervals, 
valuable information may be gained as to the absence or 
presence in the fermenting mash of infecting micro-organisms. 
When infections occur the curves both of the titratable 
acidity and of gas evolution become affected, that of the 
titratable acidity rising either continuously, or after a tem- 
porary fall, to concentrations much in excess of those at 
which the fermentation can continue its normal course. The 
curve of gas evolution shows a correspondingly marked drop 
(see Fig. 3). 

Thaysen 9 has shown that the most serious infection of the 



Hydrogen ion Concentration of fermenting mash. 




Titratable acidity of fermenting mash. 



Gas evolution. 




Titratable acidity. 



Gas evolution. 




Titratable acidity. 



168 ANAEROBIC DEHYDROGENATION OP HEXOSES 
acetone fermentation is caused by lactic acid bacteria, 
notably to one described by hi under the name of Bact. 
volutans, an organism which cannot be readily distinguished 
from Bac. acetonigenus by microscopical examination except 
after previous staining of the preparation with methylene 
blue in the cold. By this technique Bact. volutans is seen to 
contain in its cells two or three granules of purple staining 
bodies which never occur in the cells of Bac. acetonig&nus. 
The seriousness of lactic acid infections has been referred to 
also by Fred, Peterson and Mulvania, 44 and by Speakman and 
Phillips. 45 The last-named writers interpret the action of 
Bact. volutans as due to its production of an inhibitory sub- 
stance which causes Bac. acetonig&nus to produce lactic acid 
instead of its usual intermediate fermentation products. 
The experimental evidence brought forward by Speakman 
and Phillips in support of this contention is not supported 
by practical experience. 

Various methods have been recommended for counter- 
acting the harmful influence of lactic acid bacteria on the 
course of the butyl fermentation, such as lowering the fer- 
mentation temperature. In the writers' experience none of 
these recommendations have the desired effect. 

Another serious cause of failure of the butyl fermentation 
has been ascribed by Legg 46 to the occurrence of ' epidemics of 
sluggishness ' in the cultures used for inoculation, presumably 
inoUcating"'the presence of a bacteriophage in such cultures. 
This interesting observation has not yet been fully explored. 

The plant now generally chosen for the production of butyl 
alcohol is that usually adopted for pure culture fermentation 
processes and need not be described in detail. The process 
involves a preliminary sterilization of the raw material, either 
starch or maltose, followed by the introduction of the cooled 
mash into a large amount of already fermenting mash. 
Where starch mashes are used their sterilization requires 
temperatures far in excess of those usually thought necessary ; 
and an exposure of the mash for 8 hours to 144 0. is found 
to be essential. 



ANAEROBIC DEHYDROGENATION OF HEXOSES 159 
The fermentation is conducted at a temperature between 
37 C. and 40 C., and under normal conditions is com- 
pleted in 28 to 32 hours. The fermentation products, accord- 
ing to ReHly and collaborators, are : 

7 kg. of acetone 
103 kg. of n-butyl alcohol 
100-3 cubic meters of carbon dioxide 
69-1 hydrogen ^ 

12 kg. of residual acidity, from 
100 kg. of grain, containing 65 kg. of starch. 
With the increasing importance of n-butyl alcohol as a 
basis for the manufacture of dopes and varnishes, attempts 
have been made to increase the yields of butyl alcohol ob- 
tained. According to Boinot 47 this may be achieved by add- 
ing calcium lactate to the fermenting mash. 

Since the introduction of the industrial production of 
butyl alcohol by fermentation, the isolation of pure cultures 
of Bac. acetonig&n/us has become a question of considerable 
importance, the usual laboratory methods being entirely 
unsuitable for the purpose. Dorner 48 has maintained that 
this difficulty is due to the presence in ordinary culture media 
of inhibitory substances which prevent the ready develop- 
ment of Bac. acetonigenus. By the addition of substances 
such as animal charcoal, soil, or brain to dextrose broth, 
Dorner claims to have been able to overcome the inhibitory 
action of the usual laboratory media, and states that he has 
good development of a single cell by Bum's 49 Indian ink 
method. The writers have repeated Dorner's experiments, 
using glucose broth and wort with animal charcoal added. 
They did not succeed in obtaining growth of Bac. acetonigenus 
in these media when the number of cells inoculated decreased 
to less than 2,000. 

THE PEODTJOTIOlSr OP HIGHBB FATTY AOIDS 

The early investigators of the butyric acid fermentation 
record having isolated other and usually higher fatty acids 
as fermentation products. Grillone 60 and Fitz 34 obtained 



160 ANAEROBIC DEHYDROGENATION OF HEXOSES 
oaproic acid, Botkin 51 propionic acid, Locquin 52 and Wagner, 
Meyer and Dozier 63 valeric acid. 

Neuberg and Ariostein 6 paid particidar attention to these 
acids and isolated 34-9 grammes of a mixture of caproic, 
caprylio and capric acids from 3990 grammes of glucose. 
These higher fatty acids invariably contained an even number 
of carbon atoms in their chains. 

Neither Neuberg and Arinstein nor any other investigators 
have so far attempted to explain the intermediate stages 
involved in the production of these acids by micro-organisms. 
Perhaps it is justifiable to assume that the reactions involved 
in these cases are similar to those responsible for the syntheses 
to which references will be made in Chapter XV. 

LITERATURE 

1. J. Pelouze and G6hs, Ann. Ohim. Phya., 3, vol 10, p. 434, 1844. 

2. A. Fitz, Beriahte, vol. 10, p. 276, 1877. 

3. C. Buohner and J. Meisenheimer, Benchte, vol. 41, p. 1419, 1908. 

4. A. Fitz, Benchte, vol 15, p. 867, 1882. 

6. H Schade, Zeitschr. f. physikal. Chem., vol. 67, p. 1, 1907 , vol. 60, 
p. 610, 1907. 

6. C. Neuberg and B. Arinstem, Biochem. Zeits., vol. 117, p. 269, 1921. 

7. H. J L. Donker, Bijdrage tot de Kennw der Boterzuur-Bvtyl akohden 

Acetonegistingen. W. D. Meinema, Delft, 1926. 

8. J. Reilly, W. G. Hiokinbotitom, F. R. Henley and A. C. Thaysen, 

Biochem. J., vol. 14, p. 229, 1920. 

9. A. 0. Thaysen, J. Inst. Brew., vol. 27, p. 529, 1921. 

10. H. B. Speakman, J. Bid. Chem., vol. 41, p. 319, 1920. 

11. H. B. Speakman, J. Bid. Chem., vol. 43, p. 401, 1920. 

12. L. Pasteur, Com/pies rend., vol. 52, p. 344, 1861. 

13. A. Prazmowski, Baton. Zeits., vol. 37, p. 409, 1879. 

14. S. Winogradski, Zentrll.f. Bakt., Abt. n, vol. 9, p. 43, 1902. 

16. M.W. Berjennok, Verhandl. d. Korn. Akad. van WetenscJiap, Amsterdam, 
Series n, vol. 3, p. 163, 1893. 

16. R. Grassberger and A. S. Schattenfroh, Archivf. Hygiene, vol. 60, p. 40, 

1907. 

17. Ch. Weizmann, B. Pat., No. 4846, 1915. 

18. P. W. Wilson, W. H. Peterson and E. B. Fred, J. Bid. Chem., vol. 74, 

p. 496, 1927. 

19. J. H. Hall and H. B Randall, J. Infect. Dw., vol. 31, p. 326, 1922. 

20. A. P. H. Desborough, J. Reilly, A. C Thaysen and F. R. Henley, B. Pat., 

No. 128403, 1919. 



ANAEROBIC DEHYDROGENATION OF HEXOSES 161 

21. A. J. Kluyver and H. J. L. Donker, Zeitach. d. ZeUe und Qewebe, vol. 13, 

p. 134, 1926. 

22. H. Raistriok and A. B. Clark, Btochem. J., vol. 13, p. 329, 1919. 

23. C. Neuberg and E. Remfurth, Btochem. Zeits., vol. 142, p. 653, 1923. 

24. A. J. Kluyver, H. J. L. Donker and Visser't Hooft, Biochem. Zeita., 

vol. 161, p. 361, 1926. 

25. A. S. Sohattenfroh and R. Grassberger, Arch. Hygiene, vol. 37, p. 54, 

1900. 

26. F. Hlippe, Mitt. Kais. Gesund., vol. 2, p. 309, 1884. 

27. K. B. Lehmann and R. 0. Neumann, Bakteriologiache Diagnoattk, 5th. 

edition, Lehmann, Munohen, 1912. 

28. C. Dupont, Comptea rend., vol. 134, p. 1449, 1902. 

29. V. Omelianaki, Oomptea rend., vol. 121, p. 653, 1895. 

30. A. C. Thaysen and H. J. Bunker, Microbiology of Cellulose, Hemi- 

ceUuloses, Pectin and Gums, Oxford University Press, 1927. 

31. F. Boutron and E. Fre'my, Ann. de Chim. et Phys. (3), vol. 2, p. 267, 

1841. 

32. L. Pasteur, Com/pies rend., vol. 46, p. 930, 1867. 

33. C. Stricht, Jahrsber.f. Chem., vol. 21, p. 622, 1868. 

34. A. Fitz, Berickte, vol. 11, p. 42, 1878. 

35. H. Bredemann, ZentrU.f. Bakt., Abt. II, vol. 23, p. 385, 1909. 

36. Lefrano and Co., B. Pat., No. 186572, 1923. 

37. I. A. Legg, U.S. Pat., No. 1582408, 1926. 

38. H. Chick, J. Hygiene, vol. 10, p. 237, 1910. 

39. A. Fernbaoh and E. H. Strange, B. Pat., No. 16204, 1911, and B. Pat., 

No. 21073, 1912. 

40. L. Perdrix, Ann. Inst. Past., vol. 5, p. 287, 1891. 

41. 0. Emmerling, Berichte, vol. 37, p. 3535, 1904; vol. 38, p. 653, 1906. 

42. H. Pringsheim, Zentrbl. f. Bakt., Abt. II, vol. 16, p. 300, 1905 ; Berichte, 

vol. 38, p. 486, 1905. 

43. F. Ehrlich, Berichte, vol. 39, p. 4072, 1906; vol. 40, p. 1027, 1927. Jahrb. 

d. Versuch u. Lehranstaltf. Brauerei in Berlin, vol. 10, p. 516, 1907. 

44. E. B. Fred, W. H. Peterson and M. Mulvania, J. Bact., vol. 11, p. 333, 

1926. 
46. H. B. Spoakman and J. F. Phillips, J. Bact., vol. 9, p. 283, 1924. 

46. D. A. Legg, B. Pat., No. 278307, 1927. 

47. F. Boinot, U.S. Pat., Ser. B, No. 1565643, 1926. 

48. W. Corner, ZentrU.f. Bakt., Abt. H, vol. 66, p. 156, 1926. 

49. R. Bum, Das TuscheverfaJiren, Gustav Fischer, Jena, 1910. 

50. G. B. Gnllone, Annalen, vol. 166, p. 127, 1873. 

51. S. Botkin, Zeits. f. Hygiene, vol. 11, p. 423, 1892. 
62. R. Locquin, Chem Zeits., vol. 26, p. 966, 1902. 

53. E. Wagner, K. F. Meyer and C. C. Dozier, J. Bact., vol. 10, p. 321, 1925. 



M 



CHAPTER XIII 
THE FERMENTATION OF PENTOSES 

WHEN discussing the principles underlying the exposition 
given in these pages of the microbiological dehydrogenation 
of monoses it was mentioned that the fermentation of pen- 
toses would be dealt with in a separate chapter, as a separate 
subject. The decision to do so was prompted by lack of 
information on the reactions taking place, and not by any 
established dissimilarity between the methods by which 
micro-organisms utilize pentoses and hexoses. On the con- 
trary, most of the available data point to a striking similarity 
between the reactions by which pentoses and hexoses are 
decomposed by micro-organisms. To mention but a few 
examples, Bertrand 1 found that Bact. xylinum, or rather the 
closely related type the sorbose bacterium, converts xylose 
to xylinic acid, a reaction which evidently is analogous to the 
conversion of glucose by the same organism to gluconic acid. 
In his investigations on Bact. pneumoniae, Grimbert 2 observed 
that xylose was fermented with the production of ethyl 
alcohol, acetic acid, lactic acid and succinic acid, the substances 
already ascertained to be fermentation products of glucose. 
Among the more recent observations might be mentioned 
those of Peterson and Fred 8 on certain pentose fermenting 
bacteria, isolated from silage, which showed that acetalde- 
hyde was produced as an intermediate fermentation product 
from pentoses and from hexoses; and those of Northrop 
and his collaborators 4 on the action of Bac. acetoethyliciis on 
glucose and xylose . Here again the final fermentation products 
were identical, though formed in slightly varying propor- 
tions. In the case of Bac. acetonigemts, Peterson, Fred and 
Schmidt 6 came to the conclusion that the xylose fermentation 
products were identical with those of glucose, though varying 
slightly in proportion. And finally in their study of the action 
of Fusariwn lini, the wilt of flax,White and Willaman 8 observed 
the production of ethyl alcohol in quantities over and above 



THE JTERMENTATION OF PENTOSES 163 

those which could have been produced had the pentose 
molecule been broken down into one three-carbon molecule 
and one two-carbon molecule. 

There are reasons, therefore, for expecting that as experi- 
mental evidence accumulates, it will be possible to co-ordinate 
the intermediate reactions of pentose fermentations with 
those already discernible for hexoses and for anticipating 
that the formation of molecules containing three carbon 
chains must be an important stage in the breakdown of 
pentoses. 

As regards the actual stages in the fermentation, it is to be 
assumed that the pentose molecule undergoes esterification 
with phosphoric acid prior to fermentation. On this subject, 
however, there does not at present appear to exist any in- 
formation. In support of the view that intermediate products 
of molecules with three carbon chains are formed, there is 
important circumstantial evidence to be found in the fact 
that, as already mentioned, the final fermentation products 
of pentoses are frequently identical with those of hexoses, and 
that they are produced in almost equal proportions from 
both. This quantitative similarity precludes the possibility 
that the pentose molecule is converted during fermentation 
into two intermediate molecules, one containing a chain of 
three carbons and the other a chain of two carbons. As in 
the case of the hexoses the preliminary formation of but one 
intermediate with a molecule possessing chains of three 
carbon atoms appears to be the only conception fitting the 
facts so far established. This being the case it becomes 
necessary to assume that the pentose molecule undergoes 
condensation to a polysacoharide prior to fermentation, a 
polysaccharide containing a multiple of six carbon groups. 
Support for this interpretation of the early stages of pentose 
fermentation may be found in the synthetic activities of 
micro-organisms, to which reference will be made in Chap- 
ter XIV. 

The property of fermenting pentoses is probably met with 
among micro-organisms to a far greater extent than was at 

M2 



164 THE PERMENTATION OE PENTOSES 

one time anticipated, but the question has not yet received 
the attention it deserves. At a comparatively early date it 
was established by Stone and Tollens 7 that yeasts are unable 
to ferment pentoses with the production of ethyl alcohol. 
That these organisms, nevertheless, can utilize pentoses as 
a source of energy was shown by Bokorny, 8 who obtained 
reasonably good growth of yeast in culture media containing 
xylose or arabinose as the sole source of carbohydrate. Ac- 
cording to Abbott 9 traces of carbon dioxide, alcohol, and 
non- volatile acids are formed by yeast from pentoses. Many 
other lower and higher fungi are no doubt capable of fer- 
menting pentoses, but experimental evidence is meagre in the 
extreme. Czapek 10 tested Aspergillus niger for its rate of 
development on various carbohydrates and obtained good 
growth of the fungus on xylose and on arabinose, observations 
which Ekman u confirmed. White and Willaman 6 reported 
the production of ethyl alcohol as the result of the action of 
Fuaarium lini on xylose. 

Peterson, Ered and Schmidt 12 extended these observations 
to other types of fungi, notably to Asp&rgillus, Penicillium, 
and Mucor species, and found that on the whole Asp&rgillus 
and Penicillium species fermented pentoses xylose and 
arabinose more readily than Mucor species. The most 
rapidly acting types decomposed the available pentoses 
completely in 4 to 5 days, xylose being the more readily 
decomposed pentose. The fermentation products were 
represented almost solely by carbon dioxide and mycelial 
tissue. No production of alcohol or of volatile acids could be. 
detected and but a very slight trace of non- volatile acids was 
found. 

Nevertheless Amelung, 13 who examined the action of Asper- 
giUus niger on xylose and arabinose, reported the formation 
of citric acid from these carbohydrates by the fungus. 

Whether actmomycetes ferment pentoses or not has not 
yet been definitely ascertained. The frequent occurrence of 
these micro-organisms in manure and compost heaps, where 
active fermentation of pentoses and of pentosans takes place, 



THE FERMENTATION OF PENTOSES 166 

makes it almost certain that many actinomyoetes are capable 
of utilizing pentoBes as a source of carbohydrates. This is 
borne out also by unpublished observations by the writers on 
the development of aotinomycetes in xylose containing food 
solutions. 

The pentose fermenting properties of bacteria have been 
studied in considerably greater detail than those of the other 
groups of micro-organisms referred to above. It is known that 
pentoses can be utilized by bacteria belonging to most of the 
chief groups of hexose fermenting types, and that this property 
is of particular economic importance among the lactic acid 
bacteria (Kayser; 14 Gayon and Dubourg; 16 Fred, Peterson 
and Anderson; 18 Stiles, Peterson and Fred 17 ), and among 
the many insufficiently described types which are responsible 
for the destruction of hemicelluloses in the intestine and for 
the early stages of humus formation in soil and peat bogs 
(Rege 18 ). 

In the groups of Bact. typhosum Bact. paratyphosum, a 
destruction of pentoses has been recommended by Stern 19 as 
a means for differentiating closely related species. Stern 
found that xylose, but not arabinose, was fermented by Bact. 
typhosum, while Bact. paratyphosum A attacked arabinose 
and Bact. paratyphosum B both xylose and arabinose. The 
investigations of Peterson and Fred 20 have shown that pen- 
tose fermenting lactic acid bacteria actively participate in the 
silage fermentation, and the microscopic investigations of 
Henneberg 21 on the destruction of plant tissues by the in- 
testinal flora of man and of herbivorous animals have demon- 
strated that bacteria are partly responsible for the elimination 
of the pentosans of plant tissues entering the intestinal tract. 
The importance of pentoses and their corresponding poly- 
saccharides in the naturally occurring denitrification processes 
has been studied by Stoklasa 22 and by Rege, 18 who suggest 
that pentoses take part in the production of natural humus, 
a view which has been confirmed recently by Thaysen and 

Bakes. 23 

The important manure heap inhabitant Bac. mesentericus 



166 THE FERMENTATION OF PENTOSES 

ruber is reported by Ered, Peterson and Carroll 24 to be capable 

of fermenting xylose -with production of ethyl alcohol and 

acetone. 

A particularly interesting field of economic application of 
pentose-fermenting bacteria is that opened by Northrop and 
collaborators 4 with their investigations on a bacterium 
isolated by them and described under the name of Bac, 
acetoethylicus. 

The fermenting of glucose by this organism was studied in 
detail by Donker. 25 The results of this analysis were quoted 
in Table IE on page 86. According to Northrop and col- 
laborators, the yields of acetone and alcohol obtained from 
pentoses are somewhat lower than those recorded by Donker 
from glucose, but too much importance should not be attached 
to these differences, since the fermentation of carbohydrates 
by Bac. acetoethylicus is dependent to an unusual extent on 
the cultural conditions prevailing. In Northrop, Ashe and 
Senior's 4 experiments the following yields of ethyl alcohol 
and acetone were obtained from the carbohydrates men- 
tioned. 

TABLE VI 



Carbohydrate 
fermented. 


Yield expressed as percentage of 
carbohydrate fermented. 


Ethyl alcohol. 


acetone. 


Glucose 
Fructose 
Maltose 
Starch 
Xylose 
d-Arabinose 


11-23 
24-26 
23-24 
20-24 
18-20 
12-16 


9-10 
8-10 
6-7 
8-10 
4-5 
6-7 



It is not surprising that these very considerable yields of 
acetone and ethyl alcohol should have drawn the attention 
of investigators, including Northrop, to the possibility of 
utilizing the action of Bac. acetoethylicus on pentoses for the 
industrial production of power alcohol. Numerous analyses 



THE FERMENTATION OF PENTOSES 167 

have shown that pentoses, notably xylose, occur in the form 
of polysaccharides in various vegetable waste products in 
cacao shells (Churchman 26 ), in oat husks (Fred, Peterson and 
Anderson 27 ), in Nile sudd Cyp&rua papyrus (Joseph and 
Martin 28 ) and in many other similar materials, of which an 
extensive list was given by Thaysen and Galloway. 29 With a 
pentose content of 20 per cent, in such materials, a figure 
which is often reached and sometimes surpassed, Bac. acetO" 
etJiylicua should be capable of producing as much as 90 litres 
of power alcohol per 1,000 kg. of waste, a yield which would 
be of the order of that obtained from potatoes, utilized 
extensively in some countries for the production of power 
alcohol. 

The first attempts at developing a process for power alcohol 
production in which waste vegetable matter was fermented 
by Bac. acetoetJvylicus were recorded by Northrop, Ashe and 
Morgan ; 30 Peterson, Ered and Verhulst ; 31 Fred, Peterson and 
Anderson ; 27 and Juritz, 82 but none of the methods evolved 
extended beyond a laboratory scale. Subsequently Thaysen 
and Galloway 29 undertook a renewed investigation of the 
problem, and extended their work to include production in 
a small technical plant. 

One of their greatest difficulties was to devise a method by 
which the pentosans, present in the vegetable debris, could 
be converted into pentoses, a necessary step in the procedure, 
since Bac. acetoethylicus is unable to hydrolyse pentosans. 
In the investigations by previous workers this hydrolysis had 
been carried out by subjecting the vegetable waste to tem- 
peratures above 100 C. in the presence of sulphuric acid. 
But this procedure could not be adopted for work on a 
technical scale owing to the corrosive action of dilute mineral 
acids on the steel required for the construction of containers 
capable of withstanding the high temperatures required. 
Thaysen and Galloway overcame this difficulty by reducing 
the temperature required to 100 C. or below, and by in- 
creasing at the same time the hydrogen ion concentration of 
the liquid in which the waste was suspended. As the rate of 



168 THE I T ERMENTATION OF PBNTOSES 

hydrolysis was found to be directly proportional to the pre- 
vailing hydrogen ion concentration at a given temperature, 
they increased the concentration of acid used to such an 
extent that complete hydrolysis of the pentosans could be 
ensured at a temperature of 100 C. At the same time, and 
in order to avoid an increased consumption of acid, they 
decreased the bulk of the solution taken for the suspension 
of the waste during hydrolysis. 

The pentoses resulting from the hydrolysis were extracted 
under aseptic conditions with water sterilized by boiling in 
the presence of a sufficient concentration of hydroxyl ions to 
ensure complete destruction of all spores and vegetative 
cells at 100 C. By choosing an alkaline reaction for the 
mash during sterilization they introduced the simplification 
of extracting the pentoses present in the hydrolysed mash and 
of neutralizing the acid remaining in the waste in one opera- 
tion. The resulting pentose extract was cooled under aseptic 
conditions and carried with similar precautions to a suitable 
container into which a culture of Bac. acetoethylicus had 
previously been introduced. The ensuing fermentation was 
allowed to proceed at a temperature of 40 to 41 C. 

The control of the hydrogen ion concentration of the fer- 
menting mash was found to be a factor of great importance 
for the smooth conversion of the pentoses into ethyl alcohol 
and acetone. Previous investigators had recommended con- 
ducting this conversion at a reaction corresponding to a pH 
value of 6-4, a figure which may be established by addition 
of an excess of calcium carbonate. 

Confirming the observations of previous writers that the 
development of Bac. acetoethylicus proceeds faster at a some- 
what lower hydrogen ion concentration, Thaysen and Galloway 
recommended the periodic addition of a sterile calcium 
hydroxide suspension to the fermenting extract. The pH 
of the mash was thereby raised at intervals of four hours 
from the value of 6-4 to one of 7-8. By so doing they 
obtained a more rapid completion of the fermentation, with- 
out a diminution in the yield of acetone which, as Northrop, 



THE FERMENTATION OF PENTOSES 169 

Ashe and Senior had shown is greatest other conditions 
being equal in mashes where the reaction has been main- 
tained on the acid side of the neutral point. 

The yields of acetone alcohol mixture obtained by Thaysen 
and Galloway from various waste materials on a semi-techni- 
cal scale were of the order expected and indicated a complete 
conversion of the pentoses. A yield of as much as 90 litres 
per 1,000 kg. of vegetable waste was frequently recorded. 
Their process is recommended by them for the technical 
preparation of power alcohol in localities where waste vege- 
table material is available in abundance and where the price 
of other liquid fuels for internal combustion engines is suffi- 
ciently high to justify the production of power alcohol. 

Though undoubtedly useful for this purpose, Thaysen and 
Galloway's process might be improved in one direction, by 
eliminating the preliminary acid hydrolysis introduced for 
the conversion of the pentosans of the waste into soluble 
pentoses. The elimination of the acid hydrolysis, however, 
can be secured only by the use of bacteria capable of hydro- 
lysing pentosans prior to the fermentation of pentoses, and 
such types have not yet been isolated. They are probably 
found among the microflora of decomposing vegetable sub- 
stances, but an exhaustive study of this microflora has not 
yet been undertaken. 

Apart from the production of power alcohol, the use of 
pentose fermenting bacteria has been suggested (Fred, 
Peterson and Anderson 27 ) for the technical production of 
volatile acids and of lactic acid. These suggestions have not 
been further developed, and are not likely to become of 
industrial importance unless the requirements of these acids, 
notably of lactic acid, should increase to such an extent that 
the available supplies of hexose residues become inadequate 
to satisfy the demands. 

LITERATURE 

1. G. Bertrand, Comptea rend., vol. 127, p. 124, 1898. 

2. L. Grimbert, Comptea rend. Soc. biol., vol. 48, p. 191, 1896. 

3. W. H. Peterson and E. B. Fred, J. Btol. Chem , vol. 44, p. 29, 1920. 



170 THE FERMENTATION OF PENTOSES 

4. J. H. Northrop, L. H. Ashe and J. K. Senior, J. Bid. Chem., vol. 39, 

p. 1, 1919, 
6. W. H. Peterson, E. B. Fred and E. G. Schmidt, J. Biol. Ohem., vol. 60, 

p. 628, 1924. 

6. M. G. White and J. J. Willaman, Biochem. J., vol. 22, p. 683, 1928. 

7. W. E. Stone and B. Tollens, Annalen d. Ohem. und Pharm., vol. 249, 

p. 267, 1888. 
W. E. Stone, Berichte, vol. 23, p. 3791, 1890. 

8. Th. Bokorny, Zentrbl.f. Bakt., Abt. IE, vol. 47, p. 191, 1917. 

9. 0. D. Abbott, Missouri Agric. Exp. Stat. Research BuU., No. 86, 1926. 

10. F. Czapek, Hofmeister's BettrOge, vol. 3, p. 62, 1902. 

11. G. Elmrtn-Ti, abst. F. Ozapek's Biochemie der Planzen, vol. 1, p. 312, 1913. 

Gustav Fischer, Jena. 

12. W. H. Peterson, E. B. Fred and E. G. Schmidt, J. Biol. Chem., vol. 64, 

p. 19, 1922. 

13. H. Amelung, Zeits. physiol. Ghem., vol. 166, p. 161, 1927. 

14. E. Eayser, Ann. Inat. Pasteur, vol. 8, p. 736, 1894. 

16. U. Gayon and E. Dubourg, Ann. Inst. Pasteur, vol. 16, p. 627, 1901. 

16. E. B. Fred, W. H. Peterson and J. A. Anderson, J. Biol. Ohem., vol. 48, 

p. 386, 1921. 

17. H. B. Stiles, W. H. Peterson and E. B. Fred, J. Biol. Chem., vol. 64, 

p. 643, 1926. 

18. R. D. Rege, Ann. App. Biol., vol. 14, p. 1, 1927. 

19. W. Stern, Zentrblf. Bakt., Abt. I, Ong., vol. 82, p. 49, 1918. 

20. W. H. Peterson and E. B. Fred, J. Biol. Chem., vol. 41, p. 181, 1920. 

21. W. Henneberg, Zentrllf. Bakt., Abt. IE, vol. 66, p. 242, 1922. 

22. J. Stoklasa, Z. f. das landvnrtsch. Versuchswesen in Oesterreich, vol. 1, 

p. 261, 1898. 

23. A. C. Thaysen and W. E. Bakes, Biochem J., vol. 21, p. 896, 1927. 

24. E. B. Fred, W. H. Peterson and W. R. Carroll, J. Bact., vol. 10, p. 97, 

1926. 

26. H. J. L. Donker, Bijdrage tot de Kennis der Boterzuur-Butyl alcohol en 
Acetonegistingen, publ. W. D. Meinema, Delft, 1926. 

26. A. Chnrohman, J. Soc. Chem. Ind., vol. 44, p. 450 T, 1926. 

27. E. B. Fred, W. H. Peterson and J. A. Anderson, J. Ind. Eng. Chem., 

vol. 16, p. 120, 1923. 

28. A. F. Joseph and F. J. Martin, J. Soc. Chem. 2nd., vol. 69, p. 91 T, 

1920. 

29. A. C. Thaysen and L. D. Galloway, Ann. App. Biol, vol. 15, p. 392, 

1928. 

30. J. H. Northrop, L. H. Ashe and R. R. Morgan, J. Ind. Chem., vol. 11, 

p. 723, 1919. 

31. W. H. Peterson, E. B. Fred and J. H. Verhulst, J. Ind. Eng. CJum., 

vol. 13, p. 767, 1921. 

32. Ch. F. Juritz, Soitih African Journal of Industry, vol. 4, p. 906, 1921. 



PART THREE 



CHAPTEB XIV 
THE SYNTHETIC ACTIVITIES OF MICRO-ORGANISMS 

THE review given in the preceding pages of the decomposition 
of carbohydrates by micro-organisms would be incomplete 
without a reference to certain syntheses which have been 
observed to occur during the early stages of fermentation 
and which appear to be intimately connected with them. 
The subject was touched upon in the preceding chapter when 
discussing the fermentation of pentoses, but must be given 
closer attention than was there possible. 

As the study of the subject has emerged from observations 
on the microbiological synthesis of glycogen, starch, fat, and 
hemicelluloses, and is still in an early stage of development, 
it is most conveniently discussed on the basis of a knowledge 
of these observations. 

In 1899 Cremer 1 found that a yeast press juice when allowed 
to stand for some time, would lose its glycogen content and 
that glycogen could be reproduced by the juice when sufficient 
sugar, notably fructose, was added to it. Henneberg 2 con- 
firmed Cremer's observations in the case of living yeast, and 
reported that the easiest method to secure storage of glyoogen 
in yeast was to place yeast cells in a 20 per cent, saccharose 
solution. 

During the same period Macfadyen, Morris and Rowland 3 
recorded that the amount of carbon dioxide given off by a 
yeast juice from a sugar is smaller than that to be expected 
from the amount of carbohydrate which has disappeared. 
Harden and Young* confirmed this and found that the excess 
of sugar consumed by a yeast juice over and above that 
represented by carbon dioxide and alcohol amounted on an 
average to 38 per cent. They showed later 5 that this excess of 
sugar had been converted into a polysaccharide which they 
regarded as a condensation product, intermediate between 



174 THE SYNTHETIC ACTIVITIES OF MICEO-ORGANISMS 
glyoogen and hexose, possessing a higher dextrorotation than 
the latter, or even than that of hexosephosphorio esters, and 
inconvertible into glucose by hydrolysis. 

Buchner and Meisenheimer 6 repeated Harden and Young's 
work, using a German bottom yeast juice, and confirmed 
their result, though the excess amount of sugar consumed 
by the juice in this case was considerably smaller than that 
observed by Harden and Young. 

The investigations referred to had established two im- 
portant facts.' that during yeast fermentations part of the 
available sugar is condensed to a polysacoharide, related to 
or identical with glyoogen, and that this polysaocharide can 
be hydrolysed and utilized by yeast when no other carbo- 
hydrate is available, giving rise to the same fermentation 
products as the sugar from which it was formed. 

At various times papers have appeared showing that 
similar condensation products of monoses, giving a reddish 
brown coloration with iodine, are formed in the cells of many 
other micro-organisms at certain stages of their evolution. 
Most investigators, notably Errera, 7 Kayser and Boulanger, 8 
Heinze, 9 Wehmer 10 and Zikes 11 regard these condensation 
products as glycogen. Meyer 12 refers to them as carbo- 
hydrates closely related to glycogen and amylodextrin. The 
conditions favouring their production have not been studied 
in any appreciable detail. The observation of Kayser and 
Boulanger that the absence of air favours their accumulation, 
and that the presence of an acid has the opposite effect, con- 
forms with the generally expressed view. The same may be 
claimed for Zikes' 11 statement that a rise in the temperature 
of incubation increases their accumulation. 

In the case of the synthesis of polysaccharides by other 
micro-organisms, the effects of oxygen, hydrogen ion con- 
centration of the medium, and temperature may be different. 
Observations made on these points will be referred to in the 
following pages. 

The appearance in the cells of micro-organisms of starch 
or substances related to starch, staining purple to blue with 



THE SYNTHETIC ACTIVITIES OF MICEO-OBGAOTSMS 175 
iodine, has been reported by Maupas 18 in the case of infusoria ; 
by Bourquelot 14 in the case of Boletus pachypus ; by Cramer 15 
in the spores of Penicillium glcwcum ; by Alsberg and Black 16 
in the case of Penicittium puberulum ; by Dox and Neidig 17 
for Penicillium expansum ; and notably by Boas 18 for Asper- 
gillus niger and by Grey 19 for Bact. coli commune. 

The association of these substances with the presence in 
the culture medium of various carbon compounds, notably 
of monoses, was definitely established by Boas. 20 He found 
that not only glucose, fructose and mannose, but galactose, 
arabinose, glycerol, mannitol and certain organic acids, such 
as citric acid, could be converted into starch-like substances 
by Aspergillus niger. 

The significance of this fact appears to have been over- 
looked by Boas, conceivably because of his having reached 
the conviction, 21 previously expressed by Lappaleinen, 22 
that the accumulation was due to a disintegration of sub- 
stances of the cell wall of the active fungus, and not to a 
synthesis activated by an enzyme. Why Boas should have 
chosen the former interpretation in preference to the latter is 
not easy to understand, seeing that the assumption of a 
disintegration of the cell wall to starch-like substances must 
logically demand the conception of a prior synthesis by the 
organism of even higher condensation products than starch. 
The fact remains, however, that Boas has shown that in the 
presence not only of glucose and of other hexoses, but also 
of the pentose arabinose, Aspergittus niger produces starch- 
like substances. 

The conditions favouring the production of these sub- 
stances were also investigated by Boas. 23 He recommended 
the use of a culture medium containing from 6 to 10 per cent, 
of carbohydrate, 1 to 6 per cent, ammonium sulphate and 
0-2 per cent, of magnesium sulphate and potassium dihydro- 
genphosphate, and possessing a hydrogen ion concentration 
equivalent to a pH value of between 1-67 and 2-75, varying 
according to the carbohydrate selected. When cultivated in 
this medium, between the temperatures of 33 and 37 C,, 



176 THE SYNTHETIC ACTIVITIES OF MICRO-ORGANISMS 
the developing mycelium showed a positive starch reaction 
after 20 hours incubation. 

Grey, 19 on the contrary, definitely associated the production 
of starch by Bact. coli commune with the fermentative activity 
of the organism. Working with suspensions of Bact. coli 
commune grown in a glucose medium containing an excess of 
calcium carbonate, Grey reported finding that the solid 
bacterial debris which settled at the bottom of the container 
gave a blue coloration, a positive starch reaction, with iodine. 
The polysaccharide was shown to be present in the cells in 
greatest abundance after the 16th hour, but before the 40th 
hour of fermentation. The total amount isolated was equal 
to only 9 per cent, of the available glucose, but this quantity 
evidently represented the difference between starch formed 
and starch consumed. Grey deduces from his experimental 
evidence that at some stage of the fermentation of glucose 
by Bact. coli commune there is an almost complete trans- 
formation of glucose into non-reducing carbohydrates, a great 
part of which is starch. 

Beyond the addition of an excess of calcium carbonate, 
Grey does not appear to have taken specific precautions to 
ensure starch accumulation. The presence of this calcium 
carbonate shows, however, that starch synthesis would 
proceed at a less acid reaction with Bact. coli commune than 
where Aspergillus niger is used. Discussing the synthetic 
side of bacterial metabolism, Grey suggests that a very large 
proportion of the various substances which are decomposed 
by bacterial fermentation are first synthesized into more 
complex compounds within the bacterial protoplasm and are 
decomposed only subsequently into those end products which 
are finally observed. Not only his observations, but the de- 
ductions made from them by Grey are rather sweeping and 
a confirmatory investigation of the synthetic activities of 
Bact. coli commune is undoubtedly required. 

Only one paper dealing with the storage of fats has a direct 
bearing on the question of microbiological synthesis as dis- 
cussed in this chapter. Before discussing this paper by Smed- 



THE SYNTHETIC ACTIVITIES OF MICEO-OBGANISMS 177 
ley McLean and Hoffert 24 it mil be desirable to review the 
existing theories on the production of fats. 

It is generally held that fats are built up by condensation 
3f simple compounds such as acetaldehyde (Leathes, see 
Person and Raper 26 ), pyravic acid and acetaldehyde (Smedley 
md Lubrzyriska 26 ), pyravic acid (Neuberg and Aiinstein 27 ), 
Dr ethyl alcohol (Lindner 28 ). These theories have obtained a 
neasure of experimental support from Haehn and Kinttof 's 29 
)bservations on fat production from acetaldehyde, alcohol 
md pyruvic acid by Endomyces vematis. According to these 
various theories, sugars, when used as the starting-point for 
at production, are broken down in the first instance to 
bcetaldehyde, and this fermentation product is then con- 
lensed to fatty acids, which, after esterification with glycerol, 
deld fat. 

Smedley McLean and Hoffert do not deny that acetalde- 

tyde, ethyl alcohol, or pyruvic acid, can be utilized by micro- 

rganisms for the storage of fat. On the contrary they find 

hat a considerable accumulation of fat occurs in yeast in 

he presence of ethyl alcohol and an excess of oxygen. But 

hey are unable to accept these substances, and notably 

cetaldehyde, as the components from which fats are normally 

pnthesized hi yeast. In their experiments they observed that 

it synthesis from ethyl alcohol was adversely affected by 

le addition of sodium sulphite, a salt employed in biological 

jaotions for the fixation of aoetaldehyde. They concluded 

om this that acetaldehyde is an essential stage in the con- 

ersion of ethyl alcohol into fat. In those experiments in 

hich Smedley McLean and Hoffert used glucose or fructose 

3 raw material instead of ethyl alcohol, the addition of 

)dium sulphite did not diminish fat production, indicating 

lat in these cases no acetaldehyde was formed. These appa- 

mtly contradictory observations are explained by Smedley 

cLean and Hoffert on the assumption that, where ethyl 

cohol forms the raw material for fat production, it is con- 

srted into acetaldehyde and the latter synthesized into 

ucose. The glucose is then further condensed to a longer 

N 



178 THE SYNTHETIC ACTIVITIES OF MICRO- ORGANISMS 
chain structure such as required for the higher fatty acids of 
which yeast fat is composed. In the experiments in which 
glucose or fructose served as raw material, this condensation 
of the hexose is assumed to proceed direct, without a pre- 
liminary disintegration of the molecule. 

There is a certain similarity between this assumed direct 
condensation of monoses to fatty acids and the formation of 
hemioelluloses by micro-organisms, a process of synthesis 
which perhaps has been more extensively investigated than 
any other, and which in practically every case has been shown 
to be dependent on the presence of a carbohydrate. 

It may not have been experimentally proved that the 
synthesis of hemioelluloses by micro-organisms is the outcome 
of a direct condensation of monoses, but there is indirect 
evidence of this. Thus it is almost invariably observed that 
a hemicellulose which is formed as the result of microbiological 
activity is the polysaccharide corresponding to that monose 
present in the culture of the micro-organism from which it 
can be built up, and to which it can be reconverted by hydro- 
lysis with dilute acids. There appears to be no reason why 
this should be the case if the monose, prior to its synthesis, 
were converted into an enolic form or into a substance with 
a shorter carbon chain. 

Another observation supporting the assumption of a 
direct condensation of monoses to hemioelluloses is that 
hemicelluloses are formed during the early stages of the 
development of the active organism before a fermentative 
activity becomes noticeable. When fermentation has com- 
menced the polysacoharides already produced are broken 
down into the same fermentation products as the original 
monose. Had the synthesis proceeded from comparatively 
simple compounds such as aoetaldehyde or pyruvio acid, 
there would be no reason why it should be so intimately 
connected with the early stages of development, a period 
when these compounds have not yet been produced in 
measurable quantities. 
Prom the account given of the synthesis of glycogen, 



THE SYNTHETIC ACTIVITIES OF MICRO-ORGANISMS 179 
starch, starch related substances, fats and hemicelluloses, 
it will have been seen that micro-organisms which ferment 
monoses are frequently able to perform syntheses in which 
the monoses are involved. It is known that in some cases 
this synthesis affects a very large percentage of the available 
monoses, but it has not yet been conclusively proved that 
they are essential stages in the fermentation of a monose as 
suggested by Grey 19 and by Gruss. 80 

Gruss arrived at the conclusion that glucose, on being 
fermented by yeast, is first broken down to CHOH groups, 
these groups synthesized to glycogen by an enzyme of the 
yeast cell, and the glycogen subsequently hydrolysed to glucose 
and converted by enzymes into carbon dioxide and alcohol. 

Though it may be admitted that this is a somewhat ex- 
travagant conception of the reactions taking place in the 
fermentation of a monose by micro-organisms, it cannot be 
denied that cases are known which are difficult to explain 
without the assumption of a preliminary synthesis of a sub- 
stance into a more complicated compound prior to its resolu- 
tion into final fermentation products. One of the most 
striking of these cases, to which reference was made in 
Chapter ITTTTj is the conversion of a pentose into practically 
the same fermentation products quantitatively as well as 
qualitatively as those obtainable from an hexose by the 
same type of micro-organisms. The view that a preliminary 
condensation of a pentose occurs prior to its fermentation has 
undoubtedly been strengthened by the observations of Boas 31 
that starch-like substances are produced from arabinose by 
Aap&rgilkts niger. 

It has already been emphasized that the subject of the 
synthetic activity of micro-organisms has remained practically 
unexplored in the past, that little is known of the conditions 
governing the reactions, and even less of the reactions them- 
selves. Obviously they are largely of theoretical interest, 
except in so far as they affect the question of mucus pro- 
duction by micro-organisms, a subject which will be discussed 
in Chapter XV. 



180 THE SYNTHETIC ACTIVITIES OF MICRO-ORGANISMS 

LITERATURE 

1. M. Cremer, Berichte, vol. 32, p. 2002, 1891. 

2. W. Henneberg, Chem. ZentrbL, vol. 73, H, p. 1515, 1902. 

3. A. Maofadyen, G. H. Morris and S. Rowlans, Berichte, vol. 33, p. 2764, 

1900. 

4. A. Harden and W. J. Young, Berichte, vol. 37, p. 1052, 1904. 
6. A. Harden and W. J. Young, Biochem. J., vol. 7, p. 630, 1913. 

6. E. Buohner and J. Meisenheimer, Berichte, vol. 39, p. 3201, 1906. 

7. L. Errera, L'epvplasme des Ascomycetes et le gfycogene des vegdtaux. 

These, Bruxelles, 1892. 

8. E. Kayser and E. Boulanger, Chem. ZentrbL, vol. 69, H, p. 440, 1898. 

9. B. Heinze, ZentrbL /. Bald., Abt. IE, vol. 12, p. 43, 1904. 

10. C. Wehmer, Berichte d. deut. botan. OeseUech., vol. 31, p. 257, 1913. 

11. H. Zikes, ZentrbL f. Bakt., Abt. II, vol. 49, p. 353, 1917. 

12. A. Meyer, Flora, vol. 86, p. 428, 1899. 

13. E. Maupas, Comptes rend., vol. 102, p. 120, 1886. 

14. E. Bourquelot, J. Pharm. Ohim., voL 24, p. 197, 1891. 
16. E. Cramer, Arch.f. Hygiene, vol. 20, p. 197, 1894. 

16. C. L. Alsberg and 0. F. Black, U.8. Bureau of Plant Indust., Bull. No. 

270, 1913. 

17. A. W. Box and R. E. Neidig, J. Biol. Chem., vol. 18, p. 167, 1914. 

18. F. Boas, Biochem. Zevts., vol. 78, p. 308, 1917. 

19. E. C. Grey, Biochem. J., vol. 18, p. 712, 1924. 

20. F. Boas, Biochem. Zeite., vol. 81, p. 80, 1917. 

21. F. Boas, ZentrbL f. BaJet., Abt. n, vol. 56, p. 7, 1921. 

22. H. Lappaleinen, Benchte fiber die gesammte Physiologie, vol. 7, p. 233, 

1921. 

23. F. Boas, Ber. d. deut. botan. GeseUsch., vol. 37, p. 50, 1919. 

24. J. Smedley McLean and D. Hoffert, Biochem. J., vol. 20, p. 343, 1926. 
26. L. EL. Person and H. S. Raper, Biochem. J., vol. 21, p. 875, 1927. 

26. J. Smedley and E. Lubrzyriska, Biochem. J., vol. 7, p. 364, 1913. 

27. C. Neuberg and B. Arinstein, Biochem. Zeiti., vol. 117, p. 269, 1921. 

28. P. Lindner, Z.f. techn. Biologie, vol. 79, p. 100, 1921. 

29. H. Haehn and W. Kinttof, Chem. Zette und Oeioebe, vol. 12, p. 116, 1925. 

30. J. Gniss, Chem. ZentrbL, vol. 8 (5), p. 1677, 1904. 

31. F. Boas, Biochem. Zeits., vol. 86, p. 110, 1918. 



CHAPTER XV 
THE MUCUS FERMENTATIONS 

AT an early date in the annals of microbiology attention was 
drawn to the formation of cartilaginous bodies in stored beet 
and oane sugar juices, 'la fermentation visqueuse' of Des- 
fosses. 1 

These concretions, described as 'frogs' spawn' by German 
writers, accumulated at the bottom of containers holding 
the juices, and in serious cases converted the whole of the 
juices into a semi-gelatinous substance. Desfosses, and with 
him several other investigators, notably Boudrimont, 2 were 
of the opinion that the mucus had been produced by the 
nitrogen contained in the affected sugar juices. Jubert 8 
regarded the concretions as living plants and showed that 
they could be made to increase in size when placed in saccha- 
rose solutions. Durin 4 recorded similar observations, but 
failed to appreciate their significance, and, like Scheibler, 5 
considered the concretions as decomposition products of the 
cells of the sugar beet. 

The connexion of the concretions with the activity of 
bacteria was first suggested by Pasteur, 6 who attributed their 
formation to the action of a coccus forming short chains. 
Mendes, 7 Cienkowski 8 and van Tieghem 9 confirmed Pasteur's 
observation. They arrived independently at the conclusion 
that the responsible organism was related to Cohn's species, 
Ascococcua Billroihii> Cienkowski terming the new organism 
Ascococcus mesenteroides, van Tieghem Ascococcus Mendesii, 
in honour of Mendes. In many text-books Ascococcus mesente- 
roides is discussed under the name Leuconostoc mesenteroides. 
By Lehmann and Neumann 10 it is described as Strepto- 
coccus mesenteroides. Further reference will be made to this 
organism in Chapters XXI and XXII. 

Subsequently mucus production was observed in other 
carbohydrate containing liquids and was frequently found 



182 THE MUCUS FEBMENTATIONS 

to cause serious damage to industrial products and articles 
of food. In infusions of Digitalis purpurea, containing saccha- 
rose, and in other sweetened medicinal preparations, it was 
studied by Bienz, 11 Brautigam 12 and Happ, 13 and was claimed 
by Brautigam to be due, in the case examined by him, to the 
resolution of the cell wall of a coccus, found in large numbers 
in the infusions. 

In 1883 Schmidt 14 recorded a case of mucus production in 
milk. He was able to reproduce it in normal milk with a 
coccus isolated from the 'ropy' milk 

At about the same time Laurent 15 discovered mucus forma* 
tion in bread, where it had been produced by a bacterium 
termed by him Bac. panificans. His investigations were later 
confirmed and extended, notably by Vogel, 16 who found the 
micro-organisms of ropy bread to be closely related to Bac. 
mesentericus. The subject of ropy bread will be dealt with in 
greater detail in Chapter XX. 

Pasteur had described mucus production in beer and wine, 
van Laer 17 confirmed his observations, and described a rod 
Bac. viscosus, which he claimed to be responsible for the 
ropiness of beer. 

These various observations made it evident, not only that 
mucus can be produced by different types of bacteria belong- 
ing to widely separated species, but that the appearance of 
the mucus is associated with the presence of carbohydrates, 
not necessarily of saccharose, as maintained by Jubert and 
by B6champs, 18 but of lactose, glucose (Schmidt 14 ), and 
galactose (Kramer; 19 and Einmerling 20 ). Even the alcohol 
mannitol was found by Schmidt to be a suitable raw material 
for mucus production. 

More recently the mucus produced by Azotobacter croococcum 
has been shown by Stapp 21 to be a condensation product of a 
dextro-rotatory monose, and that of Bact. radicicola has been 
shown by Greig Smith 22 to be of a similar nature. 

The chemical composition of the mucus from beet juices 
was investigated in considerable detail by Scheibler, who 
describes it as a colourless substance when pure, giving a 



THE MUCUS FERMENTATIONS 153 

quantitative yield of glucose on hydrolysis. Oxidized with 
nitrio acid it yielded oxalic acid. For this reason Scheibler 
regarded it as an anhydride of glucose, to which he gave the 
name dextran. Durin, on the other hand, spoke of the mucus 
as a cellulose, while B6champs regarded it as a type of starch. 
Scheibler found that the gelatinous bodies taken direct from 
sugar beet juices were usually stained greyish black and 
contained mannitol, but repeated boiling in a solution of 
calcium hydroxide or in other alkaline solutions, in all of 
which they were found to be soluble removed the impurities 
and left a colourless mucus capable of imbibing as much as 
85 to 88 per cent, of water, in spite of its being insoluble in 
this medium. 

The origin of the mannitol found in the raw mucus was not 
discussed by Scheibler, but was subsequently associated by 
Pasteur with the activity of the mucus producing bacteria. 
Pasteur's suggestion was verified experimentally by various 
investigators. 

The discovery by Durin of appreciable quantities of fructose 
in the saccharose solutions in which mucus had been formed 
showed that its production was associated with a preliminary 
hydrolysis of the saccharose molecule. Other important 
features of the mucus formation were discussed by Burin, 
notably the favourable influence of the presence of calcium 
carbonate. That reactions close to the neutral point, and 
especially slightly on its alkaline side, are favourable, has 
been emphasized by all subsequent investigators dealing 
with this subject. Where marked acid reactions prevail 
mucus production rarely occurs. The reason for this has not 
yet been established, but can hardly be attributed, as sug- 
gested by Greig Smith, 23 to the hydrolytic action of the 
hydrogen ions present under such conditions. 

The glucose derivative investigated by Scheibler is not the 
only condensation product which bacteria synthesize from 
saccharose. It was found by Lippmann 24 that mucus obtained 
by him from saccharose yielded fructose on hydrolysis with 
dilute acids. His observations were confirmed by Greig 



184 THE MUCUS FERMENTATIONS 

Smith and by Fernbach and Sohoen, 26 the first-named terming 

the substance 'levan'. 

In these cases glucose accumulated in the saccharose 
solutions in proportions showing that fructose was the sole 
source of mucus. Similarly the mucus produced in milk could 
be shown to be a condensation product of galactose, thus 
emphasizing the conclusion usually arrived at that mioro- 
biologioally formed mucus is a condensation product of the 
one monose from which a particular organism is capable of 
synthesizing it, and that it is convertible into this monose, 
and this monose only, through simple hydrolysis with dilute 
mineral acids. The only observations which cannot be har- 
monized with this view are those of Schardinger, 26 who records 
that the galaotan investigated by him could be obtained 
from lactose, saccharose and glucose. A reinvestigation of 
his observations would be most desirable in view of the con- 
sensus of opinion outlined above on the mode of formation 
of other types of mucus. 

It will have been seen that most of the earlier publications 
on the subject associated mucus production with the activity 
of bacteria, notably with types which to-day can be recog- 
nized as belonging to the mannitol producing lactic acid 
bacteria. In time a large number of others have been added 
to the earlier types, Bact. lactis a&rogenes, for instance, by 
Emmerling, 20 an ethyl alcohol producing spore-forming 
organism, Clostridi'um gelatinoaum, by Laxa, 27 and more 
recently Bac. acetoettvylicus by Northrop and collaborators. 28 
Among the obligatory anaerobes the writers have frequently 
observed mucus formation in young cultures of Bac. acetoni- 
genus, particularly when these cultures had been prepared 
from spores kept for a prolonged period on dried sand. 

Another species spoken of as a mucus producer is Azoto- 
bacter oroococcum, one of the most important nitrogen fixing 
micro-organisms of the soil. Stapp 21 concluded from his 
investigation that the mucus produced by this organism is 
of a carbohydrate nature, yielding a dextro-rotatory monose 
on hydrolysis with dilute acids. 



V 
Jf 



THE MUCUS FERMENTATIONS 185 

Among the Phycobacteriaceae mucus production is respon- 
sible for the abnormal forms of Zoogloea ramigera produced 
by this organism, according to Zopf. 29 

In his general survey of mucus production by micro- 
organisms Beijerinok 80 states that the actinomycetes, the 
yeasts and the fungi are unable to produce this substance. 
His statement undoubtedly requires revision, at least as 
regards the yeasts and the fungi. As early as 1879 Binz u 
referred to a fungus capable of producing mucus. Meissner's 81 
mucus-producing yeasts or torulae belong here, and the same 
no doubt is the case with the Dematium pulhtZans studied 
by Lindner, 32 and the type of the same organism isolated by 
Massee 33 from a case of gummosis in Prunus japonica. In 
Aspergillus conicus, a species closely related to Aspergillus 
glaucus, mucus was observed by Dale. 34 
v^xhe isolation in pure culture of the organisms responsible 
for the production of mucus has frequently met with con- 
siderable difficulty. In most cases the raw material contains 
several species of micro-organisms which take no part in the 
production of mucus, but which are difficult to separate from 
the causative organisms by dilution in the usual way owing 
to the insolubility of the mucus. 

To secure pure cultures Liesenberg and Zopf 36 suggested 
heating the raw material to 75 C. for 15 minutes prior to 
preparation of plates in order to destroy the entangled 
secondary microflora. They assumed that the mucus pro- 
ducing micro-organisms would possess higher heat resisting 
powers than the associated flora. This method was not 
always successful, and has been replaced by the technique of 
Zettnow, 36 which is based on the observation that mucus is 
produced only on media containing carbohydrates. 

As a preliminary Zettnow triturates a small amount of raw 
material with twenty to thirty times its volume of sterile 
water and repeats this treatment three to five times. Small 
pieces of the purified raw material are then placed on firm 
saccharose gelatine plates, though saccharose agar plates 
might presumably be used for the purpose. The inoculant is 



186 THE MUCUS FERMENTATIONS 

now distributed over the surface of the plates by means of a 
sterile glass rod, suitably bent. The colonies with a slimy 
appearance developing on the plates are transferred to 
ordinary broth in which no mucus is formed and the ensuing 
culture is plated out on ordinary gelatine or agar plates. 
The colonies forming on these plates are subcultured into 
ordinary broth and the resulting culture plated out on 
ordinary gelatine or agar plates. For the purpose of identi- 
fication, the colonies appearing on the last set of plates, now 
possessing the normal consistency of bacterial colonies, are 
placed in a nutrient solution containing carbohydrates from 
which mucus can be produced by the organism. 

For the subsequent storage of the isolated strain Zettnow 
recommends the addition of calcium carbonate to the culture, 
and drying. In some cases, for instance in that of a Javanese 
mucus producing organism, Micrococcus djokjakartensis, the 
organism could be preserved in this way for three years 
without subculturing. 

Like Kramer, 19 and most of the earlier investigators, 
Beijerinck regarded the property of mucus production as a 
characteristic of certain types of bacteria, due to the presence 
in their cells of a specific enzyme 'viscosaccharase'. This 
conclusion is no longer tenable, and on further investigation 
will undoubtedly be abandoned in favour of the view referred 
to in Chapter XIV, that most, if not all carbohydrate de- 
composing micro-organisms possess synthesizing properties 
which, under favourable conditions, notably a slightly alka- 
line reaction and the presence of phosphates (Kramer, 
Glaser 87 ), and during the early stages of their development, 
give rise to the condensation of hexoses to polysacoharides. 
In oases where these polysacchandes readily imbibe water, 
a colloidal solution, i. e. a mucus, is formed. \J 

LITERATURE 

1. . Desfosses, J. Pharm. et Ohim., vol. 15, p. 602, 1829. 

2. A. Boudrimont, Oom/ptes rend., vol. 80, p. 1253, 1875. 

3. . Jubert, vide van Tieghem, Anncdes dea Sciences NatureUes (6), vol. 7, 

p. 180, 1878. 



THE MUCUS FERMENTATIONS 187 

4. E. Durin, Comptea rend., vol. 83, pp. 128 and 365, 1876. 

5. C. Soheibler, abat. Wagner's Jahresbericht der chem. Technolg., vol. 21, 

p. 790, 1875. 

6. L. Pasteur, Comptea rend., vol. 83, p. 176, 1876. 

7. T. Mendes, vide van Tieghem, Butt. Soc. Botan., vol. 26, p. 271, 1878. 

8. L. Cienkowski, Baton. Jahresbericht, vol. 1, p. 501, 1878. 

9. Ph. van Tieghem, BvM. Soc. Botan., vol. 26, p. 211, 1878. Annales 

des Sciences NatureUes (6), vol. 7, p. 180, 1878. 

10. K. B. Lehmann and R. 0. Neumann, Baktenologiache Diagnostik te 

5, Auflage, publ. J. F. Lehmann, Miinchen, 1912. 

11. C. Bienz, Pharmz Ztg., vol. 24, p. 506, 1879; vol. 46, p. 707, 1891. 

12. W. Brautigam, Pharmz. Zentralhalle, vol. 32, p. 427, 1891; vol. 33, 

p. 534, 1892. 

13. 0. Happ, Zenfrbl.f. Bakt., vol. 14, p. 175, 1893. 

14. A. Schmidt, Landioirtsch. Versiichsstat., vol. 28, p. 91, 1883. 

15. E. Laurent, Bvtt. Acad. Boyale Sci. Bdgigue. (3), vol. 10, p. 765, 1886. 

16. J. Vogel, Zeits.f. Hygiene, vol. 26, p. 398, 1897. 

17. H. van Laer, Butt. Acad. Eoyale Belgigue (3), vol. 18, p. 37, 1889. 

18. A. Bechamps, Oomptes rend., vol. 93, p. 78, 1881. 

19. E. Kramer, Monatsheftefilr Chemie, vol. 10, p. 467, 1889. 

20. 0. Emmerhng, Berichte, vol. 33, p. 2477, 1900. 

21. C. Stapp, Zentrblf. Bakt., Abt. n, vol. 61, p. 276, 1924. 

22. R. Greig Smith, J. Soc. Chem. Ind., vol. 26, p. 304, 1907. 

23. R. Greig Smith and Th. Heal, J. Chem. 2nd., vol. 21, p. 1381, 1902. 

ZentrU.f. Bakt., Abt. n, vol. 21, p. 307, 1908. 

24. E. O. v. Lippmann, Zentrblf. Bakt., Abt. II, vol. 8, p. 596, 1902. 

25. A. Fernbaoh and M. Sohoen, Comptea rend., vol. 155, p. 84, 1912. 

26. F. Schardinger, Zentrblf. Bakt., Abt. II, vol. 8, p. 144, 1902. 

27. 0. Laxa, Zentrblf. Bakt., Abt. H, vol. 8, p. 154, 1902. 

28. J. H. Northrop, L. H. Ashe and J. K. Senior, J. Biol Chem., vol. 39, 

p. 1, 1919. 

29. W. Zopf, A. Sohenck, Handbuch d. Botantk, vol. 3, sec. 1, p. 1, 1884. 

30. M. W. Beijerinck, Proceed. K. Akad. van Wetenschap, Amsterdam, Sect. 

Science, vol. 12, p. 635, 1910. 

31. R. Meissner, Landwvrtsch. Jahrbilcher, vol. 27, p. 716, 1898. 

32. P. Lindner, Wochenschr. f. Brauerei, vol. 6, p. 290, 1888. 

33. G. Massee, Kew Bulletin, p. 321, 1898. 

34. E. Dale, Annales Mycol, vol. 12, p. 33, 1914. 

35. C. laesenberg and W. Zopf., W. Zopf s Be^trage zur Physiologie und 

Morphologie niederer Organismen, 1892. 

36. E. Zettnow, Zeits.f. Hygiene, vol. 37, p. 164, 1907. ZentrU.f. Bakt., 

Abt. I, vol. 75, p. 374, 1914. 

37. F. Glaser, Zentrblf. Bakt., Abt. n, vol. 1, p. 879, 1896. 



PART FOUR 



CHAPTER XVI 

THE MICROBIOLOGY OF CEREALS AND CEREAL 
PRODUCTS 

CERTAIN aspects of the action of micro-organisms on starch, 
were dealt with in Chapter n, notably the action of fungi as 
applied in Eastern countries to the hydrolysis of starch for 
the preparation of alcohol and articles of diet. In the follow- 
ing pages attention will be concentrated on the general 
microbiology of grain and flour, on the microbiology of sizing 
materials and adhesive pastes, and on that of dough and 
bread. 

The relationship which undoubtedly exists between these 
subjects is largely due to the common origin of many of the 
organisms involved. An obvious influence is exercised also 
by the similarity in chemical composition of the substances 
involved, but on this little need be said. 

It may be recalled that all green plants possess an epiphytic 
mioroflora which normally subsists on the slight traces of 
carbohydrates, protein and inorganic salts which dissolve in 
the water exuding from, or condensing on, the epidermis of 
the host. This microflora has been studied by Burri 1 and by 
Diiggeli, 2 both of whom agree that it is able to subsist under 
the most severe climatic conditions to which the host normally 
becomes exposed, and that at periods of damp and rain it 
develops luxuriantly, spreading over the whole of the epidermis, 
including the flower and the seed. Burn points out that the 
resistance of the epiphytic mioroflora to low humidities is due 
to the production by the individual cell of an outer layer of 
mucus which retards the drying up of the cell, and incidentally 
fixes it to the surface of the plant tissues on which it lives. 

Under damp conditions, when dew or rain covers the 
epidermis of the host, the mucus dissolves and allows the 
cells to spread over the epidermis. 

The epiphytic microflora consists of comparatively few 
species, though frequently of very large numbers of cells, 



192 MICROBIOLOGY OF CEREALS AND CEREAL PRODUCTS 
sometimes more than are met with in the soil in which the 
host grows. Diiggeli, in his investigation quoted, reported 
the presence of 1,330,000 bacteria per gramme on oat grains, 
1,600,000 on barley, 126,000 on rye, and 500,000 cells per 
gramme on wheat. These figures are considerably lower in 
most oases than those previously recorded by Hoffmann 3 
for Russian, Rumanian, and German grain, but probably 
represent more correctly the numbers of the true epiphytic 
microflora than the figure given by Hoffmann, who sought 
to determine the number of micro-organisms present in 
samples of superior and inferior grades of cereals. 

Curiously enough the epiphytic mioroflora is composed 
almost exclusively of short non-spore forming rods. JBact. 
Tterbicola a. aur&um, synonymous with Bact. mesentericus 
aureus Winkler, 4 and described by Beijerinck 5 as Bact. 
agglomerans, is claimed by Diiggeli to be the preponderant 
species of the normal epiphytic flora, followed by Bact. 
fluoresc&ns liquefaciens as a second, but much less frequent 
type. 

In the writers' experience, which harmonizes with the views 
expressed by Beijerinck, 5 it is not altogether correct to ascribe 
a dominating influence to Bact. herbicola a aureum. It is 
undoubtedly true that an appreciable percentage of the 
epiphytic flora consists of short rods, 'which, when grown on 
ordinary laboratory media, produce yellow transparent 
colonies identical with or closely resembling those of Bact. 
herbicola a aureum. But such yellow colonies are produced 
by a number of species, including the yellow gas producing 
short rod found by Holliger 6 in flour, Bact. coli luteolique- 
faciens, the yellow acid producing rod isolated by Levy 7 from 
flour, and the Bact. trifolii (Pseudomonas trifolii) of Huss, 8 as 
well as several other species. 

In addition to the above there are found among the 
epiphytic microflora the short rods isolated by Burn 9 and 
by Thaysen 10 from grass which, in their morphological and 
biological characters, resemble the coli-paratyphosum groups. 
These types were regarded by Frankel 11 and by Papasotiriu 12 



MIC 



[CEOBIOLOGY OF CEREALS AND CEREAL PRODUCTS 193 
as identical with Bact. coli commune, but were later shown 
(Holliger 6 ) to differ from this organism in the composition of 
the gas produced by them and in their lack of indol production. 

It is interesting to note from Burri's and from Duggeli's 
work that the epiphytic microflora remains almost completely 
unaffected by the mioroflora of the soil in which the host is 
growing. It maintains the continuity of its existance by 
spreading to the seed, and from there to the emerging embryo 
and the new plant. 

The spread to the seed is favoured by the fact that during 
the period of flowering of the host saccharine liquids are 
secreted by the flowers, which offer favourable conditions for 
development. Even among the cereals the exudations of the 
flower, coupled with the protection against desiccation 
afforded by the presence of glumes (paleae), make these sites 
particularly suitable growth centres. In this part of the 
plant the epiphytic microflora is joined, according to 
Chrzaszcz, 13 by more accidental types brought to the flower 
with dust and particles of soil. These types comprise true 
yeasts, torula species, Dematium puttulans, PeniciUium 
species and Cladosporium herbarum, as well as occasional 
Cocci and Sarcinae. It is possible that spore bearing rods, 
notably butyric acid forming bacilli, as well as lactic acid 
producing bacteria, are also represented, but this was not 
demonstrated by Chrzaszcz. The various types, which might 
be regarded as a secondary microflora of the flower, thrive 
on exuded saccharine substances and develop beside the 
normal epiphytic flora. The secondary microflora does not 
possess special means of protection against desiccation and 
depends for its development on the presence of an abundance 
of moisture. It is not surprising, therefore, that it should be 
particularly prevalent during damp seasons. Once established 
it is shielded from destruction by the glumes which surround 
the seed of the Gramineae. The effectiveness of this protection 
is most noticeable in cereals, such as barley, where the glumes 
fuse with the grain during ripening. In such cases the exis- 
tence of micro-organisms within the normal plant cell may 

o 



194 MICROBIOLOGY OB 1 CEREALS AND CEREAL PRODUCTS 
well be thought to occur. This conclusion was actually 
arrived at by Bernheim, 14 and subsequently by Galippe, 16 who 
suggested that soil bacteria penetrated the tissues of most 
plants except garlic. However, Fernbach 16 in France and 
Buohner 17 in Germany proved that the interior of normal 
vegetable tissues are free from micro-organisms. 

Chrzaszcz describes as an internal infection the whole of 
the microflora of the cereal seed, consisting of the true 
epiphytic flora and of the secondary flora, developing in the 
flower and on the ripening grain in damp seasons, and con- 
trasts this flora with the external infection composed of an 
entirely accidental microflora which has become mixed with 
the grain with particles of dirt and soil, and which in most 
cases has had no opportunity of development. 

The external infection can be considerably reduced, and 
perhaps even completely suppressed, by removal of the soil 
and dirt adhering to the seed, or by treatment with suitable 
antiseptics. The internal infection cannot be similarly 
influenced. Wetting, whether with clean water for washing 
purposes or with dilute antiseptics, does not destroy it. On 
the contrary, wetting would in most cases induce a renewed 
activity which, as already remarked, would be favoured by 
the presence of carbohydrates. This is important as it ex- 
plains why an analogy exists between the microbiologica] 
changes occurring in grain and flour, in adhesive pastes and 
sizing materials made from flour, and in dough and bread 
In all of these materials there is available a sufficiently largt 
supply of soluble carbohydrates to ensure a rapid growth oJ 
carbohydrate decomposing micro-organisms. Under these 
conditions large populations, such as the normal interna 
infection of grain, will have every opportunity to outweigl 
more accidental infections introduced with dirt and soil, anc 
therefore to dominate the microbiological changes taking 
place. - 

An account and analysis of the observations made on th< 
microbiology of grain and flour will substantiate the correct 
ness of this statement. 



MICROBIOLOGY OF CEREALS AND CEREAL PRODUCTS 195 

LITERATURE 

1. R. Burri, Zentrbl. f. Bakt., Abt. n, vol. 10, p. 756, 1903. 

2. M. Dflggeh, Zentrbl.f. Bakt., Abt. II, vol. 13, p. 56, 1904. 

3. F. Hoffmann, Wochenschr . f. Brau&rei, vol. 13, p. 1153, 1896. 

4. W. WinMer, Zentrbl.f. Bakt , Abt n, vol. 5, p. 589, 1899. 

5. M. W. Beijerinok, Zentrbl.f. Bakt., Abt. H, vol. 15, p. 366, 1905. 

6. W. Holliger, Zentrbl.f. Bakt., Abt. H, vol. 9, p. 305, 1902. 

7. F. Levy, Arch.f. Hygiene, vol. 49, p. 62, 1904. 

8. H. HUBS, Zentrbl.f. Bakt., Abt. H, vol. 19, p. 50, 1907. 

9. R. Burri, Zentrbl.f. Bakt., Abt. II, vol. 28, p. 321, 1910. 

10. A. C. Thaysen, Zentrbl.f. Bakt , Abt. I, Orig., vol. 67, p. 1, 1912. 

11. F. Fraakel, Inaug. Dissertation, Wurzburg, 1896. 

12. J. Papasotirm, Arch.f. Hygiene, vol. 41, p. 204, 1902. 

13. T. Chrzaazoz, Wochenschnftf. Brauerei, vol. 19, p. 590, 1902. 

14. H. Bemheim, Miinchner med. Wochenachr., vol. 35, p. 743, 1888. 
16. M. Galippe, Zentrbl.f. Bakt., vol 3, p. 108, 1888. 

16. A. Fernbaoh, Ann. Inst. Pastern, vol. 2, p. 567, 1888. 

17. H. Buohner, Miinchner med. Wochenschr., vol. 35, p. 906, 1888. 



o2 



194 MICROBIOLOGY OF CEREALS AND CEREAL PRODUCTS 
well be thought to occur. This conclusion was actually 
arrived at by Bernheim, 14 and subsequently by Galippe, 16 who 
suggested that soil bacteria penetrated the tissues of most 
plants except garlic. However, Fernbaoh 10 in France and 
Buchner 17 in Germany proved that the interior of normal 
vegetable tissues are free from micro-organisms. 

Chrzaszoz describes as an internal infection the whole of 
the microflora of the cereal seed, consisting of the true 
epiphytic flora and of the secondary flora, developing in the 
flower and on the ripening grain in damp seasons, and con- 
trasts this flora with the external infection composed of an 
entirely accidental mioroflora which has become mixed with 
the grain with particles of dirt and soil, and which in most 
cases has had no opportunity of development. 

The external infection can be considerably reduced, and 
perhaps even completely suppressed, by removal of the soil 
and dirt adhering to the seed, or by treatment with suitable 
antiseptics. The internal infection cannot be similarly 
influenced. Wetting, whether with clean water for washing 
purposes or with dilute antiseptics, does not destroy it. On 
the contrary, wetting would in most cases induce a renewed 
activity which, as already remarked, would be favoured by 
the presence of carbohydrates. This is important as it ex- 
plains why an analogy exists between the microbiological 
changes occurring in grain and flour, in adhesive pastes and 
sizing materials made from flour, and in dough and bread. 
In all of these materials there is available a sufficiently large 
supply of soluble carbohydrates to ensure a rapid growth of 
carbohydrate decomposing micro-organisms. Under these 
conditions large populations, such as the normal internal 
infection of grain, will have every opportunity to outweigh 
more accidental infections introduced with dirt and soil, and 
therefore to dominate the microbiological changes taking 
place. 

An account and analysis of the observations made on the 
microbiology of grain and flour will substantiate the correct- 
ness of this statement. 



MICROBIOLOGY OF CEREALS AND CEREAL PRODUCTS 195 

LITERATURE 

1. R. Burri, Zentrbl.f. Bakt., Abt. LL vol. 10, p. 756, 1903. 

2. M. Diiggeli, Zentrbl.f. Bakt., Abt. LT, vol. 13, p. 56, 1904. 

3. F. Hoffmann, Wochenschr. f. Brauerei, vol. 13, p. 1153, 1896. 

4. W. WmMer, Zentrbl.f. Bakt., Abt. n, vol. 6, p. 669, 1899. 

5. M. W. Beijerinok, Zentrbl.f. Bakt., Abt. n, vol. 15, p. 366, 1906. 

6. W. Holhger, Zentrbl. f Bakt., Abt. H, vol. 9, p. 305, 1902. 

7. F. Levy, Areh.f. Hygiene, vol. 49, p. 62, 1904. 

8. H. HUSH, Zentrbl.f. Bakt., Abt. n, vol. 19, p. 50, 1907. 

9. R. Burri, Zentrbl.f. Bakt., Abt. n, vol 28, p. 321, 1910. 

10. A. C. Thaysen, Zentrbl f. Bakt., Abt. I, Ong., vol. 67, p. 1, 1912. 

11. F. Frankel, Inaug. Dissertation, Wurzburg, 1896. 

12. J. Papasotiriu, Areh.f. Hygiene, vol. 41, p. 204, 1902. 

13. T. Chrzaszcz, WochenscJmftf. Biau&rei, vol. 19, p. 690, 1902. 

14. H. Bernheim, M unchner med. Wochenschr., vol. 35, p. 743, 1888. 
16. M. Galippe, Zentrbl.f. Bakt., vol. 3, p. 108, 1888. 

16. A. Fernbaoh, Ann. Inst. Pasteur, vol. 2, p. 567, 1888. 

17. H. Buohner, Munchner med. Wochenschr , vol. 36, p. 906, 1888. 



02 



CHAPTER XVII 

THE MICROBIOLOGY OF GRAIN AND ITS MILLING 
PRODUCTS, BRAN AND FLOUR 

IT has been indicated that the seeds of cereals, like those of 
other plants, possess a specific microflora largely composed 
of non spore-forming short rods, but containing representa- 
tives of true yeasts, lower fungi, such as Cladosporium her- 
barum and Aspergillus and Penicillium species, and infre- 
quently also of cocci and spore-forming rods. Like all living 
cells, these types depend for their development on the presence 
of nutritive substances and of small but definite concentra- 
tions of moisture. The required Tm'm'Tnnm of water will 
frequently be exceeded while the grain is still developing, but 
normally should not be reached after ripening. Nevertheless, 
occasions frequently occur when excess moisture is present in 
the ripened grain, and when, in consequence, the microflora of 
the seed develops abnormally. In a study of the behaviour of 
the seeds of various cereals, stored in glass containers, Atter- 
berg 1 sought to establish the minimum concentration of 
moisture which allows the development of an indigenous 
microflora. He found that samples of wheat, containing up 
to 15-6 per cent, of moisture, remained unaffected by micro- 
organisms during storage, but developed mould growth when 
this moisture percentage was exceeded. In the case of barley, 
most samples remained sound when containing less than 
14-4 per cent, of moisture, though a few became slightly 
mouldy at this concentration. Samples of oats remained 
normal when containing 162 per cent, of water. For maize, 
Black and Alsberg 2 gave 12 per cent, as the maximum per- 
missible concentration. In one case Thorn and LeFevre 3 
observed development of mould on a sample of maize con- 
taining less than 13 per cent, of moisture. 

Atterberg concluded that wheat in storage should possess 
no more than 16 per cent, of moisture and barley no more 
than 14 per cent., at least not during the warmer seasons, 



MILLING PRODUCTS, BRAN AND FLOUR 197 

when the prevailing higher temperatures facilitate the de- 
velopment of micro-organisms. 

Such percentages are invariably exceeded during wet 
seasons, when moisture contents of 24 to 28 per cent, have 
been observed (Atterberg 1 ). Microbiological activity, there- 
fore, sets in at such seasons, and the grain becomes more or 
less seriously damaged. This damage has claimed the atten- 
tion of many investigators. It is often noticeable in the first 
instance as an odour of mouldiness (Emmerling 4 ), at least 
when the excess moisture is comparatively slight (KOnig, 
Spieckermann and Olig 5 ). Moisture contents of 30 per cent, 
and more give rise to bacterial activity. In extreme cases 
the damage may lead to spontaneous combustion of the 
stored grain. This was observed by Hoffmann 6 in a case of 
stored bran, and was described by him as due in the first 
instance to the respiration of the cells of the damp bran 
increasing the temperature sufficiently to allow micro- 
organisms to develop. Their activity was stated to raise the 
temperature to 70 C., a point at which the absorption of 
oxygen would proceed at a greatly increased rate, sufficient 
to bring the temperature to 160 C., when the bran would 
ignite. Hoffmann's exposition agrees in its essentials with 
Haldane and Makgill's 7 conception of the spontaneous com- 
bustion of hay. An account of Haldane and Makgill's work 
was summarized by Thaysen and Bunker 8 in their review of 
the spontaneous combustion of vegetable tissues. 

In a recent study of the spontaneous combustion of damp 
organic materials, James, Rettger and Thorn 9 refer to the 
activity of bacteria contained in damp maize meal in storage. 
They found that the most pronounced heating was observed 
when the moisture content of the flour was about 30 per cent. 
An increase of the microflora of such flour occurred during 
the time taken for the temperature to rise to 50 C. After 
that a decrease set in, which became particularly noticeable 
when the temperature of the flour had been raised by aeration 
to 62 C. When stored for 4 days at 62 C., practically the 
whole of the microflora was destroyed. 



198 THE MICROBIOLOGY OF GRAIN AND ITS 

Between the extremes of a slight mouldy odour and a 
complete charring of the material, a series of intermediate 
stages of microbiological damage have been observed in 
grain and flour. They have been studied chiefly from a 
hygienic point of view. 

As regards the action of micro-organisms on ripening 
grain, as distinct from that on stored grain and flour, it has 
been recorded that abnormally wet seasons encourage it. 
Both Eeichard 10 and Becker 11 attribute the loss of germinat- 
ing power of damp grain to the spread of an excessive micro- 
flora over the grain during ripening. Reichard emphasizes 
that this microflora is composed essentially of lower fungi. 
This, however, is by no means necessarily the case. Where 
the excess moisture is comparatively slight, fungi may pre- 
dominate, but in grain with abnormally high moisture con- 
tents, various types of bacteria, belonging to the internal 
microflora of the grain, will constitute part of the covering 
microflora. Hiltner 12 mentions that he obtained various 
bacteria from very damp grain, and refers specifically to a 
short rod with yellow colonies, Bact. herbicola a aureum (?), 
and also a butyric acid-producing bacillus and Bact. fluores- 
cens liquefaciens. Becker, in his paper referred to above, 
records that he found the bacterial content of barley to vary 
between five thousand and over twelve hundred millions per 
gramme, showing that a very dense bacterial flora can exist 
on grain. The excessive sliminess of infected grain, noted by 
Becker, also indicates that bacteria participate in the attack 
on ripening grain, as does the fact that the mucus formed 
can be removed by steeping and washing. 

The washing of infected grain was recommended by Becker 
for improving the germinating power of damaged barley. 
He found that the number of seeds capable of developing a 
normal plant could be greatly increased by this treatment. 
Reichard, who adopted a disinfection and drying of infected 
barley with alcohol and ether, or with chloroform, achieved 
an increase in germinating power of 43 per cent. Such 
methods for the improvement of the germination power are 



MILLING PRODUCTS, BRAN AND FLOUR 199 

of the greatest economic importance, not only to the maltster 
and the brewer, but to any one concerned with the protection 
of seed in storage. 

While it has been fairly conclusively proved that lack of 
germinating power in grain may be due, not necessarily 
to a destruction of the seed during its development, but to 
the action of an excessive microflora on the fully developed 
germ, it has not yet been shown how this action is to be 
explained. Becker holds the view that the mioroflora simply 
clogs the pores of the germ and thus prevents its respiration, 
without otherwise damaging the endosperm of the seed. But 
this explanation would hardly cover cases such as those in 
which the microflora possesses starch- or pectin-resolving 
enzymes, the butyric acid producing bacilli, for instance, 
observed by Hiltner, Schardinger and others, or Cladosporium 
herbarum referred to by Chrzaszcz, 18 types which almost 
certainly attack not only the endosperm but the tissues of 
the germinating plant. Future investigations are likely to 
show that the action of the microflora is a multiple one, 
depending on the actual micro-organisms comprising the 
flora ; sometimes causing little structural damage either to 
the endosperm or to the germ, but sometimes affecting one or 
both to a marked extent. In this connexion it is of interest 
to note that Hiltner 14 distinguishes between a microflora 
which affects the endosperm only, and one which attacks 
both the endosperm and the seedling. 

In the former group Hiltner places the various Penicillium 
and Asp&rgillus species met with on ripening grain. In the 
more harmful groups, attacking both endosperm and seedling, 
Hiltner includes two types, one represented by fungi such as 
Cephalotliecium roseum, Botrytis cinerea, Mucor stolonifer and 
Pyfhium deBaryanum, which attack the endosperm and the 
seedling indiscriminately, and another to which Ascochyta 
Pisi and ColUtotrichum Lindemuthianum belong, which first 
attack the endosperm and subsequently spread to the seed- 
ling. To the latter group must be added Fusarium roseum, 
which Jatschewski, 15 and subsequently Gabrilowitsch, 16 



200 THE MICROBIOLOGY OF GRAIN AND ITS 

isolated from samples of rye which had caused severe dis- 
turbances to the health of persons consuming it. The occur- 
rence of rye attacked by Fusarium roseum is common dur- 
ing damp seasons, when the fungus invades the seed during 
ripening and effects the endosperm in a way which renders it 
toxic. The toxin causes giddiness and affects the sight, with 
more serious complications on continued consumption, lead- 
ing in extreme cases to death. 

Until recently Italian investigators ascribed a similar 
origin to pellagra, a disease which affects the poorer classes 
in countries where maize is consumed as a staple food. 

Pellagra usually starts as a more or less extensive rash on 
the exposed skin, and subsequently affects both the gastro- 
intestinal canal and the nervous system. Severe cases are 
not infrequently fatal. 

The theory of the connexion of pellagra with the develop- 
ment of micro-organisms on ripening maize was first ad- 
vanced by Lombroso (see Tirelli ; 17 and Pellizzi and Tirelli 18 ), 
and was widely accepted until recently. The explanation 
given was that fungi, notably species of Aspergillus, when 
developing in maize, produced toxic protein decomposition 
products which, on continued consumption, gave rise to the 
symptoms characteristic of pellagra. A very extensive litera- 
ture exists on this aspect of the action of micro-organisms 
on ripening grain, and great efforts have been made by 
various Italian investigators to bring forward evidence in 
support of the existence of such toxins and of their specific 
action on animals and man. Summaries of this work were 
given by Serena, 19 by Bertarelli 20 and by Ceni. 21 Definite 
conclusions were not reached, however, since it was found 
(Bezzola 22 ) that guinea-pigs fed on an exclusive diet of normal 
maize showed symptoms resembling pellagra. Bezzola's 
observations, which he was anxious not to interpret as con- 
clusive evidence, have recently been confirmed by Mouri- 
quand, 23 and notably by Goldberger, Wheeler, Lillie and 
Rogers, 24 by Goldberger and Lillie 25 and by Marshall Fuidlay, 26 
all of whom attribute pellagra to an exclusive consumption 



MILLING PRODUCTS, BRAN AND !FLOUR 201 

of food deficient in the thermostable part of vitamine B 
(vitamine 'B 2 ' of the Committee of Accessory Food Factors). 
Maize, whether sound or damaged by micro-organisms, is 
notoriously deficient of this vitamine. 

Even after ripening, without being noticeably attacked by 
micro-organisms, grain and its milling products, bran and 
flour, may still become microbiologically damaged, not only 
by types belonging to the internal microflora, but by organ- 
isms introduced accidentally with soil and dust. For such 
damage to occur the presence of moisture is essential. Atter- 
berg 1 stipulated that wheat and oats, when containing more 
than 16 per cent, of moisture, became liable to attack ; rice, 
according to Haselhoff and Mach, 27 suffered damage in the 
presence of more than 10 per cent., and barley (Atterberg) 
became mouldy when containing more than 14 per cent, of 
water, provided the temperature during storage exceeded a 
certain minimum. Atterberg did not investigate the influence 
of the temperature very exhaustively, but he observed that 
barley, containing as much as 24 to 28 per cent, of moisture, 
remained sound during the winter months, while it invariably 
became attacked when warmer weather prevailed. Observa- 
tions that low temperatures are safer for the storage of damp 
grain than average room temperatures, were made also by 
Bell, 28 who nevertheless favoured a warm dry storage room, 
provided the moisture of the grain or flour had previously 
been reduced to below the minimum inducing the growth of 
micro-organisms. Such drying can be achieved in practice by 
moving the grain from one elevator to another, on endless belts 
and in a dry atmosphere, when the questionable advantage 
is incidentally secured of polishing the surface of the individual 
kernel and of removing superficial growth of mycelium and 
fungus spores. Black and Alsberg 2 draw attention to the 
drawback of this removal, and point out that the detection 
of damaged grain is rendered more difficult by the treatment. 

The methods which have been recommended for the detec- 
tion and determination of microbiological damage in stored 
grain and flour will be discussed subsequently. 



202 THE MICROBIOLOGY OF GRAIN" AND ITS 

A comparatively slight excess of moisture in stored grain 
or flour will result in the development of fungi rather than of 
bacteria, and with equal moisture contents it is generally 
noticeable (Black and Alsberg) that growth occurs more 
readily in flour or bran than in whole grain. Haselhoff and 
Mach observed growth of Asp&rgillua oryzae in rice flour 
containing no more than 10 per cent, of moisture, while 16 to 
20 per cent, were required for Penicillium glaucum to develop. 
Both species, of course, are constant members of the internal 
mioroflora of grain. In the case of rye flour, mould growth 
occurred, according to Arnoldow, 29 in samples with more than 
17 per cent, of moisture, and proceeded rapidly, the presence 
of the fungi being detectable after 24 hours, not only by a 
mouldy odour, but by the loss of several per cent, of the 
carbohydrates present, a loss which after three months' 
storage had increased to more than 60 per cent. In these 
cases little change was observed in the protein and the fat 
content of the flour, an interesting observation which was 
confirmed in the case of the protein of cotton seeds by Konig, 
Spieckermann and Olig. 5 

When bacteria, and notably putrefying bacteria, participate 
in the deterioration of stored grain and its products the pro- 
tein present undoubtedly becomes affected. This is confirmed 
by the observations of Konig, Spieokermann and Olig in the 
case of cotton seed cake. In such cases toxic protein decom- 
position products may conceivably be formed as reported by 
Cortez. 30 On the other hand, Dragendorf investigated an 
epidemic among swine, said to have been due to the feeding 
of deteriorated protein residues of a maize starch factory, 
and was unable to detect the presence of toxic products. The 
unpalatable appearance of cereal products which have been 
attacked by bacteria, as distinct from fungi, and their un- 
doubted detrimental action on the intestinal canal of man 
and beast, are probably due as much, if not more, to the 
action of butyric acid producing bacteria on the starch as to 
that of putrefying types on the protein. 

The subject of the damage of cereals and their mining 



MILLING PRODUCTS, BRAN AND FLOUR 203 

products by bacteria has received little attention in the past 
in spite of the economic interest which it undoubtedly 
possesses. It is known from the work of KOnig, Spieckermann 
and Olig, that bacteria require a higher moisture content to 
develop, 30 per cent, being the figure given by the writers 
referred to. And it has been established by Prillieux 31 and by 
Griffiths 32 that a certain type, Bact. prodigiosum, may affect 
the starch content of damp cereals and discolour them. But 
the formation of those felted nauseating lumps of grain and 
flour which may be met with under unsuitable storage con- 
ditions, and which sometimes represent a considerable pro- 
portion of a total consignment, for instance of sea-shipped 
grain or flour, have not yet been studied either by bacte- 
riologists, who might elucidate how they arise and the types 
of bacteria which participate in their formation, nor by 
chemists and physiologists, who might determine to what 
extent the damage wrought has rendered the starch present 
unsuitable not only for consumption but also for industrial 
purposes. 

There are other questions connected with the activity of 
bacteria in stored cereals and cereal products which are 
worthy of interest, for instance, the occurrence of the bacteria 
responsible for the ropiness in bread. The appearance of 
ropy bread has been definitely traced to the presence of the 
responsible bacillus, of which more will have to be said later, 
in the flour used. Laurent 33 and Thomann 34 first made the 
observation, and subsequent writers, among them Watkins, 85 
have shown that a change in the flour used in bakeries where 
an epidemic of ropy bread has broken out, will invariably 
result in the elimination of the complaint. But no informa- 
tion is available to indicate how a consignment of flour can 
become infected with the rope-producing bacillus to such an 
extent that even fractions of a gramme may suffice to initiate 
the disease in bread. 

There is also the question of the extent to which bacteria 
participate in the production of acid in stored cereals, an 
important point since the presence of such acids has been 



204 THE MICROBIOLOGY OB 1 GRAIN AND ITS 

used by various investigators as an indication of micro- 
biological activity. Bell 36 determined the changes in the 
acidity of stored flour, and suggested that lower fungi were 
responsible for an increased acidity, while bacteria caused a 
liquefaction of the flour. It is highly improbable that a 
sharply defined subdivision such as this can be justifiable in 
all cases, but the question requires investigation. 

To mention but one more subject amongst many others, 
there is at present nothing to indicate the fate of the numerous 
non-spore-f orming bacteria of the internal microflora, notably 
the gas-producing types of the paratyphosum group and the 
lactic acid bacteria, during the normal and abnormal storage 
of grain and flour. It is not known whether this flora under- 
goes changes during storage, which might be correlated with 
the chemical changes suffered by the grain, notably by its 
protein as observed by Marion, 37 and therefore might be used 
as a simple means of detecting such changes, and indirectly 
possibly the age of a stored cereal or cereal product. 

The attack of micro-organisms on stored cereals may 
occur from the surface towards the interior, when fractured 
surfaces and holes left by grain-boring insects are the ports of 
entry for the micro-organisms. Or it may start under the 
cuticle of the grain, and then usually in the cavity surrounding 
the germ, a site which, it would appear, frequently becomes 
invaded by a mycelium, notably of Penicilliuin or of A&p&r- 
gillus species. Such hidden growth of micro-organisms is 
very difficult for the untrained observer to detect. Some- 
times it can be detected only with a powerful lens, and appears 
as isolated conidiophores, with adhering spores, reaching 
from the surrounding walls towards the centre of the germ 
cavity. These cases probably indicate a growth which has 
come to a standstill prior to storage. In other cases faintly 
bluish-grey to brown spots are perceptible through the tissues 
covering the embyro cavity, indicating the growth and spread 
of a fungus. In aggravated cases these spots converge and 
cover smaller or greater parts of the whole grain. A bluish- 
grey spotting is usually due to Penicittium species, a bronze 



MILLING PRODUCTS, BRAN AND FLOUR 205 

colour to Aspergillus fumigatus, which has been reported 
present in damaged grain by various observers, most recently 
by Sartory and Sartory. 38 Where a reddish discoloration 
occurs, Black and AJsberg attribute it to Bact. prodigiosum. 
In extreme cases the whole surface of an attacked grain may 
be covered with a fine powder of fungus spores. Such cases, 
of course, are easily recognized and would require no specific 
technique for their detection. 

In his treatise on maize and maize products, Schindler 39 
stipulates that a cereal used for human or animal consump- 
tion should contain no more than 5 per cent, of mouldy 
kernels. Black and Alsberg consider this percentage too 
high, at least when taken to include the visibly damaged 
grains only. They suggest 2 to 2-6 per cent, as a maximum 
figure, a proposal which is not unreasonable. By basing the 
estimate on visibly damaged grain the necessary analyses 
are much simplified, at least when such analyses are restricted 
to an actual counting of the grains, of which 500 should be 
taken in each case. This method can in the best of oases be 
approximate only, since it gives little or no information on 
the types of deterioration in which the damage has failed to 
reach an advanced state, or where the grain has been treated 
by rubbing or polishing to remove visible signs of fungus 
growth. Nor does it suffice in the case of flour. Here other 
methods have to be adopted. 

The first of these, which was suggested by Emmerling, 4 
was very crude. It was based on the time taken for a sample 
of flour, or grain after grinding into flour, to acquire a mouldy 
odour on mixing with water to form a paste. Emmerling 
stipulated that a sound flour thus treated should retain its 
normal odour for at least 24 hours. The method was widely 
adopted for the testing of various feeding materials, in spite 
of the fact that it frequently gave inconclusive results, for 
instance in the cases reported by Gordan, 40 who used it to 
determine the soundness of various cattle foods, including 
bran. It is now universally replaced by methods which 
determine the changes of acidity occurring in stored gram, or 



206 THE MICROBIOLOGY OF GRAIN AND ITS 

flour as a result of microbiological activity. The nature of 
this acidity were studied by Dombrowski. 41 He found it due 
to the production of acetic and lactic acids. 

In their modification of these methods, Black and Alsberg 
take 500 kernels picked at random, grind them to a fine flour, 
and place 10 grs. of the flour in a 50 o.cms. glass stoppered 
flask. The flask is filled to the 50 c.oms. mark with neutral 
alcohol, of 85 per cent, by volume, and shaken. The mixture 
is allowed to stand for 24 hours, and 25 c.cms. of the filtered 
extract is then mixed with 100 to 150 c.cms. of distilled water 

and its acidity determined by titration against -^NaOH, 

zo 

using phenolphthalein as indicator. The number of o.cms. of 
alkali used, multiplied by 10, gives the acidity of 100 grs. of 
flour expressed in terms of n-alkali. The acidity of various 
grain and sound flours determined by this method is given 
below: 

TABLE VII 



Sound wheat 
rye 
barley 
oats 
white wheat 
flour 
whole meal 
flour 
maize meal 


Acidity, estimated by Black and 
Alsberg's method, and expressed 
in c.cms. of n-NaOH. 


Remarks. 


12 
4. . . 


The figures should 
be taken as approxi- 
mate and in the case 
of normal cereals 
may vary slightly on 
either side of the 
figure given. 


8 


21 
5 


15. 


9. 





Black and Alsberg's method is of considerable value in 
cases where the deterioration has led to an increase in acidity. 
It could be improved in its technique by taking 20 gms. of 
flour and a double quantity of alcohol for extraction. The 
amounts given by Black and Alsberg are barely sufficient to 
secure 25 c.cms. of the extract for titration purposes. 



MTT.LTNG PRODUCTS, BRAN AND FLOUR 207 

In addition to these general methods, other and more 
specific tests have been recommended for the detection of the 
microbiological deterioration of cereals, notably of maize, 
the Gosio 42 phenol test, for instance, and Ori's 43 catalase test. 
These methods have not been found to be reliable and need 
not, therefore, be discussed in detail. The germination test 
advocated by Sclavo, 44 and adopted officially in Italy to 
ascertain the soundness of maize, demands a TniTn'Tnmn of 
80 per cent, of germinating power, a figure which is considered 
too low by Ori, 48 who advocates 90 per cent. Sclavo's test is 
open to several objections, including the possibility of an 
otherwise sound grain having been heated too high during 
artificial drying and its germ destroyed or weakened. 

An effort to measure the deterioration of stored grain and 
flour by microbiological analysis was foreshadowed by Smith 
(see Black and AJsberg 2 ), but work in this direction does not 
appear to have been followed up. 

In spite of the apparent facility with which grain and flour 
are attacked on storage, one type of cereal at least has been 
shown to possess properties which actively inhibit the de- 
velopment of certain micro-organisms. In wheat such in- 
hibitory substances were found by Baker and Hulton. 46 
Their presence prevents brewer's yeast from being used for 
the raising of a dough. To what extent a natural resistance 
against decay occurs in seeds other than wheat has not yet 
been ascertained. 

Considerable attention has been paid to the elimination of 
micro-organisms from grain and flour. As a sterilization by 
heat cannot be adopted on account of the resulting damage 
to the protein, Wollney's cold sterilization process, based on 
treatment with ether, was tried in the case of flour by Wolffin 47 
and by Holliger, 48 who found it of considerable value, while 
Budinoff 49 and Dombrowski 41 failed to sterilize flour in this 
way, at least when spore-forming bacteria were present. 

As an alternative to ether, Vandevelde 50 recommended the 
use of chloroform in acetone, and subsequently carbondisul- 
phide, which he says causes less drastic changes in the chemical 



208 THE MICROBIOLOGY OF GRAIN AND ITS 

and physical properties of the flour than any of the other 

substances recommended. 

In the writers' experience none of these methods are entirely 
satisfactory for the sterilization of grain and flours. Various 
spore-forming rods will remain alive on grain and flour for 
periods of more than 10 months when suspended in chloro- 
form, ether, or carbondisulphide. 

To prevent microbiological damage to grain and flour on 
storage, Legendre 61 recommends the addition of small quanti- 
ties of harmless alkaline materials to the grain. He claims 
that the change in reaction thereby secured successfully 
inhibits the formation of sugars by the diastatic enzymes of 
the grain, and that a multiplication of the bacteria present 
is prevented in the absence of the resulting sugars. It is 
highly questionable whether this procedure, even if effective, 
will be found to be of practical importance. 

LITERATURE 

1. A Atterberg, Landwirtsch. Versuchsatat , vol. 39, p. 205, 1891. 

2. O. F. Black and C. L. Alsberg, U S. Dept. Agric., Bureau of Plant Ind., 

Bull. No. 199, 1910. 

3. C. Thorn and E. LeFevre, J. Agric. Res , vol. 22, p. 179, 1921. 

4. A. Emmerling, O.f. Agriculturchemie, vol. 13, p. 472, 1884. 

5. J. Konig, A. Spieokermann and A. Olig, Z. f. Untersuch. der Nahrungs- 

und Oenusam., vol. 6, p. 193, 1903. 

6. F. Hoffmann, Zeits.f. Spintua Ind., vol. 20, p 287, 1897. 

7. J. S. Haldane and R. H. Makgill, Fud, vol. 2, p. 380, 1923 

8. A. C. Thaysen and H. J. Bunker, The, microbiology of cellulose, etc., 

Oxford University Press, 1927. 

9. L. H. James, L. F. Rettger and C. Thorn, J. Bad , vol. 15, p. 117, 1928. 

10. A. Reiohard, Chem. Zeitg., vol. 21, p. 21, 1897. 

11. H. Becker, Zeits.f. d. gesam. Brauwesen, vol. 20, p. 437, 1897. 

12. L. Hiltner, Landwtrtsch. Versuchsstat., vol. 34, p. 391, 1887. 

13. T. Chrzaszcz, Wochenachnftf. Brauerei, vol. 19, p. 690, 1902. 

14. L. Hiltner, Arbeit a. d. bioL Abt. am Kais. Gesundheitsamt, vol. 3, p. 1, 

1902. 

15. A. A. Jatsohewski, Chem. Zeitg , vol 29, p. 165, Rep. 1906. 

16. 0. E. Gabrilowitsch, Btochem. Zentrbl., vol 6, p. 431, 1907. 

17. V. Tirelli, Zentrbl f. Bakt., vol. 16, p. 185, 1894. 

18. G. H. Pellizi and V. Tirelli, Zentrbl. f. Bakt., vol. 16, p. 186, 1894. 

19. . Serena, Zentrbl. f. Bakt., Abt I, Ref., vol 29, p 448, 1901. 



MILLED PRODUCTS, BRAN AND FLOUR 209 

20. B. Bertarelli, ZentrU.f. Bakt., Abt. I, Ref., vol. 34, p. 104, 1903. 

21. C. Ceni, ZentrU.f. BaU., Abt. I, Ref., vol. 39, p. 662, 1907. 

22. C. Bezzola, Z.f. Hygiene, vol. 66, p. 76, 1907. 

23. G. Mouriquand, Comptes rend., vol. 182, p. 347, 1926. 

24. J. Goldberger, G. A. Wheeler, R. D. Lfflie and L. M. Rogers, Public 

Health Report, Washington, vol. 41, p. 297, 1926. 

25. J. Goldberger and R. D. Ldllie, Public Health Reports, Washington, 

vol. 41, p. 1026, 1926. 

26. G. Marshall Frndlay, /. Pathol. and Bacterial., vol. 31, p. 663, 1928. 

27. E. Haselhoff and F. Mach, Landwirtsch.Jahrbucher, vol 36, p. 446, 1906. 

28. H. G. Bell, Operative Hitter, vol. 13, p. 691, 1909. 

29. W. A. Arnoldow, Chem. Abst., vol. 2, p. 1017, 1908. 

30. . Cortez, DraggendorfsJahresber , vol. 13, p. 616, 1878. 

31. E. Prillieux, Butt. Soc. botan. de France, vol. 21, p. 31, 1874. 

32. A. B. Griffiths, Comptes rend , vol. 116, p. 321, 1892. 

33. E. Laurent, Butt, de FAcadem. de Belgique (3), vol. 10, p 765, 1886. 

34. J. Thomann, Zentrbl.J BaU., Abt. H, vol. 6, p. 740, 1900. 
36. E. J. Watkins, J. Soc Chem. Ind., vol. 25, p. 350, 1906. 

36. H. G. Bell, American Miller, vol. 37, p. 281, 1909. 

37. F. Marion, J. Soc. Chem. Ind., vol. 28, p. 808, 1916. 

38. A. Sartory and R. Sartory, Chem. Abets., vol. 20, p. 2210, 1926. 

39. J. Schindler, Anleitung zur Beurteilung des Maises und seiner Mahl- 

producte, Kgl. und Kaiserl. Statthalterei Innsbruck, 1909. 

40. P. Gordan, Landivirtsch. Versuchsstat , vol. 60, p. 73, 1904. 

41. . Dombrowski, Arch.f. Hygiene, vol. 60, p. 97, 1904. 

42. B. Gosio, Evoista d'lgiene e Sanita Publica, vol. 7, p. 825, 1896. 

43. A. On, Rivista Crttica de Chimia Medica, vol. 6, p. 165, 1906. 

44. V. Sclavo, Gazzetta Medica di Torino, vol. 62, p. 863, 1901. 

45. J. L. Baker and H. F. E. Hulton, J. Soc. Chem. Ind., vol. 28, p. 778, 

1909. 

46. R. Wollney, ZentrU.f. Bakt., vol. 11, p. 752, 1892. 

47. A. Wolffin, Arch.f. Hygiene, vol. 21, p. 305, 1894. 

48. W. Holliger, ZentrU.f. Bakt., Abt. II, vol. 9, p. 305, 1902. 

49. L. Budinoff, ZentrU.f. BaU., Abt. n, vol. 10, p. 462, 1903. 

50. A. J. J. Vandevelde, Chem. Abuts., vol. 14, p. 3112, 1920. 

51. H. Legendre, Comptes rend., vol. 185, p. 1156, 1927. 



CHAPTER XVIII 

THE MICROBIOLOGY OF STARCH-CONTAINING 
SIZING MATERIALS AND ADHESIVES 

SIZING MATERIALS 

WHEN a fabric is woven, the warp threads are held parallel 
in the loom, whilst the weft yarn is passed to and fro by 
means of a shuttle. In order to smooth and strengthen the 
warp threads they are usually given a preliminary sizing by 
being passed through a hot paste or 'size 3 made of starch or 
flour. A film of this paste coats the yarn and to some extent 
penetrates the interstices between the individual fibres. In 
some cases the size also serves as a means of adding weighting 
materials such as china clay to the cloth. 

In the case of cloths which are to be bleached, the size is 
removed after weaving, but such cloths are in subsequent 
processes usually 'finished' with a dressing of some starch 
preparation. 

The starch preparations most commonly used for sizing 
purposes are wheat flour, potato starch (farina), maize starch, 
and sago starch. For the preparation of the size the starch- 
containing material chosen is usually agitated with cold 
water in an open storage vat until uniformly suspended. 
Steam is then admitted, and the liquid is heated and stirred 
for a time, varying from some twenty minutes to four or five 
hours. The size is next run through a pipe which communi- 
cates with the container in which the warp thread is im- 
mersed in the size. The supply of size to this container, the 
'size box' or 'sow box', is automatically controlled, and the 
size is maintained at a temperature of about 95 C. The warp 
threads pass through the hot size and thence to a drying 
drum. The quantity in the size box is renewed every six to 
seven hours, through the automatic inflow which replaces the 
amount removed with the warp. At first sight this procedure 
appears to ensure the absence of micro -biological activity 



SIZING MATERIALS AND ADHESIVES 211 

during the preparation and application of the size, and to 
guarantee that no living micro-organisms remain on the 
warp leaving the size box. 

A closer analysis of the procedure, however, as well as of 
the plant used, will show that this is not the case. The vat in 
which the size is prepared is usually made of wood, and it is so 
constructed that the last quantity of size, often amounting 
to 50 or more litres, cannot be drained off. This residue 
remains behind during the time which elapses between the 
preparation of two batches of size, a period during which 
the temperature in the storage vat sinks to approximately 
room temperature. 

As the temperature during the preparation of the size has 
never exceeded 100 C., and has usually remained somewhat 
below this figure at 95 C. the spores of many micro- 
organisms must have survived and remained viable in the 
hot size. It is almost certain that even some non-sporing 
types must have withstood the heat applied during the 
preparation of the size. In support of this may be quoted 
Thaysen's 1 observations in his account of the fermentation 
process for mn.ki.ng butyl alcohol and acetone on a lactic 
acid bacterium, Bact volutans, which withstood an exposure 
for 2 hours at a temperature of 125 C. when present in a 
5 per cent, maize mash This resistance was due perhaps to 
the protection afforded by the colloidal nature of the mash, 
since Bact . volutans was found to be destroyed in five minutes 
at 65 C., when exposed to this temperature in water. 

Many types perhaps even spores of fungi which thus 
survive the preparation of the size, would develop in the size 
remaining in the storage vat when its temperature had fallen 
below 40 C. Other types might conceivably be introduced 
from the air. Whilst the former flora would comprise butyric 
acid bacilli, capable of hydrolysing starch and of rendering 
it sour, the latter might include spore-forming soil bacilli, 
also possessed of starch hydrolysing properties and able to 
render the size an increasingly suitable food for many fer- 
menting types of the internal mioroflora of gram. 

p2 



212 THE MICROBIOLOGY OF STARCH-CONTAINING 

The whole of this microflora would become mixed with 
subsequently prepared quantities of size and would penetrate 
the pores of the wood of the tank, where it would form a 
further nucleus from which future batches of starch could be 
infected. Attention was drawn by Whewell 2 to this danger 
of an infection from the wood. 

In the size box, the temperature at which its content is 
maintained would effectively prevent a development of 
micro-organisms ; those present would be transferred to the 
sized yarn without further numerical increase and would 
remain inactive until the moisture content of the yarn had 
reached a figure suitable for growth. 

Their numbers would be correspondingly increased if, as 
sometimes happens, the operator added a quantity of un- 
treated starch or flour direct to the paste in the size box to 
thicken it. 

An attempt has not yet been made to ascertain the num- 
bers and types of micro-organisms present in the size box. 
This work could very easily be undertaken and would finally 
decide whether fungus spores and non-spore forming bacteria 
are introduced with the size to the yarn and cloth, a question 
which at present remains in dispute. 

Where wheat flour is used the size is not prepared as out- 
lined above. Practical experience has shown that a more 
suitable size can be obtained when the wheat flour is exposed 
to a preliminary fermentation process either in the absence 
of or in the presence of an antiseptic. For the latter zinc 
chloride is usually chosen. Where an antiseptic is added the 
treatment is described as a 'steeping' process, though the 
term 'steeped flour' is often loosely used as synonymous with 
fermented flour. 

For fermentation or 'steeping', wheat flour is made into 
a paste with equal weights of cold or tepid water. The paste 
is allowed to stand at room temperature for several weeks, 
two to three months being a not unusual time. During this 
period the paste is stirred frequently. The stirring would 
appear to be an essential part of the treatment. Laboratory 



SIZING MATERIAL AND ADEESIVES 213 

experiments confirm the suggestion that a certain amount of 
aeration is necessary to check putrefying micro-organisms. 

The microflora of the paste, and the resulting changes, will 
depend on whether the wheat flour is fermented in the 
absence or in the presence of an antiseptic ; one would expect 
that in the former case the whole, or at least a substantial 
part of the normal internal microflora of wheat would secure 
favourable conditions for growth, while a limitation in 
activity would occur when an antiseptic was added. 

It is not possible to give a very detailed account of the 
microbiology of the preparation of wheat flour sizes. The 
observations on the subject given by Stocks 3 are not very 
exhaustive. Stocks attributes the advantages gained by 
fermenting or 'steeping' a wheat flour prior to turning it 
into size, to the protein thereby becoming more readily 
soluble, and yielding a stronger binding agent. The simul- 
taneous production of organic acids is claimed to prevent 
putrefying bacteria from developing. Stocks also remarks 
that the first change noted, when no antiseptics are present, 
is a considerable frothing of the paste due to the action of 
yeast on sugars present. A few days later the flour-water 
mixture develops a pleasant fruity odour, while its acidity 
commences to rise owing to the formation of lactic and acetic 
acids. Though the whole of this acidity must necessarily have 
been formed from the available starch or from its dextrinous 
decomposition products, the sugars having been previously 
consumed by yeast, it is frequently asserted that even when 
the fermentation is prolonged no loss of starch occurs. Bean 
and Scarisbrick 4 deny this, and state that the introduction of an 
antiseptic is essential to prevent loss or damage to the starch. 

The following hitherto unpublished observations on the 
microbiological changes during flour fermentation were col- 
lected during the examination of a number of mill samples 
of fermenting flour, and of regular weekly samples from 
experimental vats.* 

* Quoted by courtesy of the Director of Research, British Cotton 
Industry Research Association. 



214 THE MICROBIOLOGY OF STARCH-CONTMNING 

At ordinary temperatures the fermentation was found to 
be remarkably regular in character. During the first few days 
organisms of various types yeasts and bacteria multiplied 
rapidly, and their number often reached 400 million per 
gram. There was considerable gas evolution due to the action 
of yeasts on the sugar present. After the first week, however, 
lactic acid bacteria predominated and the steadily increasing 
acidity suppressed all other organisms except a small number 
of yeasts and cocci, which persisted throughout the fermenta- 
tion. During these later stages the number of organisms per 
gram remained fairly constant and was about 100 millions. 
The process bore a general resemblance to the souring of 
normal milk, where lactic acid bacteria also rapidly pre- 
dominate and cause the suppression of putrefactive organisms. 

Originally no doubt an antiseptic was added with the idea 
of preventing all microbiological activity (Ermen 6 ). It 
certainly does not do this, though it stops the initial frothing 
of the flour-water mixture which was attributed by Stocks 
to yeast activity. 

A bacteriological analysis of 'steeped' flour during the 
early days of fermentation in the presence of 8 to 10 per cent, 
of zinc chloride reveals that a considerable increase takes 
place in the yellow short rods belonging to the internal micro- 
flora of the grain. The acid-producing species, on the other 
hand, do not appear to show much activity, and there can be 
no doubt that less severe microbiological changes take place 
in this case than in the fermentation of flour. For this reason 
steeping is strongly favoured by Bean, particularly as the 
resulting product has the same improved 'feel' as fermented 
wheat flour. 

The micro-organisms which have survived the application 
of the size to a yarn or cloth are reinforced by further infection 
consisting of fungi and bacteria, derived from the air as well 
as from other sources. The further development of these 
organisms frequently leads to discolorations, technically 
known as 'mildew', and usually due to the growth of mould 
fungi. 



SIZING MATERIAL AND ABHESIVES 216 

Mildew is of common occurrence in cotton goods, particu- 
larly of the unbleached type known as 'grey cloth '. Here the 
principal source of mildew fungi is the raw cotton itself, 
which invariably contains considerable fungal mycelium and 
spores in addition to various types of bacteria. 

The mildew fungi of cotton goods were discussed in detail 
in Thaysen and Bunker's 6 treatise on the microbiology of 
cellulose. They are principally species of Aspergillus and 
Penicillium, together with certain Fungi Imperfecti. 

In order to prevent the growth of -mildew fungi the pre- 
caution must be taken either of maintaining the moisture 
content of the yarn or cloth at a figure below the nm'm'-mTiTn at 
which the organisms can grow, or of adding an antiseptic to 
the size. 

It has long been recognized that the various sizes employed 
in the textile industries do not offer equally suitable food 
material for micro-organisms. This has been experimentally 
confirmed in a series of investigations by Morris, 7 who found 
that, taking the suitability of unfermented 'strong' wheat 
flour, i. e. wheat flour with high protein content, as a food 
substance for Asp&rgillus species as 100, other commonly 
employed starch-containing sizing materials could be placed 
in the following order : 

Rice flour 108 

Cassava flour ...... 106 

Maize dextrin (acid) 100 

Maize dextrin (heat) ..... 98 

Farina dextrin (diastase) .... 97 

Farina dextrin (acid) .... 90 

Wheat starch 89 

Soluble starch 85 

Maize starch 82 

Sago 78 

Farina 76 

Cassava starch ...... 74 

Soft wheat flour fermented for 10 weeks . 72 

Exactly the same sequence does not necessarily apply in 
the case of other fungi. Broadly speaking, however, it may 
be claimed that starches are less suitable food materials than 



&16 THE MICROBIOLOGY OF 
their corresponding raw materials, and that the fermentation 
of wheat flour very appreciably reduces its suitability as a 
food," at least for the growth of some fungi, notably Clado- 
sporium and Fusarium species. The inhibitory action of 
fermented wheat flour is less marked in the case of Asper- 
gittus, and least in that of Penicillium species. 

Washing and neutralization of a fermented flour was found 
by Morris to render it more suitable for mould growth, indi- 
cating an antiseptic value of the organic acids produced 
during fermentation. 

Similar investigations with bacteria and with actinomy- 
cetes have not yet been made. 

A reference was made above to the essential importance 
of the presence in the size of a certain Tnim'Trmm moisture 
content for micro-organisms to develop. This mmimnm was 
claimed by Armstead and Harland 8 to be approximately 
8 per cent, in the case of the Aspergillus species studied by 
them, but may be slightly higher, perhaps even 10 per cent., 
in the case of other fungi. 

To prevent growth of fungi when sufficient moisture is 
present it is essential to add an antiseptic to the size. The 
choice of the right antiseptic presents considerable difficulties 
and cannot be claimed to have been finally made. Hitherto 
zinc chloride, in the concentration of 8 to 10 per cent., calcu- 
lated on the starch in the size, has been most extensively 
used. It is not entirely satisfactory, however, as it does not 
afford complete protection and under certain conditions may 
cause damage to the cloth. 

A comprehensive review of a large number of other anti- 
septics was given by Morris, 9 who found 2:4 dibromophenol 
and tribromophenol the most effective, and thallium car- 
bonate and para-nitrophenol the most generally suitable 
antiseptics, with an effective antiseptic power of between 
six and seven times that of phenol. All of these substances, 
however, have certain drawbacks which may prevent their 
industrial application. 

Morris's investigations were carried out with fungi as test 



SIZING MATERIALS AND ADHESIVES 
organisms. The effect of these and other antiseptics 
bacteria present in the size has not yet been ascertained. 

More recently salioylanilide and certain of its compounds 
have been recommended for use in sizing, and for other 
purposes in which a powerful mildew antiseptic is required. 10 

FLOUR AND STABOH ADHESIVES 

In addition to their use in the sizing and finishing of textiles, 
starch pastes are used for a large number of other purposes ; 
in the manufacture of cigarettes, of paper bags, cardboard 
boxes, in bookbinding, for bill-posting and for paper-hanging. 
For these purposes the starches, or flours wheat and rye 
flour being the most suitable may be boiled with water or 
may be given a previous treatment which partly converts 
the starch into dextrins. Sometimes, as in the process of 
Vandenberg, 11 it may even undergo a preliminary fermenta- 
tion process similar to that used in the preparation of fer- 
mented size. 

It is probably correct to assert, as has been done by de 
Keghel, 12 that adhesives prepared from starch are less likely 
to be decomposed by micro-organisms than those made 
from flour. 

The microflora which participates in the destruction of 
adhesives includes a variety of types. When left to itself at 
room temperature a paste becomes covered with a layer of 
bacterial and mould growth, usually composed of species of 
Bac. mesenteries and of fungi of the genera Aspergillus and 
PenicilHum. The deeper layers show signs of an active 
fermentation giving rise to the production of gas and of 
esters and acids. As a result the adhesive properties of the 
paste are destroyed. A detailed analysis of the participating 
types has not been made. From the writers' experience it 
includes yellow short rods as well as many spore-forming 
types, including butyric acid bacteria. 

The antiseptics which have been recommended to check 
this microbiological activity include zinc chloride, aluminium 
sulphate, phenol and formaldehyde. The last is added by 



218 SIZING MATERIALS AND ADHESIVES 

de Keghel in a concentration of 0-2 per cent, calculated on 
the finished adhesive, at least when the adhesive is prepared 
with starch. When flour is taken, a 0-1 per cent, addition is 
recommended instead of the higher concentration which, 
according to de Keghel, might coagulate the proteins of the 
flour and destroy their adhesive properties. 

None of these antiseptics is entirely satisfactory, and the 
question of how to make starch-containing adhesives proof 
against microbiological attack under all conditions still awaits 
solution. 

LITERATURE 

1. A. C. Thaysen, J. Inst. Brewing, vol. 27, p. 629, 1921. 

2. W. H. Whewell, J. Soc. Dyers and Col, vol. 39, p. 66, 1923. 

3. H. B. Stocks, J. Soc. Dyers and Col., vol. 28, p. 148, 1912. First Report 

on Colloid Chemistry, HM. Stat. Office, London, 1917. 

4. P. Bean and F. Soorisbriok, The Chemistry and Practice of Sizing, 10th 

edition, Hutton, Hartley & Co., Manchester, 1921. 

6. W. F. A. Ennen, The Materials used in Sizing, 2nd edition. Constable 
& Co , London, 1912. 

6. A. C. Thayaen and H. J. Bunker, The microbiology of cettiilose, tt-c., 

Oxford University Press, 1927. 

7. L. E. Morris, J. Text. Inst., vol. 17, p. Tl, 1926; vol. 17, p. T23, 1926. 

8. D. Armstead and S. C. Harland, J. Text. Inst., vol. 14, p. T476, 1923. 

9. L. E. Morris, J. Text. Inst., vol. 18, p. T99, 1927. 

10. British Cotton Industry Research Assn., R G. Fargher, L. D. Galloway, 

and M. E. Probert, B. Pat., 323579. 

11. . Vandenberg, U.8. Pat., No. 621517, 1894. 

12. M. de Keghel, Fabrication des Cottes, Gauthier-Villars et C Ie , Paris, 1926. 



CHAPTER XIX 
THE MICROBIOLOGY OF BAKING 

OF the various microbiological changes which, cereals undergo 
from the time of milling to their consumption as bread by far 
the most important take place during the preparation and 
the ripening of the dough. 

The stages by which man discovered the importance of 
dough fermentation obviously cannot be traced. It is not 
unreasonable to assume, however, as suggested by Chican- 
dard, 1 that they were intimately connected with the custom 
of primitive races of consuming the bulk of their cereal food 
in the form of porridge. Like porridge, dough is in principle 
but a mixture of finely divided grain and water, cooked in 
its original form on hot stones instead of in a pot. 

Even the crudest power of observation and deduction must 
have sufficed to establish that a mixture of flour and water, 
when left for some time prior to baking, yields a lighter and 
more palatable product than one placed on hot stones im- 
mediately after TnyriTig- 

From this stage to that of retaining part of a successful 
dough as a leaven to be included in subsequent batches there 
would appear to be but a short and very natural step. The 
addition of such leavens to a dough must have been practised 
for untold generations, and is undoubtedly much older than 
that known to have prevailed in the Roman Empire of adding 
fermenting grape juice to the dough. Some writers, Lafar, 2 
for instance, consider that originally all natural leavens were 
derived from additions to the dough of fermenting grape 
juice or similar liquors, and base their assumption on some of 
Holliger's 3 experiments which showed that a mixture of flour 
and water, though capable of leavening a dough, does so 
through the activity of bacteria and not through the evolu- 
tion of gas by yeast, which, as has been demonstrated re- 
peatedly, is an essential constituent of all biological dough 
leavens used technically. 



220 THE MICROBIOLOGY OF BAKING 

Though. Holliger may have failed in his attempts to pro- 
duce a leaven, functioning through the activity of its yeast, 
when starting from a dough containing flour and water only, 
it does not necessarily follow, as Laf ar would imply, that a 
typical leaven can be produced only where saccharine liquors 
in active fermentation are added. It is known from the work 
of Chrzaszcz 4 that species of true saccharomycetes occur in 
the internal ^nicroflora of grain, types which might quite well 
have been the progenitors of the yeast met with in dough 
leavens. 

The addition to a dough of natural leavens, whatever their 
origin, is still the recognized technical procedure in the making 
of some types of bread, notably of rye bread. In the form used 
for this purpose the leaven is known as sour dough, a prepara- 
tion which will be discussed subsequently in greater detail. 

The apparent ease with which a dough can be ripened by 
the addition of a biological leaven by no means implies that 
the reactions taking place during dough fermentation are 
simple and easily controlled. More than forty years of study 
have been devoted to this subject, and yet all that can be 
claimed is that certain groups of reactions have been recog- 
nized as essential for the successful fermentation of a dough. 
Bailey and Sherwood 5 enumerated these reactions as (1) 
diastatic changes, (2) hydrolytio changes of the available 
disaccharides, (3) fermentative changes, (4) proteolytio 
changes, and (5) changes in the hydrogen ion concentration 
of the dough. 

One of the objects of the following pages will be to inquire 
into the participation of micro-organisms in these changes. 
For this purpose the subject-matter has been divided into 
four sections, one dealing with the diastatio and other hydro- 
lytio changes affecting the carbohydrates of the dough, the 
remaining three referring to Bailey and Sherwood's other 
groups of reactions. As a preliminary an account will be 
given of the principles of the technique of dough making, of 
the various micro-organisms which occur in the dough, and 
of their relationship to the normal microflora of the flour. 



THE MICROBIOLOGY OF BAKING 221 

When flour and water are mixed to form a stiff paste, and 
are left at a suitable temperature, say 20 C. to 30 C., a 
spontaneous fermentation sets in, as was shown by Dunnen- 
berger 6 and by Boutroux. 7 The dough is ripened and can be 
baked, yielding a fairly satisfactory loaf. Holliger 3 carried 
out a study of the microflora present in the dough during 
this fermentation. He found it to be dominated by two non- 
sporing bacteria, one already known under the name of Bact. 
levans, and the other a gas-producing rod forming yellow 
colonies on ordinary laboratory media. Both types may be 
claimed to belong to the normal internal microflora of grain. 

Bact. levans was first isolated and described by Wolffin, 8 who 
obtained it from sour dough and from flour , from the latter 
most readily after a preliminary incubation of a flour and 
broth mixture at 37 C. for 24 hours. 

The organism achieved considerable notice at the time, 
and was studied in detail by a number of workers, including 
Lehmann, 9 Franckel, 10 and Papasotiriu, 11 who expressed the 
view that it was closely related to, if not actually identical 
with, Bact. coli commune. This similarity, incidentally, in- 
duced Lehmann to test the leavening power of Bact. coli 
commune, which he found to be considerable. 

The relationship of Bact. levans to the most common 
intestinal inhabitant was discussed afresh by Holliger, who 
was able to show that apparent similarity to Bact. coli com- 
mune was restricted to its morphology. In its biochemical 
reactions Bact. levans differed markedly from Bact. coli 
commune, yielding no indol and producing a gas mixture 
composed of hydrogen and carbon dioxide in the proportions 
of one-third of the former to two-thirds of the latter, as against 
two-thirds of hydrogen and one-third of carbon dioxide 
produced by Bact . coli commune. 

The yellow gas-producing rod found by Holliger in spon- 
taneously ripened dough has never been described in detail. 
In morphology it is identical with the short yellow rods which 
characterize the epiphytic mioroflora of vegetable tissues. 

Observations can readily be made on the microbiological 



222 THE MICROBIOLOGY OS 1 BAKING 

changes which occur in a spontaneously fermenting dough by 
intermittent bacteriological analyses. Such analyses reveal 
that the original microflora, of a sound wheat flour for in- 
stance, which consists of short rods of the size of Bact. coli 
commune, interspersed with spore bearing types of the groups 
of aerobic soil bacilli, changes during the first 20 hours into 
one composed almost exclusively of non-sporing rods forming 
yellow colonies on ordinary media. The number of micro- 
organisms present in the dough at the end of this period may 
represent a 25,000 fold increase or more in the original flora. 
Macroscopically also certain changes are noticeable. The 
protein has softened and gas bubbles have began to appear 
in the dough. An accumulation of acid has not yet become 
noticeable and can be detected only after the fermentation 
has proceeded for 30 to 40 hours. At this stage the number 
of short rods, which dominated the microflora previously, has 
given way to some extent to types of lactic acid bacteria, 
sometimes of the Streptococcus lactis acidi type and some- 
times of the Bact. acidificans longissimum type, and to 
spore-bearing rods of the butyl alcohol-producing group. It 
is during this period that a vigorous gas evolution occurs in 
the dough. 

From this it is clear that, where a spontaneously ferment- 
ing dough is used as a leaven to initiate fermentation in a 
fresh batch of dough, the value of the addition will depend 
on the age of the leaven. Vigorous gas evolution will be 
achieved only when the leaven has reached a stage when 
spore-bearing anaerobes of the butyl alcohol-producing 
bacilli have developed. A leaven of this type is employed 
in the Southern States of the United States of America to 
prepare the so-called 'salt-risen' bread. Kohman 12 has given 
a detailed description of the preparation of salt-risen bread. 
He remarks that yeasts are seldom met with as an important 
group of the microflora. In this type of leaven, therefore, 
the active microflora is essentially of bacterial origin. 

On examination of a sample of sour dough, the type of 
leaven which in earlier days was extensively used for the 



THE MICROBIOLOGY OF BAKING 223 

raising of all types of dough, and which is still employed 
technically in the preparation of rye bread, the impression 
is gained that similar conditions prevail and that bacteria 
take an important part in the gas evolution. Microscopic 
preparations and bacteriological analyses have frequently 
been made of sour doughs and the preponderance of bacteria 
in the microflora has usually been commented upon. SchiOtz- 
Christensen, 13 for instance, found 8 bacteria to every 3-5 fungi 
and 2-6 yeast in a sour ' dough ' of Danish origin. The bacteria 
were principally lactic acid bacteria, acetic acid bacteria, and 
butyric acid producing types, while the fungi included Peni- 
cillium glaucum, Cladosporium herbarum, and Oidium lactis. 
Among the yeasts were both typical saccharomycetes and 
species of Tonda. 

Nevertheless, the activity of bacteria is not essential for 
the proper functioning of a 'sour dough', at least not for the 
production of gas, a reaction for which species of saccharo- 
mycetes are responsible. This has now been experimentally 
confirmed, but had already been indicated by Girard's 14 
observations in 1885, that the proportions of alcohol and car- 
bon dioxide given off by a 'sour dough 5 are identical with 
those produced in the fermentation of sugar by yeast. 

It is scarcely relevant to discuss in detail all the earlier 
work on the microbiology of sour dough. The observations 
made were frequently of a highly speculative character 
for instance those of Chicandard, 1 who regarded the active 
organism as a bacillus producing gas by decomposition of the 
protein of the flour or they bear unmistakable evidence of 
the technical difficulties which prevailed at the time and which 
made an exhaustive study difficult, if not impossible. This 
applies to the investigation of Engel, 15 who reported the 
presence in 'sour dough' of a specific type of yeast, Saccharo- 
myces minor, to the studies of Marcano, 16 Laurent 17 and 
Popoff, 18 who held that bacteria were responsible for the 
leavening action of sough dough, and to those of Dunnen- 
berger, 6 Boutroux 7 and Jago, 19 who regarded yeast as the sole 
active type. 



224 THE MICROBIOLOGY OF BAKING 

A more exhaustive investigation, which involved closer 
attention to technical difficulties, revealed that the action of 
sour dough leaven is due to a combined activity of yeast and 
bacteria. Peters 20 arrived at this conclusion after isolating 
three types of yeasts, including one resembling Saccharomyces 
minor, and five bacteria from samples of 'sour dough'. And 
Wolffin 8 expressed a similar opinion, finding the function of 
'sour dough ' to be due to a combined action of Saccharomyces 
minor and Bact. levans. 

This conception, however, requires that the gas given off 
by a 'sour dough 5 should contain a certain percentage of 
hydrogen, since Bact. levans produces a mixture of carbon 
dioxide and hydrogen. But hydrogen is not a component of 
the 'sour dough' gas. This has been shown both by Gerard 
and by Jago. 

In Holliger's, 3 in Burn and Holliger's, 21 and in Budinoff's 22 
investigations the function of the bacteria of 'sour dough 5 is 
considered to be of secondary importance, being limited to 
the production of acids, notably lactic acid and acetic acid, 
which, according to Lehmann, 23 are normally present in bread 
prepared from sour dough. The concentration produced dur- 
ing the leavening of rye bread is equal, SchiOtz-Christensen 
remarks, to that of a 0-75 per cent, solution of sulphuric 
acid. Assuming the acids to have been lactic and acetic 
acids, their concentration must have been equivalent to 
a pH value of 2 to 4, depending on the buffering action of 
the rye flour. The gas evolution is attributed solely to species 
of saocharomycetes, either Saccharomyces minor or other 
species of the genus, including Saccharomyces cerevisiae. 
That this view is correct is confirmed by SchiOtz-Christensen's 
statement that bakeries in Copenhagen produce a normal 
bread, when the dough has been leavened with pure cultures 
of Saccharomyces species. 

Such bread obviously must be less acid than samples in 
which considerable numbers of lactic acid and acetic acid 
bacteria have been allowed to develop, and Schiotz-Christen- 
sen does not comment on the extent to which its flavour 



THE MICROBIOLOGY OF BAKING 226 

compares with that of ordinary rye bread which Knudsen 24 
and Becoard 25 state requires the presence of acid-producing 
bacteria to develop its desired flavour. Nevertheless, the 
fact remains that the leavening action of 'sour dough', and 
notably its gas-producing properties, are due to the action of 
various species of yeast. In principle, therefore, there is little 
difference between the leavening secured by the addition of 
'sour dough' and that obtained by the introduction of active 
yeast in some other form. 

The origin of the yeast found in 'sour dough' has not yet 
been traced. That it is not necessarily identical with brewer's 
yeast or wine yeast has been established, and indicates that, 
originally, it may have been introduced with the natural 
microflora of the flour, used for the preparation of the leaven. 
Another source of yeast for leavening purposes was available 
in the Roman Empire, and probably in other wine-growing 
countries, in the form of fermenting grape juice, which, as 
already mentioned, was utilized at an early date for this 
purpose. In northern countries a similar leaven was prepared 
by the addition to a dough of fermenting beer wort, the 
'foam' or 'barm', of such beer wort. In the course of time 
numerous methods have been devised for the preparation of 
suitable barms, a subject which was discussed in some detail 
by Jago, 26 and more recently by Ellis. 27 Of these various 
methods, the only one still in practical use, at least in the 
British Isles, is that termed Parisian barm. As its micro- 
biology is more or less representative of all other types, the 
subject of barm preparation will be dealt with by describing 
the procedure in the preparation of Parisian barm. 

A mash is prepared from 2 to 3 kgs. of malt and 9 litres of 
water, previously heated to 70 C. This mash is maintained 
for 2 to 3 hours at a temperature of 60 C. and is then filtered 
through fine muslin. 

The resulting wort is warmed to about 66 C., and at this 
temperature is mixed with a handful of flour in such a way 
that the flour becomes evenly distributed and uniformly 
gelatinized. The wort is now allowed to cool and to stand 

Q 



226 THE MICROBIOLOGY OP BAKING 

for 2 days or more*at 46-5 C. During this period it becomes 
slightly sour, presumably owing to the development of lactic 
acid bacteria. When the acidity has reached the desired 
degree, the wort is inoculated with a quantity of old barm, 
usually about 3 per cent. Like sour dough this barm consists 
essentially of species of yeast which set up a vigorous fer- 
mentation in the wort. The fermentation is usually completed 
in 16 to 24 hours, when, after cooling, the fermented wort is 
ready to be mixed with flour for the preparation of dough. 
In many oases this dough is, or was, prepared in two stages 
perhaps, as suggested by Jago, 26 to f acilitate the incorporation 
of the flour. 

The final stage consisted in the mlying of the barm (perhaps 
originally the whole, but subsequently usually half or a 
quarter) with flour sufficient to form a thick paste or ' sponge '. 
This was left for 12 hours at a temperature of 28 C. to 37 C. 
By that time the remaining flour could be mixed to form the 
final dough with less effort than if the mixing had been done 
in one operation. This final dough was then allowed to stay 
in a warm place for l to 2 hours, sufficient to ensure its 
satisfactory aeration. During this tune the dough would 
usually be kneaded once to assist the fermentative changes 
taking place by introducing additional oxygen, thus favouring 
the activity of the yeast. 

During the 'resting' of the sponge a fermentation of the 
available sugars would set in and carbon dioxide be de- 
veloped. At the same time the colloidal suspension of the 
flour proteins would become more pliable, the starch slightly 
hydrolysed, and a flavour developed in the sponge; all of 
which, when imparted to the bread, would render its crumb 
lighter, more aromatic and less likely to dry up. 

As implied by the name barm, the active type of micro- 
organism in dough prepared by this method was yeast, beer- 
yeast, or 'brewer's yeast' as it is generally called, of the top 
yeast type. When barm dough was widely used this type 
of yeast was apparently found satisfactory. It has since been 
found to be much inferior to the bottom yeasts used by 



THE MICROBIOLOGY OF BAKING 227 

distillers and supplied commercially tojbakers as 'pressed 
yeast'. The reason for the superiority or the latter type has 
been shown by Baker and Hulton 28 to be the presence in 
flour of some thermolabile toxic substance to which distiller's 
yeast has become acclimatized, while brewers yeast has not. 

It is a question of some economic importance to devise 
means whereby the fermenting power of brewer's yeast, 
when growth in flour and water mixtures can be increased 
to become more nearly equal to that of distiller's yeast. 
Baker 29 suggests that one way of doing this is to cultivate 
brewer's yeast for three generations in typical distiller's mash, 
that is, in solutions which have not been heated to tempera- 
tures high enough to destroy the thermolabile flour toxin. 

In course of time barm preparations were replaced by 
distillery yeast, a change which to some extent could be 
regarded as an improvement, since it eliminated the dangers 
due to faulty barms. 

In the preparation of dough with pressed yeast the micro- 
biological aspects became restricted to the making of 'sponge ' 
and the subsequent fermentation of the dough. A further 
simplification was introduced when powerful kneading 
machines became available, and in most bakeries to-day 
direct preparation of the dough in one operation is the usual 
procedure. The risk of faulty fermentation is reduced to a 
minimum in this 'straight dough' procedure. A considerable 
saving in time is also secured, since the whole of the dough 
can be completed in 3 to 5 hours if sufficient yeast is added 
in the first instance. But in spite of these advantages the 
adoption of the straight dough procedure has not been entirely 
satisfactory. The suggestion has been made that the bread 
made by the straight dough procedure is lacking in something 
desirable in flavour and keeping properties. And, of course, 
an added expenditure is incurred by the addition of a larger 
quantity of yeast. 

It is very natural, therefore, that efforts should have been 
made to secure the advantages of the earlier methods of 
dough fermentation for the technically less complicated 

Q2 



228 THE MICROBIOLOGY OF BAKING 

straight dough procedure. The efforts made in this direction 
mil be discussed later, when an account has been given of the 
microbiological changes which take place in the straight 
dough preparation itself. 

From a microbiological standpoint these changes are 
naturally considerably simpler than those taking place in 
barms. A comparatively pure culture of yeast is utilized, 
and in quantities sufficiently large to ensure the desired 
changes being completed within a few hours. There is little 
if any opportunity therefore for other micro-organisms to 
develop. The straight dough fermentation may be regarded 
as essentially a pure yeast fermentation. 

(1) TBDB HYDROLYTIO CHANGES TAKING PLACE IN THE CARBO- 
HYDRATES OF THE FLOUR DURING DOUGH RAISING 

As yeast does not hydrolyse starch, it may be asked how 
this organism can produce a volume of carbon dioxide 
sufficient to aerate a dough which consists essentially of starch, 
water, and protein. 

It is true that most flours contain a certain percentage of 
sugars, notably saccharose and glucose, but these quantities 
are not sufficient to ensure an ample evolution of gas. Von 
Liebig 30 gives for wheat flour a sugar content of 1-0 to 1-5 per 
cent, of saccharose, and 0-1 to 0-4 per cent, of glucose ; a total 
of approximately 2 per cent, of carbohydrates fermentable 
by yeast. 

Experimentally von Liebig showed that a two-hour fer- 
mentation with yeast resulted in a loss of 1-42 to 2-05 per 
cent, of sugar, indicating that the original sugar would be 
utilized by the yeast before the dough fermentation had been 
completed. Additional sources, therefore, are required if the 
dough fermentation is to proceed normally. Von Liebig 
showed that additional sugar became available through the 
activity of the flour itself, and found that the percentage of 
reducing sugars in a dough in which micro-organisms were 
absent increased during 14 hours' incubation at 30 C. to as 
much as 4-6 per cent., calculated as glucose. The sugar 



THE MICROBIOLOGY OF BAKING 229 

formed was chiefly maltose, and its rate of production cor- 
responded to a diastatic power equal to one-seventh of that 
of a kiln-dried malt in the case of patent wheat flour, and to 
one-third in the case of whole meal wheat flour. 

It need hardly be emphasized that certain conditions must 
prevail for the amylase of wheat flour to function at its 
optimum, conditions which it is desirable, if not essential, 
to maintain during the fermentation of a dough. 

The various conditions governing the activity of the 
amylase of micro-organisms were discussed in Chapter II. 
The three most important requirements were there shown to 
be the presence of sufficient moisture, an optimum tempera- 
ture, and an optimum concentration of hydrogen ions. 
Though these requirements may be asserted to be of para- 
mount importance also for the proper functioning of the 
flour amylases, they have not yet received much attention 
by investigators of the dough fermentation. 

It has been shown by Vandevelde 31 that an increase by 
30 per cent, in the water content of the dough increases the 
fermentative power of a yeast by 6-5 per cent. But it is not 
indicated whether this increase is due to the effect of the 
added water on the diastatic activity of the flour or to an 
action on the yeast itself. 

And though the establishment of an optimum temperature 
is generally accepted as of the greatest importance for the 
successful completion of a straight dough fermentation, there 
does not appear to be any publication dealing specifically 
with the influence of temperature on the diastatic functions 
of the dough. 

The influence of a suitable hydrogen ion concentration on 
the diastatic activity of a flour has been specifically referred 
to by Bailey and Sherwood, 6 by SOrensen, 32 and by Greeve 
and Bailey, 33 the last named ascribing the chief value of this 
factor to its influence on the amylases of the flour. 

An improvement in diastatio action might of course be 
achieved also by deliberate addition of extraneous amylases, 
for instance in the form of malt, malt extract or other diastase 



230 THE MICROBIOLOGY OF BAKING 

containing substances. The effect thereby secured has been 
studied by Gore 34 in the case of sweet potatoes, which are rich 
in amylase, and bv Collatz and Raoke 35 in the case of malt 

/ * V 

extracts. The latter investigators found that too large an 
addition of malt extract tended to render the dough soft or 
'wet ' and lowered the quality of the resulting bread. 

The hydrolytio changes of disaccharides, to which Bailey 
and Sherwood refer as the second important group of re- 
actions taking place in straight dough fermentations, have 
not yet been studied in detail. Bailey and Sherwood suggest 
that these reactions are carried out by maltase and saccharase 
secreted by the yeast. 

Like the diastatic enzymes, maltase and sacoharase are 
greatly influenced in their activity by temperature and by 
hydrogen ion concentration. 

(2) JTBRMENTATIVE CHANGES IN THE DOUGH 

The actual fermentation of a straight dough, the breakdown 
of monoses to carbon dioxide, alcohol and traces of other 
substances, is due exclusively to the action of the yeast, and 
these reactions are influenced not only by temperature, but 
also by the prevailing hydrogen ion concentration. 

Before discussing the importance of these factors, it is 
desirable briefly to refer to the conditions under which the 
fermentation is carried out in general practice. After the 
flour and water have been mixed in the appropriate quanti- 
ties with the desired amount of pressed yeast and salt, the 
whole is kneaded by machine until a uniform dough results. 
This dough is incubated, or 'set', at a temperature of 26 to 
27 C., by placing it in a room kept at about 28 C., and is 
allowed to stand at this temperature until it has risen to its 
maximum volume and collapses when a finger is inserted 
into it. The time taken for this stage to be reached varies 
with the type of flour used, high protein flours 'strong' 
flours taking longer than flour of low protein content 
'weak flours'. The time is markedly influenced also by 
various factors, to which reference will be made later. Under 



THE MICROBIOLOGY OF BAKTNTG 231 

normal conditions the time will be from six to eight hours. 
The dough is now re-kneaded in order to incorporate fresh 
supplies of oxygen, is weighed off to the requisite amounts, 
and placed at a temperature of 32-37 C. for about two 
hours. During this period of 'proofing' a rapid fermentation 
ensues, which recharges the matured dough with fresh carbon 
dioxide and gives it its final volume. 

The baking is usually done between 230 C. and 248 C., 
but these temperatures are never reached in the interior of 
the loaf nor even on its surface. The actual figures will de- 
pend to some extent on the size of the loaf and on the dura- 
tion of the baking. Roussel 36 registered a maximum tempera- 
ture in the interior of the dough of 101 C. to 103-5 C., and 
on the surface of 125 C. to 140-5 C. Brewster-Morison 37 
recorded a temperature of 63 C. in the interior of a 500 
gramme loaf after 15 minutes in the oven. 

Speaking generally, the factors which influence the course 
of a dough fermentation may be divided into two groups, 
one of which affects the growth of the yeast, and the other 
influences the enzymatic changes initiated by the yeast. 

This subdivision is indicated by Neumann and Knischew- 
sky's 38 observations that actual growth of yeast takes place 
during dough fermentation. By adopting a special technique 
Neumann and Knischewsky were able to count the actual 
number of yeast cells present in a dough at various stages of 
the fermentation. They ascertained in this way that a 
noticeable cell production took place when the quantity of 
yeast incorporated was equal to, or smaller than the amount 
normally added, even after a short period of three, or some- 
times even of two hours' fermentation. On the other hand, 
where the amount of yeast was appreciably in excess of the 
normal quantity of 0-25 per cent., no reproduction occurred ; 
on the contrary, a reduction of the number of cells was often 
noticeable. 

To the growth-promoting factors belong the various food 
substances such as organic or inorganic nitrogen compounds, 
which were recommended by Kohman and collaborators 39 



232 THE MICROBIOLOGY OF BAKING 

and by Elion 40 as dough stimulants. Malt and malt extracts 

were favoured by Collatz and Racke ; K for the same purpose, 

bran extracts by Quine, 41 and by White; 42 and carbamide 

potassium chlorate magnesium sulphate mixture by 

Epstein. 43 

The effect produced by these various substances can only 
be of advantage where a reproduction of the yeast is desired 
and where the number of yeast cells finally present in the 
dough does not exceed the maximum which can be incor- 
porated without imparting a ' yeasty ' flavour. This maximum 
is usually very much in excess of the quantity of yeast nor- 
mally taken ; in the case of rye bread it was estimated by 
Holdefleiss and Wassling 44 as 4 per cent, calculated on the 
flour taken. Nevertheless it is within the bounds of possi- 
bility that it may be exceeded during fermentation unless 
the initial yeast addition is kept low. Where the initial 
addition of yeast is low the use of growth-promoting sub- 
stances is of practical importance from the point of view of 
reducing the percentage of yeast required for the normal 
raising of a dough. 

The second group of factors, the enzyme accelerating 
substances which increase the rate of gas evolution, and 
sometimes the total amount of gas produced, are controlled 
by the presence or absence of acids. Wahl 45 came to the 
conclusion that a dough, fermented by yeast in the presence 
of lactic acid bacteria of the type of Bact. Delbrucki, gave a 
more uniformly aerated and a better flavoured loaf than 
dough fermented in the absence of such bacteria. A similar, 
though not quite so pronounced effect, was observed where 
lactic acid was added instead of lactic acid-producing bacteria. 
Chabot, 46 who dealt with the subject, found that the addition 
of acid caused an increased fermentation so long as a maxi- 
mum hydrogen ion concentration was not exceeded. With 
little, if any justification, this observation has been interpreted 
as proving that an addition of acid guarantees an early 
maturing of a dough. Thus Wagner and Glaban 47 maintained 
that the changes suffered by the flour protein during dough 



THE MICROBIOLOGY OF BAKINTG 

fermentation could be brought about by the addition 
acids. This is not correct, however, and is contradicted 1 
Bailey and Johnson's 48 observations, which showed tha& 
doughs, made from flour which for some reason or other had 
acquired an abnormally high acidity, and which, according 
to Wagner and Glaban, should have matured in a shorter 
period than normal doughs, gave unsatisfactory loaf charac- 
teristics, unless fermented for the normal period. 

A number of other substances have been recommended as 
useful for accelerating the dough fermentation. Kohman 49 
recommended iodates and bromates, or their corresponding 
free acids, which he added in concentrations of 0-0005 per 
cent, calculated on the flour. Potassium salts were also found 
by Kohman and his collaborators 39 to have a slightly bene- 
ficial action. The effect of ethyl alcohol, caraway seed, onions 
and various essential oils, such as clove oil, was determined 
by Neumann and Knischewsky. 60 They found that all of 
these substances except onions, which were doubtful in their 
action, favoured the dough fermentation when added in 
very small quantities. This observation lends experimental 
evidence in support of the old custom prevailing in some 
countries of mining caraway seed with the dough. 

The interpretation given by Neumann and Knischewsky 
of the action of ethyl alcohol, that its presence checks the 
development of harmful bacteria, is probably not correct. 
In the concentration of 1 per cent, in which it was found to 
act most favourably, it could not prevent the development 
of the bacteria of the normal microflora of the flour. 

Oxidizing agents were tested by James and Huber, 61 who 
could find no improvement in the dough after their addition. 
Oxygen itself, however, is an important accelerator. Though 
no actual investigation appears to have demonstrated this, 
it is clear from experience that this is the case. It has long 
been recognized that thorough and repeated kneading, which 
introduces a fresh supply of oxygen into a dough, is of the 
greatest value for successful maturing. The action of the 
oxygen is probably that of a hydrogen acceptor, and it there- 



234 THE MICROBIOLOGY OF BAKING 

fore functions primarily in the conversion of the sugars into 
carbon dioxide and alcohol. It should be replaceable there- 
fore by other hydrogen acceptors, methylene blue for in- 
stance. It would not be without theoretical interest to 
ascertain whether this is the case. 

In this connexion the observations of Masters and Maughan 62 
on the accelerating influence on the dough fermentation of 
small quantities of fresh ox-serum are noteworthy. Additions 
of 1 per cent, of this substance were claimed to improve the 
fermentation sufficiently to cause an increase in volume of 
the finished loaf of between 15 and 16 per cent. The action 
was not due, as might have been thought, to the introduction 
of additional nitrogen, since serum more than three days old 
showed little if any action. The nature of the stimulant con- 
tained in fresh ox-serum has not yet been established. The 
agent is apparently thennolabile since it was found to be 
rapidly destroyed on boiling. Lower temperatures, 60 C. 
for instance, were withstood for some time. On standing the 
serum was inactivated after a few days ; it could be preserved, 
however, for a considerable time when precipitated as part 
of the protein precipitate obtained on saturating the serum 
with ammonium sulphate. Addition of small concentrations 
of formaldehyde to fresh ox-serum also preserved it. 

Against these observations on substances which favour 
the dough fermentation must be set the observations which 
have been made on innocuous and inhibitory substances. 

To the first group belong the chlorides, nitrites, nitrates 
and sulphates, tested by Kohman and his collaborators. 
Phosphates, at least when a sufficiency of phosphates is 
already present in the dough, were also ineffective. 

These observations of Kohman's, which wore confirmed by 
Neumann and Knischewsky, are of particular interest as 
regards the chlorides, since common salt has for years past 
been regarded as a retarder of the fermentation, checking also 
the development of undesirable bacteria and fungi. 

Vandevelde's statement that a high gluten (protein) con- 
tent in a flour affects the fermentation disadvantageously 



THE MCEOBIOLOGT OF BAKING 235 

may appear surprising. If confirmed this may be found to be 
due to the fact, elucidated by Bailey and Johnson, 48 that an 
optimum hydrogen ion concentration takes longer to establish 
in a dough with high gluten content than in one with a normal 
or small percentage, owing to the increased buffering action 
of the flour with high gluten percentages. It cannot be due 
to a direct inhibitory influence exercised by the large protein 
content. 

An inhibitory influence is undoubtedly caused by the 
addition of larger doses of certain spices or their corresponding 
essential oils and of many chemicals. It has not been de- 
termined whether the action in these cases affects the de- 
velopment of the yeast cells or the functioning of their 
enzymes. 

For the study of the dough fermentation, that is for the 
elucidation of the conditions affecting it, and of the sub- 
stances interfering with its normal course, it has been im- 
portant to devise tests which indicate a normal progress of 
the fermentation, the attainment of a maximum gas evolution, 
and the completion of the fermentation. 

In the bakehouse such tests have been empirically evolved 
and have been restricted to ascertaining when a dough was 
ready for proofing (weighing). This may be done by inserting 
a finger into the dough, which, if matured, will recede or 
collapse. 

The explanation given by Bailey and Johnson of this test 
is that the gluten of a mature dough has been softened to 
such an extent that it can no longer prevent the escape of gas, 
which, in consequence, is being lost at a rate faster than that 
at which it can be replaced, probably because of the rupturing 
of vesicles of gas at the surface of the dough and perhaps 
indirectly owing to an exhaustion of the available sugar 
supplies setting in. At this stage, therefore, the dough must 
have reached its maximum volume. 

Basing their efforts on this assumption, Bailey and Johnson 
have attempted to evolve a reliable method of ascertaining 
when a dough has matured sufficiently to be proofed and 



236 THE MICROBIOLOGY OF BAKING 

made ready for baking. They argue that, since at a given 
stage there is a rapid loss of carbon dioxide, it should be 
possible to determine this stage by measuring the carbon 
dioxide evolution of the dough at intervals. They do this 
by absorbing the gas given off with a dilute solution of alkali 
possessing a hydrogen ion concentration corresponding to a 
pH value of 7-8. The time takers for this pH value to decrease 
to 7-0, as indicated by the changes in phenol red from pink 
to yellow, is taken as a measure of the rate of carbon dioxide 
evolution. 

In their experiments Bailey and Johnson found that a 
sharp increase in gas evolution from a dough was indicated 
by this method and coincided with the time of ripening of 
the dough. A somewhat similar method has been advocated 
by James and Huber 61 for flour and water suspensions. Here 
the fermentation is allowed to proceed in a flask and the 
carbon dioxide given off is collected in an inverted tube in 
which it moves a registering arm recording the changes. 
Either of these methods appears to be superior to that by 
which the ripening of a dough is correlated with changes in 
hydrogen ion concentration. 

Since Jessen-Hansen 63 first drew attention to the connexion 
between changes in the hydrogen ion concentration and the 
ripening of a dough, the view has gained ground that once the 
optimum pH value of 5-0 has been established, the gas evolu- 
tion of a dough has reached its maximum and the fermenta- 
tion should therefore be arrested (Brewster Morison 37 ). It 
was suggested that a simple measurement of the hydrogen 
ion concentration of a dough would suffice to show when 
proofing could be commenced. It is not surprising to hear 
that more recent investigations have had to revise this view. 

The various flours used in bread-making, even various types 
of wheat flour, differ too markedly in chemical composition, 
notably as regards their protein content, to justify the con- 
clusion that the establishment of a given hydrogen ion 
concentration in a given time is unaffected by the nature of 
the flour. This is clear from the observations of Bailey and 



THE MICROBIOLOGY OF BAKING 237 

Sherwood, 6 who found that the hydrogen ion concentration 
of a dough made with wholemeal flour increases more slowly 
than that of a dough prepared from patent flour. Bailey and 
Johnson 48 recorded that, in the case of a wholemeal dough 
which is allowed to ferment until its optimum pH value of 
6-0 has been reached, the resulting bread was inferior, the 
protracted fermentation having rendered the crumb wet, 
and having greatly increased the loss of dry matter. As a 
contrast these writers record that, where the fermentation 
of a patent flour which for some reason or other had acquired 
an abnormally high acidity, was arrested through the hydro- 
gen ion concentration of the dough had reaching its optimum, 
the resulting loaf was harsh and 'unfinished'. It is hardly 
safe to assert, therefore, as has occasionally been done (Wagner 
and Glaban 47 ), that the time of fermentation of a dough may 
be shortened, and perhaps even almost completely eliminated 
by artificially establishing an optimum hydrogen ion con- 
centration in the dough. 

Apart from the attention paid to the changes in the hydro- 
gen ion concentration of a dough and to the production of 
carbon dioxide and alcohol, surprisingly little time has been 
spent on the study of the biochemical changes which take 
place as a result of the activity of yeast and perhaps of other 
micro-organisms. Undoubtedly there is here a wide field for 
future investigations. 

It is realized that the increase in hydrogen ion concentra- 
tion during fermentation is due to the production of one or 
more acids, but their exact nature has not been determined. 
It seems likely that the carbon dioxide, which has been 
known since Chicandard's time to constitute the bulk of the 
gas included in a dough, may take a part in the lowering of 
the pH value, though the chief agencies in this respect are 
probably the various acids formed during fermentation. 
The increase in pH value of a loaf which can be noticed during 
baking, and to which Bailey and Sherwood drew attention, 
indicates that part of the dough acidity must be of a volatile 
nature. The bacteria responsible for its production are 



238 THE MICROBIOLOGY OF BAKING 

identical with or related to Bact. acidificans longissimum, 

Lafar, a type which, is added in many distilleries to the wort 

to encourage the growth of the yeast and to suppress other 

bacteria. 

The appearance and disappearance of ethyl alcohol, the 
second important fermentation product of yeast, during 
dough raising and on baking respectively, has been discussed 
by some writers. Graham demonstrated very forcibly the 
possible economic importance of this question when pointing 
out, according to Pohl, 64 that more than a million and a 
quarter litres of alcohol were allowed to escape yearly from 
the ovens of the bakeries of London. 

The percentage of ethyl alcohol present in straight doughs 
may be placed at somewhat more than 1 per cent., the figure 
obtained by Snyder and Voorheer, 55 and subsequently con- 
firmed by Czapek. 66 During baking the bulk of this alcohol 
escapes, but the finished bread, nevertheless, contains 
measurable quantities. The figure obtained by Bolas 67 for 
fresh London bread was 314 per cent., and for a week old 
bread 0-120 to 0-139 per cent. These figures are possibly 
somewhat high and are probably based on analyses of bread 
made with barm or sponge. The more recent data of Pohl, 
which refer to straight dough bread, are lower and record 
0-0508 per cent, of alcohol in fresh bread. All the investi- 
gations carried out on the presence of alcohol in bread have 
confirmed Sandberg's 68 conclusion that the original alcohol 
content of fresh bread is reduced on the week's storage by as 
much as 55 per cent. 

Recently attempts have been made to recover the very 
appreciable quantities of alcohol which are produced in the 
bakehouse. Andrusiani 60 describes one such attempt which 
would appear, however, to have met with little economic 
success. It is based on an alteration to the bakeoven which 
makes it possible to withdraw and to condense the vapours 
emanatingfrom it. These vapours were claimed by Mousette 00 
to contain 1-6 per cent, of ethyl alcohol and 0-06 per cent, of 
acetic acid. 



THE MICROBIOLOGY OF BAKING 239 

Since the quantity of ethyl alcohol produced during the 
dough fermentation may amount to over 1 per cent, of 
the total weight of the dough, and knowing that the quantity 
of carbon dioxide evolved must be even greater, it will be 
appreciated that the inroad made during fermentation on the 
carbohydrates of the dough cannot be regarded as negligible. 
It was with a view to restricting this loss of carbohydrates 
that the various attempts already referred to of shortening 
the fermentation period were made. Their practical value 
has been questionable. 

A more promising method would appear to be that ad- 
vocated by Dore'e and Kirkland 61 of combining the old 
fashioned barm method of preparation of dough with the 
simplicity of the straight dough process. They attain this 
by preparing a 9 per cent, malt extract solution, and by 
fermenting it with 1 Ib. of yeast per sack of flour. The fer- 
mentation completed, the extract is mixed with the whole 
of the flour and the dough is allowed to ferment for 3 hours 
after kneading in the usual manner. In this way they utilize 
a yeast culture in a liquid medium as a starter instead of 
pressed yeast. This procedure was found by Scliber and 
Bovshik 62 to be more economical than the use of pressed 
yeast, as regards consumption of raw material for the pro- 
duction of the yeast cells required to raise a given unit of 
dough. 

An entirely different method of reducing the consumption 
of carbohydrates during dough raising is achieved in the 
making of 'salt-risen' bread, a type of bread which is exten- 
sively prepared in the Southern States of the United States 
of America. The leaven for this type of bread is made from 
maize meal, salt, soda, and milk, which are mixed into a batter 
and kept warm, usually for 15 to 20 hours, until gas begins 
to be formed. The batter is then made up with wheat flour 
into a slack dough and is allowed to ferment for 1 to 3 hours. 
According to Kohman 12 the gas evolved is a mixture of carbon 
dioxide and hydrogen in the proportions of 1 part of the 
former to 2 parts of the latter, indicating that bacteria are 



240 THE MICROBIOLOGY OF BAKING 

primarily responsible for the gas production. This is con- 
firmed by microscopic examination and bacteriological 
analysis of the dough. One of the bacteria isolated by Koh- 
man was Bact. levans. Except for small quantities of acids no 
fermentation products other than hydrogen and carbon 
dioxide are produced during salt dough raising, and less flour 
is therefore required for leavening than in the case of a yeast 
raised dough. 

Koser 63 refers to a similar method of leavening adopted by 
certain bakeries in Washington. Here an anaerobic butyric 
acid producing bacillus can be identified as the responsible 
micro-organism. Koser refers to the close relationship of this 
type to the pathogenic Bac. WekJiii, a species which he utilized 
to leaven experimental dough samples. 

(3) PROTEOLYTIO CHANGES IN THE DOUGH DURING RIPENING 

No less important than the fermentation of the available 
carbohydrates is the fourth group of reactions which occur 
during the leavening of a dough, those affecting the proteins 
present. Bailey and Sherwood refer to this group of reactions 
as the proteolysis or hydrolysis of the gluten. This proteolysis 
proceeds simultaneously with the conversion of the carbo- 
hydrates (Sharp and Schreiner 64 ) and is markedly influenced 
by the hydrogen ion concentration of the dough, a pH value 
of 6-0 representing an optimum. 

Sharp and Schreiner 64 have shown that the plasticity of the 
protein increases to a maximum as the yeast fermentation 
proceeds, but there is no definite experimental evidence to 
show that proteolytio enzymes, secreted by the yeast, axe 
responsible for this change. In fact it is still an open question 
whether the softening, or increased plasticity of the gluten 
during dough making, is due to enzymes produced by the 
yeast, by the flour, or by other agencies. Perhaps it may be 
taken as an indication that, since barms give rise to a more 
plastic protein than the straight dough fermentation, the 
proteolytic activity is governed by other factors than the 
enzymes of yeast and flour. 



THE MICROBIOLOGY OF BAKING 



241 



(4) CHANGES IN HYDROGEN ION CONCENTRATION OF THH 
DOUGH DURING RIPENING 

Turning to the last of Bailey and Sherwood's five important 
groups of reactions which governs the fermentation of a 
dough, it may be recalled that an optimum hydrogen ion 
concentration must be established in a dough for the various 
reactions to take place under favourable conditions. 

It is a remarkable and fortunate coincidence that these 
various hydrogen ion concentration requirements should be 
practically identical, and that not only the diastatio changes 
of the flour, but also the hydrolysis of the disaccharides 
thereby produced, the gas evolution, and the proteolysis of 
the gluten should proceed most favourably on the acid side 
of the neutral point. Jessen-Hansen 63 and most other in- 
vestigators give the optimum hydrogen ion concentration 
as equivalent to a pH value of 5-0 to 5-2. This reaction is 
considerably more acid than that of the flour itself, at least 
under normal conditions and in the case of patent wheat 
flours, for which Chabot 46 records a pH value of 6-0 to 6-4, 
and Fisher and Halton 65 pH 5-8. During the fermentation 
of a dough production of acid is therefore essential to establish 
an optimum reaction. 

There are in existence a number of data, determined by 
Bailey and Sherwood, which show the rate of increase in 
hydrogen ions of straight doughs prepared with wheat flour. 

TABLE VHI 



Treatment of the dough. 


Age of dough 
in 
minutes. 


Hydrogen ion 
concentration 
of dough. 


Dough mixed 
After minutes fermentation 



108 


pH value 
62 
5-88 


After minutes fermentation 


156 


578 


After minutes fermentation 


180 


5-76 


After proofing 
Bread after baking 


270 
300 


5-67 
5-75 



242 THE MICROBIOLOGY OF BAKING 

They are average figures of determinations from seven 
individual experiments. They indicate that the most suitable 
hydrogen ion concentration is not normally reached by this 
method of dough raising. 

A nearer approach to optimum conditions was obtained 
by Bailey and Sherwood when the dough was prepared by 
sponging. In this case the following hydrogen ion concentra- 
tions were recorded. 

TABLE IX 



Treatment of dough. 


Age of dough 
in 
minutes. 


Hydrogen ion 
concentration 
of dough. 


Sponge prepared 
Sponge after minutes fermentation 
Sponge after minutes fermentation 
Sponge on completion 
Dough after Tm-nng 
Dough after proofing 
Bread after baking 



120 
240 
340 
360 
425 
460 


pH value 
6-71 
4-49 
5-20 
494 
5-53 
5-34 
542 



It is not to be overlooked that the increase in acidity of a 
dough during fermentation is influenced by other factors 
than that of the mode of ripening. It is influenced also by the 
buffering properties of the flour used. Morison and Collatz 66 
give several instances of how the addition of identical quanti- 
ties of the same acid to two different flours bring about a 
greater hydrogen ion concentration in the dough made from 
one flour than in that made from the other Similar results 
were obtained by Fisher and Halton. 66 As a rule it may be 
claimed that the hydrogen ion concentration of doughs made 
from a 'clear' flour which possesses very pronounced 
buffering properties is less readily changed than those of 
doughs made from patent flour. 

There may be cases, therefore, where in practice it will be 
difficult, if not impossible, to bring the hydrogen ion concen- 
tration of a dough to the figure which until recently has been 
considered the most suitable for ripening purposes. Accord- 



THE MICROBIOLOGY OF BAKING 243 

ing to Fisher and Halton 67 this would appear to be unim- 
portant, provided that the reaction of the dough is not 
allowed to change from the acid side of the neutral point to 
the alkaline side. Also according to them it is infinitely more 
important to ascertain that the reaction of a dough does not 
fall below a pH value of 5-0, than to secure that it is main- 
tained near this figure, since their experiments have con- 
vinced them that a rapid deterioration of the dough and the 
loaf results from carrying out the dough fermentation at pH 
values of 4-8 or less. 

While this is probably correct, it is difficult to harmonize 
Fisher and Halton's statement that the hydrogen ion con- 
centration of a dough is unimportant provided it is main- 
tained on the acid side of the neutral point and does not fall 
below the pH value of 4-8, with the observations of other 
workers on the value of the addition of acids to improve the 
baking qualities of a dough. 

There are the findings of White, for instance, showing that 
an acidulated extract of bran, when added to a dough, im- 
proves the fermentation more than a neutral extract of the 
same bran, and there are the observations of Wahl 46 on the 
value of the addition of lactic acid to a dough, a value which 
is ascribed by him to the increased hydrogen ion concentra- 
tion thereby secured. 

The nature of the acids responsible for the increase in 
hydrogen ion concentration of a fermenting dough has not 
been clearly established. The fact that the acidity decreases 
during baking must be interpreted, as already mentioned, as 
proof that part at least of this increase is due to volatile 
fermentation products such as carbon dioxide and acetic 
acid, the latter of which was observed in sour doughs by 
Lehmann. 23 



244 THE MICROBIOLOGY OF BAKING 

LITERATURE 

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THE MICROBIOLOGY OF BAKING 245 

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CHAPTER XX 

DISEASES OF BREAD 

THE introduction of the ripened dough into the baking oven, 
which usually is maintained at temperatures between 230 C. 
and 248 C. (Brewster Morison 1 ), is the starting-point for far- 
reaching microbiological changes in the dough. After a brief 
interval of greatly increased activity as the temperature of 
the dough rises, all enzymatic activity comes to a standstill, 
probably at a temperature between 65 C. and 75 C. The 
moment for this cessation of the enzymatic functions will 
depend on the volume of the dough. In loaves of the usual 
commercial size it probably occurs twenty minutes after 
placing the loaf in the oven. As the baking proceeds the 
temperature of the dough increases and finally reaches a 
maximum, determined by Roussel 2 as 140-5 C. for the sur- 
face and 103 C. for the interior of the loaf. Other observers, 
Russell 3 for instance, doubt whether the internal temperature 
of the loaf ever exceeds 100 C. 

The effect of the maximum temperature on the microflora 
of the dough has not been investigated in very great detail. 
Nevertheless there are sufficient observations to show that 
though this effect is destructive it does not completely elimi- 
nate micro-organisms, at least not in the case of the spore- 
forming bacteria occurring in the crumb. On the surface of 
the dough, in the crust, no micro-organisms or their spores 
can withstand the temperature of the baking, and this part 
of the loaf leaves the oven sterile. 

The fate of fungus spores during baking, when contained 
in the interior of the crumb, was investigated by Welte, 4 who 
placed large numbers of the spores of Penicillium glaucum, 
Aspergillus nidulans and Mucor stolonifer, enclosed in filter 
paper, in the centre of a dough ready for baking. He found 
that, on removal of the test loaves from the oven, all of these 
spores had been destroyed 

The resistance to the baking temperature of non-spore- 



DISEASES OP BREAD 247 

forming bacteria enclosed in the dough was studied by 
Roussel and by von Fenyvessy and Dienes, 6 who agree that 
these forms are destroyed during baking. Roussel points out, 
however, that tubercle bacteria present in the dough retain 
their viability after baking. Nevertheless there is little doubt 
that Roussel's conclusions are essentially correct, and that 
normally non-spore-forming bacteria, as well as yeasts and 
fungi and their spores, are destroyed during baking. Types 
which may be found in the crumb comprise cocci and some- 
times lactic acid producing bacteria of the type of Strepto- 
coccus lactis acidi. These organisms can be shown occasionally 
to have survived by emulsifying a piece of the crumb of a 
newly baked loaf, collected under the strictest aseptic con- 
ditions, with sterile physiological salt solution and incubating 
the emulsion for 24 hours or more at 30 C. 

In contrast to the spores of fungi, those of spore-forming 
bacteria, notably of the group of aerobic soil bacilli, are fre- 
quently found in fresh bread. They have undoubtedly 
survived the temperature of baking. 

Where they occur, and also in the more exceptional cases 
of the survival of cocci and lactic acid bacteria, there is a 
possibility of microbiological activity starting in the interior 
of loaves which remain stored for some time, provided their 
water content does not fall appreciably below the normal 
of 45 to 48 per cent. In practice little, if any harm, is done 
by this surviving microflora, except where the crumb is 
infected with certain types of Bac. mesent&ricus, or where 
butyric acid producing bacteria have survived. In the first 
case a loaf may become ropy on storage a complaint which 
has been, and to some extent still is, of considerable economic 
importance. In the second case sour bread results, a com- 
plaint which in earlier days was regarded as a fairly common 
disease of bread (Atwater 6 ). The subject of ropy bread will 
be dealt with in some detail in this chapter. 

When a loaf has left the oven, its sterile crust becomes 
exposed to infection with micro-organisms which may be air 
borne or may be carried by insects (Roeser 7 ) or man. This 



248 DISEASES OF BREAD 

infection comprises both bacteria and fungi, the latter being 
the more important, since they develop at comparatively 
low moisture contents. Welte 4 mentions that he observed 
growth of fungi on bread containing no more than 25 per 
cent, of moisture. 

A more detailed discussion of observations made on the 
action of fungi on bread will form a section of this chapter. 
Their range of activity comprises the whole loaf, sliced bread 
and other baking products which are unprotected by a crust. 

The various products of the bakery are affected also by 
infections other than the common bread fungi, infections 
which may be either bacterial or fungal in origin. The ap- 
pearance of imaginary blood on bread and the formation of 
'chalky' bread are cases in point which may well lay claim 
to separate discussion. 

There remains to be recorded the fact that the growth of 
aotinomyoetes has not yet been associated with the deteriora- 
tion of bread and other baking products. This may well 
appear surprising in view of the ubiquitous occurrence of the 
actinomycetes and of their marked starch hydrolysing pro- 
perties. Research in this field remains to be initiated. 

ROPY BREAD 

The disease of bread, technically described as 'rope ', manifests 
itself in its early stages as a brownish to reddish-brown spotty 
discoloration of the crumb and imparts to the affected bread a 
faint sickly smell. The brownish spots subsequently coalesce 
and spread, finally turning the whole of the crumb into a 
brown, semi-liquid, mucilaginous mass which may sometimes 
be drawn out into long threads. By this time the odour of 
the crumb has become most objectionable, not unlike that of 
a mixture of rotten fruit and over-ripe cheese. 

The disease does not usually become evident until about 
12 hours after a loaf has left the oven, but once started it 
spreads quickly through the crumb and may destroy it in the 
course of a day when conditions are favourable. In this 
connexion it is noteworthy that rope has not been observed 



DISEASES OF BREAD 249 

in stale dry bread and that the most dangerous period for the 
outbreak of rope is the time during which the loaves are 
cooled subsequent to their removal from the oven. 

The first investigator to associate micro-organisms with 
the appearance of rope was Laurent, 8 who isolated a bacillus 
from affected bread and described it under the name of Bac. 
panificans. According to him this bacillus was responsible 
also for the normal fermentation of a dough, and caused rope 
only where the bread contained insufficient acid. As a means 
of preventing rope Laurent recommended the addition of 
acetic acid during periods when and in places where the 
disease occurred. 

Though subsequent investigations have shown that Sac. 
panificans is neither the specific organism of the normal 
dough fermentation nor the causative agent of rope, Laurent's 
observations have been of permanent value by connecting 
the occurrence of rope with the reaction of the affected bread. 
Without exception subsequent investigators have found that 
bread and dough possessing a definite acidity are less sus- 
ceptible to the disease of rope than neutral or slightly alkaline 
bread. The reaction which is regarded as sufficient to protect 
bread was stated by Lloyd and McCrea 9 to be equivalent to a 
hydrogen ion concentration of the pH value 5-0 to 5-5. 
Cohen, Wolbach, Henderson and Cathoart's 10 investigations 
indicated that an even higher acidity might be necessary, 
and in Morison and Collatz's 11 experiments it was shown 
that an acidity corresponding to a pH value of 4-8 to 4-9 is 
required to prevent rope. At this hydrogen ion concentration, 
however, a satisfactory loaf can no longer be produced from 
patent flour. In the best of cases, and taking Lloyd and 
McCree's highest figure, there is therefore, as Fisher and 
Halton 12 point out, only a very slight margin of safety 
between the reaction required for the prevention of rope 
and that at which bread can be produced, at least when acids 
such as acetic and lactic are used to establish the desired 
hydrogen ion concentration. Fisher and Halton would appear 
to have overcome this difficulty by selecting acid calcium 



250 DISEASES OF BREAD 

phosphate instead of free acids for the adjustment of the 
reaction. They use this salt in quantities of 2 Ib. per 280 Ib. 
of flour, or at the rate of 0-71 per cent., and claim that this 
concentration successfully prevents rope in bread without 
reducing the pH value below the figure of 6-0 ; a pH value of 
5-19 being the usual reaction observed. 

A convincing explanation of this interesting observation 
is not given by Fisher and Halton. It is made clear by them, 
however, that the action cannot be due to a specific property 
of the phosphoric acid radical. 

While the reaction of a loaf is thus of paramount impor- 
tance for its protection against rope, two additional causes 
have been elucidated which are undoubtedly of significance. 
Both the percentage of moisture in the bread and the tem- 
perature at which the loaf is stored influence the development 
of rope to a marked degree as already mentioned. Thus, it 
has repeatedly been found that a wet crumb, containing 
more than the normal 46-8 per cent, of moisture, is more 
susceptible to rope than a drier loaf. 

The influence of the temperature is perhaps less clearly 
defined than that of moisture, but is, nevertheless, very 
real. The spread of the disease within a loaf is greatly 
favoured by comparatively high temperatures, 39 C., accord- 
ing to Fisher and Halton, representing the optimum. Rapid 
development is observed also at temperatures down to 25 C., 
and only below 18 C. can a loaf be stored with what Wat- 
kins 13 considers safety. Even here, however, protection is no 
more than relative, and given time there is nothing to prevent 
the disease from appearing in bread stored at temperatures 
as low as 6 to 8 C. (Vogel 14 ). 

On the whole, however, it may be claimed that rope, which 
occurs most frequently during the damp autumn months, 
can be controlled when care is taken to ensure that the hydro- 
gen ion concentration of the bread is kept at pH values 
between 5-0 and 6-2, when the loaves are cooled rapidly on 
removal from the oven, and when they are stored in a dry 
cool store-room. 



DISEASES OF BREAD 261 

It was mentioned above that the first micro-organism to be 
associated with the appearance of rope was described by 
Laurent under the name of Sac. panificans. A few years later 
Kratschmer and Niemilowicz 16 investigated the case of ropi- 
ness in a loaf of Graham bread (wholemeal bread). They 
ascribed this to the activity of Sac. mesentericus vulgatus, 
Flugg 6 ! better known under Migula's name of Bac. vulgatus, 
a type which is closely related to, but usually larger than, 
Bac. mesentericus fuscus of Fliigge, renamed Bac. mesentericus 
by Lehmann and Neumann. 16 Another type, described by 
Flugge under the name of Bac. liodermos, which is closely 
related to Bac. vulgatus, was made responsible by UfEelmann 17 
for a case of rope in rye bread. Vogel, 14 who described two 
types of rope-producing bacteria, Bac. mesentericus panis 
viscosi I and II, and all subsequent investigators have arrived 
at the conclusion that mesentericus forms are the -causative 
agents of rope. It has not been possible, however, to allocate 
the disease to one well-defined species. 

In the light of the conception of the mucus fermentation 
of carbohydrates as given in an earlier chapter this is not 
surprising, seeing that the property of producing rope must 
be possessed by all micro-organisms which can utilize bread 
as a food material under semi-anaerobic conditions and which 
under such conditions and at alkaline reactions synthesize 
hexoses to hexosans. 

In accepting this explanation of the appearance of mucus 
in 'rope', it must be admitted that some writers, KOmg, 
Spieckermann and Tillmanns, 18 for instance, attribute the 
appearance of mucus to the dissolution of the cell walls of the 
responsible micro-organisms. The evidence in favour of this 
view is far from convincing, and usually rests on the observa- 
tion that the responsible organism fails to produce mucus in 
a medium composed of starch and peptone. Whether a 
medium such as this had the required reaction on the alkalin, 
side of the neutral point or not is not stated. Nor is it very 
convincingly proved that this medium is favourable for the 
growth of the organism in question. 



262 DISEASES OF BREAD 

Since the spores of the rope-producing types of Bac. 
mesewtericus are able to withstand the temperature of baking, 
and since the disease invariably manifests itself as a deteriora- 
tion of the crumb, it is clear that the origin of the responsible 
bacteria must be sought in the dough itself, or perhaps in 
the raw materials used for the preparation of the dough, 
rather than in an infection of the loaf subsequent to baking. 
Of the various likely raw materials, the yeast was suspected 
by Russell 3 in one case. In the great majority of cases, how- 
ever, it has been possible to trace the infection to the flour 
itself, and Watkins 18 goes so far as to suggest that the rope- 
producing mes&ntericus forms may be regarded as members 
of the normal microflora of cereals. Aa a rule rye flour has 
been found to be infected more frequently than wheat flour, 
a fact which has been emphasized both by Vogel 14 and by 
Fuhrmann. 19 

For the discovery of the rope bacillus in flour Watkins 
elaborated a technique which he claimed to be sensitive 
enough to give a positive result with as little as 0-02 gr. of 
rope infected flour. Essentially the method consists in the 
inoculation of aseptically prepared pieces of crumb of normal 
bread with increasing quantities of a 2 per cent, pasteurized 
water suspension of the suspected flour, followed by incuba- 
tion of the inoculated crumb at 28 C. for 24 hours or longer. 
If no rope develops in the test samples of crumb within 48 
hours, the suspected flour is regarded as normal. 

The value of Watkins's method is rendered doubtful by the 
fact that it does not guard sufficiently against the serious 
source of error which is introduced by omitting to adjust 
the hydrogen ion concentration of the crumb used as medium. 
This is probably the reason why Fisher and Halton, 12 in their 
experiments with Watkins's method, were sometimes unable 
to detect the rope bacillus in samples of flour known to con- 
tain it. Fisher and Halton favour the baking of a test loaf 
instead of the use of Watkins's method, a procedure which 
may meet the case where sufficient flour is available. Where 
that is not so, Watkins's method should be adjusted to 



DISEASES OF BREAD 253 

obviate the source of error referred to. It would then con- 
stitute a valuable asset to the bacteriologist endeavouring 
to trace a rope infection to its source. 

As no method is known of eliminating an infection with 
rope bacteria in flour without destroying its baking properties, 
protection against rope can be secured only by establishing 
a reaction in the dough which is unfavourable for the de- 
velopment of the causative organism, by reducing the mois- 
ture content of the bread to the lowest possible figure through 
selection of suitable flours and yeast mixtures, and by rapid 
cooling and subsequent cold storage of the loaf. 

SOUB BBBAD 

It was mentioned above that the aseptically removed crumb 
of loaves may occasionally contain lactic acid bacteria which 
have survived the baking process. 

Under storage conditions of excessive warmth and damp 
these bacteria may develop and give rise to sour bread. 
Where this complaint occurs and gives rise to dietetic dis- 
orders, however, it is not the lactic acid bacteria which are 
the cause, but butyric acid bacteria, the spores of which have 
been introduced into the dough with inferior flour or yeast, 
and remaining unaffected by the temperature of the bakmg 
process, have developed in the finished loaf. 

MOULDY BREAD 

Normally fungi do not survive the baking. This was demon- 
strated experimentally by Welte. 4 A case is known, however, 
where the spores of a fungus undoubtedly did do so and in 
which the pores of the crumb of the infected loaf became 
covered with a whitish dust of spores and mycelium. Buch- 
wald, 20 who studied this case, isolated the responsible fungus 
and identified it as Monilia variabilis, described by Lindner 21 
as the causative agent of the 'chalk disease' of bread, a 
complaint which occurs in the pores of the crumb of sliced 
bread when kept for two or three days after infection. A 
similar complaint in a loaf kept for some weeks was described 



254 DISEASES OF BREAD 

by Lindner 22 as due to Endomyces fibuliger, a, type inter- 
mediate between Willia and the true Hyphomycetes. 

In the great majority of cases the mildewing of bread is 
due to an infection of the loaf with fungus spores subsequent 
to baking. The infection spreads from the surface of the 
crust to the crumb, cracks in the former serving as places of 
entry for the hyphae. The mode of infection of the crust was 
referred to in the introduction to this chapter as being due 
either to the settling of air-borne spores adhering to flour 
dust, or to the deposition of spores by insects or man. 

According to Herter 23 the deposited fungus spores appear 
to develop more readily on the crust than in the crumb, 
possibly owing to the presence in the crust of dextrins and 
water soluble carbohydrates which represent a more readily 
absorbable food than the starch of the crumb. The germi- 
nated spore will continue to develop when the moisture content 
of the bread exceeds 25 per cent., at least where the fungus is 
Penicillium glaucum or Aspergillus nidulans (Welte 4 ). Since 
it is almost impossible to prevent infection with fungus 
spores, normal bread, with its water content of 45 to 48 per 
cent., is seriously exposed to fungus attack on storage unless, 
as suggested by Herter and Fornet, 24 it is wrapped in paper 
immediately after removal from the cooling trays. 

The time taken for a fungus infection to become visible to 
the naked eye varies with the conditions prevailing, notably 
with the temperature. Under favourable conditions it may 
be between 3 and 4 days. The problem of mildewing, there- 
fore, is no longer of such practical importance as it was when 
a supply of bread was baked, which sufficed for a week's 
consumption. 

One of the best-known epidemics of mouldy bread which 
was carefully investigated by a number of workers, was that 
discussed by Payen 25 as having affected the bread supplies 
of the military bakeries of Paris in 1842. The responsible 
fungus in this case was shown to be Oidium aurantiacum, 
which gives rise to orange red spots in the crumb, and imparts 
to it an unpleasant musty odour. The same fungus is reputed 



DISEASES OF BREAD 265 

by Scheurlen 26 to have been the cause of the infection of the 
bread supplies of the armies of Alexander the Great in his 
campaign against Tyre, when its growth was associated with 
the miraculous appearance of human blood, an interpretation 
which from the earliest days of civilization and until the 
beginning of the nineteenth century was given also to the 
appearance of Bad. prodigiosum on various articles of food. 

In addition to Oidium aurantiacum and to Penicillium 
glaucum and Aspergillua nidulans referred to above, a number 
of fungi isolated from the mouldy bread have been studied by 
Herter and Fornet. 24 They include Aspergittus candidus, 
Aspergittus fumigatus, Aspergittus glaucus, Aspergillus niger, 
Mucor pusillus, Mycoderma cerevisiae, Oospora lactis, Oospora 
variabilis, Penicillium crustaceum, and Penicillium olivac&wm. 

Mucor stolonifer has also been met with on bread (Welt*), 
and Mucor mucedo was identified by Jalade 87 as a common 
bread fungus. In the epidemic investigated by him it was 
not this fungus which was the causative agent, but Monilia 
sitophila, which imparted to the crumb a rose-coloured 
fluorescence interspersed with spots of a reddish-orange or 
yellow. The growth of the Monilia, which occurred chiefly 
on sliced bread 2 to 3 days after baking, could be completely 
checked by the addition of acetic acid sufficient to raise the 
hydrogen ion concentration of the loaf to a pH value of about 
3-1 (0-2 per cent, acetic acid), an interesting point since the 
addition of acids is not usually a suitable means for arresting 
the development of fungi. For this purpose the addition 
of antiseptics such as salicylic acid has been found more 
effective by Herter and Fornet, a procedure which to-day 
can no longer be adopted, at least in the United Kingdom. 
Nor should it be necessary, if due attention is paid to 
the handling and storage of the loaf after removal from 
the oven. In this connexion it may be recalled that Herter 23 
observed that Mucor stolonifer and Penicillium crustaceum 
were the only two of the test organisms which developed at 
a temperature below 10 C. The maximum temperature at 
which Herter observed development of fungi on bread was 



266 DISEASES OF BREAD 

60 C., at which Mucor puaittus and Aspergillus fumigotua 

were still able to grow. 

It has been stated (Chalmers Robertson 28 ) that the con- 
sumption of mouldy bread gives rise to intestinal disturbances. 
It is very doubtful, however, whether this is the case; 
Deoaisne's 29 negative feeding experiments carried out on 
rats and on himself certainly do not support it. 

DISOOLOUBBD BREAD 

Apart from the discoloration due to the development of 
Oidium aurantiocum and M onilia sitopJiila, pigmentation of 
bread and other baking produce may be due to the develop- 
ment of Bact. prodigiosum. The discoloration is then limited 
to the surface of the exposed bread, which acquires the ap- 
pearance of having been covered with a layer of coagulated 
blood. Apart from the presence of the causative organism, 
the complaint requires relatively high temperatures to de- 
velop, preferably more than 30 C., and high humidities. 

A highly interesting historical study of the occurrence of 
bleeding bread and Eucharists was published in 1896 by 
Scheurlen. 26 From this it would appear that epidemics of 
Bact. prodigiosum infections can be traced back to the early 
Egyptian civilization. 

Both Ehrenberg 30 and Scheurlen attribute the religious ban 
on the consumption of white beans, which was in force in 
Egypt and among the Pythagoreans, to the belief that not 
infrequently this article of food showed signs of bleeding, 
that is, became infected by Bact. prodigiosum. 

The first historic record of the occurrence of an epidemic 
of Bact. prodigiosum is stated by Ehrenberg to have been in 
332 B.C., when 170 women were done to death in Rome owing 
to the spontaneous appearance of blood on articles of food, 
an incident which was interpreted by the priesthood as an 
indictment against them. 

Throughout the Middle Ages persecutions or civil dis- 
turbances followed regularly on the appearance of 'bleeding* 
Hosts and amylaceous articles of food. On the last occasion, 



DISEASES OF BREAD 267 

during the summer of 1819, a peasant, living in the village 
of Legnaro, near Padua, observed the phenomenon on a dish 
of polenta maize porridge which he had kept overnight 
in a drawer. He threw the polenta away, but on the following 
day observed that a dish of rice soup, a rusk, and a cooked 
chicken had become similarly affected. Subsequently the 
phenomenon spread to the whole of the village and the 
anxiety of the populace induced the authorities to appoint a 
commission to investigate the matter. Pietro Melo, director 
of the botanical gardens of Savonara, who, with Vicenzo 
Sette, a local physician, was a member of the commission, 
ascribed the phenomenon to a spontaneous fermentation of 
the polenta, which caused the maize meal used to be trans- 
formed into a coloured mucilage. 

Sette expressed the view that the bleeding was due to the 
development of a microscopic plant, and a similar conclusion 
was arrived at by Bizio 31 in his letter to the priest Angelo 
Bellani. Bizio described the imaginary agent as Serratia 
marcescens, in honour of the Italian inventor Serafino Serrati, 
who constructed a steam-driven boat. 

Subsequently, in 1850, Cohn 82 demonstrated the true 
nature of the agent of bleeding Hosts and gave it the name 
Bact. prodigiosum. Wasserzug, 33 in his study of the organism, 
observed that the red pigment is most readily formed in the 
presence of acids, a fact which may well have added to the 
mystery surrounding the occurrence of these epidemics of 
'bleeding' on articles of food. 

LITERATURE 

1. C. Brewster Monson, J. Ind. Ung. Chem. y vol. 15, p. 1219, 1923. 

2. J. Eouasel, Bev. intend, militaire, vol. 20, p. 127, 1908. 

3. H. L. Russell, ZentrU.f Bakt., Abt. H, vol. 6, p. 234, 1899. 

4. E. Welte, Arch.f. Hygiene, vol. 24, p 84, 1895. 

5. B. von Fenyvessy and L. Dienes, Zeits. /. Hygiene, vol. 69, p. 223, 1911. 

6. H. W. Atwater, Bread and the Principles of Bread-making, U.S Dept. 

Agriculture, Farmers Bull. No. 112, 1900. 

7. P. Roeser, Arch, de mddecine et de pharm. militaire, vol. 16, p. 462, 1890. 

8. E. Laurent, Butt, de FAcad. de Belgique (3), vol. 10, p. 766, 1885. 

s 



268 DISEASES OF BREAD 

9. D. J. Lloyd and E. D. MoCrea, Soy. Soc. Report Food (War) Committee, 
No. 48, 1918. 

10. E. J. Cohn, S. B. Wolbaok, L. J. Henderson and P. H. Cathoart, 7. 

Gen. Phyaiol., vol. 1, p. 221, 1918. 

11. 0. B. Morison and F. A. Collate, Amer. Inst. Baking, Bull. No. 5, 1921. 

12. E. A. Fisher and P. Halton, Cereal Chem., vol. 6, p. 192, 1928. 

13. E. J. WatMns, J. Soc. Chem. Ind., vol. 25, p. 350, 1906. 

14. J. Vogel, Zetti.f. Hygiene, voL 26, p. 398, 1897. 

15. . Kiatsohmer and . Niemilowacz, Zentrbl. f. BaJct., vol. 6, p. 501, 

1889. 

16. K. B. Lehmann and B. 0. Neumann, Bakteriologiache Diognoatik, part n, 

6th edition. J. F. Lehmann's Verlag, Munohen, 1912. 

17. J. Uffelmann, Zentrbl.f. Bakt., vol. 8, p. 481, 1890. 

18. J. KSnig, A. Spieokermann and J. Tillmanns, Zeitach. f. Unterauch. d. 

Nohrunga- und Oenuaamittel, vol. 5, p. 36, 1902. 

19. E. Fuhrmann, Zentrbl.f. Bakt., Abt. II, vol. 16, p. 385, 1906. 

20. J. Buchwald, see F. Lafar's Handbuch d. techmscJien Mykologie, vol. 2, 

p. 628. Gustav Fischer, Jena, 1905-8. 

21. P. Lindner, Wochenschr. f. Brauerei, vol. 15, p. 209, 1898. 

22. P. Lindner, Zeitachr.f. SpmtvAinduatrie, vol. 31, p. 162, 1908. 

23. W. Herter, Angewandte Botanik, vol. 1, p. 51, 1919. 

24. W. Herter, and A. Fornet, Zentrbl. f. Bakt., Abt. II, vol. 49, p. 148, 

1919. 
26. A. Payen, Ann. de Chim. et de Phys. (3), vol. 9, p. 5, 1843. 

26. . Scherulen, Arch.f. Hygiene, vol. 26, p. 1, 1896. 

27. E. Jalade, Rev. intend, milttaire, vol. 20, p. 269, 1908. 

28. J. Chalmers Robertson, The Lancet, vol. 2, p. 618, 1887. 

29. E. Deoaisne, Comptes rend., vol. 73, p. 607, 1871. 

30. Chr. G. Ehrenberg, Ber. u. d. z. Bekanntmaclt geeign. VerJiand. der Kgl. 

Preuss. Akad. der Wtsaensch., vol. 1, p. 5, 1860. 

31. B. Bizio, see C. P. Merlino, J. Bact., vol. 9, p. 627, 1924. 

32. F. Cohn, Beitr&ge z. Biologie d. Pfianzen, vol. 1, p. 127, 1860. 

33. E. Wasserzug, Ann. Inst. Pasteur, vol. 1, p. 681, 1887. 



PART FIVE 



82 



CHAPTER XXI 

THE MICROBIOLOGY OF SUGAR-CANE, SUGAR- 
BEET, AND THEIR RAW JUICES 

Is giving an account of the microbiology of bread-making, it 
was found necessary to consider the normal mioroflora of 
grain and flour. A similar course must be adopted here in 
discussing the microbiology of sugar manufacture. As in 
bread-making, no process of sterilization is used to purify the 
raw materials, sugar-cane and sugar-beet, which are living 
tissues with a normal microflora of their own, and which are 
exposed for prolonged periods to infection from the air and 
the soil. 

In attempting to give an account of the microflora of the 
raw materials of sugar manufacture, one is faced by the 
difficulty that no exhaustive investigation has been carried 
out to establish the identity of the various micro-organisms 
which are met with on sugar-cane and sugar-beet. 

That an indigenous, epiphytic mioroflora occurs on these 
plants, or at least on sugar-cane, is clear from Wolzogen 
Kuhr's 1 experiments. He found that out of 697 samples of 
aseptioally removed healthy sugar-cane, only six, taken from 
isolated mountain districts, yielded a sterile juice when 
pressed under aseptic conditions. The remaining 591 samples 
showed a microflora consisting of various saprophytic bacteria. 
In some cases at least this microflora comprised types of 
bacteria producing yellow colonies on ordinary laboratory 
media, thus resembling Bact. herbicola a (wreum. Geerligs 2 
confirms that the cane arriving at the sugar-mill contains 
an extensive microflora on its surface, but he gives no details 
as to the nature of this flora. It is not unreasonable to expect 
that, in addition to types resembling the typical epiphyte 
Bact . herbicola a aureum, it comprises others met with among 
the normal epiphytic microflora of grain, such as Bact. 
fluorescens liquefaciens and members of the gas-producing 



262 THE MICROBIOLOGY OP SUGAR-CANE, 

paratyphosum-TeBemldlnig group already referred to in 
Chapter XVI. On the other hand, it does not necessarily 
follow from Hutohinson and Ramayyar's 8 isolation from 
fresh oane juice of a yeast of Saccharomyces cerevisiae type 
and an Aspergillus species that these micro-organisms are 
members of the normal microflora. These species, together 
with others, such as Bac. subtilis and acid-producing types, 
may have been introduced into the juice with particles of 
soil adhering to the cane. 

The epiphytic microflora of sugar-beet is even less well 
defined, probably because of an extensive contamination 
with the mioroflora of the soil adhering to the beet and its 
rootlets. 

In order to acquire further information on this subject it 
is necessary to analyse the reports on the micro-organisms 
found in freshly extracted beet-sugar juices. But even so it 
is impossible to obtain more than a vague impression of the 
types composing the epiphytic microflora of beet, since little 
has been done to identify the micro-organisms met with. 
Most satisfactory in this respect is the information compiled 
by SohOne, 4 who determined the number of micro-organisms 
not only in freshly extracted juice but on freshly washed and 
cut beet slices, technically known as 'cossettes 3 . The latter 
were found to contain from several hundreds to over four 
thousand micro-organisms per gramme sometimes of gelatine- 
liquefying types, and probably related to or identical with 
Sact. fluorescens liquefaciens. SchOne does not state whether 
his coli jpara^Aos-zm-resembling types were obtained from 
the cossettes or from the juice extracted from the beet slices. 
Their presence in several varieties appears to indicate their 
frequent occurrence, and since they are not members of the 
normal flora, it may well be assumed that they belong to the 
epiphytic microflora of sugar-beet. In addition SchOne 
records the occasional presence in fresh sugar-beet juice of 
Bact.prodigiosum, Cocci, a Torula, and two species of Monilia- 
like fungi. It is not disclosed whether some or all of these 
secondary types were derived from the beet itself or from 



SUGAR-BEET, AND THfflTTt, RAW JUICES 263 

soil, nor can this be settled as regards the two species of 
Streptococcus to which SohOne makes special reference owing 
to their mucus-producing properties. It is probable that part 
of the mioroflora mentioned must have been introduced with 
the water used for the extraction of the cossettes. 

The members of the mesentericus-suhtilis group to which 
SchOne refers as having been isolated from cossettes and 
other products of manufacture were probably introduced 
with the soil adhering to the beet rather than with the beet 
itself. 

Apart from these various micro-organisms, there is a 
possibility of the introduction into the various manufacturing 
processes of plant pathogenic organisms responsible for the 
development of disease in sugar-beet. Herzfeld 5 draws atten- 
tion to such cases, which are discussed also by SchOne. 6 

In spite of the very scanty information available on the 
microbiology of sugar-cane and sugar-beet, there is over- 
whelming evidence to show that numerous micro-organisms 
of considerable variety enter the sugar-cane mill and the beet- 
sugar factory with the raw materials employed, and that these 
various organisms play an important part, at different stages 
of manufacture, in the deterioration of sugar juices and of 
finished sugar. 

It will be the purpose of the following pages to justify the 
claim that micro-organisms are dangerous agencies in sugar 
manufacture and to support this by an analysis of the pub- 
lished information on the subject. To facilitate this analysis 
a brief description will be given of the main processes of the 
manufacture of cane- and beet-sugar. 

In cane-sugar manufacture the raw material, after removal 
of leaves and roots, is placed on specially designed crushers 
and is passed through these to squeezing rollers arranged in 
sets of three. In passing through the squeezers the crushed 
cane is exposed to a pressure of 450 to 600 tons per square 
inch and is thereby reduced to a pulp resembling saw-dust, 
while the juice contained in the cane flows off and is collected 
in vats, where stones and sand can be separated by settling 



364 THE MICROBIOLOGY OF SUGAB-CANE, 

This raw juice contains debris of the cane and other impurities, 
which necessitate its being passed through sieves before it is 
subjected to the next process of 'tempering', a process during 
which it is mixed with lime and heated to boiling-point, 
usually after a prior treatment with sulphur dioxide. After 
tempering the juice is allowed to settle for the separation of 
the impurities coagulated during boiling. The sediment is 
drawn off as a mud from the bottom of the settling- vat, while 
the clear juice is removed from the top and stored until mixed 
with the filtered residual juice recovered in a filter-press from 
the sediment. The clarified juice is evaporated under suitable 
conditions, and when it is sufficiently concentrated, left to 
crystallize. 

In beet-sugar manufacture the chief processes are similar 
in principle. The beet, usually stored in large quantities, 
arc convoyed by a stream of water and by elevators to washing- 
tanks in which they are freed from adhering stones and soil. 
After washing they are lifted to a higher floor of the building, 
weighed, and then passed on to the slicing-maohines which 
cut them into narrow strips already referred to as cossettes. 
The ooHHi'ttoR are filled into vats known as diffusers. Of these 
there aro usually 10-14 in one set, or battery. From the 
bottom of thoflo vats hot water, or preferably hot juice of a 
previous batch is rim in so as to heat the cossettes sufficiently 
to kill tho boot-colls and thus facilitate extraction of the juice. 
It is important that tho liquid used for extraction should not 
1)0 heated higher than 75 C. to 80 C., since higher tempera- 
ture timd to render the cossettea Hoft and pulpy. In such 
case they niiik into a solid mass through which the extraction 
water cannot penetrate. The extract flowing from the first 
(hlTuHor entera the second of tho battery after having been 
first heated to between 75 C. and 80" C., and thence passes 
on to tho third and so on, warmed up each time prior to 
entering a frenh diffiiHcr. In this way tho temperature of the 
juice iti maintained between 75 C. and 80 C. during the 
greater part of the extraction. Since the filling of the first 
takes no more than 10 minutes, only a comparatively 



SUGAR-BEET, AND THEIR RAW JUICES 267 

beyond that at which more-organisms can develop. How- 
ever, where extreme cleanliness is not maintained, accumula- 
tion of particles of crushed cane and other dirt occurs in 
corners and gutters of the milk and indirectly gives rise to 
inversion of saccharose. It has been observed by McCleery 11 
that the infection is more marked in a juice pressed towards 
the end of the week than in one prepared immediately after 
the cleaning of the machinery. In such cases the mill itself, 
and particularly such types of mill as facilitate the accumula- 
tion of dirt, must be partly responsible for the increase in the 
microflora of the juice. 

In trying to ascertain the connexion between time and 
increase in the mioroflora it must not be overlooked that, 
normally, the interval elapsing between the removal of a 
sample of juice from the aseptic surroundings of the cane 
cells and its arrival in the heaters, where microbiological 
activity would become arrested, is too short to allow of 
appreciable growth, at least when the micro-organisms 
present consist only of the comparatively few epiphytic 
organisms and of the types introduced with soil. Orth 10 
found that crusher juice, that is, juice which has exuded 
from the cane during its preliminary crushing prior to milling, 
deteriorated little when kept for five hours, indicating but a 
very slight microbiological activity. 

In the juice emanating from the last mill of a 'train', 
however, Orth detected a rapid hydrolysis of the saccharose 
of the juice, a hydrolysis which could be readily measured 
after 30 minutes standing of the juice at room temperature. 
Similarly, Sprankling 12 records a case in which a crude cane 
juice standing for six hours lost 14-3 per cent, of its saccha- 
rose, that is, 0-04 per cent, a minute, and in the case of sugar- 
beet juice, Neide 13 recorded a considerable loss of sugar during 
the one and three-quarter hours required by the juice to pass 
through a diffusion battery. In such cases of apparently 
heavily infected juices time has become an important factor, 
and a slight delay in the delivery of the juice to the heaters 
may occasion considerable damage. 



268 THE MICROBIOLOGY OF SUGAR-OANE, 

The time factor is of particular importance in cases where 
samples of juice either cane juice or sugar-beet diffusion 
juice have to be kept for chemical analysis. The methods 
adopted for the prevention of deterioration in such oases will 
be referred to later. 

The question of the relative importance of the micro- 
organisms found in cane and beet juices, though touched 
upon by various writers, has never received the same degree 
of attention as the subject of the respective importance of 
bacteria and fungi for the deterioration of stored crude and 
refined sugar. 

It has already been mentioned that, in raw juices, bacteria 
have been found to be far more numerous than fungi, indi- 
cating that the former are more important than the latter. 
In accepting this conclusion, however, it should not be over- 
looked that cases may occur where certain fungi predominate 
in the juice. Laxa, 14 for instance, refers to a juice in which 
yeast predominated, and it is highly probable that similar 
conditions prevail when cane-sugar juices are allowed to 
undergo spontaneous fermentation as in the West Indies, 
where this juice is used for the manufacture of vinegar 
(Sprankling 12 ). 

Another case where a fungus, Oidium terricula, prevailed 
is referred to by de Haan. 15 This organism, however, did not 
hydrolyse saccharose, but produced acids from the reducing 
sugars present. 

Of the various bacteria constituting the microflora of raw 
juices, those capable of producing mucus are particularly 
important. 

When discussing mucus fermentations in Chapter XV it 
was mentioned that the first of the bacteria known to syn- 
thesize dextran had been isolated from a sugar solution taken 
from a beet-sugar factory. Most writers emphasize the fre- 
quent occurrence of Streptococcus (Leuconostoc) mesenteroides 
and other mucus-producing types in sugar juices. Boekhout 10 
and Velich 17 suggest that these bacteria enter the raw juice 
with soil in which, according to Stoklasa and Vitek, 18 they 



SUGAB-BEET, AND THEIR RAW JUICES 260 

have their natural habitat. This is probably correct. With 
the soil, these organisms are deposited in gutters and settling 
tanks, places where they find favourable conditions for growth 
and from which they are difficult to remove, not only because 
of the adhesive properties of their mucus, but also owing to 
the protection which the mucus offers them against penetra- 
tion of heat and antiseptics. 

Though the juice contained in gutters and settling-tanks 
favours the growth of mucus-producing bacteria, it does not 
necessarily follow that the actual mucus formation is en- 
couraged. It was pointed out in Chapter XV that an alkaline 
reaction is favourable, if not actually essential, for the syn- 
thesis of dextran and levan, the carbohydrates composing 
the mucus. The reaction both of raw cane and of beet juice, 
however, is on the acid side of the neutral point, and mucus 
may not always be found, therefore, when the juice is in- 
fected by these bacteria. In such oases Streptococcus (Leu- 
conostoc) mesenteroides is present as an ordinary Streptococcus 
(SchOne 4 ), but still capable of exercising its two most im- 
portant harmful functions, the hydrolysis of the saccharose 
of the juice and the conversion of part of the resultant re- 
ducing sugars into acids, principally lactic acid. 

The same is true of the various other mucus-producing 
bacteria, with the exception that lactic acid may be replaced 
by other fermentation products, notably by a mixture of 
acetic and formic acids. It is a mistake therefore to assume, 
as did Knauer, 19 that the addition of acids to a juice prevents 
the development of Streptococcus mesenteroides. All that can 
be claimed for this treatment is that it Inhibits the synthe- 
sizing functions of the organism. 

Where an accumulation of mucus-producing bacteria is 
extensive, their influence on the juice is very marked indeed, 
and the mere passing of the juice over a heavily infected 
surface, a mill gutter or a press cloth for instance, may suffice 
to invert appreciable quantities of saccharose. Hutchinson 
and Ramayyar 3 compare such conditions to those existing on 
the filter beds of a sewage purification plant. 



270 THE MICROBIOLOGY OF SUGAR-CANE, 

The impression has prevailed in many quarters that the 
occurrence in raw sugar juices of lactic acid bacteria was 
entirely independent of and unconnected with the presence 
of mucus-forming bacteria. This is not correct. The two 
phenomena are very closely connected, inasmuch as a con- 
siderable number of mucus producers and certainly the most 
dreaded type Streptococcus mesenteroides form lactic acid as 
their chief fermentation product. In the early days of the 
study of mucus formation by bacteria, Be"champ 20 analysed 
the fermentation products of a type other than Streptococcus 
mesenteroides, and found them to comprise lactic acid and a 
small percentage of acetic acid. The frequently observed 
formation of mannitolin cultures of mucus-producing bacteria 
is a further proof that these organisms, or at least those of 
them that produce mannitol and lactic acid, are nothing but 
lactic acid bacteria in disguise not only Streptococci, but 
non-spore-f orming rods such as Bact. pediculatum Koch, and 
Hosaeus, 21 Bact. viscoswm sacchari, Kramer, 22 or Bact. gelatino- 
sum betae, Glaser. 23 

The mucus producers which do not form lactic acid have 
in practically every case been found to be spore-forming rods, 
described in the first instance as definite species such as 
Clostndium gelatinosum, Laxa, 14 and Bac. levaniformans, 
Greig-Smith and Steel, 24 but subsequently (Schttne 4 ; Owen 7 ) 
revealed as members of the group of aerobic bacilli related to 
or identical with Bac. mesentericus, Bac. liodermos, &c. These 
types, therefore, are related to the bacilli responsible for the 
production of ropiness in bread, discussed in Chapter XX. 

In normal raw juices the detection of spore-forming muous- 
produoing bacteria and of spore-forming rods as a whole is 
rendered difficult by the large number of non-spore-forming 
bacteria present. Few details are available at present on the 
properties of the latter types. It has already been mentioned 
that they comprise rods resembling or identical with Bact. 
herbicola a awewm. They include ateo paratypho8um-]ike rods 
which were found by SchOne to produce ethyl alcohol, carbon 
dioxide and certain non-specified acids. Since saccharose is 



SUGAR-BEET, AND THEIR RAW JUICES 271 

hydrolysed by them they must participate in the deteriora- 
tion of sugar juices when present in considerable numbers. 

In addition various species of Micrococcus have occasionally 
been reported present, but there is no information to show 
that they affect saccharose. Bact. xylinum, an acetic acid- 
producing rod, has been found by Browne 26 in cane-sugar 
juice. It may be the organism which is responsible for the 
spontaneous conversion of fermented cane-sugar juice into 
vinegar to which Sprankling 12 refers. Browne found that this 
acetic acid bacterium was capable of producing a substance 
resembling cellulose obviously by synthesis of saccharose, 
since, according to Hoyer, 26 Bact. xylinum is capable of hydro- 
lysing saccharose. To what extent it is normally active in 
sugar juices has not been revealed. 

In discussing the bacterial flora of crude sugar juices it 
must be mentioned that various earlier investigations as- 
sumed the presence of butyric acid bacilli. Deherain 27 en- 
deavoured to substantiate this assumption experimentally 
by mixing sugar-beet diffusion juice with soil and allowing it 
to ferment in the presence of an excess of calcium carbonate. 
As was to be expected, he found that butyric acid was actually 
formed and hydrogen evolved. Nevertheless it is remarkable 
that, as pointed out by SchOne, no case is known in which 
butyric acid has actually been isolated from fermented raw 
juice. The participation of this group of micro-organisms in 
the deterioration of raw juices is not likely therefore to be 
considerable. This is not surprising, seeing that in passing 
through the mills or diffusers to the heaters the juice is not 
exposed to the anaerobic conditions essential for the develop- 
ment of butyric acid bacteria. 

The explosive gases, presumably hydrogen, which, in the 
early days of the diffusion process, occasionally collected at 
the top of the diffusers and gave rise to serious explosions, 
must have been produced by other agencies than butyric acid 
bacteria. That they resulted from an action of acetic and 
other organic acids on the iron walls of diffusers was suggested 
by Chevron. 28 This is not a satisfactory explanation, however, 



272 THE MICROBIOLOGY OF SUGAR-CANE, 

and was only proposed because of the difficulty of reconciling 
their production in the diffuser at high temperatures with 
microbiological activity. However, if it can be assumed that, 
at the time of the occurrence of these explosions,* high 
temperatures between 70 C. and 80 C. were not reached 
in the diffusers, then it becomes probable that the production 
of the explosive gases was in whole or in part due to the 
activity of the paratyphosum-like bacteria of the epiphytic 
cossette microflora. 

Attempts have been made by some writers to ascertain 
the extent of damage, as expressed in loss of saccharose, which 
can be attributed to the microflora of the raw sugar juices 
under normal working conditions. For this purpose McCleery 11 
determined the increase in acidity of a juice from the time 
of pressing until delivery to the heaters, assuming the increase 
observed to be proportional to the saccharose destroyed. 

It is clear that a method such as this cannot give more than 
an approximate conception of what actually takes place, 
since it is based on the assumption that all saccharose hydro- 
lysing organisms found in the juice produce the same organic 
acids, at the same time and to the same extent. 

A glance at the account given in the preceding pages of 
the microflora met with will show that the types active in the 
juices are too varied to admit of this possibility. 

Efforts to estimate the extent of the deterioration by 
polarization of the juices must lead to equally unsatisfactory 
conclusions, or must at least involve highly complicated 
analyses, since the figures obtained must be influenced not 
only by the extent to which one or other of the liberated 
monoses is consumed by the microflora, but also by the degree 
to which they are utilized for the synthesis of dextran or 
levan. 

More recently Paine and Baloh 29 have advocated the use of 
inverting enzymes for the exact determination of saccharose 

* In the early days of the working of the diffusion process the tempera- 
tures utilized for the extraction of oossettes often did not exceed 60 C , 
and probably failed to rise beyond 40 C. in some of the beet shoos. 



SUGAR-BEET, AND THEIR RAW JDICES 273 

and raffinose in juices, a method which, when used for the 
estimation of saccharose after boiling of the test samples, 
should offer reasonable prospects of reliable results. Until 
such analyses have been carried out, the question, which has 
been raised on various occasions, for instance by Mtzgge 30 and 
by Neide 18 , whether microbiological activity in raw sugar 
juices can be responsible for the so-called 'undetermined 
losses' of saccharose during manufacture, must remain un- 
answered. These losses have been placed as low as 0*1 per 
cent, of the saccharose present in the juice and as high as 
1-6 per cent. Gredinger 31 in 1925 recorded from 0-38 to 0-5 
per cent, for beet-sugar juices, a figure which may well be 
taken as a conservative average. 

Whether large or small, it is obviously desirable to avoid 
losses in sugar juices which may be due to microbiological 
activity. The evolution of the methods devised for this 
purpose are not without interest and throw some light on a 
procedure which is still commonly adhered to in sugar manu- 
facturing practice, that of mamtaining an alkaline reaction 
throughout the various manufacturing processes as well as 
in the finished sugar. There is no doubt that up to 1877, 
when Gayon 32 published his observations on the phenomenon, 
the general conception of the cause of deterioration of crude 
sugar on storage was, that the acids present gave rise to an 
hydrolysis of the saccharose with liberation of reducing 
sugars. It was important, therefore, to neutralize these acids 
and to render the sugar slightly alkaline. But since the acids 
of the finished sugar had been derived, at least in part, from 
the acidic compounds responsible for the acidity of the re- 
spective raw juices,a protection of the juices against inversion 
would likewise have to be secured through neutralization. 
This was done as early as possible in manufacture by the 
addition of sufficient limestone or slaked lime, not only to 
neutralize the juices, but also to render them slightly alkaline. 

In spite of the evidence collected by Gayon and subsequent 
workers that the deterioration of stored sugar is due to the 
activity of micro-organisms, the custom of maintaining a 

T 



274 THE MICROBIOLOGY OF SUGAR-CANE, 

slight alkalinity in the finished sugar by the addition of lime 
to the raw juices has remained an important stage in manu- 
facture, no longer for the purpose of preventing deterioration, 
but to facilitate the precipitation of many colloidal impurities 
present (Bomonti 33 ). The stage of lime addition, as already 
mentioned, is technically known as 'tempering'. It IB carried 
out as soon as the raw juice has been pressed and freed from 
major impurities, such as stones, sand, pieces of oossettes and 
sugar-cane. In tempering, the pH value of the fresh crude 
juice is raised from between 4-8 (Schmidt 84 ) and 5-8 (Rao and 
Ayyar 35 ) to 8-0 (Walton, McCalip and Hornberger 36 ), or even 
8-5 (Williams and Gebelin 37 ), though one as low as 7-2 may be 
sufficient in some cases (Gebelin 38 ). Boiling of the juices at 
these reactions has been found to have a much greater de- 
structive action on the micro -organisms present than boiling 
at the original reaction of the juice, so much so in fact that, 
as will be shown in Chapter XXII, the tempered juices may, 
in many cases, be found sterile after boiling. It is important, 
therefore, that the tempering should be carried out with the 
least possible delay. 

With the practical elimination of micro-organisms from 
the juice during tempering, efforts in the direction of pre- 
venting microbiological deterioration can be concentrated on 
the period prior to tempering. 

Here addition of antiseptics and increased cleanliness are 
the only possible means of counteracting the hydrolytic 
action of the normal microflora. Both methods have been 
advocated and adopted, the former perhaps more frequently 
than the latter. 

Eor factory purposes the use of hydrofluoric acid and 
aluminium fluoride was recommended by Herzfeld and 
Paetow, 39 who found them suitable in arresting slight infec- 
tions, but incapable of preventing inversion of saccharose in 
heavily infected juices, presumably owing to their failing to 
destroy invertase which had accumulated in such cases. 

Ammonium fluoride, which Heerma van Voss 40 recom- 
mended, was considerably more efficient than the above 



SUGAR-BEET, AND THEIR RAW JUICES 271 

substances. Applied to the juice in the proportion of 10 to 15 
grammes per hectolitre, ammonium fluoride completely 
arrested the growth of micro-organisms and prevented inver- 
sion of the saccharose. In general practice, however, this 
antiseptic is too expensive and is inferior therefore to formal- 
dehyde, recommended by a number of workers, Sohott, 41 
lYiedrich, 42 Owen, 43 and Orth. 10 More recently, a proprietary 
article, a calcium hypochlorite, has been found as effective as 
formaldehyde by Haldane 44 and markedly cheaper. Haldane 
recommends its periodical addition to mill beds and gutters, 
while the crushing of cane is being carried out, using a suf- 
ficiently strong concentration to ensure the presence of 2 per 
aent. chlorine. Further, he advises that a continuous trickle 
Df a solution of the antiseptic of 1 in 500 should be allowed 
;o flow into all juice gutters where dirt and mucus-producing 
Dacteria may accumulate. 

Within the last few years Jonas 45 has drawn attention to 
-he valuable antiseptic properties of sulphur dioxide as used 
n the cane-sugar rnfll for the treatment of the raw juice 
mmediately before tempering. Jonas finds that, in con- 
entrations of from 0-05 to 0-01 per cent., it destroys Strep- 
^coccus mesenteroides instantaneously, while the spores of 
aucus-producing soil bacilli are killed by a 0-5 per cent, 
olution. 

Of interest are the efforts which have been made to prevent 
licrobiological activity in sugar juices which have to be kept 
)r the purpose of chemical analysis. A variety of antiseptics, 
icluding those already referred to as well as lead acetate 
Scheibler 46 ), have been recommended for the purpose. Most 
f them, however, suffer from the disadvantage that in one 
ay or another they affect properties of the juice which it is 
aportant or essential to preserve. One of the very few which 
D not do this is the essential oil of cinnamon recommended 
y Courtonne 47 for this purpose, and which was stated by him 
i prevent microbiological deterioration when added in pro- 
>rtions ensuring a concentration of one in a thousand. Even 
ilf of this concentration has been found sufficient by the 

T2 



276 THE MICROBIOLOGY OF SUGAR-CANE, 

writers in some oases. The methods of securing protection by 
increased cleanliness are too obvious to need any emphasis. 
They should result in ensuring the use of healthy cane and 
beet, free from adhering soil and washed in water containing 
a TnimmuTn of micro-organisms. More frequent cleaning and 
sterilization of the plant, either by antiseptics or by steam, 
is a further means by which accumulation of harmful micro- 
organisms could be prevented in the actual machinery and 
the various tanks required for the extraction of the juice. 

LITERATURE 

1. C. A. H. v. Wolzogen Kuhi, Arch. Suikerind., 31, Meddd. Proefatat. 

Java Suikerind, No. 9, p. 321, 1923. 

2. H. C. Prinsen Geerligs, Cane sugar and its Manufacture, 2nd edition. 

Normal Roger, London, 1924. 

3. C. M. Hutchinson and C. S. Ramayyar, Agric. Res. Inst. Pusa, Bull. 

No. 163, 1925. 

4. A. Sohane, Zeitsoh. dea Vereina d. D. Zuckerind. Tech. Ted, vol. 61, 

p. 453, 1901. 

6. A. Herzfeld, Zettach. dea Vereina d. D. Zuckerind. Tech. Ted, vol. 41, 
p. 44, 1891. 

6. A. SohSne, Zeitech. des Vereins d. D. ZucJcerind. Tech. Tett, vol. 56, 

p. 737, 1906. 

7. W. L. Owen, J. Ind. Eng. Ohem., vol. 3, p. 481, 1911. 

8. N. Kopeloff and L. Kopeloff, Louisiana Bull., No. 166, 1919. 

9. 0. Laxa, Zeitsch. f. Zuckennd. in Bohmen, vol. 24, p. 423, 1899-1900. 

10. W. K. Orth, Intern. Sugar J., vol. 25, p. 474, 1923. 

11. W. L. McCleery, Intern. Sugar J., vol. 27, p. 543, 1926. 

12. C. H. G. Sprankling, J. Soc. Chem. Ind., vol. 22, p. 78, 1903. 

13. E. Neide, Zeitsch. dea Vereins d. D. Zuckennd. Tech. Tett, vol. 66, p. 726, 

1906. 

14. 0. Laxa, Zeitsch. f. Zuckerind. in Bfihmen, vol. 26, p. 122, 1901-2. 

16. J. S. de Haan, Arch. v. d. Suikerind. in Ned. Ind., vol. 22, p. 1352, 1914 

16. F. W. J. Boekhout, Zentrblf. Bakt., Abt. n, vol. 6, p. 161, 1900. 

17. A. Velijjfc, Zeitschr.f. Zuckerind. in Bdhmen, vol. 27, p. 476, 1903. 

18. J. Stdtlasa and E. Vitek, ZentrU.f. Bakt., Abt. H, vol. 14, p. 102, 1904. 

19. . Knauer, Zeitsch. dea Vereina d. D. Zuckerind. Tech. Teu, vol. 38 

p. 240, 1901. 

20. A. Beohamp, Comptea rend., vol. 93, p. 78, 1881. 

21. A. Koch and H. Eosaeus, ZentrU. f. Bakt., vol. 16, p. 225, 1894. 

22. E. Kramer, Monatsch.f. Chem., vol. 10, p. 467, 1889. 

23. F. Glaser, Zentrblf. Bakt., Abt. n, vol. 1, p. 879, 1895. 

24. R. Greig-Smith and T. Steel, J. Soc. Chem. Ind., vol. 21, p. 1381, 1902. 



SUGAR-BEET, AND THEIR RAW JUICES 277 

'26. C. A. Browne, J. Amur. Chem. Soc., vol. 28, p. 473, 1906. 

26. D. P. Hoyer, Die Deutsche Esaigmduatrie, vol. 3, p. 1, 1899. 

27. P. P. Deherain, Zeitsch. dea Vereina d. D. Zuckerind. Tech. Teil, vol. 34, 

p. 269, 1884. 

28. L. Chevron, Jour, des Fabric, de Sucre, vol. 24, p. 320, 1883. 

29. H. S. Paine and R. T. Balch, J. Ind. Eng. Chem., vol. 17, p. 240, 1925. 

30. . Mugge, Zeitsch. des Veretns d. D. Zuckerind. Tech. Ted, vol. 64, 

p. 888, 1904. 

31. W. Gredinger, Zeitsch. des Vereina d. D. Zuckerind. Tech. Teil, vol. 50, 

p. 1697, 1925. 

32. U. Gayon, Com/pies rend., vol. 84, p. 606, 1877. 

33. H. F. Bomonti, Chem. Abate., vol. 21, p. 665, 1927. 

34. E. Schmidt, Zeitsch. des Vereina d. D. Zuckerind. Tech. Teil, vol. 51, 

pp. 628 and 665, 1926. 

36. T. L. Rao and G. G. Ayyar, Madras Agnc. Dept. Tear-book (1925), p. 73, 
1927. 

36. C. F. Walton, M. A. MoCalip and W. F. Hornberger, J. Ind. Eng. Chem., 

vol. 17, p. 61, 1926. 

37. W. J. Williams and J. A. Gebekn, Facts about Sugar, vol. 17, p. 202, 

1923. 

38. J. A. Gebelin, Louisiana Planter, vol. 71, p. 172, 1923. 

39. A. Herzfeld and U. Paetow, Zeitsch. des Vereina d. D. Zuckerind. Tech. 

Teil, vol. 41, p. 678, 1891. 

40. A. J. Heerma van Voss, Zeitsch. des Vereina d. D. Zuckerind. Tech. 

Teil, vol. 60, p. 438, 1900. 

41. A. Sohott, Zeitsch. dea Vereina d. D. Zuckerind. Tech. Teil, vol. 50, 

p. 434, 1900. 

42. 0. Freidrich, Q. Pat., No. 146871, 1902. 

43. W. L. Owen, Louisiana Bull., No. 153, 1916. 

44. J. H. Haldane, Intern. Sugar J., vol. 29, p. 367, 1927. 

46. W. JonaS, Z. f. d. Zuckerind. d. Cechoslov. Republ., vol. 51, p. 161, 
1927. 

46. C. Soheibler, Scheibler's Neue Zeitsch. f. Rubenzuckennd., vol. 18, p 256, 

1887. 

47. H. Courtonne, Chem. Abets , vol. 17, p. 3427, 1923. 



CHAPTER XXII 



THE MICROBIOLOGY OF CANE JUICE AND BEET 
JUICE IN MANUFACTURE 

WITH the tempering of the raw juice far-reaching changes 
occur in the microflora present. Attempts have been made 
from time to time to follow these changes experimentally 
both in the case of raw cane-sugar and in sugar-beet diffusion 
juices. Most detailed in this respect are the observations 
supplied by Owen, 1 who studied the microflora of cane juices 
in the course of conversion into sugar. 

Owen gives the following figures as the average for the 
mioroflora of nine different sets of samples taken during the 
various stages of manufacture. 

TABLE X 



Type of raw material. 


Number of micro- 
organisms per 
gramme. 


Type of micro-organisms. 


Raw juice 


280,000 


Yeast and other fungi ; bacteria 


Sulphited juioe 


35,000 


Teast and other fungi; bacteria 


Limed juice 


37,600 


Bacteria predominating 


Defecated juioe 


750"! 




(limed juioe after 


I 




boiling) 


f 


Spore-forming bacilli 


Syrup (evaporated 


400J 




juioe) 






Massecmtes 


450\ 


Spore-forming bacilli 


Sugar 


600/ 




Molasses 


35,000 


A mixed mioroflora 



The data supplied by Kopeloff and Kopeloff 2 are much less 
detailed, but confirm the impression that a striking reduction 
of the original mioroflora takes place during the stages 
through which the raw juice passes before being set aside f 01 
the crystallization of the sugar contained in it, a reduction 
which particularly affects the non-spore-forming bacteria 



CANE AND BEET JUICE IN MANUPACTUEE 279 

and the various fungi. This conclusion was arrived at also by 
Schfine, 3 who studied the microflora of sugar-beet diffusion 
juices. After liming and saturation with carbon dioxide, the 
juice was practically sterile in SchOne's experiments. 

It is clear, therefore, that the infection which is noticeable 
in all molasses must have been introduced subsequent to 
defecation, the process in which the colloidal impurities of the 
juice are precipitated by boiling in the presence of an excess 
of calcium hydroxide. 

The stage at which this infection occurs is to some extent 
revealed in Owen's experiments. During evaporation prior 
to crystallization the microflora of the juice and of the finished 
massecuites remains practically stationary (see Table X), in 
spite of the diminution in volume of the various liquors, 
showing] that neither reinfection nor development of micro- 
organisms occur at these stages. 

On the crystals formed in the massecuites the number of 
micro-organisms increased slightly, but was represented 
only by spore-forming rods of the types found in the masse- 
cuites. A reinfection, therefore, is not traceable up to this 
point. And yet the molasses, the remaining mother liquors 
after separation from the crystallizable sugar, has a large and 
varied microflora. This can only be interpreted as indicating 
that a reinfection has taken place during or after the separa- 
tion of the molasses. In order to appreciate how this is 
possible it is necessary to recall that the separation of the 
crystallized sugar from its mother liquor is carried out in 
centrifuges in which a rapidly flowing current of air, infected 
with various micro-organisms, comes into intimate contact 
with the crystals and the mother liquor during the rotation 
of the centrifuge at high speeds, and that after centrifuging, 
the crystals are washed with a small amount of water to 
improve their colour. As shown by Owen this wash water is 
not always very clean. In the case examined by him it con- 
tained no less than 25,000 micro-organisms per cubic centi- 
metre. It is clear therefore that, apart from other sources, 
the centrifuge offers possibilities for the introduction of fresh 



280 THE MICROBIOLOGY OE CANE JUICE AND 

infections into the process of sugar manufacture which are 
by no means trivial. This aspect will be dealt with in greater 
detail below. 

It is not to be assumed, however, that conditions are 
invariably as clearly defined as those recorded in Owen's 
experiments. Even assuming that at no stage of manu- 
facture subsequent to tempering does the temperature fall 
sufficiently low to allow of microbiological development, 
there must be occasions when a contamination of the juice 
can take place. This, however, has been contested in the 
past, for instance by SchOne, 3 who investigated the occurrence 
of thermophilic bacteria in sugar-beet juices. One of the 
likely occasions for contamination is when the juice passes 
the filter-press, in which the precipitate formed during tem- 
pering is removed. It will be recalled from the account given 
in Chapter XXI that after the settling in an appropriate 
tank part of the tempered juice is run off direct, while the 
sediment is treated in a filter-press to remove the adhering 
juice, the two portions of juice being subsequently mixed. 

Where the filter-press and the filter-cloths are not kept 
scrupulously clean there is an obvious danger of the juice 
becoming contaminated, a possibility which is far from 
problematic, seeing that the mioroflora of the resultant 
press-cake may be very numerous. Owen quotes 1-5 million 
micro-organisms per gramme of this cake in a case examined 
by him. 

It is on the filter-press also, and to some extent on the filters 
used for the clarification of the juice after its saturation with 
carbon dioxide and its evaporation to a syrup, that a de- 
velopment of Streptococcus mesent&roides and of other mucus- 
producing organisms frequently occurs. It is from these 
places that they are reintroduced into the now neutral or 
slightly alkaline juice. 

Such development occurs particularly where the filtration 
proceeds slowly and the temperature of the juice falls below 
60 C. Several writers, among them Laxa 4 and Gonnermann, 6 
have drawn attention to this danger. Where filtration to 



BEET JUICE IN MANUFACTURE 281 

ensure brilliancy is carried out through filters made of 
kieselguhr or of vegetable carbon a reduction of the number 
of micro-organisms may take place. Owen 8 observed that 
raw juices filtered in this way lost 99 per cent, of their micro- 
flora. Though not strictly relevant to the subject under 
discussion, it may be added here that when using cotton 
wool instead of carbon or kieselguhr, Owen found that 75 per 
cent, of the microflora was removed from raw juice, obviously 
through removal of sediment such as bagasse and other plant 
debris on which the micro-organisms were deposited. This 
experiment demonstrates very forcibly to what considerable 
extent the microflora of raw cane juice is influenced by the 
epiphytic microflora of the cane itself. 

Observations, prior to those of Owen, on the reduction of 
the microflora of a juice through filtering, were made by 
SchOne. 3 In the case of the filtration of an evaporated beet- 
sugar juice through wood shavings SchOne found a reduction 
in the microflora of about 99 per cent. 

Nevertheless, the juice leaving the press contains A con- 
siderable number of organisms even under normal working 
conditions. Church 7 records the examination of one filtered 
juice which showed 140 bacteria and 3,600 fungus spores per 
cubic centimetre. 

During the actual evaporation of the filtered juice no 
microbiological development occurs, the temperature pre- 
vailing being sufficiently high to prevent it. This is clearly 
indicated by Owen's figures quoted in Table X and is sup- 
ported by SohOne's observations on beet-sugar juices. 

On the other hand a marked sterilizing effect, beyond a 
possible destruction of fungus spores, is not achieved during 
evaporation, no doubt owing to the limitation of the micro- 
flora at this stage to spore-producing bacteria possessing 
great heat resistance in their resting stage. All workers who 
have studied the types of aerobic soil bacilli present in 
boiling and boiled juice have specifically referred to the 
resistance of these types to high temperatures. 

The microflora of the evaporated juice represents a poten- 



282 THE MICROBIOLOGY OF CANE JUICE AND 

tial danger to the preservation of the finished sugar since 
there is normally no step, from the stage of evaporation of 
the juice onwards, at which it can be eliminated. On the 
contrary, it is normally joined by additional types, once the 
massecuites have been run into the centrifuge for purposes of 
separating the mother liquid from the sugar crystals. 

The question of the infection of the raw sugar and its 
molasses during centrifuging was referred to above. It was 
mentioned that the types composing the flora present are 
introduced with the flow of infected air passing through the 
centrifuge during its revolution at high speeds and by the 
water used for washing of the sugar crystals subsequent to 
centrifuging. It was with a view to preventing this infection 
of the sugar that Shorey 8 recommended the use of high 
pressure steam instead of water for the washing of the 
centrifuged crystals. His suggestion was taken up by Kope- 
loff, Welcome and Kopeloff, 9 who elaborated a method for 
the application of superheated steam to the washing of sugar 
crystals in the centrifuge. In their experiments, carried out 
on a laboratory scale, they succeeded in reducing the bac- 
terial content of the sugar by 93 to 99-5 per cent., while the 
fungus spore content was reduced by 92 to 98 per cent. The 
flora of the mother liquor (syrup and molasses) was reduced 
by 50 to 80 per cent. In this process the superheated steam 
was allowed to pass into the centrifuge, and through the 
deposited sugar crystals while the centrifuge was rotating at 
a high speed, preferably, it need hardly be added, towards 
the end of the process. A very short exposure to the action 
of the steam, for no more than 3 minutes, caused a reduction 
of the microflora to the extent shown above. 

After separation from the crystals the mother liquor is 
again evaporated to ensure a second crop of sugar crystals, 
and, after separation, the remaining liquor, now known as 
molasses, may be given one or two further evaporations for 
the recovery of additional crops of sugar crystals. 

As the various batches of raw sugar are removed from the 
syrup and molasses the content of salts in solution materially 



BEET JUICE IN MANUFACTURE 283 

increases, and it has been frequently observed that these 
liquors show a tendency to foam on subsequent evaporation. 

This foaming, technically known as 'froth fermentation', 
occurs, according to Geerligs, 10 in low grade masseouites 
during cooling. The surface of these syrups may become 
convex owing to the uneven raising of the crust by gas pro- 
duced in the syrup. Eventually the crust may burst under 
the pressure of this gas and a brown froth ooze out of the 
crevices formed. This froth soon covers the whole of the 
surface, and continuing to rise, finally flows over the top of 
the tank. Gases containing carbon dioxide and nitrous 
oxide escape from the froth and emit an unpleasant odour. 
The reaction of such massecuites is usually markedly acid. 

It was at one time suspected that the froth fermentation 
was due to microbiological activity, observations by French 
writers, Dubrunfaut, 11 Durin 12 and others on the evolution 
of nitrous oxide in the fermentation of molasses in the dis- 
tillery having guided attention in this direction. Subse- 
quently Laxa 4 went so far as to associate a certain bacterium 
which he found capable of living in saccharose solutions of 
concentrations up to 40 per cent, and at temperatures of 
66 C. with this type of fermentation. Lafar, 14 in his account 
of this aspect of the microbiology of sugar manufacture, 
while admitting that convincing evidence in favour of a 
microbiological origin of the foam fermentation is lacking, 
is, nevertheless, inclined to favour this explanation, at least 
as regards those types of foam fermentation in which, ac- 
cording to Durin, 12 the evolution of gas is comparatively 
slight, where no caramelization of the sugar is caused, and 
where volatile acids, probably including butyric acid, are 
formed. The years which have passed since Lafar's exposi- 
tion of the foam fermentation have not supplied additional 
evidence of its microbiological origin. The present tendency 
undoubtedly inclines towards a chemical explanation of the 
phenomenon of foam fermentation. 

Quite a different matter is the occurrence of a scum on the 
surface of molasses standing at ordinary room temperatures. 



284 THE MICROBIOLOGY OF CANE JUICE AND 

This is due to the development of fungi, and is very frequently 
observed. Kopeloff, Welcome and KopelofE 9 recommended 
covering stored molasses with a layer of oil to prevent de- 
terioration. It might be desirable to ascertain experimentally 
how far this treatment will suffice to protect molasses. That 
it should be capable of preventing the formation of growth 
of micro-organisms on the surface is likely. It is by no means 
certain, however, that the deeper layers could be protected 
in this way, seeing that both Grove 16 and Hirst 16 have ob- 
served development of micro-organisms in saccharose solu- 
tions of more than 65 per cent, concentration, that is, in 
concentrations considerably higher than those of molasses. 
In a discussion on the microbiology of sugar manufacture 
there are three further subjects which must be briefly referred 
to, though they do not perhaps fall directly within the frame- 
work of the present thesis. 

One of these is the disposal of the waste waters of sugar 
manufacture, another the question of the utilization of the 
spent plant tissues from which the sugar juice has been 
extracted in other words the cossettes and the bagasse, and 
the third the utilization of the molasses. 

The problem of the elimination or purification of the waste 
waters of the sugar factory is one of considerable magnitude, 
particularly where beet sugar is being manufactured. Here 
the consumption of water is of a very high order, comprising 
per 1,000 tons of beet, according to Owen: 17 

For the flumes and washers . . 1,612,000 gallons 
diffusion and wash water . 349,440 
the pulp presses . . . 107,520 
other wash and waste purposes 20,160 
The greatest bulk of waste water, which as shown above 
comes from the flumes and washers, is fortunately not very 
difficult to purify. Besides stones, soil, beet rootlets and beet 
leaves, it contains traces of saccharose and protein in solu- 
tion. In order to remove stones and soil it is customary in 
some places to leave the water standing in ponds for some 
time, a period during which various microbiological changes 



BEET JUICE IN MAOTIFAOTUEE 285 

of the organic compounds in solution and suspension may 
be initiated. When the water, therefore, freed from stones, 
soil and larger particles, is allowed to enter a river, various 
microbiological processes may be in progress which require 
the presence of oxygen for completion. These processes, 
however, do not usually attain dangerous dimensions in the 
water from the flumes and washers, which seldom contains 
more than 0-05 to 0-1 per cent, of saccharose. Much more 
difficult is the disposal of the pulp-press water, which may 
contain as much as 0-5 per cent, of saccharose. Where this 
waste is allowed to stand for microbiological processes to be 
initiated, or even where it is discharged direct into a stream, 
the amount of oxygen required to convert the available 
carbohydrates into innocuous decomposition products is so 
large that the river water becomes incapable of supporting 
fish life owing to the removal of its dissolved oxygen. 

The sugar-containing waste water from the presses will 
also encourage the development of certain fungi, such as 
species of Fusarium and M ucor, of sheath-producing bacteria, 
such as Spha&rotilus (Lafar 18 ) and algae, notably Leptomitus 
lacteua (Kolkwitz 19 ), the growth of any of which may fre- 
quently be sufficient to congest the river into which the water 
is flowing. 

To avoid some of these disadvantages the waste waters 
must be subjected to treatment prior to their discharge into 
the river. In the case of the water from flumes and washers, 
this can be done, as already mentioned, by settling in ponds, or, 
as suggested by Owen, by carrying out a mechanical separa- 
tion of the suspended solids. For this purpose Owen recom- 
mends a rotating cylindrical drum, through which the waste 
flows prior to filtration through a suitable filter. Owen 
claims that the resultant waste water may be discharged 
direct into a river without danger to fish life. In the case of 
the filter-press waste, the introduction of a fermentation 
process has been proposed on various occasions for instance 
by Moller (see Kraisy 20 ). In Holler's process a molasses 
solution, acidified with the acid of lactic acid bacteria, is 



286 THE MICROBIOLOGY OF CANE JUICE AND 

added in quantities sufficient to increase the carbohydrate 
concentration of the waste by 1-5 per cent. The waste is 
now inoculated with a yeast and is maintained at a tempera- 
ture of 27 C. to 35 C., while flowing through a set of seven 
vats at a rate sufficiently slow to ensure the complete con- 
version of the carbohydrates present into alcohol during the 
passage of the liquid through the plant. The yeast formed 
during fermentation is recovered and is claimed to be suitable 
as cattle food. The alcohol and other fermentation products 
are not recovered. 

Methods of purification similar to those in use at sewage 
works, including septic tanks, have also been recommended 
(Grevemeyer 21 ), but in spite of the various efforts made a 
fully satisfactory method has not yet come into general use. 

In the cane-sugar mil] the consumption of water is very 
much smaller than in the beet-sugar factory, and the problem 
of the purification and disposal of the waste water is therefore 
of comparatively slight importance. 

On the subject of the disposal of the crushed cane residue, 
the bagasse, and of the extracted cossettes, very little need 
be said, since the problem has little bearing on the subjects 
under discussion. The bagasse is collected and utilized as a 
fuel for raising the steam required in the plant. The cossettes 
are valuable as a cattle food and may be consumed while 
still wet, after preliminary drying, or after conversion into 
silage. When dried the oossettes are very hygroscopic owing 
to their carbohydrate content. On storage, therefore, they 
often develop fungus growth, and become mildewed. This 
is avoided where the cossettes are ensilaged, a process of 
preservation for sugar containing plant tissues which was 
described in considerable detail by Thaysen and Bunker. 22 

The molasses accumulating in a sugar factory are usually 
disposed of by microbiological processes, through conversion 
into ethyl alcohol by fermentation with yeast. On a smaller 
scale they may be used as a cattle food, mixed with extracted 
cossettes or with specially prepared wood pulp. 

The cattle foods may suffer microbiological changes, leading 



BEET JDICE IN MANUFACTURE 287 

to excessive heating of the accumulated material, and per- 
haps even to spontaneous combustion. The microbiological 
changes giving rise to spontaneous combustion were also 
reviewed by Thaysen and Bunker. 22 

The use' of molasses for the production of ethyl alcohol by 
fermentation dates back to the early days of the manufacture 
of sugar and was a well-established industry when Reiset, 23 
Schloesing 24 and Dubrunfaut 26 drew attention in 1868 to the 
abnormal behaviour of the fermentations in cases where 
large percentages of nitrates were present in the molasses 
used. In such abnormal molasses the yeast fermentation 
was found to come to a premature standstill and to be re- 
placed by the formation of lactic acid and the evolution of 
nitrous oxide. The phenomenon was not exhaustively 
studied by the writers referred to and has received little 
attention since. It is not possible to say therefore whether 
a specific microflora is responsible for the liberation of nitro- 
genous gases from the available nitrates, or whether this 
phenomenon is associated with the evolution of lactic acid 
in the fermenting liquid. 

The remedy recommended by Reiset and Sohloesing for 
the prevention of this abnormal fermentation consisted of 
the addition to the molasses of a heavy inoculant of suitable 
yeast and of the establishment of an optimum acidity in the 
molasses prior to inoculation. 

LITERATURE 

1. W. L. Owen, J. Ind. Eng. Chem., vol. 3, p. 481, 1911. 

2. N. Kopeloff and L. Kopeloff, Louisiana Sutt., no. 166, 1919. 

3. A. SchSne, Zeitsch. des Vere%ns d. D. Zuckennd. Tech. Tetl, vol. 51, 

p. 453, 1901. 

4. 0. Laxa, Zeitsch. f. Zuckerind. in Bohmen, vol. 24, p. 423, 1899-1900. 
6. . Gronnennann, Zeitsch. des Vereins d. D. Zuckennd. Tech. Teil, 

vol. 56, p. 600, 1906. 

6. W. L. Owen, Intern. Sugar Jour., vol. 26, pp. 200 and 256, 1924. 

7. M. B. Church, Zentrll.f. Bakt , Abt. II, vol. 58, p. 538, 1923. 

8. E. C Shorey, J. Soc. Chem. Ind., vol. 17, p. 555, 1898. 

9. N. Kopeloff, C. J. Welcome and N. Kopeloff, Louisiana Butt , no. 175, 

1920. 



288 THE MICROBIOLOGY OF CANE AND BEET JUICE 

10. H. C. Prinsen Geerlings, Cane, Sugar and its Manufacture, 2nd edition. 

Normal Rodger, London, 1924. 

11. P. Dubrunfaut, Com/pies rendua, vol. 66, p. 276, 1868. 

12. E. Durin, BuU. Aesoc. des Ohim. Sucr. et Diat., vol. 1, p 134, 1884. 

13. 0. Laxa, Zentrbl.f. BaJet., Abt. H, vol. 4, p. 362, 1898. 

14. F. Lafar, Handbuch d. Tech, Mylcdlogie, vol. 2, p. 480. Gustav Fiaoher, 

Jena, 1907. 

16. 0. Grove, Annual Report Agric. and Hortic. Research Station. University 
Bristol, 1918, p. 34. 

16. F. Hirst, Annual Report Agric. and Hortic. Research Station. University 

Bristol, 1927, p. 160. 

17. B. J. Owen, Desiccation of sugar beet and the extraction of sugar. Ministry 

of Agriculture and Fisheries Publication, H.M. Stationery Office, 
1927. 

18. F. Lafar, Handbuch d. tech. Mylcdlogie, vol. 3, p. 410. Gustav Fischer* 

Jena, 1906. 

19. R. Kolkwitz, Zeitsch. des Vereins d. D. Zuckennd. Tech. Teil, vol. 54, 

p. 966, 1904. 

20. A. Kraisy, Zeitsch. des Vereins d. D. Zuckerind. Tech. Tetl, vol. 70, 

p. 163, 1920. 

21. M. Grevemeyer, Ch&m. Abats., vol. 19, p. 3033, 1925. 

22. A. C. Thaysen and H. J. Bunker, The microbiology of ceUuloee, hemv- 

cellulose, pectins and gums. Oxford University Press, 1927. 

23. J. Reiset, Comptea rend., vol. 66, p. 177, 1868. 

24. Th. Sohloesing, Comptes rend., vol. 66, p. 237, 1868. 
26. P. Dubrunfaut, Oomptea rend., vol. 66, p. 376, 1868. 



CHAPTER XXHI 

THE MICROBIOLOGICAL DETERIORATION OF 
SUGAR IN STORAGE 

IT was pointed out in the previous chapter that an infection 
Df sugar crystals may take place during their separation from 
the masseouites in the centrifuge and that in consequence 
bhe finished raw sugar may contain a considerable mioroflora. 
It -will be the object of the present chapter to investigate 
bhe nature of this mioroflora and to establish whether it affects 
jrystallized sugar kept in storage, and if so, to what extent. 

Already at the time when the introduction of sugar into 
Europe was fresh in men's minds it was shown (Ligon 1 ) that 
mgar had to be kept under dry conditions if it was to be 
Dreserved. And apparently it was known also that when this 
vas not done the sugar became discoloured, damp, and in 
extreme cases converted into a syrup. 

Towards the middle of the last century occasional observa- 

ions connected the deterioration of stored sugar with mioro- 

)iologioal activity instead of with hydrolytic action caused 

>y acids present in the sugar, as had hitherto been assumed. 

In 1829 and 1830, according to Kopeloff and Kopeloff, 2 

r an Dijk and van Beek reported their observations on a 

ample of loaf sugar which had become blackened on standing 

hrough the action of a fungus to which they referred as 

Conferva mucoroides. Payen 3 reported to the French Academy 

n 1851 on two cases of sugar deterioration in which the 

rystals had been pitted and in some oases discoloured pink 

hrough the action of a fungus. In 1869 Dubrunfaut 4 drew 

.ttention to the presence in raw beet-sugar of 'those lower 

rganisms so accurately described by Pasteur as the living 

auses of alcoholic and bacterial fermentations'. In 1880 

Jayon observed various yeasts and moulds in deteriorated 

Vest Indian sugar, while in 1898 Shorey 5 detected the 



290 THE MICROBIOLOGICAL DETERIORATION 01 
mycelium of what he describes as Penicillium glaucum on 
the crystals of four samples of damaged Hawaiian sugar, and 
attributed the deterioration to the activity of this fungus. 

!Prom that time onwards increasing interest was taken 
in the study of the action of micro-organisms on stored sugar, 
and numerous publications appeared which in the great 
majority of oases recorded it as definitely proved that micro- 
organisms were responsible for the destruction, and therefore 
endeavoured to throw light on various problems connected 
with the deterioration, rather than on accumulating fresh 
facts in support of the microbiological origin of the phe- 
nomenon. 

It is a striking fact that but few writers, Amons 6 for instance, 
and Kopeloff and Kopeloff, 7 have made an effort to ascertain 
the destructive action of the various micro-organisms isolated 
by inoculating pure cultures of them on to sterilized sugar 
crystals. The evidence in favour of a connexion between the 
deterioration of sugar on storage and microbiological activity 
is in most cases of an indirect nature and is based partly on 
the observations of the presence of exceptionally large num- 
bers of micro-organisms and partly on the failure to detect 
deterioration in samples of sugar stored under conditions 
which exclude the possibility of microbiological activity. 

At first sight it might seem surprising that micro-organisms 
should be responsible for the deterioration of stored sugar, 
seeing that such sugar rarely contains more than two per 
cent, of moisture, an amount which, in the case of most other 
organic substances, would be far too small to sustain life. 

Conditions are exceptional in the case of sugar, however, 
since the slight percentage of moisture which is found is 
concentrated on the surface of the crystals in a thin layer of 
more or less concentrated sugar solution-molasses. The 
moisture, of course, does not permeate the interior of the 
crystals, and where present, therefore, it represents a much 
higher percentage than would be expected. But even so it is 
surprising that considerable microbiological activity should 
be possible in the thin film of molasses to which the moisture 



SUGAR IN STORAGE 291 

of the sugar is limited, and which, in any case, must represent 
a highly concentrated solution of saccharose. High concen- 
trations of saccharose solution have by many been regarded 
as in the nature of an antiseptic, preventing the growth of 
micro-organisms at least when exceeding concentrations of 
60 to 70 per cent. Grove, 8 Hirst 9 and Meier 10 point out that 
the growth of most micro-organisms is prevented in such 
cases. This might imply that the types comprising the flora 
suspected as the cause of the deterioration would be limited 
to comparatively few forms. This is not confirmed by the 
available published data, however. 

It is greatly to be regretted that, in their eagerness to prove 
their assumption, the two camps of writers, one claiming 
fungi and the other claiming bacteria as the more important 
sugar destroying types, have neglected to devote attention 
to a detailed study of the whole of the mioroflora observed. 
For that reason it is not possible at present to give a compre- 
hensive review of all the types active in the deterioration of 
stored sugar. It is safe to assert, however, that they are far 
more numerous than might have been expected in view of 
the stringent conditions prevailing in the film of molasses 
in which they live. 

The numbers met with, if considered per gramme of the 
sugar, are not excessive. They seldom exceed one to two 
millions, and more often vary between a few hundreds and 
some hundreds of thousands, numbers which could hardly 
give rise to marked destruction if it were not for the fact 
that they are distributed in the thin film of molasses sur- 
rounding the crystals, the weight of which, even in the 
severest cases of deterioration, does not exceed a few per cent, 
of the total weight of the sugar. 

The concentration of micro-organisms present in this film 
is therefore very much higher than indicated by the figures 
determined in the ordinary routine estimation of numbers 
per gramme of material plated out. 

Here, as in the raw juice, the number of bacteria greatly 
exceeds that of fungi, and while the latter may be entirely 

u2 



292 THE MICROBIOLOGICAL DETERIORATION OF 
absent, even where the total number of organisms is high 
(Kopeloff,Welloome and Kopelofi 11 ), bacteria invariably occur 
in greater or smaller numbers, representing sometimes, it is 
true, a few per cent, of the total flora only. 

These facts should have been thought sufficiently con- 
vincing to justify a thorough study of the subject of the 
bacterial types present. This, however, has not been made 
and a detailed account of the bacterial flora cannot be given. 

In his study of the deterioration of stored raw beet-sugar 
Schone 12 detected no less than 34 species of bacteria. They 
included cocci, in two cases even Streptococcus meaenteroides 
as well as another saccharose-inverting coccus. In addition, 
various spore-forming rods were found, such as lactic acid 
bacteria, types described as belonging to Bact. coli commune, 
and gelatine liquefying types. The latter were probably 
related to Bact. fluorescens liquefaciens. Spore formers were 
represented by mucus-producing types of the aerobic soil 
bacilh'. The latter were carefully investigated by Owen, 18 
who states that the two types most generally met with 
resemble Bac. mesentericus vulgatus (Bac. wilgatus, Migula), 
and Bac. mesentericus fuscus (Bac. mesentericus Lehmann 
and Neumann). In his earlier publications Owen did not 
hesitate to attribute the chief role in the destruction of 
stored sugar to these bacilli, remarking that other spore 
bearers, such as Bac. liodermos, Bac. mesentericus niger and 
Bac. Megatherium, which had also been observed by him in 
deteriorating sugar, take part in the destruction. Owen 
found justification for his deductions in the observation that 
the organisms referred to produce gum from saccharose and 
therefore liberate a certain amount of reducing sugar. A 
somewhat similar argument had previously been put forward 
by Greig-Smith, 14 who regarded his mesenteries-resembling 
organism, Bac. levaniformans, as one of the most important 
infections of deteriorating raw sugar. This argument is very 
misleading. It is not the synthesis of levan by these organ- 
isms which renders them dangerous enemies of the sugar 
manufacturer, but their property of producing saccharase. 



SUGAR IN STORAGE 293 

After all, the synthesis of levan is carried out only under 
certain cultural conditions and cannot be regarded as an 
essential physiological function. The constant presence of 
these organisms in the thin fil of concentrated molasses 
surrounding the sugar crystals can only be accounted for by 
assuming that they produce as in fact they do an enzyme 
which hydrolyses saccharose and thereby sets free monoses 
which can be utilized by the organisms for the liberation of 
the energy required for their various lif e functions. Synthesis 
of levan does not set free energy, and cannot, therefore, be 
an important factor. 

In addition to the forms referred to above, attention was 
sailed by Browne 16 to another mucus-producing rod which 
ae termed Bad. inv&rtans, owing to its production of saocha- 
"ase, and more recently by Cameron and Williams 16 to the 
Dresence in sugar crystals of true thermophilic bacteria. 
Their connexion with the deterioration of sugar in storage is 
it present obscure, though it is known from SchOne's 17 ob- 
lervations that microbiological activity in stored sugar may 
each a pitch at which spontaneous combustion sets in. In 
.he case to which SchOne refers, a lot of 1,000 tons of beet- 
ugar was destroyed in this way with almost explosive force. 
The conception that bacteria are the chief type of micro- 
>rganisms responsible for deterioration of sugar on storage 
tas never gained a firm footing among workers in spite of 
he fact already referred to, that these micro-organisms 
lominate the microflora in the majority of cases, and in some 
ases may be the only type of micro-organisms present. 

It is not altogether surprising that the view of the partici- 
>ation of bacteria should have been abandoned in favour 
f allocating responsibility to various fungi. The advocates 
'ropounding the bacterial theory have failed in the past to 
ring forward proof of their contention. It will require 
snewed investigation of a more exhaustive nature to establish 
efinitely that in some cases bacteria are solely, and in other 
ases partly, responsible for the deterioration of sugar on 
borage. 



294 THE MICROBIOLOGICAL DETERIORATION OF 

The association of fungi with this phenomenon seems to 
have been much more definitely established. 

The early microscopic observations made on the deteriora- 
tion of sugar associated this phenomenon with the presence 
of fungi, and with the few exceptions referred to, practically 
every detailed study of the subject made since then has led 
to the conclusion that fungi were chiefly responsible. 

The most frequently mentioned types are species of Peni- 
cillium and Aspergillus, which are usually found in the 
majority of samples, though often in surprisingly small 
numbers. Kamerling 18 isolated no less than nineteen types 
of Penicillium from crude cane-sugar. Schone 12 noted the 
presence of Penicillium glaucum* in beet-sugar, but drew 
attention also to the importance of Torula types and to 
Mucor species. Scott 19 isolated species of Penicillium and 
Aspergillus from Brazilian, Peruvian, Jamaican and Javanese 
samples of crude sugar. More recently Browne, 16 Amons, 6 and 
Kopeloff and KopelofF have reported on the destructive 
activity of fungi found in crude sugar. In Amons 's experi- 
ments AspergiUus and Penicillium predominated, while one 
type of Mucor was also met with. Kopeloff and Kopeloff 
enumerate the following fungi as having been isolated by 
them from cane-sugar : 

Aspergillus niger 
flavus 
nidulans 
Sydowi 
repens 

in addition to some unclassified strains : 
Penicillium expansum 

divaricatum 

luteum ser. 

purpurogenum ser. near Penicillium pinophilum 

* It should be noted that the name Penicillium glaucum is often loosely 
applied rn the literature. 



SUGAR IN STORAGE 295 

PenicilUum purpurogenum ser. near Penicillvwm kUeum 

, , htteum-purpurogenwrn ser. neaxPeniciUium roseum 
Citromyces three types 
Syncepholostrum sp. 
Trichoderma 
Fusarium 

Dematium 

Monas&us purpureus ser. 
Monilia nigra and at least six further unidentified types. 

Of these the more important were present in the per- 
centages shown in Table XI. 

Aspergittus Sydowi was thus not only the type most fre- 
quently met with, but was also the most active sacoharase- 
producing species found. 

Monilia nigra, to which Kopeloff and Kopeloff refer as a 
type of minor importance, was regarded by Browne 16 as very 
destructive, being followed closely in this respect by a related 
type to which he gave the name Monilia fusca. Monilia 
nigra was found by Browne in practically pure culture in 
some of the Cuban sugar examined by him. Both this and 
Monilia fusca, particularly the latter, inverted cane-sugar 
solutions with remarkable rapidity. In one case a 21 per 
cent, saccharose solution was reduced in strength to 6-5 per 
cent, within three weeks. Like other species of Monilia 
Browne's types produced bud cells propagating like yeast 
where conditions were suitable, that is, presumably, where 
access of oxygen was restricted. It is still an open question 
whether in some cases, where species of Torula have been 
found to take an active part in the destruction of saccharose, 
it may not have been the budding growth forms of Monilia 
which were responsible, rather than Torula species as defined 
by Hansen, 20 since the latter group does not usually invert 
saccharose. Browne's own work would appear to confirm 
this assumption. The Torula communis, which he described 
as the most common fungus found in Cuban sugar, did not 
hydrolyse cane-sugar, but was restricted in its action to the 
fermentation of invert sugars, particularly fructose. From 



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SUGAR IN STOEAQE 297 

this carbohydrate it produced a slight amount of gas and 
small quantities of esters. It was a Tonda also which Owen 21 
recommended as a means of protecting stored cane-sugar 
against deterioration. Owen found that a saccharose solution 
inoculated with an inverting Hyphomycete and a Torula 
showed practically no loss on incubation, while a correspond- 
ing solution inoculated with the Hyphomycete alone showed 
marked inversion of the saccharose. 

The data referred to above on micro-organisms observed 
in, or isolated from, sugar crystals cannot have failed to 
impress the reader by the comparative variety of types, that 
is to say by the possibility that sugar deterioration may be 
due to a great variety of types not necessarily all of them 
bacteria or fungi, but bacteria and fungi combined. The 
impression is undoubtedly gaining ground, contrary to what 
was at one time assumed, that this phenomenon may be due 
to the activity of any type of organism which can find suit- 
able conditions for development in the concentrated solution 
of saccharose surrounding the individual crystal. 

In the previous chapter a partial answer at least was 
supplied to the question of how the varied microflora is intro- 
duced into the moisture film of the sugar crystal. It was 
shown that the massecuite usually contains very few micro- 
organisms beyond a few hundred spores of aerobic soil bacilli 
per gramme, but that additional types are introduced in the 
centrifuge in which the crystals are separated from their 
mother liquor, partly through the large volume of con- 
taminated air surging past the crystals at great speed and 
partly through the crystals being washed with badly con- 
taminated water. It has also been suggested (Kamerling 18 ) 
that the habit of bagging the finished sugar in infected bags 
might be the chief cause of infection of the crystals. While 
not denying the possibility of additional infections being 
derived from the bags, it is hardly safe to accept this assump- 
tion to the extent which Kamerling favours. If this infection 
actually occurred deterioration would be limited in the first 
instance to the crystals in direct contact with the bags, 



298 THE MICROBIOLOGICAL DETERIORATION OF 
whereas actual observations show that deterioration may 
have proceeded for some time in the interior before being 
noticeable on the surface. As pointed out by Browne and by 
Kopeldff and Kopeloff the distribution of moisture in the 
sugar appears to be the factor governing the initial seat of 
deterioration. 

At an early date in the study of the deterioration of sugar 
on storage the conclusion was arrived at that a sugar could 
not be expected to keep during storage when the moisture 
present appreciably exceeded the non-sugar contents of the 
sample. This led to the formulation of the so-called 'factor 
of safety' demand of the Colonial Sugar Refining Company 
of Australia. The factor of safety requires that the per- 
centage of moisture in a sugar must not exceed half that of 
the non-saccharose constituents. It is expressed in the 
following formula: 

W W 

= 0-6, or simplified =0-333; 



100-S-W * 100-8 

where W represents the moisture content, and S the saccha- 
rose content. 

As a matter of fact the figure should be put no higher than 
0-3 at least in the case of Cuban and Porto Rican sugars. 

In other words the requirement of the factor of safety 
indicates that a comparatively impure sugar may be allowed 
a considerably higher moisture content than a very pure 
sample. Thus if a sample of raw sugar, containing 90 per 
cent, of saccharose, had a moisture content of 2-4 per cent., 
the factor of safety of this sample would be : 

2-4 

=0-24; 



100-90 



while a high grade sugar of 99 per cent, saccharose content 
and with 2-4 per cent, moisture would show a safety factor of 



2-4 

- - =2-4. 
100-99 



SUGAR IN STORAGE 299 

The higher the purity of the sugar, therefore, the less moisture 
can it be suffered to contain, and in the same way the more 
will a slight change in the prevailing moisture, conditions 
affect the factor of safety. * , 

An absorption of 0-3 per cent, of additional moisture would 
make little difference in the factor of safety figure of a raw 
sugar of 90 per cent, purity and a moisture content of say 
0-2 per cent., the factor merely rising from 0-02 to 0-05. In 
a high grade sugar of the same moisture content and with 
99 per cent, of saccharose an increase of this order would 
raise the factor of safety from 0-2 to 0-5, or much beyond that 
at which the sugar could be stored with safety, assuming the 
factor of safety to be a genuine index of the keeping pro- 
perties of a sugar sample. 

But unfortunately it can only be claimed to be a genuine 
index to a limited extent. While it is correct, probably, that 
a sample of refined sugar is rendered more sensitive to 
deterioration by slight changes of moisture than low grade 
raw sugar, it is not possible to predict with certainty the 
keeping qualities of a sugar by ascertaining the factor of 
safety figure of the sample. Kopeloff and his collaborators 22 
investigated this question and arrived at the conclusion that 
the factor of safety figure was effective only in 40 per cent, 
of the oases examined. 

To some extent the reason for this would appear to be 
found in the fact that the deterioration at a given moisture 
concentration depends not only on whether micro-organisms 
are present in the surface fil of molasses or not, but also on 
the numbers present per unit of the film. For example, in 
sugars with safety factors ranging from 0-18 to 0-50, Kopeloff 
and his collaborators observed deterioration in practically 
every case when the numbers of micro-organisms per gramme 
of sugar exceeded 50,000. As the numbers decreased to about 
500 there was evidence of less deterioration at moisture 
ratios below 0-36. But even where the ratio fell below 3 
deterioration was observed in the sugar when more than 
200 micro-organisms were present per gramme. It IB in 



300 THE MICROBIOLOGICAL DETERIORATION OF 
samples of sugar with comparatively few micro-organisms 
therefore that the factor of safety is most likely to be of 
practical importance, and it should be applied only in com- 
bination with a bacteriological analysis of the samples. 

This raises the question as to how this bacteriological 
analysis is to be carried out, or rather what type of nutrient 
medium should be used for the purpose, since it is obvious 
that standard conditions must be adhered to in this respect if 
consistent results are to be expected. 

Various media have been recommended for the isolation 
of the micro-organisms developing on raw sugar. Browne 
uses a 30 per cent, solution of raw cane-sugar, Amons 6 a 
modified Czapek agar. This latter, which has been shown by 
Kopeloff and Kopeloff 7 to be of special value, contains 3 per 
cent, saccharose; 0-1 per cent, dipotassium hydrogen phos- 
phate, 0-05 per cent, potassium chloride, 0-6 per cent, mag- 
nesium sulphate, 0-001 per cent, ferrous sulphate and 0-2 per 
cent, sodium nitrate. 

Kopeloff and his collaborators finally decided on another 
modification of Czapek 's medium in which the saccharose is 
increased to 5 per cent. Peptone is introduced in a concen- 
tration of 0-6 per cent, and the sodium nitrate is in part 
replaced by ammonium nitrate, while the magnesium sul- 
phate is reduced by half. They claim that this medium is 
more effective than Czapek's for the isolation of fungi. Since 
it contains an appreciable percentage of peptone it will 
probably be found superior also for the isolation of the 
bacteria developing in sugar and might therefore be recom- 
mended as the standard nutrient medium in microbiological 
sugar analyses. 

The comparative importance of the factor of safety index 
in the demonstration of the keeping qualities of stored sugars 
will have illustrated the importance of the presence of 
moisture for the development of micro-organisms in the 
film of molasses surrounding the sugar crystal. It is not to be 
concluded, however, that, given a sufficient concentration of 
moisture in a sugar, microbiological deterioration will pro- 



SUGAR BT STORAGE 301 

oeed unchecked for an indefinite period. There are various 
factors which counteract the destruction and in time may 
bring it to a standstill. One of these factors is the temperature 
prevailing at the place of storage. 

Browne refers to a consignment of moist sugar which was 
stored in New York from October till May without suffering 
deterioration, in spite of the presence of harmful micro- 
organisms. During this period the temperature in the store- 
room did not exceed 20 C., a figure, therefore, which may 
safely be placed as the TnininvnTn at which deterioration can 
proceed within a reasonable period it should no doubt be 
added. It would be unsafe to interpret Browne's observation 
as establishing that no development at all is possible at 20 C. 
It is common knowledge that many fungi and bacteria not 
only resist, but actually develop at, temperatures down to 
the freezing-point of water. 

Another factor which may have an important action on 
the progress of the deterioration of stored sugar is the actual 
activity of the responsible micro-organisms, or rather of the 
products of metabolism of these organisms. It has already 
been mentioned that the sphere of action is restricted to the 
thin film of molasses surrounding the sugar crystals. Pro- 
longed activity, therefore, is bound to accumulate in this 
thin layer a considerable concentration of products of meta- 
bolism, not only invert sugars, but also various fermentation 
products, including esters, alcohols and volatile and non- 
volatile acids, which in the great majority of oases cannot be 
removed, and therefore must arrest the activity of the various 
enzymes responsible for their production. That this actually 
occurs, at least where deterioration proceeds in a sample of 
sugar which is stored under conditions preventing the ab- 
sorption of moisture, has frequently been observed. Lewton- 
Brain and Deerr 23 report on a case of this type where a sugar 
stored for some years had, in time, become more or less sterile. 

On the other hand, when an opportunity arrives for the 
absorption of additional moisture, a reduction in the con- 
centration of the accumulated products of metabolism will 



302 THE MICROBIOLOGICAL DETERIORATION OF 
result and the progress of destruction can continue, other 
factors being favourable. This happens in many storage 
places, partLoularly in damp climates or at sea, and here it 
is no unusual sight to find a thick syrup oozing from the 
bags, spreading an infection not only from bag to bag, but 
through the intermediary of workmen to other consignments 
in storage. 

Such conditions are highly dangerous and should certainly 
be avoided. The most obvious method of doing this might 
be thought to be the reduction of the humidities in the storage 
sheds. This may not be practicable, however, since the 
absorption of minute percentages of moisture suffices to 
support microbiological activity in sugar. Additional pre- 
cautions will usually be found necessary, notably the reduc- 
tion of the number of micro-organisms present. To some 
extent this can be ensured by the introduction of increased 
cleanliness in the sugar factory, particularly during those 
processes which start with the separation of the sugar crystals 
in the centrifuge. Where superheated steam, as suggested 
by Kopeloff and his collaborators, 11 is utilized instead of 
water for the removal of the adhering molasses, the very 
startling reduction in the microflora thereby secured, com- 
bined with the added facility of drying the hot sugar, should 
materially assist in securing a sugar of high resistance to 
decay during storage. 

The economic importance of the deterioration of sugar on 
storage hasjbeen ascertained on various occasions and has 
been shown to be of considerable magnitude. For Cuban 
raw sugar alone it amounted to 200,000 annually, according 
to Browne. 16 It cannot be an exaggeration to claim, therefore, 
that the total world loss must be more than two million 
pounds sterling annually. 

A destruction of saccharose occurs occasionally in chocolate- 
coated creams, a subject which' has been studied by Weinzirl, 24 
and more recently by Church, Paine and Hamilton. 26 Though 
this type of deterioration may not be one of great importance, 
it is interesting to know that it has been attributed in both 



SUGAR IN STORAGE 303 

cases to the activity of Bac. sporogenes, an anaerobic butyric 
acid producing bacillus, and of yeasts. Paine, Birokner and 
Hamilton 26 find that the destruction is prevented when a 
certain percentage of invert sugar is present in the creams. 
They secure this by the addition of sacoharase to the sugar. 

LITERATURE^ 

1. 0. Ligon, History of the Island of Barbados, London, 1673 (see N. and L. 

Kopeloff, Abst. of Bacteriology, vol. 6, p. 221, 1922). 

2. N. and L. Kopeloff, Abst. of Bacteriology, vol. 6, p. 221, 1922. 

3. A. Payen, Comptes rend., vol. 33, p. 393, 1851. 

4. P. Dubrunfaut, Comptes rend., vol. 68, p. 663, 1869. 

5. E. C. Shorey, J. Soc. Ohem. Ind., vol. 17, p. 655, 1898. 

6. W. J. T. Amons, Arch. u. Suikerind. Ned. Ind., vol. 26, p. 1226, 1917. 

7. N. and L. Kopeloff, Louisiana BuU., no. 166, 1919. 

8. 0. Grove, Ann. Rep. Agrio. and Eortic. Research Stat., University Bristol, 

1918, p. 34. 

9. F. Hirst, Ann.Eep.Agric. and Horttc. Research Stat., University Bristol, 

1927, p. 150. 

10. A. Meier, ZentrU.f. Bakt., Abt. n, vol. 64, p. 241, 1926. 

11. N. Kopeloff, C. J. Welcome and L. Kopeloff, Louisiana Butt., no. 175, 

1920. 

12. A. Schfine, ZentrU.f. Bakt., Abt. n, vol. 17, p. 663, 1907. 

13. W. L. Owen, Louisiana Bull, No. 126, 1911. 

14. R. Greig-Smith, Proc. Linnean Soc. N.S.W., vol. 26, p. 602, 1902. 
16. C. A. Browne, J. Ind. Eng. Chem., vol. 10, p. 178, 1918. 

16. E. J. Cameron and 0. 0. Williams, Zentrbl. f. Bakt., Abt. II, vol. 76, 

p. 28, 1928-29. 

17. A. Schane, Die Deutsche Zuckerindustrie, vol. 36, p. 247, 1911. 

18. Z. Kamerling, Veralag over de botanische en physiologiscJte Werksaamhe- 

den, Proefstat. v. Suikerind in West Java, Kapok, pp. 97-104, 
1899. 

19. J. Scott, Intern. Sugar J., vol. 14, p. 582, 1912. 

20. H. Chr. Hanson, Comptes rend. d. Laboratoire de Carlsberg, vol. 2, p. 143, 

1888. 

21. W. L. Owen, Chimie et Industrie, vol. 11, p. 761, 1924. 

22. N. and L. Kopeloff, Louisiana BuU., no. 170, 1920. 

23. L. Lewton-Brain and N. Deerr, Louisiana Planter, vol. 54, p. 282, 1915. 

24. J. Weinzirl, J. Bacteriology, vol. 7, p. 699, 1922. 

26. M. B. Church, H. S. Paine and J. Hamilton, J. Ind. Eng. Chem., vol. 19, 

p. 353, 1927. 
26. H. S. Paine, V. Birckner and J. Hamilton, J. Ind. Eng. Chem., vol. 19, 

p. 358, 1927. 



Z205 



INDEX OF AUTHORS 



Aagaard, T , see Bridel, M. 
Abbott, 0. D., decomposition of 

pentoses by yeasts, 164. 
Abderhalden, E., assimilation of 

food substances, 40. 
Albus, W. R., see Whittier, E. 0. 
Alekhine, A., hydrolysis of mele- 

zitose, 45. 

Allemonn, O.,see Th8ni, I. 
Alsberg, 0. L., see also Black, O. F. 
Alsberg, C. L., and Black, O. F., 
synthesis of starch by PenwMium 
puberulum, 175. 
Alvarez, E., hydrolysis of indican, 

60. 

Amelung, H., decomposition of 

pentoses by Aspergtilua mger, 164. 

Amons, W. J. T., deterioration of 

sugar by micro-organisms, 290. 
, media for bacteriological analysis 

of sugar, 300. 

Anderson, J. A., see Fred, E. B. 
Anderson, E. S., see Nelson, J. M. 
Andrusiani, M., recovery of alcohol 

from bread, 238. 
Annstem, B., see Neuberg, 0. 
Armstead, D., and Harland, S. 0., 
moisture requirements of mildew, 
216. 
Armstrong, H. E., and Armstrong, 

E. F., action of saccharose, 52. 
t t effect of glyoine on sacoharase, 

63. 
Arnoldow, W. A., mould growth in 

stored rye, 202. 

Aronowski, A., see Pnngsheim, H. 
Ashe, L. H., see Northrop, J. H., 

and Kohman H. A. 
Atkinson, A. W., preparation of 

Kon, 23. 

Atterberg, A., effect of moisture 
content on microflora of stored 
gram, 196, 197, 201. 
Atwater, H., sour bread, 247. 
Aubel, E., see also Cambier, R 
Aubel, E., decomposition of glucose 

by Bact pyocyaneum, 112. 
, decomposition of hexoses by 

facultative anaerobes, 112. 
, decomposition of methylglyoxal 

by Bact. coh commune, 118. 
, enzymes of Bact. coh commune, 
116, 118. 



Aubel, E , pyruvio acid in bacterial 
fermentations, 106,- 107, 119. 

Aubry, A., see also Bourquelot, E. 

, action of maltase on a-gluoosides, 
67. 

Avery, 0. E., lactic acid manu- 
facture, 131. 

Ayyar, G. G., see Rao, T. L. 



Baohrach, E., and Cardot, H., opti- 
mum pH for lactic acid bacteria, 
132. 

Bailey, C. H., see also Greeve, E. 
Bailey, C. H , and Johnson, A. H , 
dough, method of testing, 235-6. 
, , dough fermentation, effects 

of pH on, 233, 237. 
, , dough fermentation, gluten 

in, 235. 

Bailey, 0. H., and Sherwood, R. C , 
effect of pH on diastatio activity 
of flour, 229. 
, , dough fermentation, 220. 
, , dough fermentation, effect 

on disaccharides of, 230. 
, , dough fermentation, pH 

changes during, 236-7, 241-2. 
, , dough fermentation, protein 

changes in, 240. 

Bailey, G. C., and Potter, R. S , 
fumario acid for synthesis of 
indigo, 100. 

Banner, S., see Sartory, A. 
Baker, J. L., treatment of brewers' 

yeast for breadmaking, 227. 
Baker, J. L., and Hulton, H. F. E., 
substances in flour toxic to yeast, 
207, 227. 

Bakes, W. E , see Thaysen, A. C. 
Baloh, R. T., see Paine, H. S. 
Baldwin, M. E., see Sherman, H C. 
Banning, F., production of oxalic 

acid by micro-organisms, 99. 
Barendrecht, H. P., isolation of 

lactase, 59. 

Barlund, B., see Virtanen, A. I. 
Barthel, 0., lactic acid bacteria, 126. 
Baudnmont, , yeast saccharase, 

47. 
Bean, P., and Scansbnck, F., flour 

fermentation, 213, 214. 
Beocard, E., production of rye 
bread, 225. 



306 



INDEX OF AUTHORS 



Bechamps, A., mucus production, 

182-3. 
Becker, H., microflora of damp 

gram, 198. 
van Beek, , sugar deterioration 

by fungi, 289. 
Behrens, J., action of Boo. amylo- 

bacter on starch, 18. 
Beijerinck, M. W., constitution of 

amylase, 12. 

, Bac. saccharobidyricus, 146. 
, Bact. agglom&rana, 192. 
, Bact. lactqfermentum, 126. 
, indican, action of plant enzymes 

on, 68. 

, laotase, isolation of, 69. 
, lactase in yeasts, 58. 
, lactic acid bacteria, 127-8, 

133. 

, mucus production, 186, 186. 
, starch decomposition by bacteria, 

17, 19. 

Beiser, A., eee Pnngsheim, H. 
Bell, H. G., stored grain, acid pro- 
duction in, 204. 
, stored grain, optimum conditions 

for, 201. 
Bellinger, P., and Delaval, H., pH 

range of Mucor Rouocii, 32, 34 
Benson, A., lactic acid manufacture, 

131, 133. 
Bergey, D. H., laotase in Actino- 

mycetes, 69. 
Bergtheil, 0., hydrolysis of mdican 

by enzymes, 68 
Berlin, H., constitution of gentio- 

biose, 44. 
Bernhauer, K., fungi, production of 

citric acid by, 98, 103. 
, fungi, production of gluconic 

acid by, 96, 98-9. 
Bernhauer, K., and Schon, K., acet- 

aldehyde formation by Asper- 

gittus ntger, 97. 
Bernhauer and Wolf, H., influence 

of phosphoric acid on citric acid 

production, 97. 
Bernheim, H., micro - organisms 

within plant tissues, 194. 
Bertarelh, E., pellagra, 200. 
Berthelot, M. P , action of yeast on 

saccharose, 47. 
Bertrand, G., Bact. acylinum, action 

on glycerol, 96, 107 
, Bact. xylinum, action on xylose, 

162. 

, dihydroxyacetone as interme- 
diate product, 81, 107. 



Bertrand, G., and Compton, A., 

ceUobiase, 68. 
Bertrand, G., and Holderer, M., 

hydrolysis of cellobiose, 68. 
Bertrand, G., and Rosenblatt, M , 

factors affecting saccharose, 63, 

65. 
Bertrand, G., and Weissweiler, G., 

Bact bulgaricum, 125, 128. 
, , optical properties of fermen- 
tation lactic acid, 129. 
Bettmger, M., saocharification of 

starch by Bac. burdigalense, 21. 
van Beyma thoe Kingma, see Falck, 

B. 

Bezzola, 0., pellagra, 200. 
Bidzmski, Z., see Ohrzaszcz, T. 
Bienz, 0., mucus production by 

fungi, 185. 
Bienz, C , Brautigam, W., and 

Happ, 0., mucus production in 

infusions of Digitalis, 182. 
Bierry, H , and Coupin, F., Aspcr- 

ffillus niger lactase, 59, 60. 
Birckner, V., see Paine, H. S. 
Bishop, R. O., and Feik, G. R., nco 

fermentation by M wear Rouom, 24. 
Bitter, H., bacterial decomposition 

of starch, 17, 19 

Bizio, B , 'blood' on bread, 257. 
Black, O. F., sec also Alsberg, C. L 
Black, O. F., and AJsberg, 0. L., 

Bact. prodigiosum in, 205. 
, , stored gram, deterioration 

of, 196, 201, 202, 206, 207. 
Blagowestschenski, A , synthesis of 

saccharose, 55. 

Blake, A. E., see Kohman, H. A. 
von Blucher, H., manufacture of 

lactic acid, 131. 
Boas, F., starch synthesis by Aspor- 

gdlus mger, 176, 179. 
Boekhout, F. W. J., mucus produc- 
tion in sugar manufacture, 268. 
Boidin, A , amylo process, 36. 
, desizmg of textiles, 21. 
Boidin, A., and Effront, J., desizmg 

of textiles, 21. 

Boinot, F., butyl alcohol fermenta- 
tion, 159. 
Bokorny, T., assimilation of maltose, 

57. 
, assimilation of pentoses by yeast, 

164. 

, effect of pH on lactaso, 69. 
Bolas, T., alcohol in bread, 238. 
Bomonti, H. F., use of lime in sugar 

manufacture, 274. 



INDEX OF AUTHORS 



307 



Bondi, J., see Pnngsheim, H. 

Boselli, J., uxulase, 36. 

Botkin, S ,propionic acid production 
by bacteria, 138, 160. 

Boudrimont, A., mucus production, 
181. 

Boulonger, E., see Kayser, E. 

Bourquelot, E., decomposition of 
gentianose and rafflnose by As- 
p&rgfilua niger, 44. 

, decomposition of glucosides by 
fungi, 65. 

, decomposition of maltose by 
micro-organisms, 66. 

, effect of glycerin on saccharase, 
62. 

, mulin from Aapergillus niger, 36. 

, isolation of trehalose, 46. 

, starch synthesis by Boletus 
pochypus, 175. 

Bourquelot, E , and Aubrey, A , 

effect of pH on gluoosidase, 67. 
Bourquelot, E., and Bridel, M., 
assimilation of rafflnose, gentia- 
nose and staqhyose, 55. 
, , effect of alcohol on saccharase, 

65. 

Jourquelot, E., and H6nssey, H., 
decomposition of gentianose by 
AspergiUus niger, 43, 44. 
-, , decomposition of glucosides 

by fungi, 66 

-, , lactose in Polyporua sul- 
phureus, 59. 

-, , melezitase in Aspergillus 
niger, 45. 

loutron, F., and Fre'my, E., lactic 
acid fermentation, 130, 160-1. 
outroux, L., glucomc acid produc- 
tion by micro-organisms, 93. 
-, oxygluconie acid production by 
bacteria, 96. 

-, spontaneous fermentation of 
dough, 221. 

-, yeasts in sour dough, 223. 
ovshik, G., see Schiber, G. 
oysen- Jensen, P., dihydroxyace- 
tone as intermediate product of 
fermentation, 81. 
rttutigam, W., see Beinz, C. 
r^audat, L., action of plant en- 
zymes on indican, 68. 
'edemann, G , decomposition of 
starch by Sao. amylobacter, 18. 
, butyric acid bacteria, 151. 
idel, M , see also Bourquelot, E. 
idel, M., and Aagaard, T., con- 
stitution of melezitose, 45. 



British Cotton Industry .Research 
Association, Fargher, B. G., 
Galloway, L. D., and Probert, 
M. E., antiseptic properties of 
salicylanilide, 217. 

Brown, A. J., action of saccharose, 
52. 

, gluconio acid production by 
Bact. Pasteurianum, 93. 

Brown, H. T., and Heron, J., malt 
amylase, 11. 

Brown, H. T., and Morns, G. H., 
malt amylase, 11. 

Browne, 0. A., Bact xyUnum, 271. 

, sugar deterioration, 301, 302. 

, sugar deterioration, organisms 
causing, 293, 295, 300. 

Brunstem, A., decomposition of 
glucosides by fungi, 65. 

Buchner, H., absence of micro- 
organisms in plant tissues, 194. 

Buohner, E., and Meisenheimer, J , 
butyric acid bacteria, 142-3, 
149. 

, , metabolism of yeast, 174. 

Buohner, E., and Bapp, B., glyoo- 
genase in yeast, 13. 

Buohner, E., and Wustenfeld, H , 
citric acid fermentation, 103. 

Buchwald, J., Mondw vanabiha in 
bread, 253. 

Budmoff, L , microflora of sour 
dough, 224. 

, sterilization of cereal products, 
207. 

Bunker, H. J., see Thaysen, A. C. 

Burn, B., Bact. coh-typhosum group, 
60. 

, Bact. coH-typhosum, saccharase 
production by, 48, 60. 

, epiphytic microflora of plants, 
191, 192. 

, Indian ink method, 159. 

Burri, B., and Holhger, W., micro- 
flora of sour dough, 224. 

Butkewitsoh, W., citric acid produc- 
tion of fungi, 06, 102, 103. 

Butkewitsch, W., and Fodoroff, 
M. W., fumoric and citric acid 
production by Mucor stolomfer, 
100. 

C 

Calmetto, A , arayloso of Mucor 

Rouxii, 30, 32. 
Cambior, B , and Aubol, E , pyruvio 

acid as fermentation product, 107, 

119 



X2 



308 



INDEX OF AUTHORS 



Cameron, E. J., and Williams, 0. 0., 
thennophilio bacteria in stored 
sugar, 293. 

Cardot, H., see Bachrach, E. 
Carroll, W. B., see Fred, E. B. 
Castellfuu, A., hydrolysis of gluco- 

sides by bacteria, 65. 
Castellani, A., and Taylor, F. E., 
inulase of M omlia macedontenms, 
36. 

Gathoart, P. H., see Cohn, E. J. 

Oeni, C., pellagra, 200. 

Chabot, G., pfi of flour and dough, 
232, 241. S 

Challenger, F., Subramaniam, V., 
and Walker, T. K., saccharic acid 
production by Aapergti&us niaer, 
97. 

Chalmers, 0. H., see Gray, P. H. H. 

Chaudun, A., gee Cohn, H. 

Chevron, L., hydrogen evolution in 
sugar manufacture, 271. 

Chicandard, G.,doughfennentation, 
219, 223. 

Chick, H., pH and sterilization, 162. 

Chrzaszcz, T., amylase of Mucor 
Cambodia, 32. 

, Gladosponum Jierbarum in gram, 
199. 

, epiphytic bacteria of plants, 193. 

, lactic acid production by Mucor 
Rcmxti, 134. 

, yeasts in gram, 220. 

Chrzaszcz, T., Bidzinski, Z., and 
EJrause, A., pH range of amylases, 
27. 

Chrzaszoz, T., and Tinkow, D., 
oxalic acid formation by micro- 
organisms, 99. 

Church, M. B., see also May, O. E., 
and Thorn, C. 

Church, M. B., mioroflora of sugar 
juices, 281. 

Church, M. B , Paine, H. B., and 
Hamilton, J., decomposition of sac- 
charose in chocolate creams, 302 

Churchman, A., pentoses in cacao 
shells, 167. 

Cienkowski, L., mucus production, 
181. 

Claflm, A. A., lactic acid manu- 
facture, 131. 

Clark, A. B., see Baistnck, H. 

Clayson, D. F. H., and Schryver, 
S. B., hemicelluloses in starch, 6. 

Cohn, E. J., Wolbach, S. B., Hen- 
derson, L. J., and Cathcart, P. H , 
ropy bread, 249. 



Cohn, F., Bact. prodigiosum or 

bread, 257. 

Cohn, R., fermentation of saccha- 
rose, 42. 
Cohn, H., and Chaudun, A., action 

of saccharose, 52. 

Oollatz, F. A., see also Morison, C. B 
Collatz, F. A., and Baoke, O. C. 

malt extracts in bread-making, 

230, 231. 

Compton, A., see Bertrand, G. 
Ooolhaas, 0., decomposition ol 

starch by thermophilio bacteria 

18, 22. 
Cortez, > toxins in stored grain 

202. 

Coupin, F., see also Bierry, H. 
Coupin, F., laotase, 60. 
Courtonne, H., cinnamon oil ai 

antiseptic, 275. 
Cramer, H., see von Euler, H. 
Cremer, M., action of amylase on 

glycogen, 13. 
, synthesis of glycogen by yeast 

juice, 173. 
Currie, J. N., citric acid production 

by fungi, 102. 

Curtis, R. E., see Waksman, S. A. 
Czapek, F., alcohol in brood, 238. 
, decomposition of pentoses by 

Aspergillits mger, 164. 
, dextrins, 9. 
, inuhn in algae, 13. 



Dakin, H. D., and Dudley, H. W., 
methylglyoxal as precursor of 
lactic acid, 118. 

Dale, E., Aspergittua comcus, 185. 

Davis, W. A., hydrolysis of mdican 
by micro-organisms, 69-70. 

Dean, A. L , inulase production, 36. 

Decaisne, E., mouldy broad as food- 
stuff, 256. 

Deerr, N., see Lewton-Brain, L. 

Dehdrain, P. P , butyric fermenla- 
tion of beet sugar, 271. 

Delaval, H., see Bellinger, P. 

Desborough, A. P. H., addition of 
acetic acid to acetone fermenta- 
tion, 114, 147. 

Desfosses, , mucus production, 
181. 

Desmots, H., hexose decomposition 
by aerobic bacilli, 106. 

Dienes, L., see von Fenyvossy, B. 

Dombrowski, , ether sterilization 
of cereal products, 207. 



INDEX OF AUTHOES 



309 



Dombrowski, , nature of acids in 

stored gram, 206. 
Donker, H. J. L., see also KHuyver, 

A.J. 
, Boo. acetoefhyUcus, 86, 113, 114, 

166. 
, Bac. acetonigenus, 114, 147, 148, 

153. 

, Bac. Pasteunamts, 145. 
, butyric fermentation, 144, 147, 

161. 
Dore'e, C., and Kirkland, J., dough 

fermentation, 230. 
Dorner, W , Bac. acetonigemis, 159. 
Dox, A. W., and Neidig, R. E , syn- 
thesis of starch by Peniffkttvum 

eccpansum, 175. 
Dox, A W., and Plaisance, G. P., 

Tnn.rmit.nl in silage, 133 

Dozier, 0. 0., see Wagner, E. 

Draggendorf, G., manmtol produc- 
tion, 133. 

, toxic substances in stored grain, 
202. 

Dubourg, E., see Gayon, U. 

Dubrunfaut, P., action of yeast on 
saccharose, 47. 

, bacterial deterioration of stored 
sugar, 289. 

, effect of nitrates on molasses 
fermentation, 287. 

, 'froth fermentation' of sugar, 
283. 

, maltose, 3. 

Duclaux, E., decomposition of starch 
by moulds, 29. 

, effect of medium on enzyme 
production, 31. 

, production of gallic acid from 
gall apples, 71. 

, technique of enzyme isolation, 
30. 

Dudley, H. W., see Dakm, H D. 

Duggeh, M , epiphytic microflora of 
plants, 102. 

Dunnenberger, C., dough fermenta- 
tion, 221, 223. 

Dupont, 0., Bac. mesentencua ruber, 
160. 

, bacterial decomposition of starch, 

18, 19, 20. 
Durandard, M., amylase of Mucor 

stolomfer, 32. 

Durieux, O., effect of heat on sac- 
charase, 54. 
)unn, E., 'froth fermentation' of 

sugar, 283. 
, mucus production, 181, 183. 



E 



Effront, J., aee also Boidin, A. 

, action of electrolyte on amylases, 

27. 

, bacterial amylases, 20. 
, isolation of amylases from Bac. 

mesentencua, 17. 
Ehrenberg, C. G., Bact. prodtffiosum 

on bread, 256. 
Ehrhch, F., action of yeasts on 

glutaimc and succimc acids, 110. 
, action of yeasts on proteins, 141. 
, fumaric acid production by 

Mucor stolonifer, 100. 
, fusel oil, 163. 

Eijkmann, C., bacterial decom- 
position of starch, 18. 
, lactic acid production by Mucor 

Itouxi^, 134. 

Eissler, F., see Pringsheim, H. 
Ekman, G., decomposition of pen- 

toses by Aepergtttua ntger, 164. 
Ehasberg, P., see Kostytschew, S. 
Ehon, L , dough stimulants, 232. 
Ellis, D., preparation of leavens, 225. 
Emmerlmg, O., fusel oil, 153. 
, mouldmess of stored cereals, 107, 

205. 

, mucus production, 182, 184. 
Emmerlmg, O., and Reiser, O., 

action of Bact. fluorescens lique- 

facnens on starch, 18 
Engel, , Saccharomyces minor in 

sour dough, 223. 
Epstein, A. K., dough stimulants, 

232. 
Errera, L , synthesis of glycogen, 

174. 

von Euler, H , see also Myrback, K. 
von Euler, H., saooharase produc- 
tion, 42, 40, 50, 51. 
von Euler, H., and Cramer, H., 

effect of mannose on saccharase 

production by yeast, 61. 
von Euler, H., and Josephson, K., 

substances affecting saccharase, 

53. 

von Euler, H., and Laurin, T., opti- 
mum pH of saccharase, 51. 
von Euler, H., and Myrback, K., 

action of saccharose, 52. 
von Euler, H., and Svanberg, O., 

bacterial decomposition of starch, 

10 
, , saccharase production by 

yeast, 51, 52. 
, , substances affecting sacoha- 

rase, 54. 



310 



INDEX OF AUTHORS 



F 



Falok, B., and van Beyma thoe 

Kmgma, citric acid production 

by fungi, 102. 
, , gluconio aoid production by 

fungi, 96. 
Falck, Ei., and Kapur, 8. N., gluconio 

acid production by fungi, 96. 
Pales, H. A., and Kelson, J. M., 

effect of salt onKojisacchaxase, 53. 
Fargher, B. G., see British Cotton 

Industry Research Association. 
FedorofE , W. S , see Butkewitsch, 

M.W. 

Feik, G. B., see Bishop, B. O 
von Fenyvessy, B , and Dienes, L., 

eHect of baking on bacteria in 

dough, 247. 
Fermi, C., sacoharase production by 

bacteria, 48. 
, starch decomposition by bacteria, 

17-20. 
Fermi, C , and Montesano, G , gluco- 

sidase production by bacteria, 67. 
, , saccharose decomposition by 

bacteria, 48, 51, 52. 
Fernbach, A., see also Wolff, J. 
, bacteria absent in plant tissues, 

J04. 
-, formaldehyde production by 

aerobic bacilli, 108 
, hexose decomposition by aerobic 

bacilli, 106. 
, starch decomposition by Asper- 

gillusmger, 29. 
, starch decomposition by Bac. 

tenuia, 18. 

Fernbach, A., and Schoen, M., con- 
stitution of mucus, 184. 
Fernbach, A., and Strange, E. H., 

acetone fermentation, 152, 153. 
Fernbach, A , Yuill, J. L., Bowntree 

& Co., citric acid production by 

fungi, 102. 

le Fevre, B , see Thorn, C. 
le Fevre, A. J., see also de Graaf, 

W.C. 
, glyceraldehyde decomposition by 

Soct.coh, 118. 
Findlay, G M., pellagra, 200. 
Fischer, E., effect of pH on gluco- 

sidases, 67. 

, kephir lactase, 59, 60. 
, maltase and sacoharase, 66. 
, trehalase in yeast, 46. 
Fischer, E., and Lindner, P., de- 
composition of mehbiose by yeasts, 

60. 



Fischer, E., and Lindner, P., saocha- 

rase in Moniha candtda, 49. 
Fischer, E., and Zemplen, Q., emul- 

sm and oellobiase, 58. 
Fisher, E. A., and Halton, P., buffer 

action of flours, 242. 
, , optimum pH for dough fer- 
mentation, 243. 
, , pH of flours, 241. 
, , ^rope' in bread, 249, 262. 
Fitz, A., butyric acid production by 

bacteria, 142, 151. 
, caproic acid as fermentation 

product, 160. 
, origin of fusel oil, 152. 
, propionio aoid production by 

bacteria, 136. 

, starch decomposition by bac- 
teria, 16, 17. 
Fleming, W. L., and Neill, J. M, 

starch decomposition by -Bac. 

WeloJtU, 18. 

Fornet, A., see Herter, W. 
Fouard, E., phosphorus content of 

starch, 6. 

Fox, J., see Frankland, P. F. 
Frankel, F., Boot, levans, 221. 
, epiphytic bacteria of plants, 192. 
Fr&nkel, S , see Kerry, B 
Frankland, H., see Frankland, P. F. 
Frankland, P. F., Frankland, H., 

and Fox, J., fermentation, by 

Bad. pneumomae and Bac. etha- 

ceticus, 113. 
Frankland, P. F., andLumsden, J. S., 

fermentation by Bact. pneumomae 

and Bac. etltaceticus, 113. 
Frankland, P. F., Stanley, A., and 

Frew, W., fermentation by Bact. 

pneumomae and Bac. ethaceticus, 

113. 
, , , fermentation of raffino&e 

by Bact Fnedlander, 45. 
Fred, E. B., see also Pederson, C. S., 

Peterson, W. H., Stiles, H. B., 

and Wilson, P. W. 
Fred, E. B , Peterson, W. H , and 

Anderson, J. A., Bac. acetoethyh- 

cus, 167. 
, , , pentose decomposition by 

lactic bacteria, 165, 169. 
, , , pentoses in oat husks, 167. 
, , , raffinose decomposition 

by Bact. lactt arabinosum, 45. 
Fred, E. B , Peterson, W. H., and 

Carroll, W. B , xylose decom- 
position by Bac meaenter-icus 
nfler, 166. 



INDEX OF AUTHORS 



311 



Fred, E. B., Peterson, W. H., and 
Mulvania, M., lactic infections of 
acetone fermentation, 158. 

Fr&ny, E., see Boutron, F. 

von Freudenreioh, E., propionio 
acid bactena, 136-7. 

Frew, W., see Frankland, P. F. 

Frey, L., see Kostytsehew, S. 

Friedrich, 0., use of formaldehyde 
in sugar manufacture, 275. 

Frou, G , inulase in Morchetta, 36. 

Frouin, A., and Guillaumie, M., 
trehalose, 47. 

Fuhrmann, E., Sac. mesentericus in 
flours, 252. 

Funke, G. I., action of takadiastase, 
28. 

G 

Gabnlowitsch, O. E., Fusanum 
roseum m rye, 199. 

Galippe, M., micro-organisms in 
plant tissues, 194. 

Galle, E , amylo process, 32. 

Galloway, L. D., see Thaysen, A. C., 
and British Cotton Industry Be- 
search Association. 

Gasohing, P , see Tisaier, H. 

Gatm-Gruzewska, Z., amylose and 
amylopectin, 6. 

Gayon, U., effect of sucoimc acid on 
saccharose, 48. 

, starch decomposition by Asper- 
g/iMus mger, 29. 

, sugar deterioration by micro- 
organisms, 273, 289. 

Gayon and Dubourg, E., lactic bac- 
teria in wine, 126. 

, , mannitol baotena, 42, 126. 

, , pentose decomposition by 
lactic bacteria, 166. 

, , starch decomposition by 
Mucor, 30. 

Gebelin, J. A., see also Williams, 
W. J. 

, pH m sugar manufacture, 274. 

Geerhgs, H. C. P., 'froth fermenta- 
tion' of sugar, 283. 

, nucroflora of sugar cane, 261. 

Gelis, , see Pelouze, J. 

Ge'rard, E , glucoside decomposition 
by PenvyMtum glaucum, 65. 

Gillot, H., raffinose decomposition 
by Aspergittus niger, 44. 

Girard, A , absence of hydrogen 
evolution m sour dough, 224 

Glaban, C. A., see Wagner, T B 

Glaser, F , mucus production, 1 86, 
270. 



Gobley, , lactic acid manufacture, 

131. 

Godfrey, T. M., see Kohman, H. A. 
Goldberger, J., and lalhe, B. D., 

pellagra, 200. 
Qoldberger, J., Wheeler, G. A., 

Lillie, B. D., and Bogers, L. M , 

pellagra, 200. 
Gonnermann, , mioroflora of 

sugar juices, 280. 
Gordan, P., testing of cattle foods, 

206. 
Gore, H. 0., use of sweet potatoes 

in bread-making, 230. 
Gorr, G., see Neuberg, C. 
Gosio, B., see also Solavo, . 
, phenol test for damaged grain, 

207. 
Gottheil, O., starch decomposition 

by bactena, 17, 18. 
Goupil, B., sucoimo acid production 

by Mucor Rouxvi, 99. 
de Graaf, W. C., and le Fevre, A. J., 

fermentation by Bact. coh com- 
mune, 118. 

Graham, , see Pohl, O. 
Gramenitzki, M. J., effect of heat on 

amylases, 35. 
Grassberger, B., see also Schatten- 

froh, A. S. 
Grassberger, B., and Sohattenfroh, 

A. S., butyric acid fermentation, 

146, 161. 
Gray, P. H H., and Chalmers, C. H , 

Microapvra agarliquefaciena, 67. 
Gredmger, W., sugar losses in manu- 
facture, 273. 
Green, J. B M mulase of Jerusalem 

artichoke, 36. 
Greeve, E , and Bailey, C. H., effect 

of pH on diastase, 229. 
Greig-Smith, B , Bac. levaniformans, 

292. 

, Bact. radi<ytcola, 182. 
Greig-Smith, B., and Steel, T , 

mucus production, 183 
, , Bac. levamformans, 270. 
Grevemeyer, M , purification of 

waste water m sugar manufacture, 

286. 
Grey, E. C., Bact. coh commune, 114, 

116-17. 
, starch synthesis by Bact coh 

commune, 120, 176, 176. 
, synthetic activity of micro- 
organisms, 189 
Grezes, G , action of takadiastase, 

28. 



312 



INDEX OF AUTHOES 



Grazes, Gt., saooharase of AspergUlus 
rwger, 61. 

Griffiths, A. B , Bact. prodigioeum 
in cereal products, 203. 

Gngoroff, S., Bact. bvlgaricwn, 125. 

Grillone, G B., caproio acid pro- 
duction, 159. 

Grimbert, L., production of Z-lactic 
acid, 113. 

, xylose decomposition by Bact. 
pneumoniae, 162. 

Grove, O., micro-organisms in strong 
sugar solutions, 284, 291. 

Gruber, T., starch decomposition by 
JBao. polymyaxt, 18. 

von Grunoherr, G. E , see Kuhn, B. 

Gruss, J., mulase in Ustttago Maydts, 
36. 

, saccharic acid production by 
yeast, 96. 

, synthetic activity of micro- 
organisms, 179. 

GuiUaumie, M., see Frouin, A. 

Gunther, O., and Thierfelder, H., 
production of rf- lactic acid, 129. 

Guyot, H., beverage from QenMana 
roots, 44. 

H 

de Haan, J. S , Oidwim temcola in 

sugar cane juice, 268. 
Haehn, H., and Kmttof, W., syn- 
thesis of fats by Endomyces ver- 

ncdia, 177. 

Haerdtl, H., see Sameo, M. 
Haldane, J. H., use of calcium hypo- 
chloride in sugar manufacture, 

275. 
Haldane, J. S., and Makgill, B. H., 

spontaneous combustion of hay, 

197. 
Hall, J. H., and Bandall, H. B., 

Boc. Welohn, 147. 
Halton, P , see Fisher, E. A. 
Hamilton, J., see Church, M. B., and 

Paine, H. S. 
Hansen, H. C., systematic position 

of Torula, 296. 
Happ, C., mucus production m 

ihgttahs infusions, 182. 
Harden, A., aoetylmethylearbinol 

production, 109, 121. 
, fermentation by Bact. coli-ty- 

phoaum group, 116. 
, optical properties of fermentation 

lactic acid, 129. 
Harden, A., and Penfold, W. J., 

enzymes of Boot, ooh, 116. 



Harden, A., and Walpole, G. S. 
Bact. lochs aerogenee, 120. 

Harden, A., and Young, W. J. 
action of amylase on glyoogen, 13 

, , hexosediphosphoiio esters 
80. 

, , synthetic activity of yeast 
173, 174. 

Harland, S. 0., see Armstead, D. 

Haselhofi, E., and Mach, E., de 
tenoration of stored rice, 201. 

Hauzawa, J., mulase of Rhizoput 
Delamar, 36. 

, lactase of RMzopus, 59. 

Heal, T., see Greig-Smith, B. 

Hefferan, M., Bact. ruber indtcwn, 
112. 

, Bact. prodiffioatan, 112. 

Hememann, P. G., optical properties 
of fermentation lactic acid, 129. 

Heinze, B., glyoogenase in micro- 
organisms, 36. 

, synthesis of glycogen, 308. 

Henderson, L. J., see Conn, E. J. 

Henley, F. B., see Beilly, J. 

Henneberg, W., acetic acid bacteria, 
94. 

, acetic acid, action on starch of, 
18. 

, acetic acid, gluconic acid pro- 
duction by, 93. 

, intestinal decomposition of plant 
tissues, 165. 

, synthesis of glycogen, 173. 

Heiissey, H., see Bourquelot, E. 

Hermann, T. S., Bact. glucomcum, 
94. 

Heron, J., see Brown, H. T. 

Hemck, H. T., see also May, O. E. 

Herriok, H. T., and May, O. E., 
gluoonic acid production by fungi, 
96. 

Herter, W., mf action of bread by 
fungi, 254, 255. 

Herter, W , and Fornet, A., infec- 
tion of bread by fungi, 254, 255. 

Herzfeld, A., plant pathogenic bac- 
teria m beet sugar, 263. 

Herzfeld, A , and Lanart, G , use of 
glucomo acid in foods, 94. 

Herzfeld, A., and Paetow, U., use of 
fluorides m sugar manufacture, 
274. 

Hess, K., Weltzien, W., andMessner, 
E., cello biose production, 11. 

Hiokenbottom, W. G , see Beilly, J. 

Hiltner, L., microflora of damp grain, 
198, 199. 



INDEX OF AUTHORS 



313 



Hirst, F., micro-organisms in strong 
sugar solutions, 284, 291. 

von Hoefft, F., see Sameo, M. 

Hoffert, D., see Smedley, J. 

Hoffmann, C., see Kohman, H. A. 

Hoffman, F., bacteria in stored bran, 
197. 

, epiphytic bacteria of gram, 192. 

Holdefleiss, P., and Wesslmg, B., 
amount of yeast required for rye 
bread, 232. 

Holderer, M., see Bertrand, G. 

Holland, D. T., starch, decomposi- 
tion by Bac. bottdinus, 18. 

.Holliger, W., Batik, coh-typhosum 
group in plants, 192. 

, bacteria in dough and leaven, 
219-21, 224. 

, ether sterilization of cereal pro- 
ducts, 207. 

Hopkins, B., hydrogen activation, 
78. 

Homberger, W. F., see Walton, 
0. F. 

Hosaeus, H., see Koch, A. 

Hoyer, D. P., Bact. xyhnum, 94, 271. 

, hydrogen acceptors, 93. 

Huber, L. X., see James, T. B. 

Hudson, C. 8 , and Paine, H. 8., 
synthesis of saccharose, 55. 

Hulton, H. F. B., see Baker, J. L. 

HUBS, H., Bact. tnfofa, 192. 

Hutchinson, C. M , mdioan hydro- 
lysis by micro-organisms, 69. 

Hutchmson, C. M., and Bamayyar, 
C. 8 , fermentations by fungi, 24. 

, , inversion of saccharose by 
mucus bacteria, 269. 

, , microflora of cane sugar, 262. 



Ingersoll, 0. D., action of saocharase, 

52. 
Inghilleri, , amygdalase of Boat 

coli-fyphosum group, 66. 
Irvine, J. C., structure of inulin, 13. 
Irvine, J. 0., and Macdonald, J., 

constitution of starch, 8. 
Ivanov, N. N., trehalose in Myxo- 

mycetea, 46. 



Jago, W., absence of hydrogen evo- 
lution from sour dough, 224. 
, preparation of leavens, 225, 226. 
, yeasts m sour dough, 223. 
Jahn, B., see Piotet, A. 



Jalade, E., fungi in bread, 255. 
James, L. H., Bettger, L. F., and 

Thorn, C., bacteria m stored 

maize, 197. 
James, T. B., and Huber, L. X., 

effect of oxidizing agents on dough 

fermentation, 233. 
, , testing flour and water sus- 
pensions, 236. 
JatschewHki, A. A., Fusarium ro- 

seum m rye, 199, 
Javilher, M., and Tsohernoroutzky, 

H., glucoside decomposition by 

fungi, 66. 
Jensen, 0., see also von Freuden- 

reioh, E. 
Jensen, O., lactic acid bacteria, 127, 

133. 

, propiomc acid bacteria, 137. 
Jessen-Hansen, H., pH during dough 

fermentation, 26, 241. 
Johnson, A. H., see Bailey, C. H. 
Jonas, W., use of sulphur dioxide m 

sugar manufacture, 275. 
Jones, H. N., and Wise, L. E , cello- 

biose decomposition, 58. 
Joseph, A. F., and Martin, F. J., 

pentoses in Oyperus papyrus, 167. 
Josephson, K., see also von Euler, H. 
, substances affecting action of 

saccharase, 53. 

Joszt, A., amylocoagylase, 12. 
Joucla, H., amylase of Bac. burdi- 

galense, 21. 

, amylase of Mucor eloeis, 32. 
Jubert, , mucus production, 181, 

182. 
Juntz, C. F., Bac. acetoethylwus, 167. 



Kamerhng, Z., deterioration of 
sugar, 294, 297. 

Kapur, S. W., see Falok, B. 

Karrer, P., structure of inulin, 13. 

Karrer, P., and Nageli, C., depoly- 
merization of starch, 8. 

Katz, J., amylase production, 31. 

, takadiastase, 28. 

Kayser, E., lactic acid bacteria, 125. 

, melezitase production by bac- 
teria, 45. 

, pentose decomposition by bac- 
teria, 165. 

Kayser, E., and Boulanger, E., 
synthesis of glycogen, 174. 

de Keghel, M., preparation of ad- 
hesives, 217, 218. 

Kellner, O., Moi, Y., and Nagaoka, 



314 



INDEX OF AUTHORS 



M., effect of salt on Koji saccha- 

raae, 53. 

Kerb, J., see Neuberg, C. 
Kennaok, W. 0., Lambie, C. G., and 

Slater, R. H., insulin hypoglyi 

oaemia, 81. 

Kerry, R., and Franks!, S., conver- 
sion of starch to dextrins, 18, 19. 
Kertesz, Z. I., sacoharase of Peni- 

eilhum glaucum, 50. 
Kinttof, W., see Haehn, H. 
Kirchoff, S. R , acid hydrolysis of 

starch, S. 

Kirkland, J., see Dore, C. 
Kita, G., protection of amylases, 27. 
Kita, G., and Kyoto, E., effect of 

calcium salts on amylases, 27. 
Kluyver, A. J., glucose assimilation 

by yeasts, 40. 
Kluyver, A. J., and Donker, H. J. L., 

acetone production from acetio 

acid, 148. 

, , classification of fermenta- 
tions, 81-9. 
, , direct dehydrogenation of 

glucose, 93. 

, , glyoeraldehyde decomposi- 
tion, 118. 

, , hexose decomposition, 111. 
, , hydrogen activation, 78. 
, , lactic acid bacteria, 128. 
, , methylglyoxal decomposi- 
tion, 107. 

, , pyruvio acid production, 119. 
Kluyver, A. J., Donker, H. J. L., 

and Visser't Hooft, P., acetone 

production, 148. 
, , , acetylmethylcarbinol, 

108, 109, 121. 
, , , 2.3 butylene glycol, 109, 

121. 
Kluyver, A J., and Struyk, A. P., 

phosphoric esters, 80. 
Knauer, H., Streptococcus mesente- 

roides, 269. 

Kmschewski, O.,see Neumann, M. P. 
Knudsen, L., tannin fermentation, 

72 
Knudsen, S., manufacture of rye 

bread, 225. 
Koch, A., and Hosaeus, H., Boat. 

pedicukttum, 270. 
Kodama, H., and Takeda, H , starch 

decomposition by Vibno choleras, 

20. 
Kohman, H. A , 'salt rising' bread, 

222, 239, 240. 
, dough improvers, 233. 



Kohman, H. A., Hoffmann, 0., 
Godfrey, T. M., Ashe, L. H., and 
Blake, A. E., dough improvers, 
231, 233, 234. 

Kohnstamm, P., glucoside decom- 
position by fungi, 65. 

Kolkwitz, R., algae in sugar waste 
water, 285. 

KOnig, J., Spieokermarm, A., and 
Ohg, A., deterioration of cotton 
seeds in storage, 202, 203. 

, , , mouldiness in stored 
grain, 197. 

Kdnig, JT., Spieokermann, A., and 
TUJmanns, J., mucus production, 
251. 

Kopaczewsky, W., effect of acids on 
maltase, 57. 

Kopeloff, N., and Kopeloff, L., de- 
terioration of cane sugar, 289, 290. 

, , 'factor of safety' for sugar, 
299. 

, , microflora of cane sugar, 265, 
266, 278, 294, 295, 299. 

, , media for bacterial analysis 
of sugar, 300. 

Kopeloff, N., Welcome, C. J., and 
Kopeloff, L., microflora of cane 
sugar, 284, 292. 

, , , use of superheated steam 
on stored sugar, 282. 

Korschelt, , preparation of Koji, 
23. 

Koser, S. A., bacteria in leaven, 240. 

, cellobiose decomposition, 58. 

, trehalose decomposition, 40. 

Kostytsohew, S., and Eh'asberg, P., 
sacoharase of Mucor raoemosus, 
48. 

Kostytschew, S., and Frey, L., pro- 
tease of yeasts, 141. 

Kostytsohew, S., and Soldatenkov, 
S , pyruvio acid production by 
lactic bacteria, 128 

Kostytschew, S., and Tschesnokov, 
V., oxalic acid production, 99. 

Kozai, Y., optical properties of fer- 
mentation lactic acid, 129. 

, protein decomposition by bac- 
teria, 141. 

, use of fungi in the East, 22. 

Kramsky, A., starch decomposition 
by Actinomycet.es, 22. 

, disposal of sugar wastes, 285. 

Kramer, E., mucus production, 182, 
186, 270. 

Kratschmer, , and Niemilowiez, 
, rope in bread, 261. 



INDEX OF AUTHORS 



315 



Krause, A., see Chrzaszcz, T. 
Kr6ber, B., see Lintner, C J. 
Kruis, K., and Raymann, B., lactic 

bacteria in malt, 126. 
Kuhn, R., see also Willst&tter, R. 
, amylase production, 11. 
, isomaltose, 4. 
Kuhn, R., and von Gnmdherr, G. E., 

constitution of melezitose, 45. 
Kuhn, R., and Munch, H., fruoto- 

and gluco-sacoharasea, 53. 
Kyoto, E., see Kita, G. 



Lachmann, S., see Neuberg, C. 

van Laer, H., mucus production, 
182. 

Lafar, F., bacteria in sugar waste 
water, 285. 

Lafar, F., 'froth fermentation' of 
sugar, 283. 

, origin of leavens, 219, 220. 

Lambie, C. G., see also Kermack, 
W. 0. 

, insulin hypoglycaemia, 81. 

Lanart, see Herzfeld, A. 

Langhans, A., see Pringaheim, H. 

Lappaleinen, H., starch -like sub- 
stances m fungi, 175. 

Laurent, E., roprness m bread, 182, 
203, 249, 251. 

, sour dough, 223. 

Laurin, T., see von Euler, H. 

Laxa, , Olostrid&um gelatmosum, 
184, 270. 

, 'froth fermentation 1 of sugar, 
283. 

, mioronora of sugar juices, 206, 
268, 280. 

oLaybourn, R. L., starch decomposi- 
tion by Bact. lactis aerogenes, 18 

Leathes, , see Person, L K , and 
Raper, H S. 

van der Leek, G., glucoside decom- 
position by bacteria, 67. 

Lefranc et Che, butyric acid fermen- 
tation, 151 

Legendre, H., deterioration of 
cereals in storage, 208. 

Legg, D. A., acetone fermentation, 
168. 

, butyric acid fermentation, 162 

Lehmann, K. B , bacteria in dough, 
221, 224. 

, acid production in dough, 224, 
243. 

Lehmann, K. B , and Neumann, 
R. O , Bac butylicus, 160 



Lehmann, K. B., and Neumann, 
R. 0., Streptococcus mesenteroides, 
181. 

Lehrmarm, L., see Taylor, F. C. 

Leibowitz, J., see Pringsheim, H. 

Leichmann, G., Mactic acid by fer- 
mentation, 129. 

Lemoigne, M., acetylmethylcarbmol 
and 2:3 butyleneglyool, 108, 109, 
111. 

, Bac. aubtiUa, 107, 109. 

, Bact. prodagwsum, 112. 

, hexose decomposition by aerobic 
bacilli, 106, 107. 

Levy, F., bacteria in flour, 192. 

Lewkowitsoh, J., optically inactive 
lactic acid, 129. 

Lewton-Bram, L., and Deerr, N., 
sugar deterioration, by micro- 
organisms, 301. 

Lichtenstein, S., see Fnngsheim, H. 

von Liebig, H. J , sugars in flour, 
228. 

Liesenberg, 0., and Zopf, W., isola- 
tion of mucus producing bacteria, 
185. 

Ligon, 0., effect of moisture on sugar 
deterioration, 289. 

Lilhe, R. D., see Goldberger, J. 

Lindner, P., see also Fischer, E. 

, amylase production m fungi, 34. 

, Dematoum pulltdans, 185. 

, diseases of bread, 253, 254 

, synthesis of fats, 177. 

Lindner, P., and Saito, K., assimila- 
tion of glucose by yeasts, 40. 

Lindstrdm, B., see Virtanen, A. I. 

Ling, A. R., amylopectm and hexa- 
amylose, 5. 

Ling, A. R , and Nanji, D. R , con- 
stitution of starch, 8, 12 

, , isomaltose, 4. 

Lintner, C. J., determination of 
diastatic power, 28. 

, isomaltose, 4. 

von Lippmann, E. O , constitution 
of mucus, 183. 

, isolation of mehbiose, 60. 

Lloyd, D J , and McCrea, E. D , 
ropmess in bread, 249. 

Locqum, R., valeric acid production, 
160. 

Lohnis, F., lactic acid bacteria, 143. 

Loiseau, D., isolation of raffinose, 44. 

Lombroso, , pellagra, 200. 

Lowry, C D , see Willstatter, R. 

Lubrzyriska, E , synthesis of fats, 
177. 



316 



INDEX OF AUTHORS 



Lumsden, J. 8., Boat. ethaceticus, 

113. 
, Bact. pneumoniae, 113. 

M 

MeCahp, M. A., see Walton, 0. F. 

McCleery, W. L., cane sugar manu- 
facture, 267, 272. 

MoCrea, E. D , see Lloyd, D. J. 

Macdonald, J., see Inane, J. C. 

Maofadyen, A., Moms, G. H., and 
Rowland, S , carbon dioxide pro- 
duction by yeast, 173. 

Mach, F., see Haselhoff, E. 

MoKenzie, A , optical properties of 
fermentation lactic acid, 129. 

McLean, J. S., and Hoffert, D , fat 
synthesis, 177. 

Maknnov, I. A , starch decomposi- 
tion by Sac. amylobacter, 18. 

Maquenne, L., and Roux, E., amylo- 
biose, 7. 

, , amylose and amylopeotin, 
4,8. 

, , pH and amylase action, 11. 

Maroano, V., bacteria in dough, 223. 

, starch decomposition of bacteria, 
17. 

Marion, F., protein decomposition 
in stored gram, 204. 

Martin, F. J., see Joseph, A. F. 

Massee, G., Dematium pullulans, 
185. 

Massmi, R., Bact. coli - typlioaum 
group, 60. 

, laotase production by bacteria, 
48. 

Masters, H., and Maughan, M , 
dough improvers, 234. 

Maughan, M., see Masters, H. 

Maumus, , starch decomposition 
by Boc. anthrocts, 18. 

Maupas, E., starch synthesis by 
infusoria, 175. 

Maurer, K , propiomc acid bacteria, 
140. 

May, O. E , see also Hemck, H. T. 

May, O. E., Hemok, H. T., Thorn, 
C , and Church, M. B., citric acid 
production by fungi, 102 

, , , , glucomc acid produc- 
tion by fungi, 96. 

Mayer, A., lactic acid manufacture, 
133 

Meier, A., micro-organisms in strong 
sugar solutions, 291. 

Meisenheimer, J., see Buchner, C. 

Meisaner, R., mucus production, 185. 



Melo, P., Bact. prodigiosum, 257. 

Mendes, T., mucus production, 181. 

Mering, , see Musoulus, . 

Messner, E., see Hess, K. 

Meyer, A., a- and /3-amylose, 5. 

, isolation of gentianose, 43. 

, synthesis of glycogen, 174. 

Meyer, F., methylglyoxal produc- 
tion, 80 

Meyer, K. F., see Wagner, E. 

Michaehs, L , and Pechstein, H., 
effect of glycerin on saccharose, 52. 

Michaehs, L , and Rona, P., opti- 
mum pH for maltose, 56. 

, , solubility of saccharase, 53. 

Moi, Y , see KeUner, O. 

Mohsch, H., hydrolysis of indican, 
68. 

, test for glucosidase, 67. 

Moller, , see Kraisy, A. 

Molhard, M., glucomc acid produc- 
tion by fungi, 96. 

Montesano, G., see Fermi, 0. 

Morgan, R. R., see Northrop, J. H. 

Morison, C. B., temperature of 
baking, 231, 246. 

Morison, 0. B., and Collate, F. A., 
buffer action of flour, 242. 

, , pH during dough fermenta- 
tion, 242. 

, , ropmess in bread, 249 

Moms, G. H., see Maofadyen, A., 
and Brown, H. T. 

Moms, L. E , antiseptics and mil- 
dew fungi, 216. 

, liability of sizing materials to 
mildew, 215-16. 

Mouriquand, G., pellagra, 200. 

Mousette, , alcohol in bread, 238. 

Mugge, , losses in sugar manu- 
facture, 273. 

Muller, D , glucomc acid production 
by moulds, 92, 95. 

Muller-Thurgau, H., and Oster- 
walder, A., lactic acid bacteria in 
wine, 176. 

, manmtol bacteria, 126. 

Mulvania, M , see Fred, E. B. 

Munch, H., see Kuhn, R. 

Musculus, , Mering, , action of 
amylase on glycogen, 13. 

Myrback, K., see also von Euler, H. 

Myrbftok, K , and von Euler, H., 
ooenzyme of lactic acid bacteria, 
128. 

, , fermentation by Bact. lactis 
aerogenes, 123. 

, , propionic acid bacteria, 140. 



INDEX OF AUTHORS 



317 



N 



Nagaoka, M., see Kellner, O. 
Nftgeli, 0., see Karrer, P. 
Nageh, W., see Oppenheimer, 0. 
, granulose and starch-cellulose, 5. 
Nakazawa, B., amylase of Mucor 

batatas, 32. 

, laotase of Rhtzopus, 59. 
Nanji, D. B., see Ling, A. B. 
Nef, J. TJ., methylglyoxal produc- 
tion, 118. 

Neide, E., losses in cane sugar manu- 
facture, 267, 273. 
Neidig, B. E., see Box, A. W. 
Neill, J. M., see Fleming, W. L. 
Neisser, M., Bact. coh-typhosum 

group, 60. 

, laotase in bacteria, 48. 
Nelson, J. M., see Fales, H. A. 
Nelson, J. M., and Anderson, B. S., 
substances affecting glucose hy- 
drolysis, 53. 

Nelson, J. M., and Post, C. J., sub- 
stances affecting saccharose hy- 
drolysis, 53. 
Nenoki, M., and Sieber, N., cZ-lactio 

acid by fermentation, 129. 
Neuberg, 0., see also Oppenheimer, 

C. 

, decomposition of raffinose, 44. 
Neuberg, 0., and Armstein, B., 

butyric acid bacteria, 143. 
, , production of higher fatty 

acids, 160. 
, , pyruvio acid in fat synthesis, 

177. 

Neuberg, 0., and Qorr, G., methyl- 
glyoxal decomposition, 118, 122, 
128, 130. 

Neuberg, C., and Kerb, J., methyl- 
glyoxal production, 80. 
Neuberg, C., and Lachmann, S., 
action of kephir enzymes on 
staohyose, 43. 
Neuberg, C., and Nord, F. T , Bact. 

coh commune, 117, 119. 
Neuberg, C., and Bemfurth, E., 

acetone fermentation, 148. 
, , aoetylmethylcarbinol pro- 
duction by yeast, 108, 121. 
Neuberg, C , and Bmger, M., pro- 
tern decomposition by yeasts, 141. 
, , suooinio and ketoglutamio 

acids, 119. 
Neuberg, C., and Simon, E , Bact 

ascendens, 94. 

Neumann, M. P , and Kruschewski, 
O , growth of yeast in dough, 231. 



Neumann, M. P., and Kmsohewski, 
O., substances affecting dough fer- 
mentation, 233, 234. 

Neumann, B. O., see Lehmann, K. B. 

van Niel, C. M., propiomc acid bac- 
teria, 137-8, 140. 

Niennlowicz, , see Kratsohmer, . 

Nill, W., amylase of BMzopus, 24. 

Nishimura, S., amylase produc- 
tion, 9. 

, takadiastase, 28. 

Niflhiwaki, Y., M onilia stiophila, 25. 

Nord, F. T , see Neuberg, C. 

Norns, B. U., glyoogenase, 13. 

Northrop, J. H., Ashe, L. H., and 
Morgan, B. B., Bac. acetoeihylwus, 
167. 

Northrop, J. H., Ashe, L. H , and 
Senior, J. K., Bac. acetoeOvylwus, 
113, 114, 162, 166, 168, 169, 184. 

O 
OkazaM, K., starch decomposition 

by Asp&rgttlus, 29. 
Ohg, A., see Komg, J. 
Olsson, U., substances affecting 

amylase, 10. 

OmehansM, V., cellulose decomposi- 
tion by bacteria, 150. 
, starch decomposition by Azoto- 

bacter croococcum, 18. 
Oppenheimer, C., hydrogen activa- 
tion, 78. 

, lactase production, 58. 
, oxidation reductions, 79 
Oppenheimer, C., and Neuberg, C , 

desmolases, 77. 

Oppenheimer, T., see Willstatter, B. 
On, A., catalase test for damaged 

gram, 207. 

Orth, W. K., cane sugar manufac- 
ture, 267, 275. 

Ortved, , takadiastase manufac- 
ture, 25. 

Oshima, K., see also Takamme, Jr , J. 
Oshima, K., use of antiseptics in 

diastase manufacture, 25. 
Osterwalder, A., aeeMuller-Thurgau, 

H. 

O'Sulhvan, C , maltose, 3. 
, and Thompson, F. W., action of 

saccharose, 52. 
Otto, G , see Pringsheim, H. 
Owen, B. J., disposal of beet sugar 

wastes, 284, 285. 
Owen, W. L , microflora of cane 

sugar juices, 285, 278-81. 
, mucus production, 270, 292 



318 



INDEX OF AUTHORS 



Owen, W. L., protection of stored 
sugar by inoculation, 297. 

, use of formaldehyde in sugar 
manufacture, 276". 



Paetow, U., see Herzfeld, A. 
Fame, H. S , see also Church, M. B., 

and Hudson, C. 8. 
Paine, H. S., and Balch, R. T., 

analysis of sugar juices, 272. 
Paine, H. S., Birckner, V., and 

Hamilton, J., saccharose decom- 
position in chocolate creams, 303. 
Papasotirm, J., Bact. coh-typhosum 

group, 192. 
, Bact. levans, 221. 
Pasteur, L., Bact. Pasteurianum, 93. 
, butyric acid fermentation, 161. 
, mucus production, 181, 182. 
, saccharose decomposition by 

yeast, 47. 

, Vtbrion bitfynque, 146. 
Payen, A., Oidium aurantoacum in 

bread, 264. 

, sugar deterioration by fungi, 289. 
Payen, A., and Persoz, , isolation 

of diastase, 3. 

Pechstein, H., see Michaelis, L. 
Pederson, C. S , Peterson, W. H., 

and Fred, E. B., optical properties 

of fermentation lactic acid, 130. 
Pelhzzi, G. H., and Tirelli, V., pel- 

lagra, 200. 
Pelouze, J., and Gehs, , butyric 

acid bacteria, 142, 150. 
Penfold, W. J., see Harden, A. 
Perdrix, L., origin of fusel oil, 162. 
, starch decomposition by Boo. 

amylozyma, 18 
Per6, A., formaldehyde production, 

108. 

, formic acid production, 108. 
, hexose decomposition by aerobic 

bacteria, 106. 

, optical properties of fermenta- 
tion lactic acid, 129. 
, valeric acid production, 108. 
Person, L. K., and Raper, H. S., 

acetaldehyde in fat synthesis, 177. 
Persoz, , see Payen, A 
Peters, W. L., microflora of sour 

dough, 224. 
Peterson, G. Troili, propionio acid 

bacteria, 137. 
Peterson, W. H., see also Fred, E. B., 

Pederson, C. S., Stiles, H. R., and 

Wilson, P. W. 



Peterson, W. H , and Fred, E B., 

acetaldehyde production by Boo. 

acetoethyhcus, 114. 
, , lactic acid bacteria, 126. 
, , pentose decomposition by 

bacteria, 162. 
, , silage, 166 
Peterson, W. H , Fred, E. B., and 

Schmidt, E. G., pentose decom- 
position by fungi, 162, 164. 
Peterson, W. H., Fred, E. B., and 

Verhulst, J. H., Bac. acetoethyU- 

cus, 167. 

Phillips, J. F , see Speaktnan, H. B. 
Pick, F., glycogonase m animal 

tissues, 13. 
Pictet, A., and John, R., depoly- 

merization of starch, 7. 
Plaisance, G. P., see Dox, A. W. 
von Planta, A., and Schulze, E., 

isolation of stachyose, 42. 
Pohl, O., alcohol in bread-making, 

238. 
Popoff, M., bacteria in sour dough, 

223. 

Post, C. J., see Nelson, J. M. 
Potter, R. S., see Bailey, G. C. 
Pottevm, H., dextrinase, 9. 
, laotase of Asperg-illus mger, 69, 

60. 

Prazmowski, A., Bac. butyncits, 146. 
Pwllieux, E., Bact. prodigwswn m 

stored cereals, 203. 
, starch decomposition by bac- 
teria, 17. 

Fringsheim, H., omylase, 9, 10. 
, amylopectm, 4. 
, Bact coh-typhosum group, 60. 
, origin of fusel oil, 153. 
, polyamyloses, 7. 
, polysacchandes, 40. 
Pnngsheim, H., and Aronowski, A., 

constitution of inuhn, 13 
Pringshoma, H., and Beiser, A., 

amylase, 9, 10. 

, , constitution of glyoogen, 13. 
Pringsheim, H., Bondi, J , and 

Thilo, E., action of amylase, 10. 
Pnngsheim, H., and Eissler, F., 

polyamylose, 7. 
Pnngsheim, H , and Langhans, A., 

a- and fl-amy loses, 7. 
Pnngsheun, H., and Leibowitz, J., 

a- and /3-amylases, 12. 
, , amylobiose and amylotnose, 

7. 
Pringsheim, H., andLichtenstein, S., 

glyoogenase in bacteria, 36. 



INDEX OF AUTHORS 



310 



Pringsheun, H., and Otto, G., taka- 
diastase, 28. 

, and Wolfsohn, K., amylose and 
amylopectdn, 5. 

, , polyamyloses, 7. 

, and Zempleii, G , saccharose 
assimilation by fungi, 41. 

Probert, M. E , aee British Cotton 
Industry Research Association. 

Punewitech, K., glucoside decom- 
position by fungi, 66. 



Quastel, J. H., electrical theory of 
enzyme action, 77. 

, pyruvio acid production, 107, 
119. 

Quastel, J. H., and Stephenson, 
M. D., synthetic activity of micro- 
organisms, 78. 

, , and Whetham, M. D., ' activa- 
tion 1 by micro-organisms, 78. 

, , , utilization of lactic acid 
by aerobic bacteria, 107. 

Quastel, J. H., and Whetham, M. D , 
hexose decomposition by faculta- 
tive anaerobes, 112. 

Quastel, J. H., Whetham, M.D., 
Wooldridge, W. R., and Stephen- 
son, M., enzymes of Soct. coli 
commune, 116-17. 

Quastel, J. H., and Wooldridge, 
W. R., Boct. wdgare, 111. 

, , hexose decomposition by 
facultative anaerobes, 112. 

Quevenne, , saccharose decom- 
position by yeast, 47. 

Quine, J. H., bran extract as dough 
improver, 232. 

R 

Racke, O. 0., see Collatz, F. A. 
Raistnck, H., and Clark, A. B., 

acetoacetic acid in acetone fer- 
mentation, 148. 
, , Aspergittus niger, 123. 
, , citric acid production by 

Aspergittus niger, 98. 
, , oxalic acid production by 

micro-organisms, 99 
Ramayyar, 0. S., see Hutchmson, 

C.M. 

Randall, H. B., see Hall, J. H. 
Rao, T. L , and Ayyar, G. G., pH in 

sugar manufacture, 274. 
Raper, H. S., aee Person, L. K. 
Rapkine, L., and Wurmster, R., 

hydrogen activation, 78. 



Rapp, R., see Buchner, E. 

Rege, R. D., formation of humus, 
165. 

Reiohard, A., mioroflora of damp 
grain, 198. 

Reilly, J., Hickenbottom, W. J., 
Henley, F. R., and Thaysen, A. G., 
chemistry of acetone fermenta- 
tion, 144, 147, 153, 169. 

Reinfurth, E., aee Neuberg, C. 

Reiset, J., effect of nitrates on 
molasses fermentation, 287. 

Rettger, L. F., see James, L. H. 

Ringer, M., see Neuberg, C. 

Robertson, J. C., mouldy bread as 
foodstuff, 256. 

Robiquet, E., action of plant en- 
zymes on tannin, 71. 

Robison, R., hexosemonophosphono 
ester, 80. 

Roeser, P., infection of bread, 247. 

Rohmann, F., action of saccharase 
on maltose, 56. 

Rons, P., aee Miohaelis, L. 

Rose, L., Endomycea Mognuaii, 40. 

Rosenblatt, M., see Bertrand, G. 

Roussel, J., temperature of bread 
during baking, 231, 246. 

, bacteria in dough during baking, 
247. 

Roux, E., see Maquenne, L. 

Rowland, 8., aee Macfadyen, A. 

Rowntree, Messrs., see Fernbach, A. 

Rullmann, W., starch decomposi- 
tion by Actinomycea odonfera, 22. 

Russell, H. L., ropmess in bread, 
252. 

, temperature of bread during 
baking, 246. 



von Saitcew, J., lactic acid manu- 
facture, 132. 

Saito, K., see also Lindner, P. 

Saito, K., AspergiUus batatae, 24. 

, lactic acid production by Mucor 
chinensis, 134. 

, soya manufacture, 25. 

, starch decomposition by Asper- 
gilh, 29. 

Salabartan, J., see Aubel, E. 

Salkowski, E , saccharase in yeast, 
50. 

Sandberg, I., alcohol in bread, 238. 

Sanguinetti, J., starch decomposi- 
tion by AspergiUus oryzae, 29 

Saruec, M , constitution of starch, 5. 

Samec, M., von Hoefit, F., Haerdtl, 



320 



INDEX OF AUTHOBS 



H., Meyer, A., constitution of 

starch, 6, 9. 

Sartory, A., and Baimer, S , charac- 
ters of Oitoramyces, PenidMum 

and Aepergittus, 101. 
Sartory, A., and Sartory, R. t Asper- 

gittus fumigatus in gram, 205. 
de Saussure, T., decomposition of 

starch by micro-organisms, 3, 16. 
Scales, F. M., starch decomposition 

by AspergiUua terncola, 29. 
Scarisbnok, F., see Bean, F. 
von Scent-Qyorgyi, A., hydrogen 

activation, 78. 
Sohardinger, F., bacteria m damp 

gram, 199. 
, Bac. macerans, 6. 
, galaotan production, 184. 
, starch decomposition by bac- 
teria, 6, 18, 19. 
Sohattenfroh, A. S., see also Grass- 

berger, B. 
Sohattenfroh, A. S., and Grass- 

berger, B., aerobic butyric acid 

fermentation, 150. 
Soheele, K. W., occurrence of gallic 

acid, 71. 
Soheibler, C., mucus production, 

181, 182, 183. 

, preservation of sugar juices, 275. 
Scheurlen, , Bact. prodigwsum on 

bread, 256. 
, Oidmm aurantiacum on bread, 

255. 
Sohindler, J., mouldmess in maize, 

205. 

Schidtz-Ohristensen, L. A., micro- 
flora of sour dough, 223. 
, rye bread, 223, 224. 
Sohloesmg, T., effect of nitrates on 

molasses fermentation, 287. 
Schmidt, A., mucus production in 

muk, 182. 

Schmidt, B., pH in sugar manufac- 
ture, 274. 

Schmidt, E. G., see Peterson, W. H. 
Schneider, K., see Willstatter, B. 
Sohoen, M., see Fernbach, A. 
Sohon, , see Bernhauer, K. 
Schdne, A., butyric acid bacteria m 

sugar, 271. 

, fungi in stored sugar, 293. 
, microflora of beet sugar, 262-3, 

265, 279, 280, 292. 
, mucus production, 270. 
, spontaneous combustion of 

sugar, 293. 
, Streptococcus mesenteroides, 269. 



Sohott, A., use of formaldehyde in 

sugar manufacture, 275. 
Sohottelius, M., carbon dioxide pro- 
duction by Bact. prodigiosum, 112. 
Sohreiner, O. M., see Sharp, P. F. 
von Schrenk, H., melezitose m 

Polyporaceae, 45. 
Schreyer, B., Aspergittus fwnancus, 

99, 100. 
Schryver, S. B., see also Clayson, 

D. H. F. 
Schryver, S. B., and Thomas, E. M., 

hemicelluloses in starch, 6. 
Sohulze, E., isolation of stachyose, 42. 
Sclavo, , germination test of 

damaged gram, 207. 
Solavo, , and Gosio, B., amylase 

in bacteria, 19, 20. 
, , starch decomposition by 

Bact. vulgare, 19. 
Schber, G , and Bovshik, G., dough 

fermentation, 239. 
Senior, J. K., see Northrop, J. H. 
Serena, , pellagra, 200. 
Sette, V., Bact. prodigiosum, 257. 
Sharp, P. F., and Schremer, 0. M., 

protein changes during dough 

fermentation, 240. 
Shaw, B. H , see Sherman, J. M. 
Sherman, H. 0., and Tanberg, A. P., 

takadiastase, 26, 29. 
Sherman, H. 0., Thomas, A. W., 

and Baldwin, M. E., pH range of 

amylase, 26. 
Sherman, J. M., see also Whittier, 

E.G. 

Sherman, J. M., propiomc acid bac- 
teria, 137, 140. 
Sherman, J. M., and Shaw, B. H., 

propiomo acid bacteria, 137. 
Sherwood, B. C., see Bailey, 0. H. 
Shorey, E. C., sugar deterioration by 

fungi, 289. 
, use of high pressure steam in 

sugar manufacture, 282. 
Sieber, N., see Nencki, M. 
Siedish, A. S , dextrins by bacterial 

action on starch, 19. 
Simon, E , see Neuberg, C. 
Slater, B. H., see Kermaok, W 0. 
Smedley, J , and Lubrzyriska, E , 

fat synthesis, 177. 
Smit, J , manmtol production by 

bacteria, 126. 
Smith, T , glucose decomposition by 

Bact. vulgare, 111, 112. 
Snyder, H., and Voorheer, L. A., 

alcohol m bread, 238. 



INDEX OF AUTHORS 



321 



oldatenkov, S., see Kostytsohew, S. 
foensen, S. P. L., effect of pH on 
diastatic activity of flour, 229 
peakman, H. B., acetone fermenta- 
tion, 144, 147, 148, 153. 
peakman, H. B , and Phillips, J. F., 
lactic infection in acetone fer- 
mentation, 158. 
pieckermann, A , see Konig, J. 
prankbng, C. H G., losses in cane 
sugar manufacture, 267, 268. 
-, spontaneous fermentation of 
cane sugar to vinegar, 271. 
banley, A., see Frankland, P. F. 
bapp, C., mucus production by 
Azotobacter croococcum, 182, 184. 
teel, T., Sac. levaniformans, 270. 
m Steenberge, P., lactic acid bac- 
teria, 127, 133. 
-, manmtol bacteria, 126, 127. 
.eibelt, W., see Willstatter, R. 
^ephenson, M., see also Quastel, 
J. H. 

/ephenson, M , and Whetham, 
M. D., Sact. coU commune, 118. 
^ern, W , pentose decomposition by 
Sact. typhosum, 165. 
-lies, H. R., Peterson, W. H., and 
Fred, E. B., pentose decomposi- 
tion by lactic acid bacteria, 165. 
<ocks, H. B., flour fermentation, 
213. 

lOklasa, J., pentoses in denitnfica- 
tion processes, 165. 
-, and Vitek, E., mucus forming 
bacteria in soil, 268. 
-one, W. E., and Tollens, B., in- 
ability of yeasts to decompose 
pentoses, 164. 

orange, E. H., see Fernbach, A. 
Tecker, A., propionio acid bac- 
teria, 136. 

, manmtol bacteria, 133. 
orient, C., butyric acid bacteria, 
161. 

ruyk, A. P , see Kluyver, A J. 
ibramaniam, V , see Challenger, F. 
ranberg, 0., see also von Euler, H. 
, effect of temperature on saccha- 
rase production, 51. 



ikamme, J., preparation of taka- 
diastase, 25 

, commercial utilization of fungi, 23. 
ikamme, J., and Oshuna, K., 
effect of temperature on 'poly- 
zime', 27. 



Takeda, H., see also Kodama, H. 
Takeda, Y., utilization of Rhizopus, 

24. 

Tanberg, A. P., see Sherman, H. C. 
Tanret, C., amylose and amylopeotin, 

6. 
, inulm-like substances in plants, 

14. 
- , stachyose decomposition by 

Aspergdlus niger, 43. 
Tata, G., lactic acid bacteria, 126, 

129. 
Taylor, F. C., and Lehrmann, L., 

fatty acids in maize starch, 6. 
Taylor, F. E., see Castellam, A. 
Thaysen, A. C., see also Beilly, J". 
, acetone fermentation, 144, 153, 

154. 

, mioroflora of grass, 192. 
, saccharase in bacteria, 48. 
, sterilization of maize mash, 211. 
Thaysen, A. C., and Bakes, W. E., 

pentoses in humus formation, 165. 
Thaysen, A. 0., and Bunker, H. J., 

cellulose decomposition by butyno 

bacteria, 150. 

, , mildew organisms, 216. 
, , silage, 286. 
, , spontaneous combustion, 286. 
, , wood destruction by fungi, 

34. 
Thaysen, A. C., and Galloway, L. D., 

Bac. acetoethyltcus, 167-9. 
, , hydrolysis of pentosans, 167. 
, , occurrence of pentosans in 

nature, 167. 

Thierfelder, H., see Gunther, C. 
Thilo, E., see Pringsheim, H. 
Thorn, C , see also James, L. H., and 

May, O. E. 
Thorn, C., and Church, M. B., posi- 

sion of Ctiromyces, 102. 
Thorn, C., and Le Fevre, E., mould 

growth in maize, 196. 
Thomann, J , ropiness in bread, 203. 
Thomas, A. W., see Sherman, H. C. 
Thomas, E. M., see Schryver, S. B. 
Thompson, F. W., see O'Sulhvan, C. 
Thom, I., and Allemann, O., pro- 

piomo acid bacteria, 137. 
van Tieghem, P., gallic acid from 

tannin, 71. 

, mucus production, 181. 
Tiilmanns, J , mucus production, 

251. 

Tmkow, D., see Chrzaszcz, C. 
Tirelh, V , see also Pelhzzi, G. H. 
Tirelh, V., pellagra, 200. 



322 



INDEX OF AUTHORS 



Tissier, H , and Gaschrng, P., pro- 

piomo acid bacteria, 138. 
Tollens, B., see Stone, W. E. 
Tnbot, G., effect of magnesia on 

saccharase, 54. 
Tsohernoroutzky, H., see Javillier, 

M. 

Tschesnokov, V., see Kostytchew, S. 
Twort, F. W., gluoosidase in bao- 

tena, 66. 

TJ 

Uffelmann, J., ropiness in rye bread, 
261. 



Vandenberg, , , preparation of 
adhesives, 217. 

Vandevelde, A. J. J., effect of gluten 
on dough fermentation, 234. 

, effect of moisture on dough fer- 
mentation, 229. 

, sterilization of cereal products, 
207. 

Vandevelde, G., glucose decomposi- 
tion by Bact. subtilts, 109. 

Vedder, B. B., starch decomposition 
by Micrococcus gonorrhoeae, 19. 

Vehoh, A , mucus production, 268. 

Verhulst, J. H., see Peterson, W. H. 

Villiers, A , melezitose in manna, 
45. 

, starch decomposition by bac- 
tena, 18, 19. 

Vmtilesco, J., stachyose decomposi- 
tion by Aspergilkis rwger, 43. 

Viollo, H , direct assimilation of 
saccharose, 41 

Virtanen, A. I., Bact lactisaerogenes, 
123. 

, propionio acid bacteria, 138, 139, 
140. 

, Buecimo acid decomposition, 119. 

Virtanen, A. I., and Barlund, B., 
dihydroxyacetone production, 96. 

Virtanen, A. I., Wichmann, B , and 
Lmdstrbm, B., optimum pH for 
lactase, 132. 

Visser't Hooft, F., see Kluyver, A J. 

Vissier, A. W , synthesis of saccha- 
rose, 55. 

Vitek, E., see Stoklasa, J 

Vogel, J , ropineBs in bread, 182, 
250, 252. 

Voorheer, L. A., see Snyder, H. 

van Voss, H., use of ammonium 
fluoride in sugar manufacture, 
274. 



W 



Wagner, E., Meyer, K. F., and 
Dozier, 0. C., valeric acid pro 
duotion, 160. 

Wagner, T. B., and Glaban, C. A., 
effect of acidity on dough fer- 
mentation, 232, 237. 

Wahl, A., effect of lactic acid on 
dough fermentation, 232, 243. 

Waksman, S. A., enzymes of taka 
diastase, 26. 

, estimation of diastatio power, 28 

, saccharose decomposition by 
Acttnomyces, 22. 

Waksman, 8. A., and Curtis, B. E., 
starch decomposition by Actino 
myces, 49. 

Walker, T. K., see Challenger, F. 

Walpole, G. S , see also Harden, A. 

Walpole, G. S , acetylmethylcarbinol 
production, 121. 

Walton, C. F., McCalip, M. A., and 
Hornberger, W F., effect of pH 
in sugar manufacture, 274. 

Wasserzug, E , Bact prodigiosum, 
257. 

, saccharase of Fuaanwn, 51. 

Wassling, R., see Holdefleiss, P. 

Watkms, E. J., ropiness in bread, 
203, 260, 252. 

Wehmer, C , citric acid production, 
101-3. 

, fumario acid production, 98, 100. 

, glucomo acid production, 98, 99. 

, mulase in Muoor, 36 

, laotase in fungi, 59. 

, oxalic acid production by fungi, 
101. 

, synthesis of glycogen, 174. 

Weidenhagen, R., substances affect- 
ing saccharase production, 53. 

Wemztrl, J , saccharose decomposi- 
tion in chocolate creams, 302. 

Weissweiler, G , see Bertrand, G. 

Weizmann, C., acetone fermenta- 
tion, 147, 153. 

Welcome, C. J , see Kopeloff, N. 

Welte, E., fungi in bread, 246, 248, 
253, 254, 255. 

Weltzien, W , see Hess, K. 

Went, F. A F. C , Manilla sitophila, 
45, 46, 57. 

Wenzel, E., see Willstatter, R. 

Wheeler, G. A., see Goldberger, J. 

Whetham, M I)., see Quastel, J. H., 
and Stephenson, M. 

Whewell, W H , infection of sizing 
material, 212 



INDEX OF AUTHORS 



323 



rite, H. L., bran extract as dough 

mprover, 232, 243. 

iite, M G , and Willaman, J. J., 

Dentose decomposition by Fusa- 

vwm hm, 162, 164. 

iittier, E. O , Sherman, J. M., and 

\lbus, W. R., propiomo acid bao- 

na, 137. 

nTiTrmnrij E , see Virtanen, A I. 

eland, C., chemistry of respira- 

.ion, 77, 78. 

Ilamann, J. J., see White, M. G. 

Uiams, C. C., see Cameron, E. J. 

Ihams, W. J., and Gebehn, J. A., 

iff ect of pH in sugar manuf aotnre, 

!74. 

Ustatter, R , isolation of saocha- 

ase, 40. 

Ustatter, R., and Kuhn, R., 

affinase, 45. 

Ustfttter, R , and Lowry, C. D., 

lirect fermentation of saccharose, 

2. 

Ustatter, R , Lowry, C. D., and 

Ichneider, K., substances affect- 

ig saccharase production, 51. 

Istatter, R., Oppenheimer, T., 

nd Steibelt, W., diffusion of 

laltase through cell wall, 57. 

Istatter, R., Schneider, K., and 

7enzel, E., isolation of saccharase 

'om yeast, 50. 

son, P. W., Peterson, W. H , and 

'red, E. B., acetone fermentation, 

47, 148. 

ikler, W., Bact. mes&ntericus 

wr&us, 192. 

logradski, S , Bac. Pasteunanus, 

46. 

e, L. E., see. Jones, H. N. 



Wohlgemuth, J., determination of 

diastatio power, 28. 
Wolbaoh, S. B., see Cohn, E. J. 
Wolf, H., see Bernhauer, K. 
Wolff, J., and Fernbach, A., amylo- 

ooagulase, 12. 

Wolfan, A., Bact. levans, 221. 
, mioroflora of sour dough, 234. 
Wolfsohn, K., see Pnagsheim, H. 
Wollmann, E., starch decomposition 

by CFlycobacter, 10. 
Wolzogen Ruhr, C. A. H., micro- 

flor^jf sugar cane, 261, 266. 
Wooldndge, W. R., see Quastel, 

J.H. 
Wortmann, J., amylase in bacteria, 

16-17. 

Wurmster, R., see Rapkme, L. 
Wustenfeld, H., see Buchner, E. 



Yamagashi, H., starch decomposi- 
tion by takadiastase, 28. 
Young, W. J., see Harden, A. 
Ytull, J L , see Fernbach, A. 



Zeidler, A., acetic acid bacteria, 04. 
Zellner, J., amylase in higher fungi, 

34. 
Zemplen, G., see Fischer, E., and 

Pnngsheim, H. 

Zettnow, E., mucus production, 185. 
Zikes, H., glycogen synthesis, 174. 
Zopf, W., see also Liesenberg, C. 
, mucus production by Zoogloea 

ramigera, 185. 
, oxalic acid production by acetic 

bacteria, 101. 



Y2 



$( LIBRARY 




Aoetaldehyde, effect on yeast fer- 
mentation, 122. 

, formation by bacteria, 84-7, 
108, 109, 111-13, 117, 119, 122, 
123, 128, 129, 138, 140, 143, 145, 
146, 148, 149. 

, from methyglyoxal, 107. 

hydrate, 84. 

, occurrence in fat synthesis, 177. 

, hemioellulose synthesis, 

178. 

Acetic acid, see also 57, 68. 

, addition to acetone fermenta- 
tion, 147. 

, formation by bacteria, 84, 

86, 88, 90, 106-9, 112-14, 116- 
22, 126, 127, 129, 136-40, 142-6. 

, occurrence in flour and 
dough fermentation, 213, 224, 243. 

, in stored grain, 206. 

, use in bread-making, 249, 255. 

Acetoooetio acid, 85, 148. 
Acetone, formation by bacteria, 86, 

113, 144, 146-7, 166-8. 
, from acetic acid, 114, 147-8. 
, from butyric acid, 148. 
Acetyl bromide, action on starch 

and polyamyloses, 8. 
Acetylmethylcarbinol, formation by 

bacteria, 84, 86, 106, 108, 109, 

111-13, 118, 121-2, 139, 145, 

148, 153. 

. by yeast, 108, 121. 
Achroodextrms, 9. 
Acids, see also Acetic, Lactic, &o. 
, effect on takadiastase, 27. 
, formation by aerobic bacilli, 

108. 
, in dough fermentation, 237, 

243. 

, in indigo fermentation, 69-70. 
, in stored cereal products, 

203-6. 

, in sugar manufacture, 271-2. 
, use in Amylo process, 32, 34. 
Actinomyces boms, 22. 

diastatwua, 22. 

odonfera, 22. 

spp., lactase in, 59. 

starch decomposition by, 22. 

, see alao, 49, 66, 67, 164, 248. 
Adhesives, 191, 194, 217-18. 
, phenol as antiseptic for, 217. 



Aesouhn, 65, 67. 

Aldol, suggested formation by bac 

tena, 143, 146. 

Algae, amylolytic properties, 16. 
, in waste from sugar manufac 

tore, 285. 

, occurrence of inulin in, 13. 
Alhogi camelorum, manna of, 45. 
Aluminium sulphate, use as anti 

septic, 217. 
Amphierma rubra, production o 

saccharic acid by, 96. 
Amygdalase, 66. 
Amygdalin, 66-7 
Amylase, a- and /?-, 11, 12. 
, bacterial, 17-36. 
, destruction by heat, 20. 
, , optimum temperature, 20 

21, 27. 

, effect of pH on, 17, 20, 26, 32. 
, of temperature on, 32, 35. 
, in Asperffittus oryzae, 26-7. 
, in flour, 229 
, liquefying enzymes in, 10, 26 

27, 35. 

, in malt, 26, 229. 
, in pancreas, 11, 26. 
, isolation of, 3, 20. 
, saccharifying enzymes in, 10 

12, 19, 26, 27, 35. 
, starch decomposition by, 9. 
, use m bread-making, 229. 
Amylo process, 32, 36. 
Amylobiose, 7. 
Amylocoagulase, 12. 
Amylodextrins, 9, 174. 
Amylomyces Rowoii, see Mucoi 

Bouxii. 

Amylopectm, 4-12. 
, relationship to glycogen, 13, 36. 
Amylose, 4-12. 
, a- and /J-, 6, 6. 
Amylotriose, 11. 
Antiseptics, effect on bacterial 

amylase, 20. 

, use in adhesives, 217. 
, in preparation of takadia- 
stase, 25. 

, in sizing of textiles, 213-17. 
, m sugar manufacture, 274. 
Arabmose, 164, 165. 
, decomposition by Bac. ctcetoeihy- 

hcus, 166 
, synthesis of starch from, 175. 



SUBJECT INDEX 



325 



'butin, 65. 
Kochyta Pisi, 199. 
KOCOCCUS Bittroihn, 181. 
Mendeeit, 181. 

mesenterotdea, see Streptococcus 
mesenteroidea. 
ipergiMua albus, 29. 
batatae, 24, 29. 
candtdua, 29, 255. 
conicua, 185. 
arpanawm, 296. 
flavua, 266, 294, 296. 
/wmoj-fctw, 98-100. 
Jumtgotus, 205, 255, 256. 
gktucua, 29, 66, 185, 255. 
nidulans, 246, 254, 255, 294. 
niger, 29, 31, 36, 43-6, 61, 53, 
57-9, 66, 66, 78, 97, 98, 101, 123, 
164, 175, 266, 266, 294, 296. 
Okazdhii, 29. 
oryzae, 23-5, 27-9, 31. 
pseudoflavua, 29. 
repena, 265, 294. 

pergtttus 8pp., compared with 
JitromyceaandPemcittium, 101-2. 
occurrence on bread, 254-5. 

gram, 196, 199, 202, 204, 

SOS. 

sizing materials and 
bdhesives, 215. 

sugar, 262, 265, 296. 

Sydovn, 265, 294. 

temcola, 29. 

bolysis, of yeast, 10. 

>tobacter croococcum, 18, 182, 184. 



ocetoethylicua, 86-90, 111, 
13-14, 145, 162, 166-9, 184. 
ocetonigenua, 114, 143-4, 147, 
50, 153-9, 162, 184. 
yddi propionvn, 136-7. 
vnylobocter app., 18. 
imylozymua, 162. 
inGiracM, 18. 
isterosporus, 18. 
'jotuhnuf, 18. 
mrdigalense, 21. 

butyltcua, see Bac. acetomgenus. 
miyrtcua, 146. 
thoceticua, 90, 113, 114. 
Vitzianus, 18. 
'oaaicularum, 160. 
rranM^oiac/er pecftnoioruwi, 147. 
raveolens, 18. 
nwertona, 293. 

ZactopropyZofeirfynoiw non-ltgue- 
ciens, 138. 



Bacillus levaniformans, 251, 270, 
292. 

bodermoe, 251, 270. 

maceran#, 6, 18. 

Megatherium, 18, 31, 67, 107, 
150, 292. 

mzsentencus-ffubt'ilia group, 263. 

mesentencus, 17, 21, 106, 150, 
217, 247, 262, 266, 270. 

aureus, see Bact. herbicola a 

aureum. 
fuscus, see Boo. mesenterious 

wdgatus. 

niger, 106, 292. 

pams viscon I and II, 251. 

ruber, 18, 106, 150, 166. 

wdgatus, 106, 251, 292. 

methamgenes, 150. 

myccndes, 18. 

oedematis mahgni, 18. 

panvflcans, 182, 249. 

Pasteunanus, 145, 146. 

perfnngens, 138. 

petaaites, 18. 

polymyxa, 18. 

rumtnatus, 18. 

saccharobutynous, 146. 

sporogenes, 303. 

au&fcZw, 18, 21, 106, 107, 109, 
262, 266. 

tenuts, 18, 21, 106. 

thermoamylolytijyus, 18, 22. 

wscosus, 182. 

vulgatus, see ac. mesentencua 
vulgotua. 

Welchvi, 147, 240. 

Bacteria, butyric acid, causing 
diseases of bread, 247, 253. 

, classification of, 150-1. 

, epiphytic on plants, 193, 

, in adhesives, 217. 

, in chocolate creams, 303. 

, in faeces, 143, 151. 

, in flour fermentation, 

222-3. 

, in germinating grain, 222. 

, in lactic acid manufacture, 

131. 

, in stored grain, 202. 

in adhesives, 217. 

in dough, 221-4. 

effect of baking on, 246-7. 

in grain, 196-204 

in indigo manufacture, 68-9. 

in leaven, 219-24, 240. 

in raw sugar juices, 270-1. 

in size, 211-12 

in stored sugar, 289-93. 



326 



SUBJECT INDEX 



Bacteria, in sugar beet, 262. 
, m sugar cane, 261-2. 
, in textiles, 214. 
, in waste waters of sugar manu- 
facture, 285. 

, indican decomposition by, 69. 
, infecting bread after baking, 

248. 

, lactase in, 59. 

, lactic acid, 41, 45, 46, 90, 126- 
34, 270. 

, , coenzyme of 123, 128. 

, , economic importance of, 

130-34. 

, , epiphytic'on plants, 193. 

, , fermentation by, 129. 

, , in bread, 263. 

, , in bread-making, 232. 

, , in flour fermentation, 214, 

222-5. 

, , in malt, 126. 

, , in manure, 126. 

, , medicine, 130. 

, , in Parisian barm, 226. 

, , m silage, 126. 

, , in stored flour and gram, 

204. 

, , in stored sugar, 292. 

, , m wine, 126. 

, , infection of acetone fer- 
mentation by, 158. 
, mucus production by, 181-6, 

270, 292. 

, pentose decomposition by, 166-9. 
, propiomo acid, 89, 91, 136-7, 

139. 

, saccharose in, 48. 
, soil, 126, 211, 292. 
, starch decomposition by, 16- 

22. 

, starch synthesis by, 175-6 
, theories of fermentation by, 79- 

91 

, thermophilie, 22, 197. 
, yellow pigmented, in adhesives, 
217. 

, , in dough, 221. 

, , in steeped flour, 214. 

Bacterium actdtficans longwsvmum, 
132, 222, 238. 

agglomerans, see Boot, herbwola a 
aureum. 

alkaligenes, 140. 

ascendens, 94. 

bulgancum, 125, 128. 

casei, 140. 

coli-typkoaum group, 60, 66, 192, 
262. 



Bacterium coU commune, 48, 68, 
85, 116-22, 126, 130, 175, 1 
193, 221, 292. 

DelbrQcM, 132, 232. 

dysentenae, 18. 

enteritidw, 46. 

fluorescens Uquejuciens, 18, 
111, 112, 192, 198, 261-2, 2 
292. 

Fnedldnder, 45. 

gelatinoaum betae, 270. 

glucontcum, 94. 

herbicola a aureum, 192, 198, 21 
270. 

mdigogenum, 69. 

industrium, 18. 

lactt-arabtnoauin, 46. 

loctis acidi, 132. 

aerogenes, 18, 68, 67, 116, 1J 

3, 184. 

lactofermentum, 126. 

levanformans, 270. 

levans, 221, 224, 240. 

pamflcans, 251. 

paratyphosum, 46, 118, 120, ll 
204, 270, 272. 

Pasteunanum, 93. 

pedwulatum, 270. 

peatw, 18. 

phosphorescent, 18 

pneumonias, 111, 113, 162. 

prodiffiosum, 90, 111, 112, 2( 
206, 266-7, 262, 266. 

pyocyaneum, 112. 

pyogenes foetidum, 19. 

radwcola, 137, 182. 

ruber vndwum, 112. 

SchottmHtten, 46. 

suboxidans, 95. 

smpesttfer, 46. 

termo, 16, 19. 

tnfohi, 192. 

typhoaivm, 19, 116, 118, 120, IS 

woloceoe, 19. 

vtacosum socchart, 270. 

volutans, 158, 211. 

wdgare, 16, 17, 19, 111, 140. 

xylinum, 94, 96, 107, 162, 271. 

Zopfit, 19 

Bagasse, utilization of, 286. 

'Bakhar', 24. 

Banana starch, 5. 

Barley, bacterial content of, 198. 

, deterioration of stored, 201. 

, epiphytic bacteria of, 192 

, minimum moisture content f< 

mould growth, 196. 
Barm, preparation of, 225-6. 



SUBJECT INDEX 



327 



Bean flour, use in preparation of 

Koji, 25 

Beans, Bact. prodigiosum on, 256. 
Beer, mucus production in, 182. 
Beer wort, use as leaven, 225. 
Beet sugar, see Sugar. 
'Butyn', 24. 
Boletus pachypus, starch synthesis 

by, 175. 

Botrytis einerea, effect on germinat- 
ing grain, 199. 
Boulard process, 32. 
Bran, 132, 169. 

, as dough improver, 232, 243. 
, bacterial development in, 197. 
, fungal development in, 202. 
, microflora of, 201-2. 
, use in takadiastase preparation, 

26. 

Bread, 'blood' on, 248, 265-7. 
, chalk disease of, 248, 253. 
, discoloured, 256. 
, diseases of, 246-57. 
, infection after baking, 247-8. 
, by msects, 247. 
, mioroflora of, 194. 
, , effect of moisture content 

on, 247, 260. 

, moisture content of, 247-8. 
, mouldy, 253-6. 
, , as foodstuff, 256. 
, roprness in, 182, 203, 248-63. 

, , control of, 249-50. 

, , detection of organisms 

causing, 252. 
, , influence of moisture 

content on, 250 

, , of pH on, 249. 

, , of storage temperature 

on, 250. 

, , organisms causing, 251. 

, rye, 220, 223, 224, 232, 251. 

, salt rising, 222, 239-40. 

, sour, 247, 263. 

, temperature of baking, 231, 246. 

, of loaf during baking, 246. 

, wrapping of, 264. 

Bread-making, microbiology of, 219- 

43 

Bromates, as dough improvers, 233 
Butyl alcohol, formation by bac- 
teria, 84, 89, 91, 113-14, 138, 142- 

52. 
, production by fermentation 

process, 152-9 
2 3 Butylene glycol, 84, 86, 106, 

108, 109, 111, 113-14, 118, 121-2, 

146, 148, 154. 



Butyric acid, addition to acetone 

fermentation, 148. 
, as hydrogen acceptor, 149. 

fermentation, influence of pH 
on, 144, 152. 

, formation by bacteria, 84, 109, 

138, 142-60. 

, formation from lactic acid, 151 
Buffer action, 27, 242. 



Cacao shells, pentosans in, 167. 

Cannizarro reaction, 119. 

Capnc acid, formation by bacteria, 
160. 

Caproio acid, formation by bac- 
teria, 109, 160. 

Caprylio acid, formation by bac- 
tena, 160. 

Caraway seed, effect on dough 
fermentation, 233. 

Carbamide, in dough improver, 232. 

Carbohydrates, reserve, 6, 12-13. 

Carbon dioxide, production by bac- 
tena, 84-6, 111-13, 116-19, 121-2, 
127, 136, 139, 142, 144, 149, 154, 
221. 

, by fungi, 164. 

, by yeasts, 164, 173, 179. 

, in bread-making, 226, 227, 
239. 

, in indigo fermentation, 69. 

Casein, in lactic acid manufacture, 
131. 

Cassava, 216. 

Catalase, absence of, in anaerobic 
organisms, 79. 

, , in lactic bacteria, 127. 

, m propionio bacteria, 137. 

Cellase, 58. 

Cellobiase, 68. 

Cellobiose, decomposition by micro- 
organisms, 58 

, formation from glucose anhy- 
dride, 11. 

Cellulose, effect on maltase forma- 
tion by Monilw wtoplwla, 67. 

, relation of mucus to, 183. 

Qephaloihecium roeeum, effect on 
germinating gram, 199. 

Cereal products, see also Grain, 
Flour, &c 

, addition of alkaline sub- 
stances to stored, 207 

, chemical sterilization of, 
207-8 

, microbiology of, 191-4 

, tests for damage to, 207. 



328 



SUBJECT INDEX 



Cereals, amylocoagulase in, 12. 
, epiphytic microflora of, 193-4, 

106. 

, Bhizopus spp. in, 24-6. 
Charcoal, use in isolation of Sac. 

acetonigenua, 159. 
Cheese, lactic bacteria in, 137. 
, propionio bacteria in, 136-7, 139. 
Chestnut starch, 5. 
China day, 210. 
'Chinese' yeast, 30. 
Cinnamon oil, as antiseptic, 275. 
Citric acid, production by fungi, 

92, 97, 98, 101-3, 164. 

, medium for, 103. 

, starch synthesis from, 175. 

Oitromyces glaber, 101. 

, in raw cane sugar, 265, 295-6. 

Pfeffenanua, 101. 

spp; 101-2. 

Oladosponum, epiphytic on plants, 
193. 

growth on sizing materials, 216. 

in gram, 196, 199. 

in raw cane sugar, 265, 296. 

in sour dough, 223. 

in Soya, 26. 

Glvstndium acetobutyhcum, see Sac. 
aoetonigewus. 

acetontgenum, see Bac. acetoni- 



gdattnosum, mucus production 
by, 184, 270. 

Pastffimanum, see Bac. Pasteuri- 
anus. 

Clove oil, effect on dough fermenta- 
tion, 233. 

Cocci, epiphytic on plants, 193. 
, in bread, 247. 

in fermented flour, 214. 

in grain, 196. 

in sugar juices, 262, 265, 271. 

mucus formation by, 182. 

oxygluconic acid formation by, 
96 

Coenzyme, 9, 10, 123, 128, 140, 149. 

Oolletotnchum Lindemutkiamifm, ef- 
fect on germinating grain, 199. 

Complement, 28. 

, in yeast extract, 10. 

Compost, pentose decomposition in, 
164. 

Condensation, preliminary, in fer- 
mentation, 179. 

Conferva rrmcorvidea in stored, sugar, 
289. 

Conifenn, 65. 

Goryneboctenum diphthenae, 22, 67. 



Cossettes, 264. 

, microflpra of, 262-3, 265. 

, mildewing of, 286. 

, utilization of, 286. 

Cotton goods, mildew in, 214-16 

, seeds, fungal damage to, 202. 

Crotomo aldehyde, 143. 

Cyperus papyrus, 167. 

D 
Dairy practice, lactic bacteria i 

130. 
Dehydrogenation, aerobic, of he 

oses, 106-10. 

, anaerobic, of hexoses, 111-60. 
, direct, 89, 93, 96. 
Dematium, epiphytic on plants, 19 
, gurrrmosia caused by, 185. 
, in cane sugar, 295. 
Denitrification processes, impo 

tance of pentoses in, 165. 
Desizing of textiles, 210. 
Desmolases, 77. 
Dextran, 183, 268, 272. 
Dextrms, 4, 6, 9, 21, 217. 
, formation from glycogen, 13. 
, from starch, 19. 
, residual, 10. 
Diastase, see Amylase 
Diastatic power, determination o 

28. 

Dibromophenol, 216. , 
Diffusers, in beet sugar manufai 

ture, 264. 
Digitalin, 65. 
Ihgitalw purpurea, mucus in it 

fusions of, 182. 
Digitoxin, 65. 

Dihydroxyacetone, 83, 85, 113, 111 
, as substitute for glucose i 

insulin hypoglycaemia, 81. 
, formation by bacteria, 95, 10< 

107, 112, 114, 119, 145, 149. 
Dwspyros KaJei, 25. 
Disaccharides, decomposition of, 4( 

46-60. 

Dopes, use of butyl alcohol for, 16! 
Dough, 191. 
, acids in, 237, 243 

fermentation, 219, 230. 
, acid formation during, 23( 

241-3. 

, alcohol formation durinj 
238-9. 

, effect of acids on, 232, 243. 

, oxidizing agents or 

233. 
, enzymatic factors in, 232-1 



SUBJECT INDEX 



329 



Dough fermentation, growth factors 

in, 231-2. 
, loss of carbohydrates during, 

239. 

, methods of testing, 235-6. 

, nucroflora of, 221-5. 

, spontaneous, microflora of, 

221-2. 

, pH changes during, 230. 
236-7, 241-3. 

, proteolysis during, 240-1. 

, substances inhibiting, 235. 

, not affecting, 234. 

improvers, 231-4. 

microflora of, 104. 

, effect of baking on, 246-7. 

'proofing' of, 235. 

sour, 220, 222-5. 

straight, method of bread- 
making, 227. 
, , preparation of, 230. 

E 
Electrolytes, effect on glycogen 

decomposition, 13. 
, on takadiastase, 27. 
Emulsin, 11, 43, 44, 58. 
Endomycea flbuhger, 254. 

Magnusvi, 40. 

vernalis, 177. 

Enzymes, extraction by Duclaux's 
method, 30. 

, plant, indican decomposition 
by, 69. 

, , tannin decomposition by, 71. 

, number of, in micro-organisms, 
72. 

Erythrodextrin, 9, 13, 31. 

Ethyl alcohol, effect on dough fer- 
mentation, 233. 

, in fat synthesis, 177. 

, production by Aspergillua 
oryzae, 29-30. 

, by bacteria, 106, 129, 138, 

142, 144, 147. 
, by Bac. acetoethylioua, 84, 

86, 88, 113, 166-9. 

, by Boc. ocetontgenus, 148. 

, by Boc. ethocetwms, 113. 

, by Boo. meaentencua 
ruber, 165-6. 

, by Boct. oacendena, 94. 

, by Boct. coh commune, 85, 

116-18, 119. 
, by Bact . lactte aerogenes, 

120. 

, Bact.pneumoniae, 113, 162. 

, by Bact. prodigiosum, 112 



Ethyl alcohol, production by Bact. 
pyocyaneum, 112. 

by Bact. nfoerindwum, 112. 

by Olostridium gelafano- 
sum, 184. 

by Ftisarium lini, 162. 

by Mucor spp., 32. 

by yeast, 164, 179, 286. 

from molasses, 281. 

from pentoses, 167, 169. 
recovery from bread, 138-9. 

Ethylmethylcarbinol, formation by 
Bac. acetoethyltcus, 114. 

F 

Farina, 210, 215. 

Fat, synthesis of, 173, 176-8. 

Fatty acids, formation of higher, 
159-60. 

, in fat synthesis, 177. 

'Fermentation visqueuse' of sugar, 
181. 

Flax wilt, 162-4. 

Flour, acid production in, 222. 

, buffering action of, 242. 

, diaatatic action in, 229. 

, fermented, 213-14. 

, fungi m damp, 202. 

, infected, as cause of ropy 
bread, 252. 

, microflora of, 130, 192, 194, 
201, 221-2. 

, steeped, 214. 

, strong and weak, 230. 

, substances toxic to yeast in, 
227. 

, sugar content of, 228. 

, use for adhesivea, 217. 

, wholemeal, dough from, 237. 

Fluorescence, production by bac- 
teria, 109 

Fluorides, as antiseptics, 274. 

Formaldehyde, as antiseptic, 217. 

, formation by bacteria, 106, 108. 

Formates, effect on fermentation 
by Bact. coli, 117. 

Formic acid, 113, 120, 122, 125-8. 

, effect of, on saccharose, 53. 

, production by bacteria, 84-7, 

107-9, 112-13, 116-28, 138-49, 
269. 

'Frog spawn' in sugar, 181. 

Froth fermentation, in sugar manu- 
facture, 283. 

Fructo-sacoharase, 53 

Fructose, 41-5, 52, 55, 84, 126. 

, conversion to mannitol, 125. 

, to lactic acid, 127. 



330 



SUBJECT INDEX 



Fructose, decomposition by acetic 
acid bacteria, 94 

by Hoc. acetoethykcits, 166. 

Bac. vidgare, 111. 

effect on hydrolysis of saccha- 
rose, 53. 

hydrolysis of inuhn to, 13. 

in mucus, 1834. 

synthesis of fats from, 178. 

of glyoogen from, 173. 

of starch from, 176, 179. 

Fumarates, activation by Bac. 

vulgar e, 111. 
Fumario acid, formation by fungi, 

92, 97-100. 
, growth of Aspergillua nig&r 

in, 98. 
Fungi, see also Fumaric acid, 

Oxalic acid, &c. 

, assimilation of sugar by, 40-1. 
, decomposition of glucosides by, 

66-6. 

, of pentoses by, 164. 
, of starch by, 22-35. 
, of tannin by, 71-2. 
, direct dehydrogenation by, 90. 
, higher, 34. 
, m adhesives, 217. 
, m beet sugar, 266-6. 
, m beet sugar residues, 286. 
, in bread, 248, 254. 
, in cane sugar, 265, 291-7. 
, m cane sugar juices, 278-87. 
, m gram, 196-208. 
, in molasses, 283-4. 
, in stored sugar, 289-90. 
, m sour dough, 223. 
, m textiles, 214-15. 
, in waste water from sugar 

manufacture, 285. 
, lactase in, 59. 
, mucus production by, 186. 
, pellagra attributed to, 200. 
, sacoharase in, 48. 
, starch synthesis by, 176. 
, trehalose m, 46. 
Fusanum hm, pentose decomposi- 
tion by, 162-4. 

rosewm, effect on germinating 
gram, 199. 

, toxic effect in rye, 199-200. 

spp., growth of, on sizing 
materials, 216. 

, in cane sugar, 295. 
, in waste water from sugar manu- 
facture, 286. 

saccharase m, 61. 
Fusel oils, origin of, 153. 



G 



Galactan, 184. 

Galactose, 424. 

, mucus production from, 182, 
184. 

, starch synthesis from, 175. 

Gallic acid, production by fermenta- 
tion of tannin, 71-2. 

Gelatimzation of starch, 4. 

Oentmna, alcoholic beverage from, 
44. 

Gentianose, 43-4. 

, action of saccharase on, 55. 

, decomposition by micro-organ- 
isms, 44. 

Gentiobiose, 44, 58. 

Germinating power, of grain, 198-9. 

Glucomo acid, 92-7. 

, formation by bacteria, 93, 

107. 

, by fungi, 92, 96, 98-9. 

, in Kombucha, 94. 

, nutritive value of, 94. 

Glucosaccharase, 53. 

Glucose, 28, 40, 42, 44, 45, 62, 53, 
65, 126-7, 162-3. 

, as source of lactic acid, 132. 

, conversion of cellobiose to, 58. 

, of cellulose to, 160. 

, of mucus to, 182-3. 

, of starch to, 19-20. 

, decomposition by bacteria, 109, 
111, 112, 129, 140, 146, 166. 

, effect on rate of hydrolysis of 
saccharose, 53. 

, on amylase production, 31. 

, formation of butyric acid from, 
142-4, 152. 

, of citric acid from, 103. 

, of fats from, 177. 

, of galactan from, 184. 

, of gluconic acid from, 92. 

, of higher fatty acids from, 
160. 

, of mucus from, 182-3. 

, of propiomc acid from, 139. 

, of starch from, 176-6, 179. 

, in flour, 228. 

Glucosides, 57. 

, decomposition by micro-organ- 
isms, 65-72. 

Glumes, of cereals, 193. 

Glutamic acid, 119. 

Gluten, 152, 240 

Glyoeraldehyde, 82, 85, 118-19. 

, anhydride, 82-3. 

, decomposition by Bact. coh, 
118. 



SUBJECT INDEX 



331 



Glyoeraldehyde, formation by bac- 
teria, 106, 109, 112, 119, 129, 139. 

Glycerol, as fermentation product, 
127. 

, effect on saccharose, 51-2. 

, decomposition by Sac tennis, 
106. 

by acetic bacteria, 95. 

, by butyne bacteria, 151. 

> by propiomo bacteria, 137. 

, formation of butyric acid from, 
142-3. 

, of dihydroxyacetone from, 
95. 

, of starch from, 176. 

, in synthesis of fats, 177. 

Glycme, effect on saccharose, 53. 

Qfycobacter, 19. 

Glycogen, 12-13. 

, decomposition by micro-organ- 
isms, 35-6. 

, occurrence of in Saccharomy- 
cetes, 35. 

, synthesis of, 173-4, 179. 

Grain, acid production in, 203-4, 
206. 

, chemical sterilization of, 207-8. 

, deterioration in storage, 196- 
208. 

, , effect of temperature on, 

201. 

, effect of moisture on microflora 
of, 196-7. 

, epiphytic flora of, 192-4, 196. 

, fungi in stored, 202. 

, germinating power of, 1989. 

, infection of, from soil, &c., 
201. 

, microflora of, 194, 202-3. 

, protein decomposition in stored, 
202, 204 

, toxic substances in stored, 202. 

Granulcibacter, 19. 

saccharobutyncum, see Sac. sac- 
charobtctyncus. 

Granulose, 5. 

Grape juice, use as leaven, 226. 

H 

Haemolysin, in takadiastase, 31. 
Hay, spontaneous combustion of, 

197. 

Hehcrn, 66. 

Hemicelluloses, in starch, 6. 
, synthesis by micro-organisms, 

173, 178. 
Hexosans, 8. 
Humus, formation by bacteria, 165. 



Hydrogen acceptors, 78, 79, 92-3. 

mdigotm as, 93. 

methylene blue, 78, 93. 

nitrates as, 78, 107. 

organic acids as, 139, 146. 

oxygen as, 93, 234. 

selenium and tellurium salts 

as, 127. 
, activation of, 78, 82, 89. 

donator, 78, 80. 

evolution m salt-rising bread, 
239 

in sugar manufacture, 271. 

formation by bacteria, 85-7, 
111-13, 116-17, 119-22, 142, 146, 
147, 149, 154, 221. 

peroxide, 80, 128. 
j3-hydroxybutaldehyde, 84. 
fl-hydroxybutyric acid, 108-9. 
Hypochlorites, use in sugar manu- 
facture, 275. 



Indican, 65, 67, 68. 
Indigo brown, 70-1. 
gluten, 70-1. 
, manufacture of, 68-71. 
Indigotin, 68-71, 93. 
Indoxyl, 68-70. 

Infusoria, starch synthesis by, 175. 
Inulase, 36. 
Inulin, 13-14. 

, decomposition by micro-organ- 
isms, 36. 

Invertase, see Saccharose. 
Isomaltose, 4, 8, 44. 



'Kephir', 43. 
, lactase in, 59. 
Koji, 23-6. 

'Kombucha', preparation from tea, 
94. 



Lactase, 58-60. 

, effect of pH on, 59. 

, in Kephir, 43, 60. 

, isolation of, 69. 

Lactate, calcium, decomposition by 
bacteria, 113, 136, 142, 159. 

Lactic acid bacteria, see Bacteria 

, decomposition by bacteria, 

58-60, 138 

, effect on dough fermenta- 
tion, 243 

, formation by of Bad. coh, 

117-18. 



332 



SUBJECT INDEX 



Lactic aoid, formation by bacteria, 
83, 85, 90, 107, 109, 113, 116, 
119-22, 125-44, 149, 151, 162, 169, 
269-70. 

, by fungi, 134. 

, in bread, 224. 

, in leather industry, 133. 

, in molasses fermentation, 

287. 

, in mordanting, 133. 

, in stored grain, 206. 

, in textile industry, 133. 

manufacture, importance of 

pH, 132. 

, optical properties of, 113, 

129-30. 

Lactobacterium jermentum, see Bact. 
lactqfermentum. 

Lactose, decomposition by bac- 
teria, 94, 109, 137, 140. 

, effect on saccharose production, 
51. 

, formation of galactan from, 184. 

, of mucus from, 182. 

Lead acetate, use for preserving 
sugar solution, 275. 

Leptormtus lacteua, 285. 

Leuconoatoc mesentermdes, see Strep- 
tococcus meaenterowLes. 

Levan, 184, 272, 292. 

Lime, use in sugar manufacture, 
264-5, 273-4, 279. 

, in lactic aoid manufacture, 
132. 

Litmus, as hydrogen acceptor, 87, 93. 

, reduction by bacteria, 109. 

M 

Maize, Boat, prodigwsum in, 257. 
, butyric aoid from, 151. 
, fungi in, 205. 
, pellagra attributed to, 200-1. 

starch, fatty acids in, 6. 
, hemicelluloses in, 6. 

, thennophiko bacteria in, 197. 
Malates, activation of, 111. 
Malt, 126, 232. 

diastase, 3-4, 7, 11-12, 27-8. 

extract, use in bread-making, 229- 
30. 

Maltase, 52. 

action on staohyose, 43. 

destruction by alcohol, 66. 

effect of acids on, 66. 
of pH on, 56, 230. 

of temperature on, 56, 230. 

in bread-making, 230. 

in yeast 66, 56. 



Maltose, butyric acid production 
from, 151. 

, decomposition by micro-organ- 
isms, 40, 66-7, 94, 137, 166 

, effect on amylase production, 31. 

, on saccharase production, 5L 

, formation from amylose and 
amylopectin, 10-11. 

, from glycogen, 13. 

, from starch, 19, 21-2. 

, m flour, 228-9. 

, isolation of, 3. 

, lactic acid production from, 132. 

Manna, 45. 

Manmtol, formation by bacteria, 
42, 84, 125-7, 133. 

, in mucus, 183. 

, mucus production from, 182. 

, starch synthesis from, 175. 

Mannose, 61. 

, starch synthesis from, 176, 179. 

Mannotriose, 42. 

Manure, pentose fermentation in, 
164, 165. 

Massecuite, beet sugar, 265. 

Melezitose, 45. 

Mehbiose, 44, 60. 

Mehtnose, see Raffinose 

Mercuric chloride, effect on saccha- 
rose, 54. 

Methane, formation by bacteria, 121. 

Methylene blue, effect on dough 
fermentation, 234. 

, on yeast fermentation, 

122. 

, reduction by bacteria, 127, 

137. 

Methylglucoside, 53. 

Methylglyoxal, formation by bac- 
teria, 80-7, 106-14, 118-19, 129, 
139, 149. 

, hydrate, 83-4. 

Microooccus dgofcjakartenaw, 186. 

gonorrheae, 19-21. 

maattfidts gangraenosae, 19. 

pyogenes tenuw, 67. 

tetoragemus, 19. 

Mwro&pvra agar-hquefacwns, 57. 

Mildew in textiles, 214-15. 

, moisture requirements of, 215-16. 

Milk, acid production in, 109. 

, butyric bacteria m, 161. 

, lactic acid production from, 131. 

, ropmess in, 182 

Molasses, butyric acid from, 151. 

, disposal of, 286 

, lactic acid from, 132. 

Monaacua purpureus, 295. 



SUBJECT INDEX 



333 



Wonttia Candida, saooharase of, 49. 

fusca, 295. 

macedoniensis, 36. 

nigra, 295. 

sitophila, 25, 45, 46, 57, 256. 

spp., 266. 

in sugar beet manufacture, 

262. 

tfonoses, fermentation of, 77-91. 
MorcheUa, 36. 
Mucor aUernana, 30. 

batatae, 32. 

Cambodia, 32. 

Chinen&is, 134. 

circindloides, 30. 

etoew, 21, 32. 

mucedo, 255. 

pvnfonms, 102. 

pusittus, 255-6. 

racemosua, 48. 

Eouxii, 21, 24, 30, 32, 33, 59, 99, 
134. 

spp., 119. 

, decomposition of inulin by, 

36. 

, pentosea by, 164. 

, starch by, 30. 

, in sugar manufacture, 285, 

294. 
, use of, in distillery practice, 

32 

stolomfer, 32, 100, 199, 246, 256. 
Mucus, composition of, 182-3. 

, formation by micro-organisms, 
90, 181-6, 263, 268-70, 280. 

, influence of pH on, 183. 

, influence of SO 8 on, 275. 

hydrolysis of, by acids, 181. 

in bread, 251. 

in gram, 198. 

in milk, 182. 

in stored sugar, 292-3. 
Mycoboctenum tuberculosis, 22, 47. 
Mycoderma acett, 93. 

cerevmoe, 255. 

N 

Nitrates, as hydrogen acceptors, 78, 
93, 107, 111, 120. 

, influence on molasses fermenta- 
tion, 287. 

Nitric acid, effect on saccharase, 53. 

Nitrogen, influence on citric acid 
formation, 103. 

, on gluconio acid formation, 
96. 

, on lactic acid formation, 129, 
132 



O 



Oat husks, pentoses in, 167. 
Oats, epiphytic bacteria of, 192. 
, deterioration of stored, 201. 
Ovhum auranttacum, 254-6. 

Zociw, 223. 

temcola, 268. 

Onions, effect on dough fermenta- 
tion, 233. 
Oospora lactos, 255. 

vanabilis, 255. 

Oxalic acid, formation by bacteria, 

97, 101. 

, _ by fungi, 92, 97-9, 101. 

, , influence of pH on, 99. 

, from mucus, 183. 

Oxidation-reductions, 83-5. 
Oxygen, as hydrogen acceptor, 78- 

9, 89-90. 
, effect on lactic fermentation, 

125. 
Oxygluconic acid, 96. 



Pancreas, amylase of, 26. 
Paramtrophenol, 216. 
Pectin, decomposition of, in ger- 
minating gram, 199. 
Pellagra, 200-1. 
Pemcittium citnnum, 102. 

crustaceum, 256. 

dwancatum, 102, 265, 294. 

expansum, 175, 294, 296. 

glaucum, 25, 29, 31, 50, 61, 102, 
175, 223, 290. 

htteum, 102, 294, 296. 

Ivtewn-pwrpurogerwvm ser., 96, 
294-5. 

ohvaceum, 255. 

pmophUum, 294. 

pubemhim, 175. 

roseum, 295. 

ruguLosum, 72. 

Pemcilhum s-pp. , acid production by, 
92, 95-7, 102. 

compared with Gitiromycea, 101-2. 
, decomposition of glucosides by, 

66. 

of pentoses by, 164. 

of saccharose by, 48, 60-1. 

of tannin by, 72. 
epiphytic on plants, 193, 196. 
in adhesives, 217-18. 

in bread and dough, 223, 255. 
in grain, 196, 199, 204. 
in sugar, 265, 290, 294-6 
in textile industry, 215-16. 



334 



SUBJECT INDEX 



Pentosans, decomposition by bac- 
teria, 169. 

, hydrolysis of, 167-8. 

, occurrence in nature, 167. 

Pentoaea, condensation of, prior to 
fermentation, 163. 

, decomposition by micro-organ- 
isms, 90, 162-9. 

Peptone, effect on amylase produc- 
tion, 31. 

Phosphates, effect on acetone fer- 
mentation, 149. 

Phosphoric acid, effect onsaccharase, 
63. 

esters, 6, 79-86, 93, 97, 107, 109, 
112, 114, 119, 122, 128-9, 139-40, 
163, 174. 

Plants, epiphytic microflora of, 120, 
191. 

Polyamyloses, 7. 

Potyporaceae, melezitose in, 46. 

Polyporus avlphur&us, laotase in. 
69. 

Polysacoharides, hydrolysis of, 40- 
60. 

'Polyzime', 27. 

Popuhn, 66. 

Potassium, salts of, as dough 
improvers, 233 

Potatoes, production of alcohol 
from, 167 

, sweet, use in bread-making, 230. 

Proofing of dough, 231. 

Propiomc acid, detection of, 138. 

, effect on saccharose, 53. 

, formation by mioro-organ- 

isms, 136-40. 

Propyl alcohol, formation by Boo 
acetoethyhcus, 160. 

Proteins, decomposition of, by Bac. 
acetomgemia, 151. 

, , in bread-making, 226. 

, effect of formaldehyde on, 218. 

, on acetone fermentation, 
149. 

, precipitation of, m indigo 
manufacture, 70. 

Prunua joponica, gummosis in, 185. 

Pseudomonas tnfoln, see Bad. 
tnfohi. 

Pyruvic acid, 84, 113, 122, 177. 

, as hydrogen acceptor, 138. 

, formation by micro-organ- 
isms, 106-7, 109, 119-20, 128-9, 
138-9, 144, 177-8. 

, m synthetic processes, 107, 

177-8. 

Pyffmun deBaryanum, 199. 



Hafnnose, action of sacoharase on 
55. 

, decomposition by micro-organ- 
isms, 44, 94. 

, effect on maltase production. 
57. 

, estimation of, by use of invert- 
ing enzymes, 273. 

Rhizopus Delemar, 36. 

japorwcua, 25. 

spp., 24. 

, laotase in, 69. 

tonfcinensw, 24. 

Rice, Boct. prodigioaum on, 257. 

beer, preparation of, 24. 

, deterioration of, by micro- 
organisms, 2012. 

, hemioelluloses in, 6. 

, preparation of sak from, 23-4. 

, production of butyric acid from. 
161. 

Bye, see also Bread. 

, epiphytic bacteria of, 192. 

, flour, Bac. meaentenaua in, 262. 

, fungi in stored, 202. 

, toxic effect of Fuaanum rose/urn 
in, 200. 

S 

Sacoharase, 41-66. 
, effect of various substances on, 

62-5. 

, extraction from yeast, 50. 
, in broad-making, 230. 

of Aspergilh, 43, 48, 295. 

, of bacteria infecting stored 
sugar, 292-3. 

, optimum pH for, 61, 63. 

, temperature for, 51, 54. 

, regeneration of, 64. 

, reversible action of, 55. 

Saccharic acid, formation by micro- 
organisms, 92, 97. 

Saccharomyccs cerewsiae, 224, 262. 

minor, 223-4 

spp , 224, 226. 
Saccharose, see also Sugar. 

, decomposition by bacteria, 47- 
55, 94, 112, 137, 271 

direct assimilation by fungi, 40-2. 

effect on amylaso production, 31. 

estimation of, 272-3 

formation of butyric acid from, 
161. 

of citric aoid from, 103 

of galactau from, 184. 
of glycogen from, 174 



SUBJECT INDEX 



335 



Saccharose, formation of lactic acid 
from, 131-2. 

, of manmtol from, 133. 

, of mucus from, 181-3. 

, in flour, 228. 

, loss of, in sugar manufacture, 
269, 272-3. 

, synthesis of, 66. 

, use in tannin fermentation, 72. 

Sake, 23. 

Salioin, 66. 

Sahcylanihde, as fungicide, 217. 

Sambucm, 65. 

Sarcmae, epiphytic on plants, 103. 

Sauerkraut, 46. 

Selenium salts, reduction by manm'- 
tol bacteria, 127. 

Serratia marcescens, see Bact. prodi- 
giosum. 

Serum, ox, as dough improver, 234. 

Silage, mannitol production from, 
133. 

, pentose-fermenting bacteria in, 
162, 186. 

, propiomo bacteria in, 139. 

, utilization of cossettes for, 286. 

Silver nitrate, effect on saccharase, 
64 

Size, for textiles, preparation of, 210. 

, , source of microflora of, 

211-12. 

Sizing materials, 191, 210. 

, microbiology of, 210-17. 

, , relative liability to mildew, 
215. 

Sodium chloride, effect on Koji 
sacoharase, 53 

Soil bacteria, 120 

, formation of humus by, 166. 

, of mucus by, 268. 

, in milling products, 201. 

, in plants, 193-4. 

, use of, in isolation of Bac. 
acetonigenus, 159. 

Sorbitol, decomposition by Bact. 
xyhnum, 95. 

Sorbose bacterium, see Bact. xy- 
hnum. 

Sough dough, see Dough. 

Soya, manufacture of, 23, 25. 

Sphaerottlus, in waste water of 
sugar manufacture, 285. 

Sponge, preparation of, in bread- 
making, 226-7 

Stachyose, action of sacoharase on, 
55 

, decomposition by micro-organ- 
isms, 43. 



Stocky s tubifera, 42. 
StapTiylococcua pyogenea awreus, 19. 
Starch, constitution of, 3-12. 
, decomposition by acid, 3. 
, by Actinomycetes, 22. 
, by bacteria, 16-22, 160-2, 

166. 

, by fungi, 22-36. 
, by heat, 7 
, spontaneous, 16. 
, in bread-making, 226. 
, in germinating grain, 199. 
, depolymenzation of, 7-9. 
, deterioration in storage, 34. 
, effect on maltase production, 57. 
, gelatinization of, 4. 
, lactic acid production from, 132. 
, methylation of, 8. 
, phosphorus content of, 6, 9. 
, relation of mucus to, 183. 
, soluble, 215 
, synthesis by micro-organisms, 

120, 173"-6. 
Starch-cellulose, 5. 
Sterilization, chemical, of cereal 

products, 207-8. 

, influence of pH on, 162, 168. 
Streptococcus lactw acidi, 132, 137, 

222, 247. 

mesenterotdes, 126, 181, 265, 
288-70, 275, 280, 292. 

app., 41, 263, 265. 

Succimc acid, formation by micro- 
organisms, 47, 97-100, 113-21, 
126-7, 140, 162. 

Sudd, Nile, pentosans of, 167. 

Sugar, bacterial analysis of, 300. 

, beet, microflora of, 261-2, 266-6. 

, cane, mieroflora of, 261-2, 265. 

, deterioration of, 291-2, 297, 
299-300, 302 

, , influence of moisture on, 
289-91, 298, 300-2 

, , of temperature on, 301. 

, , inoculation to prevent, 297. 

, , micro-organisms causing, 
289-303. 

, growth of organisms in concen- 
trated solutions of, 291. 

, juices, microflora of, 266-6, 275, 
278-87. 

, manufacture, 263. 

, , disposal of waste water, 
from, 284-6. 

, influence of pH in, 266, 273-4. 

, losses in, 267, 272-3. 

, polarization of raw juices in, 
272 



336 



SUBJECT INDEX 



Sugar, mucus production in, 181, 

268-9. 

, safety factor of, 298-9 
, spontaneous decomposition of, 

293. 

y-Sugars, in inulin, 14. 
Sulphites, fixation of acetaldehyde 

by, 143. 
Sulphur dioxide, use of, in sugar 

manufacture, 275. 
Sulphuric acid, hydrolysis of pen- 

tosans by, 167. 
Syncephalastrum, 265, 295-6. 
Synthetic processes of micro-organ- 
isms, 173-9. 

T 

Takadiastase, 11, 25-8. 
, enzymes of, 6, 26-7, 31, 48. 
, precipitation of, 29. 
, regeneration of heated, 35. 
, rennet in, 31. 
Tannin, 65, 68. 

, effect on amylase production, 3 1 . 
, fermentation, 71-2. 
'Tempering' in sugar manufacture, 

264, 274. 
Tetrasacchandes, hydrolysis of, 40, 

42-3. 
Textiles, desizing of, by bacterial 

enzymes, 21. 
Thallium carbonate, toxicity to 

mould fungi, 216. 
Tonda communis, 296, 297. 
spp., 265-6. 

epiphytic on plants, 193. 

in sour dough, 223. 

in sugar, 262, 266, 295-7. 

Trehalase, 46. 
Trehalose, 46. 
Tribromophenol, toxic to mould 

fungi, 216. 

Trichloracetio acid, 67. 
Tnglucosan, 10. 
Triglucose, 10. 
Trihexosan, 10. 
Tnsacchandes, hydrolysis of, 40, 

43-5. 

Turanose, 45. 
Tyrothrix tenuw, see Sac. tennis. 

U 
Ustdago May die, 36. 

V 
Valeric acid, formation by bacteria, 

106, 108, 160, 160. 
Varnishes, use of " 
159. 




Vibrio bvtyricus, 146. 

cholerae, 19, 20. 

Metchmkqff, 21, 67. 

Vinegar, spontaneous conversion oi 

saccharose to, 271. 
Visoosaccharase, 186. 
Vitamin, pellagra attributed to 

deficiency of, 201. 
Voges-Proskauer reaction, 109, 121. 

W 
Weaving, preparation of warp for, 

210. 
Wheat, deterioration in storage, 

196, 201. 

, , substances inhibiting, 207. 

, epiphytic bacteria of, 192. 

flour, fermentation of, 212-14. 
, Koji from, 26 

, liability to mildew, 215. 

, steeping of, 212, 214. 

Willia spp., 254. 

Wine, lactic bacteria in, 126. 

, mucus production in, 182. 

Woollen industry, lactic acid in, 
133. 

X 

Xyhmc acid, formation by bac- 
teria, 162. 

Xylose, see Pentose. 

Y 

Yeast, coenzymes of, 123. 
, epiphytic on plants, 193. 
, fat synthesis by, 177. 
, in bread-making, 222-7, 231-2. 
, in chocolate creams, 303. 
, in fermented flour, 214. 
, in grain, 220. 

, in lactic acid manufacture, 131. 
, in sugar, 266, 278, 286-7, 289. 
, in tannin fermentation, 72. 
, lactase of, 59. 
, maltase of, 55-7. 
, mehbiase of, 60. 
, molasses fermentation by, 286-7. 
, pentose decomposition by, 164. 
, saccharase of, 47-55. 
, substances in flour toxic to, 

227. 

, trehalase of, 46. 
Yoghurt, 125. 

Z 

Zinc chloride, as antiseptic, 213-17. 
, effect on glucosidase, 66. 
Zoogloea, production by propiomo 

bacteria, 137. 

loea ramigera, 185.