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UNIVERSITY  OF  CALIFORNIA 
-RTMENT   OF  CIVIL   ENGINEERING 
"ERKELEY.  CALIFORNIA 


UNIVERSITY  OF  CALIFORNIA 

DEPARTMENT   OF   CIVIL   ENGINEERING 

BERKELEY,  CALIFORNIA 


AN   INTRODUCTION 

TO 

BACTEKIOLOGICAL  AND  ENZYME  CHEMISTRY 


AN  INTRODUCTION 

TO 

BACTERIOLOGICAL 


AND 


ENZYME    CHEMISTRY 


BY 

GILBERT   J.   FOWLER,  D.Sc.,  RIO. 

iv 

LECTURER  IN  BACTERIOLOGICAL  CHEMISTRY  IN  THE  VICTORIA  UNIVERSITY 

OF  MANCHESTER  ;   EXAMINER  IN  BIOLOGICAL  CHEMISTRY    TO  THE 

INSTITUTE  OF  CHEMISTRY  OF  GREAT  BRITAIN  AND  IRELAND 


SECOND-  JMPRESSJOT*.; 


LONDON 
EDWARD    ARNOLD 

[J.  H   rights   reserve dj 


Engineering 


TO 

A.  H.  F. 


PKEEACE 


THE  subject  of  bacteriological  and  enzyme  chemistry  is 
becoming  year  by  year  of  increasing  importance.  A  know- 
ledge of  it  is  now  necessary  for  the  scientific  conduct  of  many 
industrial  processes  of  great  magnitude. 

Apart  from  its  well-known  applications  in  the  fermen- 
tation industries,  a  scientific  understanding  of  this  branch 
of  chemistry  is  likely  to  exercise  considerable  influence  upon 
the  future  development  of  agriculture. 

Recent  advances  in  sanitation,  especially  the  provision  of 
pure  water,  and  the  inoffensive  disposal  of  sewage,  call  for 
the  co-operation  of  the  engineer  and  the  biological  chemist. 

The  Institute  of  Chemistry  has  recognised  these  require- 
ments in  the  special  examination  in  biological  chemistry, 
which  it  has  conducted  for  a  number  of  years  past.  The 
author's  students  have  frequently  asked  him  to  recommend 
an  elementary  book,  which  should  serve  as  an  introduction 
to  the  somewhat  overwhelming  literature  of  the  subject. 

The  difficulty  of  pointing  to  any  one  work  which  satisfied 
these  requirements  led  the  author  to  attempt  himself  to 
supply  the  deficiency. 

In  writing  the  book  he  has  had  in  mind,  not  only  the 
purely  chemical  student,  but  also  members  of  other  pro- 
fessions, with  whom  he  has  frequent  occasion  to  co-operate, 
notably  the  engineer  and  medical  officer  of  health,  as  well 
as  the  general  reader,  to  whom  the  subject  offers  many 
attractions. 

«>  w  w»— 

o77  i 


VI  PREFACE 

On  this  account  certain  chapters  of  the  book  especially, 
e.g.,  those  on  the  principles  of  organic  chemistry,  have  been 
written  in  a  more  elementary  manner  than  would  be  called 
for  by  the  pure  chemist.  On  the  other  hand,  the  methods 
of  experiment  and  research  employed  in  bacteriological  and 
enzyme  chemistry  have  been  illustrated  in  some  detail  by 
typical  examples. 

The  endeavour  has  also  been  made  to  keep  the  style  in- 
teresting and  readable,  without  sacrificing  scientific  accuracy, 
How  far  this  object  has  been  attained  it  will  be  for  the  reader 
to  judge. 

It  is  impossible  for  any  one  scientific  worker  to  be  a 
specialist  in  more  than,  at  most,  a  very  few  branches  of 
study.  The  author  has  been  fortunate  in  obtaining  valuable 
assistance  in  the  writing  of  this  book  from  many  of  his 
scientific  colleagues.  In  particular  he  would  gratefully 
acknowledge  the  help  which  he  has  received  from  Professor 
Adrian  Brown  of  the  University  of  Birmingham,  Dr.  A.  Harden 
of  the  Lister  Institute,  Dr.  E.  J.  Kussell  of  the  Rothamsted 
Experimental  Station,  Dr.  H.  H.  Mann,  Chemist  to  the 
Indian  Tea  Association,  and  Mr.  S.  H.  Davies,  Chemist  to 
Messrs.  Rowntree  and  Co. 

Other  references  will  be  found  in  the  body  of  the  book, 
or  in  the  short  bibliography  at  the  end.  The  latter,  while 
comprising  only  important  text-books,  and  original  papers 
of  fundamental  interest,  will,  it  is  hoped,  enable  the  student 
to  continue  his  reading,  and  to  follow  up  the  subject  in  any 
direction,  by  means  of  the  fuller  bibliographies  in  the  works 
cited. 

In  conclusion  it  is  only  right  to  mention  the  great  assistance 
the  author  has  received  from  his  wife,  who  has  acted  as  his 
amanuensis,  and  to  whom  this  book  is  dedicated 

G.  J.  F. 

January,  1911. 


CONTENTS 


CHAPTER 

I.     THE   CHARACTERISTICS   or   CHEMICAL   ACTION  IN  LIVING 

MATTER      .........  1 

IL     OUTLINES  OF  BACTERIOLOGICAL  TECHNIQUE      ...  16 

HI.    SOME  LEADING  CONCEPTIONS  IN  ORGANIC  CHEMISTRY        .  35 

IV.    SPACE- ISOMERISM  AND  THE  CHEMISTRY  OF  THE  SUGARS       .  65 

V.    THE  HYDROLYSIS  OF  STARCH  BY  AMYLASE       .         .         .  100 

VL    THE    CONDITIONS    OF    FORMATION    OF   AMYLASE   IN   THE 

LIVING  CELL 118 

VII.     INVERTASE  AND  MALTASE 126 

VIII.    THE  ALCOHOLIC  FERMENTATION  OF  GRAPE  SUGAR      .         .131 

IX.    THE  ACID  FERMENTATION  OF  ALCOHOLS  AND  CARBOHYDRAT  ES  145 

X.    THE  FERMENTATION  OF  CELLULOSE  AND  ALLIED  BODIES      .  159 

XL    MISCELLANEOUS  FERMENTATIONS,  FAT- SPLITTING  ENZYMES, 

OXIDASES,  CLOTTING  ENZYMES 169 

XII.     OUTLINES  OF  THE  CHEMISTRY  OF  ALBUMINS  OR  PROTEINS     .  181 

XIII.  THE  NITROGEN  CYCLE     ....                           .  212 

XIV.  THE  SULPHUR  CYCLE 236 

XV.    FERMENTATION    OF    INDIGO,    TEA,    COCOA,    COFFEE,    AND 

TOBACCO 245 

XVI.    BACTERIOLOGICAL   AND   ENZYME  CHEMISTRY  IN   RELATION 

TO  AGRICULTURE 256 

XVII.    THE  CHEMISTRY  OF  SEWAGE  PURIFICATION      .         .         .  280 

BIBLIOGRAPHY 312 

INDEX    .         .         .         .         .         .  318 


LIST  OF  PLATES 

PLATE  Facing  page 

L         STARCHES •     100 

II.  (i)  ROOT  NODULES  OF  PEA          .  118 
(ii)  SECTION  OF  BARLEY  GRAIN     ..  9) 

III.  (i)  INDIGO  VATS  NEAR  MIRZAPUR,  INDIA       .         .  .     252 
(ii)  FERMENTING  BOXES  FOR  COCOA       ...  ,; 

IV.  (i)  SEWAGE  WORKS  AT  MATUNGA,  NEAR  BOMBAY  .     284 
(ii)  PERCOLATING  FILTERS  AT  ACCRINGTON  „ 


AN  INTRODUCTION 


TO 


BACTERIOLOGICAL  AND  ENZYME 
CHEMISTRY 

CHAPTER  I 

THE  CHARACTERISTICS   OF  CHEMICAL  ACTION  IN 
LIVING  MATTER 

THE  student  of  chemistry  must  always  be  impressed  with  the 
extraordinary  ease  with  which  complicated  chemical  changes 
take  place  in  living  matter.  By  comparison  the  methods 
used  in  the  laboratory  to  effect  the  artificial  preparation  of 
natural  products  appear  cumbersome  and  violent. 

Thus,  e.g.,  to  take  a  fairly  simple  case,  the  colouring 
matter  alizarine  is  produced  in  the  madder  plant  under 
natural  conditions  of  growth ;  at  temperatures,  that  is, 
much  below  the  boiling-point  of  water  and  without  the 
production  of  any  excessive  alkalinity  or  acidity. 

To  prepare  this  substance  artificially  a  hydrocarbon  an- 
thracene is  made  use  of,  itself  produced  by  the  distillation  of 
coal  tar  at  a  high  temperature.  This  is  first  violently  oxidised 
by  reagents  such  as  bichromate  of  potash  and  glacial  acetic 
acid  ;  the  resulting  oxidised  product  anthraquinone  is  then 
dissolved  in  concentrated  acid,  the  sulphonic  acid  so  obtained 


2        BACTERIOLOGICAL  AND  ENZYME   CHEMISTEY 

converted  into  a  lime  salt  by  the  addition  of  lime,  and  the  lime 
salt  finally  fused  with  caustic  soda,  producing  the  sodium  salt 
of  di-hydroxy-anthraquinone  or  alizarine. 

The  artificial  preparation  of  such  substances  as  indigo, 
camphor  and  terpenes,  uric  acid,  etc.,  is  even  more  com- 
plicated, although  the  actual  chemical  reactions  may  not 
always  be  of  so  drastic  a  character. 

The  same  contrast  between  natural  and  artificial  processes 
is  observable  when  the  change  results  in  the  decomposition  of 
substances.  Thus  to  saponify  a  fat,  i.e.,  to  split  it  up  into  its 
constituents,  viz.,  a  fatty  acid  and  glycerine,  by  chemical 
means,  high  pressure  steam  or  strong  acid  or  alkali  is  neces- 
sary, a  condition  of  things  which  obviously  does  not  obtain  in 
the  ordinary  processes  of  fat  digestion  in  the  body.  Moreover 
certain  chemical  changes  which  have  so  far  not  been  artificially 
produced  are  brought  about  with  the  greatest  ease  by  living 
matter  ;  thus,  e.g.,  cellulose,  a  carbohydrate  of  the  general 
formula  (C6H1005)n  can  be  split  up  by  fermentation  into  marsh 
gas,  CH4,  hydrogen,  H,  and  carbon  dioxide,  C02,  and  various 
subsidiary  products.  This  change  can  be  observed  in  nearly 
any  green  stagnant  pond,  the  mud  on  the  bottom  of  which 
generally  yields  copious  bubbles  of  gas  if  stirred,  and  one  of 
the  famous  frescoes  by  Ford  Madox  Brown  in  the  Man- 
chester Town  Hall  represents  John  Dalton,  Manchester's 
great  chemical  philosopher,  collecting  marsh  gas  in  this  way. 

The  well-known  and  extremely  important  alcoholic  fer- 
mentation of  grape  sugar  is  similarly  instructive.  By  the 
action  of  yeast  this  readily  yields  alcohol  and  carbon  dioxide 
roughly  in  accordance  with  the  following  equation  : — 

C6H1206  =  2C2H5OH  +  2C02 

In  this  case  also,  simple  as  the  change  appears,  it  has  not 
been  hitherto  possible  to  bring  it  about  under  strictly  artificial 
conditions. 

In  seeking  to  elucidate  the  conditions  under  which  these 


CATALYSIS  3 

chemical  changes  take  place  in  nature  they  may  be  compared 
in  the  fir^t  place  with  ordinary  chemical  changes,  which  can 
be  effected  in  the  laboratory  with  a  minimum  of  assistance 
from  external  chemical  or  physical  energy.  Examples  of 
such  changes  are  frequent  in  the  category  of  so-called  catalytic 
actions. 

The  little  cigar  lighter,  a  smoker's  toy  which  is  often  to  be 
seen  in  tobacconists'  shops,  is  a  good  illustration  of  the  chemical 
action  brought  about  by  catalysis.  In  this  case  the  warmth 
of  the  hand  causes  a  little  alcohol  vapour  to  evaporate  from 
the  metal  box  and  to  impinge  on  a  small  knob  of  spongy 
platinum  which  acts  as  the  catalyst.  Its  precise  mode  of 
action  is  not  fully  known,  but  it  greatly  accelerates  the  rate 
of  combination  of  the  alcohol  vapour  with  the  oxygen  of  the 
air,  with  the  result  that  the  alcohol  bursts  into  flame.  Platinum 
in  a  state  of  fine  division,  such  as  may  be  obtained,  for  example, 
by  soaking  asbestos  in  platinum  chloride  and  driving  off  the 
chlorine  by  heat,  is  thus  capable  of  bringing  about  a  number 
of  changes  at  temperatures  much  below  those  at  which  they 
would  normally  take  place.  If  a  thread  of  asbestos,  covered 
with  platinum  in  the  manner  above  described,  is  warmed 
and  then  held  in  a  stream  of  coal-gas  escaping,  for  example, 
from  an  unlit  Bunsen  burner,  the  platinised  asbestos  will 
glow. 

A  technical  process  of  importance,  viz.,  the  manufacture  of 
highly  concentrated  sulphuric  acid,  consists  in  passing  sulphur 
dioxide  (S02),  obtained  by  burning  pyrites  or  sulphur,  together 
with  oxygen,  or  air,  over  heated  platinum  in  a  fine  state  of 
division.  The  two  gases  then  combine  in  accordance  with  the 
simple  equation : — 

S02  +  0  =  S03 

This  combination  takes  place  only  to  an  infinitesimal  extent 
without  the  presence  of  a  substance  like  the  platinum,  which 
acts  as  a  catalyst. 

Bi 


4         BACTERIOLOGICAL   AND   ENZYME  CHEMISTRY 

In  the  case  of  spongy  platinum  and  other  finely  divided 
metals  the  chemical  change  is  accelerated  in  a  large  degree  by 
physical  causes  ;  a  finely  divided  metal  presents  an  extended 
surface  on  which  the  reacting  substances  are  brought  into 
intimate  union.  Chemical  agencies  may  be  at  work  at  the 
same  time,  e.g.,  the  formation  of  unstable  intermediate 
compounds  such  as  oxides  or  hydrides ;  but  the  physical 
conditions  are  probably  the  governing  factor. 

It  is  otherwise  with  certain  other  catalytic  changes,  notably, 
e.g.,  the  combination  of  sulphur  dioxide  with  oxygen  through 
the  intervention  of  nitric  oxide,  which  is  the  basis  of  the  cham- 
ber process  for  the  manufacture  of  sulphuric  acid.  Sulphur 
dioxide  does  not  combine  directly  with  oxygen,  but  when 
oxygen  is  presented  to  it  in  combination  as  nitrogen  peroxide, 
it  is  easily  oxidised  with  simultaneous  formation  of  nitric 
oxide.  Nitric  oxide,  on  the  other  hand,  readily  combines 
with  the  oxygen  of  the  air,  again  producing  nitrogen  peroxide. 
The  changes  are  expressed  in  the  following  equations  : — 

NO  +  0  -  N02 

S02  +  N02  =  S03  +  NO 

It  will  thus  be  seen  that  in  presence  of  oxygen,  or  of  course 
of  air,  a  very  small  amount  of  nitric  oxide  (NO)  is  capable 
of  converting  an  indefinite  quantity  of  S02  into  S03,  itself 
remaining  unchanged  at  the  end  of  the  process. 

On  the  large  scale  this  change  takes  place  in  the  vast 
leaden  chambers  which  cannot  fail  to  be  noticed  in  centres  of 
chemical  industry,  such  as  Widnes.  The  various  gases  are 
introduced  into  these  chambers,  together  with  steam.  The 
steam,  H20,  and  S03  together  form  sulphuric  acid,  H2S04, 
which  collects  on  the  floor  of  the  chamber. 

The  catalytic  action  of  nitrous  .fumes  can  be  readily  shown 
in  the  laboratory  by  shaking  a  solution  of  ferrous  sulphate 
(green  copperas)  with  a  little  nitrite  of  soda  and  sulphuric 
acid  in  a  bottle  nine-tenths  full  of  air.  The  green  colour 


CATALYSIS  0 

of  the  copperas  solution  quickly  changes  to  yellow,  owing  to 
the  formation  of  ferric  sulphate,  according  to  the  following 
equation  : — 

2FeS04  +  H,S04  +  N02  =  Fe2(S04)3  +  NO  +  H20 

The  NO  combines  with  the  oxygen  in  the  air  present  to 
form  N02  and  so  continues  the  reaction.  This  process  has 
been  made  the  subject  of  a  patent,  and  is  used  to  prepare  ferric 
salts  on  the  large  scale  for  the  purpose  of  precipitating  sewage. 

Another  important  case  of  catalytic  action  is  the  action 
of  manganese  dioxide  on  the  decomposition  of  potassium 
chlorate  by  heat ;  the  temperature  at  which  oxygen  is  evolved 
from  potassium  chlorate  on  heating  is  very  much  reduced  by 
the  addition  of  a  comparatively  small  amount  of  manganese 
dioxide.  In  this  case  also  it  has  been  shown  by  McLeod,  the 
present  writer  and  others,  that  the  action  of  the  manganese 
dioxide  is  probably  due  to  the  formation  and  decomposition 
of  intermediate  substances. 

The  reactions  which  take  place  in  living  matter  come,  in 
many  cases,  under  the  order  of  catalytic  reactions.  The 
nature  of  the  catalyst  is  one  of  the  problems  for  consideration. 
These  catalysts  occurring  in  living  matter  are  known  as 
enzymes  or  ferments,  and  their  varying  effects  form  the  chief 
subject-matter  of  this  book. 

Many  of  the  reactions  which  take  place  in  nature  can  be 
imitated  in  the  laboratory  by  fairly  simple  methods ;  thus, 
e.g.,  cane  sugar  is  easily  converted  into  grape  sugar  by  warming 
for  some  time  with  dilute  acid,  according  to  the  following 
equation  : — 

C12H22On  +  H,0  =  2C6H1206 

Ethereal  salts  or  esters  of  the  simpler  fatty  acids,  such  as, 
e.g.,  ethyl  acetate,  can  be  broken  up  by  warming  with  dilute 
acid  or  alkali,  yielding  alcohol  and  acetic  acid.  Such  a 
reaction  does  not  proceed  to  completeness  under  ordinary 


6        BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 

conditions,  but  ceases  when  a  certain  definite  proportion  of 
the  ester  has  been  broken  up.  Such  a  reaction  is  known  as  a 
reversible  reaction,  and  is  generally  written  thus  : — 

CH3COOC2H5  +  H20  •+  C2H5OH  +  CH3COOH 

The  changes  above  described  are  typical  of  a  series  of 
reactions  characterised  by  the  absorption  of  the  elements  of 
water  ;  such  a  process  is  generally  referred  to  as  hydrolysis. 

A  great  many  fermentative  changes  are  hydrolytic  in 
their  character  and  consequently  of  a  very  simple  order. 
It  was  at  one  time  considered  that  under  natural  conditions 
only  changes  took  place  which  were  essentially  of  this  order, 
and  in  which  there  was  always  a  liberation  of  heat  as  a  result. 
Recent  research  has,  however,  shown  that  this  generalisation 
does  not  hold,  it  being  possible  to  build  up  substances  by  the 
action  of  enzymes,  as  well  as  to  break  them  down,  It  is 
probably  more  correct  to  say  that  enzyme  actions  are,  strictly 
speaking,  reversible,  but  that  the  reaction  takes  place  in  both 
directions  only  under  special  conditions. 

Besides  the  multiplicity  of  chemical  agents  already 
mentioned,  the  chemist  has  at  his  disposal  means  for  varying 
at  will  within  wide  limits  the  physical  conditions  of  reaction. 

Temperature  and  concentration  have  already  been 
mentioned,  but  it  is  also  possible  to  remove  one  or  more  of 
the  reacting  bodies  from  the  sphere  of  action  by  distillation, 
either  at  the  ordinary  or  at  reduced  pressure.  Filtration 
through  various  kinds  of  filtering  media  is  possible,  or  separa- 
tion by  varying  solubilities. 

Under  natural  conditions  the  choice  of  methods  is  obviously 
much  more  restricted,  and  therefore  before  going  farther  it 
will  be  well  to  consider  more  closely  the  conditions  under 
which  chemical  actions  actually  do  take  place  in  nature,  and 
for  this  purpose  to  devote  some  attention  to  what  may  be 
termed  nature's  ultimate  laboratory,  that  is,  a  living  cell. 

The  unit  of  all  living  matter  is  the  cell.    Broadly  speaking, 


THE    CHEMISTRY    OF   THE    CELL  7 

the  cell  consists  of  an  envelope  which  can  be  described  as 
semi-permeable,  that  is,  permeable  to  one  class  of  bodies  but 
not  to  another.    The  contents  of  the  envelope 
consist  of  liquid  plasma  or  sap,  throughout 
which,  and  lining  the  interior  of  the  envelope, 
is  a  semi-fluid  mucilaginous  substance  referred 
to  generally  as  protoplasm.     This  is  in  a  con- 
tinual state  of  movement  and  of  chemical 
change ;   and  in  the  midst  of  it  is  a   cell 
nucleus. 

The  substances  entering  or  leaving  the  cell 
must  obviously  be  possessed  of  certain  physi- 
cal  properties  if  they  are  to  pass  through 
the  semi-permeable  membrane.  It  is  necessary,  therefore,  to 
consider  the  different  conditions  which  the  matter  composing 
the  various  substances  entering  and  leaving  the  cell  labora- 
tory may  assume.  There  are  first  solid  insoluble  bodies ; 
these,  of  course,  are  not  likely  to  pass  through  the  cell  mem- 
brane ;  on  the  other  hand,  substances  in  true  solution,  such  as, 
e.g.,  salt  dissolved  in  water,  will  as  a  rule  pass  freely  through. 
There  are,  however,  intermediate  conditions  in  which  matter 
can  exist. 

Colloids. — It  was  first  shown  by  Graham  that  by  appro- 
priate means  solutions  could  be  obtained,  which,  while  devoid 
of  visible  particles,  were  incapable  of  passing  unchanged 
through  a  parchment  membrane.  Substances  which  were 
soluble  and  which  would  pass  while  in  solution  through  a 
parchment  membrane  Graham  termed  crystalloids  ;  substances 
which  while  soluble  as  judged  by  ordinary  physical  tests  would 
yet  not  pass  through  a  parchment  membrane  he  termed 
colloids.  A  typical  case  illustrating  the  difference  between  a 
colloid  and  a  crystalloid  is  the  one  selected  by  Graham,  viz., 
silicate  of  soda.  If  a  dilute  solution  of  silicate  of  soda  is 
carefully  acidified  with  hydrochloric  acid,  no  precipitation 


8         BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

takes  place ;  if  the  solution  is  now  placed  in  a  cylindrical 
vessel  one  end  of  which  is  closed  by  a  parchment  diaphragm 
and  the  whole  immersed  in  clean  water,  which  is  renewed 
from  time  to  time,  the  sodium  chloride  formed  by  the  action 
of  the  hydrochloric  acid  on  the  sodium  silicate  will  diffuse 
through  the  parchment  and  eventually  be  completely  re- 
moved. The  silicic  acid  will  remain  behind  in  the  cylinder. 
The  sodium  chloride  in  this  case  is  the  crystalloid,  the  silicic 
acid  the  colloid.  The  apparatus  used  in  the  experiment  is 
known  as  a  dialyser  and  the  process  as  dialysis. 

A  large  amount  of  work  has  been  done  of  recent  years  on 
the  chemistry  of  colloids.  It  has  been  shown  that  no  very 
marked  line  can  be  drawn  between  the  two  extremes  of 
matter  in  the  solid  insoluble  condition  and  matter  in  true  and 
crystalloid  solution.  The  following  are,  however,  typical 
properties  of  colloids  : — 

(1)  When  examined  by  an  instrument  known  as  the 
ultramicroseope,  colloidal  solutions  are  all  found  to 
contain  particulate  matter,  that  is,  matter  in  an  extremely 
divided  state  but  still  existing  as  separate  particles. 

The  ultramicroseope  is  an  instrument  whose  design  is 
founded  upon  what  is  known  as  the  '  Tyndall  phenomenon/ 
The  lighting  up  by  a  sunbeam  of  the  dust  in  the  atmosphere  of 
a  room  is  a  matter  of  common  observation.  Tyndall  found 
that  if  a  closed  space  was  rendered  e  optically  empty '  by 
smearing  its  sides  with  glycerine  and  allowing  all  particles  to 
subside  and  be  caught  by  the  glycerine,  a  beam  of  light  on 
passing  through  was  invisible  when  viewed  at  right  angles  to 
its  path.  On  admitting  a  little  smoke  the  path  of  the  beam  at 
once  became  visible.  The  same  phenomenon  is  observed  with 
solutions.  A  solution  perfectly  free  from  suspended  particles 
allows  a  beam  of  light  to  pass  through  and  remain  invisible. 
On  introducing  a  colloid  substance  such  as  gum-mastic  into  the 
solution  the  path  of  the  beam  at  once  lights  up. 

The   application   of   this   phenomenon   to   the   study   of 


COLLOIDS 


9 


colloidal  matter  has  been  the  subject  of  very  interesting 
researches  by  Zsigmondy  and  Siedentopf.  Fig.  2  clearly 
illustrates  the  principle  of  the  ultramicroscope.  The  solution 
to  be  examined  is  placed  in  the  glass  cell  at  b  and  is  strongly 
illuminated  by  a  converging  beam  of  light.  On  observing 
the  lighted-up  solution  by  the  microscope  at  right  angles  to  the 
path  of  the  beam  the  colloid  substance  present  in  the  solution 
is  visible  as  brightly  illuminated  particles.  The  methods  of 


FIG.  2. — THE  PRINCIPLE  OF  THE  ULTRAMICROSCOPE.1 


producing  a  brilliant  converging  beam  of  light,  and  the 
construction  of  the  observation  cell  have  in  practice  been 
improved  and  rendered  more  compact  and  precise,  but  Fig.  2 
sufficiently  illustrates  the  principle  employed. 

By  means  of  the  ultramicroscope  particles  are  rendered 
visible  which  are  far  smaller  than  any  that  can  be  seen  under 
the  ordinary  microscope.  Thus,  for  example,  if  an  ordinary 
blood-corpuscle  be  represented  by  a  circle  three  inches  in 

1  Reproduced   by  permission  from   Zsigmondy's  wcrk,  Zur   Erkennt- 

niss  der  Kolloide. 


10      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

diameter,  a  particle  of  colloidal  gold  to  the  same  magnification 
would  be  barely  visible  as  a  minute  dot,  but  yet  it  can  be 
distinguished  by  means  of  the  ultramicroscope. 

(2)  Colloids  can  readily  be  precipitated  from  solution, 
usually  by  acidification,  by  the  addition  of  solutions  of  various 
salts  or  by  the  introduction  of  other  colloids. 

Certain  colloids  when  once  so  precipitated  are  only  brought 
into  solution  again  with  difficulty,  others  readily  pass  into 
solution  if  the  precipitating  agent  is  removed,  e.g.,  by  dialysis, 
or  if  its  effect  is  diminished  by  dilution.  These  two  classes  of 
colloids  are  distinguished  as  irreversible  and  reversible  respec- 
tively. The  difference  in  behaviour  is  probably  mainly  due 
to  differences  in  the  sizes  of  the  precipitated  particles. 

A  characteristic  example  of  an  irreversible  colloid  is  the 
soluble  silica  already  referred  to.  On  addition  of  hydro- 
chloric acid  or  salt  solution  to  the  aqueous  solution  of  silica 
the  latter  gelatinises  and  cannot  readily  be  brought  into 
solution  again. 

A  colloid  when  in  solution  in  water,  as  in  the  case  of  the 
unprecipitated  silica,  is  frequently  referred  to  as  a  hydrosol ; 
when  precipitated  in  a  gelatinous  or  anhydrous  form  it  is 
known  as  a  hydrogel. 

Many  enzymes  are  typical  reversible  colloids.  They 
can  be  precipitated  from  their  aqueous  solutions  by  means  of 
alcohol,  but  redissolve  in  water  if  the  alcohol  is  removed  by 
filtration. 

True  colloids  conduct  electricity  very  slightly,  if  at  all ;  in 
fact,  under  the  influence  of  the  electric  current,  they  move  as 
a  whole  towards  one  pole  or  the  other. 

The  precipitation  of  one  colloid  by  another  has  been  shown 
to  be  connected  with  the  electrical  condition  of  the  respective 
substances.  An  electro-positive  colloid  will  precipitate  an 
electro-negative  colloid,  and  vice  versa. 

The  precipitation  of  organic  colloids  by  gelatinous  mineral 
hydroxides  which  is  made  use  of  in  the  clarification  of  sewage 


OSMOTIC    PRESSURE  11 

is  an  interesting  instance  of  the  mutual  precipitation  of 
colloids. 

The  interaction  of  '  toxins '  and  '  antitoxins  *  in  serum 
therapy  further  illustrates  the  same  property. 

It  is  probable  that  we  have  here  also  to  do  with  the  attrac- 
tive action  of  extended  surfaces,  such  as  are  presented  by 
gelatinous  precipitates,  whereby  not  only  colloids  but  also  to 
a  certain  extent  crystalloids  are  withdrawn  from  solution. 
This  attractive  effect  is  known  generally  as  absorption. 

Colloids  exercise  a  very  low  osmotic  pressure,  and  conse- 
quently are  assumed  to  have  a  very  high  molecular  weight. 

The  latter  characteristic  is  of  considerable  importance  in 
considering  the  changes  taking  place  in  a  cell.  Modern  research 
has  shown  that  substances,  such  as  ordinary  salt,  which  allow 
the  passage  of  electricity  when  they  are  dissolved  in  water 
exist,  at  any  rate  in  dilute  solution,  in  a  state  of  dissociation, 
and  the  dissociated  ions,  as  they  are  termed,  obey  in  dilute 
solution  the  laws  of  gaseous  particles.  They  will  tend  rapidly 
to  diffuse  throughout  the  solution. 

If,  therefore,  a  dilute  solution  of  salt  is  enclosed  in  a  vessel 
with  semi-permeable  walls,1  i.e.,  walls  which  are  permeable  to 
the  molecules  of  the  solvent  but  not  to  those  of  the  dissolved 
substance,  there  is  a  tendency  for  the  ions  to  extend,  they 
cannot  pass  through  the  sides  of  the  vessel ;  but  if  the  latter  be 
placed  in  clean  water  there  will  be  a  tendency  for  the  water  to 
enter  and  thus  a  pressure  will  be  created  in  the  ulterior  of  the 
vessel ;  this  is  known  as  the  osmotic  pressure.  This  will 
obviously  depend  on  the  concentration  of  the  salt  solution. 
In  dilute  solutions  it  is  proportional  to  the  number  of  the 
molecules  of  the  dissolved  salt  present  in  a  given  volume  of  the 
solution. 

In  order  that  chemical  activity  may  go  on  in  the  cell,  it  is 
evident  that  it  must  be  possible  for  crystalloidal  bodies  to 

1  A  porous  pot,  in  the  pores  of  which  copper  ferrocyanide  has  been 
precipitated,  forms  such  a  semi-permeable  septum. 


12      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

enter  and  leave  the  cell  through  the  cell  wall ;  the  rate  of 
interchange  of  substances  will  depend  on  the  difference  of 
osmotic  pressure  within  and  without  the  cell. 

In  fact,  a  very  delicate  method  of  determining  differences 
of  osmotic  pressure  consists  in  immersing  certain  plant  cells 
in  different  solutions  and  examining  the  cells  microscopically. 

If  the  osmotic  pressure  of  the  solution  is  greater  than  that 
of  the  cell  contents,  the  cell  protoplasm  will  contract  and  leave 
the  walls  of  the  cell,  a  phenomenon  known  as  plasmolysis. 

If  the  protoplasm  within  the  cells  just  begins  to  show 
signs  of  contracting,  it  may  be  taken  that  the  osmotic  pressure 
is  equal  on  the  two  sides  of  the  cell  wall.  Such  solutions  are 
said  to  be  isotonic,  i.e.,  the  number  of  molecules  present  in 
equal  volumes  of  the  solutions  within  and  without  the  cell,  or 
the  molecular  concentration  of  the  dissolved  substances,  is 
such  that  they  exercise  the  same  osmotic  pressure.  The 
changes  taking  place  in  the  cell  must  consist  in  the  breaking 
down  of  colloidal  substances,  notably  albumin,  into  crystal- 
loidal  substances  which  escape  from  the  cell,  and  the  building 
up  of  complex  matter  from  other  cry  stall  oidal  substances 
which  find  entry  to  the  cell.  Further,  it  is  obvious  that 
these  changes  must  be  analogous  to  those  chemical  changes 
which  require  the  least  complexity  of  chemical  conditions, 
i.e.,  they  must  be  of  the  nature  of  catalysis. 

It  is  important,  however,  to  note  that  while  the  chemical 
changes  are  such  as  can  be  produced  in  many  cases  in  the 
laboratory,  if  not  by  ordinary  chemical  reagents,  at  any  rate 
by  products  or  enzymes  extracted  from  the  living  cell,  they 
only  take  place  in  nature  when  the  cell  is  alive.  The  precise 
definition  of  what  is  meant  by  vital  action  cannot  here  be 
attempted;  it  may,  however,  be  stated  that  the  cell  can 
be  looked  upon  as  an  energy  transformer,  in  which  the  energy 
which  is  characteristic  of  living  matter,  and  which  may  be 
termed  biotic  energy,  is  transformed  into  chemical  activity 
and  eventually  into  heat  in  the  cell  processes. 


THE    CHEMISTRY    OF   THE    CELL  13 

The  simplest  kind  of  living  organism  is  a  bacterium  or 
what  is  popularly  known  as  a  microbe  ;  this  is  a  unicellular 
organism  and  as  a  rule  specially  fitted  to  bring  about  certain 
defined  chemical  changes.  In  more  complex  organisms 
separate  cells  are  found  to  have  separate  functions  ;  thus  the 
cells  of  the  lining  of  the  stomach  bring  about  changes  which 
take  place  best  in  an  acid  medium.  In  the  pancreas,  on  the 
other  hand,  chemical  change  takes  place  under  alkaline  con- 
ditions. From  many  species  of  cells  it  is  possible  to  isolate 
the  catalytic  substance  or  enzyme  which  helps  to  bring  about 
the  change. 

While  a  large  number  of  fermentations  are  known  which 
can  be  produced  by  the  action  of  enzymes,  there  are  others 
which  so  far  have  only  been  produced  by  the  action  of  living 
organisms,  such  as  for  example  the  butyric  acid  fermentation 
and  the  nitrification  of  ammonia.  The  alcoholic  fermenta- 
tion of  sugar  was  at  one  time  thought  to  belong  to  this 
class  of  fermentation.  But  the  experiments  of  Buchner 
showed  that  it  was  possible  to  extract  a  substance  from 
yeast  cells  which  brought  about  the  formation  of  alcohol  and 
carbonic  acid  when  added  to  grape  sugar ;  this  substance 
he  termed  zymase,  and  recent  researches  by  Harden  and 
others  have  elucidated  in  a  very  interesting  way  the  con- 
ditions of  its  activity. 

It  is  probable  that  other  cases  where  the  active  enzyme 
has  not  yet  been  discovered  will  be  found  on  further  investiga- 
tion to  resolve  themselves  in  a  similar  manner. 

At  the  same  time  it  should  be  pointed  out  that  the  activity 
of  the  cell  is  of  a  complex  nature,  and  it  is  probable  that  the 
living  organism  is  concerned  in  two  distinct  modes  of  activity, 
i.e.,  in  maintaining  its  body  substance  and  in  developing 
energy  for  growth  and  reproduction.  Thus,  broadly  speaking, 
in  the  animal  body  the  processes  of  digestion  are  concerned 
with  the  maintenance  of  the  body  substance,  the  processes 
of  respiration  with  the  maintenance  of  energy.  In.  both 


14      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

cases  the  chemical  action  is  probably  resolvable  ultimately 
into  similar  factors,  though  the  nature  of  the  products  and 
the  energy  or  heat  changes  are  different  in  the  two  cases. 

A  simple  case  which  illustrates  the  difference  between 
what  may  be  termed  digestive  and  respiratory  fermentation 
is  afforded  by  the  decomposition  of  urea  in  the  presence  of 
micro-organisms.  The  simple  fermentative  change  consists 
in  the  transformation  of  the  urea  into  ammonium  carbonate 
by  the  addition  of  a  molecule  of  water,  as  in  the  following 
equation  : — 

CO(NH2)2  +  2H20  =  (NH4)2C03 

At  the  same  time  a  portion  of  the  nitrogen  is  found  to  be 
taken  up  by  the  organism  with  simultaneous  production  of 
C02.  The  second  is  a  much  more  complex  change  than  the 
first  and  its  conditions  are  not  so  fully  understood,  but  it 
is  probable  that  here  also  we  have  to  do  with  a  chemical  change 
in  which  intermediate  loosely  compounded  complexes  are 
formed,  as  in  the  simpler  purely  chemical  reactions  mentioned 
in  the  earlier  part  of  the  chapter. 

Finally,  it  has  been  found  possible,  as  already  stated,  not 
only  to  break  down  substances  in  the  manner  indicated 
through  the  agency  of  enzymes,  but  also  to  effect  syntheses 
of  more  complex  from  less  complex  compounds.  Thus,  e.g., 
Croft  Hill  has  been  able  to  produce  isomaltose  by  the  action 
of  the  enzyme  maltase  upon  dextrose,  as  follows  : — 

2C6H1206— H20  =  C12H22On 

This  discovery  is  of  very  far  reaching  importance  and 
opens  up  a  wide  field  of  possibilities.  Already  Emil  Fischer 
and  his  co-workers  have  announced  the  synthesis  of  certain 
decomposition  products  of  albumin  by  means  of  enzyme  action. 

In  the  following  pages  the  attempt  will  be  made,  by  means 
of  typical  examples,  to  render  clear  the  methods  of  investiga- 
tion which  are  used  in  the  study  of  the  chemistry  of  changes 


THE    CHEMISTRY    OF   THE    CELL  15 

brought  about  by  enzymes  or  bacteria.  Though  it  will  be 
necessary  to  refer  to  certain  organisms,  the  subject  will  be 
approached  primarily  from  a  chemical  standpoint,  fermenta- 
tion being  defined  as  the  chemical  change  produced  by  the 
agency  of  protoplasm  or  of  a  secretion  prepared  from  it. 

NOTE  TO  PAGE  11. — The  word  «  absorption  '  is  used  advisedly  in 
the  sense  employed  by  Dunbar  (Principles  of  Sewage  Treatment,  p.  142) 
to  signify  the  power  possessed  by  gelatinous  films  of  withdrawing  certain 
substances  from  solution,  and  which  is  more  of  the  nature  of  suction 
than  of  mere  surface  attraction,  or  '  absorption.' 


CHAPTER  n 

OUTLINES  OF  BACTERIOLOGICAL  TECHNIQUE 

BACTERIOLOGICAL  and  enzyme  chemistry  is  essentially  the 
chemistry  of  the  single  cell :  biological  chemistry  and  physio- 
logical chemistry  in  the  wider  sense  deal  with  the  changes 
taking  place  in  higher  organisms,  which  consist  of  collections 
of  ceDs  of  varying  and  interdependent  functions.  We  have 
tfcffffmr  only  to  consider  the  chemical  changes  brought 
about  by  the  simplest  organisms,  which  if  not  actually 
unicellular  are  only  very  slightly  differentiated ;  or  with 
tie  chemistry  of  specific  cells  of  higher  organisms.  More- 
over, from  the  chemical  point  of  view,  the  form  of  the 
organism,  and  its  method  of  growth  and  development,  are 
of  leas  importance  than  the  chemical  changes  it  brings 
about. 

The  following  pages  deal  with  the  methods  of  recognition 
and  cultivation  of  the  simplest  organisms,  the  subject  being 
treated  in  quite  a  general  manner.  For  the  detailed  methods 
used  in  the  irrognitinn  of  specific  organisms,  text  books  on 
bacteriology  should  be  consulted. 

The  micro-organisms  whose  chemical  activities  have  to  be 
studied  may  be  divided  into  three  groups,  viz. : — 

L  Bacteria; 
IL  Yeasts  ; 
IIL  Moulds. 

Bacteria  (Fig.  3, 1.  and  IA).— These  are  the  lowest  forms  of 


BACTERIOLOGICAL  TECHNIQUE 


17 


vegetable  life.  Under  a  high-power  microscope  they  appear 
as  minute  round  dots,  rods  or  threads  ;  they  multiply  either  by 
splitting  into  two  (that  is,  by  fission  or  cell  division),  or  by  the 


o 

o        o 

0  o°  o 


I.  BACTERIA. 


IL  YEASTS. 


III.  MOULDS. 


IA.  BEGGIATOA. 


FIG.  3. — BACTERIA,  YEASTS,  MOULDS,  AND  BEGGIATOA. 


production  of  small  protuberances,  which  separate  eventually 
from  the  main  organism  and  develop  into  fresh  organisms 
similar  to  the  parent  organism  ;  this  method  of  reproduction 
is  known  as  spore  formation.  Bacteria  are  colourless,  that  k», 


18     BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

they  contain  no  chlorophyll  ;  they  all  possess  an  envelope  or 
capsule  consisting  probably  of  cellulose  or  allied  substances. 
For  these  various  reasons  they  are  classed  among  the  fission 
fungi,  and  from  their  method  of  reproduction  are  known  as 
schizomycetes. 

They  are  further  divided  according  to  their  main  differences 
in  form  into  the  following  subdivisions  :  — 

1.  Coccaceae,  round  cells  ; 

2.  Bacteriaceae,  rods  and  threads  ; 

3.  Leptotricheae  ") 
4. 


Yeasts  (Fig.  3,  II.).—  These  are  closely  allied  to  the 
bacteria,  differing  mainly  in  their  method  of  reproduction. 
This  consists  in  the  formation  of  small  daughter  cells  or  buds 
which  are  extruded  from  the  parent  cell,  a  process  known  as 
budding.  Their  chemical  functions  are  also  more  complex, 
a  single  yeast  cell  being  able  to  bring  about  a  number 
of  different  chemical  changes.  As  they  are  mainly  capable 
of  growing  in  a  saccharine  medium,  they  are  known  generally 
as  saccharomycetes.  The  characteristic  form  of  yeast  cells 
with  buds  is  shown  in  Fig.  3,  II. 

Moulds  (Fig.  3,  III.).  —  These  are  still  more  highly  organ- 
ised than  the  bacteria  or  yeasts  ;  they  are  sporing  organisms. 
The  spores  or  conidia  give  rise  to  long  threads  of  cylindrical 
cells  forming  a  network  known  as  mycelium.  The  individual 
threads  are  known  as  hyphae.  From  these  organs  the  moulds 
derive  their  general  name  of  hyphomycetes. 

A  mould  which  at  first  has  a  fine  thread-like  appear- 
ance, on  further  growth  will  be  seen  to  be  covered  with  minute 
dots,  which  are  often  darker  than  the  mycelium  ;  these  on 
microscopical  examination  will  be  found  to  be  clusters  of  spores 
or  conidia  ;  in  the  case,  e.g.,  of  aspergillus  niger  they  are 


BACTERIOLOGICAL   TECHNIQUE  19 

black.     According  to  the  form  of  the  hyphae  the  moulds  are 
divided  into  four  divisions,  viz. : — 

1.  Mucorineae  ; 

2.  Aspergillinae ; 

3.  Penicilliaceae ; 

4.  Oidaceae. 

All  three  classes  of  organisms,  bacteria,  yeasts,  and  moulds, 
occur  very  widely  distributed  in  nature.  They  are  always 
most  abundant  where  there  is  the  needful  food  supply.  It  is 
a  matter  of  common  knowledge  that  meat  goes  bad  if  long 
exposed  to  the  air,  that  jam  if  uncovered  develops  mould, 
that  milk  becomes  sour,  that  sewage  or  excretal  matter  becomes 
offensive  if  allowed  to  accumulate.  These  changes  are  due  to 
micro-organisms  either  originally  present  in  the  decomposing 
substance,  or  carried  in  air  and  deposited  on  substances  capable 
of  putrefactive  change,  which  themselves  thus  become  sources 
of  infection.  The  presence  of  bacteria  in  the  air  can  be  demon- 
strated by  exposing  a  slice  of  potato  for  some  time  in  a  room. 
In  the  course  of  forty-eight  hours  or  so  small  spots  or 
centres  of  growth  will  appear,  which  can  be  recognised  as 
colonies  of  bacteria  or  as  moulds  by  methods  shortly  to  be 
described. 

Certain  organisms  are  capable  of  producing  chemical 
changes  in  the  bodies  of  higher  living  organisms,  and  have  been 
found  to  accompany  the  development  of  specific  diseases ;  such 
organisms  are  termed  pathogenic. 

Other  organisms  perform  exceedingly  useful  functions. 
It  is  scarcely  necessary  to  refer  to  the  technical  importance  of 
yeasts  in  the  brewing  industries.  Special  varieties  of  bacteria 
are  concerned  in  the  production  of  vinegar  and  the  ripening 
of  cheese,  or  are  useful  at  certain  stages  in  the  manufacture  of 
leather  in  the  tannery.  The  harmless  disposal  of  refuse  matter 
from  men  and  animals  is  effected  largely  by  the  activity  of 
bacteria,  and  the  processes  of  agriculture  are  increasingly 

found  to  depend  upon  the  activity  of  the  organisms  in  the  soil ; 

c2 


20      BACTERIOLOGICAL   AND    ENZYME   CHEMISTRY 

they  are  therefore  well  described  by  Percy  Frankland  as  '  our 
secret  friends  and  foes.' 

For  the  purpose  of  studying  the  precise  chemical  changes 
effected  by  a  single  organism  it  is  necessary  to  obtain  it  in 
pure  culture,  that  is,  free  from  admixture  of  any  other  organism. 
The  earliest  method  for  accomplishing  this,  such  as  was 
used  by  Pasteur  and  Lister,  was  the  method  of  dilution.  A 
small  portion  of  the  solution  containing  the  mixture  of 
organisms  was  transferred  to  a  second  portion  of  the  same 
solution  rendered  sterile  by  heat,  and  after  development  of 
the  organisms,  a  small  portion  of  this  solution  was  again  trans- 
planted, and  so  on,  until  a  growth  was  obtained  consisting 
of  only  one  species  of  organism,  arrived  at  through  a  process 
of  natural  selection.  Such  a  method  is  exceedingly  tedious, 
but  it  is  surprising  what  great  advances  in  knowledge  were 
made  by  its  means.  The  method  of  plate  culture  described 
by  Koch  in  1881  is  much  more  rapid  and  certain.  Koch 
introduced  a  solution  containing  bacteria  into  a  mixture 
of  suitable  nutritive  substances  thickened  with  gelatine,  the 
mixture  being  kept  at  a  temperature  slightly  above  the  melt- 
ing point  of  gelatine  ;  on  pouring  the  gelatine  culture  medium 
on  to  a  plate  and  allowing  the  gelatine  to  set,  wherever  a 
micro-organism  was  present  it  developed  in  situ,  forming  a 
small  cluster  or  colony,  which  could  be  picked  out  and  trans- 
ferred to  a  similar  gelatine  culture  medium,  and  if  necessary 
re-plated  until  only  one  species  of  organism  was  found  to 
be  present  upon  the  gelatine  plate.  The  form  of  plate  now 
generally  used  is  known  from  its  inventor  as  a  Petri  dish, 
and  consists,  as  shown  in  Fig.  5  a,  of  two  shallow  glass  dishes, 
fitting  into  one  another,  the  larger  serving  as  a  cover  for  the 
smaller,  into  which  the  gelatine  is  poured. 

Different  culture  media  have  been  found  to  be  necessary 
for  different  organisms,  but  all  require  nitrogen  in  some  form 
together  with  certain  mineral  salts,  especially  phosphates. 

It  is  of  the  greatest  importance  in  the  preparation  of 


BACTERIOLOGICAL   TECHNIQUE 


21 


culture  media,  and  in  all  operations  concerned  with  the  in- 
vestigations of  micro-organisms,  to  be  able  to  insure  sterility, 


FIG.  4. — (a)  STERILISER  (IN  SECTION);    (b)  WATER-BATH. 

that  is,  to  insure  that  no  organisms  are  present  in,  or  gain 
access  to,  the  medium  except  those  which  it  is  intended  to 
study.  A  culture  medium  can  generally  be  effectively 


22       BACTEKIOLOGICAL  AND  ENZYME  CHEMISTRY 

sterilised  by  exposure  to  moist  steam  for  about  twenty  minutes, 
especially  if  trie  operation  is  repeated  in  forty-eight  hours. 
In  this  way  any  spores  which  are  specially  resistant  to  sterilisa- 
tion, and  which  may  have  escaped  the  first  heating,  will  have 
had  time  to  develop,  and  the  adult  organism  will  be  killed 
by  the  second  heating.  For  sterilising  media  or  apparatus 
in  this  manner,  a  very  simple  form  of  steriliser  will  suffice, 
which  is  illustrated  in  Fig.  4  a.  It  consists  of  a  large 
semicircular  tin  can,  with  ordinary  cover,  and  provided  with  a 
perforated  false  bottom  of  tin  plate  about  an  inch  from  the 
bottom  of  the  can.  About  half-an-inch  in  depth  of  water 
is  placed  in  the  bottom  of  the  can,  which  can  be  quickly 
boiled  by  the  flame  of  a  Bunsen  burner  beneath,  the  whole 
can  being  thus  filled  with  moist  steam. 

Culture  Media  for  Bacteria 

Broth  or  bouillon. — The  basis  of  most  media,  suitable 
for  cultivating  bacteria,  is  broth  or  '  bouillon/  This  is  made 
by  boiling  up  one  pound  of  finely  minced  lean  beef  free  from 
fat  or  gristle  with  one  litre  of  water  in  a  large  flask  and  strain- 
ing through  muslin ;  five  grammes  of  salt  (sodium  chloride)  and 
ten  grammes  of  peptone  are  added,  and  the  mixture  boiled  for 
five  minutes.  The  liquid  is  rendered  very  faintly  alkaline 
with  carbonate  of  soda,  made  up  to  a  litre  if  necessary  with 
fresh  water,  the  neck  of  the  flask  plugged  with  cotton  wool, 
and  the  whole  sterilised. 

Nutrient  gelatine  is  made  by  dissolving  100  grammes  (or 
150  grammes  if  a  rather  high  melting-point  is  required)  of 
gelatine  in  1000  c.c.  of  broth.  The  gelatine  should  be  first 
soaked  in  water  to  render  it  easily  soluble  and  the  whole 
volume  of  gelatine  broth  made  up  to  1100  c.c.  If  necessary, 
the  solution  after  addition  of  the  gelatine  can  be  clarified  by 
warming  on  the  water  bath  with  the  white  of  one  egg.  The 
whole  is  then  filtered  through  a  pleated  filter  paper  in  a  hot 


BACTERIOLOGICAL   TECHNIQUE  23 

funnel  into  a  sterile  flask,  the  neck  of  which  is  packed 
with  cotton  wool.  Such  a  medium  is  known  as  G.P.B., 
gelatine  peptone  bouillon,  10  to  15  per  cent.,  according  to 
the  gelatine  added.  A  medium  of  this  composition  will  fur- 
nish nitrogen  and  carbon  from  the  albumen  and  peptone; 
the  necessary  salts  are  also  present  in  the  meat  extract. 

Instead  of  using  actual  minced  beef  '  bouillon/  it  is  often 
more  convenient  to  make  up  a  medium  directly  with  Liebig's 
Extract  of  Meat.  The  following  formula  has  been  found 
satisfactory  for  occasional  investigation  in  a  Sewage  Works 
Laboratory.  Ingredients : — 

Liebig's  Meat  Extract     . .         . .         9  grammes 

Witte's  Peptone 9 

Sodium  chloride   . .         . .         . .         4*5     „ 

Distilled  water 900 

Gelatine 100 

The  meat  extract,  vpeptone,  salt  and  water  are  boiled  for 
a  quarter  of  an  hour,  and  the  gelatine  gradually  added  as 
it  dissolves.  The  whole  is  allowed  to  cool  (to  50°  C. 
approx.)  and  neutralised  with  about  30  c.c.  of  a  4  per  cent, 
solution  of  caustic  soda  (NaOH).  The  white  of  an  egg  is 
mixed  with  an  equal  volume  of  water  and  added  to  the 
neutralised  liquid.  The  mixture  is  placed  in  the  steam  bath 
for  one  hour  and  1*5  grammes  soda  crystals  added. 

After  a  further  forty  minutes  in  the  steam  bath  the 
liquid  is  filtered  through  a  hot  water  filter  as  described.1 

The  melted  medium  is  carefully  poured,  preferably  from 
a  separating  funnel,  into  a  series  of  sterile  test  tubes  (cf. 
Fig.  5  6) ;  about  10  c.c.  are  added  to  each  test  tube,  care 

1  For  very  exact  work,  e.g.  differentiation  of  species,  etc.,  very  careful 
neutralisation  of  the  media  is  necessary,  for  the  details  of  which  special 
text  books  should  be  consulted.  It  may  be  mentioned  that  the  alkalinity 
or  acidity  of  a  medium  is  often  expressed  in  the  number  of  c.c.  of  noimal 
acid  or  soda  required  for  neutralisation,  a  —  sign  being  used  to  denote 
alkalinity  and  a  +  sign  to  denote  scidity. 


24     BACTERIOLOGICAL  AND  ENZYME   CHEMISTRY 

being  taken  not  to  allow  any  medium  to  run  down  the  sides. 
The  test  tubes  are  plugged  with  cotton  wool,  stacked  in  wire 
cages  and  sterilised  in  the  steam  bath  for  twenty  minutes  on 


FIG.  5. — APPARATUS  FOB  BACTERIOLOGICAL  CULTURE. 

three  successive  days.  The  medium  should  remain  perfectly 
clear  after  sterilisation,  and  the  tubes  are  then  ready  for  use. 
Agar  medium. — For  cultures  to  be  grown  at  a  high  tem- 
perature, agar  agar,  a  Japanese  product  made  from  a  species 
of  marine  alga,  is  used  instead  of  gelatine  in  the  above  process. 
This  medium  can  be  heated  to  40°  C.  without  melting. 


BACTERIOLOGICAL   TECHNIQUE  25 

Starch  gelatine. — For  the  purpose  of  detection  of  the 
enzyme  amylase  among  the  products  of  bacteria  or  other 
growing  cells,  2  per  cent,  of  soluble  starch  is  thoroughly 
mixed  with  the  melted  gelatine  medium.  The  starch  in  this 
case  should  first  be  boiled  with  water  to  a  clear  paste  in  order 
to  obtain  a  homogeneous  mixture  with  the  nutrient  gelatine. 

Silica  jelly. — Certain  organisms  will  not  grow  on  ordinary 
nutrient  gelatine,  and  a  method  was  devised  by  Kiihne  and 
by  Percy  Frankland  in  which  gelatinous  silica  is  used  instead  of 
gelatine,  the  medium  being  entirely  free  from  organic  matter. 

The  method  of  preparation  is  as  follows  : — 

Two  solutions  of  the  following  composition  are  prepared  :— 

(a)  Ammonium  sulphate . .  . .       0'4    gramme 

Magnesium  sulphate  . .  . .       0'05         „ 

Calcium  chloride        . .  .  .       trace 

Distilled  water  .  .  . . x  50'0    c.c. 

(6)  Potassium  phosphate .  .  .  .       O'l     gramme 

Sodium  carbonate      . .  . .       0'75         „ 

Distilled  water  . .  . .     50'0    c.c. 

These  two  solutions  are  rendered  sterile,  and  then  mixed. 

A  sterile  solution  of  dialysed  silicic  acid  is  now  prepared 
as  follows  :  A  solution  of  potassium  or  sodium  silicate  (1.05- 
1.06  sp.  gr.)  is  poured  into  an  equal  volume  of  dilute  hydro- 
chloric acid  (1.1  sp.  gr.) ;  the  mixture  is  then  placed  in  a 
dialyser,  the  outside  of  which  is  kept  surrounded  with  running 
water  during  the  first  day,  and  subsequently  with  distilled 
water,  which  is  frequently  changed  until  it  yields  no  trace  of 
turbidity  with  silver  nitrate,  thus  showing  the  whole  of  the 
chlorides  to  have  been  extracted.  The  contents  of  the  dialyser, 
if  the  solution  of  alkaline  silicate  originally  employed  was  not 
too  strong,  will  be  quite  clear.  This  liquid  is  then  poured  into 
a  flask  and  concentrated  by  boiling  until  it  is  of  such  a  strength 
that  it  is  found  that,  on  cooling  a  little  of  the  solution  and 
mixing  it  with  one- third  of  its  volume  of  the  above  mixed 


26       BACTERIOLOGICAL  AND  ENZYME  CHEMISTRY 

alkaline  solution,  it  readily  gelatinises  on  standing.  When 
the  solution  of  silicic  acid  is  found  to  give  this  result,  it  is 
cooled,  and  one-third  to  one-half  of  its  volume  of  the  mixed 
alkaline  solutions  (a  and  6)  are  added,  the  solutions  well  mixed 
and  at  once  poured  into  Petri  dishes  or  flat-bottomed  flasks. 
The  medium  should  gelatinise  in  from  five  to  fifteen  minutes. 
The  material  containing  the  organisms  for  examination  is 
introduced  and  thoroughly  mixed,  before  gelatinisation  takes 
place  ;  or  a  streak  culture  may  be  made  on  the  surface  after  the 
medium  has  solidified. 

As  this  method  has  been  used  for  the  study  of  the  very 
important  organisms  of  nitrification,  its  method  of  preparation 
is  of  special  interest. 

It  will  be  understood  that  for  the  study  of  special  organisms 
various  additions  to  the  typical  gelatine  or  agar  media  can 
be  made.  Thus  it  is  characteristic  of  certain  bacteria,  especially, 
e.g.,  of  B.  coli,  the  typical  organism  of  sewage  pollution,  to 
produce  acid  from  glucose  and  other  sugars ;  when  therefore 
glucose  and  litmus  are  added  to  the  medium  the  reddening  of 
the  litmus  indicates  acid  formation. 

The  following  medium  has  been  suggested  by  Dr.  Mac- 
Conkey,  and  has  been  largely  used  for  the  detection  of  Bacillus 
coli  in  polluted  water  : — 

Sodium  taurocholate     . .         . .     0'5  gramme 
Glucose  . .         . .         . .     05        „ 

Peptone  2'0 

Water     ..  lOO'O  c.c. 

The  constituents  are  heated  together,  filtered  and  tinted 
with  litmus  solution.  The  medium  is  then  poured  into  test 
tubes  and  a  small  inverted  fermentation  tube  placed  in 
each,  to  serve  as  a  trap  for  any  gas  evolved.  The  tubes  are 
then  sterilised  in  the  usual  way. 

A  certain  number  of  bacteria  are  found  only  to  develop 
in  absence  of  air ;  such  organisms  are  classed  as  anaerobic  in 


BACTERIOLOGICAL  TECHNIQUE  27 

contradistinction  to  those  which  thrive  in  presence  of  oxygen 
or  air.  In  order  to  cultivate  such  bacteria  it  is  necessary  to 
remove  the  oxygen  from  above  the  medium ;  this  can  be 
done  most  simply  by  enclosing  the  culture  tube  in  a  larger 
tube  (Fig.  5  c)  or  receptacle  containing  alkaline  pyrogallate 
of  soda,  which  has  the  property  of  rapidly  absorbing  oxygen. 
An  even  simpler  method  is  to  fill  the  tuBe  nearly  to  the  top 
with  medium,  and  after  inoculation  to  fill  up  the  remaining 
space  with  vaseline. 

Culture  Media  for  Yeasts. — In  the  case  of  yeasts,  wort 
gelatine  is  a  more  suitable  medium  than  ordinary  nutrient 
gelatine ;  in  this  case,  instead  of  bouillon,  boiled  hot  wort, 
obtainable  from  a  brewery,  may  be  used  advantageously; 
the  wort  should  be  diluted  with  water  to  a  specific  gravity  of 
about  1050.  The  wort  must  be  filtered  until  it  is  quite  bright, 
and  should  remain  free  from  deposit  after  sterilisation.  To 
prepare  wort  gelatine  100  grammes  of  gelatine  are  added  to  a 
litre  of  the  wort  and  the  whole  clarified,  filtered  and  sterilised 
in  the  same  manner  as  ordinary  G.P.B. 

Culture  Medium  for  Moulds.— Moulds  will  grow  on  nearly 

all  the  media  so  far  considered.  A  solution  specially  suited 

for  their  development  is  known  as  Raulin's  solution.     It  is 
prepared  as  follows  : — 

Water            . .         . .  . .     1500      grammes 

Cane-sugar     . .         . .  . .         70           „ 

Tartaric  acid .-         . .  . .           4           „ 

Ammonium  phosphate  . .           0*60       „ 

Magnesium  carbonate  . .           0'40      „ 

Ammonium  sulphate  . .           0*25      „ 

Zinc  sulphate            . .  . .           0'07 

Ferrous  sulphate       . .  . .           0*07      „ 

Potassium  silicate     . .  . .           0'07      „ 

To  prepare  a  Pure  Culture  of  Bacteria. — In  transferring 


28       BACTERIOLOGICAL  AND  ENZYME  CHEMISTRY 

small  quantities  of  material  from  one  medium  to  another, 
that  is  for  purposes  of  inoculation,  short  lengths  of  platinum 
wire  mounted  in  glass  rods  as  in  Fig.  6  a  are  used;  for 
small  quantities  of  liquid  a  wire  with  a  small  loop  at  the  end 
is  employed.  With  a  little  care  loops  can  be  made  which  will 
take  up  almost  exactly  a  milligram,  that  isO'OOl  c.c.  of  liquid. 


FIG.  6. — (a)  METHOD   OP  INOCULATING  THE  CULTURE  MEDIUM  ; 
(6)  FIXING. 

For  transferring  colonies  of  bacteria  a  small  hook  is  made 
at  the  end  of  the  wire.  To  inoculate  a  test  tube  of  gelatine 
the  cotton-wool  plug  is  first  sterilised  by  singeing  in  the 
Bunsen  flame,  is  removed  by  a  pair  of  forceps  similarly  steri- 
lised, and  held  between  the  first  and  second  fingers  of  the 
left  hand,  while  the  test  tube  is  held  between  the  first  finger 
and  thumb  (Fig.  6  a).  The  platinum  wire,  after  having 


BACTERIOLOGICAL  TECHNIQUE 


29 


been  sterilised  by  passing  through  the  flame,  is  dipped  into  the 
solution  to  be  examined  and  then  inserted  into  the  gelatine 
to  about  half  the  depth  and  then  withdrawn,  the  plug  of 
cotton  wool  again  singed  and  then  replaced.  Such  a  culture 
is  known  as  a  stab  culture,  and  is  chiefly  useful  when  inocu- 
lating from  a  pure  cultivation.  If  the  culture  is  a  mixed 
one,  the  gelatine  is  melted  before  removing  the  cotton-wool 
plug,  by  allowing  the  tube  to  stand  for  a  few  minutes  in  a 
beaker  of  water  which  has  been  heated  to  a  temperature 
some  ten  or  twenty  degrees  above  the  melting-point  of  the 
gelatine.  After  inoculation  and  mixing  the  culture  with 
the  melted  gelatine,  the  latter  is  poured  into  a  sterile 
Petri  dish. 

The  gelatine  is  allowed  to  set  in  the  Petri  dish,  which  is 
then  placed  in  a  moist  chamber. 
The  latter  is  a  similar  glass 
vessel  of  a  much  larger  size,  in 
which  some  moist  blotting-paper 
or  a  small  Petri  dish  of  water 
has  been  placed. 

In  order  to  accelerate  the 
growth  of  organisms  on  the 
gelatine  in  the  Petri  dish  it 
may  be  necessary  to  place  the 
latter  in  an  incubator. 

The  incubator  consists  essen- 
tially of  a  water- jacketed  cham- 
ber heated  by  a  gas  flame,  the 

size  of  which,  and  consequently 

. !  i        -,  FIG.    7.  —  HEAKSON  INCUBATOR 

the  temperature  produced,    can       WITH    THERMO  .  REGTILATOB. 


(Messrs.    Flatters    &   Garnett, 
Ltd.). 


be  very  exactly  regulated  by  a 
thermostat.  A  very  satisfactory 
form  of  incubator  is  the  Hearson 

incubator  shown  in  Fig.  7,  though  less  expensive  arrange- 
ments are  obtainable.  A  set  of  instructions  for  adjusting  the 
temperature  of  the  Hearson  incubator  is  issued  with  the 


30     BACTERIOLOGICAL  AND  ENZYME  CHEMISTRY 

apparatus.  After  twenty-four  hours  the  Petri  dish  should  be 
examined,  and  signs  of  the  development  of  colonies  will  then 
be  probably  apparent,  though  it  is  generally  necessary  to 
allow  at  least  two  days  to  elapse  before  making  the  sub- 
culture. Specific  subcultures  are  best  made  when  the 
number  of  colonies  on  the  plate  does  not  exceed  100;  it  is 
generally,  therefore,  best  to  make  two  or  three  plates  by 
transferring  a  loop  full  of  the  inoculated  and  melted  gelatine 
from  the  first  culture  tube  to  a  second  and  similarly  to  a 
third,  plates  being  poured  in  each  case.  Well-defined  colonies 
having  been  obtained  on  the  plate  culture,  separate  colonies 
can  be  removed  by  means  of  the  platinum  hook  and  transferred 
to  a  tube  of  gelatine,  there  to  develop. 

For  the  proper  carrying  out  of  these  operations,  manipula- 
tive practice  is  necessary,  in  order  to  avoid  accidental  infection 
by  extraneous  organisms  from  the  air,  etc.,  and  also  to  acquire 
rapidity  and  dexterity  of  handling.  It  is  wise  to  consider 
always  that  everything  not  actually  sterilised  is  liable  to  be  a 
source  of  infection ;  thus  a  platinum  wire  after  being  laid  down 
on  the  bench  must  be  re-sterilised,  and  cotton-wool  plugs 
re-singed  after  being  held  between  the  fingers.  Such  manipula- 
tive details  soon  become  a  matter  of  habit. 

Examination  of  Bacteria  under  the  Microscope. — As 

already  mentioned,  a  high  power  is  necessar}T  for  a  satisfactory 
examination  of  bacteria.  Under  a  -p2-  inch  oil-immersion  lens 
it  is  possible  to  observe  them  either  in  the  living  condition, 
in  a  drop  culture  or  as  a  stained  preparation.  To  examine 
them  in  drop  culture  a  small  portion  of  growth  either  from  a 
plate  or  tube  culture  is  removed  by  means  of  the  platinum 
loop,  and  quickly  mixed  with  a  drop  of  water  on  the  under 
side  of  an  ordinary  microscopic  cover  glass,  which  is  then 
placed  on  a  specially  made  slide  with  a  depression  ground  into 
it  (Fig.  5  d).  On  placing  the  cover  slide  with  the  drop  on 
the  under  side  over  the  depression,  the  bacteria  can  be  observed. 


BACTERIOLOGICAL  TECHNIQUE  31 

This  method  of  examination  is  particularly  useful  to  determine 
whether  the  bacteria  are  capable  of  movement  or  not,  that  is, 
whether  they  belong  to  the  class  of  motile  bacteria.  Bacteria 
are  more  simply  observed  when  they  are  dried  and  stained 
with  suitable  dyes,  which  render  them  more  clearly  observable. 
There  are  a  number  of  methods  in  use  for  staining  bacteria, 
varying  according  to  the  medium  in  which  they  are  observed, 
especially,  e.g.,  in  tissues,  and  also  for  the  purpose  of  bringing 
out  such  features  as  the  flagellae  or  thread-like  processes, 
which  are  characteristic  of  certain  organisms,  e.g.,  the  typhoid 
bacillus.  Special  methods  also  are  necessary  for  staining 
spores.  It  will  be  sufficient  here  briefly  to  indicate  a  simple 
method  of  staining  a  pure  culture.  A  carefully  cleaned  cover 
glass  is  taken,  and  held  in  a  pair  of  specially  constructed 
forceps,  a  drop  of  clean  water  is  placed  on  the  slip  and  a  small 
portion  of  the  culture  mixed  with  the  water  and  spread  in  a 
thin  film  over  the  glass  by  means  of  a  sterile  platinum  wire  ; 
the  film  is  now  carefully  dried  by  passing  the  glass  several 
times  through  a  Bunsen  flame  with  the  film  uppermost 
(Fig.  6  6).  The  cover  glass  should  never  be  made  hotter  than 
can  be  easily  borne  by  the  finger  if  the  under  side  of  the  glass 
is  pressed  down  on  it.  When  the  film  is  dry  a  drop  of  stain  is 
placed  on  the  slide,  ordinary  magenta  (rose-aniline)  or  gentian 
violet  are  commonly  used.  The  stain  is  allowed  to  remain 
for  a  minute  or  two  in  contact  with  the  glass  and  then  washed 
off  in  a  gentle  stream  of  water  or  by  immersion  in  a  large 
volume  of  clean  water.  The  preparation  is  again  carefully 
dried,  and  a  drop  of  Canada  balsam  placed  on  the  film  side  of 
the  cover  glass,  which  is  then  carefully  placed  in  contact  \vith 
the  ordinary  mounting  slide.  With  a  iittle  care  only  such  a 
quantity  of  Canada  balsam  is  dropped  on  to  the  cover  glass 
as  will  just  suffice  to  reach  to  its  edge  when  it  is  pressed 
down  upon  the  mounting  slide.  Care  should  be  taken  to 
remove  all  air  bubbles  from  between  the  cover  glass  and  the 
slide. 


32     BACTEKIOLOGICAL  AND  ENZYME  CHEMISTRY 

Preparation  of  a  Pure  Culture  of  Yeast.— It  is  possible  in 
the  case  of  yeast  actually  to  separate  a  single  cell  from  the 
rest  of  the  culture  and  inoculate  suitable  media  from  this 
single  cell.  This  method,  which  is  of  great  technical  importance 
in  the  control  of  the  various  fermentations  due  to  yeast,  was 
introduced  by  Hansen.  The  following  description  is  based 
upon  that  given  in  Brown's  '  Laboratory  Studies/  p.  160. 


FIG.  8. — FREUDENREICH  FLASK,  SQUARED  COVER  GLASS,  AND  MOIST 
CHAMBER,  FOR  YEAST  CULTURE. 

The  following  are  the  requisites  for  the  method  : — 

A  sterilised  glass  plate  and  bell  jar,  or  other  cover. 
Sterilised  glass  rods. 
Sterilised  Botcher  chamber. 

Sterilised  cover  glass  divided  into  numbered  sqaures. 
Freudenreich  flasks   of   sterilised  wort  gelatine   and   of 
sterilised  water. 

The  Freudenreich  flask,  the  moist  chamber  and  the  squared 
cover  glass  are  shown  in  Fig.  8.  Mix  a  drop  of  fresh  yeast 
with  sterilised  water  in  a  Freudenreich  flask,  shake  well  and 
dilute  still  further  by  transferring  a  drop  of  the  mixture  to  a 


BACTERIOLOGICAL  TECHNIQUE  33 

second  flask  of  water.  Again  mix  by  shaking,  and  if  the  liquid 
then  appears  slightly  opalescent  the  right  dilution  has  probably 
been  obtained ;  transfer  a  drop  of  the  mixture  to  a  Freuden- 
reich  flask  containing  wort  gelatine  and  mix  thoroughly.  Then 
spread  a  drop  of  the  wort  gelatine  mixture  in  a  thin  layer  on 
the  cover  glass  by  means  of  a  glass  rod,  and  place  the  glass  on 
the  glass  plate  underneath  the  bell  jar  and  leave  until  the 
gelatine  is  set.  Prepare  a  Botcher  chamber  by  placing  a 
small  drop  of  water  at  the  bottom  of  the  well  and  smearing 
the  edge  of  the  ring  with  vaseline,  next  reverse  the  glass  with 
the  gelatine  film  and  adjust  it  to  the  ring  of  the  chamber ; 
the  preparation  should  then  be  transferred  to  the  microscope 
for  examination.  The  lowest-power  objective  with  which  the 
yeast  cells  can  be  distinctly  seen  should  be  employed.  For 
the  purpose  of  obtaining  colonies  those  cells  are  chosen  which 
are  several  millimetres  apart  from  other  cells,  and  their  position 
must  be  carefully  recorded,  a  diagram  being  made  to  indicate 
the  position  of  the  cells  chosen. 

After  marking  the  position  of  several  cells  keep  the  culture 
at  a  temperature  of  about  20°,  and  examine  it  from  day  to  day 
with  the  microscope,  as  the  cells  multiply,  in  order  to  be  sure 
that  no  cells  in  the  immediate  vicinity  of  the  colonies  have 
been  overlooked.  When  the  colonies  are  large  enough  a  pure 
culture  in  wort  may  be  obtained  from  each  colony  by  inocu- 
lation in  the  manner  described  for  gelatine  plate  culture. 

Permanent  preparations  sufficient  to  show  the  general  form 
of  the  yeast  cells  can  be  stained  and  mounted  in  a  similar 
manner  to  bacteria  ;  special  methods  are  necessary  to  render 
clearly  visible  the  inner  structure  of  the  cell  and  to  stain 
spores. 

Examination  of  Mould  Culture.— Suitable  culture  media 
can  be  inoculated  with  moulds  in  a  manner  similar  to  the 
methods  used  for  bacteria.  As  moulds  are  aerobic  organisms, 
the  method  of  inoculation  on  gelatine  may  be  used,  in  which 


34      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

case  a  slight  scratch  is  made  on  the  surface  of  the  gelatine 
slope  (Fig.  5  6,  p.  24)  by  means  of  a  platinum  hook  infected 
with  the  organisms,  i.e.,  what  is  called  a  *  streak  culture.' 
Growths  are  of  course  best  obtained  when  the  hyphae  are 
well  matured ;  mould  cultivations  can  be  examined  in  the 
hanging  drop  and  their  stages  of  growth  and  developments 
studied  therein. 

For  the  preparation  of  permanent  specimens  of  moulds 
some  modifications  are  necessary  in  the  usual  staining  process. 
Owing  to  the  presence  on  their  surface  of  a  very  thin  layer  of 
fat,  moulds  are  not  easily  moistened  with  water.  Before 
mounting,  therefore,  a  portion  of  the  mould  intended  for 
examination  is  immersed  in  alcohol,  to  which  a  little  ammonia 
has  been  added ;  the  mould  can  then  be  stained  with  methylene 
blue,  the  filaments  of  the  mycelium  and  hyphae  taking  up  the 
colour  while  the  spores  remain  unstained.  Special  care  must 
be  taken  not  to  overheat  the  specimens  by  too  rapid  drying. 

Instead  of  Canada  balsam  it  is  better  to  use  glycerine 
in  the  case  of  organisms  such  as  moulds  and  algae,  infusoria, 
etc.,  the  cover  glass  being  attached  to  the  slide  by  a  ring  of 
shellac  varnish. 


CHAPTEK  III 

SOME    LEADING   CONCEPTIONS   IN   ORGANIC    CHEMISTRY 

THE  number  of  chemical  substances  dealt  with  in  this  book  is 
not  large,  and  the  chemical  reactions  involved  are  not  really 
difficult  to  follow,  even  for  those  who  do  not  possess  an  ex- 
tensive acquaintance  with  organic  chemistry,  but  some 
understanding  of  the  principles  which  underly  the  formulae 
employed  for  expressing  the  composition  and  structure  of 
organic  compounds,  and  of  certain  general  reactions  which 
these  latter  undergo,  is  essential  if  the  following  chapters  are 
to  be  properly  understood. 

For  the  benefit,  therefore,  of  the  general  reader  and  of  those 
whose  studies  have  been  mainly  confined  to  other  branches 
of  knowledge,  some  space  may  be  usefully  devoted  to  the  con- 
sideration of  certain  fundamental  conceptions  in  the  science 
of  organic  chemistry,  and  to  the  description  of  certain  typical 
substances  and  their  characteristic  reactions. 

According  to  the  atomic  theory  of  the  structure  of  matter, 
all  material  substances  are  supposed  to  consist  ultimately 
of  atoms.  A  substance  which  can  by  some  method  be 
divided  into  two  or  more  kinds  of  matter  differing  from 
one  another  and  from  the  original  substance  is  evidently  a 
compound  of  more  elementary  substances.  But  a  substance 
which  has  never  yet  been  subdivided  into  other  kinds  of 
matter  having  properties  different  from  its  own  is  regarded 
as  an  element.  A  few  such  substances  are  known,  and  out 
of  them  all  others  are  found  to  be  built  up.  If,  then,  we 
imagine  a  particle  of  one  of  these  '  elements/  e.g.,  of  iron,  to 

D2 


36      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

be  continuously  subdivided  until  upon  further  subdivision 
it  ceases  to  exist  in  the  form  known  to  us  as  iron,  at  that 
point  we  may  be  said  to  have  reached  an  *  atom/  one  of  the 
ultimate  components  of  matter. 

Recent  physical  researches  suggest  that  the  atom  itself 
can  be  further  subdivided  into  still  smaller  particles  known 
as  electrons,  but  setting  aside  this  possibility,  for  the  purposes 
of  the  chemist  it  suffices  to  define  the  atom  as  the  smallest 
existing  particle  of  an  element. 

This  idea  of  the  atomic  structure  of  matter  is  a  very  old  one 
and  was  held  by  the  ancients,  and  entered  largely  into  the 
conceptions  of  Robert  Boyle  and  other  chemical  philosophers. 

It  is  to  the  genius  of  Dalton  that  we  owe  a  development 
of  the  atomic  theory,  which  converted  it  from  a  more  or  less 
barren  speculation  into  a  fundamental  and  fruitful  conception. 
Dalton  was  able  to  show  that  the  atom  of  any  given  element 
was  characterised  by  a  definite  and  unalterable  weight  which, 
while  too  small  to  be  expressed  by  absolute  numbers,  could  be 
referred  to  in  terms  of  the  weight  of  the  lightest  then  known 
element,  viz.,  hydrogen,  which  was  taken  as  unity  ;  thus  the 
atom  of  iron,  e.g.,  has  been  found  to  be  56  times  as  heavy  as 
the  atom  of  hydrogen. 

Dalton  used  symbols,  somewhat  akin  to  the  old  alchemical 
symbols,  viz.,  circles,  semicircles  and  the  like,  for  expressing 
the  ultimate  atoms  and  elements.  It  was  the  great  Swedish 
chemist,  Berzelius,  who  introduced  the  much  more  convenient 
method  of  referring  to  elements,  either  by  their  initial  letters, 
or  by  the  initial  letter  together  with  a  second  significant 
letter.  These  are  known  as  the  symbols  of  the  elements ;  thus 
the  symbol  H  signifies  one  part  by  weight  of  hydrogen,  the 
symbol  0  sixteen  parts  by  weight  of  oxygen. 

In  order  to  obtain  true  values  for  these  relative  weights 
of  the  elements,  which  should  really  express  the  weights  of 
their  atoms  as  compared  with  the  weight  of  an  atom 
of  hydrogen,  it  was  necessary  to  extend  the  conception  of 


THE   PRINCIPLES   OP   ORGANIC   CHEMISTRY         37 

Dalton  and  to  conceive  of  chemical  substances  as  being  made 
up  of  aggregations  of  atoms  which  are  known  as  molecules. 

Now  in  considering  the  various  states  of  matter  it  is 
evident  that  it  is  in  the  gaseous  state  that  the  molecules  or 
atoms  are  most  widely  separated  ;  thus,  e.g.,  we  know  that  a 
comparatively  small  volume  of  water  will  give  rise  on  boiling  to 
a  considerable  volume  of  steam.  And  it  is  from  the  study  of 
chemical  substances  in  the  gaseous  state  that  our  fundamental 
conceptions  of  the  properties  of  atoms  and  molecules  and  of 
their  relative  weights  have  been  chiefly  derived. 

Before  Dalton's  time  Boyle  discovered  that  various  gases, 
though  they  might  differ  in  composition,  obeyed  certain 
simple  laws.  Thus  Boyle  found  that  if  the  pressure  upon 
a  gas  was  doubled,  its  volume  at  the  same  temperature  was 
halved,  and  the  statement  that  the  volume  of  a  gas  varies 
inversely  with  the  pressure  is  known  as  Boyle's  law.  The 
same  generalisation  was  made  by  the  Frenchman  Mariotte. 
It  was  further  found  by  Gay  Lussac  that  all  gases  expanded 
equally  for  equal  increments  of  temperature. 

Although  later  researches  have  shown  that  the  laws  of 
Boyle  and  Mariotte  and  of  Gay  Lussac  only  hold  strictly 
within  certain  limits  of  temperature  and  pressure,  yet  they 
afford  clear  evidence  that  gases  possess  essentially  the  same 
general  physical  properties  whatever  be  their  composition. 

When  it  was  further  discovered  by  Gay  Lussac  that  a 
given  volume  of  oxygen,  say,  when  compared  with  a  given 
volume  of  hydrogen  under  the  same  conditions  of  tempera- 
ture and  pressure,  was  always  sixteen  times  the  weight  of 
the  hydrogen,  the  conclusion  was  inevitable  that  a  definite 
relation  existed  between  the  volume  of  the  gas  and  the 
number  of  atoms  in  it. 

A  satisfactory  explanation  of  the  properties  of  gases,  and 
of  the  relations  which  exist  between  the  weights  of  equal 
volumes  of  gases  differing  in  composition,  was  afforded  by  the 
Italian  chemist,  Avogadro,  who  enunciated  the  law  that  equal 


38      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

volumes  of  all  gases  under  the  same  conditions  of  temperature 
and  pressure  contain  the  same  number  of  molecules.  Avogadro's 
conception  of  molecules  served  to  explain  certain  discrepancies 
met  with  when  comparing  the  weights  of  equal  volumes  of 
different  gases :  thus,  e.g.,  if  the  weights  of  equal  volumes 
of  hydrogen  and  oxygen  and  of  steam  be  compared — always,  of 
course,  under  the  same  conditions  of  temperature  and  pressure 
— it  will  be  found  that  the  ratio  of  the  weights  is  as  follows, 
viz.,  H  =  1,  0  =  16,  and  steam  =  9. 

It  was  further  found  that  two  volumes  of  hydrogen  com- 
bined with  one  volume  of  oxygen  to  form  two  volumes  of 
steam.  Now  it  is  evident  that  each  of  the  two  volumes 
of  steam  contains  an  equal  proportion  of  oxygen,  inasmuch 
as  their  weights  and  physical  properties  are  identical.  By 
introducing  the  conception  of  molecules,  Avogadro  enabled 
a  clear  conception  to  be  formed  of  the  action  taking  place. 
He  assumed  that  the  molecule  of  oxygen  contained  at  least 
two  atoms,  one  of  which  combined  with  hydrogen  to  form 
a  molecule  of  steam.  We  may  represent  the  union  of  two 
volumes  of  hydrogen  with  one  volume  of  oxygen  to  form  two 
volumes  of  steam  in  the  following  manner : — 


18 


Taking  hydrogen  as  unit,  the  weights  of  the  molecules 
will  be  represented  by  the  figures  below  the  squares,  and  we 
thus  see  how  it  is  that  if  a  volume  of  hydrogen  is  taken  as 
weighing  1,  the  same  volume  of  oxygen  will  weigh  16,  and 
the  same  volume  of  steam  9.  Assuming  the  molecule  of 
hydrogen  to  contain  two  atoms,  the  molecular  weight  of  all 
other  substances  will  be  represented  by  the  weight  of  their 
vapour  when  compared  under  identical  conditions  with  an 
equal  volume  of  hydrogen  whose  weight  is  taken  as  two. 


THE    PRINCIPLES   OF   ORGANIC   CHEMISTRY       39 

We  thus  reach,  a  very  important  fundamental  conception, 
viz.,  that  of  the  weight  of  a  molecule  of  a  substance  in  terms 
of  the  weight  of  a  molecule  of  hydrogen. 

The  difference  between  molecules  and  atoms  receives 
confirmation  from  the  properties  of  elements  in  what  is  called 
the  nascent  state,  i.e.,  at  the  moment  of  their  release  from 
combination. 

Thus  if  gaseous  hydrogen  is  passed,  e.g.,  through  a  yellow 
solution  of  ferric  chloride,  no  change  takes  place ;  if,  however, 
the  hydrogen  is  evolved  actually  in  the  solution  by  inserting, 
e.g.,  a  strip  of  zinc,  the  ferric  chloride  is  rapidly  reduced  with 
formation  of  a  colourless  ferrous  salt  containing  less  chlorine 
than  the  ferric  chloride.  The  hydrogen  in  the  nascent  state 
combines  with  the  chlorine  of  the  latter  according  to  the 
following  equation  : — 

FeCl3  +  H  =  FeCl2  +  HC1 

Yellow  ferric  Colourless 

chloride  ferrous  chloride 

This  is  a  typical  instance  of  a  process  known  generally  as 
reduction,  when  oxygen,  or  its  equivalent,  is  removed  from 
a  compound. 

The  oxidising  properties  of  such  substances  as  ozone  and 
hydrogen  peroxide  are  due  to  the  liberation  of  oxygen  from 
them  in  the  nascent  state.  Ozone  is  considered  to  be  a 
condensed  form  of  oxygen  containing  three  atoms  in  the 
molecule ;  on  coming  in  contact  with  oxidisable  matter  the 
third  atom  of  oxygen  is  liberated  and  ordinary  oxygen  with 
two  atoms  in  the  molecule  is  set  free,  thus  : — 

03  +  metal  =  02  +  metallic  oxide 

Similarly  hydrogen  peroxide  (H202)  readily  loses  one  atom 
of  oxygen  with  formation  of  ordinary  water,  H20. 

As  a  matter  of  fact  ozone  and  hydrogen  peroxide  are 
mutually  destructive  when  they  are  brought  together,  for  the 


40     BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

loosely  combined  oxygen  atoms  in  the  respective  molecules 
combine  together  to  form  a  molecule  of  ordinary  oxygen, 
thus : — 

03  +  H202  =  H20  +  202 

These  are  typical  cases  of  oxidation,  the  opposite  process 
to  reduction. 

We  shall  see  later  that  this  special  activity  of  nascent 
oxygen  is  of  very  great  importance  in  connection  with  a  set 
of  changes  brought  about  by  a  class  of  enzymes  known  as 
oxidases. 

The  study  of  the  action  of  elements  in  the  nascent  state 
leads  to  the  conclusion  that  the  atom  of  an  element  is  in 
general  incapable  of  a  separate  existence,  and  the  atom  has 
therefore  been  defined  as  the  smallest  portion  of  an  element 
which  can  enter  into  or  be  expelled  from  a  compound. 

A  molecule  is  defined  as  the  smallest  portion  of  an  element 
or  compound  which  is  capable  of  a  separate  existence. 

Certain  exceptional  cases  exist  where  the  molecular 
weight  of  an  element  is  found  to  be  identical  with  its 
atomic  weight,  but  these  do  not  affect  the  general  conclusions. 

We  may  now  proceed  to  the  application  of  these  funda- 
mental chemical  laws  to  that  branch  of  the  science  known  as 
organic  chemistry,  so  called  because  it  deals  with  the  sub- 
stances elaborated  to  a  large  extent  by  living  or  organic 
matter,  as  distinguished  from  the  constituents  of  the  inorganic 
or  mineral  world. 

It  was  at  one  time  thought  that  organic  compounds,  pro- 
perly speaking,  could  only  be  produced  by  vital  energy.  The 
synthesis  of  a  characteristic  vital  product,  viz.,  urea,  by  Wohler 
in  1828  broke  down  this  distinction,  and  since  then,  out  of  the 
countless  substances  included  under  the  science  of  organic 
chemistry,  although  many  are  natural  products,  many  have 
only  been  prepared  in  the  laboratory  and  are  of  purely 
scientific  interest.  One  characteristic  all  these  substances 


THE   PRINCIPLES   OF   ORGANIC   CHEMISTRY        41 

possess  in  common,  they  all  contain  carbon,  and  perhaps  the 
best  definition  of  organic  chemistry  is,  the  chemistry  of  the 
carbon  compounds.  It  is  remarkable  that  the  compounds  of 
carbon  by  far  exceed  in  number  the  compounds  of  all  the  other 
elements,  and  the  reason  for  this  is  to  be  sought  in  the  nature 
of  the  carbon  atom  itself.  In  order  to  understand  this  we  must 
consider  a  further  general  property  of  atoms,  viz.,  what  is 
known  as  their  valency,  and  for  this  purpose  we  must  clearly 
understand  the  meaning  of,  and  the  method  of  determining, 
a  molecular  formula. 

We  have  already  seen  how,  by  determining  the  weights 
of  equal  volumes  of  substances  in  the  gaseous  state,  as 
compared  with  the  weight  of  an  equal  volume  of  hydrogen, 
it  is  possible  to  determine  the  weight  of  a  molecule  of  the 
substance.  By  suitable  methods  of  analysis  we  can  de- 
termine also  the  proportion  by  weight  of  any  element  in 
that  compound  and  thus  obtain  its  molecular  formula, 
just  as  we  have  found  that  the  molecular  formula  for 
steam  is  H20.  Again,  by  burning  a  known  weight  of  car- 
bon in  oxygen,  determining  the  weight  of  carbon  dioxide 
produced,  and  by  knowing  also  the  weight  of  a  volume 
of  this  gas  as  compared  with  the  weight  of  an  equal 
volume  of  oxygen,  we  find,  that  12  parts  of  carbon 
unite  with  32  parts  by  weight  of  oxygen  to  form  a  gas 
the  molecular  weight  of  which  is  44,  and  consequently  its 
molecular  formula  is  C02.  Knowing  thus  the  molecular 
weight  of  steam  and  of  carbon  dioxide  and  their  molecular 
formulae,  viz.,  H20  and  C02,  we  are  in  a  position  to  determine 
the  molecular  formulae  of  many  organic  compounds. 

On  burning  a  given  weight  of  a  substance  containing  carbon 
and  hydrogen,  the  carbon  is  burnt  to  C02,  and  the  hydrogen 
to  H20,  which  may  be  respectively  weighed ;  from  the 
weights  of  C02  and  H20  formed,  we  can  calculate  the  weight 
of  carbon  found  in  the  original  compound  taken,  and  thus 
obtain  its  percentage  composition.  This  method  of  analysis 


42      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 


is  carried  out  in  practice  by  heating  a  weighed  quantity  of  the 
substance  to  be  analysed  in  a  small  porcelain  boat  placed  in 
a  tube  about  a  yard  long  (Fig.   9)  filled  with 
granulated  oxide  of  copper,  and  through  which  a 
current  of  oxygen  or  air  can  be  passed.     The 
whole   tube   is   heated    in    a  furnace,  and  any 
partially  burned  vapour  of  the  substance  which 
o5    escapes  direct  combustion  is  finally  oxidised  by 
passing  over    the    red-hot  copper   oxide.    The 
water  is  retained  in  a  tube  containing  calcium 
^    chloride,  which    readily  absorbs  moisture,  and 
8    the  C02  is  retained   in  specially  devised    bulbs 
|    filled  with  caustic  potash,   which  are  weighed 
g    before  and  after  the  analysis.     This  process  is 
^    known  as  combustion  analysis  and  is  regularly 
2    employed   in   laboratories  devoted    to    organic 
g    chemistry.    Special  methods,  of  course,  are  made 
g    use  of  in  the  determination  of  elements  other 
jjj    than  carbon  and  hydrogen,  e.g.,  nitrogen,  phos- 
"I    phorus,  or  sulphur.     Oxygen  is  usually  deter- 
®    mined    by    difference,    i.e.,    by   deducting    the 
g    weights  of  all  the  other  elements  present  from 
the  weight  of  the  substance    originally  taken, 
when  the  remainder,  if  any,  is  assumed  to  be 
oxygen.     The  determination   of  the  percentage 
composition  of  the  substance  from  combustion 
analysis  will  be  made  clear  by   the  following 
example : — 

0-2  grm.  of  a  substance  yielded  on  analysis  0'290  grm. 
C02  and  012  grm.  H20. 

Now  in    every    44    parts   C02   there  are    12    parts    C, 
therefore  in  0'29  grm.  C02  there  will  be : — 


0-29  X  12 
44 


=  0-079  parts  C 


THE   PRINCIPLES    OP   ORGANIC   CHEMISTRY        43 

Similarly  in  every  18  parts  H20  there  are  2  parts  H ; 
therefore  in  0*12  grm.  H20  there  will  be  : — 


=  0-013  parte  H 


Together  the  C  and  H  make  up  0'079  +  0'013  =  0-092  of  the 
total  weight,  0'2  grm.,  of  substance  taken ;  the  remainder, 
0'108,  is  assumed  to  be  oxygen. 

Converting  these  proportions  to  percentages  we  have : — 

0-079  x  100 

jr- =39*5  per  cent,  carbon. 

0-013  x  100 

=  6*5      „      „    hydrogen. 


0-2 
0-108  x  100 


=  54-0    „      „    oxygen, 


0-2 

From  the  percentage  composition  we  can  readily  calculate 
the  empirical  formula  of  the  substance,  i.e.,  the  simple  ratio  of 
the  number  of  atoms  of  each  element  to  each  other,  by  cal- 
culating how  many  times  12  parts  by  weight  of  carbon, 
1  part  by  weight  of  hydrogen  or  16  parts  of  oxygen,  etc., 
are  contained  in  the  percentage  amounts,  viz. : — 

39-5     QQ  ,      , 

— _  =3*3  parts  of  carbon. 

MM 

—  =  6*5  parts  of  hydrogen. 

54-6 

=  3'4  parts  of  oxygen. 

16 

The  lowest  ratio  of  these  numbers,  i.e.,  the  empirical 
formula,  is  obviously  CH20,  the  slight  errors  in  the  experi- 
mental results  being  neglected. 

In  order  now  to  determine  the  molecular  formula  of  a  com- 
pound we  need  to  know  its  molecular  weight ;  this  is  readily 
obtained  by  determining  the  weight  of  a  known  volume  of  its 


44      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

vapour  as  compared  with  the  weight  of  an  equal  volume  of 
hydrogen.1  The  number  of  molecules  being  the  same  in  the  two 
equal  volumes  according  to  Avogadro's  law,  then,  assuming 
the  weight  of  a  molecule  of  hydrogen  to  be  2,  the  molecular 
weight  of  the  substance  is  twice  the  vapour  density,  and  the 
molecular  formula  can  therefore  now  be  readily  deduced  from 
the  empirical  formula.  Thus,  supposing  that  the  vapour 
density  of  the  substance,  whose  empirical  formula  was  calcu- 
lated above,  was  found  to  be  44,  this  gives  a  molecular 
weight  of  44  X  2  =  88.  The  molecular  formula  will  be 
CoHiOa  as  the  atoms  are  in  the  same  ratio  as  in  the  empirical 
formula,  and  the  sum  of  their  atomic  weights  equals  88. 

There  are  of  course  a  great  many  substances  which  cannot 
be  vaporised  without  decomposition ;  in  such  cases  it  is 
impossible  to  determine  their  molecular  weights,  and  conse- 
quently their  molecular  formula),  by  measurement  of  their 
vapour  density  as  compared  with  hydrogen.  It  has,  however, 
been  shown  by  the  experiments  of  van't  Hoff,  Raoult  and 
others,  that  in  dilute  solutions  the  molecules  of  the  dissolved 
substance  behave  as  if  they  were  in  the  gaseous  state,  and  a 
specific  effect  is  produced  on  the  melting  and  boiling-point  of 
the  solvent,  proportional  to  the  molecular  weight  of  the  dis- 
solved substance.  By  determining  the  rise  of  boiling-point,  or 
the  lowering  of  the  melting-point  of  a  solvent,  produced  by  a 
known  weight  of  the  dissolved  substance,  and  comparing  these 
values  with  those  obtained  when  an  equal  weight  of  a  substance 
of  known  molecular  weight  is  dissolved,  the  molecular  weight 
of  the  first  substance  can  be  deduced. 

Various  other  means  of  a  somewhat  indirect  character  are 
made  use  of  in  certain  special  cases ;  e.g.,  the  determination  of 
the  osmotic  pressure  of  a  solution  of  known  concentration  may 
be  employed  as  indicated  in  Chapter  I. 

1  In  practice  a  given  weight  of  liquid  is  converted  into  vapour  and 
the  volume  of  this  vapour  measured  by  allowing  it  to  replace  an  equal 
volume  of  air. 


THE   PRINCIPLES   OF   ORGANIC   CHEMISTRY        45 

This  somewhat  lengthy  description  of  the  methods  and 
arguments  involved  in  arriving  at  the  molecular  formula  for 
an  organic  compound  has  been  entered  into,  because  it  appears 
of  fundamental  importance  that  the  real  meaning  of  a  mole- 
cular formula  should  be  properly  understood,  as  all  other 
developments  in  regard  to  the  molecular  structure  of  com- 
pounds depend  upon  this. 

A  molecular  formula  tells  us  how  many  atoms  of  each  con- 
stituent element  are  present  in  the  molecule  of  the  compound. 
It  tells  us  nothing,  however,  as  to  the  way  in  which  these 
atoms  may  be  combined  within  a  molecule.  The  extra- 
ordinary advances  which  modern  chemistry  has  made  in  the 
study  of  the  arrangement  of  the  atoms  within  the  molecule, 
a  study  which  must  necessarily  precede  a  systematic  attempt 
to  build  up  these  molecules  from  their  constituent  elements, 
naturally  had  to  begin  with  the  study  of  the  simplest  com- 
pounds. Supposing  we  take  the  following  simple  compounds 
of  carbon,  whose  molecular  weight  and  molecular  formulae  are 
easily  ascertained  by  the  methods  already  indicated  : — 

Carbon  monoxide,  CO ; 
Carbon  dioxide,  C02 ; 
Methane,  CH* ; 
Chloroform,  CHC13 ; 
Hydrocyanic  acid,  HCN ; 

we  see  that  one  atom  of  carbon  is  able  to  combine  with  one  or 
two  atoms  of  oxygen  ;  with  four  atoms  of  hydrogen  ;  or  with 
one  atom  of  hydrogen  and  one  atom  of  nitrogen.  We  also 
know  that  one  atom  of  oxygen  combines  with  two  atoms  of 
hydrogen  to  form  water,  H20 ;  that  one  atom  of  hydrogen 
combines  with  one  atom  of  chlorine  to  form  hydrochloric  acid 
gas,  HC1 ;  further  that  one  atom  of  nitrogen  combines  with 
three  atoms  of  hydrogen  to  form  ammonia,  NHs. 

If  we  study  the  formulae  of  the  five  compounds  of  carbon 
given  in  the  above  list  in  the  light  of  these  facts,  we  shall  see 


46      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

that  the  carbon  is  attached  to  elements  which  are  equivalent 
in  every  case  but  one  to  four  atoms  of  hydrogen  ;  the  exception 
is  carbon  monoxide,  where  only  one  atom  of  oxygen,  equivalent, 
that  is,  to  two  atoms  of  hydrogen,  is  attached  to  the  carbon. 
Carbon  monoxide,  however,  as  is  commonly  known,  is  a  com- 
bustible gas,  burning  with  a  blue  flame  to  form  C02,  a  com- 
pound in  which  again  the  carbon  is  attached  to  two  other 
atoms,  together  equivalent  to  four  atoms  of  hydrogen.  More- 
over carbon  monoxide  can  combine  with  two  atoms  of  chlorine 
to  form  a  compound  known  as  carbonyl  chloride,  COC12, 
where  again  the  carbon  is  combined  with  atoms  which  are 
together  equivalent  to  four  atoms  of  hydrogen.  Such  examples 
might  be  multiplied,  with  the  result  that  it  can  be  shown  that 
one  atom  of  carbon  is  always  capable  of  combining  with  four 
atoms  of  hydrogen  or  their  equivalent.  Incidentally  we  have 
learnt  also  that  one  atom  of  chlorine  is  capable  of  taking  the 
place  of  one  atom  of  hydrogen  ;  one  atom  of  oxygen  is  capable 
of  taking  the  place  of  two  atoms  of  hydrogen ;  one  atom  of 
nitrogen  is  capable  of  taking  the  place  of  three  atoms  of  hydro- 
gen. This  atom-replacing  power  of  the  elements  is  known 
as  their  valency.  We  speak  of  chlorine  and  hydrogen  as 
being  monovalent,  of  oxygen  as  divalent,  of  nitrogen  as  tri- 
valent,  and  of  carbon  as  tetravalent.  Where  the  atom  of  an 
element  does  not  exercise  its  full  valency,  an  unsaturated  com- 
pound results,  such,  e.g.,  as  carbon  monoxide. 

Throughout  the  vast  range  of  organic  chemistry  the  carbon 
atom  is  always  tetravalent ;  where  it  apparently  is  not  tetra- 
valent, further  atoms  can  always  be  taken  into  combination 
till  saturation  results.  Victor  Meyer  indeed  was  accustomed 
to  define  organic  chemistry  as  *  the  chemistry  of  constant 
valency/  because  such  constancy  is  not  so  apparent  among  the 
elements  which  build  up  the  mineral  kingdom. 

We  must  now  consider  the  second  very  important  property 
of  the  carbon  atom.  Not  only  will  the  carbon  atom,  as  we 
have  seen,  combine  with  hydrogen,  with  chlorine,  etc.,  it  will 


THE   PRINCIPLES    OF   ORGANIC    CHEMISTRY         47 

also  combine  with  itself.  This  fact  lies  at  the  foundation  of 
Kekule's  law  of  the  linking  of  atoms,  which  is  one  of  the  main 
foundation  stones  of  modern  organic  chemistry.  The  genesis 
of  this  idea  of  Kekule's  was  singular.  He  tells  us  that  it 
came  to  him  more  or  less  as  a  dream.  As  he  was  sitting  half 
asleep  by  the  fire,  he  seemed  to  see  the  atoms  executing  a 
mazy  dance,  till  suddenly  some  of  them  separated  themselves 
into  chains,  while  others  joined  themselves  in  rings.  He  sat 
up  all  night  working  out  the  consequences  of  this  dream. 
Very  briefly  it  came  to  this,  that  if  we  consider  a  single  carbon 
atom  with  its  tetrad  valency,  exercising  a  power  of  combination 
with  four  atoms  of  hydrogen  or  their  equivalent,  it  may  be 

I 

symbolically  written  thus,  — C — ;  if  another  atom  joins  itself 

I 

to  this,  a  compound  will  be  formed  with  a  skeleton  structure 


of  this  kind,  viz.,  and  so  on.    Each  of  the  vacant 

— c— 

I 

'  bonds,'  as  they  may  be  termed,  can  be  combined  with 
hydrogen  or  other  elements,  and  we  can  easily  see  that  as  we 
go  on  adding  carbon  atoms,  for  each  carbon  atom  two  hydrogen 
atoms  or  their  equivalent  can  be  also  added.  Thus  we  get 
what  is  known  as  an  homologous  series.  Supposing  the  bonds 
in  the  above  case  to  be  combined  with  hydrogen,  we  obtain 
the  series,  CnH2n  +  2;  this  is  the  series  of  paraffin  hydro- 
carbons, the  initial  member  of  which  is  : — 

Methane,  C  H4,  followed  by 
Ethane,    C2H6, 
Propane,  C3H8, 
Butane,    C4H10,  etc. 

If  two  adjacent  carbon  bonds  in  such  a  chain  be  left 


48      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

unsaturated,  we  then  get  the  series  of  the  olefine  hydrocarbons 
of  the  general  formula  C7lH2/l,  e.g. : — 

Ethylene,  C2H4, 
Propylene,  C3H6, 
Butylene,  C4H8,  etc. 

The  initial  member  of  this  series  should  of  course  be 
methylene  CH2,  but  all  efforts  to  prepare  it  result  in  the 
formation  of  ethylene  or  dimethylene. 

A  further  elimination  of  hydrogen  results  in  the  series 
CnH2n  _2,  the  initial  member  of  which  is  acetylene,  C2H2. 

The  next  great  series  resulting  from  Kekule's  generalisa- 
tion are  the  ring  hydrocarbons,  of  which  the  best  known 
member  is  benzene.  Kekule  represented  benzene  by  the 
following  formula  : — 

CH 


HC 


-CH 


CH 


The  proof  of  the  ring  formation  in  benzene  is  a  very 
beautiful  instance  of  the  method  of  determining  what  is 
known  as  the  constitutional  formula  of  an  organic  compound. 
Inasmuch  as  the  structure  of  benzene  as  indicated  by  Kekule's 
formula  is  a  symmetrical  one,  it  should  follow  that  whichever 
of  the  hydrogen  atoms  is  replaced  by  chlorine  the  same 
monochlorbenzene  should  result.  As  a  matter  of  fact,  how- 
ever monochlorbenzene  is  prepared,  only  one  monochlor- 
benzene has  ever  been  obtained.  It  has  indeed  been  possible 
by  a  series  of  reactions,  too  complex  to  be  here  considered, 
systematically  to  replace  one  atom  of  hydrogen  after  another 
in  benzene,  and,  as  has  been  stated,  whichever  atom  is  re- 
placed only  one  monochlorbenzene  results. 

A  formula  such  as  Kekule's  formula  for  benzene,  which 


THE   PRINCIPLES   OF   ORGANIC   CHEMISTRY        49 

gives  a  symbolic  representation  of  the  relation  of  the  atoms  in 
the  molecule  one  to  another,  is  known  as  a  constitutional 
formula.  That  these  formulae  do,  as  a  matter  of  fact,  bear 
some  relation  to  an  actual  reality  in  nature,  is  shown  by  the 
circumstance  that,  once  a  constitutional  formula  has  been 
correctly  established,  the  artificial  production  of  the  sub- 
stance is  generally  only  a  matter  of  time.  Thus,  to  take  the 
case  of  benzene  itself,  its  formula  suggests  that  if  three  mole- 
cules of  acetylene  C2H2  could  be  induced  to  combine,  benzene 
C6H6  would  result.  On  passing  acetylene  through  a  red-hot 
tube  benzene  is  actually  produced,  the  reaction  being  repre- 
sented as  follows : — 

CH 

CH 

III        = 
HC          CH 
% 
CH 

It  goes  without  saying  that  before  any  conclusion  can 
be  drawn  as  to  the  composition  or  constitution  of  a  com- 
pound, it  is  essential  that  it  should  be  obtained  pure.  The 
methods  in  use  in  organic  chemistry  for  obtaining  compounds 
in  the  pure  state  resolve  themselves  into  crystallisation  and 
distillation. 

Crystallisation  is  effected  by  evaporating  a  solution  of 
the  substance  in  suitable  solvents  either  at  the  ordinary 
atmospheric  pressure  or  in  vacuo.  The  crystals  first  de- 
posited are  usually  the  purest ;  by  redissolving  these  and  re- 
peating the  process  pure  crystals  are  eventually  obtained.  This 
process  is  known  as  fractional  crystallisation.  Crystallisation 
is  often  brought  about  by  combining  the  substance  to  be 
purified  with  some  other  body  with  which  it  will  form  a 
crystallisable  compound.  A  notable  instance  of  this  method 
is  the  case  of  many  of  the  sugars,  which  by  themselves  form 


50      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

difficultly  crystallisable  syrups  ;  they  can  be  combined  with 
a  substance  known  as  phenyl  hydrazine  to  form  well-defined 
crystalline  compounds. 

The  crystalline  compound  is  pure  when  it  has  a  constant 
melting-point ;  that  is,  if  the  melting-point  of  the  substance 
is  determined  and  it  is  redissolved  and  recrystallised,  and 
the  melting-point  of  the  crystals  again  determined,  the  two 
melting-point  determinations  should  be  the  same. 

To  purify  a  substance  by  distillation,  it  can  be  distilled 
either  at  the  ordinary  atmospheric  pressure,  or  under  reduced 
pressure,  so  long  as  the  temperature  of  the  vapour  remains 
constant;  if  a  rise  of  the  thermometer  is  observed  during 
distillation,  it  means  that  some  substance  other  than  the 
lower  boiling  substance  is  being  distilled  over.  By  repeating 
the  distillation  of  the  portions  distilled  over  between  various 
limits  of  temperature,  a  distillate  is  finally  obtained  having  a 
constant  boiling-point ;  such  a  process  is  known  as  fractional 
distillation.  The  separation  of  the  products  of  petroleum  by 
distillation  on  the  large  scale  is  a  good  instance  of  this  process. 
It  is  characteristic  of  a  pure  compound  that  it  has  a  constant 
boiling-point. 

It  may  not  be  superfluous  here  to  emphasise  the  fact  that  to 
the  chemist  a  substance  can  only  be  considered  to  be  a  definite 
chemical  entity  when  it  satisfies  one  of  three  conditions  : — 

1.  It  has  a  definite  crystalline  form, 
or 

2.  It  has  a  constant  melting-point, 
or 

3.  It  has  a  constant  boiling-point. 

Many  of  the  substances  met  with  in  the  chemistry  of 
vital  processes,  more  especially  the  derivatives  of  albumin, 
do  not  satisfy  these  conditions.  Such  substances  can  be 
differentiated  one  from  another  by  their  general  chemical  and 
physical  properties,  and  by  the  products  of  their  decom- 


THE   PRINCIPLES   OF   OEGANIC   CHEMISTRY         51 

position  under  defined  conditions,  but  they  cannot  be  looked 
upon  as  chemical  individuals  in  the  same  sense  as  compounds 
which  fulfil  one  of  the  above-mentioned  requirements. 

The  determination  of  the  constitutional  formula  of  the 
countless  substances  met  with  in  the  study  of  organic  chemistry 
shows  that  they  can  be  classified  under  three  heads. 

1.  Aliphatic  compounds,  viz.,  all  open  chain  compounds  both 
saturated  and  unsaturated,  viz.,  the  paraffin,  olefine,  acetylene, 
etc.,  hydrocarbons  already  referred  to,  and  their  derivatives. 

2.  Isocyclic  compounds.  —  All  compounds  containing  closed 
chains  formed  by  the  union  of  carbon  atoms  only,  viz.,  deri- 
vatives of  polymethylene  hydrocarbons,  consisting   of  rings 
formed  by  three  or  more  CHg  groups  ;    thus  :  — 

CH 

trimethylene 


or  substances  derived  from  benzene 

CH 


CH 


and  from  hydrocarbons  containing  more  than  one  ring  such 
as  naphthalene,  anthracene,  etc. 

3.  Heterocyclic  compounds. — All  compounds  containing 
closed  chains,  having  other  atoms  in  addition  to  carbon  atoms, 
viz.  : — 

PTT 
HC CH  _X 

L  pyridine 


thiophene 


HC    CH 

V 

N 

£2 


52      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

CH   CH 

A  /\ 
HC     C     CH 

quinoline 

HC     C     CH 


N      CH 

etc.,  and  their  derivatives. 

From  all  of  these  root  compounds  derivatives  can  be 
built  up  by  well-defined  processes,  and  these  derivatives  are 
characterised  by  containing  certain  groups  of  atoms  which  are 
easily  recognisable  by  their  reactions. 

It  will  be  useful  at  this  stage  to  consider  the  more  important 
classes  of  derivatives  and  their  reactions  in  a  highly  general 
manner.  A  knowledge  of  organic  chemistry  really  consists  in 
being  familiar  with  certain  general  reactions  typical  of  certain 
specific  atomic  groups,  rather  than  in  a  detailed  acquaintance 
with  individual  compounds.  In  what  follows,  therefore, 
reference  will  be  made  mainly  to  those  atomic  groupings,  the 
knowledge  of  whose  properties  will  be  useful  in  the  study 
of  the  substances  to  be  considered  in  the  later  chapters  of 
the  book. 

Alcohols. — These  are  derivatives  of  aliphatic  hydrocarbons 
characterised  by  the  presence  of  the  group  — OH,  known  as 
the  hydroxyl  group.  The  simplest  alcohol  is  methyl  alcohol, 
CH3OH,  a  hydroxyl  derivative  of  methane,  CH4.  Ordinary 
alcohol  is  the  next  member  of  the  series,  viz.,  hydroxy-ethane 
or  ethyl  alcohol,  CH3CH2OH  (or  C2H5OH).  Alcohols  may  be 
divided  into  three  classes  : — 

Primary  alcohols  of  the  general  formula  E — CH2OH ; 
Secondary  alcohols  of  the  general  formula  K2=CHOH ; 
Tertiary  alcohols  of  the  general  formula  K3^C — OH. 


THE   PRINCIPLES    OF   ORGANIC    CHEMISTRY         53 

Alcohols  are  capable  of  combining  with  mineral  acids  to 
form  salts,  thus  :  — 

RCH2OH  +  HC1  =  RC1  +  H20 

Aldehydes  and  Ketones.—  The  first  product  of  the  oxida- 
tion of  an  alcohol  is  either  an  aldehyde  or  a  Jcetone.  Primary 
alcohols  yield  aldehydes,  thus  :  — 

/» 

R-CH2OH  +  0  =  R-C^    +  H20 

Primary  0 

alcohol  Aldehyde 

Secondary  alcohols  yield  ketones  :  — 

R2=CHOH  +  0  =  R2=C=0  +  H20 

Tertiary  alcohols  yield  mixtures  of  aldehydes  and  ketones. 

It  will  be  noted  that  both  aldehydes  and  ketones  contain 
the  group  ^C=0  which  is  known  as  carbonyl  ;  in  fact  alde- 
hydes differ  only  from  ketones  in  that  a  complex  residue  R 
replaces  hydrogen  in  the  latter.  The  group  ^>C=0  is  a 
highly  reactive  group  ;  the  German  word  reactionsfahig,  or 
capable  of  reaction,  is  perhaps  more  expressive. 

As  this  group  occurs  in  most  of  the  carbohydrates,  certainly 
in  most  of  the  sugars,  and  possibly  in  cellulose,  it  is  important 
that  its  general  reactions  should  be  understood.  The  more 
commonly  used  are  the  following. 

With  ammonia  an  ammo  compound  is  produced  thus  :  — 


NH3  = 

\NH2 

With  hydrocyanic  acid  we  have  the  following  :  — 

/-OH 
R=C=0  +  HCN  =  R^C 

NGN 


54      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

With   phenyl  hydrazine   a  compound  of  the  following 

formula  C6  H5  N— NH2  we  have  :— 

R=C=0  +  H2N— NHC6H5  =  RCN— NHC6H5  +  H20 

Acids. — Upon  oxidation  the  CO  group  gives  rise  to  an  acid, 
the  exact  composition  of  which  depends  on  the  elements  or 
groups  attached  to  the  carbon.  Thus  an  aldehyde  oxidises 
as  follows  : — 

R  R 

l_  l_ 

I  I 

H  OH 

A  ketone  gives  a  mixture  of  acids  according  to  a  rather 
more  complex  reaction. 

The  group  C02H,  which  is  a  shortened  form  of  the  group 

OTT 
""""       "    as  written  above,  is  known  as  the  carboxyl  group, 


and  is  characteristic  of  all  organic  acids  which  may  be  written 
according  to  the  general  formula  RCOOH ;  thus  in  acetic  acid 
R  is  represented  by  the  group  CH3  or  methyl,  and  the  formula 
of  the  acid  is  CH3COOH.  The  substance  used  as  an  illustra- 
tion of  the  determination  of  a  molecular  formula  on  p.  44  was 
acetic  acid.  On  reduction  with  nascent  hydrogen  the  group 

/H 

C02H  is  reconverted  to  — C=0  and  — CH2OH,  i.e.,  acids 

give  on  reduction  aldehydes  and  alcohols. 

Esters. — Alcohols  combine  with  organic  acids  to  form  what 
are  known  as  esters  or  ethereal  salts ;  thus  ethyl  alcohol  com- 
bines with  acetic  acid  according  to  the  following  equation  : — 

C2H5OH  +  CH3COOH  =  CH3COOC2H5  +  H20 
which  may  be  generalised  as  follows  : — 

ROH  +  RCOOH  =  RCOOR  +  H20 


THE    PRINCIPLES    OF    ORGANIC    CHEMISTRY        55 

It  should  be  noted  that  these  reactions  in  which  salts  are 
formed  from  alcohols  with  elimination  of  water  are  typical 
examples  of  what  are  known  as  reversible  reactions  ;  that  is, 
when  a  certain  amount  of  water  and  salt  is  formed,  an  equili- 
brium is  attained,  and  the  reverse  action  tends  to  take  place, 
resulting  in  the  formation  of  acid  and  alcohol.  Such  reactions 
are  generally  written  thus  :  — 

KOH  +  RCOOH  £  RCOOR  +  H20 

If  it  is  desired  that  the  reaction  should  become  complete 
it  is  necessary  to  add  some  substance  such  as  strong  sulphuric 
acid  or  chloride  of  zinc  which  will  take  up  water  as  it  is 
formed. 

It  is  probable  that  under  specific  conditions  nearly  all 
chemical  reactions  are  reversible.  The  case  of  the  esters 
is  interesting  as  a  simple  one,  which  has  been  carefully 
studied. 

Ethers.  —  Esters  should  not  be  confused  with  ethers,  which 

T> 

are  bodies  of  the  general  formula  -n^O,  R  m  this  case 

representing  a  hydrocarbon  residue  ;   thus,  in  ordinary  ether 

C  H 
R  =  the  group  C2H5  or  ethyl,  and  its  formula  is  f?*r 


Phenols.  —  When  the  group  OH  is  connected  directly 
with  a  benzene  ring,  substances  known  generally  as  phenolic 
compounds  are  produced,  the  simplest  of  which  is  ordinary 

OH 

/\ 
carbolic  acid  or  phenol,  |       I.    On  oxidation  these  substances 


yield  somewhat  complicated  mixtures  and  are  thus  distin- 
guished from  ordinary  alcohols. 


56      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

Groups  containing  Nitrogen.—  The  simplest  compound  of 
nitrogen  is  of  course  ordinary  ammonia,  which  has  the  com- 
position NH3.  Each  of  these  three  atoms  of  hydrogen  is 
capable  of  being  replaced  by  complex  groups  of  various  kinds  ; 
moreover,  just  as  ammonia,  NH3,  combines  with  acids,  e.g.,  HC1, 
to  form  ammonium  chloride,  NH3HC1  (or  NH4C1  as  it  is  gener- 
ally written),  so  organic  derivatives  of  ammonia  also  are  capable 
of  acting  as  bases  in  this  way.  Ordinary  sulphate  of  quinine 
is  a  case  in  point.  Ammonia  derivatives  are  possessed  of 
different  properties  according,  on  the  one  hand,  to  the  number 
of  hydrogen  atoms  replaced  or,  on  the  other,  to  the  character 
of  there  placing  groups.  E.g.,  if  one  of  the  hydrogen  atoms  is 
replaced  by  a  hydrocarbon  residue  we  have  what  are  known 
as  amino  derivatives,  thus  :  — 

CH3NH2  is  methyl-amine. 

C6H5NH2  is  phenylamine  or  amino-benzene,  commonly 
known  as  aniline. 

CH2NH2 

is    amino-acetic-acid,     glycocol     or    glycin,     a 

COOH 
very  important  member  of  the  series  of  amino  acids. 

If  the  replacement  is  effected  by  an  acid  residue  an  acid 
amide  results  ;    thus  CH3CONH2  is  known  as  acetamide. 
The  well-known  substance  urea  is  an  amide  of  carbonic 

,  and  has  the  formula  C 


The  group  NH2,  which  is  thus  seen  to  be  formed  by  the 
replacement  of  one  hydrogen  in  ammonia  by  a  complex  group, 
is  known  as  the  amino  group,  and  like  other  well-defined 
groups  it  can  be  recognised  in  a  compound  by  its  specific  re- 
actions ;  one  of  the  most  important  of  these  is  its  reaction  with 
nitrous  acid,  which  results  in  the  elimination  of  nitrogen  and 


THE   PRINCIPLES   OF   ORGANIC   CHEMISTRY        57 

the  replacement  of  the  NH2  group  by  the  hydroxyl  group 
—  0  —  H,  thus  :— 

R-NH2  +  HONO  =  K—  OH  +  N2  +  H20 

When  two  atoms  of  hydrogen  in  ammonia  are  replaced 
the  group  NH  is  left,  which  is  known  as  the  imino  group. 
This  also  is  characterised  by  its  reaction  with  nitrous  acid 
when  substances  known  as  oximes  are  obtained,  thus  :  — 

R2NH  +  HONO  =  R2N—  NO  +  H20 

'Finally  all  three  hydrogen  atoms  in  ammonia  may  be 
replaced  and  we  obtain  a  tertiary  amine,  R3N. 

Compounds  are  known  which  are  derived  from  a  com- 
bination of  two  amino  groups  joined  thus  :  — 

H2N—  NH2 

This  substance  has  been  prepared  and  is  known  as  hydra- 
zine  ;  its  phenyl  derivative  C6H5HN  —  NH2  has  already  been 
mentioned  more  than  once,  and  is  a  substance  of  great  import- 
ance because  of  its  property  of  combining  with  the  carbonyl 
group  which  occurs  in  numerous  sugars,  and  of  thus  giving 
rise  to  crystallisable  compounds. 

Cyanides.  —  The  group  CN  is  an  important  one  because  of 
the  facility  with  which  on  treatment  with  water  (in  presence 
of  acid  or  alkali)  it  gives  rise  to  the  group  COOH,  that  is,  to 
acids,  thus  :  — 

RON  +  2H20  =  RCOOH  +  NH3 

Such  a  process  in  which  one  or  more  molecules  of  water 
take  part  is  generally  known  as  hydrolysis. 

Moreover,  on  treatment  with  nascent  hydrogen,  it  is 
reduced,  forming  an  amino  derivative,  thus  :  — 


XNH2 


58      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

In  the  foregoing  paragraphs  are  given  some  of  the  more 
important  atomic  groupings  which  are  met  with  in  the  sub- 
stances which  form  the  subject  matter  of  bacteriological  and 
enzyme  chemistry  ;  the  point  must  be  emphasised  that  where- 
ever  they  occur,  and  however  complicated  the  atomic  groupings 
may  be  with  which  they  may  be  associated,  they  can  always 
be  recognised  by  their  specific  reactions. 

A  short  table  summarising  the  reactions  of  the  few  typical 
groupings  which  have  been  considered  may  therefore  be  found 
useful  (Table  I).  The  unsaturated  linkings,  it  must  be 
understood,  may  combine  with  groups  of  atoms  of  greater 
or  less  complexity  symbolised  by  R. 

Constitutional  Formulae.— In  determining  the  constitution 
of  a  compound  the  main  problem  consists  in  ascertaining 
by  the  reactions  given  what  atomic  groupings  are  present. 
To  take  a  simple  case,  a  substance  is  found  by  the  methods 
already  indicated  to  have  the  molecular  formula  CH40.  Upon 
oxidation  it  is  found  to  yield  an  aldehyde  and  finally  an  acid. 
We  conclude,  therefore,  that  it  contains  the  group  CH2OH, 
and  bearing  in  mind  that  the  carbon  atom  is  uniformly 
tetravalent,  we  assign  the  constitution  CH3OH  and  write  the 
equation  expressing  its  oxidation  as  follows  : — 

HCH.OH  +  0  =  HCHO  +  H20 
HCHO-f  0=HCOOH 

The  substance  is,  of  course,  methyl  alcohol  yielding  on 
oxidation  formaldehyde  and  formic  acid. 

Isomerism. — A  little  reflection  will  already  have  suggested 
that  it  is  possible,  even  though  the  number  of  atoms  in  a 
molecule  may  be  identical,  that  the  arrangement  of  atoms 
within  the  molecule  may  differ  in  different  cases.  In  con- 
sidering the  constitutional  formula  for  benzene,  the  assump- 
tion of  a  ring  arrangement  of  the  carbon  atoms  in  the  molecule 


TABLE  I 


GROUP 


TYPICAL  REACTION 


—  CH.OH 

Primary  alcohol  group 

=CHOH 

Secondary  alcohol  group 


Tertiary  alcohol  group 

=0=0  * 

Carbonyl  group 


XOH 
Carboxyl  group 


— NH2 

Amino  group 


— C=N 
Cyanogen  group 


H 


Oxidises     to     aldehyde  —  C'          and     acid 
— C02H  ^O 

Oxidises  to  ketone  =CO  and  mixture  of  acids. 
Oxidises  to  mixture  of  acids. 


OH 


With  ammonia  forms  =C 


With  hydrocyanic  acid  forms  =( 
With  phenyl  hydrazine  forms 


.OH 


Yields  on  reduction  — 


and  —  C—  OH 


Combines  with  alcohols  to  form  esters  of  the 
general  formula  — COOR 

Replaces  H  in  hydrocarbons  to  form  a 
primary  amine,  e.g.,  CH3NH2,  methylamine  ; 
CgHj — NH2,  phenylamine  or  aniline. 

Replaces  hydrogen  in  an  acid  to  form  amino 
acids  thus,  CHoNH2,  amino-acetic. 

C02H 

Replaces  OH  in  the  carboxyl  group  of  an 
acid  to  form  an  amide  thus,  CH3CONH2, 
acetamide. 

Treated    with    nitrous    acid    yields    nitrogen 
and  an  alcohol,  thus : — 
RNH2  +  HONO  =  ROH  +  N2  +  H20 
The   hydrogen   in   the   NH2  can   be   further 
replaced,  yielding : — 

R2NH  R.,N 

Secondary  amine  Tertiary  amine 

On    hydrolysis    yields    the    carboxyl    group 

—  C02H 
On  reduction  yields  an  amine,  RCH2NH2 


60      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

was  seen  to  be  justified  by  the  fact  that  only  one  monochlor- 
benzene  could  be  obtained.  Many  other  consequences  follow 
from  the  ring  formation,  but  the  above  is  one  of  the  simplest, 
and  suffices  to  distinguish  benzene  from  another  possible 
arrangement  of  six  carbon  atoms  and  six  hydrogen  atoms  to 
form  the  molecule  C6H6  which  might  be  conceived  as  follows, 
the  atoms  of  carbon  forming  a  chain  : — 

CHfeC— CH2— CH2— C^CH 

Such  a  substance  does  as  a  matter  of  fact  exist,  and  is 
known  as  dipropinyl ;  it  differs,  however,  from  benzene  in  that 
it  forms  two  monochlor  derivatives  according  as  chlorine  is 
attached  to  the  first  or  third  carbon  atom  from  the  end  of  the 
chain.  Moreover  it  will  be  seen  that  dipropinyl  is  an  acetylene 
hydrocarbon  that  readily  combines  with  bromine,  the  bromine 
being  added  to  the  compound,  which  then  becomes  saturated. 
The  first  action  of  bromine  upon  benzene  is  one  of  substitution. 

Two  compounds  such  as  benzene  and  dipropinyl,  which 
have  the  same  number  of  atoms  in  the  molecule  but  whose  atoms 
are  differently  arranged,  are  known  as  isomeric  substances,  and 
the  phenomenon  is  spoken  of  generally  as  isomerism.  In  order 
to  determine  the  arrangement  of  the  atoms  in  the  molecule 
and  thus  to  distinguish  between  isomeric  substances,  a  syste- 
matic study  must  be  made  of  the  typical  reactions  of  such 
substances. 

A  simple  case  may  be  taken  to  illustrate  the  determination 
of  the  constitutional  formula  in  the  case  of  two  substances 
having  the  molecular  formula  C2H3N.  When  these  substances 
are  treated  with  potash  one  of  them  yields  potassium  acetate 
and  ammonia,  while  the  other  yields  methylamine  and 
potassium  formate.  These  reactions  point  clearly  to  the 
conclusion  that  in  the  one  case  the  two  carbon  atoms  must 
be  closely  connected,  as  they  reappear  together  in  the  molecule 
of  acetic  acid ;  in  the  other  case,  one  of  them  is  separated  from 


THE   PRINCIPLES   OF   ORGANIC   CHEMISTRY        61 

the  other  and  joined  to  nitrogen,  and  so  reappears  as  methyl- 
amine.  Bearing  in  mind  the  underlying  assumption  that  the 
hydrogen  atoms  are  always  monovalent,  the  carbon  atoms 
always  tetravalent,  and  the  nitrogen  either  trivalent  or  penta- 
valent,  the  above  reactions  find  satisfactory  explanation  in  the 
following  formulae  and  equations  :  — 

OK 

H3C—  f|=N  +  KOH  +  H20  =  H3C-C=0  +  NH3 

Cyanide. 

OK 
H3C-N=C  +  KOH  +  H20  =  H3C—  N=H3  +  HC=0 

Iso-cyanide. 

The  first  compound  is  termed  a  cyanide,  the  other  an  iso~ 
cyanide. 

Another  simple  but  important  instance  of  isomerism  may 
be  referred  to  in  illustration,  viz.,  the  case  of  the  lactic_acid$. 
Ordinary  lactic  acid  is  produced  by  the  fermentation  of  milk 
sugar  or  lactose  ;  it  has  the  molecular  formula  C3H603.  Another 
acid  of  the  same  molecular  formula  exists  whose  chemical 
properties  are  quite  different  from  those  possessed  by  the 
fermentation  lactic  acid.  The  difference  between  these  two 
acids  finds  an  explanation  in  the  reactions  by  which  they 
have  been  artificially  prepared,  and  in  the  products  to  which 
they  give  rise  on  oxidation,  etc.  An  acid  having  chemical 
properties  identical  with  the  fermentation  acid  is  obtained 
from  acetaldehyde  by  the  following  typical  reactions.  By 
the  action  of  hydrocyanic  acid  on  aldehyde  a  cyanhydrin 
is  formed  :  — 

CH3 

CH3  | 


0=0  CN 


62      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

Upon  hydrolysis,  according  to  the  general  reaction  several 
times  referred  to,  a  salt  of  lactic  acid  is  formed  thus  : — 

CH3  CH3 

I  1 

CHOH  +  KOH  -  CHOH  +  NH3  +  H20 

CN  COOK 

I 
This  acid  contains,  it  will  be  seen,  the  group  CHOH  ;  upon 

'  l 

oxidation,  therefore,  this  will  yield  the  group  =C=0,  and  a 
ketonic  acid  is  formed,  thus  : — 

CH3  CH3 

I  I 

CHOH+0  =  C=0  +  H20 

I  1 

COOH  COOH 

It  should  here  be  noted  that  it  is  customary  for  the  con- 
venient nomenclature  of  open  chain  compounds  to  refer  to  the 
C  atoms  in  order  as  a,  ft,  7,  etc.,  according  as  they  are  one, 
two,  three,  etc.,  removes  from  the  end  of  the  chain.  The  above 
lactic  acid  is  therefore  known  as  a  lactic  acid.  It  is  obvious 
that  the  hydroxyl  OH  group  might  be  attached  to  the  second 
carbon  atom,  when  a  ft  acid  would  be  obtained.  We  thus  have : 

CH3  CH2OH 

(a)  CHOH  (ft)  CH2 

C02H  C02H 

As  a  matter  of  fact  the  latter  is  the  second  lactic  acid 
above  referred  to.  It  is  obtained  from  ft  iodopropionic  acid, 
which  is  known  to  have  the  constitutional  formula 


THE   PRINCIPLES   OP   ORGANIC   CHEMISTRY       63 


ca, 


the  iodine  being  replaced  by  th6  group  OH,  through  the  action 
of  moist  silver  oxide,  AgOH. 

The  constitutional  formula  of  /3-lactic  acid  is  further  con- 
firmed by  the  fact  that  on  oxidation,  as  would  be  expected,  the 
group  CH2OH  yields  finally  a  carboxyl  group  COgH,  and  a 
dibasic  acid  known  as  malonic  acid  is  formed,  thus  :  — 


CH,,  +  02  =  CH  +  H20 


In  the  next  chapter  reference  will  be  made  to  certain  other 
isomeric  varieties  of  lactic  acid  which  cannot  be  distinguished 
by  any  difference  in  the  products  of  their  reactions  or  in  the 
methods  of  their  preparation  ;  the  present  chapter  may,  how- 
ever, fitly  end  at  this  point  with  a  few  words  of  summary  and 
emphasis. 

It  will  have  been  sufficiently  evident  that  in  such  a  limited 
space  only  a  few  simple  examples  have  been  made  use  of  to 
illustrate  the  general  principles  of  the  science  of  organic 
chemistry.  It  is  of  the  greatest  importance  for  the  proper 
understanding  of  any  subject  involving  the  use  of  organic  com- 
pounds, and  the  expression  of  the  construction  of  the  com- 
pounds by  formulae,  that  the  real  meaning  of  these  formulae 
should  be  once  for  all  properly  understood.  For  this  reason 
rather  disproportionate  space  has  been  taken  in  the  endeavour 
to  make  clear  the  meaning  and  the  methods  of  determining 
successively  molecular  weights  and  molecular  formulae.  To 


64      BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 

discuss  constitutional  formulse  at  any  length  would  involve 
writing  a  text-book  on  organic  chemistry,  but  emphasis  has 
been  laid  on  the  importance  of  a  knowledge  of  the  reactions  of 
certain  fairly  simple  groups  of  atoms  which  occur  again  and 
again  in  the  numberless  substances  which  form  the  subject 
matter  of  this  science.  Finally,  one  or  two  very  simple 
instances  of  the  determination  of  constitutional  formulae 
have  been  given.  It  is  thus  hoped  that  even  those  readers 
whose  knowledge  of  organic  chemistry  is  limited,  may  yet  be 
able  easily  to  follow  the  subsequent  chapters  of  this  book. 


CHAPTER  IV 

SPACE- ISOMERISM  AND  THE  CHEMISTRY  OF 
THE  SUGARS 

TOWARDS  the  end  of  the  preceding  chapter  reference  was 
made  to  certain  varieties  of  lactic  acid  which  could  not  be 
distinguished  by  their  chemical  reactions  and  yet  whose 
physical  properties  were  not  identical.  It  is  found,  e.g., 
that  if  ordinary  lactic  acid  produced  by  fermentation  is  com- 
bined with  strychnine,  which  has  the  properties  of  a  base  and 
thus  forms  salts  with  acids,  and  if  the  strychnine  compound 
is  allowed  to  crystallise  slowly  from  solution,  the  first  portions 
of  salt  which  crystallise  out  will  differ  in  physical  properties 
from  those  which  are  obtained  later ;  the  most  important 
difference  is  in  regard  to  the  action  of  their  solutions  upon 
polarised  light. 

This  property  of  affecting  polarised  light  is  one  of  very 
great  importance,  and  its  study  has  led  to  great  extensions 
in  our  conceptions  of  molecular  structure.  Moreover,  the 
effect  of  certain  solutions  upon  polarised  light  affords  a 
means  of  determining  the  quantity  of  dissolved  substances 
present  in  solution.  For  all  these  reasons  it  is  important 
that  the  fermentation  chemist  should  possess  some  elementary 
knowledge  at  any  rate  of  the  subject  of  the  polarisation 
of  light,  and  of  the  practical  use  of  the  polarimeter,  and 
at  this  point,  therefore,  it  will  be  well  to  make  a  digression 
and  devote  some  space  to  the  theory  and  use  of  the  polari- 
meter. 


66      BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 

The  Theory  of  the  Polarimeter 

Light  is  one  of  the  primary  forms  of  energy  which  reaches 
this  earth  from  the  sun.  It  is  well  known  that  the  atmosphere 
of  air  surrounding  the  earth  becomes  more  and  more  attenuated 
as  the  distance  from  the  earth  increases,  and,  in  fact,  does  not 
extend  even  in  a  rarefied  form  to  a  greater  distance  than 
approximately  200  miles  from  the  surface  of  the  earth  ;  some 
medium  other  than  the  atmosphere  must,  therefore,  be  con- 
ceived of  as  a  means  of  transmitting  light  and  other  forms  of 
energy  from  the  sun  to  the  earth.  This  medium,  which  is 
thought  of  as  filling  all  space,  has  been  termed  the  luminiferous 
ether,  for  the  reason  just  mentioned.  Light  is  conceived  of 
physically  as  a  wave  motion  set  up  in  this  all-pervading  ether. 
The  essential  features  of  wave  motion  can  be  readily  studied 
by  carefully  observing  the  ripples  formed  when  a  pebble  is 
thrown  into  a  still  pool  of  water ;  it  will  be  seen  that  the 
water  does  not  move  as  a  whole  from  the  point  where  the 
pebble  plunged,  but  that  a  series  of  up  and  down  motions 
takes  place  in  successive  portions  of  water.  This  can  be 
easily  verified  by  throwing  in  a  few  light  match  stalks,  which 
will  be  seen  merely  to  move  up  and  down  and  not  to  approach 
the  edge  to  any  appreciable  extent.  The  same  essential 
feature  of  wave  motion  is  clearly  seen  when  the  wind  blows 
over  a  field  of  wheat ;  obviously,  here  individual  ears  of  wheat 
cannot  move  beyond  certain  limits,  and  there  is  only  a  to 
and  fro,  or  up  and  down  movement.  The  regular  movement 
within  certain  limits,  such  as  the  water  particles  or  the  ears  of 
wheat  exhibit  under  the  above  circumstances,  is  known  as  a 
vibration ;  the  extent  of  the  displacement  of  any  given  vibrat- 
ing particle  from  its  position  of  rest  is  known  as  the  amplitude 
of  the  vibration,  and  a  motion  such  as  we  have  been  con- 
sidering is  known  generally  as  wave  motion. 

A  wave  length  is  measured  from  crest  to  crest  or  from 
hollow  to  hollow  of  the  wave.  If  it  were  possible  to  set  two 


THE   POLARIMETER 


67 


waves  in  motion  in  the  same  direction,  the  crests  of  one 
corresponding  to  the  hollows  of  the  other,  the  vibrations  of 
the  wave  particles  would  obviously  neutralise  one  another 
and  all  motion  would  be  stopped.  Such  a  phenomenon  is 
termed  the  interference  of  waves.  This  interference  may  be 
complete,  as  in  the  case  just  mentioned,  which  would  occur 
when  one  wave  was  exactly  half  a  wave  length  behind  the 


1  Wave-length 


1  Wave-length 


FIG.  10.— WAVE  MOTION. 

other;  it  would  be  less  complete  if  the  one  wave  were  a 
quarter  or  three-quarters  of  a  wave  length  behind  the  other, 
whereas  a  difference  of  a  whole  wave  length  would  mean 
that  crest  reinforced  crest  and  hollow  reinforced  hollow,  and 
the  amplitude  of  the  vibrating  particles  would  be  doubled. 
This  is  clearly  seen  from  Fig.  10. 

It  has  been  found  that  the  physical  properties  of  light 
receive  their  full  explanation   if  it  is  assumed  that  light 

F2 


68       BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 


consists  in  a  wave  motion  of  the  luminiferous  ether;  the 
intensity  of  light  depends  on  the  amplitude  of  the  vibra- 
tions, the  colour  of  the  light  depends  on  the  wave  length, 
which  is  generally  referred  to  as  X. 

Now  in  an  ordinary  ray  of  light  the  waves  are  conceived 
of  as  following  each  other  in  very  rapid  succession,  with 
constantly  varying  planes  of  vibration ;  thus,  e.g.,  if  we 
imagine  a  wave  motion  (Fig.  11)  vibrating  in  one  instant  of 
time  parallel  to  AB,  the  following  wave  may  vibrate  along 
A'B'  and  the  next  along  A"B"  and  so 
A  //  on.  Such  a  ray  of  light,  therefore, 
/  /  has  no  two-sidedness,  that  is,  the 
/  /  plane  of  vibration  of  its  waves  can- 
/  not  be  determined  ;  on  the  other 
/  hand,  a  ray  of  light  all  of  whose  waves 

pass  through  AB  would  be  referable 
definitely  to  this  plane,  and  such  a 
ray  is  said  to  be  polarised. 

The  unassisted  eye  is  unable  to 
distinguish  between  polarised  light 
and  ordinary  light ;  it  is  conceivable 
that  if  we  could  construct  a  barred 
screen  of  sufficient  fineness  to  pre- 
vent the  passage  of  all  waves  except 
those  undulating  in  a  plane  parallel 
to  the  bars,  we  should  know  that  the 
light  passing  through  the  screen  was 
polarised  in  that  plane.  Now  the  structure  of  certain 
crystals  is  such  that  they  act  somewhat  in  the  manner  of 
such  a  screen,  and  compel  the  waves  of  light  passing  through 
them  to  vibrate  in  defined  planes.  Such  a  crystal  is  tour- 
maline ;  if  two  pieces  of  tourmaline  cut  parallel  to  the  long 
axis  of  the  crystal  are  placed  at  right  angles  one  to  the 
other,  opacity  results. 

For  an  explanation  of  this  property  it  will  be  necessary 


FIG.  11. 


THE    POLARIMETEK  69 

more  closely  to  consider  what  happens  when  a  ray  of  light 
passes  through  the  crystalline  medium,  and  for  this  purpose 
we  may  study  a  crystal  of  calcspar.  Cfclcspar  crystallises  in 
beautiful  rhombs  which  are  colourless  and  transparent ; 
unlike  a  rhomb  of  glass,  however,  we  shall  find  that  if  a 
crystal  of  calcspar  is  placed  over  an  inkspot  on  a  piece  of 
white  paper  two  inkspots  will  be  seen  :  this  is  known  as  the 
phenomenon  of  double  refraction.  Calcspar,  like  tourmaline, 
belongs  to  a  class  of  crystals  whose  density,  or  the  packing  of 
whose  particles,  is  different  in  different  directions.  Now  it 
is  a  simple  consequence  of  the  undulatory  theory  of  light,  that 
the  velocity  of  propagation  of  a  wave  varies  according  to  the 
density  of  the  medium,  and  further  that,  owing  to  this  altera- 
tion of  velocity  as  the  wave  passes  from  one  medium  to 
another  of  differing  density,  alteration  of  the  direction  of  the 
wave  takes  place.  As  the  density  of  the  calcspar  crystal  is 
different  in  different  directions  the  rays  vibrating  along  one  axis 
will  emerge  from  the  crystal  in  a  direction  differing  somewhat 
from  those  vibrating  in  the  plane  of  the  other  axis  ;  thus  we 
have  either  two  images  in  the  case  of  the  inkspot,  or,  if  we 
direct  a  ray  of  light  upon  the  face  of  the  crystal,  two  beams 
will  emerge.  A  further  phenomenon  is  observed;  if  we 
slowly  rotate  the  crystal  over  the  inkspot  one  spot  will  be 
found  to  maintain  its  position,  the  other  moves  round  with 
the  crystal,  and  similarly  with  the  two  rays  of  light.  The 
ray  whose  position  remains  unaltered  as  we  move  the  crystal 
is  known  as  the  ordinary  ray,  because  it  obeys  the  ordinary 
laws  of  refraction,  that  is,  a  constant  relation  always  obtains 
between  the  angle  of  incidence  and  the  angle  of  refraction. 
This  is  not  the  case  with  the  ray  giving  rise  to  the  movable 
image.  This  ray  is  therefore  referred  to  as  the  extraordinary 
ray. 

The  peculiarity  of  tourmaline  is,  that  while  it  breaks  up 
the  ray  into  two,  in  the  same  way  as  the  rhomb  of  calcspar 
does,  the  planes  of  vibration  being  likewise  at  right  angles 


70      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 


to  each  other,  it  has  the  property  of  diverting  the  ordinary 
ray  and  only  allowing  the  extraordinary  ray  to  pass,  whose 
vibrations  are  parallel  to  the  long  axis  of  the  crystal.  Such 
a  ray  is  polarised,  and  a  ray  which  is  polarised  at  right  angles 
to  this  direction  will  not  pass  through  the  crystal.  Con- 
sequently when  two  plates  of  tourmaline  identical  in  structure 
are  held  at  right  angles  no  light  passes,  and  it  follows  also 
that  a  plate  of  tourmaline  is  capable,  as  already  indicated,  of 
enabling  us  to  recognise  whether  a  ray  of  light  is  polarised  or 
not.  By  means  of  such  a  plate  of  tourmaline  it  can  be  shown 
that  the  rays  issuing  from  the  rhomb  of 
calcspar  are  polarised  at  right  angles  one 
to  the  other.  The  use  of  tourmaline  for 
studying  polarised  light  is  unsatisfactory, 
owing  to  the  green  colour  of  the  crystal 
and  the  consequent  loss  of  intensity  in  the 
light  passing.  Obviously  a  better  source 
of  polarised  light  would  be  one  of  the 
emergent  rays  from  calcspar.  If  means 
could  be  found  to  cut  off  one  of  these  rays, 
the  other  would  remain  as  a  ray  of  undi- 
minished  intensity  whose  direction  of  vibra- 
tion was  known.  Such  a  means  is  found 
in  the  Nicol  prism.  This  prism  is  made  by 
taking  an  elongated  rhomb  of  calcspar  and 
dividing  it  so  that  the  plane  of  division  aaf 
forms  an  angle  of  68°  with  the  vertical  sides 
of  the  rhomb  as  in  Fig.  12,  and  the  two  portions  are  then 
reunited  by  a  film  of  Canada  balsam.  If  now  a  ray  of  light 
cd  impinges  upon  the  shorter  face  of  the  prism,  double  refrac- 
tion will  take  place,  but  the  ordinary  ray  suffers  total  reflec- 
tion at  the  surface  of  the  Canada  balsam  and  so  passes  out  of 
the  crystal  in  the  direction  hi.  The  extraordinary  ray  def,  on 
the  other  hand,  suffers  no  refraction  on  the  surface  of  the 
Canada  balsam,  and  so  passes  on  with  its  direction  unaltered, 


9 

FIG.  12. 

NICOL  PRISM. 


THE   POLAKIMETER 


71 


and  we  have  thus  an  emergent  polarised  ray  fg.  In  practice 
the  absorption  of  the  ordinary  ray  is  effected  by  mounting  the 
Nicol  prism  in  a  black  mounting.  Such  a  prism  is  known  as 
a  polariser.  A  second  similar  prism  placed  parallel  to  the  first 
will  of  course  allow  the  ray  similarly  to  pass  through  ;  if  held 
at  right  angles,  on  the  other  hand,  it  will  act  in  a  similar  way 


FIG.  13. — POLABIMETEB  (Schmidt  and  Haensch  pattern). 

to  the  cross  tourmaline  plate,  i.e.,  as  a  hypothetical  barred 
screen,  and  will  extinguish  the  ray.  The  second  Nicol  prism, 
as  it  enables  us  to  recognise  the  polarised  ray  emerging  from 
the  polariser,  is  known  as  the  analyser.  An  instrument  fitted 
with  these  two  prisms,  together  with  suitable  lenses  for  observ- 
ing the  ray  and  with  a  tube  between  the  prisms  in  which 
substances  can  be  placed  to  observe  their  effect  on  the  polarised 
ray,  is  known  as  a  polarimeter  (Fig.  13).  Certain  details  of 


72      BACTERIOLOGICAL   AND    ENZYME   CHEMISTRY 


construction  and  methods  of  use  are  necessary  in  such  an 
instrument  if  accurate  results  are  to  be  obtained.  It  is  only, 
it  must  be  remembered,  with  rays  of  a  given  wave  length 
that  absolute  darkness  will,  as  a  rule,  be  obtained  when  the 
Nicols  are  crossed,  because  the  angle  of  refraction  is  different 
for  rays  of  differing  wave  length,  so  that  a  prism  that  was  cut 
in  such  a  way  that  the  ordinary  violet  ray  was  just  totally 
reflected  might  not  completely  cut  off  the  ordinary  ray  of  red 
light,  and  so  a  small  proportion  would  come  through  even 
when  the  Nicols  were  crossed.  It  is  better,  therefore,  always  to 
use  light  of  a  definite  wave  length,  and  for  this  purpose  the 
yellow  light  obtained  when  a  compound  of  sodium,  such  as 
a  bit  of  melted  carbonate  of  soda,  is 
•y  held  in  the  flame  of  a  Bunsen  burner,  is 
employed  ;  even  then  the  point  of  com- 
plete darkness  is  not  altogether  easy 
to  distinguish.  It  must  be  remem- 
bered that  unless  the  Nicols  are  exactly 
at  right  angles  a  certain  component  of 
the  vibration  will  pass  through,  in- 
creasing in  amount  in  proportion  as  the 
Nicols  become  more  nearly  parallel, 
B/  This  may  be  rendered  clearer  by  the; 

FIG.  14.  following  diagram;  keeping  to  the   an- 

alogy of  the  barred  screen,  if  we  assume- 
the  barred  screen  (Fig.  14)  placed  at  an  angle  to  the 
polarised  ray  vibrating  along  AB  this  vibration  will  be- 
resolved  into  two,  one,  ba,  parallel  to  the  bars  which  will  pass- 
through  and  the  other,  6c,  at  right  angles  which  will  be* 
extinguished.  Obviously  the  component  passing  through  will 
depend  on  the  angle  of  the  barred  screen  to  the  polarised  rayy 
and  in  the  diagram  db  represents  the  portion  of  light  passing 
through. 

In  order  sharply  to  define  the  point  of  darkness  in  the 
polarimeter,  half  the  field  of  view  of  the  instrument  is  taken 


THE    POLARIMETER  73 

Up  with  a  semicircular  plate  of  quartz,  cut  in  such  a  direction 
in  reference  to  the  optic  axis  of  the  crystal,  and  of  such  a 
thickness  that  it  retards  the  light  passing  through  by  half  a 
wave  length.  We  thus  obtain  two  beams  of  polarised  light, 
differing  in  phase  by  half  a  wave  length.  At  a  certain  angle, 
therefore,  interference  will  take  place,  as  explained  earlier,  in 
the  case  of  the  rays  passing  through  the  quartz  plate,  and  one 
side  of  the  field  will  appear  completely  black  and  the  other 
completely  bright.  A  position  can,  however,  be  found  when 
both  sides  are  completely  bright  or,  on  the  other  hand,  com- 
pletely dark.  By  a  differential  arrangement  of  this  sort  it  is 
much  easier  to  distinguish  the  alteration  of  illumination  which 
occurs  on  moving  the  analyser,  and  we  can  thus  make  exact 
observations  of  the  effect  upon  the  polarised  ray  of  substances 
placed  between  the  two  prisms. 

The  effect  of  the  quartz  plate  just  referred  to  is  not  only 
to  retard  the  wave  by  half  a  length,  it  also  alters  its  plane  of 
vibration,  and,  therefore,  if  such  a  plate  is  inserted  between 
crossed  Nicols,  a  certain  component  of  the  light  passing 
through  the  quartz  will  also  pass  through  the  analyser.  In 
order  to  produce  interference  and  consequent  darkness,  it  is 
necessary  to  rotate  the  analyser  through  a  certain  angle  in 
order  that  the  rays  passing  through  the  quartz  should  be 
brought  into  the  same  plane  as  those  passing  through  the 
analyser.  Other  substances  besides  quartz  are  capable  of 
altering  or  rotating  the  plane  of  polarisation,  even  when  their 
solutions  are  placed  in  the  polarimeter,  between  the  two  prisms. 

For  observing  the  effect  of  such  solutions,  a  glass  tube 
closed  by  thick  glass  discs  and  screw  caps  is  made  use  of, 
tubes  of  different  lengths  being  used  according  to  the 
concentration  of  the  liquid  to  be  examined. 

In  Fig.  15 l  are  given  the  essentials  of  construction  of 
the  Laurent  polarimeter. 

1  Adapted  by  permission  from  Dr.  A.  Findlay's  Practical  Physical 
Chemistry. 


74      BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 

L  represents  a  Bunsen  flame  in  which  is  inserted  a  bead 
of  carbonate  of  soda  to  obtain  monochromatic  light.  A  is 
a  lens  to  render  the  rays  of  light  parallel,  B  is  the  polariser, 
C  and  C'  the  quartz  plate.  0  is  the  tube  containing  the 
solution  to  be  observed,  D  the  analyser,  and  EF  the 
telescope. 

The  Lippich  model  of  polarimeter  differs  only  from 
the  Laurent  in  having  a  small  Nicol  prism  to  produce  the 
half  shadow  instead  of  the  quartz  plate.  The  outward 
appearance  of  the  two  instruments  is  identical,  and  is  shown 
in  Fig.  13.  In  both  cases  a  light  filter  consisting  either  of 
a  solution  of  potassium  bichromate  or  a  crystal  of  this  salt 
is  placed  in  front  of  the  lens  A. 


n>    „    0 


FIG.  15. — DIAGRAM  OF  POLABIMETEB. 

After  passing  through  the  polariser  and  the  quartz  plate, 
the  light  emerges  as  two  beams  of  polarised  light,  differing 
in  phase  by  half  a  wave  length.  If  the  polariser  is  rotated 
so  that  the  plane  of  polarisation  forms  an  angle  (0)  with  the 
quartz  plate,  the  planes  of  polarisation  of  the  two  beams 
will  also  be  inclined  at  an  angle,  equal  to  20.  This  is  the 
half-shadow  angle.  On  rotating  the  analyser,  a  position 
will  be  found  at  which  the  one  beam  will  be  completely, 
the  other  only  partially,  extinguished.  The  one  half  of  the 
field  of  view,  therefore,  will  appear  dark,  while  the  other 
half  will  still  remain  light. 

The  details  of  practical  use  of  the  polarimeter  will  be  better 
considered  in  a  later  chapter ;  it  should  here  be  stated  that 
the  angle  of  rotation  of  the  analyser  can  be  accurately  measured 


THE    POLAEIMETER  75 

on  a  circular  scale.  Further,  those  substances  whose  solutions 
give  a  right-hand  twist  to  the  plane  of  polarisation  looked  at 
from  the  eye  of  the  observer  are  known  as  dextro-rotatory, 
those  which  twist  it  in  the  opposite  direction  are  known  as 
Iccvo-rotatory.  The  property  of  rotating  the  plane  of  polarisa- 
tion in  this  way  is  known  as  optical  activity. 


The  Relations  between  Optical  Activity  and 
Molecular  Structure 

The  effect  of  a  solution  of  tartaric  acid  and  sundry  other 
organic  substances  upon  the  plane  of  polarisation  was  observed 


FIG.  16. — EXANTIOMOEPHOUS  CRYSTALS. 

first  by  Biot  in  1838,  and  he  also  showed  that  racemic  acid, 
which  has  the  same  composition  as  tartaric  acid,  does  not 
possess  this  optical  property.  This  was  confirmed  by  Mitscher- 
lich  in  1844,  but  it  was  Pasteur  in  1848  who  made  the  first 
great  step  in  unravelling  the  cause  of  this  difference.  He  found 
that  by  careful  crystallisation  of  sodium  ammonium  racemate, 
a  salt  which  in  itself  has  no  action  upon  polarised  light,  it  was 


76      BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 

possible  to  pick  out  crystals  which  differed  from  each  other 
in  structure  only  as  the  image  in  the  mirror  differs  from  its 
real  object,  or  as  the  right  hand  differs  from  the  left ;  thus 
certain  small  faces  could  be  seen  on  one  set  of  crystals  on  the 
right  hand,  whereas  the  corresponding  set  of  faces  on  the 
other  crystal  were  on  the  left  (Fig.  16). l  One  of  these  crystal- 
line forms  turned  the  plane  of  polarisation  to  the  right,  the 
other  to  the  left,  and  the  crystals  were  derivatives  respectively 
of  dextro  and  Isevo  tartaric  acid.  When  these  two  forms  were 
crystallised  together  to  form,  racemate,  optical  inactivity 
resulted. 

The  optical  difference  in  these  two  modifications  of  tartaric 
acid  was  here  clearly  referred  to  a  difference  in  crystalline 
form.  Pasteur  at  the  same  time  suggested  that  the  cause  of 
the  difference  lay  deeper,  viz.,  in  the  actual  molecular  structure 
of  the  two  acids,  that  is  upon  the  arrangement  of  the  atoms 
in  their  respective  molecules.  This  illuminating  suggestion 
of  Pasteur  found  its  full  development  in  the  theory  of  van't 
Hoff  and  Le  Bel. 

These  investigators  found  that  every  optically  active 
substance  contained  within  its  molecule  a  carbon  atom  to 
which  were  attached  four  dissimilar  groups ;  such  a  carbon 
atom  they  referred  to  as  an  asymmetric  carbon  atom.  In  order 
to  explain  why  such  a  grouping  should  give  rise  to  actual 
physical  asymmetry  they  suggested  that  the  arrangement  of 
the  groups  must  be  considered  as  occurring  in  three  dimen- 
sions. Now  all  investigation  goes  to  show  that  the  four  com- 
bining units,  bonds,  directions  of  affinity,  or  whatever  term 
may  be  used  to  express  what  is  symbolised  by  the  four  lines 
attached  to  the  C  in  the  formula  of  an  organic  compound,  are 
strictly  equivalent.  The  only  way  to  express  this  fact  in 
three  dimensions  is  to  consider  the  carbon  element  as  being 
at  the  centre  C  of  a  regular  tetrahedron  (Fig.  17),  with  its 

1  Reproduced  from  Dr.   C.   A.  Reane's  Modern  Organic  Chemistry,  by 
permission  of  the  publishers. 


SPACE-ISOMERISM 


77 


four  affinities  (shown  by  the  full  lines)  directed  towards  the 
four  solid  angles  (shown  dotted),  thus  : — 

a 


b  c, 

FIG.  17. — THE  TETRAHEDRON  OP  THE  CARBON  ATOM,  IN  PERSPECTIVE 

If  now  the  four  different  groups  be  attached  at  each  of  the 
four  angles  of  the  tetrahedron,  say,  a,  b,  c  and  d,  it  will  be  seen 
that  a  right-hand  and  left-hand  arrangement  can  be  produced 
thus  (Fig.  18)  :— 


c       c  b 

FIG.  18. — RIGHT-  AND  LEFT-HAND  ARRANGEMENT.  l 

1  The  following  useful  suggestion  is  taken  from  F.  J.  Moore's  Out- 
lines of  Organic  Chemistry  (p.  150).  The  student  can  readily  con 
struct  tetrahedral  models  from  paste- 
board in  the  following  manner.  An 
equilateral  triangle  is  drawn,  each  of 
the  three  sides  bisected  and  the 
middle  points  joined  up  as  shown  in 
Fig.  19.  The  large  triangle  is  cut 
out  and  the  corners  folded  along  the 
dotted  lines  of  the  smaller  one ;  the 
points  at  the  top  are  joined  up  by 
fine  wire  or  gum  paper.  By  marking 
the  corners  differently  the  simpler 
relations  of  space  isomerism  can  easily 
be  studied.  FIG.  19. 


78       BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

These  two  arrangements  cannot  be  symmetrically  super- 
posed ;  they  are  what  is  known  as  enantiomorphous.  Such  a 
difference  can  only  occur  when  all  four  replacing  groups  are 
different,  that  is  to  say,  when  there  is  an  asymmetric  carbon 
atom  in  the  molecule,  as  in  the  example  just  given,  a,  6,  c 
and  d,  representing  the  replacing  groups. 

If  the  substitution  takes  place  by  groups  a,  a,  b,  c,  thus 
(Fig.  20)- 


a  CL      a,  & 

FIG.  20. 

it  is  easy  to  see  that  by  turning  the  tetrahedron  the  two 
forms  are  superposable,  so  that  there  is  no  essential  differ- 
ence between  them. 

We  are  now  in  a  position  more  fully  to  understand  how 
there  exists  more  than  one  form  of  lactic  acid  and  of  tartaric 
acid,  even  though  the  constitutional  formulae  as  determined 
by  chemical  reactions  may  be  the  same ;  thus  in  the  last 
chapter  it  was  shown  that  fermentation  lactic  acid  or  a-lactic 
acid  had  the  formula  CH3  C  HOHC03H,  the  centre  carbon 
atom  is  attached  to  four  different  groups  and  is  therefore 
asymmetrical. 

It  is  possible,  therefore,  to  obtain  a  dextro,  a  Icevo,  and  an 
inactive  lactic  acid.  As  already  stated,  these  different  forms 
can  be  obtained  by  fractional  crystallisation  of  the  strychnine 
salts,  and  also  from  the  zinc  salts.  In  writing  what  are  known 
as  stereo-chemical  formulae,  i.e.,  formulae  expressive  of  the  space 
arrangement  of  the  atoms  in  the  molecule,  it  is  convenient, 


SPACE-ISOMERISM 


79 


instead  of  drawing  the  actual  tetrahedral  perspective,  to 
write  a  projection  of  the  formula  on  the  plane  of  the  paper 
(Fig.  21),  the  asymmetric  carbon  atom  being  distinguished  by 
a  circle  round  it  or  by  heavy  type.  Thus  the  dextro  and 
Isevo  lactic  acid  can  be  written  as  follows  : — 


CH< 


HO 


C02H     C02H 
Fio.  21. 


OH 


or  in  projection  looked  at  from  above  :  — 

CI^  CHg 

HOCH  HCOH 


The  case  of  the  tartaric  acids  is  somewhat  more  complex,  as 
there  are  here  two  asymmetric  carbon  atoms  in  the  molecules  ; 
the  following  configurations  are  therefore  possible  :  — 


(1) 
COOH 


(2) 
COOH 


(3) 
COOH 


H-C-OH  HO-C-H  H— C-OH 

I  i  I 

HO— C-H  H— C-OH  H-C-OH 

I  I  I 

COOH  COOH  COOH 


80      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

If  the  upper  and  lower  halves  of  each  molecule  be  con- 
sidered, it  can  be  seen  that  in  formulae  (1)  and  (2)  the  upper 
and  the  lower  halves  are  not  mirror  images  of  each  other ;  both 
upper  and  lower  halves  therefore  represent  the  same  optical 
isomer.  Which  is  actually  the  formula  for  dextro  or  for  Ia3vo 
tartaric  acid  is  a  matter  of  indifference,  but  both  will  be 
optically  active  and  their  mixture  will  form  racemic  acid. 
On  the  other  hand,  in  formula  (3)  the  upper  and  lower  halves 
of  the  molecule  are  related  as  object  and  mirror  image  and 
represent  therefore  optically  opposite  groups.  We  have  here 
intra-molecular  compensation  and  such  an  acid  is  optically 
inactive  ;  it  is  known  as  meso-tartaric  acid.  These  relations 
of  the  four  acids  can  be  summarised  as  follows,  d  and  I 
being  the  opposite  optical  activities  of  the  two  portions  of  the 
molecule  : — 

d  I  did 

II  III 

d  I  d        I  I 

Dextro-rotatory  Lsevo-rotatory  Eacemio  Meso-tartario 

The  cases  of  the  lactic  and  tartaric  acids  will  serve  to 
illustrate  the  character  of  the  isomerism  which  is  to  be  found 
in  more  complex  substances  and  especially  among  the  sugars, 
a  field  of  organic  chemistry  which  has  been  worked  out  in 
great  detail,  mainly  by  Emil  Fischer. 

Before  passing  on  to  a  brief  sketch  of  the  chemistry  of  the 
sugars  it  is  important  that  the  reader  should  understand  that 
although  the  conception  of  space-isomerism  owes  its  origin 
to  observations  connected  with  the  optical  activity  of  sub- 
stances, yet  once  the  spatial  arrangement  of  the  atoms  is 
conceded,  and  the  carbon  atom  considered  always  as  being 
the  centre  of  a  tetrahedral  space,  a  number  of  conclusions 
follow,  quite  unconnected  with  the  subject  of  optical  activity. 
Remarkable  relations  have  been  found  to  obtain  between  the 
structure  of  compounds  and  their  stability,  which  become 


SPACE-ISOMERISM  81 

clear  when  actual  models  are  built  up  in  which  the  tetrahedral 
arrangement  of  the  carbon  affinities  is  retained. 

The  explosive  nature  of  acetylene  derivatives  appears  to 
bear  some  relation  to  the  space  formula  for  carbon,  as  can 
be  seen  by  the  following  space  formula  for  acetylene  : — 


C 


There  is  evidently  a  condition  of  strain  between  the  two  carbon 
atoms,  the  line  of  attraction  not  being  direct  between  carbon 
and  carbon  as  in  the  case,  e.g.,  of  a  saturated  compound,  the 
space  formula  for  ethane  being — 


Moreover,  when  a  series  of  carbon  atoms  is  thus  joined  into 
a  ring,  it  is  found  that  a  differing  amount  of  strain  is  put  upon 
the  bonds,  considered  for  the  moment  as  semi-rigid  links, 
according  as  the  ring  contains  a  different  number  of  carbon 
atoms.  Thus  the  pentamethylene  ring,  which  may  be  shortly 
written  thus  — 


V 
CH3 


82       BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

is  found  to  be  the  most  stable  arrangement,  while  rings  of  more 
than  seven  atoms  are  difficult  if  not  impossible  to  prepare. 

Further  it  has  been  found  that  differences  in  the  constitu- 
tion of  certain  nitrogen  compounds  find  their  best  explanation 
on  the  assumption  of  a  varying  arrangement  in  three-dimen- 
sional space  of  the  groups  attached  to  the  nitrogen. 

Recent  researches  by  Pope  and  by  Kipping  have  extended 
the  idea  of  space-isomerism  to  the  derivatives  of  silicon,  tin 
and  other  elements.  Pope  has  also  recently  developed  a 
theory  according  to  which  the  crystalline  form  of  every  sub- 
stance is  minutely  dependent  upon  its  molecular  structure,  thus 
confirming  Pasteur's  original  suggestion,  while  approaching 
the  subject  from  the  side  of  the  molecule  rather  than  from  the 
side  of  the  crystal. 

These  references  to  recent  developments  in  space- 
isomerism,  or  stereo-isomerism,  have  been  made  because 
remarkable  relationships  have  been  found  to  exist  between 
the  actions  of  enzymes  and  the  stereo-chemical  configura- 
tion of  the  molecules  of  the  substances  upon  which  they 
act ;  in  fact,  a  very  common  method  for  obtaining  an 
active  substance  from  the  inactive  mixture,  which  results 
from  ordinary  methods  of  preparation,  consists  in  submitting 
such  an  inactive  mixture  to  the  action  of  certain  organisms 
or  the  enzymes  secreted  by  them,  when  one  modification  is 
generally  attacked  at  a  different  rate  from  the  other.  Further 
references  to  this  subject,  and  also  to  the  theory  of  the  natural 
production  of  optically  active  substances,  will  be  made  later. 
We  have  now  to  consider  a  class  of  substances  which  perhaps 
more  than  any  other  serve  as  the  basis  for  extremely  important 
fermentation  processes,  viz.,  the  sugars. 

INTRODUCTION  TO  THE  CHEMISTRY  OP  THE  SUGARS 

The  term  '  sugar '  as  popularly  understood  generally  refers 
to  cane  sugar  or  preparations  made  from  it.  Chemically 
speaking,  however,  the  word  has  a  much  wider  application, 


THE  CHEMISTRY  OF  THE  SUGARS        83 

and  comprises  a  large  number  of  substances  which  are  classified 
as  carbohydrates.  A  carbohydrate  is  a  compound  of  carbon 
with  hydrogen  and  oxygen,  the  last  two  elements  being  in 
the  proportion  to  form  water ;  the  simplest  carbohydrate, 
therefore,  would  be  CH20.  As  a  matter  of  fact  this  is 

H 

formaldehyde    with     the    constitutional     formula      |  /H. 

<0 

There  is  considerable  evidence  for  believing  that  the  great 
family  of  carbohydrates  as  found  in  nature  may  originate 
in  the  first  instance  from  formaldehyde.  A  suggestion  of  this 
sort  appears  reasonable  even  when  we  simply  look  at  the 
empirical  formulae  of  the  three  great  classes  of  carbohydrates 
generally  termed  the  mono-saccharoses,  the  di-saccharoses, 
and  the  poly-saccharoses.  These  terms  are  not  altogether 
satisfactory,  because  the  so-called  mono-saccharoses  include  a 
large  number  of  substances  of  differing  molecular  weight  and 
molecular  formulae,  all  of  which  have  the  general  formula 
CnH2nOn ;  the  best  known  members  of  this  group  are,  however, 
the  hexoses  of  the  general  formula  C6H1206,  and  the  di- 
saccharoses  are  so  named  because  by  addition  of  a  molecule  of 
water  they  give  rise  to  two  molecules  of  a  hexose.  The  general 
formula,  therefore,  of  the  di-saccharoses  is  C12H220U.  The 
poly-saccharoses  are  much  more  complicated  substances  whose 
molecular  formulae  are  unknown,  but  they  have  the  general 
formula  (C6H1005)n. 

Taking  now,  for  the  sake  of  comparison,  an  even  number 
of  carbon  atoms  in  all  three  cases  we  get  the  following  relation- 
ship : — 

mono-saccharoses  (two  or  more  molecules)  C^H^O^ 
di-saccharoses  Ci2H22On 

poly-saccharoses    (n  =  2)  C12H20010 

We  can  thus  see  at  a  glance  how  these  important  naturally 

02 


84       BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

occurring  groups  are  generally  related.  The  mono-saccharoses 
can  evidently  be  considered  as  built  up  by  the  combination 
of  a  number  of  molecules  of  formaldehyde.  By  elimination  of 
water  from  two  or  more  molecules  of  mono-saccharoses,  a 
di-saccharose  results,  and  by  further  elimination  of  water 
poly-saccharoses  are  obtained.  Among  the  more  important 
members  of  these  various  groups  may  be  mentioned,  among 
the  saccharoses,  grape  sugar  or  glucose,  and  fruit  sugar  or 
Icevulose  ;  among  the  di-saccharoses,  cane  sugar  (or  beet  sugar, 
which  has  the  same  composition)  and  milk  sugar ;  among  the 
poly-saccharoses,  starch  and  cellulose. 

Our  knowledge  of  the  molecular  structure  of  carbohydrates 
is  naturally  greatest  in  regard  to  the  simplest  group,  viz.,  the 
mono-saccharoses,  and  inasmuch  as  sugars  belonging  to  this 
group  are  produced  by  the  addition  of  the  elements  of  water 
to  both  di-saccharoses  and  poly-saccharoses,  it  is  evident 
that  a  knowledge  of  the  simpler  substances  must  be  of  great 
help  towards  the  ultimate  unravelling  of  the  much  more 
complicated  chemistry  of  such  substances  as  starch  and 
cellulose.  We  may,  therefore,  proceed  to  consider  the 
general  properties  of  the  carbohydrates  of  this  group. 

Mono-saccharoses. — The  members  of  this  group  of 
carbohydrates  may  be  described  as  the  first  oxidation  products 
of  alcohols  containing  more  than  one  carbon  atom  each  of 
which  has  an  OH  group  attached. 

The  simplest  alcohol  is  of  course  methyl  alcohol,  HCH2OH, 

H 

its  first  oxidation  product  is  fwmaldehyde,    \  /H,  and,  as 

0 

already  stated,  formaldehyde  is  the  simplest  carbohydrate, 
and  may  be  looked  upon  as  the  basal  substance  of  the  sugars, 
although  it  does  not  itself  exactly  fall  within  the  above 
definition  of  a  mono-saccharose. 


THE    CHEMISTRY    OF   THE    SUGARS  85 

The  first  alcohol  which  fulfils  the  definition  given  above 

CH2OH 

is   glycol,  ,  so    named    from    its    sweet   taste,  and 

CH9OH 

CH2OH 
the  corresponding  sugar  is  glycol-aldehvde,  | 

CHO 

The  next  member  of  the  series  of  alcohols  is  glycerol  or 
ordinary  glycerine,  whose  sweet  taste  is  a  matter  of  common 

CH2OH 

knowledge.    The  formula  for  glycerol  is  CHOH,   from  which 

CH2OH 

it  is  evident  that  two  first  products  of  oxidation  can  be 
obtained,  that  is,  the  CH2OH  group  may  oxidise  to  an  aldehyde 

/H 
group  — C         or   the    CHOH   to  ^rCO,  the    characteristic 

^0 
ketonic  group,  and  thus  we  have  the  following  relationship  : — 

CH2OH  CH9OH  CH2OH 

I  I  I 

CHOH  CHOH  CO 

I  I  I 

CH2OH  CHO  CH2OH 

Glycerol  Glycerolaldehyde  Di-oxy-acetone 

These  last  two  substances  are  the  first  representatives  of 
two  important  groups  of  the  mono-saccharoses,  viz.,  the 
aldoses  and  the  kctoses,  the  former  containing  the  aldehyde 

/" 

group  — C       and   the   latter   the    ketonic   group   ^>C=0. 

P 

The  sugars  it  will  be  noted  end  in  ose,  and  according  to  the 

number  of  carbon  atoms  they  are  referred  to  as  biose,  triose, 


86       BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 


etc.,  while  the  corresponding  alcohols  are  generally  given  the 
termination  ite  or  ol ;  we  have,  therefore,  the  following  series 
of  alcohols  and  corresponding  sugars,  the  aldose  and  ketose 
form  being  given  in  each  case  : — 


CH2OH 
CH2OH 

Methylene  glycol 

CHoOH 
CHO 


CH2OH 

CHOH 

CH2OH 

Glycerol 


CHOH      CO 


Glycol  aldehyde 
or 


CH2OH 
CHOH 


CH2OH 

Tetrite 


CHO 

Aldose 


CH2OH 

Ketose 


Glycerose  or  Triose 

CH,OH  CH2OH 


(CHOH)3 
CH2OH 

Pentite 


(CHOH)4 


CH2OH 

Hexite 


CH2OH    CH2OH        CH2OH    CH2OH        CH2OH    CH2OH 

I  I  II  II 

(CHOH)2  CHOH        (CHOH)3  (CHOH)2    (CHOH)4  (CHOH)3 


CHO        CO 


CHO        CO 


CHO        CO 


Aldotetrose       CH2OH         Aldopentose       CH2OH        Aldohexose          CH2OH 

Ketopentose 


Ketotetrose 


Ketohexose 


Sugars  containing  seven,  eight  or  nine  atoms  of  carbon 
have  been  obtained,  viz.,  heptoses,  octoses  and  nonoses,  but 
the  above  list  includes  those  sugars  which  are  met  with  in 


THE    CHEMISTRY    OF   THE    SUGARS  87 

nature  and  which  have  a  practical  as  well  as  a  scientific  import- 
ance, and  it  is  unnecessary,  therefore,  to  extend  it  further. 

All  these  aldoses  and  ketoses  have  certain  general  reactions 
by  which  they  can  be  readily  identified  :  — 

1.  They  are  readily  reduced  by  nascent  hydrogen  to  the 
corresponding  alcohols,  the  aldehyde  and  ketone  group  being 
attacked,  thus  :  — 

CH2OH        CE^OH  CILjOH 

I  I 

=  (CHOH)4     (CHOH)3  (CHOH)S 

I  I  |  +H,=    | 

CHO  CH2OH        CO  CHOH 

I  I 

CH2OH  CH2OH 

2.  Dilute  nitric  acid  oxidises  aldoses  to  oxycarboxylic  acids, 
thus  :  — 

CH2OH(CHOH)4CHO  +  0  =  CH2OH(CHOH)4C02H 

With  ketoses  the  chain  is  broken  on  oxidation  :  — 

CH2OH(CHOH)3COCH2OH  +  30  = 

C02H(CHOH)2C02H  +  C02HCH2OH 

Tartario  acid  Glycollic  add 

3.  Phenyl-hydrazine  converts  both  aldoses  and  ketoses  into 
hydrazones  and  finally  into  osazones  by  the  following  impor- 
tant reactions  :  — 

Aldose  — 
i.  - 


Aldose  group  Phenyl-hydrazine 

=   -CHOHCH=N-NHC6H5  +  H20 

Hydrazone 

u,  —  CHOHCH=N-NHC6H5  +  C6H5NHNH2 

=    -COCH=N-NHC6H5  +  C6H5NH2-f 

Carbonyl  compound 


88      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 


iii.  -CCH-CH^N-NHCeHg  + 
=   -CCH=NNHC6H5 


NNHC6H5 

Osazone 


Ketose  — 


i.  -COCH2OH  +  H2NNHC6H5  =   -CCH2OH 

Ketose  group  ||  + 

NNHC6H5 

Hydrazone 

ii.  -CCHjOH 

||  +  C6H5NHNH2 

NNHC.H, 

—  CCHO 

=     ||  +C6H5NH3  + 

NNHC6H5 

Aldehyde  compound 

in.  -CCHHO+'Kj-NNHC6H5 

II 
NNHC6H5  -CCH=NNC6H5 


Osazone 

In  both  cases  it  will  be  seen  that  the  osazone  grouping  is 
the  same  whether  derived  from  an  aldose  or  a  ketose  ;  if  two 
sugars,  therefore,  yield  different  osazones  it  is  a  proof  that  they 
differ  in  constitution  in  portions  of  the  molecule  other  than  the 
aldose  or  ketose  group. 

As  the  osazones  are  mainly  soluble,  crystallisable  compounds 
with  definite  melting-points,  they  are  exceedingly  useful  both 
in  isolating  and  identifying  the  various  sugars. 

Sugars  can  be  obtained  from  osazones  by  the  action  of 
strong  hydrochloric  acid  which  eliminates  the  phenyl-  hydra- 


THE  CHEMISTRY  OF  THE  SUGARS        89 

zine  group,  forming  a  ketone-aldehyde  which  on  reduction 
yields  a  sugar.  Thus  in  the  case  of  glucose-osazone  we  have 
the  following  sequence  of  compounds  : — 

CH2OH  CH2OH  CH2OH 

I  I  I 

(CHOH)3  (CHOH)3  (CHOH)3 

C=NNHC6H6  CO  CO 

I  i  I 

CH=N-NHC6H5       CHO  CH2OH 

Glucose-osazone  Glucose-osone  Ketose 

4.  By  successive  treatment  with  hydrocyanic  acid  and 
hydrochloric  acid,  acids  are  formed  as  follows  : — 

OH 
R-CHO  +  HCN  =  R-CH/ 

XCN 
OH 

K-CH/        +  2H20  =  R-CHOHC02H  +  NH3 
XCN 

On  reduction  by  nascent  hydrogen  of  the  acid  so  formed, 
an  aldose  containing  one  more  carbon  atom  than  the  sugar  first 
taken  is  produced. 

R-CHOHCOOH  +  H,  =  R-CHOHCHO  +  H20 

It  will  be  noted  that  an  additional  carbon  atom  is  in  this  way 
attached  to  the  chain,  and  so  a  means  is  afforded  of  producing 
a  series  of  sugars,  each  member  of  which  contains  one  carbon 
atom  more  than  the  preceding  one.  By  this  method  the  sugars 
above  referred  to  containing  seven,  eight,  and  nine  carbon 
atoms  have  been  produced.  This  reaction,  which  is  of  great 
importance,  was  discovered  by  Kiliani,  whose  name  it  bears. 

5.  Inasmuch  as  the  aldoses  and  ketoses  are  capable  of 
oxidation,  they  themselves  act  as  reducing  agents,  and  so  they 


90      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

are  capable  of  reducing  certain  metallic  salts  with  production 
of  the  metal  or  a  lower  oxide  of  the  metal.  Thus,  an  ammoniacal 
solution  of  nitrate  of  silver  when  warmed  with  ordinary  glucose 
(grape  sugar)  yields  a  brilhant  mirror  of  silver. 

On  warming  with  an  alkaline  solution  of  copper  potassium 
tartrate  (known  as  Fehling  solution)  a  red  precipitate  of 
cuprous  oxide,  Cu20,  is  produced.  This  is  an  important 
reaction  which  can  be  used  for  the  quantitative  determina- 
tion of  the  amount  of  reducing  sugar  present  in  a  solution. 

Stereo-isomerism  of  the  Ketoses  and  Aldoses. — If  the 

formula  for  an  aldose  or  ketose  containing  more  than  two 
carbon  atoms  be  carefully  studied,  it  will  be  seen  that  in  most 
cases  one  or  more  asymmetric  carbon  atoms  are  present  in  the 
molecule.  Thus  to  take  the  simplest  case,  viz.,  the  aldose 
form  of  glycerose  CH2OH  C  HOHCHO,  the  centre  carbon 
atom  is  combined  with  four  different  atoms  or  groups,  and 
consequently  a  right-hand  and  left-hand  and  also  an  inactive 
form  of  this  sugar  are  capable  of  existence. 

In  the  case  of  a  hexose  the  number  of  asymmetric  carbon 
atoms,  and  consequently  of  right-hand  and  left-hand  forms, 
becomes  considerable  ;  thus  a  ketohexose  contains  three  asym- 
metric carbon  atoms  : — 

CH2OH  C  HOH  C  HOH  C  HOHCOCH2OH 

An  aldohexose  contains  four  asymmetric  carbon  atoms : — 
CH2OH  C  HOH  C  HOH  C  HOH  C  HOHCHO 

The  separation  and  identification  of  the  large  number  of 
possible  ketohexoses  and  aldohexoses  is  a  very  complicated 
task  ;  mainly  by  the  exertions  of  Emil  Fischer  and  his  pupils 
it  has  to  a  great  extent  been  accomplished. 

It  would  lead  too  far,  and  would  be  foreign  to  the  subject 
of  the  present  work,  to  consider  the  methods  of  preparation  and 
identification  of  all  these  compounds  in  detail,  It  may  be 


THE   CHEMISTRY   OF  THE   SUGARS  91 

stated  generally  that  the  researches  have  been  conducted  on 
the  following  lines  : — 

(a)  The  production  of  aldoses  or  ketoses  from  naturally 
occurring  substances  whose  ordinary  constitutional  formulae 
and  specific  optical  activity  are  known.  Thus  dextro  mannite 
or  mannitol,  which  can  be  readily  obtained  from  naturally 
occurring  manna,  yields  dextro  mannose  on  oxidation. 

(6)  The  building  up  of  sugars  from  compounds  of  known 
constitution  by  Kiliani's  reactions.  Thus  arabinose  yields 
eventually  Z-glucose  or  Z-mannose  as  follows  : — 

CH2OH  CH2OH  CHjOH  CH2OH  CH2OH 

(CHOH)3  -»    (CHOH)3     and     (CHOH)3     ->     (CHOH)3   and    (CHOH)3 

CHO          H— C— OH        HO— C— H  H— C— OH      HO— C— H 

I  I  I  I 

ON  CN  CHO  CHO 


Arabinose  Arabinose  cyanhydrin  /-Glucose  and  /-Mannose 

(c)  Passing  from  ketose  to  aldose  or  vice  versa  by  means  of 
the  phenyl-hydrazine  compounds ;  the  example  already  given 
on  p.  87  will  illustrate  this. 

(d)  Kesolving  inactive  compounds  by  fractional  crystallisa- 
tion of  suitable  salts,  or  by  the  action  of  enzymes. 

It  will  be  useful  briefly  to  describe  the  chief  properties  of 
one  or  two  well-known  members  of  the  hexose  group  and  of 
certain  related  compounds,  which  are  of  interest  from  the  fact 
that  they  have  been  used  as  a  means  of  differentiating  certain 
bacteria  one  from  another,  by  the  capacity  or  otherwise  which 
these  may  possess  of  fermenting  the  substance  in  question. 

Mannite  or  Mannitol  has  already  been  referred  to ;  it  is  a  6 
carbon  alcohol  of  the  general  formula  d^OHCCHOH^CILjOH. 
It  occurs  in  manna,  which  consists  of  the  evaporated  sap 
exuding  from  various  species  of  ash  cultivated  in  southern 
Europe ;  it  also  occurs  widely  distributed  in  the  vegetable 


92      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

kingdom,  e.g.,  in  the  roots  of  celery,  in  the  sugar  cane  and  in 
various  algae  and  fungoid  growths  ;  it  can  be  extracted  from 
manna  by  boiling  out  with  dilute  alcohol  and  recrystallising. 
It  has  a  pleasant  sweet  taste,  and  is  sparingly  soluble  in  cold 
but  readily  in  hot  water. 

Dulcite  or  Dulcitol  is  isomeric  with  mannite  ;  it  occurs  in 
Madagascar  manna,  from  which  it  can  be  extracted  by  hot 
water.  Dulcite  is  not  so  sweet  tasting  as  manna  and  is  less 
soluble  in  water.  It  is  important  to  note  that  both  mannite 
and  dulcite  yield  secondary  hexyl  iodide,  CH3(CH2)3CHICH3, 
when  treated  with  concentrated  hydriodic  acid. 

Glucose,  also  known  as  dextrose  or  grape  sugar,  is  found  in 
large  quantities  in  grapes.  As  already  mentioned,  it  is  a 
characteristic  member  of  the  aldohexose  group  ;  it  occurs 
frequently,  together  with  laevulose,  also  called  fructose  or  fruit 
sugar,  which  is  the  corresponding  ketohexose  (see  pp.  84,  85),  in 
the  juice  of  sweet  fruits  and  in  honey.  The  mixture  of  the  two, 
dextrose  and  laevulose,  is  generally  known  as  invert  sugar. 
Dextrose  and  laevulose  can  be  obtained  from  invert  sugar  by 
the  crystallisation  of  the  dextrose  from  an  alcoholic  solution  ; 
or  by  the  preparation  of  an  insoluble  lime  compound  of  laevu- 
lose,  which  is  decomposed  by  suspending  it  in  water  and 
passing  carbon  dioxide  through  the  mixture.  On  filtering 
off  the  calcium  carbonate  and  evaporating  the  filtrate  the 
laevulose  is  obtained  as  a  syrup  which  can  be  crystallised 
from  alcohol. 

Inosite. — This  is  a  somewhat  rare  substance  l  obtained  as 
an  extract  from  the  heart  or  lungs  of  the  ox  by  a  complicated 
process ;  it  crystallises  from  dilute  alcohol  with  2H20. 

Galactose  is  a  sugar  formed  along  with  dextrose  when 
milk  sugar  is  boiled  with  dilute  sulphuric  acid ;  it  is  also 
formed  when  gum  arabic  is  similarly  treated.  It  is  less  soluble 

1  Formerly  classed  as  a  sugar  from  its  empirical  composition  ;  it  is 
however  a  ring  compound,  viz.,  hexa-hydroxy-cyclohexane. 


THE  CHEMISTRY  OF  THE  SUGAES        93 

than  dextrose  and  can  therefore  be  separated  from  it  by 
crystallisation. 

The  relationship  of  these  and  other  related  sugars,  together 
with  their  corresponding  alcohols,  will  be  rendered  clear  from 
the  following  Table  II,  based  on  the  researches  of  Emil  Fischer 
and  other  workers  : — 

TABLE   II 

Five-carbon  Alcohols 

CHoOH  CH2OH 

I  I 

H— C— OH  H— C— OH 

H— C— OH  HO— C— H  HO— C— H 

H— 0— OH  H— C— '  H  HO— C— H 

I  I  I 

CH2OH  CH3OH  CH2OH 

Adonite  Xylite  J-Arabite 

Fiw-carbon  Sugars  (aldopentoses) 
CHO  CHO  CHO 

I  I  I 

H— C— OH  H— C— OH  H— C— OH 

I  I  f 

H— 0-OH  HO— C— H  HO— C— H 

I  I  I 

H— C— OH  H— C— OH  HO— C— H 

I  I  I 

CH2OH  CH2OH  CH2OH 

f-Bibose  J-Xylose  J-Arabinose 

Six-carbon  Akohols 
CHoOH  CH2OH  CH->OH  CH2OH 

I  i  I 

H— C-OH  HO— CH  HO— C— H        H— C-OH 

I  I  I  I 

H— C— OH  HO— CH  H— C-OH  HO— C— H 

I  I  I 

HO— C— H        H— C— OH    HO— C— H 

I  I  I 

HO— C— H        H— C— OH     HO— G-H 

I  I  I 

CH2OH  CH2OH  CH2OH 

i-Mannite  d-Mannite  /-iJorbite 


94      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

Six-carbon  Sugars  (aldohexoses) 

CHO  CHO  CHO  CHO  CHO 

I  I  I  I  I 

H— C— OH  HO— C— H      HO— C— H       H— C— OH     H— C— OH 

I  I  I  I  I 

H— C— OH   HO— C— H         H— C— OH  HO— C— H      HO— C— H 

I  I  I  1  I 

HO— C— H        H— C— OH    HO— C— H       H— C— OH  HO— C— H 

I  I  I  I  I 

HO— C— H        H— C— OH    HO— C— H       H— C— OH      H— C— OH 

I  I  I  I  I 

CH2OH  CH2OH  CH2OH  CH2OH  CH2OH 

f-Mannose  d-Mannose  /-Glucose  d-Glucose  d-Galactose 

The  Di-saccharoses.— The  chief  members  of  this  group  are 
cane  sugar,  milk  sugar,  and  malt  sugar.  By  the  action  of 
dilute  acids,  or,  as  will  be  shown  later,  by  the  activity  of  certain 
organisms  or  enzymes,  they  are  converted  into  hexoses  accord- 
ing to  the  general  equation 

C12H220U  +  H20  =  C6H1306  +  C6H1206 

This  splitting  up  of  the  di-hexose  into  two  hexoses  is 
generally  known  as  inversion,  because  in  the  case  of  cane  sugar 
which  is  dextro-rotatory,  the  resultant  mixture  of  sugars 
is  laevo-rotatory,  owing  to  the  fact  that  the  Isevo-rotatory 
power  of  fructose  or  Isevulose  is  greater  than  the  dextro- 
rotatory power  of  glucose  or  dextrose. 

Cane  sugar  forms  glucose  and  fructose,  lactose  forms 
glucose  and  galactose,  and  maltose  two  molecules  of  glucose. 

The  following  are  a  few  interesting  facts  in  regard  to  the 
three  sugars  above  mentioned  : — 

Cane  Sugar. — Saccharose  or  sucrose  occurs  in  large  quan- 
tities in  the  sugar  cane  and  in  beetroot,  from  which  two 
sources  the  world  derives  practically  the  whole  of  its  sugar. 
Both  in  the  case  of  sugar  cane  and  beetroot,  the  general 
methods  of  extraction  are  much  the  same.  The  material 


THE  CHEMISTRY  OF  THE  SUGARS       95 

is  either  crushed  in  presses  and  the  juice  thus  forced  out,  or 
the  sugar  is  systematically  extracted  by  water ;  the  extract 
is  clarified  with  lime,  decolourised  with  animal  char- 
coal, filtered  and  evaporated  in  vacuum  pans  till  the  sugar 
crystallises.  The  dark  mother  liquor  is  known  as  molasses 
or  treacle,  the  sugar  can  be  obtained  from  it  by  precipitation 
with  strontium  hydroxide ;  from  this  precipitate  the  sugar 
is  recovered  by  suspending  in  water,  passing  carbon 
dioxide  through,  filtering  from  the  strontium  carbonate  and 
evaporating. 

Cane  sugar  crystallises  from  water  in  hard  four-sided  prisms ; 
it  is  generally  purified  for  purposes  of  scientific  investigation 
by  recrystallisation  from  hot  alcohol.  It  melts  at  about 
160°  C.  and  is  dextro-rotatory ;  it  does  not  reduce  Fehling 
solution  ;  it  also  does  not  combine  with  phenyl-hydrazine. 

Maltose  is  obtained  from  starch  by  the  action  of  the 
enzyme  known  as  amylase ;  this  reaction  forms  the  subject 
of  Chapters  V  and  VI  of  this  book. 

Maltose  is  more  soluble  in  water  than  is  cane  sugar,  and 
is  more  strongly  dextro-rotatory ;  it  also  reduces  Fehling 
solution  and  combines  with  phenyl  hydrazine.  A  further 
distinction  from  cane  sugar  lies  in  the  fact  that  it  directly 
ferments  with  yeast,  which  is  not  the  case  with  cane  sugar. 

Milk  sugar  or  lactose  occurs  in  the  milk  of  all  mammals 
to  the  extent  of  about  4  per  cent. 

In  the  manufacture  of  cheese,  milk  is  treated  with  a 
clotting  enzyme  known  as  '  rennet '  which  coagulates  the 
casein,  milk  sugar  remaining  in  solution ;  it  can  be  readily 
crystallised  from  this  solution  on  evaporation,  the  crystals 
containing  one  molecule  of  water  of  crystallisation.  It  is 
much  less  sweet  than  cane  sugar  and  is  dextro-rotatory, 
though  to  a  less  extent  than  cane  sugar ;  it  reduces 
Fehling  solution  slowly  and  forms  a  phenyl-hydrasine: 


96      BACTERIOLOGICAL  AND   ENZYME   CHEMISTRY 

compound,  but  like  cane  sugar  it  does  not  ferment  with 
pure  yeast. 

The  Poly-saccharoses.— Of  these,  starch  and  cellulose  will 
be  more  usefully  considered  separately  in  the  chapters  devoted 
to  their  decomposition  by  enzyme  or  bacterial  action.  It 
will  be  understood  that  their  molecular  structure  is  much 
more  complicated  than  that  of  the  carbohydrates  belonging  to 
the  two  preceding  classes. 

Glucosides. — A  class  of  substance  occurs  in  nature, 
generally  in  the  leaves  of  plants  or  bark  of  trees,  which  on 
treatment  with  acid,  or  by  the  action  of  certain  enzymes, 
yields  a  sugar  together  with  another  organic  compound.  In 
the  majority  of  cases  the  sugar  present  is  glucose,  and  these 
bodies,  therefore,  are  termed  glucosides. 

One  of  the  earliest  and,  at  the  same  time,  best  known  of 
the  glucosides  is  amygdalin,  which  occurs  in  bitter  almonds 
and  in  the  kernels  of  apricots,  peaches  and  plums. 

Liebig  and  Wohler  in  1837  isolated  an  enzyme  which  they 
termed  emulsin.  They  found  that  on  crushing  bitter  almonds 
the  amygdalin  was  decomposed  according  to  the  following 
equation  : — 

0^0^  +  2:0,0    =    C7H60  +  HCN+2C6H1206 

Amygdalin  Benzaldehyde     Hydrogen  Glucose 

cyanide 

Kecent  researches  by  Fischer,  Caldwell  and  Courtauld  and 
others  have  shown  that  amygdalin  can  be  split  up  at  several 
centres  marked  x  y  z  in  the  formula 

NC  .  CHC6H5 .  0  .  C6H1004  .  0  .  C6Hn06 
z  x  y 

which  are  attackable  only  by  specific  enzymes ;  thus  an 
infusion  of  yeast  only  splits  off  one  molecule  of  grape  sugar 
at  y,  leaving  a  residue  termed  almond  nitril  glucoside  which  is 
capable  of  being  completely  split  up  by  emulsin. 


THE  CHEMISTRY  OF  THE  SUGARS 


97 


The  following  are  a  few  typical  naturally  occurring  gluco- 
sides, together  with  their  products  of  hydrolysis  : — 
Arbutin 


C12H1607 
Salicin 


yields  glucose  plus     7    ?n™  °] 


fsalicyl  alcohol 
lC6H4OHCH2OH 
/coniferl  alcohol 


Coniferin  ) 

C16H2208  j 

Helicin  ) 

Indican  ) 
CMH1706N 

Sinigrin  | 


/ 


OCH, 


CH=CH2OH 

I  salicylaldehyde 
OH 


XCHO 

( indoxyl 

(C8H7ON 

fallyl  mustard  oil 

tC3H5CNS 


-f  KHS04 


C10H1609NS2KJ 

The  naturally  occurring  glucosides  are  accompanied,  in 
most  cases  at  any  rate,  by  the  enzyme  which  is  capable  of 
effecting  their  hydrolysis.  The  enzyme  and  the  glucoside 
occur  in  separate  cells,  and  they  only  act  upon  one  another 
when  the  cell  contents  are  brought  together  by  crushing,  as  for 
instance,  when  bitter  almonds  are  pounded  in  a  mortar ;  the 
emulsin  in  this  case  is  probably  contained  in  the  skin  of  the 
almond.  It  is  possible  that  glucosides  form  a  reserve  of 
food  material  for  the  plant,  their  constituents  being  only 
capable  of  assimilation  after  hydrolysis,  that  is,  when  brought 
in  contact  with  the  enzyme. 

It  has  been  possible  to  prepare  a  certain  number  of  sub- 
stances artificially  which  belong  to  the  same  class  as  glucosides. 
When  glucose  is  dissolved  in  water  and  the  freshly  prepared 
solution  examined  in  the  polarimeter,  the  optical  activitr 
observed  immediately  after  solution  is  found  gradually  to 
diminish,  and  after  about  six  hours  becomes  constant  at  a 


98       BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 


value  rather  less  than  half  that  of  the  original  solution.  It 
has  been  concluded  that  the  glucose  molecules  when  in  solution 
exist  both  in  an  aldehyde  form  and  in  an  oxide  form,  viz.  : — 


CHO 


CHOH 


CHOH 


CHOH 


CHOH 


CHOH 


CHOH 


CH2OH 

Aldehyde  form 


CH2OH 

Oxide  form 


It  will  be  seen  that  in  the  oxide  formula  the  terminal 
carbon  atom  attached  to  the  oxygen  is  asymmetric,  and  con- 
sequently two  different  derivatives  are  possible.  By  acting 
upon  glucose  with  methyl  alcohol  in  presence  of  hydrochloric 
acid  two  methyl  esters  have  been  obtained,  which  are  really 
the  simplest  members  of  the  glucosides,  their  formulae  being  as 
follows  : — 


H— C-OCH3 


CHOH 


CHOH 


CHOH 


CH30-C-H 


CHOH 


CH2OH 

-Methyl  glucoside 


CH2OH 

/8— Methyl  glucoside 


THE    CHEMISTRY    OF   THE    SUGARS 


99 


A  number  of  similar  compounds  have  been  prepared  by 
Emil  Fischer,  and  it  has  been  found  by  him  and  by  E.  F. 
Armstrong  and  others  that  the  a  and  £  glucosides  show 
well-defined  differences  in  their  resistance  to  the  action  of 
enzymes.  It  has  been  further  found  that  if  the  hexose  result- 
ing from  decomposition  of  the  glucoside  is  added  to  the 
reacting  mixture  of  glucoside  and  enzyme  the  action  is  re- 
tarded. These  investigations  suggest  that  the  decomposition 
of  glucosides  which  is  effected  by  enzymes  is  first  preceded 
by  a  combination  of  the  glucoside  with  the  specific  enzyme. 
They  would  indicate  that  enzymes  are  also  asymmetric 
products,  and  in  the  words  of  Emil  Fischer :  '  Enzyme  and 
glucoside  must  fit  each  other  like  key  and  lock  in  order  that 
the  one  may  exercise  a  chemical  action  on  the  other.' 

The  di-saccharoses  have  probably  a  glucosidic  structure, 
saccharose  or  cane  sugar  being  the  glucoside  of  glucose,  with 
the  following  probable  formula  : — 


caps: 


The  enzymic  hydrolysis  of  the  di-saccharoses  and  poly- 
saccharoses  is  of  great  technical  and  scientific  importance  and 
will  be  dealt  with  in  separate  chapters  in  the  following  pages. 


H  2 


CHAPTER  V 

THE  HYDROLYSIS  OF  STARCH  BY  AMYLASE 

STARCH  occurs  widely  in  the  vegetable  world,  being  the  first 
visible  product  of  assimilation  in  plants  containing  chlorophyll. 
Starch  is  usually  manufactured  in  Europe,  from  potatoes,  rice, 
wheat  and  maize  ;  and  in  tropical  countries,  from  the  palm 
and  from  tubers  of  various  plants. 

Examined  under  the  microscope,  starch,  which  in  the 
mass  is  a  white  powder,  is  seen  to  consist  of  small  granules 
which  have  the  power  of  polarising  light.  Different  species 
of  starch  vary  greatly  in  the  size  of  their  granules.  This  is 
clearly  seen  in  Plate  I,  reproduced  from  actual  photographs, 
the  same  magnification  being  used  in  every  case.  Under  high 
magnification,  especially  after  treatment  with  dilute  alkali, 
the  starch  granules  can  be  seen  to  consist  of  a  series  of  layers 
arranged  round  a  nucleus. 

Starch  is  found  to  consist  of  several  isomeric  compounds, 
the  chief  portion  being  starch  proper,  termed  either  amylum 
or  granulose,  the  remainder  consisting  of  starch  cellulose  or 
farinose.  The  starch  cellulose  is  not  readily  attacked  by 
enzyme  action  or  by  acids.  Soluble  starch  consists  of  granu- 
lose from  which  the  less  soluble  starch  cellulose  has  been 
removed. 

If  a  solution  of  starch  is  boiled  for  some  time  with  dilute 
acid  the  solution  will  become  clear  and  it  will  be  possible  by 
suitable  tests  to  recognise  the  presence  of  a  sugar  in  the 
solution.  The  same  reaction  is  brought  about  if  a  small 


5  ^ — =-^  6 

[Photo  by  Messrs.  Flatters  <£•  Garnett,  Ltd. 
PLATE  I.— STARCHES  (Magnification    x    70  diam.). 


1.  Rice. 

4.  Arrowroot. 


2.  Maize. 
5.  Sago. 


3.  Wheat. 
6.  Potato. 


THE   ACTION   OF   AMYLASE  ON  ;STAE£H:         101 

amount  of  extract  of  malt  is  added  to  the  starch  solution. 
This  reaction,  which  is  known  as  the  hydrolysis,  or  saccharifica- 
tion  of  starch,  has  been  found  to  be  due  to  an  enzyme  which 
has  been  termed  amylase.  Before  studying  the  reaction 
further  it  will  be  useful  briefly  to  describe  the  characteristics 
and  method  of  preparation  of  malt.  Malt  is  barley  which 
has  been  allowed  to  germinate  up  to  a  certain  point,  after 
which  the  process  is  arrested  by  heat. 

The  processes  in  the  manufacture  of  malt  are  as  follows  : — 

(1)  Preliminary  cleaning,  grading,  etc. — This  is  effected 
in  ordinary  screening  and  winnowing  apparatus,  much  dust 
and  dirt  having  frequently  to  be  removed  from  certain  classes 
of  barley,  especially  those  sent  from  the  East. 

(2)  Steeping. — The   clean   barley  grains   are   steeped   in 
water  in  vats  until  quite  soft. 

(3)  Malting. — The  steeped  grains  are  placed  on  floors  and 
constantly  turned  over  until  they  begin  to  germinate. 

(4)  When  germination  has  gone  far  enough  the  grains  are 
placed  on  drying  floors  and  finally  heated  in  kilns  to  a  tem- 
perature   not    exceeding   230°  F.      This  process  has  to  be 
carefully  conducted  and  lasts  some  days ;    the  malt  is  then 
screened  to  remove  the  dry  rootlets  and  finally  stored. 

Malt  will  be  seen  on  inspection  to  differ  from  barley  in  the 
following  particulars  :  The  barley  grain  is  hard  and  difficult 
to  break  with  the  teeth  and  has  no  special  taste  or  smell ; 
the  malt,  on  the  other  hand,  is  friable,  has  a  pleasant  odour 
and  sweet  taste.  On  separating  the  barley  grain,  especially  if  it 
has  been  previously  soaked  in  water,  the  germ  will  be  readily 
distinguished  at  the  base  of  the  grain  (cf.  Plate  II).  In 
the  case  of  the  malt  the  germ  will  be  found  to  have  developed 
some  two-thirds  of  the  length  of  the  grain.  It  is  now  known 
as  the  acrospire.  The  precise  change  taking  place  in  the  barley 
grain  during  the  process  of  malting  will  be  more  fully  studied 
in  the  next  chapter ;  for  the  moment  it  will  suffice  that  in 
malt  we  have  a  substance  containing  a  store  of  amylase 


102    ^BACTEMOLOQICAL  'AND    ENZYME   CHEMISTRY 


which  will  enable  us  to  study  the  action  of  this  enzyme  upon 
starch  and  thereby  to  obtain  a  knowledge  of  the  conditions 
of  enzyme  action  in  general. 

In  the  first  place  it  will  be  necessary  to  prepare  some 
malt  extract,  and  it  may  here  at  once  be  stated  that  it  is  of 
fundamental  importance  in  all  work  connected  with  the 
preparation  and  study  of  enzymes  that  the  conditions  of 
experiment  should  be  very  carefully  under  control,  more 
especially  the  temperature.  For  this  purpose  constant- 
temperature  incubators  (see  Fig.  7)  are  essential.  It  is  also 
better  to  use  constant  temperature  water-baths  ;  Fig.  4  b  shows 
a  convenient  type  of  water-bath  for  this  kind  of  work.  Small 
beakers  capable  of  holding  conveniently  about  100  c.c.  of 
solution  can  be  fitted  neatly  into  this  bath  by  means  of  flat 
rings  ;  test-tubes  can  also  be  held  in  position,  or  stacked  in 
the  beakers. 

To  prepare  a  cold  water  extract  of  malt  100  grams  of 
ground  malt  are  mixed  with  250  c.c.  of  water  and  the  mixture 
allowed  to  stand  with  frequent  stirring  for  about  five  hours. 
For  the  purpose  of  grinding  the  malt  a  small  hand-mill  similar 
to  a  coffee  grinding  mill  can  be  used. 

The  various  starches  differ  considerably  in  the  ease  with 
which  they  are  attacked  by  amylase,  and  for  the  purpose  of 
experiment  it  is  better  to  use  so-called  soluble  starch,  which 
is  prepared  after  the  manner  described  below.1 

1  Cf.  Brown,  Laboratory  Studies,  p.  65.  Preparation  of  Soluble  Starch.  — 
Introduce  about  fifty  grams,  of  potato  starch  into  a  500  c.c.  flask,  and  half 
fill  the  flask  with  a  7  '5  per  cent,  solution  of  hydrochloric  acid  made  by 
diluting  125  c.c.  of  the  concentrated  acid  to  500  c.c.  with  distilled  water. 
Allow  the  starch  to  digest  with  the  dilute  acid  at  the  ordinary  room  tempera- 
ture for  seven  or  eight  days.  The  acid  should  then  be  poured  off  and  the 
starch  washed  repeatedly  with  distilled  water  by  decantation  until  the 
granules  no  longer  give  an  acid  reaction  when  placed  on  blue  litmus  -paper. 
One  or  two  drops  of  dilute  ammonia  should  then  be  added,  and  the  starch 
again  washed  until  every  trace  of  ammonia  is  removed.  Drain  the  starch 
thoroughly  on  a  filter,  and  spread  it  on  filter-paper  to  air-dry  at  a  temperature 
of  about  25°  C.  (77°  F.). 


THE    ACTION    OF    AMYLASE    ON    STARCH          103 

The  action  of  malt  extract  upon  starch  may  now  be  studied 
as  follows :  A  3  per  cent,  solution  of  starch  paste  (i.e., 
six  grams  of  starch  to  200  c.c.  of  water)  is  first  prepared.  In 
preparing  starch  paste  the  starch  should  first  be  rubbed 
down  in  a  mortar  to  a  thin  cream  with  a  portion  of  the  water 
used,  while  the  remaining  volume  of  water  is  heated  to  boiling ; 
the  starch  cream  is  then  carefully  added,  stirring  the  while.  A 
solution  of  soluble  starch  prepared  as  above  contains  no 
visible  undissolved  particles. 

With  the  extract  of  malt  and  the  cold  starch  solution  it  is 
now  possible  qualitatively  to  examine  the  changes  which 
occur  when  the  two  are  brought  together.  For  this  purpose, 
say,  six  small  beakers  or  large  test-tubes  may  be  used  and 
about  10  c.c.  of  starch  solution  placed  in  eauh,  together 
with  -J-  c.c.  of  the  filtered  malt  extract.  The  test-tubes  are 
then  placed  in  a  constant-temperature  water-bath  at  a  tem- 
perature of  60°  C.  The  test-tubes  can  now  be  observed  from 
time  to  time.  The  first  change  to  be  noted  is  the  clarification 
of  the  starch ;  simultaneously  with  this  it  may  be  found  that 
the  ordinary  blue  colour  is  no  longer  given  when  a  drop  of 
dilute  iodine  solution  is  added  to  the  solution.  On  removing 
the  second  test-tube  after  the  lapse  of  a  further  period,  the 
colour  of  the  iodine  will  be  found  to  have  become  distinctly  red. 
If  simultaneous  tests  are  made  by  the  addition  of  Fehling 
solution,  a  gradually  increasing  amount  of  precipitation  will 
be  noted  until  finally  no  reaction  is  given  by  the  iodine,  while 
a  copious  red  precipitate  is  formed  on  boiling  with  the  Fehling 
solution. 

This  experiment  indicates  that  the  action  of  the  malt 
extract  upon  the  starch  solution  is  progressive.  In  the  first 
place  a  substance  is  formed  which  gives  a  purple  colour  with 
iodine  but  does  not  reduce  Fehling  solution.  Later  on  sub- 
stances are  formed  which  give  a  red  coloration  with  iodine, 
and  eventually  only  the  sugar  or  Fehling  reducing  substance 
can  be  detected.  A  more  exact  investigation  of  the  first 


104      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

change  will  be  attempted  later ;  it  is  sufficient  here  to  say  that 
the  substances  which  give  colour  reactions  with  iodine  are 
known  as  dextrins,  owing  to  their  effect  on  polarised  light ;  the 
sugar  can  be  shown  to  be  maltose. 

The  nature  of  the  substance  present  in  the  malt  extract 
which  brings  about  the  change  has  now  to  be  considered.  If 
the  extract  is  added  to  alcohol,  a  white  precipitate  is  formed. 
This  precipitate  can  be  shown  to  contain  the  active  substance 
or  enzyme  in  question,  which,  as  it  is  concerned  in  the  breaking 
down  of  starch,  is  known  as  amylase  (or  frequently  '  diastase'). 
Its  preparation  and  investigation  will  illustrate  very  well 
the  properties  and  method  of  preparation  of  enzymes  in 
general. 

The  following  method  may  be  used  for  the  preparation 
of  the  amylase  of  malt :  100  grams  of  ground  malt  (preferably 
air  dried)  are  digested  with  250  c.c.  of  20  per  cent,  alcohol 
for  four  hours  and  then  filtered.  Strong  alcohol  is  added  to 
the  filtrate  so  long  as  a  white  flocculent  precipitate  is  formed ; 
this  precipitate  contains  the  amylase,  it  is  allowed  to  stand 
and  the  supernatant  liquid  poured  off.  The  precipitate  is 
washed  by  decantation  with  a  little  strong  alcohol,  and  after- 
wards transferred  to  a  smooth  hardened  filter  and  washed 
repeatedly  with  small  quantities  of  absolute  alcohol.  Portions 
of  the  precipitate  may  now  be  examined  in  various  ways. 
By  warming  as  much  as  will  go  on  the  end  of  a  knife  blade 
with  about  20  c.c.  of  starch  solution  the  above  described 
changes  in  the  starch  solution  will  be  found  to  take  place. 
The  following  reactions  are  characteristic  of  enzymes  in 
general : — 

1.  A  small  portion  of  the  precipitate  is  dissolved  in  the 
least  possible  quantity  of  water  and  a  few  drops  of  an  alcoholic 
solution  of  guaiacum  resin  are  added,  together  with  a  little 
hydrogen  peroxide.  An  intense  greenish  blue  colour  is  obtained. 
By  taking  different  quantities  of  the  aqueous  solution  it  will 
be  found  that  the  blue  colour  varies  in  proportion  to  the  con- 


THE    ACTION    OF    AMYLASE    ON   STARCH  105 

centration  ;  in  this  way  the  actual  amount  of  enzyme  present 
in  the  solution  may  be  roughly  determined.  This  method 
is  very  useful  for  quickly  following  the  rate  at  which  an 
enzyme  is  developed  under  different  conditions,  e.g.,  at  the 
different  stages  of  growth  of  the  roots  or  leaves  of  plants,  or 
during  the  progress  of  a  technical  process,  e.g.,  the  withering  of 
tea  leaves. 

2.  A  small  portion  of  the  precipitate  is  warmed  with  strong 
caustic  soda  ;  the  presence  of  ammonia  can  be  recognised  by 
the  smell  and  by  introducing  red  litmus  paper  into  the  upper 
portion  of  the  test-tube. 

3.  A  small  portion  of  the  precipitate  is  dissolved  in  strong 
caustic  soda  and  a  few  drops  of  very  dilute  copper  sulphate 
solution  added  ;  a  violet  colour  is  produced.    This  is  known 
as  the  biuret  reaction,  as  it  is  given  by  biuret,  a  substance 
produced  by  heating  urea. 

4.  To  the  aqueous  solution  of  a  portion  of  the  precipitate 
a  few  drops  of  Millon's  reagent  are  added ;  a  white  precipitate 
is  obtained.     Millon's  reagent  is  a  solution  of  mercuric  nitrate 
containing  free  nitric  acid.     It  is  prepared  by  dissolving  one 
part  of  mercury  in  two  parts  of  strong  nitric  acid  and  diluting 
the  solution  with  twice  its  bulk  of  water ;  after  standing  some 
time  the  supernatant  liquid  is  decanted  from  the  precipitate. 

5.  A  portion  of  the  precipitate  is  warmed  in  a  small 
porcelain  dish  with  a  little  concentrated  nitric  acid,  and  the 
excess  of  acid  gently  evaporated ;    on  addition  of  a  drop  or 
two  of  strong  ammonia  a  bright  orange  colour  is  obtained. 
This  is  known  as  the  Xanthoproteic  reaction. 

All  the  above  reactions,  with  the  exception  of  the  colour 
reaction  with  guaiacum  resin  and  hydrogen  peroxide,  are 
characteristic  of  albumin  and  its  derivatives.  Enzymes, 
therefore,  can  be  broadly  described  chemically  as  complex 
nitrogenous  substances  akin  to  albumin.  Many  attempts 
have  been  made  to  obtain  enzymes  in  the  pure  state,  but 
with  little  success.  Like  all  complicated  nitrogenous  bodies  of 


106      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

this  class  they  tend  to  carry  down  with  them  other  substances 
which  are  present  in  solution,  especially  inorganic  salts  ;  it  is 
therefore  very  difficult  to  obtain  them  free  from  ash.  More- 
over, in  the  course  of  the  operations  necessary  to  prepare  them 
in  an  approximately  pure  state,  they  tend  to  suffer  a  loss  in 
activity.  So  difficult  indeed  is  it  to  obtain  them  as  definite 
chemical  compounds  that  it  has  been  seriously  suggested 
that  enzyme  action  is  really  a  property  of  matter,  such,  for 
example,  as  radio-activity  or  static  electrical  potential. 

The  following  method  will,  however,  serve  to  illustrate 
the  preparation  of  amylase  in  an  approximately  pure 
condition. 

The  precipitate,  formed  as  already  described  by  adding  the 
malt  extract  to  alcohol,  contains,  besides  the  active  enzyme, 
a  quantity  of  carbohydrate  (dextrin  and  sugar)  together  with 
albuminoid  matter  and  salts.  It  is  possible  to  eliminate  the 
carbohydrate  and  the  albuminoid  impurity  to  a  large  extent 
by  the  action  of  yeast,  if  the  latter  has  been  previously 
starved  of  nitrogen,  by  allowing  it  to  remain  for  twenty-four 
hours  in  a  10  per  cent,  solution  of  sugar.  To  prepare  the 
amylase  in  this  way,  100  grams  of  crushed  malt  are  macerated 
with  300  c.c.  of  water  at  a  temperature  of  30°  C.  for  eighteen 
hours,  stirring  at  half-hour  intervals.  The  mass  is  filtered  and 
pressed  and  thoroughly  washed  with  water,  the  washings  being 
mixed  with  the  original  extract.  After  filtering  the  solution 
is  made  up  to  300  c.c.  with  water,  ten  grams  of  beer  yeast 
added  and  left  at  a  temperature  of  28°  C.  for  forty-eight  hours. 
The  solution  is  then  filtered  and  700  c.c.  of  alcohol  added  to 
the  clear  liquid  ;  the  precipitated  amylase  is  filtered  through 
a  hardened  filter  paper,  washed  with  small  quantities  of 
absolute  alcohol,  and  finally  dried  in  a  vacuum  desiccator. 
About  three  grams  of  a  white  powder  are  obtained  which  has 
about  80  per  cent,  of  the  activity  of  the  original  extract. 

A  product  of  diminished  purity  and  activity  can  be  obtained 
if  the  treatment  with  yeast  is  omitted  and  the  precipitate 


THE    ACTION   OF    AMYLASE    ON    STARCH  107 

with  alcohol  simply  filtered,  re-dissolved  in  water,  re-precipi- 
tated with  alcohol,  washed  with  alcohol  and  dried  in  vacuo. 


THE   QUANTITATIVE    STUDY    OF    THE    ACTION    OF    AMYLASE 
ON  STARCH 

It  is  evident  from  the  foregoing  experiments  that,  to  follow 
exactly  the  course  of  the  change  which  takes  place  when 
malt  extract  acts  upon  a  solution  of  starch,  it  is  necessary  to 
make  use  of  methods  which  will  enable  the  change  to  be 
followed  when  all  the  bodies  concerned  are  present  in  solution 
together,  since  to  isolate  any  one  of  them  will  be  likely  to 
decompose  the  others.  The  following  properties  are  therefore 
made  use  of  in  studying  the  reaction  : — 

(1)  Specific  gravity ; 

(2)  Optical  activity ; 

(3)  Cupric  oxide  reducing  power. 

(1)  Specific  Gravity.— It  is  possible  to  determine,  e.g.,  the 
amount  of  sugar  present  in  a  solution  by  comparing  the 
specific  gravity  of  the  solution  with  that  of  a  solution  of  sugar 
of  known  strength. 

The  specific  gravity  is  best  determined  by  means  of  the 
specific  gravity  bottle.  For  this  purpose  a  50  c.c.  specific 
gravity  bottle  with  a  perforated  stopper  is  required  ;  the  bottle 
must  be  cleaned  thoroughly  by  washing  with  distilled  water 
and  rinsing  out  with  a  little  strong  alcohol.  The  bottle  is 
then  gently  warmed  over  a  flame  and  air  sucked  through  by 
means  of  a  glass  tube  or  blown  through  with  the  foot-bellows 
until  it  is  quite  dry ;  it  is  then  allowed  to  cool  in  a  desiccator 
and  accurately  weighed.  The  bottle  is  now  filled  with 
distilled  water  at  a  temperature  of  15'5°  C.  (which  is  the 
temperature  of  graduation  of  the  bottle)  by  completely 
immersing  it  in  a  beaker  of  distilled  water  which  has  been 


108      BACTERIOLOGICAL   AND    EN?ArME    CHEMISTEY 

carefully  brought  to  this  temperature  in  a  constant  tempera- 
ture water-bath.  The  stopper  is  inserted,  care  being  taken 
that  no  air  bubbles  are  enclosed.  The  bottle  is  allowed 
to  cool  somewhat  and  then  quickly  wiped  dry  with  a  soft 
cloth  and  immediately  weighed.  Consecutive  weighings  in 
this  manner  should  not  differ  by  more  than  a  milligram. 

It  is  probable  that  the  weight  of  water  will  not  be  exactly 
fifty  grams,  but  the  specific  gravity  of  any  liquid  can  be  deter- 
mined by  filling  the  bottle  with  the  liquid  in  a  similar  manner, 
weighing  and  dividing  the  weight  of  water  in  the  bottle  into  the 
weight  of  the  liquid. 

In  working  with  solutions  of  sugar  or  similar  bodies,  in 
order  to  determine  the  amount  of  sugar  present  from  the 
specific  gravity,  a  factor  known  as  the  solution  factor  is  made 
use  of. 

Thus  it  has  been  found  that  ten  grams  of  maltose  made  up 
to  100  c.c.  at  60°  F.  has  a  specific  gravity  of  1038'5. 

The  amount  of  maltose  contained  in  100  c.c.  of  specific 

IQKK 1000 

gravity  1055  will  be          0  OK         =  14'285    grams.      The 

3*85 

number  3 '85  is  termed  the  solution  factor  for  maltose ;  dextrin 
has  the  same  factor. 

The  specific  gravity  and,  consequently,  the  solution  factor 
are  not  the  same  for  every  carbohydrate,  and  an  allowance 
must  be  made  for  this  in  specific  cases. 

(2)  Optical  Activity. — The  subject  of  optical  activity  has 
been  already  dealt  with  in  a  general  manner  in  Chapter  IV. 

The  polariscope  of  Fig.  13  is  adjusted  as  follows  l :  When 
the  apparatus  is  well  illuminated  by  the  sodium  flame,  the 
zero  position  (the  starting  point  of  all  experiments)  must 
first  be  found :  this  is  indicated  by  the  two  halves  of  the 

1  Based,  by  permission  from  Messrs.  Baird  and  Tatlock  (London),  Ltd., 
on  the  instructions  issued  for  use  with  the  Lippich  model  half -shadow 
polariscope. 


THE    ACTION    OF    AMYLASE    ON    STARCH  109 

field  appearing  equally  illumined  (equal  half -shadows).  For 
this  purpose  the  telescope  F  is  focussed  on  the  quartz  plate,1 
so  that  the  field  presents  a  perfectly  clear  round  circle  divided 
into  two  equal  parts  by  a  sharply  defined  vertical  line.  If 
the  graduated  dial  is  turned  through  three  or  four  degrees  to 
either  the  right  or  the  left  of  the  zero  line,  it  will  be  seen  that 
one  half  of  the  field  will  become  lighter,  the  other  half  darker. 

In  the  first  place,  the  zero  position  is  so  adjusted  that  the 
zero  line  of  the  circle  coincides  with  the  zero  line  of  the 
vernier.  The  half -shadow  can  now  be  made  lighter  or  darker 
(according  as  the  polariser  is  turned  to  the  right  or  left  of  the 
zero  line)  by  means  of  the  pointer  reaching  from  the  dial 
segment.  When  the  pointer  h  is  in  the  zero  position,  and  at 
the  same  time  the  analyser  A  is  placed  in  the  zero  position, 
both  halves  of  the  field  of  view  appear  black.  The  nearer 
the  pointer  is  to  the  zero  line,  the  darker  the  half-shadow  will 
become,  and  the  more  sensitive  the  apparatus ;  but  when 
the  solutions  are  not  quite  transparent,  the  pointer  must  be 
moved  more  or  less  away  from  the  zero  line,  so  that  the  field 
is  clear.  For  the  majority  of  experiments  the  position  of  the 
pointer  at  7|°  is  most  suitable,  therefore  the  apparatus  is 
usually  so  adjusted  that  in  this  position  the  dial  and  vernier 
read  exactly  0.  When  the  pointer  is  moved,  of  course  the 
zero  point  of  the  apparatus  changes,  and  no  longer  corre- 
sponds with  the  zero  line  of  the  dial.  The  difference  between 
the  latter  and  the  zero  position  of  the  apparatus  must  either 
be  taken  into  account  (the  simpler  way),  or  else  after  the 
graduated  dial  has  been  moved  to  0  the  apparatus  must  be 
again  placed  in  the  zero  position :  to  do  this,  the  analysing 
Nicol  prism  is  turned,  by  means  of  the  screws  A,  to  the  right 
or  left  until  the  half-shadows  are  equal  in  tint. 

Special  attention  must  be  called  to  the  following  circum- 
stance, which,  if  not  noted,  may  lead  to  considerable  confusion. 

When  the  circle  has  been  turned  too  far,  and  has  gone 
1  Or  small  Nicol  prism  in  the  Lippich  model. 


110      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

beyond  the  sensitive  range  of  the  apparatus,  the  light,  on 
comparison,  appears  to  a  certain  extent  of  the  same  intensity 
on  either  side  of  the  vertical  line,  and  this  point  may  be 
mistaken  for  the  zero  position.  Under  these  circumstances, 
even  if  the  circle  is  turned  through  ten,  fifteen,  or  even  a 
greater  number  of  degrees,  hardly  any  change  will  be  observed. 

It  is  a  matter,  therefore,  of  the  greatest  importance,  parti- 
cularly after  the  sample  to  be  examined  has  been  placed  in 
the  apparatus,  to  see  that  when  the  circle  has  been  turned  a 
few  degrees  on  either  side  of  the  zero  line,  the  transition  from 
light  to  shade,  and  vice  versa,  is  instantaneous. 

On  placing  the  sample  to  be  tested  within  the  apparatus, 
the  first  thing  to  do  is  accurately  to  adjust  the  telescope  so 
that  the  field  is  quite  clear  and  equally  divided  by  the  vertical 
line  ;  then  the  circle  is  turned  until  the  shades  are  exactly  of 
the  same  intensity  on  either  side  of  the  line. 

The  angle  rotated  by  a  column  of  10  per  cent,  solution  of 
a  sugar  ten  decimetres  long  is  known  as  the  specific  rotatory 
power  of  the  sugar. 

In  the  case  of  a  10  per  cent,  solution  of  pure  cane  sugar 
ten  decimetres  long  the  angle  is  66*5  degrees  when  sodium 
light  is  used  ;  this  is  generally  known  as  [a]D.  In  practice 
it  is  convenient  to  use  tubes  one  or  two  decimetres  long. 

If  the  quantity  of  sugar  per  100  c.c.  is  known,  the  specific 
rotatory  power  is  given  in  the  following  equation  :  When  E, 
=  the  reading  of  the  polarimeter,  L  the  length  of  tube,  and  C 
the  number  of  grams  per  100  c.c.,  then 

R 


On  the  other  hand,  if  the  specific  rotatory  power  of  the 
solution  is  known,  the  quantity  present  can  be  calculated 
from  a  determination  of  the  specific  rotatory  power,  thus  :  — 
C_  B 

100 


THE    ACTION   OF   AMYLASE    ON   STARCH          111 

Further,  if  the  weight  of  original  substance  present,  e.g., 
starch,  is  known,  and  the  specific  rotatory  power  of  dextrin 
and  maltose  respectively,  then  the  amount  of  conversion  which 
has  taken  place  after  the  first  appearance  of  dextrin  can  be 
determined  by  an  observation  of  the  specific  rotatory  power 
of  the  mixture,  e.g.,  the  specific  rotatory  power  of  dextrin  is 
195,  of  maltose  135*4.  If  the  specific  rotatory  power  of  the 
mixture  is,  say  165 '2,  the  relative  proportions  present  can 
be  calculated  from  the  following  equation : — - 

195z  +  135-4  (l-x)=  165-2 

x  in  this  case  will  be  found  to  equal  0'5,  i.e.,  the  dextrin  and 
maltose  were  present  in  equal  quantities. 

(3)  Cuprie  Oxide  reducing  Power.— The  cupric  oxide  re- 
ducing power  of  sugars  is  conveniently  referred  to  a  typical 
sugar  taken  as  a  standard.  This  standard  is  generally  known 
as  K,  the  amount  of  CuO  reduced  by  one  gram  of  glucose 
being  taken  as  100. 

The  actual  amount  of  CuO  reduced  from  Fehling  solution 
by  one  gram  of  glucose  is  2 -205  grams ;  the  sugar  reducing  say 
1*345  grams  CuO  per  gram  would  give  the  value  for  K  as 
61,  thus  :— 

2-205  :  1-345  ::  100  :  61 

In  the  case  of  starch  it  is  usual  to  take  maltose  as  a 
standard,  in  which  case  the  letter  B,  is  used  instead  of  K  ;  thus 
a  substance  with  three-quarters  the  reducing  power  of  maltose 
would  be  considered  to  have  a  reducing  power  K75  instead  of 
K45.75.  A  rapid  method  for  determining  cupric  oxide  reducing 
power  is  given  in  the  following  paragraphs,  based  on  the 
Report  of  the  Malt  Analysis  Committee  to  the  Council  of  the 
Institute  of  Brewing. 

The  method  is  there  used  for  determining  the  diastatic 
activity  of  malt.  It  may  be  used  generally  for  determining 


112      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

the  amount  of  copper  oxide  reducing  sugar  present  in  any 
solution. 

Briefly,  the  process  consists  in  adding  successive  small 
measured  quantities  of  the  sugar  solution  to  a  given  volume 
of  Fehling  solution  till  complete  reduction  takes  place,  the 
end  of  the  reaction  being  determined  by  means  of  a  special 
indicator. 

The  Fehling  solution  is  prepared  as  follows  : — 

(a)  Copper  Solution. — Kecrystallised  copper  sulphate  (69'2 
grams)  is  dissolved  in  water  and  the  solution  made  up  to 
one  litre  at  60°  F.  with  distilled  water. 

(b)  Alkaline  Tartrate  Solution. — Rochelle  salt,  i.e.,  sodium 
potassium  tartrate  (346  grams)  and  caustic  soda  (130  grams) 
are  dissolved  in  about  600  c.c.  of  distilled  water,  the  solution 
cooled   and  made  up  to  one  litre  at  60°  F.  with  distilled 
water. 

The  two  solutions  are  to  be  kept  separate,  and  equal 
volumes  mixed  for  each  day's  work,  from  which  mixture  the 
volumes  specified  in  the  analytical  method  are  measured  out 
at  60°  F. 

Preparation  of  the  Indicator. — One  gram  of  ferrous 
ammonium  sulphate  and  the  same  quantity  of  ammonium 
thiocyanate  are  dissolved  in  10  c.c.  of  water  at  a  moderate 
temperature,  say  120°  F.,  and  immediately  cooled  ;  5  c.c.  of 
concentrated  hydrochloric  acid  are  then  added.  The  solution 
so  obtained  has  invariably  a  brownish-red  colour,  due  to  the 
presence  of  ferric  salt,  which  latter  must  be  reduced.  For 
this  purpose  zinc  dust  is  the  most  satisfactory  reagent  to 
employ,  and  a  mere  trace  is  sufficient  to  decolourise  the 
solution  if  pure  reagents  have  been  employed. 

When  kept  for  some  hours,  the  indicator  develops  the 
red  coloration  by  atmospheric  oxidation.  It  may,  however, 
be  decolourised  by  the  addition  of  a  further  quantity  of  zinc 
dust,  but  its  delicacy  is  decreased  after  it  has  been  decolourised 
several  times.  For  practical  purposes  the  indicator  may  be 


THE    ACTION   OF   AMYLASE    ON   STARCH          113 

too  delicate,  and  it  is  recommended  to  prepare  it  the  day 
before  it  is  required  for  use,  as  it  gives  the  best  results  after 
the  second  decoloration. 

The  titration  is  carried  out  as  follows  : — 

The  Fehling  solution  must  first  be  standardised  by 
taking,  say,  1  gram  of  pure  dextrose,  and  dissolving  in  200  c.c. 
of  distilled  water. 

Five  c.c.  of  the  Fehling  solution  are  accurately  measured 
into  a  150  c.c.  boiling  flask,  and  raised  to  boiling  over  a 
small  naked  Bunsen  flame.  The  sugar  solution  obtained  as 
above  is  added  from  a  burette  in  small  quantities  at  first 
of  about  5  c.c.,  the  mixture  being  kept  rotated  and  boiled  after 
each  addition  until  reduction  of  the  copper  is  complete, 
which  is  ascertained  by  rapidly  withdrawing  a  drop  of  the 
liquid  by  a  glass  rod,  and  bringing  it  at  once  into  contact 
with  a  drop  of  the  indicator  on  a  porcelain  or  opal  slab. 

The  reduction  is  complete  as  soon  as  no  red  coloration, 
due  to  the  formation  of  ferric  thiocyanate,  is  produced. 

Having  once  standardised  the  Fehling  solution,  the 
amount  of  reducing  sugar  present  in  any  given  solution  can 
be  simply  determined,  care  being  taken  in  the  case  of  a 
solution  containing  an  active  enzyme  to  stop  the  action  of 
the  latter  by  addition  of  caustic  soda  (say  10  c.c.  •§•  caustic 
soda  to  100  c.c.  of  the  solution),  so  that  alteration  in  the 
composition  of  the  solution  may  not  take  place  in  the  course 
of  the  titration  operations. 

It  is  possible  in  this  way  to  follow  the  course  of  change, 
say  when  starch  solution  is  acted  upon  by  amylase,  by  with- 
drawing portions  of  the  solution  from  time  to  time,  stopping 
the  reaction  with  caustic  soda  and  titrating  as  above. 

The  presence  of  maltose  in  the  products  of  the  action  of 
amylase  on  starch  can  be  demonstrated  by  the  preparation 
of  an  osazone,  by  the  reaction  described  on  p.  87. 

In  order  to  prepare  an  osazone,  to  O'Ol  gram  of  the  sugar 
in  about  half  a  test-tube  full  of  water,  O'l  gram  of  phenyl 


114      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

hydrazine  is  added,  together  with  0'2  gram  of  sodium  acetate  ; 
the  whole  is  warmed  until  solution  takes  place  and  then 
heated  half  an  hour  on  a  boiling  water-bath. 

Glucosazone  formed  in  this  way  from  glucose  is  almost 
insoluble  in  water,  and  has  a  melting-point  225°  C.  The 
osazone  of  maltose  is  soluble  in  75  parts  of  water  at  100°  C., 
and  its  melting-point  is  205°  C.  Maltose  is  further  distin- 
guished from  glucose  by  its  specific  rotatory  power,  which 
is  140  degrees  compared  with  52  '5.  The  cupric  oxide  reducing 
power  is  two-thirds  that  of  glucose. 

The  methods  of  investigation  which  have  just  been 
described  render  it  possible  quantitatively  to  follow  the 
changes  taking  place  in  the  course  of  the  action  of  amylase 
upon  starch.  A  large  number  of  investigators  have  pub- 
lished researches  on  this  subject,  the  general  result  of  which 
has  been  to  lead  to  the  conclusion  that  the  starch  molecule 
breaks  down  by  a  series  of  hydrations  and  subsequent  decom- 
positions, maltose  being  formed  at  each  splitting,  together 
with  a  dextrin  of  less  molecular  weight.  Certain  of  these 
dextrins,  as  the  qualitative  examination  of  the  reaction 
showed,  give  characteristic  colour  reactions  with  iodine,  the 
red  colour,  e.g.,  being  due  to  a  dextrin  termed  erythro-dextrin. 

Brown  and  Morris  noted  that  when  80  per  cent,  of  maltose 
and  20  per  cent,  of  dextrin  had  been  formed,  the  last  20  per 
cent,  hydrolised  with  difficulty,  and  they  assumed  the  forma- 
tion of  a  body  intermediate  between  maltose  and  dextrin 
which  they  termed  malto-dextrin.  Their  theory  to  account 
for  this  assumes  that  the  starch  molecule  breaks  up  into  a 
stable  dextrin  and  so-called  amylin  groups  which  are  capable 
of  gradual  hydrolysis  to  maltose  ;  we  have  thus  the  following 
equations  :— 


5[(C12H20010)20]  = 

Starch  Stable  dextrin  Amylin  groups 


(C12H20010)20  +  H20  =         1223"    eto-.  etc. 


THE   ACTION  OF  AMYLASE  ON  STARCH  115 


The  Conditions  of  Action  of  Amylase.—  The  study  of 
the  conditions  under  which  the  characteristic  activity  of 
amylase  is  manifested  will  serve  as  an  example  for  the  mode 
of  action  of  enzymes  in  general  ;  in  many  respects  the  action 
of  the  enzyme  resembles  the  activity  of  a  living  organism, 
e.g.  :— 

1.  Enzymes  are  destroyed  by  heat. 

2.  They  have  an  optimum  temperature  of  reaction. 

3.  They  are  not  exhausted  by  continuous  activity. 

4.  They  are  greatly  affected  by  alterations  in  the  medium 
in  which  they  act,  e.g.,  by  certain  antiseptics  and  poisons. 

The  following  experiments  will  serve  to  illustrate  the 
above  statements  :  — 

Experiment.  —  Two  lots  of  20  c.c.  each  of  3  per  cent. 
starch  solution  are  taken  ;  to  one  is  added  1  c.c.  of  unboiled 
malt  extract,  to  the  other  1  c.c.  of  boiled  extract,  and  the 
two  solutions  warmed  to  50°  C.  On  testing  with  iodine 
and  Fehling  solution  saccharification  will  be  found  to  have 
taken  place  in  the  case  of  the  solution  to  which  the  unboiled 
extract  was  added,  while  no  change  takes  place  in  the  second 
mixture. 

Experiment.  —  A  number  of  test-tubes  may  be  taken  contain- 
ing, say,  20  c.c.  of  3  per  cent,  starch  solution  and  1  c.c.  of  malt 
extract,  and  kept  for  an  equal  time  at  different  temperatures, 
say,  the  ordinary  laboratory  temperature,  an  incubator  at 
20°  C.  and  water-baths  at  50°  to  80°  C.  respectively.  At 
the  end  of,  say,  ten  minutes  all  the  solutions  are  quickly 
brought  to  the  boiling-point  and  titrated  with  Fehling  solution 
and  ferrous  sulphocyanate.  It  will  be  found  that  the  greatest 
amount  of  sugar  has  been  formed  at  50°  C.  By  making  a 
larger  number  of  similar  trials  at  different  temperatures  the 


116      BACTERIOLOGICAL   AND    ENZYME   CHEMISTRY 

exact  optimum  temperature  for  a  given  enzyme  can  be 
determined. 

Experiment. — The  following  experiment  was  devised  by 
Effront  to  show  the  continuous  activity  of  amylase :  200  c.c.  of 
starch  paste  are  mixed  with  3  c.c.  of  malt  extract  and  left  for 
four  hours  at  30°  C.  The  liquid  is  now  diluted  with  distilled 
water  to  a  volume  of  300  c.c.,  100  c.c.  of  this  mixture  is 
mixed  with  a  further  200  c.c.  of  starch  solution  and  heated 
for  one  hour  to  50°  C. ;  call  this  solution  A.  A  second 
100  c.c.  of  the  original  mixture  is  taken  and  boiled  and  after- 
wards added  to  another  200  c.c.  of  starch  solution,  together 
with  1  c.c.  of  the  original  malt  extract.  This  mixture  is  heated 
to  50°  C.  for  one  hour  ;  it  may  be  called  solution  B. 

Upon  titration  with  Fehling  solution  the  two  solutions 
A  and  B  will  be  found  to  give  practically  identical  results, 
which  indicates  that  100  c.c.  of  starch  mixture  will  do  as 
much  work  as  1  c.c.  of  fresh  malt  extract,  that  is,  that  the 
amylase  is  not  exhausted  by  continuous  activity. 

Experiment. — Three  lots  of  starch  solution  of  200  c.c.  each 
are  taken,  to  one  of  them  0'25  gram  of  potassium  or  ammonium 
alum  is  added,  to  the  second  a  few  drops  of  strong  solution 
of  potash,  while  the  third  is  left  without  any  addition.  To 
each  solution  1  c.c.  of  malt  extract  is  added  and  the  three 
solutions  are  warmed  for  one  hour  at  50°  C.  Upon  titra- 
tion with  Fehling  solution  the  greatest  action  will  be 
found  to  have  taken  place  in  the  solution  to  which  the 
alum  has  been  added,  while  the  action  has  been  practically 
inhibited  by  the  potash. 

Effront  has  studied  the  effect  of  a  number  of  salts,  such  as 
phosphates  and  acetates  and  of  organic  bases,  e.g.,  asparagin, 
upon  the  action  of  amylase  and  has  found  that  in  most  cases  a 
considerable  acceleration  of  the  action  takes  place.  These 
results  are  of  special  interest  in  view  of  the  effect  of  phosphates 
upon  the  fermentation  brought  about  by  the  enzymes  of 
yeast,  which  has  been  studied  by  Harden  and  his  colleagues. 


THE  ACTION  OF  AMYLASE  ON  STARCH  117 

The  above  experiments  show  the  analogy  which  exists 
between  the  action  of  enzymes  and  the  action  of  organisms. 
They  can,  however,  be  differentiated  by  certain  other  pro- 
perties. Thus  some  enzymes,  e.g.,  invertase,  will  pass  readily 
through  a  porous  porcelain  filter,  which  under  similar  con- 
ditions will  retain  all  living  organisms.  Further,  certain 
antiseptics  which  inhibit  the  action  of  micro-organisms  are 
without  effect  on  enzymes  ;  among  these  the  most  frequently 
used  are  thymol,  chloroform,  and  especially  toluene. 

It  has  also  been  found  that  when  the  amount  of  substance 
to  be  acted  upon  is  large  in  proportion  to  the  quantity  of 
enzyme  used,  then  the  amount  of  reaction  taking  place  is 
proportional  to  the  quantity  of  enzyme  present ;  this  is 
known  as  the  law  of  proportionality.  The  following  experi- 
ments may  serve  to  illustrate  it : — 

Two  lots  of  300  c.c.  each  of  3  per  cent,  starch  solution 
are  taken;  to  one  is  added  \  c.c.  of  malt  extract,  to  the 
other  2  c.c.  of  the  same  extract,  and  the  two  mixtures  heated 
for  a  quarter  of  an  hour  at  50°  C.  Upon  titration  with 
Fehling  solution  it  will  be  found  that  the  greater  amount  of 
reaction  has  taken  place  in  the  solution  to  which  the  larger 
quantity  of  malt  extract  was  added. 


CHAPTEE  VI 

THE  CONDITIONS   OF  FORMATION  OF   AMYLASE  IN   THE 
LIVING  CELL 

IT  has  been  possible  in  the  case  of  amylase  more  than  with 
many  other  ferments  carefully  to  study  the  conditions  under 
which  it  is  produced,  and  it  is  therefore  instructive  to  repeat, 
in  a  simple  way,  some  of  the  experiments  which  have  been 
made  and  so  to  obtain  an  insight  into  the  methods  of  research 
made  use  of  in  this  class  of  study. 

In  the  first  place  then,  as  the  chief  source  of  amylase  so  far 
considered  is  the  malted  barley  grain,  it  will  be  well  to  study 
more  carefully  the  structure  of  the  barley  grain,  and  note  the 
difference  between  it  in  its  original  condition  and  after  the 
process  of  germination  or  conversion  into  malt  has  taken 
place.  In  order  to  examine  the  barley  grain  microscopically 
it  is  necessary  first  to  soften  it  by  immersion  in  water,  possibly 
for  a  day  or  two,  until  it  can  easily  be  cut  through  with  a 
knife  ;  there  is  then  no  difficulty  in  separating  the  outer  skin 
or  husk  and  in  dividing  the  two  halves  of  the  grain.  At  the 
base  of  the  grain  in  the  cleft  of  the  two  halves  will  be  noted 
the  embryo. 

So  much  can  readily  be  discerned  by  the  naked  eye.  In 
order  to  obtain  sections  suitable  for  microscopical  examination 
the  following  implements  and  reagents  will  be  necessary  : — 

Some  blocks  of  paraffin ; 

A  sharp  razor  or  microtome  ; 

One  or  two  mounted  needles ; 


PLATE    II. 


(i)  ROOT  NODULES  OF  PEA. 


a 


(ii)  SECTION  OF  BARLEY  GRAIN. 

[Photos  by  Messrs.  Flatters  &  Garnett,  Ltd. 


• 
AMYLASE    IN   THE    LIVING    CELL  119 

A  few  watch  glasses  ; 

Absolute  alcohol ; 

Clove  oil; 

Alcoholic  iodine  solution  ; 

Microscope  slides  and  cover  glasses ; 

Xylol ; 

Canada  balsam ;  and 

Shellac  varnish. 

In  order  to  make  a  section  laterally  through  the  embryo, 
a  softened  barley  grain  should  be  embedded  laterally  into  a 
block  of  paraffin,  say  one  inch  cube,  if  the  razor  and  not  the 
microtome  is  to  be  used.  The  grain  is  easily  embedded  by 
melting  a  little  of  the  paraffin  in  the  middle  of  one  of  the  sides 
of  the  block  with  a  hot  glass  rod,  carefully  placing  the  grain 
in  the  little  melted  pool  of  paraffin  and  allowing  it  to  set 
thoroughly  hard.  With  a  little  practice  it  is  possible  with  a 
sharp  razor  to  cut  very  fairly  accurate  thin  lateral  sections  of 
the  grain ;  a  number  of  these  can  be  cut  until  the  embryo 
is  fully  exposed,  when  the  section  of  the  grain  will  have  the 
appearance  roughly  as  shown  in  Plate  II  (ii).  A  number  of 
these  sections  should  be  cut  and  immersed  in  a  little  absolute 
alcohol,  contained  in  one  of  the  watch  glasses,  in  order  to  harden. 
As  the  moisture  in  the  section  is  reduced  by  alcohol  the  section 
becomes  hard.  It  is  next  transferred  to  a  watch  glass  containing 
clove  oil,  in  order  to  clear  it  and  render  it  transparent ;  it  is 
then  immersed  in  xylol  to  remove  the  excess  of  clove  oil,  placed 
on  a  microscope  slide,  covered  by  a  cover  slip  and  examined. 
If  a  permanent  preparation  is  required  it  may  be  dipped 
into  an  alcoholic  solution  of  iodine  and  then  into  picric 
blue,  and  the  excess  of  iodine  and  of  colour  washed  out 
with  alcohol.  The  iodine  stains  the  starch  granules  purplish 
blue,  and  the  aleurone  (or  albuminoid)  layer  yellow.  The 
remaining  tissues  are  coloured  blue  by  picric  blue. 

The  section  is  now  ready  for  mounting ;  it  is  placed  in  the 
centre  of  a  microscope  slide,  covered  with  a  drop  of  Canada 


120      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

balsam  and  the  cover  glass  pressed  down  over  it,  any  excess 
of  Canada  balsam  exuding  from  the  edges  of  the  cover  glass 
being  carefully  wiped  away  with  a  clean  rag.  As  soon  as  the 
Canada  balsam  is  set,  the  section  is  ready  for  examination 
under  the  microscope. 

Before  making  a  permanent  preparation  it  is  well  to 
examine  a  number  of  sections  in  order  to  obtain  one  which  is 
really  characteristic.  A  good  section  will  show  the  structure 
of  the  grain  as  in  Plate  II  (ii).  Here  a  is  the  germ,  b  the 
scutellar  epithelium  which  divides  the  germ  from  the  endosperm 
c,  while  d  is  the  husk. 

If  the  section  of  a  barley  grain  so  obtained  be  compared 
with  a  section  of  undried  malt,  in  the  first  place  by  simple 
examination  of  the  grain  with  the  naked  eye,  it  will  be  seen 
that  the  germ  has  grown  very  considerably  and  that  the  cells  of 
the  endosperm  are  broken  down,  so  that  the  main  bulk  of  the 
grain  is  soft  and  friable,  and  it  is  extremely  difficult  to  make 
a  microscopic  section  of  it  in  this  condition.  As  a  matter  of  fact 
the  cellulose  walls  of  the  starch-containing  cells  have  been 
broken  down  in  the  first  stage  by  a  cellulose  dissolving  enzyme, 
and  afterwards  the  amylase  has  penetrated  the  bulk  of  the 
endosperm  and  has  largely  saccharified  the  starch  present. 

The  difference  in  the  distribution  of  amylase  in  the  un- 
malted  and  malted  barley  grain  can  be  seen  if  a  section 
through  the  median  line  is  treated  with  a  small  quantity  of 
guaiacum  resin  and  hydrogen  peroxide,  when  the  blue  colour 
will  be  found  to  extend  all  over  the  grain  in  the  case  of  the  malt, 
but  to  be  only  noticeable  in  the  neighbourhood  of  the  embryo 
in  the  case  of  the  barley.  This  observation  suggests  that  the 
seat  of  production  of  amylase  is  in  the  embryo ;  this  can  be 
proved  by  the  following  experiment  first  made  by  Brown  and 
Morris. 

Some  starch  gelatine  is  prepared  by  adding  7  grams  of 
gelatine  to  100  c.c.  of  a  1  per  cent,  solution  of  soluble  starch  in 
water,  warming  until  the  gelatine  is  uniformly  dissolved  and 


AMYLASE    IN   THE    LIVING    CELL  121 

sterilising  in  a  steam  steriliser.  With  this  starch  gelatine  a 
number  of  cultivation  tubes  and  plates  may  be  prepared  in 
order  to  determine  the  production  of  arnylase  under  different 
conditions. 

For  the  determination  of  the  production  of  amylase  by  the 
growing  embryo  of  the  barley  grain,  a  small  deep  Petri  dish 
may  be  taken,  and  the  starch  gelatine  poured  in  to  the  depth 
of  about  J  inch  and  allowed  to  set.  By  means  of  a  sterile 
needle  or  knife  blade  an  embryo  may  be  detached  from  the 
grain,  previously  softened  in  water,  and  placed  on  the  surface 
of  the  starch  gelatine.  It  can  be  brought  into  close  contact 
with  the  starch  gelatine  by  melting  a  minute  portion  of  the 
jelly  immediately  under  the  embryo  with  a  warm  sterile  needle. 
Several  embryos  may  thus  be  set  up  and  allowed  to  remain  at 
a  temperature  of  about  18°  C.  for  a  day  or  two.  At  the  end 
of  that  time  sections  of  the  jelly  a  little  wider  than  the  embryo 
may  be  cut  out  so  that  the  jelly  immediately  below  the  embryo 
can  be  observed.  On  treating  the  slices  of  jelly  with  a  little 
dilute  iodine  solution  it  will  be  found  that  a  semicircular  space 
below  the  embryo  is  colourless,  thus  showing  that  the  embryo 
has  secreted  amylase,  which  has  saccharified  the  starch  in  its 
immediate  vicinity. 

By  making  similar  observations  with  the  other  embryos 
used,  at  intervals,  say,  of  twenty-four  hours,  it  will  be  seen 
that  the  area  affected  increases  as  the  embryo  develops. 

Brown  and  Morris  have  shown  that  embryos  separated  from 
the  barley  grain  in  this  way  can  be  grown  on  quite  a  variety 
of  different  media.  Thus,  e.g.,  barley  embryos  could  be  grown 
in  the  endosperm  of  a  wheat  grain,  the  embryo  of  the  latter 
being  removed.  They  can  also  grow  in  solutions  of  sugar  or 
even  on  moist  filter  paper,  their  action  in  the  last  two  cases 
being  very  probably  due  to  the  secretion  of  enzymes  other 
than  amylase.  Careful  experiment  has  shown  that  the  amy- 
lase is  secreted  mainly  by  cells  in  the  neighbourhood  of  the 
scutellar  epithelium  (6,  Plate  II  (ii)). 


122      BACTEKIOLOGICAL    AND    ENZYME    CHEMISTRY 

By  means  of  further  observations,  using  starch  gelatine  as 
a  cultivation  medium,  it  can  be  shown  that  various  micro- 
organisms are  capable  of  secreting  amylase.  Thus,  e.g.,  an 
ordinary  Petri  dish  may  be  taken  and  a  thin  layer  of  melted 
starch  gelatine  poured  into  it  and  allowed  to  set.  A  few  drops 
of  ordinary  sewage  diluted  ten  times  with  water  can  then  be 
run  over  the  surface  of  the  jelly,  any  excess  being  poured  off  ; 
at  the  end  of  twenty-four  hours  a  number  of  colonies  will 
probably  have  appeared.  On  pouring  a  dilute  solution  of 
iodine  on  and  off  the  plate,  a  number  of  colonies  will  be  found  to 
be  surrounded  with  white  rings,  showing  that  the  starch  has 
been  saccharified  in  their  immediate  neighbourhood,  i.e.,  that 
the  particular  organism  forming  the  colony  has  the  power 
of  secreting  amylase.  It  is  possible,  of  course,  to  take  out 
such  colonies  with  a  sterile  platinum  wire  and  prepare  streak 
cultures  in  starch  gelatine  tubes. 

In  order  to  be  sure  that  the  white  ring  observable  on  addi- 
tion of  iodine  is  not  simply  due  to  the  production  of  alkalinity 
in  the  medium  at  that  point,  the  plate  may  be  treated  with 
dilute  hydrochloric  acid  prior  to  the  addition  of  iodine,  but 
in  this  case  there  is  danger  that  the  colonies  may  be  sterilised. 

Among  the  bacteria  which  produce  amylase  Koch's  cholera 
bacillus  may  be  mentioned,  also  B.  anihracis,  B.  megatherium, 
and  B.  lactis  aerogenes,  which  is  a  characteristic  sewage 
organism.  B.  coli  communis  does  not,  however,  secrete  amy- 
lase ;  in  fact,  this  organism  can  be  used  as  an  elegant  test  for 
the  production  of  sugar  by  an  amylase-secreting  organism, 
such  as  the  bacillus  of  cholera  or  anthrax,  by  growing  the 
latter  in  starch  gelatine  and  then  incubating  with  B.  coli,  when 
the  characteristic  gas  formation  due  to  the  fermentation  of 
sugar  by  this  organism  will  be  noted. 

That  the  saccharification  of  the  starch  is  really  due  to  the 
formation  of  amylase  by  the  organism,  and  that  it  is  not  due 
simply  to  its  ordinary  developmental  activity,  may  be  proved 
by  taking  a  little  of  the  converted  starch  gelatine,  melting 


AMYLASE    IN  THE    LIVING   CELL  123 

with  a  little  thymol  in  order  to  inhibit  vital  phenomena,  and 
adding  the  mixture  to  a  further  quantity  of  starch  gelatine, 
when  saccharification  will  continue,  showing  that  the  change 
is  due  to  an  enzyme  secreted  by  the  organism  which  is  capable 
of  acting  whether  the  organism  be  alive  or  not. 

Besides  numerous  bacteria  a  certain  number  of  moulds  are 
also  capable  of  secreting  amylase,  e.g.,  Aspergillus  niger ;  this 
can  readily  be  shown  by  making  a  streak  culture  (Fig.  5  6.)  of 
this  organism  in  a  tube  of  starch  gelatine.  After  some  days, 
when  a  vigorous  growth  of  the  mould  has  taken  place,  the 
gelatine  may  be  melted,  dissolved  in  warm  water,  and  filtered 
from  the  mould  and  the  filtrate  tested  with  Fehling's  solution 
for  the  presence  of  maltose. 

All  the  foregoing  experiments  necessitate  care  in  manipula- 
tion in  order  to  prevent  infection  by  extraneous  organisms, 
but  with  a  little  practice  in  bacteriological  technique  they  are 
not  difficult  to  carry  out  and  are  highly  instructive.  The 
secretion  of  an  enzyme,  such  as  amylase,  is  analogous  to  the 
secretion  of  toxins  by  pathogenic  organisms,  and  the  chemical 
problems  involved  in  all  these  cases  are  of  a  similar  nature.  It 
will  be  shown  later  that  certain  organisms,  e.g.,  Aspergillus 
niger,  are  capable  of  secreting  enzymes  suitable  to  the  conditions 
of  their  environment.  Thus,  e.g.,  Aspergillus  niger,  is  capable, 
not  only  of  saccharifying  starch,  but  also  of  inverting  cane 
sugar  and  of  splitting  up  fats ;  in  fact,  it  has  been  shown  by 
Delepine  that  this  organism  can  derive  sus  enance  from 
almost  every  conceivable  organic  medium.  Similarly,  a  yeast 
cell  can  bring  about  quite  a  number  of  different  chemical 
changes.  Organisms  of  simpler  structure  and  function,  such 
as  bacteria,  are  more  limited  in  their  range  of  activity,  but  there 
is  no  doubt  that  they  too  are  capable  of  bringing  about  a 
variety  of  changes  according  to  their  differing  environments. 
A  possible  explanation  suggests  itself  here  of  the  difference  in 
pathogenic  effect,  which  is  observed  when  the  same  organism 
is  cultivated  under  differing  conditions. 


124      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

There  is  evidence  also  that  the  amylase  secreted,  e.g.,  by 
the  growing  plant  embryo  and  by  growing  micro-organisms, 
is  somewhat  different  chemically  from  the  amylase  secreted 
by  the  purely  vegetative  organs  of  plants,  e.g.,  the  leaf  cells,  and 
by  animal  cells.  Thus,  an  amylase  is  secreted  by  the  salivary 
gland,  and  its  presence  can  be  demonstrated  by  warming  a 
little  3  per  cent,  starch  solution  with  a  few  drops  of  saliva, 
and  testing  with  iodine  and  Fehling  solution.  The  enzyme 
can  be  precipitated  from  saliva  in  the  usual  way  by  means  of 
alcohol.  If  necessary  the  secretion  of  saliva  can  be  stimulated 
by  inhaling  a  little  ether. 

The  presence  of  amylase  can  also  be  demonstrated  in 
pancreatic  extract. 

Brown  and  Morris  have  exhaustively  investigated  the 
conditions  of  formation  of  amylase  in  foliage  leaves,  and  the 
following  description  from  their  paper1  will  serve  as  a  very 
good  example  of  the  methods  used  in  this  kind  of  research, 
and  will  usefully  illustrate  the  application  of  the  analytical 
processes  described  in  Chapter  V. 

A  quantity  of  leaves  of  tropaeolum  majus  were  dried  in  a 
steam  oven  and  ten  grams  of  the  dried  leaves  were  treated  with 
boiling  water.  The  solution  was  cooled  to  50°  C.  and  digested 
with  a  little  amylase  for  two  hours.  The  mixture  was  then 
filtered  and  the  filtrate  and  washings  made  up  to  144  c.c. 

The  optical  activity  in  a  10  cm.  tube  was  then  found  to 
amount  to  1*9  divisions.  100  c.c.  of  the  solution  also  re- 
duced 0-532  gram  CuO,  which  is  equivalent  to  0'395  gram 
maltose. 

This  amount  of  maltose  in  a  10  cm.  tube  will  rotate  the 
polarised  ray  through  T54  divisions  of  the  scale.  The 
difference  between  this  value  and  the  observed  value,  viz., 
1'90  —  1*54  =  0*36,  must  be  due  to  dextrin,  amounting  in 
weight  to  0'064  grm. 

1  '  A  Contribution  to  the  Chemistry  and  Physiology  of  Foliage  Leaves/ 
Journ.  Chem.  Soc.  Trans.  1893,  p.  629. 


AMYLASE   IN  THE  LIVING  CELL  125 

The  total  144  c.c.  of  solution  or  ten  grams  of  leaf  have 
therefore  yielded : — 

Maltose  05688  grm.  =  0'5486  grm.  starch 
Dextrin  0'0922  grm.  =  0'0922     „ 

Total  =  0-6408     „ 

Ten  grams  of  leaf  therefore  contain  0'6408  grm.  starch  or 
6' 408  per  cent,  of  their  weight. 

If  an  appreciable  amount  of  malt  extract  has  been  used,  a 
correction  must  of  course  be  made  for  it  by  determining  its 
optical  activity  and  copper-oxide  reducing  power  in  a  similar 
manner. 

The  determination  of  the  actual  amount  of  amylase 
present  is  not  possible,  but  comparative  determinations  can 
be  made  by  measuring  the  amount  of  saccharification  which  a 
given  amount  of  tissue  can  perform  under  standard  conditions 
in  a  given  time.  The  starch,  it  must  be  remembered,  in  order 
that  the  '  law  of  proportionality '  may  obtain,  must  always 
be  in  large  excess. 

Brown  and  Morris  investigated  thirty-four  species  of 
plants ;  they  found  that  all  of  them  contained  a  measurable 
amount  of  amylase,  the  greatest  quantities  being  obtained 
from  kguminosae,  especially  the  common  pea.  They  found 
that  the  amount  of  amylase  present  varied  with  the  environ- 
ment, the  greatest  quantity  being  found  when  the  plant  was 
kept  in  darkness ;  on  exposure  to  light  diminution  in  the 
quantity  of  amylase  present  took  place.  It  is,  of  course, 
well  known  that  starch  formation  in  the  leaf  cells  takes  place 
in  presence  of  light ;  it  appears,  therefore,  that  the  digestion 
of  the  starch  and  consequent  formation  of  amylase  takes 
place  in  darkness ;  thus  the  starch  and  amylase  are  present 
in  inverse  proportion^ 


CHAPTER  VII 

INVERTASE    AND    MALTASE 

Invertase.  —  Invertase,  or  sucrase,  is  the  enzyme  which 
brings  about  the  inversion  of  cane  sugar  according  to  the 
following  equation  :  — 


CAOn  +  H20  =  C6H1206  + 

Cane  sugar  Dextrose  Lsevulose 

Invertase  is  most  readily  prepared  from  beer  yeast.  A 
quantity,  say  ten  grams,  is  thoroughly  washed  with  water 
with  the  aid  of  a  filter  pump  ;  it  is  then  mixed  with  100  c.c. 
of  water  and  about  1  c.c.  of  chloroform;  the  chloroform 
prevents  the  growth  of  the  yeast.  On  warming  the  mixture 
for  a  few  hours  at  about  30°  C.  and  filtering,  a  solution  is 
obtained  which  contains  the  enzyme  invertase.  This  can  be 
proved  by  adding,  say,  5  c.c.  of  the  solution  to  50  c.c.  of  a 
10  per  cent,  solution  cane  sugar  and  warming  to  about  30°  C. 
for  an  hour.  Before  testing  for  the  presence  of  invert  sugar, 
the  solution  should  be  boiled  to  remove  the  chloroform  present, 
which  otherwise  would  tend  to  reduce  the  Fehling  solution. 
The  boiled  solution  should  be  cooled  and  made  up  to  its 
original  volume  with  distilled  water,  and  the  invert  sugar 
determined  by  means  of  Fehling  solution  and  by  the  polari- 
meter  in  the  usual  way. 

It  will  be  seen  that  the  separation  of  invertase  from  the1 
yeast  has  not  necessitated  the  breaking  up  of  the  yeast  cell  ; 
simple  diffusion  has  been  sufficient  to  extract  it.  Invertase 


INVERTASE  127 

is,  therefore,  an  enzyme  capable  of  passing  through  the  cell 
wall,  and  thus  belongs  to  the  class  known  as  extra-cellular 
enzymes,  in  contradistinction  to  other  enzymes  found  in 
yeast  such  as  maltose  and  zymase,  which  are  only  obtained  if 
the  cell  wall  is  partially,  at  any  rate,  broken  down. 

Invertase  can  be  separated  from  solution  in  the  ordinary 
way  by  precipitation  with  alcohol,  as  was  first  shown  by 
Berthelot.  It  has  been  exhaustively  investigated  by  O'Sulli- 
van  and  Thompson.  The  best  yield  of  invertase  was  obtained 
from  yeast  liquor,  which  results  when  well  pressed  sound 
yeast  is  allowed  to  stand  for  some  time.  A  process  of  self- 
digestion  then  sets  in,  the  yeast  being  converted  into  a  dark- 
coloured  liquid  with  a  characteristic  but  not  unpleasant 
smell.  An  addition  of  47  per  cent,  of  alcohol  to  this  liquid 
gives  a  good  precipitate  of  invertase.  They  found  that  the 
action  of  invertase  on  cane  sugar  proceeded  in  accordance 
with  the  law  which  has  been  found  to  obtain  in  purely 
chemical  reactions,  in  which  no  condition  varies  except 
the  diminution  of  the  changing  substance ;  i.e.,  if  the 
quantities  of  sugar  inverted  were  plotted  as  ordinates  to 
a  curve,  and  the  corresponding  times  as  abscissae,  a  definite 
time  curve  resulted. 

The  speed  of  the  reaction  was  found  to  increase  with  the 
temperature  up  to  55°-60°  C.,  but  at  75°  C.  the  enzyme  is 
destroyed. 

Caustic  alkalis  were  found  to  be  instantly  destructive  of 
the  enzyme,  whereas  minute  quantities  of  sulphuric  acid  were 
favourable  to  its  action.  Any  excess  of  acid  above  a  defined 
minimum  was,  however,  detrimental  in  its  effect. 

There  appeared  to  be  no  limit  to  the  activity  of  the 
enzyme,  as  a  sample  of  invertase  which  had  inverted  100,000 
times  its  own  weight  of  sugar  was  still  active. 

It  is  noteworthy,  in  view  of  the  more  recent  work  on 
maltase,  that  the  products  of  the  reaction  appeared  to  have 
no  effect  on  its  rate ;  on  the  other  hand,  the  enzyme  can 


128      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

withstand  a  temperature  25°  higher  in  presence  of  sugar 
than  when  heated  by  itself. 

The  secretion  of  invertase  by  a  typical  mould,  e.g.,  Asper- 
gillus  niger,  can  be  demonstrated  by  the  following  method 
described  by  Duclaux. 

A  quantity  of  Kaulin's  solution  (see  p.  27)  should  be 
made  up  and  sterilised  by  heating  several  times  in  the  steam 
steriliser.  A  large  sterile  Petri  dish  about  20  cm.  in  diameter 
is  taken  and  filled  to  a  depth  of  one  centimetre  with  the  sterile 
solution.  It  is  then  inoculated  by  means  of  a  sterile  platinum 
wire  with  a  pure  cultivation  of  Aspergillus  niger  and  the  whole 
is  allowed  to  develop  for  some  days ;  a  voluminous  growth 
quickly  takes  place.  When  the  mould  has  developed  over 
the  surface  and  has  acquired  a  green  or  brownish  colour, 
the  liquid  can  be  carefully  siphoned  off  from  beneath  it 
and  the  solution  replaced  by  sterile  water.  On  repeating 
this  operation  at  the  end,  say,  of  two  days,  practically  no 
sugar  will  be  found  to  be  present  on  testing  with  Fehling 
solution.  On  filtering  the  solution  and  warming,  say  10  c.c. 
with  50  c.c.  of  a  10  per  cent,  solution  of  cane  sugar,  inversion 
will  be  found  to  take  place,  showing  that  invertase  has  been 
secreted  by  the  organism,  and  has  gone  into  solution. 

Maltase  or  Glucase. — This  enzyme  converts  maltose  into 
dextrose  according  to  the  following  equation  : — 

CuH«Ou  +  H20  =  2C6Hla06 

Maltase  is  an  enzyme  which  occurs  in  yeast,  but  whoso  pre- 
sence is  not  so  easy  to  demonstrate  as  that  of  invertase.  The 
following  method  is  described  by  Croft  Hill  (see  also  Brown's 
*  Laboratory  Studies/  p.  142) :  A  quantity  of  ordinary  pressed 
brewer's  yeast  is  well  washed  by  decantation  and  drained  and 
pressed  over  the  filter  pump  ;  it  is  then  finely  crumbled  in 
a  mortar  and  further  drained  from  moisture  if  necessary. 
About  twenty  grams  are  then  taken,  spread  in  a  thin  layer  on 
a  porous  plate  and  dried  in  a  vacuum  desiccator  over  sulphuric 


MALTASE  129 

acid  for  two  or  three  days.  The  dry  mass  is  then  powdered 
very  finely  in  a  mortar  and  transferred  to  an  air-bath,  the 
temperature  of  which  must  be  raised  very  slowly  (in  about 
two  hours)  to  50°  C.,  at  which  point  it  must  be  kept  for 
one  hour. 

To  demonstrate  the  presence  of  maltase  in  the  prepared 
yeast,  add  about  0*5  gram  of  the  powder  to  100  c.c.  of  a  solution 
of  about  5  per  cent,  of  maltose  of  known  rotatory  power 
containing  0'5  c.c.  of  toluene  as  an  antiseptic  (chloroform 
must  not  be  used,  as  it  prevents  the  action  of  maltase).  Cork 
the  flask  containing  this  solution  and  keep  it  at  a  temperature 
of  35°  C.  for  three  or  four  hours.  The  solution  is  then  filtered 
and  examined  in  the  polarimeter.  A  considerable  fall  in  the 
rotation  will  be  found  to  have  taken  place,  due  to  the  formation 
of  dextrose ;  the  presence  of  dextrose  may  be  confirmed  by 
preparing  its  osazone. 

The  action  of  maltase  upon  maltose  is  of  very  special 
interest,  as  it  is  the  first  case  of  a  reversible  enzyme  action 
that  has  been  studied.  Croft  Hill  found  that  if  maltase  was 
added  to  a  very  concentrated  solution  of  dextrose  a  disaccha- 
ride  was  formed.  He  at  first  thought  that  this  was  a  simple 
reconversion  of  dextrose  into  maltose,  but  further  research 
showed  that  the  sugar  formed  was  isomeric  with  maltose.  The 
essential  fact  remained  that  while  in  dilute  solutions  there  was 
a  breaking  down  of  larger  into  smaller  molecules,  in  concen- 
trated solutions  there  was  a  building  up  or  synthesis  of  the 
simpler  molecules  into  more  complex.  This  would  seem  to 
indicate  that  all  enzyme  actions  are  potentially  reversible,  and 
the  direction  of  the  reaction  depends  on  the  concentration  of 
the  solution  and  the  relative  masses  of  the  reacting  bodies ; 
thus  in  solutions  of  less  than  four  per  cent,  of  dextrose  no 
formation  of  disaccharose  occurred. 

Subsequent  to  Croft  Hill's  researches  other  instances  of 
reversible  enzyme  action  have  been  discovered.  Thus 
Fischer  and  Armstrong  have  found  that  isolactose  can  be 


130      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

synthesised  in  the  presence  of  the  enzyme  lactase  from  a 
mixture  of  equal  proportions  of  glucose  and  galactose  ;  and 
certain  fat-splitting  enzymes  have  been  found  to  act  reversibly, 
but  the  difficulty  of  working  with  very  concentrated  solutions 
limits  the  number  of  successful  experiments  in  this  direction. 
The  importance  of  such  synthetic  reactions  cannot  be 
over-estimated,  as  we  see  here  a  possibility  of  bringing  about 
reactions  by  methods  closely  akin  to  those  by  which  the 
synthesis  of  natural  substances  is  effected  by  the  living 
organisms,  whether  plant  or  animal. 


CHAPTEE  VIII 

THE  ALCOHOLIC  FERMENTATION  OF  GRAPE  SUGAR 

IT  has  already  been  shown  how  by  the  action  of  the  enzyme 
invertase,  secreted  by  the  yeast  cell,  ordinary  cane  sugar  takes 
up  the  elements  of  water  to  form  a  molecule  of  dextrose  and  a 
molecule  of  laevulose  according  to  the  equation  :  — 


Dextrose  Laevulose 

and  it  was  shown  how  this  enzyme  could  readily  be  extracted 
from  the  yeast.  If  yeast  is  allowed  to  develop  in  a  solution 
of  sugar  an  entirely  different  and  more  profound  change 
takes  place.  This  may  be  demonstrated  by  the  following 
experiment. 

About  eight  grams  of  cane  sugar  are  added  to  about  200  c.c. 
of  water  in  an  ordinary  half-litre  flask,  and  about  1  c.c.  of 
fresh  brewer's  yeast  added.  The  flask  is  then  placed  in  an 
incubator  at  a  temperature  of  24°  C.,  and  after  some  time  an 
effervescence  of  gas  takes  place.  If  a  stopper  with  a  bent 
tube  is  attached  to  the  flask  and  the  tube  led  below  the 
surface  of  a  little  lime  water,  the  latter  will  turn  milky,  showing 
that  the  gas  evolved  consists  of  carbon  dioxide.  The  contents 
of  the  flask  after  fermentation  has  continued  for  some  time 
will  be  found  to  have  an  alcoholic  smell.  If  the  flask  is  now 
attached  to  a  Liebig's  condenser,  and  placed  on  a  water-bath, 
the  alcohol  can  be  distilled  over.  It  is  possible  more  simply 
to  demonstrate  its  presence  by  attaching  a  long  tube  to  the 

K2 


132     BACTERIOLOGICAL   AND    ENZYME   CHEMISTRY 

flask ;  on  heating  the  latter  on  the  water-bath  alcohol  will 
be  seen  first  of  all  to  condense  in  the  tube,  and  afterwards  to 
pass  off  as  vapour,  which  can  be  easily  detected  by  applying 
a  light,  when  the  characteristic  non-luminous  flame  of  alcohol 
is  produced.  This  is  the  alcoholic  fermentation  of  sugar  which 
is  the  foundation  of  the  great  brewing  and  distilling  industries. 
As  it  is  of  great  technical  and,  one  might  add,  social  importance, 
it  has  been  studied  from  the  very  earliest  times,  and  only 
recently  great  additions  have  been  made  to  our  knowledge  of 
it.  The  history  of  the  subject  is  very  largely  the  history  of 
fermentation,  and  some  brief  account  of  the  older  theories 
of  this  process  will  not  only  be  of  interest  in  itself,  but  may 
enable  the  full  bearing  of  modern  investigations  to  be  better 
understood. 

Alcoholic  fermentation  has  been  known  from  the  very 
earliest  times ;  the  preparation  of  beer  from  barley,  of  wine 
from  grapes  and  the  leavening  of  dough  are  mentioned  in  the 
oldest  known  writings.  By  the  alchemists  alcoholic  fermenta- 
tion was  much  studied ;  the  Philosopher's  Stone  was  considered 
to  be  a  kind  of  ferment.  No  very  clear  ideas  were,  however, 
possessed  by  the  alchemists  in  regard  to  what  took  place,  and 
a  confusion  existed  in  their  time  between  fermentation  and 
effervescence,  which  were  not  properly  distinguished  till  the 
middle  of  the  seventeenth  century. 

The  great  medical  chemist  Libavius  (1595)  considered  that 
fermentation  was  a  process  akin  to  digestion,  a  guess  the  true 
bearing  of  which  it  is  hardly  likely  that  its  author  properly 
appreciated. 

An  even  more  happy  suggestion  was  made  in  1648  by  Van 
Helmont,  who  stated  that  out  of  the  ferment  something  passes 
into  the  fermenting  liquid,  which  grows  in  it  as  a  seed. 

The  authors  of  the  phlogistic  theory  of  combustion,  Becher 
and  Stahl,  paid  attention  to  alcoholic  fermentation.  Becher 
showed  that  the  juice  of  grapes  does  not  ferment  if  the  skin  of 
the  grape  is  unruptured,  and  thus  showed  that  alcohol  was  not 


ALCOHOLIC    FERMENTATION    OF   SUGAR          133 

pre-existent  in  grape  juice  as  had  been  imagined,  e.g.,  by  the 
alchemist  Basil  Valentine.  Becher  considered  that  air  was 
necessary  for  the  process  ;  according  to  the  phlogistic  theory, 
an  unknown  substance,  phlogiston,  was  set  free  on  combustion. 
As  air  was  necessary,  according  to  his  theory,  for  fermentation, 
he  regarded  it  as  a  species  of  combustion  in  which  likewise 
phlogiston  disappeared.  The  exact  methods  of  Lavoisier 
and  Cavendish  threw  light  upon  this  problem,  as  upon  the 
simpler  problems  of  combustion  and  all  chemical  combinations 
in  general.  Cavendish  determined  the  amount  of  carbon 
dioxide  given  off  from  a  known  weight  of  sugar.  Lavoisier 
weighed  both  the  alcohol  and  the  carbon  dioxide.  He 
thought  at  first  that  they  exactly  made  up  the  weight  of  the 
sugar  taken,  and,  his  mind  filled  with  the  chemistry  of  oxygen 
and  the  formation  of  oxides  by  combustion,  he  regarded  sugar 
also  as  an  oxide  splitting  off  into  two  simpler  oxides,  that  is 
alcohol  and  carbonic  acid,  by  fermentation.  He  thought  at 
first  that  the  yeast  suffered  no  change,  but  found  that  this 
was  not  the  case,  and  recognised  further  that  the  breaking 
up  of  the  sugar  was  not  so  simple  as  he  had  at  first  imagined, 
certain  by-products  in  addition  to  the  main  substance  formed 
being  always  present. 

Gay  Lussac  in  1810  contributed  some  very  interesting 
experiments ;  although  these  did  not,  in  the  light  of  subse- 
quent investigations,  confirm  the  conclusions  which  he  drew 
from  them,  yet  they  are  highly  instructive.  He  exposed 
some  grapes  with  unbroken  skins  to  hydrogen  gas  so  as  to 
eliminate  all  oxygen  from  their  surface,  he  then  expressed  the 
juice  into  a  vessel  over  mercury  in  such  a  way  that  no  air 
could  gain  access.  So  long  as  air  was  not  present,  no  fermen- 
tation took  place,  but  immediately  oxygen  was  pumped  into 
the  vessel,  fermentation  arose.  He  was  also  able  to  prevent 
fermentation  of  grape  juice  by  confining  it  in  an  atmosphere 
of  sulphur  dioxide ;  he  naturally  concluded  that  oxygen  was 
an  essential  factor  in  the  fermentation  process,  and  that  in 


134      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

its  absence  no  change  would  take  place.  He  considered 
that  oxygen  set  up,  as  it  were,  a  movement  among  the  particles 
of  the  ferment  which  was  communicated  throughout  the 
liquid.  The  true  explanation  of  Gay  Lussac's  results  was 
reserved  for  later  investigators. 

It  was  Cagniard  de  Latour  who  made  a  careful  examination 
of  the  fermentation  process  and  suggested  that  the  decom- 
position of  the  sugar  was  due  to  the  growth  of  yeast.  Shortly 
after  this  began  the  long  conflict  of  opinion  between  the 
supporters  of  the  purely  biological  and  of  the  purely  chemical 
theory  of  fermentation.  It  was  in  1836-37  that  Schwann 
furnished  his  famous  experiment  of  passing  air  through 
red-hot  tubes  and  afterwards  into  fermentable  solutions, 
when  no  change  took  place.  Gay  Lussac's  notion  that 
fermentation  was  due  to  oxygen  was  thus  shown  to  be 
untenable.  Schwann  concluded  that  fermentation  must  be 
due  to  living  organisms  suspended  in  the  air,  which  were 
destroyed  when  they  passed  through  a  red-hot  tube. 

The  great  authority  of  Liebig  was  thrown  on  the  side  of 
the  purely  chemical  explanation  of  fermentation.  It  was  he 
who  developed  the  idea  of  catalysis,  a  word  already  invented 
by  Berzelius.  Liebig  compared  fermentation  changes  to  such 
catalytic  actions  as  have  been  mentioned  in  the  first  chapter 
of  this  book,  e.g.,  the  effect  of  finely  divided  platinum  in 
accelerating  the  union  of  gases  at  low  temperatures,  etc. 
He  considered  the  ferment  or  catalyst  to  be  itself  in  a  state 
of  unstable  equilibrium  or  decomposition,  which  it  communi- 
cated to  its  surroundings,  producing  chemical  change,  as 
the  additional  snowflake  may  precipitate  an  avalanche.  To 
Liebig' s  purely  chemical  explanation  were  opposed  the  famous 
researches  of  Pasteur  and  Tyndall  on  the  possibilities  of 
spontaneous  generation.  Briefly  Pasteur's  method  was  to  boil 
fermentable  solutions  in  flasks  provided  with  finely  drawn 
out  necks,  which  after  the  solution  was  boiled  would  either 
be  sealed  or  bent  in  such  a  way  that  germs  could  not  enter. 


ALCOHOLIC    FERMENTATION    OF   SUGAR  135 

Tyndall  allowed  the  open  ends  of  flasks  containing  boiled 
fermentable  solutions  to  communicate  with  a  chamber  whose 
walls  were  coated  with  glycerine,  and  the  air  in  which  had 
been  allowed  to  be  at  rest  for  some  time ;  in  this  way  all  the 
germs  present  settled  and  were  fixed  by  the  glycerine.  That 
the  space  was  free  from  germs  was  proved  by  passing  a 
strong  beam  of  light,  as  explained  on  p.  8. 

The  researches  of  Pasteur  and  Tyndall  corroborated  one 
another :  no  fermentation  took  place  in  Pasteur's  boiled  flasks 
when  the  precaution  was  taken  to  prevent  subsequent  access 
of  germs ;  similarly  no  fermentation  took  place  in  TyndalPs 
flasks  when  the  beam  of  light  showed  the  air  above  them  to 
be  germ  free.  Pasteur,  therefore,  contended  that  no  fermen- 
tation took  place  without  an  organism,  and  he  even  went 
further,  and  stated  that  for  any  given  fermentation  a  specific 
organism  must  be  present.  Liebig  remained  unconvinced  ; 
he  found  that  while  no  fermentation  occurred  in  a  solution 
seeded  with  yeast  after  filtration  through  a  membrane, 
yet  an  extract  of  meat  similarly  filtered  became  putrid. 
Moreover,  Liebig  quoted  his  own  experiments,  in  conjunction 
with  "Wohler,  on  the  decomposition  of  oil  of  bitter  almonds 
into  benzaldehyde  and  grape  sugar  by  a  substance  contained 
in  the  almond,  which  we  should  now  call  an  enzyme.  He 
considered  that  a  substance  of  a  like  character  must  be 
secreted  by  the  yeast,  and  that  the  only  connection  between 
the  physiological  development  of  the  yeast  and  the  phenomena 
of  fermentation  is  the  production  in  the  living  cell  of  a  substance 
which,  acting  as  a  ferment  or  catalyst,  effects  the  decom- 
position of  the  sugar.1 

Liebig  died  in  1873  before  the  publication  of  the  recent 
researches,  which  have  provided  an  explanation  of  the  apparent 
contradiction  between  the  purely  vital  or  physiological 
theories  of  Pasteur  and  his  own  purely  chemical  point  of  view. 

The  development  of  enzyme  chemistry  has  been  to  a  large 
1  Ann.  Chem.  Pharm.,  153,  1870,  p.  0. 


136      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

extent  independent  of  advances  in  bacteriology,  being  more 
intimately  related  with  physiology,  both  animal  and  vegetable ; 
thus  the  gastric  juice  of  birds  was  studied  by  Reaumur  and  the 
Abbe  Spallanzani  in  the  latter  part  of  the  eighteenth  century. 

In  1822  Dubrunfaut  published  experiments  showing  that 
the  saccharification  of  starch  was  due  to  a  small  quantity 
of  active  substance  secreted  by  the  barley  grain  ;  he,  in  fact, 
discovered  the  existence  of  what  we  now  term  amylase.  This 
work  was  followed  up  later  in  1833  by  Payen  and  Persoz, 
who  discovered  the  method  of  precipitation  by  alcohol  now 
generally  used  for  the  preparation  of  enzymes.  Allusion 
has  also  been  made  to  the  decomposition  of  the  glucoside 
amygdalin  by  an  enzyme  which  is  known  as  emulsin.  All 
these,  it  will  be  seen,  are  products  of  the  activity  of  cells  of 
highly  organised  animals  or  plants.  The  earliest  instance 
of  the  isolation  of  an  enzyme  from  a  micro-organism  is  the 
case  of  urease,  or  the  ferment  which  converts  urea  into 
ammonium  carbonate,  and  which  was  shown  by  Musculus  to 
be  present  in  the  dead  cells  of  the  organism  micrococcus  ureae, 
which  develops  in  putrid  urine. 

The  isolation  of  invertase  from  yeast  was  dealt  with  in 
Chapter  VII.  It  was  originally  discovered  in  the  early  part 
of  the  nineteenth  century  by  Dobereiner  and  Mitscherlich,  and 
isolated  later  by  Berthelot  by  precipitation  with  alcohol. 

It  is  only  comparatively  recently,  however,  that  an 
enzyme  has  been  discovered  capable  of  producing  alcoholic 
fermentation  in  solutions  of  grape  sugar.  Invertase  is 
capable  to  a  large  extent  of  being  washed  out  of  the  yeast 
cell  without  rupture  of  the  cell  wall.  In  1897  Buchner  of 
Munich,  by  employing  drastic  measures  for  breaking  down 
the  yeast  cells  and  expressing  the  juice,  was  enabled  to  prepare 
a  solution  which  would  cause  alcoholic  fermentation  to  take 
place  in  solutions  of  cane  sugar. 

Buchner's  method  was  as  follows  :  1000  grams  of  brewer's 
yeast  were  carefully  mixed  with  an  equal  weight  of  quartz 


ALCOHOLIC    FERMENTATION   OF   SUGAR  137 

sand  and  250  grams  of  infusorial  earth  generally  known  as 
Kieselguhr,  and  the  mixture  was  ground  together  till  plastic 
and  damp ;  100  grams  of  water  were  added  to  the  mixture,  and 
it  was  then  wrapped  up  in  a  press  cloth  and  put  in  a  filter  press 
capable  of  exerting  a  pressure  of  400  to  500  atmospheres. 
About  300  c.c.  of  juice  were  thus  obtained.  The  remaining 
cake  was  ground  up  again,  sieved,  and  another  100  c.c.  of 
water  added  ;  on  again  pressing  a  further  150  c.c.  of  juice  were 
obtained.  The  whole  volume  of  juice  was  clarified  by  shaking 
with  Kieselguhr  and  filtering. 

Thus  prepared,  the  juice  is  a  clear  opalescent  liquid  of  a 
pleasant  yeast  smell,  with  a  specific  gravity  at  17°  C.  of 
T0416.  On  boiling,  a  quantity  of  albuminoid  matter  separates 
and  the  liquid  becomes  nearly  solid. 

If  the  unboiled  juice  is  mixed  with  an  equal  volume  of  con- 
centrated cane  sugar  solution,  an  evolution  of  carbon  dioxide 
begins  after  a  period  varying  from  a  quarter  of  an  hour  to  an 
hour,  and  the  evolution  continues  for  about  twenty-four  hours. 

Similar  results  are  obtained  from  grape  sugar  and  from 
fructose,  but  not  from  lactose  or  mannitol ;  this  corresponds 
with  the  activity  of  the  living  yeast.  Every  precaution  was 
taken  to  work  aseptically,  and  no  yeast  cells  could  be  found  on 
microscopical  examination  of  the  liquid.  Moreover,  the  action 
is  not  stopped  by  chloroform,  nor  by  passage  of  the  liquid 
through  a  Berkefeld  filter  or  through  a  dialysing  membrane. 
Hydrocyanic  acid  stops  the  action,  but  this  recommences  if 
air  is  passed  through  to  drive  off  the  HCN,  showing  that  the 
effect  of  the  latter  is  not  due  to  the  poisoning  of  the  living 
organism,  but  more  probably  to  the  formation  of  a  loose  com- 
pound between  HCN  and  the  enzyme. 

The  fermentation  is  a  true  alcoholic  fermentation  in  that 
alcohol  and  C02  are  produced  in  the  same  proportion  as  when 
living  yeast  is  used;  by-products  such  as  succinic  acid 
and  glycerine  are  also  produced.  The  enzyme  which  is 
present  in  the  solution  has  been  termed  by  Buchner  zymase. 


138      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

The  preparation  of  yeast  juice  by  Buchner's  method  requires 
special  apparatus  for  obtaining  high  pressures  which  is  not  to 
be  found  in  every  laboratory.  It  is  possible,  however,  to 
demonstrate  the  power  of  alcoholic  fermentation,  which  is 
possessed  by  yeast  apart  from  its  ordinary  vital  activity,  by 
making  use  of  a  preparation  described  by  Albert  in  1900,  and 
known  as  permanent  yeast  ('Dauerhefe')  or  more  recently 
zymin.  This  is  prepared  in  the  following  way  :  Yeast  is 
rubbed  into  a  powder  and  brought  into  a  mixture  of  alcohol 
and  ether,  filtered  over  the  filter  pump,  and  again  submitted  to 
the  same  process  of  digestion  with  alcohol  and  ether  and  filter- 
ing. It  is  then  washed  with  alcohol  and  ether  and  finally  with 
dry  ether  ;  on  allowing  the  ether-wet  substance  to  dry  at  air 
temperature  zymin  is  obtained  as  a  fine  impalpable  powder. 
On  examination  under  a  high-power  microscope  it  will  be 
found  that  the  finer  structure  of  the  yeast  cell  has  disappeared. 

If,  now,  a  small  quantity  of  this  powder  is  ground  up  with 
a  few  c.c.  of  a  warm  solution  of  sugar  and  a  little  sand,  and  the 
mass  poured  into  a  narrow  tube,  say  about  5  mm.wide  and  20 
cm.  long,  the  whole  being  then  placed  in  the  incubator  and 
kept  at  a  temperature  of  about  27°  C.,  an  evolution  of  gas  will 
be  observed  in  about  half  an  hour,  and  with  larger  quantities 
the  presence  of  alcohol  can  be  detected  in  the  usual  way. 

This  preparation  of  zymin  is  termed  permanent  yeast, 
because,  in  contradistinction  to  the  yeast  juice  of  Buchner,  it 
will  retain  its  activity  for  a  prolonged  period.  Yeast  juice,  on 
the  other  hand,  rapidly  loses  its  activity  on  standing,  and  such 
inactive  yeast  juice  is  further  characterised  by  the  fact  that 
it  gives  no  precipitate  on  boiling,  that  is  to  say,  that  the 
albumin  content  of  the  juice  has  been  broken  down.  It  would 
appear,  therefore,  that  in  addition  to  zymase  the  yeast  juice 
contains  another  enzyme  which  is  capable  of  digesting  albumin, 
that  is,  a  proteolytic,  or,  to  use  Armstrong's  nomenclature, 
proteoclastic  enzyme  ;  this  enzyme  would  seem  to  digest  not 
only  the  albumin  present  but  also  the  zymase. 


ALCOHOLIC   FERMENTATION   OF   SUGAR  139 

These  facts  have  led  to  the  very  interesting  series  of  re- 
searches by  Harden  and  Young.  Harden  showed  that  if  an 
equal  volume  of  blood  serum  was  added  to  the  yeast  juice, 
digestion  of  the  yeast  albumin  did  not  proceed  so  rapidly  and 
the  activity  of  the  zymase  was  increased,  that  is,  there  was  a 
more  prolonged  alcoholic  fermentation.  Harden  and  Young 
further  found  that,  besides  serum,  boiled  yeast  juice  greatly 
increased  the  alcoholic  fermentation ;  thus  the  total  fermenta- 
tion produced  by  25  c.c.  of  yeast  juice  acting  on  2'5  grams 
of  glucose,  was  on  the  average  doubled  by  the  addition  of  an 
equal  volume  of  boiled  juice,  and  increased  to  a  maximum  when 
three  to  five  volumes  were  added,  after  which  it  decreased. 

It  might  be  contended  with  equal  justice  either  that  this 
increase  of  fermentation  was  due  to  an  increase  in  the  activity 
of  the  zymase,  to  decrease  in  the  activity  of  the  proteolytic 
enzyme,  or  to  a  combination  of  these  causes.  As  a  matter  of 
fact,  the  true  cause  is  being  found  to  lie  rather  deeper  than 
might  at  first  sight  be  concluded.  When  fresh  yeast  juice  is 
boiled  there  is,  as  has  been  stated,  a  heavy  precipitation  of 
albuminous  matter ;  if  this  is  filtered  off,  the  filtered  juice 
still  increases  the  activity  of  the  zymase ;  this  would  seem  to 
indicate  that  the  increased  activity  was  not  an  enzyme  effect, 
as  enzymes  in  general  are  destroyed  by  boiling.  The  unknown 
substance  is  besides  capable  of  passing  through  a  dialyser, 
but,  on  the  other  hand,  is  precipitated  by  75  per  cent,  alcohol. 

It  was  possible  by  an  ingenious  experiment  to  show  that 
the  alcoholic  fermentation  certainly  depends  on  two  substances, 
neither  of  which  is  capable  alone  of  causing  fermentation. 
By  soaking  an  ordinary  Chamberland  filter  candle  (such  as  is 
often  attached  to  household  water  taps  for  the  purpose  of 
removing  organisms  from  the  water  before  drinking)  in  melted 
gelatine  and  allowing  the  latter  to  set  in  the  pores  of  the  filter, 
it  is  possible  to  obtain  a  fairly  rapid  dialysis  of  colloidal 
matter  by  filtering  a  solution  containing  such  matter  through 
this  gelatine  filter,  under  high  pressure.  This  method  of 


140      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

dialysis  under  pressure  was  suggested  by  Dr.  Martin,  the 
director  of  the  Lister  Institute,  and  the  filter  is  known  generally 
as  a  Martin  filter.  If,  now,  yeast  juice  is  passed  through  such  a 
filter  a  residue  is  obtained  soluble  in  water,  and  it  is  found  that 
neither  this  residue  nor  the  liquid  which  passes  through  the 
filter  are  either  of  them  separately  capable  of  causing  alcoholic 
fermentation.  On  the  other  hand,  when  brought  together  the 
mixture  produces  fermentation  almost  equal  to  that  in  the 
original  juice. 


Fia.  22. — APPARATUS  FOR  MEASURING  RATE  OF 
EVOLUTION  OF  C02. 

The  proteolytic  enzyme,  as  might  be  expected,  remains 
behind  on  the  filter  with  the  rest  of  the  colloidal  matter,  and 
on  adding  the  residue  to  water,  digestion  of  the  albumin 
rapidly  proceeds.  The  addition  of  the  filtered  juice  does  not 
increase  this  effect.  So  far,  then,  it  is  clear  that  the  alcoholic 
fermentation  is  due  to  at  least  two  substances,  one  of  a  colloidal 
and  the  other  of  a  crystalloidal  nature. 

It  should  be  explained  that  in  studying  the  amount  and 
rate  of  alcoholic  fermentation,  the  evolution  of  C02  is  taken  as 
a  measure  of  this  change.  This  rate  of  evolution  is  measured  in 
the  special  apparatus  (Fig.  22). 


ALCOHOLIC   FERMENTATION    OF   SUGAR 


141 


The  fermenting  mixture  is  contained  in  the  round- 
bottomed  flask,  placed  in  a  constant  temperature  water- 
bath.  The  C02  is  collected  over  mercury  in  the  graduated 
burette,  a  constant  pressure  being  maintained  by  the  simple 
compensating  arrangement  shown  in  the  diagram. 

Table  III  will  illustrate  the  results  already  described  : — 

TABLE  in 
(i)  The  effect  of  the  addition  of  boiled  juice 


CO2  evolved  in  presence  of 


Volume  added 

Water 

Boiled  Juice 

1 

gram 
0-19 

gram 
0-33 

4 

0-17 

0-53 

6 

0-14 

0-65 

(ii)  Experiment  on  the  filtration  of  yeast  juice  : — 


C02  evolved  in  presence  of 


No.  of 
Experi- 
ment 

Original 
juice 

Residue 

Filtrate 

Mixture  of 
Residue  and 
Filtrate 

1 

gram 

gram 
0-013 

gram 
0 

gram 
0-068 

2 

0-0704 

0 

o-ooi 

0-051 

3 

0-0704 

0-001 

0-008 

0-064 

4 

— 

0 

0107 

0-040 

The  question  now  remains,  what  is  the  nature  of  the 
dialysable  matter,  or  co-ferment,  as  it  has  been  termed, 
which  is  necessary  for  the  activity  of  the  enzyme,  which 


142      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

latter  presumably  is  contained  in  the  nondialysable  residue 
and  is  capable  of  digestion  by  the  proteolytic  enzyme  also 
present  therein.  Harden  and  Young's  further  researches 
have  shown  that  phosphoric  acid  is  at  any  rate  a  necessary 
constituent  of  this  dialysable  substance.  On  addition  of  a 
phosphate  to  unfiltered  yeast  juice  a  great  increase  of  fer- 
mentation is  obtained.  On  the  other  hand,  this  effect  is  not 
produced  if  the  phosphate  is  added  to  the  residue  or  to  the 
filtrate  separately,  and  consequently  the  phosphate,  though 
apparently  necessary  to  the  reaction,  is  not  the  initially  active 
agent.  Moreover,  the  phosphate  does  not  affect  living  yeast. 
It  appears  that  the  phosphate  actually  takes  part  in  the 
fermentation  reaction,  and  that  for  every  molecule  of  sugar 
which  is  broken  down  into  alcohol  and  carbon  dioxide,  a 
molecule  of  a  complex  hexose  phosphate,  a  compound  of 
a  sugar  molecule  with  two  of  phosphate,  is  simultaneously 
formed.  This  compound  has  actually  been  isolated.  The 
ordinary  fermentation  involves  the  phosphate  present  in 
ordinary  yeast  juice,  either  as  hexose  phosphate  or  free 
phosphate,  and  this  phosphate  passes  repeatedly  through 
the  cycle  of  changes  represented  in  the  following  equations :— 

(1)  2C6H1206  +  2R2HP04 

=  2C02  +  2CyS60  +  C6H1004(P04R2)2  +  2H20 

(2)  C6H1004(P04R2)2  +  2H20  =  C6H1206  +  2K2HP04 

The  hexose  phosphate  as  is  shown  in  equation  (2)  is 
hydrolysed  with  the  production  of  free  phosphate,  which 
again  undergoes  reaction  (1),  partly  with  the  sugar  formed 
at  the  same  time,  and  partly  with  fresh  sugar  from  the 
solution.  The  rate  at  which  the  second  of  these  reactions 
occurs  determines  the  rate  of  fermentation  observed  when 
glucose  is  fermented  by  yeast  juice,  which  is  therefore  a 
measure  of  the  rate  at  which  phosphate  is  being  formed  in 
the  juice. 


ALCOHOLIC   FERMENTATION   OF   SUGAR  143 

The  fermentation  of  mannose  and  fructose  in  the  presence 
of  yeast  juice  has  also  been  examined  by  Harden  and 
Young.  They  discovered  that  while  mannose  behaves  towards 
yeast  juice  in  the  same  manner  as  glucose  both  in  presence 
and  absence  of  added  phosphates,  fructose  is  much  more 
rapidly  fermented  in  the  presence  of  phosphates  than  either 
of  the  other  two  sugars. 

An  excess  of  phosphates  lowers  the  rate  of  fermentation 
of  glucose  and  mannose  by  yeast  juice,  but  an  addition  of 
fructose  to  the  fermenting  mixture  under  these  conditions  has 
the  effect  of  inducing  a  rapid  fermentation  of  the  other  sugars. 
Fructose  in  this  case  appears  to  act  as  a  catalyst.  The  addition 
of  glucose  or  mannose  under  similar  circumstances  has  no 
similar  effect. 

The  precise  part  played  by  the  fructose  in  this  interesting 
change  is  not  yet  fully  elucidated. 

The  fermentation  produced  by  yeast  juice  is,  of  course, 
not  exactly  the  same  thing  as  the  fermentation  which  results 
from  the  activity  of  the  living  yeast  cell.  The  respiratory,  as 
distinct  from  the  fermentative,  activity  of  the  yeast  has  also 
to  be  considered.  While  Harden  and  Young's  researches  are 
of  the  highest  value  as  an  intimate  study  of  a  detached 
portion  of  the  problem,  the  study  of  the  conditions  of  activity 
of  the  living  cell  is,  of  course,  also  necessary  for  a  complete 
solution  of  the  question. 

The  labours  of  earlier  workers  in  this  field  have  been 
supplemented  in  recent  years  by  the  researches  of  Slator. 

He  determined  the  rate  of  fermentation  by  measuring  the 
change  of  pressure  due  to  evolution  of  carbon  dioxide. 

He  found  that  in  comparatively  small  intervals  of  time  the 
rate  of  fermentation  was  proportional  to  the  amount  of  yeast 
taken,  and  was  independent  of  the  concentration  of  the  sugar 
except  in  very  dilute  solutions. 

The  interesting  observation  was  made  that  while  galactose 
is  not  fermented  by  yeasts  grown  in  other  solutions,  it  is 


144      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

possible  to  acclimatise  certain  yeasts  by  growing  them  in  a 
mixture  of  this  sugar  and  dextrose,  after  which  they  will  attack 
galactose  readily. 

The  results  of  the  experiments  on  the  fermentation  of 
different  sugars  by  yeasts  lead  to  the  conclusion  that  the 
enzyme  of  the  yeast  combines  with  the  sugar,  and  that  the 
velocity  of  formation  of  carbon  dioxide  is  determined  by 
the  rate  of  decomposition  of  the  compound  formed. 

It  is  still  somewhat  an  open  question  whether  there  are 
present  in  yeast  cells  a  large  number  of  enzymes,  each  capable 
of  exerting  its  own  specific  action,  or  whether  only  a  few 
enzymes  are  present,  and  that  the  same  enzyme  can  promote 
different  chemical  actions. 

From  the  foregoing  pages  it  is  evident  that  the  chemistry 
of  the  yeast  cell  has  been  a  fruitful  subject  of  inquiry.  The 
researches  that  have  been  considered  are  of  great  scientific 
interest  in  showing  the  complexity  of  the  reactions  which  take 
place  even  under  the  comparatively  simple  conditions  afforded 
by  a  single  cell  of  yeast. 

They  have  also  a  very  important  bearing  on  the  fermenta- 
tion industries,  which  have  for  their  object  the  preparation  of 
various  forms  of  alcoholic  beverages. 

While  it  is  impossible  usefully  to  consider  these  in  this 
book,  owing  to  the  complexity  of  their  purely  technical  detail, 
mention  should  be  made  of  the  great  advance  made  in  the 
brewing  industry  by  the  use  of  pure  cultures  of  yeasts  intro- 
duced by  Hansen. 

His  method  of  obtaining  these  on  a  small  scale  has  been 
described  in  Chapter  II.  By  successive  inoculation  into 
larger  and  larger  volumes  of  sterile  wort  it  has  been  possible 
to  brew  beer  by  means  of  one  culture  only.  The  brewer  is 
enabled  thus  to  conduct  the  process  of  fermentation  under 
rigidly  controlled  conditions. 


CHAPTER  IX 

THE  ACID  FERMENTATION  OF  ALCOHOLS  AND 
CARBOHYDRATES 

IT  is  probable  that  the  earliest  fermentation  known  to  man 
was  the  souring  of  milk  ;  this  we  now  know  to  be  due  to  the 
fermentation  of  milk  sugar,  and  it  is  one  of  the  more  important 
of  a  class  of  fermentation  changes,  all  of  which  essentially 
consist  in  the  oxidation  of  the  characteristic  alcohol  group 
CH2OH  to  the  group  characteristic  of  acids,  viz.  C02H  or 
carboxyl,  either  by  addition  of  oxygen  or  by  intra-molecular 
change. 

The  simpler  carbohydrates  or  sugars  are,  as  we  have  learnt, 
ketone  or  aldehyde  alcohols,  and  therefore  lend  themselves  to 
this  change. 

The  oxidation  of  the  alcohol  group  can  of  course  be  brought 
about  by  purely  chemical  reactions.  The  chemical  method 
which  is  of  most  interest  in  the  present  connection  is  the 
oxidation  of  alcohols  by  means  of  platinum  black  ;  the  latter 
is  obtained  as  a  black  precipitate  when  solutions  of  platinum 
salts  are  treated  with  certain  reducing  agents.  This  finely 
divided  platinum  has  the  power  (see  p.  3)  of  enormously 
accelerating  the  rate  of  combination  of  oxidisable  vapours 
with  oxygen  when  the  two  are  led  over  it  together ; 
thus,  e.g.,  ordinary  formalin  or  formaldehyde  is  prepared  by 
bubbling  air  through  methyl  alcohol,  and  leading  the  mixture 
of  air  and  methyl  alcohol  vapour  over  platinum  black.  In 
this  case  indeed  it  is  sufficient  to  heat  a  spiral  of  platinum  wire, 
and  plunge  it  into  the  mixture  of  methyl  alcohol  vapour  and 

L 


146      BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 


air,  for  the  reaction  to  begin.     The  wire  continues  to  glow  so 
long  as  the  gases  pass  over  it. 

The  oxidation  of  alcohol  vapour  by  means  of  platinum 
black  can  be  shown  by  the  following  simple  experiment. 

A  wide  shallow  porcelain  dish  is 
placed  upon  a  water-bath  and  a  little 
alcohol  poured  in,  about  a  gram  of 
platinum  black  is  placed  in  a  watch 
glass  resting  on  a  small  tripod,  the  whole 
is  covered  by  a  large  inverted  funnel, 
through  the  neck  of  which  a  piece  of 
blue  litmus  paper  is  suspended  (Fig.  23). 
On  gently  warming  the  alcohol  it 
vapourises  and  oxidation  takes  place 
at  the  surface  of  the  platinum  black ; 
aldehyde,  and  finally  acetic  acid,  being  ob- 
tained, the  presence  of  which  is  rendered 
evident  by  the  reddening  of  the  litmus 
paper.  Care  must  be  taken  to  vapourise 
the  alcohol  very  slowly,  or  oxidation 
may  take  place  with  explosive  violence. 
The  oxidation  of  alcohols  by  means  of 
platinum  black  has  been  dwelt  on  at 
some  length  because  it  offers  the  nearest 
analogy  to  bacteriological  or  enzyme  re- 
actions. There  are  good  reasons  for  think- 
ing that  the  progressive  oxidation  of  an 
alcohol  to  an  acid  takes  place  by  addition  of  oxygen,  through 
the  formation  of  additional  hydroxyl  groups,  and  subsequent 
elimination  of  water.  Thus  the  addition  of  oxygen  to  the 
group  — CH2OH  may  be  considered  to  result  first  in  the  forma- 

H 

tion     of    the    group    — C— OH ;     such    a    combination    is 

\H 


FIG.  23.— THE  OXI- 
DATION or  AL- 
COHOL VAPOUR. 


ACID   FERMENTATION  147 

unstable,  and  water  is  eliminated  with  formation  of    the 
aldehyde  group. 

H 


-C-OH  =  -C          +  H20 


Further  addition  of  oxygen  gives  rise  to  an  acid,  thus  :  — 

/0H 
+  0  =  -C 


If  the  fermentation  is  carried  still  further,  hydrocarbons  and 
carbon  dioxide  (C02)  generally  result.  E.g.,  calcium  acetate 
undergoes  fermentation  with  formation  of  calcium  carbonate 
and  marsh  gas,  thus  :  — 

CH3C02 

)Ca  +  H20  =  CaC03  +  2CH4  +  C02 
CH3CO/ 

Buchner  and  Meisenheimer  and  others  l  have  shown  by 
methods  similar  to  those  employed  in  the  case  of  zymase, 
that  the  acetic  acid  and  lactic  acid  fermentations  can  take 
place  in  the  absence  of  living  bacteria  ;  only  small  quantities 
of  material  are  however  thus  transformed  compared  with  the 
corresponding  activity  of  living  organisms.  Few,  if  any,  of 
these  reactions  are  confined  to  specific  bacteria,  consequently 
the  oxidation  of  an  alcohol  through  the  intervention  of  the 
living  organism  is  a  highly  complex  process,  generally  resulting 
in  a  number  of  secondary  products.  The  action  of  an  organism, 
as  has  been  frequently  stated,  may  be  broadly  described  as 
respiratory  and  fermentative.  It  consumes  a  certain  amount 
of  the  medium  for  building  up  its  own  structure  ;  in  such  a 

1  Fuhrmann,  Vorlesungen  iiber  Bakterienenzyme. 


148      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

case  ultimate  products,  such  as  C02  and  other  gases,  result. 
Incidentally,  as  it  were,  more  of  the  medium  has  to  be  broken 
up  than  actually  suffices  for  the  food  of  the  organism,  and  we 
thus  get  the  normal  products  of  fermentation.  The  course  of 
reaction,  therefore,  in  every  case  depends  on  several  factors,  viz. : 

1.  The  nature  and  molecular  constitution  of  the  ferment- 
able substance,  whether  an  alcohol,  aldehyde  or  ketone,  etc. 

2.  Whether  any  other  food  supply  is  present,  thus,  e.g., 
the  character  of  the  decomposition  of  a  sugar  has  been  found 
to  vary  according  to  the  presence  or  otherwise  of  peptone  in 
the  nutrient  mixture. 

3.  The  species  and  state  of  growth  of  the  organism  ;   for 
instance,  results  will  vary  according  as  the  culture  is  or  is  not 
of  recent  growth,  or  according  to  whether  it  comes  from 
strains  which  have  been  transplanted  from  time  to  time  in  the 
laboratory. 

A  complete  account  of  all  the  oxidation  changes  of  the 
type  under  consideration,  and  of  the  bacteria  concerned 
therein,  would  lead  too  far  and  would  be  of  doubtful  utility, 
inasmuch  as  many  of  them  have  not  been  worked  out  in 
detail.  Keference  will  be  made  in  the  first  place  to  three 
fermentations  of  technical  importance,  and  afterwards  some 
account  will  be  given  of  the  detailed  work  in  the  case  of 
specific  organisms  which  will  serve  to  illustrate  the  method 
of  research  used  in  this  class  of  inquiry. 

The  Oxidation  of  Alcohol  to  Acetic  Acid. — The  simple 
equation  expressing  this  reaction  is  as  follows  : — 

CH3CH2OH  +  02  =  CH3C02H  +  H20 

In  reality,  for  reasons  mentioned  above,  the  bacterial 
oxidation  of  alcohol  is  by  no  means  capable  of  so  simple  an 
expression. 

It  is  well  known,  of  course,  that  alcoholic  liquids  such  as 
wine  and  beer,  on  exposure  to  air,  gradually  become  sour. 


ACID    FERMENTATION  149 

The  true  explanation  of  this  phenomenon  was  afforded  by 
the  researches  of  Pasteur,  though  others,  e.g.,  Person  in  1822, 
had  noticed  the  growth  of  organisms  as  a  fine  film  on  the 
surface  of  such  a  liquid  and  had  given  the  name  Mycoderma 
aceti  to  the  growth. 

Pasteur  showed  that  certain  rod-like  bacteria  were  the  true 
causes  of  the  formation  of  acetic  acid,  while  other  organisms 
which  might  be  present,  such  as  yeasts,  etc.,  carried  the  oxida- 
tion further  to  C02  and  H/).  Hansen  was  the  first  to  obtain 
pure  cultivations  of  Mycoderma  aceti,  and  discovered  also 
further  species  capable  of  bringing  about  the  same  change. 
As  a  matter  of  fact,  as  already  indicated,  quite  a  large  number 
of  organisms  can  effect  the  formation  of  acetic  acid,  not  only 
from  ethyl  alcohol,  but  from  other  alcohols  and  carbohydrates 
which  contain  the  characteristic  group — CHgOHgOH. 

It  should  be  pointed  out  that  the  formation  of  acetic  acid 
by  bacterial  action  can  only  take  place  within  certain  limits 
of  concentration,  and  in  presence  of  the  essential  ingredients  of 
bacterial  food,  that  is,  nitrogen  must  be  present  in  some  form, 
e.g.,  as  peptone  or  albumin,  and  phosphorus  as  phosphate. 

The  Lactic  Acid  Fermentation.— As  already  mentioned, 
the  souring  of  milk  is  due  to  the  formation  of  lactic  acid  by 
decomposition  of  milk  sugar.  The  simple  chemical  equation 
in  this  case  is  as  follows :  The  milk  sugar  is  first  inverted, 
forming  two  hexose  molecules — 

C12H22011  +  H20  =  20^,0. 

By  a  simple  molecular  decomposition  one  hexose  molecule 
yields  two  molecules  of  lactic  acid,  thus  : — 

0.^0.  =  2C3H603 

The  production  of  lactic  acid  from  milk  can  be  brought 
about  by  the  addition  of  a  small  quantity  of  previously  soured 
milk.  The  reaction  quickly  reaches  a  limit  if  the  solution  is 
allowed  to  become  too  acid,  and  therefore  chalk  is  generally 


150      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

added  to  neutralise  the  free  acid  as  it  is  formed,  calcium 
lactate  being  the  result. 

Under  certain  conditions  a  further  decomposition  of  the 
lactic  acid  occurs,  forming  butyric  acid  according  to  the  follow- 
ing equation  : — 

2C3H603  =   C4H802  +  2C02  +  2H2 

Lactic  acid  Butyric  acid        Carbonic          Free 

acid  gas        hydrogen 

This  butyric  fermentation  is  brought  about  by  a  number 
of  organisms,  some  of  which  are  anaerobic. 

The  equation  on  p.  149,  representing  the  formation  of 
lactic  acid  from  a  hexose,  must  only  be  taken  as  a  part  of  what 
actually  occurs.  Moreover,  in  the  case  of  lactic  acid,  there  are 
in  this  simple  equation  further  possibilities  because,  as  already 
explained,  lactic  acid  contains  an  asymmetric  carbon  atom, 
and  therefore  exists  in  three  possible  forms,  viz.,  a  right-handed 
and  left-handed,  and  an  inactive  modification.  Which  of  these 
forms  remains  at  the  end  of  the  reaction  depends  on  the  con- 
ditions of  experiment.  It  will  be  remembered  that  the  lactic 
acid  above  referred  to  is  the  a-acid  CH3  C  HOHC02H,  the 
central  carbon  atom  being  asymmetric  ;  the  inactive  form  is 
almost  always  met  with  as  the  result  of  lactic  acid  fermenta- 
tion.1 There  is  also,  it  may  be  remembered,  another  form  of 
lactic  acid,  viz.  /3-lactic  acid,  CH2OHCH2C02H,  the  production 
of  which  is  a  further  possibility.  Its  production  has  been 
stated  to  occur  when  inosite  is  fermented  r.nder  certain  condi- 
tions,but  the  evidence  of  its  occurrence  is  somewhat  conflicting. 

The  chemical  interest  of  the  lactic  acid  fermentation  centres, 
therefore,  generally  round  the  conditions  of  production  of  the 
right-handed  and  left-handed  modifications,  and  reference  may 
therefore  be  made  to  the  experiments  of  Frankland  and 
MacGregor,  which  indicate  that  the  inactive  or  racemic  form 
of  acid  may  in  certain  cases  be  produced,  after  which  a  pre- 
ferential decomposition  of  one  of  the  modifications  takes  place. 

Frankland  and  MacGregor  experimented  with  a  bacterial 

1  An  active  form  known  as  sarcolactic  acid  can  be  obtained  from  meat 
juice, 


ACID    FERMENTATION  151 

growth  which  had  the  power  of  exciting  a  vigorous  fermenta- 
tion in  suitable  solutions  of  calcium  lactate.  The  composition 
of  the  medium  was  as  follows  : — 


Calcium  lactate,         3     grams 
Peptone  solution,       0'3  gram 
Salt  solution,  30     c.c. 

Calcium  carbonate,    3     grams; 


made  up  to  300  c.c.  with 
distilled  water. 


This  solution  was  inoculated  with  a  minute  quantity  of 
calcium  lactate  solution  in  active  fermentation. 

The  quantity  of  nutrient  solution,  its  concentration  (3  per 
cent,  instead  of  1  per  cent.,  as  above,  being  occasionally 
used),  and  the  duration  of  the  fermentation  were  varied  in 
different  cases.  At  the  end  of  each  experiment  the  calcium 
carbonate  was  filtered  off  and  the  filtrate  concentrated  and 
examined  in  the  polarimeter.  The  calcium  was  removed  from 
solution  by  means  of  oxalic  acid,  and  the  filtrate  from  the 
calcium  oxalate  evaporated  on  a  water-bath  to  remove  volatile 
acids.  The  lactic  acid  remaining  was  separated  from  other 
impurities  by  precipitation  with  lead  acetate,  decomposition  of 
the  lead  salt  with  H^S,  evaporation  of  the  nitrate  from  the  lead 
sulphide,  and  extraction  with  ether.  The  residue  after  eva- 
poration of  the  ether  was  converted  into  the  zinc  salt  by  boiling 
with  zinc  carbonate,  and  the  solution  of  the  zinc  salt  was  again 
examined  in  the  polarimeter.  This  zinc  salt  was  found  to  be 
pure  laevo-rotatory  zinc  lactate.  The  calcium  lactate  originally 
taken  was  inactive,  so  that  there  had  evidently  been  prefer- 
ential decomposition  of  the  dextro  salt.  If  the  fermentation 
was  stopped  at  too  early  a  stage,  the  active  lactate  was  found 
to  be  mixed  with  a  large  quantity  of  inactive  lactate,  whilst 
when  the  fermentation  was  too  long  continued,  the  active 
lactate  was  also  destroyed.  The  above  description  will  serve 
to  illustrate  the  kind  of  investigation  necessary  for  determining 
the  precise  products  of  a  reaction,  when  there  is  a  possibility  of 
one  or  another  stereo  -  chemical  modification  being  produced. 


152      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

The  production  of  an  inactive  or  active  modification  depended 
in  this  case  on  the  organism.  With  different  conditions  of 
experiment  different  results  are  obtained,  and  the  production 
of  a  dextro  or  laevo  form  depends  quite  as  much  on  the 
fermenting  medium  as  on  the  organism  producing  the  fermen- 
tation. The  dependence  of  the  products  of  the  reaction  upon 
the  constitution  of  the  fermenting  molecule  has  been  the 
subject  of  a  very  interesting  research  by  Harden,  who  has 


FIG.  24. — APPAEATUS  USED  IN  DR.  HARDEN'S  EXPERIMENTS  ON 
B.  coli  communis. 

studied  the  chemical  action  of  B.  coli  commums  and  similar 
organisms  on  carbohydrates  and  allied  compounds. 

The  apparatus  shown  in  Fig.  24  was  made  use  of  ;  one  litre 
of  the  solution  was  placed  in  a  large  flask  provided  with  a 
side  tube  and  an  indiarubber  stopper,  through  which  passed  a 
straight  glass  tube  leading  to  the  bottom  of  the  flask,  the  side 
tube  and  vertical  tube  were  plugged  with  cotton  wool  and  the 
flask  then  sterilised.  The  side  tube  was  then  attached  to  a 
piece  of  bent  tubing  (A)  on  which  a  small  bulb  was  blown  near 
tjie  bend?  a  drop  of  mercury  was  placed  in  the  tube  and  served 


ACID    FERMENTATION  153 

to  seal  the  apparatus  and  prevent  diffusion,  whilst  at  the  same 
time  it  readily  allowed  gases  to  pass  out  of  the  flask.  After 
inoculation,  which  was  effected  by  removing  the  stopper 
and  introducing  a  loop  full  of  the  culture,  the  air  of  the  flask 
was  displaced  by  nitrogen,  prepared  by  the  action  of  ammonia 
solution  on  copper. 

After  passing  nitrogen  for  about  one  to  two  hours,  the 
flask  was  removed  and  the  long  vertical  tube  sealed  off  at  a 
constriction  previously  made  near  the  top.  It  was  then  placed 
in  an  incubator  (Fig.  24),  the  side  of  which  was  pierced  by  a 
brass  tube,  with  which  the  tube  A  is  connected  by  indiarubber 
tubing.  The  apparatus  for  collecting  and  measuring  the  gas 
was  connected  with  the  other  end  of  the  brass  tube.  It 
consisted  of  a  Winchester  quart  bottle  (B),  fitted  as  an  aspirat- 
ing bottle,  and  provided  with  a  long  piece  of  indiarubber 
tubing  passing  to  the  bottom  of  a  second  bottle,  graduated  in 
volumes  of  100  c.c.  on  a  piece  of  paper  pasted  to  the  glass.  On 
the  tube  between  the  flask  and  the  collecting  bottle  was 
placed  a  three-way  tap  (D),  by  means  of  which  samples  of 
gas  can  be  withdrawn  either  directly  from  the  flask  or  from 
the  collecting  bottle.  The  collecting  bottle  was  filled  with 
saturated  brine,  on  the  surface  of  which  a  little  oil  was  poured, 
to  prevent  absorption  of  carbonic  acid  gas.  Direct  experiment 
showed  that  a  mixture  of  carbonic  acid  gas  and  air  could  be 
preserved  over  this  liquid  for  a  considerable  time  without 
undergoing  any  perceptible  alteration  in  composition. 

About  100  c.c.  of  brine  were  placed  in  C,  and  the  connecting 
rubber  tube  was  also  filled  with  brine  so  that  the  volume  of  gas 
evolved  could  be  measured  by  that  of  the  liquid  displaced. 

During  the  period  of  incubation  the  flask  was  agitated  at 
frequent  intervals  in  order  to  secure  the  neutralisation  of  the 
acid  produced,  and  the  volume  of  the  liquid  displaced  was 
read  off,  the  measuring  bottle  being  raised  or  lowered  until 
the  surface  of  the  liquid  in  it  was  at  the  same  level  as  that  in  the 
collecting  bottle, 


154      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

As  soon  as  about  two  litres  of  gas  had  been  collected  a 
sample  of  about  500  c.c.  was  taken  for  analysis.  The  remainder 
of  the  gas  was  swept  out  through  the  three-way  tap  by  raising 
the  measuring  bottle,  and  the  apparatus  then  arranged  as  before 
for  the  collection  of  a  fresh  quantity  of  gas. 

At  the  close  of  about  fourteen  days  the  flask  was  removed 
from  the  incubator,  and  a  culture  made  on  agar,  which  was 
examined  and  in  every  case  found  to  give  the  usual  tests  for 
normal  B.  coli  communis,  or  other  organism  studied.  The 
solution  was  then  measured,  and  aliquot  portions  removed 
for  the  estimation  of  the  various  constituents. 

These  products  comprised  :— 

Lactic  acid, 
Succinic  acid, 
Acetic  acid, 
Ethyl  alcohol, 
Formic  acid, 
Carbon  dioxide, 
Hydrogen. 

The  effect  of  various  nitrogenous  products  serving  as 
sources  of  nitrogen  for  the  organism  was  also  studied.  It 
was  found  that  Witte's  peptone  was  the  best  source  of  nitrogen 
to  employ,  as  the  products  of  its  decomposition  are  not 
sufficient  to  interfere  with  the  estimation  of  those  produced 
from  the  special  compound  under  examination. 

The  general  method  of  preparation  of  the  medium  under 
examination  was  as  follows  :  10  grams  of  Witte's  peptone 
were  boiled  with  tap  water,  20  grams  of  the  sugar  or  other 
compound  to  be  examined  were  added,  together  with  10 
grams  of  pure  calcium  carbonate,  the  whole  being  made  up 
to  one  litre  ;  in  some  cases  2  grams  of  calcium  phosphate 
were  added,  but  no  beneficial  effect  could  be  observed.  Harden 
found  that  glucose  yielded  chiefly  Isevo-lactic  acid  together 
with  5*25  per  cent,  of  the  inactive  form ;  fructose,  arabinose 
and  galactose  behave  similarly.  On  the  other  hand,  mannite 


ACID    FERMENTATION  155 

yields  a  greater  percentage  of  laevo  acid  and  is  especially 
distinguished  by  the  fact  that  nearly  25  per  cent,  of  the  weight 
of  mannite  fermented  appears  as  ethyl  alcohol,  or  more  than 
twice  as  much  as  in  the  case  of  the  sugars  studied. 

The  general  equation  for  the  decomposition  of  glucose 
which  may  be  considered  as  typical  is  as  follows  :  — 

2C6H1206+H20  =  2C3H603+C2H402  +  C2H60+2C02+2H2 

Harden  concludes  that  the  products  of  the  reaction  can  be 
referred  to  the  constitution  of  the  fermenting  substances. 
Thus  the  yield  of  alcohol  depends  essentially  on  the  presence 
of  the  group,  CHL2OHCHOH  ;  this  only  occurs  once  in  glucose, 
whose  formula  it  will  be  remembered  is 


On  the  other  hand,  mannite  contains  the  alcohol-producing 
group  twice,  thus  :  — 


and  consequently  is  capable  of  yielding  a  greater  proportion 
of  alcohol. 

The  mechanism  of  the  fermentation  of  glucose  as  effected 
by  B.  coli  is  shown,  according  to  Harden,  by  the  following 
scheme  :  — 


CH2OH 
CHQH 


C02 


CHOH  CHOH 

I  I 

CHOH  CHOH       =   lactic  acid,  succinic  acid,  etc. 

I     i 
CHOH  CHOH 

COH  CHOH 
COH 


156       BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

The  reaction  in  the  case  of  mannite  would  be  represented 
as  follows  : — 

CH2OH 

|  =  CH3-CH0OH  +  C09  +  H2 

CHOH  CH2OH 

CHOH  CHOH 

!        I 

CHOH  CHOH 

I  I 

CHOH  CHOH 


CH2OH  CHOH 


CH2OH 


=  CH3-CH2OH  +  C02  +  H2 


The  precise  modification  of  lactic  acid  which  may  be 
produced  will,  according  to  this  theory,  depend  on  the  con- 
figuration of  the  three  centre  CHOH  groups  and  also  on  the 
particular  organism  taking  part  in  the  reaction. 

Harden's  suggestion  as  to  the  dependence  of  the  alcohol 
formation  on  the  presence  of  a  terminal  group  CH2OHCHOH 
finds  further  confirmation  in  his  studies  in  conjunction  with 
Walpole  on  the  action  of  B.  lactis  aerogenes  on  glucose  and 
mannite.  They  found  that,  in  addition  to  the  usual  products 
of  this  class  of  fermentation,  glucose  yields  butylene  glycol, 
CH3CH(OH)CH(OH)CH3.  Mannite,  on  the  other  hand,  gives 
similar  products,  but  less  butylene  glycol  and  more  alcohol. 

The  instances  given  in  the  foregoing  chapter  will  suffi- 
ciently indicate  the  complexity  of  the  problem  involved  in 
obtaining  a  full  explanation  of  the  manner  in  which  carbo- 
hydrates break  down  under  the  action  of  bacteria.  Com- 
paratively few  cases  have  been  worked  out  in  a  rigorous 
manner  as  in  the  researches  referred  to  above  ;  it  is,  however, 
only  by  systematic  quantitative  work  of  this  kind,  with  sub- 


ACID   FERMENTATION  157 

stances  whose  chemical  constitution  can  be  determined,  that 
a  sure  advance  in  our  knowledge  of  the  chemistry  of  vital 
action  is  likely  to  be  attained. 

The  technical  applications  of  the  activity  of  acid-forming 
bacteria  are  numerous  and  important. 

The  production  of  vinegar  is  due  to  the  activity  of  various 
species  of  bacteria  which  bring  about  the  oxidation  of  alcohol 
to  acetic  acid.  Different  qualities  of  vinegar  are  obtained 
according  to  the  process  used.  In  France  wine  is  allowed  to 
become  sour  in  vats  which  are  first  filled  with  vinegar,  wine 
being  gradually  added,  with  simultaneous  withdrawal  of  a 
portion  of  the  vinegar.  The  wine  becomes  charged  with 
acetic  acid  bacteria  and  is  rapidly  converted  into  vinegar, 
when  the  withdrawal  of  the  vinegar  formed  and  the  addition 
of  more  wine  is  repeated. 

A  more  rapid  process  is  in  use  in  Germany  and  also  in 
England,  according  to  which  dilute  alcohol  is  slowly  passed 
over  beech  wood  shavings  contained  in  large  vats,  suitably 
ventilated  to  allow  free  passage  of  air.  The  shavings  are 
previously  sown  with  acetic  acid  bacteria.  A  rapid  oxidation 
of  the  alcohol  takes  place. 

In  the  tannery  acid-forming  bacteria  also  play  their  part. 
In  order  to  remove  hair  from  hides,  they  are  generally  first 
soaked  in  lime,  which  has  to  be  thoroughly  removed  from 
the  skin  before  the  tanning  process.  This  removal  takes 
place  partly  in  what  is  known  as  the  '  puering '  or  '  bating ' 
process  and  partly  in  the  subsequent  '  drenching.'  In  the 
'  puering '  process  the  skins  are  placed  in  a  bath  of  dog's  dung 
or  similar  material,  when,  in  addition  to  many  other  changes, 
e.g.,  the  action  of  proteolytic  enzymes  on  the  albumin  con- 
stituents of  the  skin,  ammonium  salts  of  butyric  and  other 
acids  are  formed,  which  exercise  a  solvent  action  on  the 
lime.  This  is  completed  in  the  '  drenching '  process,  where 
the  skins  from  the  bate,  after  washing,  are  placed  in  an 
infusion  of  bran.  A  mixture  of  organic  acids,  chiefly  lactic, 


158      BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 

is  produced  from  the  fermenting  bran,  which  removes  the 
last  traces  of  lime. 

The  very  important  application  of  the  lactic  fermentation 
to  dairy  practice  will  be  referred  to  in  the  special  chapter  on 
the  applications  of  bacteriological  chemistry  to  agriculture. 


CHAPTER  X 

THE  FERMENTATION  OF  CELLULOSE  AND  ALLIED  BODIES 

CELLULOSE,  broadly  speaking,  constitutes  the  framework  of 
the  vegetable  world,  and  when  the  vast  quantity  of  vegetable 
matter  on  the  face  of  the  globe  is  considered,  a  knowledge 
of  the  changes  which  accompany  its  decomposition  and 
absorption  into  the  cycle  of  life  is  seen  to  be  of  the  first  import- 
ance. Before  considering  these  changes  and  the  conditions 
of  their  operation,  some  brief  description  must  be  given  of 
cellulose  and  its  allied  substances. 

Cellulose  can  be  obtained  as  a  residue  after  dissolving 
out  the  other  constituents  of  plants,  by  the  following  experi- 
ment : — 

Dissolve  30  grams  of  powdered  chlorate  of  potash  in 
520  c.c.  of  cold  nitric  acid  (s.g.  I'l).  Suspend  in  this  mixture 
a  number  of  leaves,  stems,  etc.,  and  allow  them  to  remain 
undisturbed  at  a  temperature  not  above  20°  C.  until  they  are 
perfectly  whitened.  This  may  require  from  two  to  three 
weeks. 

Pure  Swedish  filter  paper  (acido  hydrochlorico  et  fluorico 
extracto)  is  practically  pure  cellulose. 

We  are  indebted  for  our  knowledge  of  the  chemistry  of 
cellulose  in  large  measure  to  the  long-continued  and  careful 
researches  of  Cross  and  Bevan,  from  whose  works  the  follow- 
ing information  is  largely  derived. 

From  its  empirical  composition  cellulose  is  found  to  belong 
to  the  carbohydrates  and  its  empirical  formula  is  (C6H1005)n. 


160      BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 

The  composition  of  the  actual  cell  wall  of  plants  varies  greatly, 
as  there  is  a  large  variety  of  substances  known  generically  as 
cellulose,  and  having  the  same  empirical  composition,  but 
which  yet  exhibit  considerable  differences  in  their  physical 
properties  and  in  their  behaviour  towards  reagents. 

Cross  and  Bevan  divided  celluloses  into  three  classes 
according  to  their  behaviour  with  reagents.  The  main 
reagents  used  in  cellulose  investigation  are  strong  acids  and 
alkalies,  which  bring  about  conversion  into  sugar  by  the 
ordinary  hydrolytic  change,  that  is  :  — 

C6H1005  +  H20   =  C6H1206 

Acetic  anhydride  combines  with  any  OH  groups  which 
may  be  present  according  to  a  general  equation  :  — 


2R-OH  +  ^3°  }  0  =  2R-OC2H30  +  H20 

^2^3^  ) 

According,  therefore,  to  their  behaviour  with  these  and 
other  reagents  cellulose  bodies  are  classified  as  follows  :— 

1.  Those  which  offer  a  maximum  resistance  to  hydrolytic 
action  and  which  contain  in  their  molecule  no  directly  active 
CO  groups,  i.e.,  the  CO  is  not  easily  oxidised  and  does  not 
combine,  e.g.,  with  phenyl  hydrazine.     These  are  represented 
by  the  cellulose  of  cotton  fibre. 

2.  Those  of  less  resistance  to  hydrolysis  which  contain 
active  CO  groups,  i.e.,  which  will  give  osazones  with  phenyl- 
hydrazine.     These  are  perhaps  best  regarded  as  oxycelluloses. 
They  appear  to  constitute  the  main  mass  of  the  tissue  of 
flowering  plants  and  they  exist  in  conjunction  with  a  sub- 
stance called  lignine  in  the  walls  of  wheat  cells. 

3.  Those  that  hydrolyse  with  some  facility,  being  more  or 
less  soluble  in  alkalies  and  easily  decomposed  by  acid,  with 
formation  of  carbohydrates  of  low  molecular  weight.     In- 
cluded among  these  is  the  cellulose  of  the  walls  of  the  cells  of 
seeds.     It  will  be  remembered  that  in  the  preparation  of 


FERMENTATION   OF   CELLULOSE  161 

soluble  starch,  the  starch  cellulose  enveloping  the  starch 
granules  was  destroyed  by  digesting  with  dilute  hydrochloric 
acid. 

Allied  with  cellulose  are  kindred  bodies  belonging  to  the 
pectin  group. 

Pectose  is  the  name  given  to  the  parent  substance  of  bodies 
such  as  pectin,  pectic  acid,  etc. 

Pectin  can  be  obtained  by  filtering  the  juice  of  a  ripe 
apple  or  pear  through  muslin,  and  adding  an  equal  bulk  of 
alcohol.  The  pectin  is  precipitated  as  a  stringy  gelatinous 
mass,  which  can  be  reduced  to  a  white  powder  soluble  in 
water. 

A  solution  of  pectin  gelatinises  on  standing,  probably 
by  the  action  of  the  enzyme  pectase  contained  in  the  fruit 
juice. 

The  members  of  the  pectose  group  have  chiefly  been  in- 
vestigated by  the  French  chemist  Mangin,  who  divides  these 
bodies  into  two  series  : — 

(1)  Neutral   bodies   which   vary   in   their   solubility   in 
water.    At  one  extreme  we  have  the  substance  pectose,  which 
is  insoluble  in  water  and  closely  associated  with  cellulose  ;  at 
the  other  extreme  the  substance  known  as  pectin,  which  is 
soluble  in  water  but  tends  to  form  a  jelly  fairly  readily.    Inter- 
mediate between  these  are  bodies  of  a  gelatinous  nature. 

(2)  Substances  allied  to  this  group  are  feeble  acids,  the 
chief  member  being  pectic  acid,  which  occurs  as  calcium 
pectate ;    the  latter  forms  a  binding  substance  between  the 
fibre  of  many  plants. 

Pectose  bodies  differ  from  cellulose  derivatives  in  being  in- 
soluble in  Schweitzer's  reagent.  This  is  obtained  as  follows  : — 

A  saturated  solution  in  water  is  made  of  equal  parts  of 
copper  sulphate  and  ammonium  chloride.  Strong  caustic  soda 
is  added  till  no  further  precipitate  is  formed.  This  precipi- 
tate of  hydrated  copper  oxide  is  dissolved  in  strong  ammonia 
solution  as  required. 


162      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

The  solubility  of  cellulose  in  this  reagent  can  be  tested 
by  warming  a  few  strips  of  filter  paper  in  half  a  test-tube 
full  of  Schweitzer's  reagent  until  solution  is  practically 
complete ;  on  acidification  cellulose  will  be  precipitated  as  a 
flocculent  precipitate.  One  of  the  technical  processes  for  the 
production  of  artificial  silk  is  based  on  the  solution  of  cellulose 
in  copper-ammonium  solutions,  and  its  re-precipitation  under 
conditions  resulting  in  the  production  of  fine  fibres. 

It  is  possible  to  distinguish  microscopically  in  a  plant 
section  between  cellulose  and  pectose,  by  dissolving  out  the 
cellulose  with  a  few  drops  of  Schweitzer's  reagent. 

The  cellulose  can  be  further  distinguished  from  the  pectose 
by  treatment  with  dilute  iodine  ;  partially  hydrated  cellulose, 
such  as  can  be  obtained  by  treatment  of  ordinary  cellu- 
lose with  alkali,  is  stained  blue  by  iodine.  E.g.,  the  cellulose 
precipitated  from  solution  in  the  foregoing  experiment  can 
be  coloured  thus ;  pectose  bodies  give  no  coloration. 

Coming  now  to  the  method  by  which  cellulose  and  pectose 
bodies  are  broken  down  in  nature,  we  find  in  the  case  of  cellu- 
lose that  this  occurs  by  three  well-defined  processes  : — 

1.  By  the  action  of  the  enzyme  cytase  which  is  secreted 
by  cells  and  by  various  organisms. 

2.  By   fermentation    under    anaerobic    conditions,    that 
is,  in  absence  of  air,  through  the  action  of  certain  specific 
bacteria. 

3.  By  decomposition  under  aerobic  conditions,  through  the 
action  of  certain  bacteria  and  moulds  in  presence  generally  of 
nitrates. 

It  is  probable,  of  course,  that  the  action  of  the  organism  in 
the  last  two  cases  is  due  to  secretion  of  a  cellulose- dissolving 
enzyme,  but  this  has  not  so  far  been  actually  isolated. 

1.  We  may  take  these  cases  in  order.  It  will  be  re- 
membered that  in  the  process  of  saccharification  of  starch, 
which  takes  place  in  the  development  of  the  barley  grain,  the 
cell  walls  of  the  endosperm  are  broken  down  and  the  interior 


FERMENTATION   OF   CELLULOSE  163 

of  the  grain  of  malt  rendered  quite  friable,  and  that  for  this 
reason  it  is  almost  impossible  to  obtain  a  section  of  such  a 
grain.  This  is  due  to  a  cellulose-dissolving  enzyme  known 
as  cytase  being  secreted  by  the  growing  embryo,  and  the  action 
of  this  secretion  must  precede  the  action  of  amylase,  if  the 
latter  is  to  obtain  access  to  the  starch  grains  confined  within 
the  cells  of  the  endosperm.  This  can  be  demonstrated  by 
careful  microscopic  observation  of  the  germinating  barley 
grain,  but  more  simply  by  the  following  experiment,  which 
depends  on  the  fact  that  cytase  is  destroyed  at  a  temperature 
above  60°  C. 

A  solution  of  malt  extract  is  taken  and  divided  into  two 
portions,  say  of  50  c.c.  each.  One  of  these  is  heated  for  half 
an  hour  at  70°  C.  In  each  of  the  solutions  a  thin  slice 
of  potato  is  suspended  by  means  of  a  thin  copper  wire 
attached  to  a  glass  rod  or  match  stalk  placed  across  the 
top  of  the  small  beaker  used  for  the  experiment.  A  little 
thymol  is  added  to  each  of  the  solutions  to  prevent  the 
development  of  moulds  or  bacteria,  and  the  two  beakers 
placed  in  an  incubator  at  40°  C.  for  some  days.  It  will 
soon  be  noticed  that  the  slice  of  potato  in  the  solution 
in  which  the  cytase  has  not  been  destroyed  becomes  soft 
and  pulpy,  the  other  slice  remains  quite  hard,  and  the  cell 
walls  can  be  seen  by  microscopical  observation  to  be  quite 
unattacked. 

A  similar  result  can  be  obtained  if  an  infusion  of  raw  oats 
is  used,  and  careful  investigation  by  Horace  T.  Brown  has 
shown  that  the  power  of  grain-feeding  animals  to  digest  such 
food  depends  on  the  enzyme  contained  in  the  food,  and  not 
on  any  cellulose-dissolving  power  possessed  by  the  secretion 
of  the  stomach  of  the  animal. 

2.  The  decomposition  of  cellulose  by  bacteria  in  absence 
of  air  can  be  demonstrated  as  follows  : — 

Some  strips  of  filter  paper  are  placed  in  a  small  flask  and 
a  few  c.c.  of  deposit  from  an  ordinary  sewage  septic  tank,  or 

M2 


164      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

of  mud  from  the  bottom  of  a  stagnant  pond  in  which  fermenta- 
tion has  been  shown  to  take  place  by  the  production  of  gas 
on  stirring  the  deposit  on  the  bottom,  are  added.  The  flask 
is  filled  up  with  water  and  attached  to  a  Hempel  gas  burette. 
On  keeping  the  flask  for  some  days  at  a  temperature  of  about 
35°  C.  gas  will  be  evolved,  and  the  filter  paper  will  show  signs 
of  pitting,  and  after  the  expiration  of  possibly  some  weeks 
will  finally  be  completely  disintegrated.  On  testing  the  gas  it 
will  be  found  to  be  inflammable,  burning  with  a  non-luminous 
bluish  flame,  and  if  analysed  can  be  shown  to  consist  mainly  of 
marsh  gas,  CH4,  together  with  smaller  quantities  of  hydrogen, 
H,  and  carbon  dioxide,  C02. 

This  fermentation  has  been  very  carefully  worked  out  by  the 
Russian  chemist,  Omelianski.  For  the  purpose  of  his  investi- 
gation he  used  Neva  mud  and  pure  Swedish  filter  paper ;  he 
was  able  to  isolate  two  different  organisms,  one  of  which  pro- 
duced marsh  gas  and  the  other  hydrogen.  His  method  of 
separation  depended  on  the  fact  that  both  organisms  formed 
spores,  and  the  spores  of  the  hydrogen  organism  were  able 
to  withstand  a  higher  temperature  than  those  of  the  marsh- 
gas  organism.  On  starting  the  fermentation  the  marsh-gas 
fermentation  is  predominant ;  by  heating  the  mixture  for 
fifteen  minutes  to  75°  C.  at  this  stage,  the  marsh-gas  organism 
was  killed,  but  the  spores  of  the  hydrogen  organism  were 
unaffected.  On  re-inoculating  a  fresh  quantity  of  filter  paper 
from  the  heated  solution,  the  hydrogen  organism  mainly 
developed,  and  by  a  succession  of  similar  operations  he  suc- 
ceeded in  obtaining  pure  cultivations  of  the  two  bacteria. 
They  were  found  to  be  almost  identical  in  appearance 
and  both  produced  spores.  They  differed  only  in  their 
optimum  temperature  of  reaction  and  in  their  resulting 
products. 

He  was  able  to  show  that  the  products  obtained  completely 
accounted  for  the  weight  of  paper  originally  taken,  certain 
fatty  acids  being  produced  together  with  the  gas.  Thus  in  the 


FERMENTATION   OF   CELLULOSE  165 

case  of  the  hydrogen  bacillus  the  following  products  were 
obtained  from  the  original  weight  of  3' 3471  grams  of  paper  : — 

Fatty  acid          . .         . .     2'2402 

C02         -9722 

H  -0138 

3-2262  grams 

The  marsh-gas  fermentation  yielded  the  following  products 
from  2'0065  grams  of  paper  : — 

Methane 0'1372 

C02         0-8678 

Volatile  acids     . .         . .     T0023 

2-0073 

The  fatty  acids  consisted  mainly  of  acetic  acid,  together 
with  smaller  quantities  of  butyric  acid. 

3.  The  fermentation  of  cellulose  above  described  takes 
place  in  absence  of  air.  It  is  obvious,  however,  that  much  of 
the  natural  destruction  of  cellulose,  e.g.,  the  mass  of  dead 
leaves  which  fall  each  autumn,  must  take  place  in  presence 
of  air. 

Researches  by  van  Iterson  have  indicated  various  methods 
by  which  this  breaking  up  can  take  place.  He  found  that  in 
presence  of  nitrates  certain  organisms  are  capable  of  oxidising 
cellulose,  utilising  the  oxygen  of  the  nitrate  which  is  simul- 
taneously reduced.  The  following  experiment  will  illustrate 
this  action  : — 

100  c.c.  of  tap  water  are  placed  in  a  200  c.c.  flask  together 
with  2  grams  of  Swedish  filter  paper,  0'25  gram  potassium 
nitrate,  0'05  gram  potassium  hydrogen  phosphate  (K2HP04), 
a  few  c.c.  of  sewage  and  a  little  leaf  mould.  The  flask  is  then 
filled  to  the  neck,  plugged  with  cotton  wool  and  placed  in  an 


166      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

incubator  at  35°  C.  In  fifteen  days,  if  the  conditions  of  the 
experiment  are  successfully  realised,  all  nitrite  and  nitrate  will 
have  disappeared  :  100  c.c.  of  the  solution  are  then  poured  off 
and  a  further  100  c.c.  of  tap  water,  containing  the  same 
quantities  of  potassium  nitrate  and  potassium  hydrogen  phos- 
phate as  originally  used,  are  added.  On  incubating,  the  nitrate 
will  be  found  to  disappear  much  more  rapidly,  and  on  further 
repetitions  of  the  process  van  Iterson  was  able  to  reduce 
0'5  gram  potassium  nitrate  in  one  or  two  days  ;  the  paper  in 
the  meanwhile  disintegrates  and  disappears,  and  potassium 
carbonate  or  bicarbonate  is  found  in  solution.  The  evolution 
of  nitrogen  was  observed,  but  no  trace  of  hydrogen,  methane 
or  nitrous  oxide.  The  equations  representing  this  change  are 
given  by  van  Iterson  as  follows  :  — 


5C6H1005  +  24KN03  =  24KHC03  +  12N2+6C02+  13H.O 
C6H1005  +  8KN02  =  4KHC03  +  2K2C03  +  4N2+3H20 

The  evolution  of  gas  during  this  reaction  can  be  demon- 
strated, as  in  the  case  of  the  anaerobic  decomposition  of  cellu- 
lose, by  attaching  a  flask  to  the  end  of  the  Hempel  gas  burette, 
the  flask  being  kept  meanwhile  in  a  constant  temperature 
water-bath. 

For  the  qualitative  demonstration  of  the  evolution  of 
gas  in  decompositions  of  this  sort,  and  to  obtain  a  rough 
idea  of  its  rate  of  evolution,  it  is  only  necessary  to  provide 
the  evolution  flask  with  a  suitably  stoppered  inlet  and 
outlet  tube,  the  latter  reaching  nearly  to  the  bottom  of 
the  flask,  and  being  bent  twice  at  right  angles.  As  the 
gas  is  evolved,  the  liquid  is  pushed  out,  and  can  be  measured 
from  time  to  time,  its  volume  obviously  being  equal  to  the 
volume  of  the  gas  evolved.  At  the  end  of  the  experiment 
the  exit  tube  can  be  connected  to  a  cylinder  of  water,  and 
the  gas  in  the  original  vessel  drawn  out  by  the  inlet  tube 
into  a  Hempel  burette  and  examined,  water  entering  to 
replace  the  gas.  The  arrangement  has  the  disadvantage  that 


FERMENTATION  OF  CELLULOSE  167 

liquid  is  removed  from  the  fermentation  flask,  and  so  the  con- 
ditions of  the  experiment  are  altered  as  it  proceeds.  But  if 
the  fermentation  is  mainly  confined,  as  in  the  case  of  cellulose, 
to  the  deposited  matter  at  the  bottom  of  the  flask,  the  removal 
of  the  liquid  is  not  of  such  serious  moment,  and  it  is  convenient 
to  have  an  apparatus  the  whole  of  which  can  be  placed  in  an 
ordinary  incubator. 

Van  Iterson  found  that  the  decomposition  of  cellulose  in 
presence  of  nitrate,  as  above  described,  went  on  at  much  the 
same  rate  as  the  anaerobic  change  studied  by  Omelianski.  He 
drew  attention  to  the  fact  that  the  nitrite  produced  is  readily 
re-oxidised  by  the  organisms  of  nitrification,  and  consequently 
that  in  presence  of  air  the  nitrate  is  being  continually  repro- 
duced, and  the  conditions  for  the  destruction  of  cellulose  are 
therefore  constantly  maintained.  This  observation  is  of  great 
importance  in  connection  with  the  destruction  of  cellulose  in 
the  bacterial  filter  beds  employed  in  the  purification  of  sewage. 

Van  Iterson  found  further  that  the  spores  and  mycelia  of 
higher  fungi  were  also  active  in  breaking  down  cellulose  in 
presence  of  air.  Thus,  if  a  little  leaf  mould  is  placed  in  contact 
with  moist  filter  paper  in  a  moist  chamber,  rotting  of  the  filter 
paper  takes  place  with  production  in  general  of  yellow  stains. 
This  is  probably  a  complex  process  wherein  various  moulds 
together  with  chromogenic  or  pigmenting  bacteria  take  part. 
It  appears  that  woody  fibre  resists  decomposition  under  these 
circumstances,  and  may  remain  practically  intact  for  a  long 
period  of  time  in  a  disintegrated  condition,  in  such  end  pro- 
ducts as  peat,  lignite,  etc.  These  contain  the  somewhat  ill- 
defined  substance  known  as  humus,  which  is  also  formed  by 
prolonged  boiling  of  sugars  with  dilute  acids.  Humus  bodies 
are  generally  of  an  acid  character,  dissolving  in  alkalis  to 
form  brown  solutions. 

The  Decomposition  of  Pectose  Bodies.— This  fermen- 
tation has  been  studied  by  Winogradski  and  his  pupils,  and 


168     BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

has  been  found  to  be  due  to  an  anaerobic  bacillus,  which  will 
decompose  pectin  and  calcium  pectate,  but  has  no  action  on 
cellulose.  This  fermentation  is  of  great  importance  in  con- 
nection with  the  retting  of  flax  and  other  fibre.  In  such  a 
process  it  is  necessary  to  separate  the  fibres,  which  are  held 
together  by  an  integument  consisting  largely  of  calcium 
pectate.  It  is  necessary  to  disintegrate  this  without  injury 
to  the  fibre,  and  the  object  is  best  accomplished  by  a  fermen- 
tation or  retting  process,  which  decomposes  the  integument 
while  leaving  the  fibre  intact. 


CHAPTER  XI 

MISCELLANEOUS    FERMENTATIONS,    FAT-SPLITTING 
ENZYMES,    OXEDASES,  CLOTTING  ENZYMES 

Fat-Splitting  Enzymes. — It  is  a  matter  of  common  observa- 
tion that  household  fat,  if  allowed  to  accumulate,  becomes 
what  is  termed  rancid  and  evil  smelling.  This  is  due  to 
fermentation  of  the  fat  with  production,  amongst  other 
substances,  of  free  fatty  acids,  which  have  an  unpleasant  smell. 

Fats  are  denned  chemically  as  esters  of  the  so-called  fatty 
acids  with  glycerine.  Glycerine  is  an  alcohol  containing  three 
hydroxyl  groups  with  the  formula  CH2OHCHOHCH2OH. 

Mutton  or  beef  fat  or  stearin  is  a  compound  of  glycerine 
and  stearic  acid,  the  latter  having  the  formula  C^H^COOH. 
Stearin  therefore,  being  a  glycerol  ester  of  stearic  acid,  has 
the  formula  C^C^CO^. 

Soap  is  formed  by  the  decomposition  of  fats  by  means  of 
alkali,  glycerine  being  obtained  as  a  by-product,  while  the 
soap  is  the  alkali  salt  of  the  fatty  acid.  Thus,  e.g.,  if  stearin 
is  heated  with  caustic  soda  the  following  reaction  takes  place  : 

C17H35C03— CH2  C17H35C02Na      CH2OH 

I  I 

C17H35C02-CH+  3NaOH  =  C17H35C02Na  +  CHOH 

C17H35C02-CH2  C17H35C02Na      CH2OH 

Stearin  Caustic  Soda  Sodium  Stearate  Glycerol 

(Soap) 

This  process  of  splitting  up  of  fat  with  formation  of  a  soap 


170     BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

is  known  as  the  saponification  of  a  fat.  The  term  saponi- 
fication  has  come  to  be  a  general  one  applied  to  all  processes 
whereby  a  fat  is  split  up,  yielding  a  fatty  acid  and  glycerine  ; 
the  process,  indeed,  is  essentially  one  of  hydrolysis  and  may 
be  expressed  in  general  terms  in  the  following  equation,  where 
K  =  the  residue  of  a  fatty  acid  : — 

R3C3H5  +  3H20  =  3RH  +  C3H5(OH)3 

Such  a  reaction  can,  e.g.,  be  brought  about  by  heating  a 
fat  with  a  mineral  acid,  or  even  by  the  action  of  steam  under 
pressure. 

Nature's  method,  however,  for  effecting  this  change,  which 
is  of  primary  importance  in  the  assimilation  of  fat  by  living 
organisms,  is  as  usual  a  much  less  drastic  one.  In  the  plant 
or  animal  which  uses  fat  to  build  up  its  body  substances, 
enzymes  are  produced  known  as  lipolytic  or  fat-splitting  en- 
zymes, which  are  generally  referred  to  as  an  individual  enzyme 
under  the  term  lipase  or  steapsin. 

The  decomposition  of  animal  fats  by  lipase  may  be  illus- 
trated by  taking  butter  fat  as  an  example  which  is  a  compound 
of  glycerine  and  butyric  acid.  This  is  readily  obtained  by 
melting  a  small  quantity  of  butter  in  an  evaporating  dish 
over  a  water-bath  and  pouring  off  the  liquid  portion,  leaving 
the  solid  residue  of  casein ;  or  more  exactly  by  warming  the 
butter  with  ether,  filtering  through  filter  paper,  and  distilling 
off  the  ether.  The  butter  fat  is  a  neutral  yellow  liquid,  as 
can  be  ascertained  by  testing  the  ethereal  solution  with 
litmus  paper. 

To  determine  the  action  of  lipase  upon  it  liquor  pancreaticus 
may  be  utilised.  It  was  shown  by  Claude  Bernard  that 
digestion  of  fat  was  mainly  brought  about  by  pancreatic  juice. 

A  few  c.c.  of  butter  fat  may  therefore  be  placed  in  a  test- 
tube  and  thoroughly  shaken  with  a  few  drops  of  '  liquor 
pancreaticus/  when  an  emulsion  is  formed.  On  warming 
this  emulsion  on  a  water-bath  or  incubator  at  40°  C.  for  some 


PAT-SPLITTING   ENZYMES  171 

hours  and  testing  again  with  litmus  the  mixture  will  be  found 
to  have  become  acid. 

A  similar  tube  of  butter  fat  incubated  without  the  addition 
of  the  pancreatic  extract  will  be  found  to  be  unchanged.  If 
the  action  of  the  pancreatic  juice  is  sufficiently  prolonged,  the 
peculiar  unpleasant  smell  of  butyric  acid  can  be  recognised. 

In  order  to  demonstrate  the  action  of  lipase  upon  a  vegetable 
fat,  castor  oil  seeds  may  be  made  use  of.  Just  as  the  barley 
plant  derives  its  nutriment  from  starch  during  the  early 
stages  of  growth,  and  for  that  purpose  secretes  during  germina- 
tion an  amylase  which  hydrolyses  the  starch  in  the  grain,  so  the 
germ  of  the  castor  oil  plant  secretes  a  lipase  which  hydrolyses 
the  oil  (a  glyceride  of  ricinoleic  acid)  contained  in  its  seeds. 
To  demonstrate  this,  therefore,  castor  oil  seeds  are  allowed 
to  germinate  for  some  days  by  embedding  them  in  moist  sand 
placed  in  a  small  dish,  which  again  can  be  placed  in  a  moist 
chamber,  and  the  whole  incubated  at  a  moderate  temperature. 
When  the  seeds  show  signs  of  sprouting  they  may  be  thoroughly 
ground  up  in  a  mortar  and  the  enzyme  investigated  in  one  of 
two  ways. 

1.  The  fat  may  be  extracted  by  grinding  up  with  ether 
and  filtering,  the  operation  being  repeated  several  times  till 
no  more  fat  is  extracted,  as  can  be  readily  ascertained  by 
evaporating  a  little  of  the  ethereal  solution  on   a  watch 
glass  :   when  the  fat  extraction  is  complete,  the  ether  should 
leave   no  residue   on   evaporation.     The   fat   may   also   be 
removed  by  continuous  extraction  with   ether  in  a  Sohxlet 
apparatus,  but  it  is  probable  that  an  extraction  at  the  ordinary 
temperature  gives  a  more  active  product ;   in  each  case  the 
residue  after  extraction  of  the  fat  is  freed  from  ether  by 
allowing  the  latter  to  evaporate  spontaneously  in  the  air 
without  heat.     The  ether-free  residue  contains  the  lipase. 

2.  The  germinated  seeds  are  ground  in  a  mortar  with  a 
solution  containing  5  per  cent,  of  sodium  chloride  and  0'2  per 
cent,  potassium  cyanide,  which  is  allowed  to  stand  in  contact 


172      BACTERIOLOGICAL   AND    ENZYME   CHEMISTRY 

with  the  ground  seeds  for  twenty-four  hours.  The  solution  is 
then  filtered  and  can  be  tested  for  the  presence  of  lipase. 

To  test  for  the  presence  of  lipase,  either  in  the  ether  extracted 
residue  of  the  seeds,  or  in  the  solution  obtained  as  described, 
an  emulsion  of  castor  oil  is  made  by  thoroughly  shaking, 
say,  5  c.c.  of  the  oil  with  a  little  gum  arabic.  Six  test-tubes 
may  now  be  made  up  as  follows  :  to  each  of  them  2  c.c.  of  a 
castor  oil  emulsion  may  be  added  together  with  a  drop  or  two 
of  neutral  litmus,  to  two  a  few  centigrams  of  the  residue 
from  the  ether  extraction  of  the  seeds  may  be  added,  to  two 
others,  say,  one  c.c.  of  the  sodium  chloride  extract,  while  the 
remaining  two  test-tubes  are  left  as  controls.  One  test-tube 
from  each  pair  is  now  boiled,  and  after  cooling  all  six  test-tubes 
are  incubated  for  some  hours  at  a  temperature  of  35°  C.  In 
the  case  of  the  tubes  containing  the  unboiled  enzyme  the 
formation  of  acid  will  be  evident  from  the  reddening  of  the 
litmus,  while  the  boiled  liquids,  and  the  unboiled  liquid  to 
which  no  enzyme  has  been  added,  remain  unchanged. 

The  actual  amount  of  acid  produced  can  be  determined 
by  adding  dilute  standard  caustic  soda,  say  f^,  till  the  blue 
colour  of  the  litmus  is  restored. 

Not  only  is  lipasa  capable  of  splitting  up  fats  properly 
so-called,  but  it  can  also  decompose  simpler  esters,  and  the 
reaction  in  such  a  case,  owing  to  the  more  complete  solu- 
bility of  the  products,  is  capable  of  being  more  exactly  studied. 
For  this  purpose  ethyl  butyrate,  which  has  the  formula 
C3H7C02C2H5,  has  been  utilised  by  several  investigators. 
Among  these  researches  those  of  Armstrong  and  Ormerod  in 
England  and  Kastel  and  Loevenhart  in  America  may  be 
specially  mentioned.  Armstrong  and  Ormerod  made  use  of  the 
dried  residue  obtained  on  extracting  the  castor  oil  with  ether  ; 
they  found  that  the  action  of  the  lipase  was  increased  by  the 
presence  of  dilute  acid.  Their  investigations  were  directed 
towards  finding  some  chemical  explanation  of  the  action  of 
the  enzyme,  and  for  this  purpose  they  investigated  a  number 


. 

FAT-SPLITTING   ENZYMES  173 

of  esters  both  of  mono-basic  and  of  di-basic  acids.  They 
proposed  a  provisional  hypothesis,  according  to  which  the 
hydrolysis  of  ethereal  salts  by  lipase  involves  the  direct 
association  of  the  enzyme  with  the  carboxyl  group.  Hydro- 
lysis appears  to  take  place  more  readily  when  OH  groups 


are  absent  ;    thus   ethyl    succinate,    |  ,    is  more 

CR^02C^i5 

CHOHC02C2H5 

readily  broken  up  than  ethyl  tartrate,  |  ,  while 


ethyl    malate,     |  ,    occupies    an    intermediate 


position. 

They  concluded  that  the  difference  between  animal  and 
vegetable  lipase  is  one  of  degree,  and  if  sufficient  enzyme 
is  used  almost  all  esters  are  more  or  less  attacked. 

Kastel  and  Loevenhart  made  use  of  animal  lipase  in  the 
following  way  :  they  macerated  fresh  pancreas  with  coarse 
sand,  extracted  the  enzyme  with  water  or  glycerine,  1  c.c. 
of  the  extract  from  either  10,  20  or  50  grams  of  tissue 
was  diluted  to  100  c.c.  and  allowed  to  act  for  forty  minutes 
on  a  mixture  of  4  c.c.  of  water,  O'Ol  c.c.  toluene,  and  0*25  c.c. 
of  ethyl  butyrate  at  40°C.,  the  mixture  being  afterwards 
titrated  with  ^  potash  solution.  They  found  that  the 
enzyme  was  destroyed  at  a  temperature  of  60°  to  70°  C., 
and  that  most  antiseptics  had  an  injurious  effect  on  it, 
especially  sodium  fluoride  and  mineral  acids. 

By  titrating  the  solutions  at  definite  intervals  of  time  they 
obtained  results  which  led  them  to  the  following  conclusions  :  — 


174      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

1.  The  velocity  of  the  reaction  was  not  proportional  to  the 
amount  of  ester  present. 

2.  The  velocity  of  the  reaction  was  nearly  proportional  to 
the  concentration  of  the  enzyme. 

3.  The  reaction  in  general  did  not  attain  completion  ; 
only  when  a  large  quantity  of  enzyme  was  present  in  proportion 
to    the   ester  was   the  decomposition   of  the  latter  nearly 
complete. 

4.  The  coefficient  of  velocity  of  the  reaction,  that  is,  the 
ratio  of  decomposed  ester  to  undecomposed  ester  per  unit 
of  time,  was  not  constant  but  decreased  with  the  progress 
of  the  reaction. 

These  results  indicate  that  the  reaction  belongs  to  the  class 
of  changes  known  as  reversible,  and  that  there  is  a  tendency 
for  an  equilibrium  to  be  established  between  the  action  result- 
ing in  the  decomposition  of  the  ester,  and  the  reverse  action 
tending  to  combination  of  the  free  acid  and  alcohol.  It  will 
be  remembered  that  a  similar  case  was  met  with  by  Croft 
Hill  when  studying  the  decomposition  of  maltose,  and  Kastel 
and  Loevenhart  have  added  to  the  number  of  synthetic 
enzyme  actions  by  effecting  a  synthesis  of  ethyl  butyrate 
by  the  bringing  together  of  ethyl  alcohol  and  butyric  acid  in 
the  presence  of  lipase. 

Secretion  of  Lipase  by  Micro-organisms.— The  secretion 
of  lipase  by  micro-organisms  can  be  demonstrated  in  a  similar 
manner  to  the  secretion  of  amylase,  viz.,  e.g.,  by  growing 
Aspergillus  niger  on  a  substratum  of  suet  or  butter.  Moreover, 
if  a  little  butter  be  melted  in  a  Petri  dish  and  allowed  to 
set  and  some  dilute  sewage  be  poured  over  it,  liquefaction 
and  accompanying  rancidity  will  soon  be  observable.  These 
reactions  are  of  importance  in  connection  with  the  treatment 
of  sewage  by  anaerobic  processes. 

The  destruction  of  fat  under  aerobic  conditions  is  very 
probably  the  work  of  higher  organisms  such  as  worms. 


OXIDASES  175 


OXIDASES 

It  is  again  a  matter  of  common  observation  that  if,  e.g., 
an  apple  is  cut  open  and  the  interior  is  left  exposed  to 
air,  in  a  short  time  it  becomes  brown.  Everyone,  too,  must 
have  been  struck  by  the  difference  in  appearance  between 
mushrooms  as  bought  in  the  shop  and  the  same  when 
freshly  gathered ;  the  dark  brown  appearance,  especially  of 
the  under  surface,  is  an  unpleasant  change  from  the  delicate 
white  and  pink  they  exhibited  whilst  growing.  These  and 
many  other  similar  changes  are  due  to  oxidation  brought 
about  by  a  class  of  enzymes  known  as  oxidases  ;  that  the 
change  is  due  to  the  presence  of  oxygen  can  be  shown 
by  leaving  freshly-cut  slices  of  apple  in  vacuo  or  in  an 
inert  atmosphere  such  as  hydrogen,  when  no  browning  takes 
place. 

Oxidases  are  very  widely  distributed  enzymes,  and  for 
this  reason  a  great  many  vegetable  extracts  and  juices 
tend  to  darken  on  standing.  A  notable  instance  of  such 
a  change  is  the  case  of  the  juice  of  the  lac  tree,  which 
furnishes  the  raw  material  of  Japanese  lacquer ;  this  juice 
is  a  clear  yellow  when  first  drawn,  exposed  to  air  it  rapidly 
turns  brown  and  finally  black.  It  has  been  discovered 
that  this  is  due  to  an  oxidising  enzyme  which  has  been 
termed  laccase. 

The  browning  of  wine  which  takes  place  in  course  of  time, 
and  which  is  known  as  ageing,  is  due  to  the  oxidation  and  pre- 
cipitation of  the  colouring  matter  ;  this  can  be  accelerated  by 
the  addition  of  an  oxidase. 

These  enzymes  have  been  studied  in  the  same  manner 
as  other  cases  already  considered ;  the  following  instances 
from  numerous  researches  will  serve  to  illustrate  the  methods 
employed.  Laccase  was  investigated  by  Yoshida,  who  dis- 
covered in  Japanese  lac  an  acid,  urushic  acid,  which  is 


176      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

capable  of  oxidation  to  the  substance  known  as  oxiurushic 
acid  thus : — 

2C14H1902+  30  =  2C14H1803  +  H20 

Some  ten  years  later  Bertrand  separated  the  juice  into 
laccol,  an  alcohol  derivative  which  was  soluble  in  alcohol, 
and  into  the  enzyme  laccase  which  was  insoluble  in  alcohol. 
Laccol  was  found  to  oxidise  spontaneously,  but  the  rate 
of  oxidation  was  greatly  accelerated  when  laccase  was 
present. 

An  enzyme  with  the  same  properties  was  obtained  from 
many  vegetables,  especially  members  of  the  mushroom  family. 
The  same  enzyme  also  will  oxidise  numerous  hydroxy  and 
amido  derivatives  of  benzene  to  quinone  :  thus  in  the  case  of 
hydroquinone  the  following  reaction  takes  place  : — 

C6H4(OH)2  +  0  =  =  C6H40,  +  H20 

While  the  action  of  laccase,  or  an  enzyme  akin  to  it,  is  not 
specific,  in  the  sense  that  one  reaction  and  one  only  can  be 
brought  about  by  its  intervention,  yet  it  has  its  limitations, 
and  will  only  oxidise  such  bodies  as  are  capable  of  yielding 
quinols.  It  does  not,  therefore,  oxidise  tyrosin,  the  formula 
of  which,  it  may  be  remembered,  is 

C6H4OHCH2CHNH2C02H 

and  which  would  therefore  require  to  be  broken  up  completely 
before  a  quinol  could  result  from  its  oxidation.  Tyrosin  can, 
however,  be  oxidised  by  a  specific  enzyme  known  as  tyrosinase, 
which  has  quite  recently  been  investigated  by  Gortner.  The 
source  of  Gortner's  enzyme  was  the  meal  worm.  To  obtain 
the  enzyme  the  larvae  were  ground  in  a  mortar  with  chloroform 
water,  and  the  milky  liquid  strained  through  a  cheese  cloth :  the 
milky  extract  if  kept  a  short  time  in  the  air  rapidly  darkens 
on  the  surface,  it  remains  white  where  not  in  contact  with 
oxygen.  It  was  found  that  a  soluble  and  insoluble  tyrosinase 


OXIDASES  177 

was  present  in  this  extract :  the  soluble  tyrosinase  could 
be  precipitated  with  ammonium  sulphate  from  the  filtrate 
left  on  filtering  off  the  insoluble  enzyme.  The  soluble  enzyme 
was  capable  of  colouring  tyrosin  dark  violet  black,  with  the 
final  formation  of  a  precipitate,  within  twenty-four  hours ;  this 
reaction  did  not  take  place  if  the  extract  was  previously 
heated  to  90°  C.  The  insoluble  tyrosinase  caused  the  tyrosin 
solution  to  undergo  a  series  of  colour  changes  ranging  through 
pink,  rose,  violet  and  blue-black  to  a  deposition  of  a  black 
pigment-like  substance,  leaving  the  supernatant  liquid 
completely  decolourised.  That  a  small  quantity  of  the 
enzyme  was  able  to  affect  a  large  quantity  of  tyrosin  was 
proved  by  pouring  away  the  colourless  supernatant  liquid 
and  adding  more  tyrosin  solution,  when  the  series  of  colour 
changes  was  repeated.  This  operation  of  pouring  off  the 
colourless  solution  and  adding  more  tyrosin  was  done  seven 
times  with  one  specimen  of  insoluble  tyrosinase,  weighing 
approximately  O'Ol  gram.  Tyrosinase,  therefore,  has  the 
property  of  continuous  activity  which  is  characteristic  of 
enzymes  in  general. 

The  brown  colour  of  tea  is  due  to  an  oxidase  formed  in 
the  growing  leaf.  This  has  been  investigated  by  Mann,  who 
extracted  the  enzyme  in  the  following  way  : — 

Ten  grams  of  fresh,  or  6 '6  grams  of  the  withered  leaf,  were 
ground  to  pulp,  5  grams  of  hide  powder  were  added  in 
order  to  precipitate  the  tannin,  and  the  mixture  ground 
together  with  a  known  quantity  of  water  in  which  the  enzyme 
is  soluble.  After  standing  two  hours  the  mixture  was  pressed 
through  a  cloth  and  precipitated  by  alcohol,  the  amount  of 
enzyme  present  in  the  solution  could  be  determined  by  the 
intensity  of  the  blue  colour  produced  in  a  known  amount  of 
the  solution  by  guaiacum  tincture ;  a  certain  proportion  of 
the  enzyme  gave  a  blue  colour  with  guaiacum  resin  alone, 
while  another  portion  required  the  addition  of  hydrogen 
peroxide  before  the  blue  colour  was  obtained.  Only  those 


178      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

enzymes  which  give  a  blue  colour  with  guaiacum  tincture 
without  hydrogen  peroxide  are  true  oxidases. 


THE   CLOTTING   ENZYMES 

Rennet. — This  enzyme,  which  is  also  sometimes  referred 
to  as  lab  or  chymosin,  has  the  property  of  curdling  or  clotting 
milk.  It  is  generally  prepared  from  the  stomach  of  the  calf  ; 
an  impure  product  can  be  obtained  by  macerating  the  stomach 
with  water  or  with  a  5  to  10  per  cent,  solution  of  sodium 
chloride  in  presence  of  a  little  acid. 

It  is  obtained  in  a  purer  state  by  digestion  with  sodium 
chloride  solution  0'5  per  cent,  strength  at  30°  C.  for  twenty- 
four  hours.  On  filtering  the  solution  and  adding  acid  up  to 
0*1  per  cent,  a  precipitate  of  mucous  matter  is  obtained  which 
can  be  filtered  off;  acid  is  then  further  added  to  the  filtrate 
up  to  0'5  per  cent.,  and  the  solution  saturated  with  sodium 
chloride.  On  standing  and  stirring  for  two  or  three  days  and 
gradually  raising  the  temperature  to  30°  or  35°  C.  a  flocculent 
scum  of  rennet  separates  which  is  soluble  in  water. 

An  enzyme  capable  of  clotting  milk  occurs  in  many  animal 
and  plant  extracts,  e.g.,  in  germinating  castor  oil  seeds; 
certain  bacteria  also  secrete  a  clotting  enzyme. 

The  chemical  action  of  rennet  upon  milk  is  of  considerable 
interest.  On  addition  of  rennet  to  milk  a  curd  separates  out, 
but  the  whey  still  contains  an  albumin,  which  differs  from 
lact-albumin  in  that  it  is  not  precipitated  by  boiling.  The 
curd  also  is  different  from  the  precipitate  produced  by  acids, 
as  this  can  be  redissolved  on  neutralisation,  while  the  curd 
produced  by  rennet  is  insoluble.  It  has  been  found  that 
curd  contains  calcium  phosphate,  which  is  consequently  present 
in  cheese.  If  calcium  phosphate  is  dialysed  out  of  milk, 
curdling  is  no  longer  obtained  by  addition  of  rennet.  It 
would  appear  that  the  greater  part  of  the  albumin  of  milk 


CLOTTING   ENZYMES  179 

exists  as  a  body  which  may  be  termed  caseinogen  ;  the  action 
of  rennet  is  to  break  up  this  substance,  a  portion  of  which 
remains  in  solution,  the  remainder  being  precipitated,  together 
with  calcium  phosphate,  as  casein.  This  theory  of  the  action 
of  rennet  derives  support  from  the  following  experiment : 
the  caseinogen  as  a  whole  may  be  precipitated  by  acetic  acid, 
washed  and  dissolved  in  lime  water.  On  neutralising  with 
phosphoric  acid,  a  milky-looking  liquid  is  obtained  which 
forms  a  clot  on  addition  of  rennet. 

Another  method  of  exhibiting  the  same  phenomenon  is 
to  dissolve  the  caseinogen  in  acid  sodium  phosphate,  add  the 
rennet  to  the  solution  and  allow  it  to  act  for,  say,  half  an  hour. 
No  clotting  occurs ;  the  solution  is  then  boiled  to  destroy  the 
activity  of  the  enzyme  ;  on  adding  calcium  chloride,  clotting  at 
once  takes  place. 

This  last  experiment,  which  was  devised  by  Hammersten, 
would  indicate  that  the  action  of  the  rennet  is  simply  to 
break  up  the  caseinogen,  the  subsequent  clotting  being  due  to 
calcium  salts. 

The  addition  of  peptone  to  milk  inhibits  the  clotting  efiect 
of  rennet,  probably  owing  to  its  affinity  for  calcium  salts.  It 
may  be  finally  mentioned  that  the  optimum  temperature  for 
the  action  of  rennet  is  40°  C.,  while  its  activity  is  destroyed 
above  70°  C. 

Other  important  clotting  enzymes  are  the  fibrin  ferment 
or  thrombase,  which  causes  the  clotting  of  the  blood,  and 
pectase,  which  gelatinises  fruit  juices  containing  pectin ;  in 
both  these  cases,  as  in  the  case  of  rennet,  calcium  salts  play 
an  important  part. 

The  investigation  of  the  action  of  thrombase  indicates 
that  a  substance  which  has  been  termed  fibrinogen  occurs 
in  unshed  blood.  The  action  of  thrombase  is  to  precipi- 
tate fibrin,  which  carries  down  with  it  the  red  corpuscles  of 
the  blood,  leaving  a  globulin  dissolved  in  the  clear  serum. 
This  phenomenon  does  not  occur  in  the  absence  of  calcium 

vl 


180      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

salts,  especially  calcium  sulphate.  Thrombase  can  hardly  be 
present  in  the  free  state  in  the  body,  but  apparently  must  be 
looked  upon  as  entering  into  combination  with  some  other 
substance  to  form  a  zymogen,  from  which  it  is  separated 
apart  from  the  body  in  presence  of  calcium  salts. 


CHAPTER  XII 

OUTLINES    OF   THE   CHEMISTRY   OF   ALBUMINS 
OR  PROTEINS 

ALL  living  organisms  contain  as  an  essential  constituent  a 
highly  complex  nitrogen-containing  substance  known  gene- 
rally as  protoplasm.  The  simplest  of  all  organisms,  e.g.,  the 
amoeba,  is  virtually  a  simple  mass  of  protoplasm ;  it  has  the 
property  when  alive  of  dividing  into  smaller  living  portions, 
and  of  building  itself  up  from  elements  absorbed  from  its 
external  surroundings. 

The  most  highly  developed  animal,  chemically  considered, 
is  a  vast  aggregation  of  cells  of  different  structure  and  function, 
but  all  of  them  containing  protoplasm  in  some  form  or  other. 
Protoplasm  is  in  no  sense  a  chemical  entity  with  a  definite 
composition  such  as  may  be  ascribed  to  even  highly  com- 
plicated organic  substances  ;  it  possesses  structure  visible 
under  the  microscope,  and  must  be  looked  upon  when  alive 
as  a  constantly  changing  complex,  wherein  loose  combina- 
tions are  constantly  being  formed  and  decompositions  taking 
place.  Protoplasm  may  indeed  be  regarded  as  a  factory  where 
raw  material  of  various  kinds  is  taken  in,  where  finished 
products  are  delivered,  and  where  a  certain  amount  of  waste 
material  is  produced. 

It  would  obviously  be  of  little  help  to  the  understanding 
of  the  operations  of  such  a  factory  simply  to  know  the  materials 
of  which  it  is  composed,  or  even  the  bare  enumeration  of  its 
contents  in  terms  of  iron  and  steel  and  bricks  and  mortar  or 


182      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

weight  of  stores.  The  ultimate  chemical  composition  of 
protoplasm,  therefore,  can  tell  us  little  of  its  real  nature  ;  it 
is  of  interest  to  know  that  its  invariable  constituents  include 
carbon  and  nitrogen,  and  almost  universally  phosphorus.  It 
will  obviously  be  more  instructive  to  describe  it  as  it  were  by 
stages,  classifying  the  chemical  contents  into  substances  of 
gradually  decreasing  complexity.  Even  then  we  shall  only 
have  obtained  a  vague  idea  of  the  constituents  of  dead  proto- 
plasm, as  we  might  make  an  inventory  of  the  contents  of 
our  hypothetical  factory  after  business  had  been  shut  down. 
Of  the  course  of  operations,  or  the  economic  conduct  of  the 
factory,  we  should  know  little  or  nothing.  Having  obtained 
such  an  inventory,  however,  and  presuming  the  factory  began 
work  again,  by  taking  careful  note  of  the  material  entering 
and  leaving  the  factory,  we  could  form  a  much  better  idea  of 
the  nature  of  the  processes  carried  on  therein.  The  task 
which  confronts  the  chemist  is  to  investigate  in  this  kind  of 
way  the  chemistry  of  protoplasm,  which  in  other  words  is  the 
chemistry  of  life.  In  the  present  chapter  an  attempt  will 
be  made  broadly  to  indicate  essential  facts  with  reference  to 
the  products  of  the  activity  of  protoplasm.  The  substances 
which  have  been  isolated  as  more  or  less  definite  chemical 
entities  belong  to  the  class  known  generally  as  albumins, 
proteid  bodies,  or  more  recently  as  proteins. 

It  will  probably  be  simplest  to  take  one  or  two  of  the 
most  characteristic  of  these  substances  and  study  their  pro- 
perties and  products  of  decomposition  ;  afterwards  will  be 
given  in  brief  summary  an  account  of  the  principal  bodies  of 
this  class  which  are  known,  together  with  their  decomposition 
products.  At  the  same  time  occasion  will  be  taken  to  indicate 
certain  of  the  main  lines  of  investigation  which  are  at  present 
being  made  use  of  in  regard  to  them. 

As  a  typical  albumin  ordinary  white  of  egg  may  be  made  use 
of,  and  the  following  experiments  carried  out : — 

Experiment.— About  1  c.c.  of  white  of  egg  may  be  poured 


THE   CHEMISTRY   OF   ALBUMINS  183 

into  50  c.c.  of  water,  stirring  meanwhile  ;  a  white  precipitate 
is  formed.  This  can  be  filtered  off  and  a  portion  of  the  filtrate 
boiled,  when  a  further  precipitate  is  obtained. 

It  will  thus  be  seen  that  the  egg-white  can  be  separated 
readily  into  two  substances,  one  soluble,  the  other  insoluble, 
in  cold  water.  The  insoluble  portion  is  known  as  globulin,  the 
soluble  substance  is  albumin. 

As  the  word  albumin  is  also  used  in  a  more  or  less  generic 
sense,  it  is  better  perhaps  to  refer  to  this  body  as  egg-albumin. 
A  related  substance  can  be  obtained  from  blood  serum  and 
is  known  as  serum  albumin,  and  also  from  milk,  when  it  is 
known  as  lact-albumin. 

A  larger  quantity  of  egg-albumin  solution  may  now  be 
prepared  by  adding  further  quantities  of  egg-white  to  water, 
stirring,  and  filtering  off  the  globulin ;  the  solution  of  egg- 
albumin  can  then  be  used  for  investigating  certain  typical 
properties  of  this  class  of  substance.  In  the  first  place 
a  number  of  simple  qualitative  tests  may  be  carried  out, 
which  will  indicate  the  presence  of  certain  elements  in 
albumin,  and  the  class  of  chemical  substances  to  which  it 
may  be  referred. 

Experiment. — 5  c.c.  of  the  solution  may  be  warmed  with  a 
little  strong  caustic  soda ;  an  evolution  of  ammonia  can 
readily  be  detected  which  indicates  the  presence  of  nitrogen, 
and  further  that  it  is  most  probably  present,  in  part  at  least, 
in  combination  as  a  so-called  amino  group  or  NHg. 

Experiment. — A  few  drops  of  lead  acetate  are  added  to  a 
20  per  cent,  solution  of  caustic  soda  ;  a  precipitate  is  formed 
which  readily  redissolves.  If  a  little  of  this  solution  is  boiled 
with  a  solution  of  egg-albumin,  it  rapidly  darkens  owing  to 
the  formation  of  sulphide  of  lead  ;  this  indicates  the  presence 
of  sulphur  in  the  p-gg-a.1br.min. 

Experiment. — On  warming  with  strong  caustic  soda  and 
adding  a  few  drops  of  dilute  copper  sulphate  solution  a  violet 
colour  is  obtained.  This  is  known  as  Piotrowski's  reaction.  If 


184     BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

the  boiling  with  caustic  soda  is  prolonged,  a  rose-pink  colour  is 
obtained  on  addition  of  copper  sulphate,  i.e.,  the  biuret  re- 
action. This  has  been  already  observed,  as  a  characteristic 
reaction  for  enzymes,  when  examining  the  properties  of 
amylase.  The  formation  of  this  colour  is  due  to  the  production 
of  biuret  or  an  allied  substance  ;  the  biuret  group,  it  will  be 
noted,  therefore  is  characteristic  of  the  decomposition  pro- 
ducts of  albumin,  and  the  biuret  reaction  is  a  useful  indicator 
of  the  extent  to  which  decomposition  has  taken  place. 

Experiment. — The  Xanthoproteic  reaction,  i.e.,  the  orange 
colour  obtained  on  warming  with  strong  nitric  acid  and  subse- 
quently adding  ammonia,  is  a  general  reaction  for  albumins  as 
they  are  broken  up  by  the  strong  nitric  acid. 

Experiment. — Milton's  reagent  will  be  found  to  give  a  brick- 
red  precipitate  on  boiling  with  the  albumin  solution. 

It  will  already  have  been  noticed,  and  it  is  of  course  common 
knowledge,  that  egg-albumin  is  coagulated  on  heating;  this 
would  suggest  that  egg-albumin  belongs  to  the  class  of  sub- 
stances known  as  colloids.  This  can  be  demonstrated  by 
enclosing  a  solution  of  albumin  in  a  parchment  cylinder, 
adding  a  little  thymol  to  prevent  putrefaction,  and  immersing 
the  cylinder  in  water.  On  boiling  a  portion  of  the  external 
water  at  intervals  no  coagulation  will  take  place  ;  the  albumin, 
will  be  found  still  present  in  the  interior  of  the  parchment/ 
cylinder,  and  capable  of  coagulation. 

Like  other  colloids,  egg-albumin  can  be  precipitated  by* 
the  addition  of  certain  salts.  Thus  ordinary  sodium  chloride,, 
magnesium  sulphate,  zinc  acetate,  may  be  employed  for  this-; 
purpose,  and  especially  ammonium  sulphate. 

The  chief  methods  in  use  for  the  separation  of  albumin 
substances  consist  in  fractional  precipitation  by  means  of 
certain  salts. 

It  is  possible  to  obtain  certain  albumins  in  an  approxi- 
mately crystalline  state.  Egg-albumin  may  be  taken  as  an 
example.  The  following  description  of  the  method  of  pre- 


THE    CHEMISTRY   OF   ALBUMINS  185 

paration  of  crystalline  albumin  is  taken,  with  some  modifica- 
tion, from  the  monograph  on  '  The  General  Characters  of  the 
Proteins  '  by  Dr.  Schryver,  p.  20. 

Egg-white  is  beaten  to  a  froth  (to  break  up  the  membranes) 
with  exactly  its  own  bulk  of  saturated  ammonium  sulphate 
solution.  The  mixture,  after  standing  overnight,  or  at  least 
for  some  hours,  is  filtered  from  the  precipitated  globulin. 
The  filtrate  is  now  measured.  Ten  per  cent,  acetic  acid 
(glacial  acetic  acid  diluted  to  ten  times  its  bulk)  is  then 
very  gradually  added  from  a  burette,  until  a  well-marked 
precipitate  forms.  The  object  of  the  addition  of  acid  is 
to  neutralise  the  alkalinity  which  is  developed  in  the 
ammonium  sulphate  solution  on  standing.  The  formation 
of  a  precipitate  indicates  the  point  of  neutralisation.  A 
further  quantity  of  acid  is  now  added,  1  c.c.  for  each  100  c.c. 
of  the  filtered  mixture  as  already  measured.  A  bulky  precipi- 
tate is  thus  produced,  which  is  at  first  amorphous,  but  which 
becomes  crystalline  in  the  course  of  four  or  five  hours,  if  shaken 
from  time  to  time.  To  obtain  the  full  yield,  the  material 
should  stand  for  twenty-four  hours.  The  precipitate  can  then 
be  filtered  off,  and  allowed  to  drain  on  a  plate  of  porous 
porcelain.  The  precipitate  will  probably  contain  ammonium 
sulphate,  from  which  indeed  it  is  not  easy  completely  to  free  it ; 
but  it  can  be  obtained  in  a  purer  state  by  redissolving  in  water, 
adding  half-saturated  ammonium  sulphate,  containing  acetic 
acid  in  the  proportion  of  1  per  1000,  till  a  permanent  pre- 
cipitate forms,  and  finally  a  further  2  c.c.  of  ammonium 
sulphate  in  excess  of  this. 

Albumin  substances  belong  as  a  rule  to  the  class  of  com- 
pounds known  as  amphoteric,  that  is,  they  are  capable  of  acting 
both  as  weak  acids  and  as  weak  bases.  A  solution  of  albumin 
in  dilute  alkali  is  sometimes  known  as  alkali-albumin.  If 
acid  is  added  very  carefully  to  such  a  solution,  the  albumin 
is  first  precipitated  and  then  redissolved,  forming  so-called 


186      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

acid-albumin*  Thus  albumin,  it  will  be  seen,  is  capable  of 
forming  salts  and  of  combining  both  with  acids  and  bases. 

Hydrated  oxides  such  as  aluminium  hydroxide,  A12(OH)6, 
or  ferric  hydroxide,  Fe2(OH)6,  are  capable  of  precipitating 
albumin  from  solution.  This  phenomenon  is  no  doubt  partly 
physical  and  partly  chemical ;  physical  in  that  one  colloid 
body  on  separating  from  solution  tends  to  attract  other 
colloids  by  a  process  known  as  adsorption ;  and  chemical 
in  that  the  metallic  hydroxide  actually  combines  with  the 
albumin.  This  property  by  which  colloidal  precipitates  tend 
to  carry  out  of  solution  other  colloids,  especially  those  related 
to  albumin,  finds  application  on  the  large  scale  in  the  chemical 
precipitation  of  sewage  and  other  polluted  liquids. 

The  well-known  household  cookery  receipt  for  clarifying 
soup,  etc.,  by  means  of  white  of  egg  is  an  illustration  of  the 
same  property. 

It  has  been  pointed  out  by  the  writer  of  this  book  and 
others,  that  by  carefully  conducted  precipitation,  either  with 
hydra  ted  alumina  or  ferric  hydroxide,  it  is  possible  to  remove 
from  solution  all  colloidal  matter,  and  to  obtain  results  similar 
to  those  which  are  obtained  by  dialysis.  The  method  may  be 
illustrated  by  the  following  example  of  what  has  been  termed 
the  clarification  test. 

Two  hundred  cubic  centimetres  of  the  liquid  to  be 
examined,  e.g.,  a  sample  of  sewage  (freed  from  grosser  solid 
matter  by  settlement  and  decantation),  or  a  solution  contain- 
ing albumin,  is  treated  with  2  c.c.  of  10  per  cent,  solution  of 
iron  or  aluminium  alum,  together  with  2  c.c.  of  a  10  per 
cent,  solution  of  sodium  acetate,  and  boiled  vigorously  for 
two  minutes ;  on  cooling  and  filtering  through  filter  paper 
a  crystal  clear  solution  is  obtained.  By  making  a  suitable 
analytical  estimation,  e.g.,  of  the  amount  of  oxygen  absorbed 
from  an  acid  solution  of  potassium  permanganate  of  known 
strength,  or  by  boiling  a  known  amount  of  the  clarified 
and  unclarified  liquid  respectively  with  alkaline  perman- 


THE    CHEMISTRY   OF   ALBUMINS  187 

ganate  and  determining  the  so-called  '  albuminoid  ammonia  ' 
evolved,  a  measure  is  obtained  of  the  quantity  of  albumin  sub- 
stance removed  from  solution.  It  should  be  noted,  of  course, 
that  in  a  complicated  substance  like  sewage  other  substances 
besides  albumins,  notably  e.g.  fats,  are  carried  down  by  this 
process. 

Besides  hydroxides  of  aluminium  and  iron,  hydrated  copper 
oxide  combines  readily  with  albumin,  and  copper  salts  have 
been  used  on  a  large  scale  in  the  treatment  of  water  supplies, 
more  especially  with  the  object  of  preventing  the  growth  of 
algae  in  reservoirs.  It  is  probable  that  the  toxic  action  of 
copper  in  this  respect,  and  also  its  analogous  action  as  a  germi- 
cide, is  due  to  the  readiness  with  which  insoluble  compounds  of 
copper  and  albumin  are  formed. 

Albumin  can  be  recovered  from  its  compounds  with 
metallic  oxides  by  treatment  with  acids,  when  the  metal  goes 
into  solution  and  the  albumin  is  precipitated.  By  careful 
treatment  of  a  copper  compound  it  has  been  possible  to  obtain 
albumin  in  a  form  which  is  not  crystalline,  which  is  almost 
completely  soluble  in  alcohol  and  which  does  not  coagulate 
on  boiling.  The  following  description  is  given  by  Harnack 
(Ber.  XXII.  ii.  pp.  30^46)  :  A  clear  solution  of  albumin  is 
obtained  by  dissolving  egg-albumin  in  water  and  filtering  off 
the  globulin  ;  acetic  acid  is  added  and  the  precipitate  obtained 
filtered  off.  The  filtrate  is  exactly  neutralised  and  again 
filtered ;  in  this  way  the  remaining  portions  of  globulin  are 
removed.  The  neutral  solution  is  now  precipitated  with 
copper  sulphate  and  the  precipitate  thoroughly  washed,  then 
suspended  in  water,  dissolved  in  a  few  drops  of  caustic  soda 
and  reprecipitated  with  acetic  acid.  The  precipitate  is  again 
washed,  redissolved  in  caustic  soda  and  precipitated  with 
acetic  acid,  and  again  thoroughly  washed.  It  is  then  dis- 
solved in  excess  of  caustic  soda  and  the  dark  violet-blue  jelly 
allowed  to  stand  twenty-four  hours,  when  it  is  precipitated 
with  hydrochloric  acid,  the  copper  in  this  case  going  into 


188      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

solution.  The  precipitated  albumin  is  carefully  washed  on  a 
filter  pump,  and  finally  dried  in  a  platinum  dish  at  a  tempera- 
ture not  exceeding  100°  C.  It  is  necessary  in  this  process  to 
use  plenty  of  material  to  start  with,  as  the  losses  by  washing, 
especially  in  the  final  removal  of  the  copper,  are  apt  to  be  con- 
siderable. The  preparation,  however,  is  of  much  interest  as 
affording  a  means  of  obtaining  albumin  in  a  pure  state  and  in 
a  form  more  convenient  for  investigation  than  that  in  which  it 
is  commonly  found. 

In  the  foregoing  paragraphs  the  properties  of  a  typical 
albumin  have  been  considered  in  some  detail,  apart  from  the 
study  of  the  products  obtained  when  it  is  submitted  to  partial 
decomposition.  This  study  may  now  be  followed  up,  keeping 
always  to  the  one  typical  substance,  viz.,  egg-albumin.  In  the 
light  of  the  information  thus  obtained  it  will  be  easier  to  follow 
the  subsequent  general  description  of  other  substances  of  a 
similar  nature. 

It  has  already  been  shown  by  qualitative  examination  that 
on  violently  attacking  albumin  by  such  substances  as  strong 
caustic  soda,  the  presence  of  end  products  such  as  ammonia, 
biuret  and  sulphuretted  hydrogen  could  be  detected.  It  is 
obvious,  however,  that  such  a  procedure  gives  us  but  little 
information.  Determinations  by  physical  methods  would 
indicate  that  the  molecular  weight  of  albumin  is  probably 
somewhere  in  the  neighbourhood  of  15,000.  Its  composition, 
according  to  ultimate  analysis,  can  be  expressed  within  the 
following  limits : — 

Carbon 50    to  55  per  cent. 

Hydrogen      6'9  „    7'3     „ 

Nitrogen        15     ,,19       „ 

Oxygen 19     „  24       „ 

Sulphur 0-3,,    2-4    „ 

The  information  given  by  these  figures  is  the  same  kind  of 
information  that  would  be  obtained  in  regard  to  the  construe- 


THE    CHEMISTRY    OF    ALBUMINS  189 

tion  of  a  watch,  if  it  were  stated  to  be  made  up  of  a  certain 
weight  of  glass,  of  silver,  of  gold,  of  brass  and  of  steel,  together 
with  a  few  precious  stones.  It  is  obviously  necessary  that,  in 
order  to  get  some  idea  of  the  construction  of  the  watch,  it 
must  be  taken  to  pieces  carefully  and  each  independent 
portion  separately  described.  Similarly,  in  order  to  obtain 
even  an  approximate  idea  of  the  structure  of  the  albumin 
molecule,  means  must  be  found  to  take  it  to  pieces  gradually, 
and  to  identify  the  products  thus  obtained.  In  order  to 
accomplish  this  two  means  are  at  our  disposal,  viz.,  in  the  first 
place  the  action  of  acids,  in  the  second  place,  and  especially,  the 
action  of  so-called  proteolytic  enzymes,  that  is,  enzymes  which 
are  capable  of  breaking  up  protein  substances.  Of  these  the 
two  chief  are  pepsin  and  trypsin.  The  methods  of  preparation 
of  these  and  their  characteristic  modes  of  action  may  now  be 
usefully  considered. 

Pepsin. — This  is  a  characteristic  enzyme  of  the  gastric 
juice.  Ordinary  *  liquor  pepticus  '  is  prepared  by  macerating 
the  mucous  membrane  of  the  stomach  of  a  dog  or  pig  with 
dilute  hydrochloric  acid,  0*2  per  cent.,  and  filtering  the 
solution.  The  filtered  solution  contains  pepsin. 

By  extraction  with  glycerine  in  absence  of  acid  a  purer 
but  less  active  product  is  obtained.  The  enzyme  can  be 
further  purified  by  precipitation  with  sodium  phosphate  and 
calcium  chloride,  the  calcium  phosphate  formed  carrying  down 
the  enzyme.  The  enzyme  is  separated  from  the  precipitate 
by  solution  in  hydrochloric  acid,  and  the  mineral  salts  removed 
by  dialysis,  the  salts  passing  through  the  parchment  mem- 
brane, leaving  a  solution  of  the  enzyme  in  the  dialyser. 

Trypsin. — Trypsin  is  the  enzyme  of  the  pancreatic  juice 
and  is  obtained  in  a  similar  manner  to  pepsin,  by  digesting 
pancreatic  tissue  with  dilute  acid  or  glycerine  at  35°  to 
40°  C.  The  preparation  of  the  pure  enzyme  is  an  exceedingly 
complex  process. 


190      BACTEEIOLOGICAL   AND    ENZYME    CHEMISTRY 

The  characteristic  difference  between  pepsin  and  trypsin  is 
that  pepsin  acts  in  dilute  act'c?  solution,  and  trypsin  in  dilute  alka- 
line solution.  The  following  experiments  may  usefully  be  made 
to  illustrate  the  characteristic  properties  of  these  enzymes. 

A  quantity  of  hard-boiled  egg-white  may  be  cut  up  into 
strips  of  approximately  2  cm.  x  5  mm.  x  1  mm.  dimensions 
and  one  of  these  placed  in  each  of  eleven  test-tubes,  to  which 
the  following  additions  are  made  in  order,  about  10  c.c.  of 
solution  being  taken  in  each  case  : — 

1.  Water; 

2.  Hydrochloric  acid,1  0*2  per  cent. ; 

3.  Water  -}-  J  c.c.  of  '  liquor  pepticus  ' ; 

4.  Hydrochloric    acid,    0'2    per    cent.  -(-  J    c.c.    '  liquor 

pepticus ' ; 

5.  Hydrochloric    acid,    0*2    per    cent.  +  J    c.c.    '  liquor 

pepticus ' ; 

6.  One  per  cent,  sodium  carbonate  solution  ; 

7.  One    per    cent,    sodium    carbonate    solution  -f-  J  c.c. 

'  liquor  pepticus  ' ; 

8.  Hydrochloric  acid,  0'2  per  cent,  -f  J  c.c.  '  liquor  pan- 

creaticus ' ; 

9.  Water  -f  J  c.c.  '  liquor  pancreaticus  ' ; 

10.  Sodium  carbonate  solution  1  per  cent,  -f-  J  c.c.  '  liquor 

pancreaticus ' ; 

11.  Sodium  carbonate  solution  1  per  cent.  +  \  c.c.  '  liquor 

pancreaticus.' 

All  of  these  are  now  placed  in  a  water-bath  at  40°  C.,  with 
the  exception  of  numbers  5  and  11,  which  are  boiled. 

At  the  end  of  some  hours  the  following  results  will  be 
observed  :  the  strips  of  egg-white  will  be  virtually  unattacked 
either  by  water,  by  dilute  acid  or  alkali,  by  pepsin  and  alkali 
together,  or  by  trypsin  and  acid  together.  On  the  other  hand, 
some  digestion  will  probably  be  observed  in  the  case  of  both 

TT(~n 

1  110  c.c.  of  —    made  up  to  200  c.c.  gives  a  solution  of  this  strength. 


THE    CHEMISTEY    OF   ALBUMINS  191 

pepsin  and  trypsin  alone,  while  in  the  case  of  the  mixture  of 
pepsin  and  acid,  and  of  trypsin  and  alkali,  digestion  will  be 
almost  complete. 

This  demonstrates  the  fact  that  pepsin  is  most  active  in 
presence  of  dilute  acid,  while  trypsin  is  most  active  in  the 
presence  of  dilute  alkali.  In  order  to  investigate  the  products 
of  decomposition  in  each  of  these  cases  larger  quantities  of 
egg-white  must  of  course  be  taken  ;  if  this  is  done,  the  products 
present  in  solution  can  then  be  investigated  in  the  manner  to  be 
described. 

In  following  the  reaction  it  will  be  advisable  to  make 
observations  from  time  to  time,  as  the  reaction  is  progressive, 
products  of  decreasing  complexity  being  obtained  as  it 
proceeds.  If  to  a  portion  of  the  solution  shortly  after  the 
beginning  of  the  reaction  strong  alcohol  or  a  saturated  solution 
of  ammonium  sulphate  is  added,  a  precipitate  is  formed ;  the 
substances  thus  precipitated  are  known  as  albumoses.  At 
a  further  stage  ammonium  sulphate  is  added ;  no  precipitate 
will  be  obtained,  but  a  precipitate  will  still  be  formed  if  alcohol 
is  added.  These  products  of  decomposition  of  albumin,  which 
are  soluble  in  water  and  precipitated  by  alcohol  but  not  by 
ammonium  sulphate,  are  known  as  peptones.  It  will  be  found 
on  testing  that  they  still  give  the  biuret  reaction,  showing 
that  a  complex  residue  containing  amino  (NH2)  and  imino 
(NH)  groups  is  still  present.  The  red  substance  of  the  biuret 
reaction  is  believed  by  Schiff  to  be  a  copper  potassium  com- 
pound having  the  following  constitution  : — 

0  OH  OH     0 

II      I  I        II 

C-NH2-Cu NH2-C 

NH/  ^ 

XC-NH,-K  K-NH2-C 

II      I  I        II 

0   OH  OH    0 


192      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

The  further  decomposition  of  peptones  results  in  the 
formation  first  of  substances  which  have  still  a  complicated 
composition,  and  which  are  known  as  polypeptides,  and 
finally  into  substances  of  a  simpler  character,  viz.,  ammo 
acids,  of  which  amino-acetic  acid  or  glycocoll,  CH2NH2COOH, 
is  a  prototype.  The  separation  and  investigation  of  these 
is  a  task  for  the  experienced  organic  chemist,  and  its  details 
cannot  profitably  be  fully  discussed  here. 

The  whole  subject  has  been  brilliantly  investigated  by 
Emil  Fischer  and  his  colleagues,  who  have  not  only  devised 
methods  for  separating  and  identifying  amino-acids,  but 
have  also  been  enabled  to  synthesise  a  number  of  polypeptides, 
whose  complexity  approaches  in  certain  cases  the  complexity 
of  the  peptone  molecule,  and  which  are  even  capable  of  being 
broken  down  again  into  simpler  substances  by  the  action  of 
trypsin. 

Certain  American  investigators  have  announced  that  they 
were  able  to  synthesise  peptone-like  bodies  by  the  action  of 
trypsin  on  polypeptides.  However  this  may  be,  it  is  clear 
that  in  this  direction  we  must  look  for  any  definite  know- 
ledge as  to  the  ultimate  structure  of  the  albumin  molecule 
or  its  derivatives,  and  a  brief  account  of  the  chief  products 
separated  or  prepared  by  Emil  Fischer  and  others,  and  of  the 
methods  used  in  their  researches,  will  be  of  interest  and  value 
as  affording  a  basis  for  the  classification  of  the  very  numerous 
bodies  related  to  albumin. 


PRIMARY  DISINTEGRATION  PRODUCTS  OP  ALBUMIN 

Fischer  made  use  of  three  chief  methods  for  separating 
amino-acids : — 

1.  The  acids  are  converted  into  ethyl  esters  which  are 
separated  by  fractional  distillation  under  the  lowest  possible 
pressure.  The  following  description  will  indicate  in  outline 
the  practical  carrying  out  of  the  method. 


THE    CHEMISTRY    OF   ALBUMINS  193 

The  solution  containing  the  mixture  of  amino-acids  is 
carefully  evaporated  at  reduced  pressure  and  at  a  temperature 
not  exceeding  40°  C.  The  syrupy  residue  is  dissolved  in 
absolute  alcohol,  and  gaseous  hydrochloric  acid  passed  into 
the  solution  to  saturation,  the  hydrochlorides  of  the  esters 
being  thus  formed.  The  excess  of  alcohol  is  evaporated  off 
under  diminished  pressure.  Strong  caustic  soda  solution  is 
carefully  added  to  the  residue,  until  the  hydrochloric  acid  is 
neutralised. 

The  esters  thus  set  free  are  separated  by  solution  in  ether. 
The  ethereal  solution  is  then  fractionally  distilled  in  a  specially 
designed  apparatus  in  which  the  pressure  is  reduced  to  less 
than  1  mm. 

A  number  of  precautions  in  detail  are  necessary  if  the  best 
yields  are  to  be  obtained. 

2.  The  acids  are  converted  into  their  y8-naphthalene-sulpho 
derivatives,  which  are  sparingly  soluble  compounds. 

The  following  equation  indicates  the  formation  of  the 
y9-naphthalene-sulpho  derivative  of  serin  by  the  action  of 
/3-naphthalene-sulpho-chloride : — 

CH2OH 
C10H7S02C1  +  H2NCH/ 

COOH 

CH2OH 
=  HC1+  C10H7S02NHCH/ 

XCOOH 

3.  The  acids  are  combined  with  phenyl  isocyanate,  which 
gives  characteristic  compounds. 

The  equation  representing  the  formation  of  the  glycocoll 
compound  is  as  follows  : — 

C6H5-NCO  +  NH2CH2COOH  =  C6H5NHCONHCH2COOH 

The  chief  end  products  obtained  by  taking  to  pieces,  as 
it  were,  the  molecule  of  albumin,  may  be  roughly  classified  as 
follows : — 


194      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

Mono-amino  acids  ; 
Hydroxy-amino  acids  ; 
Di-amino  acids  ; 
Amino-dicarboxylic  acids ; 
Sulphur  derivatives ; 
Purin  bases ; 
Ptomaines ; 
Carbohydrates. 

In  addition,  simple  substances  such  as  sulphuretted  hydro- 
gen, carbonic  acid,  ammonia,  sundry  fatty  and  other  acids, 
etc.,  are  produced. 

Mono-amino  Acids 

The  following  are  the  chief  mono-ammo  acids  : — 
1.  Glycocoll  or  ammo-acetic  acid. — This  is  the  simplest 
member  of  the  mono-amino  acids ;  it  is  frequently  termed 
glycin  for  the  sake  of  convenience  in  describing  its  numerous 
derivatives  among  the  polypeptides.  Glycocoll  is  related, 
to  what  is  believed  to  be  the  mother  substance  of  sJcatol, 
a  substance  occurring  in  excreta,  the  unpleasant  smell  of 
which  is  largely  due  to  it.  Skatol  can  be  recognised  in 
concentrated  fresh  sewage  by  the  pink  colour  which  is  obtained 
on  warming  with  strong  sulphuric  acid.  It  has  been  shown 
to  be  /3-methyl-indol,  the  relation  of  the  two  bodies  skatol 
and  indol  beirg  given  by  the  following  formulae  : — 


THE    CHEMISTRY   OF   ALBUMINS  195 

It  probably  occurs  as  a  decomposition  product  of  albumin, 
in  the  form  of  skatol-amino-acetic  acid  :  — 


CH 


C.CH(NH2).COOH 


H 


2.  Alanin  or  a-amino-propionic  add,  CH3CHNH2COOH. 
This  acid  has  been  shown  by  Emil  Fischer  to  be  widely 
distributed  as  a  decomposition  product  of  albuminoids.  Its 
derivatives  have  many  of  them  been  well  known  for  some  time, 
especially  phenyl-alanin,  that  is  C6H5CH2CHNH2COOH,  and 
para-hydroxy-phenyl-alanin,  more  commonly  known  as  tyrosin> 


CH2CHNH2COOH 

Tyrosin  is  easily  isolated  on  account  of  its  sparing  solubility  ; 
it  is  one  of  the  products  of  excretion  of  the  animal  body  and 
occurs  together  with  leucin. 

Other  derivatives  of  alanin  give  rise  to  the  very  important 
substances  indol  and  skatol  already  mentioned.  A  sub- 
stance termed  tryptophane  has  been  isolated  from  the  mixture 
of  substances  produced  by  the  action  of  trypsin  on  albumin. 
Some  amount  of  discussion  has  taken  place  as  to  the  constitu- 
tion of  this  body ;  it  appears  certainly  to  be  an  indol-amino- 
propionic  acid.  Indol-amino-propionic  acid  or  tryptophane 
will  therefore  have  either  of  the  two  following  formulae  :— 

02 


196       BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

COOH 


CH 


or 


-N 

1 
H 

—  a-  CH2 .  CH(NH2)  COOH 


H 

In  this  way  we  can  see  how  indol  may  be  produced  by  the 
decomposition  of  albumin  substances.  It  is  of  interest  to  note 
that  the  formation  of  indol,  recognised  by  the  red  coloration 
it  produces  with  nitrous  acid,  is  a  characteristic  reaction  for 
certain  bacteria,  notably  B.  coli,  and  serves  to  distinguish  this 
from  the  more  dangerous  typhoid  bacillus.  The  cholera-red 
reaction  given  by  the  cholera  organism  depends  also  on  the 
formation  of  indol. 

In  both  cases  the  red  coloration  is  due  to  the  formation 


— CH 


of  nitroso-indol, 


!— N-NO 


formed    by    the  action    of 


nitrous  acid  on  the  imino  group  present. 

3.  Amino  -  valerianic  acid,  CH3CH2CHoCHNH2COOH. 
This  acid  is  of  interest  mainly  on  account  of  its  derivatives, 
the  chief  of  which  is  known  as  arginin,  to  which  reference 
will  be  made  later,  and  leucin  or  isobutyl-a-amino-acetic  acid, 
which  has  the  following  formula  : — 


THE    CHEMISTRY    OF   ALBUMINS  197 


H  H   NH2 
C_C_C-0 

H3C'         H  H       N)H 


This  is  one  of  the  earliest  known  of  the  decomposition  pro- 
ducts of  albumin,  and  readily  crystallises  in  scales  or  nodules 
with  very  characteristic  appearance. 


Hydroxy-amino  Acids 

Serin,  a-amino-y^-hydroxy-propionic  acid, 


0 


H    NH, 


HO— C-C— C 
H  H      \ 

OH 

This  acid  is  of  special  interest  as  being  one  of  the  chief 
decomposition  products  of  silk.  Emil  Fischer  has  shown 
that  it  is  a  general  product  of  the  breaking  up  of  albumin. 


Di-amino  Acids 

There  are  three  important  di-amino  acids  which,  according 
to  Kossel,  occur  in  all  albumins  in  greater  or  less  amount, 
and  whose  relative  preponderance  can  therefore  serve  as  a 
means  of  classification  of  albumin  bodies.  These  three 
di-amino  acids  are  termed  by  Kossel  hexone  bases,  as  they  all 
contain  six  carbon  atoms,  and  the  basic  character  which  is 
characteristic  of  all  amino  acids  predominates.  These  three 
substances  are  arginin,  lysin,  and  histidin.  Arginin  is  the 


198     BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

guanidine-a-amino-valerianic  acid  ;  guanidine  has  the  formula 
NH=C(NH2)2,  and  arginin,  therefore,  may  be  written 

NH2  NH2      0 

|  H  H   H     |       / 

NH=  C—  N—  C—  C—  C—  C—  C 
H   H  H  H   H      \ 

OH 

Arginin  is  really  a  compound  of  guanidine  with  ornithin  a 
derivative  of  which  is  found  in  the  urine  of  birds.  Ornithin 
is  a-S-di-amino-valerianic  acid. 

Lysin  is  a-e-di-amino-normal-caproic  acid,  i.e. 

CH2NH2CH2CH2CH2CHNH2C02H 
It  occurs  in  greatest  quantities  in  casein  and  gelatine. 

Histidin  has  a  rather  more  complex  constitution  than 
either  of  the  other  two  hexone  bases.  It  is  probably  a 
condensation  product  formed  by  elimination  of  NH3  from 
arginin  and  its  constitutional  formula  may  be  provisionally 
written  thus  :  — 

CH—  N 


C—  HN 

I 
CH2 

CH—  NH2 
COOH 

Amino-dicarboxylic  Acids 

Of  these  the  following  may  be  mentioned,  asparlic  acid  or 

CHNH2C02H 
amino-succinic  acid,  ,  and   glutaminic  acid  or 

CH2C09H 
a-amino-glutaric  acid,  COOHCHNH2CH2CH2C03H. 


THE    CHEMISTRY    OP    ALBUMINS  199 

Pyrollidin-carboxylic  Acid  or  Prolin 

This  has  been  obtained  as  a  product  of  hydrolysis  of  casein 
and  is  of  interest  from  the  point  of  view  of  the  synthetical 
experiments  of  Emil  Fischer  and  others.  It  has  the  follow- 
ing constitution : — 


H2C 


CH2 


H2C         CH—  COOH 
NH 

Sulphur  Bodies 

Cystin.  —  This  is  a  very  interesting  substance  as  it  is  prob- 
ably the  parent  body  of  the  isomeric  forms  a-cystin  and 
/3-cystin,  which  are  very  likely  the  parent  bodies  of  the  un- 
pleasant smelling  sulphur  derivatives  of  albumin.  These  two 
bodies  are  differentiated  according  to  the  products  obtained 
when  they  are  treated  with  hydrochloric  acid  under  pressure,  as 
indicated  by  the  following  formulae  :  — 


CH2SH-CHNH2-COOH  -+  C^ 

a-cystin  ->  a-alanin  +  sulphuretted 

hydrogen 

CH2NH2-CHSH-COOH  ->  CHg-CHSH-COOH+NHg 

j8-cystin  o-thiolactic  add  +  ammonia 

Probably  both  a-  and  /3-cystin  contain  at  least  two  groups 
as  given  in  the  above  equations,  joined  in  each  case  by  sulphur 
thus  :  — 

SCH2-  CHNH2-  COOH  CH2NH2-  CHS-  COOH 

I  I 

SCH3-CHNH2-COOH  CH2NH2-  CHS-  COOH 

a-cystin  /3-cystin 


200     BACTERIOLOGICAL   AND 


CHEMISTRY 


Purin  Bases 

"These  important  substances  are  obtained  as  decomposition 
products  of  nucleic  acid,  produced  in  its  turn  from  so-called 
nucleo  albumins.  They  are  derived  from  a  parent  substance, 
prepared  by  Emil  Fischer,  which  he  termed  purin.  The 
relation  of  the  purin  bases  to  purin  is  shown  by  the  following 
formulae  :— 


Purin 


Guanin 


=         HC    C-NH 


N-C-N 


X 


CH 


C5H5ON5 


HN-CO 

!    I 

NH2C    C-NH 


Xanthin     ..    C5H,02N4    = 


Hypoxanthin     C5H4ON4    = 


HN-CO 

I      I 
OC    C-NH 


\ 


CH 


HN-CO 

I      I 
HC     C-NH 


\ 


CH 


N=CNH 


Adenin       ..    C5H5N5        =         HC   C-NH 


N-C-N 


CH 


THE    CHEMISTRY    OF   ALBUMINS  201 


Ptomaines 

These  bodies  are  products  of  putrefactive  decomposition 
of  albumin  and  are  mostly  strong  bases  ;  they  can  be  obtained 
by  splitting  off  C02  from  amino  acids.  Thus  leucin  gives  rise 
in  this  way  to  pentamethylene-diamine  or  cadaverin  according 
to  the  following  equation  : — 

CH2NH2-(CH2)3-CHNH2COOH 

=  CH2NH2-(CH2)3-CH2NH2+C02 

while  argenin  gives  rise  to  putrestin,  cyanamide  being  formed 
at  the  same  time  : — 

NH2C(NH)NH-CH2-(CH2)2CHNH2-COOH 

=  NH2-CN  +  C03  +  NH2CH2-(CH2)2-CH2NH2 

Cyanamide  Putrescin 


Carbohydrates 

These  occur  among  the  decomposition  products  of  certain 
albumins  in  the  form  of  amino  derivatives  of  which 
glucosamin,  CH2OHCHOHCHOHCHOHCHNH2CHO,  is  a 
characteristic  example. 

Synthesis  of  Disintegration  Products.— We  are  now  in  a 

position  to  understand  something  of  the  significance  of  the 
syntheses  of  the  complicated  bodies  known  as  polypeptides, 
from  the  starting  point  of  the  disintegration  products  which 
have  just  been  described.  It  would  lead  too  far  to  attempt  to 
give  these  in  any  detail,  but  the  simplest  case  will  suffice  to 
indicate  the  principle  on  which  more  complex  substances  may 
be  built  up.  Glycocoll  or  glycin  may  be  taken  as  a  starting 
point. 

The  ethyl  ester  is  first  prepared  ;  on  standing,  condensation 


202      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

takes  place,  with  formation  of   a  ring  compound  known  as 
di-aci-piperazin,  or  di-glycocoll  anhydride, 

/CH2-NHX 
NH2CH2COOH->  NH2CH2COOC2H5->  0-  V  /C=  0 


Qlycocoll  Glycocoll  ethyl  ester  Diacipiperazin 

On  saturating  a  boiling  solution  of  this  compound  with 
gaseous  hydrochloric  acid,  it  is  split  up  with  formation 
of  the  simplest  polypeptide,  known  as  glycyl-glycin  or 
NH2CH2CO-NHCH2COOH,  the  group  NH2CH2CO  being 
termed  glycyl.  The  reaction  is  expressed  as  follows  :  — 


Onx 
—  ^\  \J=\J   ~T  boiling ~^V/=V; 

XNH-CH2/  XNH-CH2-C02H 

Diacipiperazin  Glycyl-glycin 

It  is  readily  seen  that  if  glycyl-glycin  is  taken  in  its  turn 
as  a  starting  point,  and  a  similar  set  of  reactions  carried  out, 
further  similar  complexes  of  higher  molecular  weight  could  be 
obtained.  The  most  complex  polypeptide  so  far  synthesised 
has  the  constitution  : — 

rNH2CH(C4H9)CO[NHCH2CO]3NHCH(C4H9)CO[NHCH2CO]3-i 
L  NHCH(C4H9)CO[NHCH2CO]8NHCH2COOH 

It  is  termed  Meucyl-triglycyl-Meucyl-triglycyl-Meucyl- 
octaglycyl  glycin. 

It  is  an  octadecapeptide,  containing  no  less  than  18  amino- 
acid  residues,  giving  it  a  molecular  weight  of  1213. 

Compounds  such  as  this  give  the  biuret  reaction,  and  are 
capable  of  being  partially  split  up  by  ferments,  such  as  trypsin  ; 
they  are  in  fact  nearly  akin  to  peptones,  which,  as  we  have 
seen,  are  some  way  on  to  the  complexity  of  albumin. 


THE    CHEMISTRY    OF    ALBUMINS  203 

The  Constitution  of  Albumins.— The  investigation  of  the 
properties  of  the  amino  acids,  the  synthetical  work  of  Fischer 
on  the  polypeptides,  and  other  researches  in  similar  directions, 
have  led  to  the  conception  of  the  albumin  molecule  as  con- 
sisting of  a  complex  of  amino-acid  residues,  linked  together  by 
the  condensation  of  a-amino  groups  with  carboxyl  groups. 
The  following  complex  will  serve  to  illustrate  the  theory  which 
has  been  propounded  by  Hoffmeister  :— 

R  R'  R"  R'" 

I  ._ ,      I  , ,     I  . ,      I 

COOHCH— NH  H  OH  |COCH— NHj  H  OH  JCOCH— NH|  H  OH  |COCHNH2 

On  condensation  this  yields — 

R  R'  R"  R"' 

I  I  I  I 

COOH— CHNH— COCH— NHCOCH— NHCOCHNH3 

The  groups  K,  R',  R",  etc.,  represent  various  residues  which, 
on  splitting  off,  give  the  various  characteristic  decomposition 
products  of  albumin.  Thus,  the  following  typical  examples 
will  serve  for  illustration  : — 

CH(CH3)2        C^OH  COOH  CH2NH2 

I  I  I  I 

CH2  CH2  CH2  (CH2)3 

— NH— CHCO— NH— CHCO— NH— CHCO— NH— CHCO— NH— 

1.  Leucin          2.  Tyrosin  3.  Aspartic  acid  4.  Lysin 

It  can  easily  be  seen  how  by  simple  hydrolytic  changes 
the  various  substances  leucin,  tyrosin,  aspartic  acid,  or 
lysin  can  be  split  off  from  such  a  complex.  On  oxidation 
with  permanganate,  these  side  chains  are  finally  converted 
into  oxalic  acid  and  ammonia. 

In  ordinary  animal  metabolism,  hydrolysis  and  oxidation 
go  on  together,  with  formation  of  urea  as  an  end  product. 
The  constitution  of  individual  albumins  is  by  no  means 


204     BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

sufficiently  well  known  to  permit  of  a  strict  chemical  classifica- 
tion according  to  their  decomposition  products.  An  attempt 
has,  however,  been  made  by  Kossel,  who  divides  albumins  into 
four  classes,  according  to  their  yield  of  the  so-called  hexone 
bases  already  referred  to,  viz.,  lysin,  arginin,  and  histidin. 
Kossel's  classification  was  as  follows  : — 

1.  Protamins — All  rich    in  arginin,  but   differing  in   the 
amounts  of  other  bases  and  of  mono-amino  acids. 

2.  Histories — Kelatively  high  in  arginin. 

3.  Vegetable  albumins. — Poor  in  arginin  and  no  lysin. 

4.  All  others  containing  all  three  hexone  bases  and  most 
amino  acids. 

The  Separation  and  Extraction  of  Albumins.— It  has 

already  been  seen  when  studying  the  properties  of  ordinary 
egg-albumin  that  it  was  possible  to  separate  it,  e.g.,  from  the 
associated  substance  globulin,  by  the  insolubility  of  the  latter 
in  water.  Further,  it  was  found  that  whereas  albumoses 
were  precipitated  by  both  alcohol  and  ammonium  sulphate, 
peptones  were  precipitated  by  alcohol,  and  not  by  ammonium 
sulphate.  The  method  of  precipitation  by  suitable  salts  and 
other  substances,  if  carried  out  with  care,  can  be  used  for 
separating  the  various  albumins  one  from  another.  Such  a 
process  is  known  as  salting  out. 

The  salts  chiefly  used  for  separation  of  the  albumins  are 
as  follows,  beginning  with  the  least  effective  : — 

Class  I.     Sodium  chloride  ; 

Sodium  sulphate ; 

Sodium  acetate ; 

Sodium  nitrate ; 

Magnesium  sulphate. 
Class   II.     Potassium  acetate. 
Class  III.     Ammonium  sulphate ; 

Zinc  sulphate. 


THE    CHEMISTRY   OF   ALBUMINS  205 

The  members  of  each  class  are  more  or  less  equivalent  in 
precipitating  power,  but  whereas,  e.g.,  sodium  chloride  will 
not  precipitate  egg-albumin,  ammonium  sulphate  will  not 
only  precipitate  egg-albumin,  but  also  its  primary  disintegra- 
tion products,  viz. ,  albumoses. 

In  making  salting-out  experiments  it  is  important  that  the 
concentration  of  the  albumin  solution  shall  not  be  altered. 
Thus,  for  example,  to  study  the  effect  of  various  concentra- 
tions of  any  salt  on  an  albumin  solution,  a  number  of  test- 
tubes,  each  containing  2  c.c.  of  the  albumin  solution,  may  be 
taken,  and  8  c.c.  of  a  mixture,  in  varying  proportions,  of 
distilled  water  and  a  saturated  solution  of  the  salt  under 
observation. 

By  experiments  of  this  sort  it  has  been  found  that  the 
operation  of  salting  out  is  subject  to  the  following  well-defined 
laws: — 

1.  The  degree  of  concentration  of  any  salt  necessary  for 
the  precipitation  of   any  particular  albumin   is  character- 
istic  for  that   body.    If,  for  example,  a  serum  solution  is 
precipitated  with   ammonium   sulphate,  it   has   been  found 
that  the  globulin  begins  to  come  down  when   ammonium 
sulphate   is  present  to  the  extent  of   24-29  per  cent,  of 
complete  saturation.      The   albumin  does  not  begin  to  be 
precipitated  until  the   degree   of  saturation  reaches  about 
64  per  cent. 

2.  If  one  albumin  is  precipitated  by  a  lower  degree    of 
concentration    than    others   of    any  given   salt,  a  propor- 
tionally lower  concentration  will  also  be  effective  with  other 
salts. 

Thus,  in  the  example  just  given,  if  zinc  sulphate  were  used 
instead  of  ammonium  sulphate,  less  of  it  would  be  required 
to  precipitate  the  globulin  than  the  albumin. 

3.  The   limits   between   which  precipitation   commences 
and  finishes  on  addition  of  a  salt  to  a  solution  are  numbers 
characteristic  for  each  albumin.      Thus  the  precipitation  of 


206      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

globulin  in  a  serum  solution  by  means  of  ammonium  sulphate 
begins  at  24-29  per  cent,  of  complete  saturation,  and  is  com- 
pletely thrown  out  of  solution  when  the  saturation  reaches 
46  per  cent.  The  corresponding  limits  for  serum  albumin  are 
64  and  90  per  cent,  of  saturation. 

In  addition  to  the  salts  above  mentioned,  albumin 
can  be  precipitated,  as  we  have  seen,  by  colloidal  metallic 
hydroxides. 

Albumins  also  combine  with  numerous  organic  colouring 
matters,  and  advantage  is  taken  of  this  in  the  various  methods 
for  staining  tissues  for  microscopical  examination.  Many  of 
the  naturally  occurring  colouring  matters  exist  in  combination 
with  albumin,  from  which  they  have  to  be  separated  if  the  pure 
colouring  matter  is  required.  In  the  indigo  plant,  for  example, 
a  portion  of  the  indigo  probably  occurs  in  combination  with 
indigo-gluten  ;  and  there  is  evidence  that  laccainic  acid,  the 
colouring  matter  of  lac  dye,  exists  in  the  body  of  the  lac 
insect  as  an  insoluble  albumin  compound.  These  facts 
have  their  practical  importance  in  connection  with  dyeing. 
The  reason  that  wool  can  be  dyed  with  certain  colouring 
matters  which  are  not  taken  up  by  cotton,  that  is  by 
cellulose,  is  that  wool  is  chemically  related  to  albumin, 
and  is  therefore  capable  of  combining  with  colouring 
matters,  more  especially  those  of  an  acid  character.  Further, 
various  albumins,  especially,  e.g.,  serum  albumin,  as  being 
obtainable  in  large  quantity  from  the  blood  of  slaughtered 
animals,  is  used  as  a  mordant  for  fixing  certain  colours  in 
calico  printing. 

For  the  precipitation  of  peptones — and  to  these  may  be 
added  enzymes,  which  we  have  seen  have  many  of  the  properties 
of  peptones  and  are  allied  to  them  in  composition — substances 
such  as  phosphotungstic  and  phosphomolybdic  acids  may 
be  used.  Metaphosphoric  acid,  and  also  a  mixture  of  potas- 
sium ferrocyanide  and  concentrated  acetic  acid,  can  also  be 
used  for  precipitation  of  bodies  of  this  class  ;  it  may  be 


THE  CHEMISTRY    OF    ALBUMINS  207 

remembered  that  metaphosphoric  acid  was  used  for  the  pre- 
cipitation of  the  amylase  of  saliva. 

Tannic  acid  also  precipitates  peptone  bodies,  and  it  is 
probable  that  the  difficulty  of  extracting  certain  enzymes 
from  plants  depends  on  the  fact  that  they  exist  in  the  plant 
in  combination  with  tannic  acid.  It  was  for  this  reason  that 
Brown  and  Morris,  in  their  research  on  the  amylase  of  foliage 
leaves,  obtained  better  results  by  using  powdered  dry  leaf 
than  by  using  a  watery  extract. 

In  order  to  illustrate  the  preparation  of  a  specific  albumin 
from  its  natural  source,  and  the  separation  of  other  bodies,  the 
following  description  of  the  preparation  of  a  typical  vegetable 
albumin,  viz.,  edestin,  may  here  be  given. 

A  quantity,  say  500  grams,  of  hemp-seed  is  ground  up  and 
the  fat  thoroughly  extracted  by  shaking  in  a  large  flask  with 
b'ght  petroleum  and  pouring  off  the  solution.  After  draining 
off  as  much  as  possible  of  the  petroleum,  the  remainder  may 
be  allowed  to  spontaneously  evaporate.  The  residue  is  then 
digested  at  60°  C.  with  350  c.c.  of  5  per  cent,  salt  solution,  with 
continual  stirring.  The  liquid  is  filtered  through  calico  and 
allowed  to  cool.  A  precipitate  forms,  which  can  be  washed 
by  decantation  with  distilled  water.  It  is  redissolved  in 
250  c.c.  of  5  per  cent,  salt  solution,  and  the  solution  filtered 
through  a  warm  filter.  On  cooling  crystals  of  edestin  separate, 
which  can  be  washed  successively  with  cold  5  per  cent,  salt 
solution,  distilled  water,  alcohol  and  ether. 


CLASSIFICATION   OF  ALBUMINS 

We  are  now  in  a  position  better  to  appreciate  the  following 
classification  of  albumins  and  related  substances.  Where 
the  name  of  the  substance  does  not  indicate  its  source  or 
characteristic  properties,  short  explanatory  notes  are  added. 


208      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 


Group  I 

Albumins  Proper.— These  are  naturally  occurring  sub- 
stances and  are  all  typical  colloids : — 

1.  Serum  albumin,  lact-albumin,  egg-albumin. 

2.  Serum  globulin,  lacto-globulin,  cell  globulin. 

3.  Plant  globulins  and  vitellins. 

4.  Fibrinogen.     (Occurs  in  the  blood  plasma  of  all  verte- 
brates.) 

5.  Myosin  and  allied  substances.     (Derived  from  muscle.) 

6.  Phosphorus-containing  albumins,  casein,  vitellins  and 
the  nucleo  albumins  of  the  cell  protoplasms. 

7.  Protamines.     (These    occur    in    the    spermatozoa    of 
fishes,  etc.) 

8.  Histones.     (These  do  not  occur  in  the  free  state  but  in 
combination  with  other  complexes,  to  form  substances  such 
as  haemoglobin.) 

Group  II 

Disintegration  Products  of  Group  I  :— 

1.  Acid  albumins,  alkali  albumins. 

2.  Albumoses,  peptones  and  peptides. 

3.  Halogen  compounds  of  albumins,  etc. 

Group  III 

Proteids. — These  are  compounds  of  albumins  with  other 
complex  groups  which  have  been  termed  prosthetic  groups  : — 

1.  Nucleo  proteids,  compounds  of  albumin  with  nucleic 
acid. 

2.  Haemoglobin  and  allied  substances.      (Haemoglobin   is 


THE    CHEMISTRY    OF    ALBUMINS  209 

the  red  colouring  matter  of  the  blood,  and  consists  of  an 
albumin  compound  with  a  prosthetic  group,  which  in  this 
case  gives  rise  to  colouring  matter  and  is  therefore  called  a 
chroma togenic  group.) 

3.  Glycoproteid  sand  mucins  occurring  in  mucus.  In 
this  case  the  prosthetic  group  is  a  residue  of  a  carbohydrate. 

Group  IV 

Albuminoids. — These  are  many  of  them  rather  ill-defined 
bodies  which  form  part  of  the  skeletal  structure  of  the  animal 
or  plant  organism.  The  classification  is  mainly  anatomical. 

1.  Collagin,    gelatine.     (The    sub-stratum    of    bone   and 
cartilage  consists  of  collagin ;  on  boiling  with  water  it  yields 
gelatine  or  glue.) 

2.  Keratin.     (The  chief  constituents  of  the  horny  sub- 
stances of  mammals  and  birds.) 

3.  Elastin.     (Occurs  in  certain  fibrous  animal  tissue.) 

4.  Fibroin.     (Occurs  in  raw  silk.) 

5.  Spongin.     (Forms  the  frame- work  of  the  bath-sponge.) 

6.  Amyloid.     (A  pathological  product,  sometimes  found  in 
the  brain,  liver,  etc.) 

7.  Albumoids.     (Sundry    substances    found    in    various 
animals,  membranes,  etc.,  difficult  to  classify.) 

8.  Colouring  matters  derived  from  albumins,  e.g.,  mela- 
nin, the  pigment   substance  of   the   skin   of    dark-skinned 
races. 

It  may  be  useful  shortly  to  summarise  the  information  in 
the  foregoing  chapter  as  follows  : — 

Albumins  or  Proteins  are  complex  nitrogenous  colloidal 
substances  occurring  in  animal  and  vegetable  protoplasm,  etc., 
and  capable  of  being  separated  by  their  varying  solubility  in 
solutions  of  certain  salts  (pp.  204-207). 


210     BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

These  yield,  on  treatment  with  dilute  acids  or  alkalis, 
solutions  containing  acid  or  alkali-albumins  (p.  185). 

By  heating  with  acids  or  by  the  action  of  enzymes  such 
as  pepsin  or  trypsin,  albumins  are  gradually  broken  down, 
yielding  successively : — 

1.  Albumoses  precipitated  by  alcohol  and  by  ammonium 
sulphate. 

2.  Peptones  precipitated  by  alcohol  but  not  by  ammonium 
sulphate. 

3.  Polypeptides,  compounds  which  still  give   the   biuret 
reaction,  are  capable  of  synthesis  by  the  condensation  of 
amino-acids,  and  can  be  further  broken  down  to  (pp.  201-202) 

4.  Amino-acids  and  related  substances,  known  as  primary 
disintegration  products  (pp.  192-201). 

BACTERIA  AND   PROTEOLYSIS 

If  an  ordinary  plate  culture  is  made  from  a  small  quantity 
of  sewage  the  gelatine  will  be  found  to  liquefy  round  several 
of  the  colonies.  This  liquefying  action  is  not  infrequently 
so  rapid  and  intense  that  a  few  liquefying  organisms  will 
cause  the  whole  plate  to  become  liquid,  before  the  remaining 
colonies  have  time  to  develop. 

Such  organisms  manifestly  play  an  important  part  in  the 
disintegration  of  albuminous  matter. 

The  action  of  these  and  other  bacteria  on  the  organic 
matter  of  sewage  has  been  the  subject  of  a  research  by  Messrs. 
Clark  and  Gage  of  the  Massachusetts  State  Board  of  Health. 
They  compared  some  300  cultures  of  sewage  bacteria,  as  regards 
their  ability  to  produce  ammonia  in  peptone  solution,  to 
reduce  nitrates  in  nitrated  peptone  solution,  and  to  liquefy 
organic  matter  in  the  form  of  gelatine,  during  an  incubation 
period  of  seven  days. 

The  peptone  solution  consisted  of  O'l  per  cent.  Witte's 
peptone  in  distilled  water,  which  gave  an  organic  nitrogen 


THE    CHEMISTRY    OF    ALBUMINS  211 

value  (determined  by  the  Kjeldahl  method)  of  14  parts  nitro- 
gen per  100,000.  The  nitrated  peptone  solution  contained,  in 
addition,  nitrate  equivalent  to  10  parts  nitrogen  per  100,000. 
The  liquefying  power  was  determined  by  taking  test-tubes  of 
uniform  bore  filled  to  a  depth  of  100  mm.  with  standard  beef 
peptone  gelatine.  The  entire  surface  was  inoculated,  and 
the  depth  of  liquefaction  was  measured  after  a  given  time. 

The  general  result  of  these  researches  was  to  show  that,  as 
a  rule,  the  liquefying  power  was  synonymous  with  increased 
ability  to  reduce  nitrates  and  to  ammoniafy  peptone. 

In  order  to  determine  whether  a  liquefying  organism 
secretes  a  proteolytic  enzyme,  about  0'5  per  cent,  of  thymol 
may  be  added  to  the  liquefied  gelatine,  to  inhibit  further 
bacterial  activity,  and  a  measured  quantity  of  the  liquid 
thus  obtained,  say  O'l  c.c.,  placed  on  the  surface  of  nutrient 
gelatine,  containing  also  0*5  per  cent,  of  thymol,  in  a  tube  of 
uniform  bore.  The  liquefaction  of  the  gelatine  can  be  readily 
observed,  and  by  taking  different  strengths  of  the  liquid  con- 
taining the  enzyme,  quantitative  measurements  can  be  made. 

Reference  may  here  be  made  to  the  activity  of  proteolytic 
organisms  in  the  so-called  '  bating '  or  '  puering  '  process  in 
the  tannery.  In  this  process  the  skins,  which  have  been  '  de- 
haired  '  by  lime,  are  immersed  in  a  bath  or  '  bate  '  of  pigeon's 
or  dog's  dung.  The  bacteria  present  produce  digestive  enzymes, 
which  have  a  solvent  action  on  the  fibres  of  the  skin,  rendering 
it  more  supple.  At  the  same  time  the  acids,  ammonia  and 
amines  which  are  produced  assist  in  the  solution  of  the 
lime  remaining  in  the  skin  from  the  de-hairing  operation. 

In  order  to  avoid  the  use  of  the  unpleasant '  bate '  or '  puer ' 
above  mentioned,  and  with  the  object  also  of  more  accurately 
controlling  the  process,  J.  T.  Wood,  in  conjunction  with  Popp 
and  Becker,  has  successfully  made  use  of  a  puer-substitute, 
termed  '  erodin/  which  consists  of  a  culture  medium  of 
peptonised  gelatinous  tissue,  with  a  special  mixed  culture  of 
selected  bacteria. 

P2 


CHAPTEE  XIII 

THE  NITROGEN  CYCLE 

WE  have  seen  in  the  chapter  on  the  chemistry  of  albumins 
that  substances  comprised  under  this  term  constitute  the 
basis  of  both  animal  and  vegetable  living  matter.  We  know 
that  the  nitrogen  in  our  food  stuffs  occurs  mainly  in  the  form 
of  albumin,  either  animal  or  vegetable.  The  vegetarian,  if  he 
does  not  consume  eggs,  must  at  any  rate  add  to  his  diet  a 
considerable  proportion  of  beans  and  peas,  which  are  rich  in 
vegetable  albumin.  The  actual  amount  of  nitrogenous  food 
needed  for  useful  work  is  a  vexed  question  and  need  not  here 
be  considered,  our  object  being  confined  to  following  out  the 
chemical  history  of  the  nitrogen  whether  large  or  small 
in  quantity.  Used  as  food  we  have  already  learned  that 
peptic  and  tryptic  digestion  of  albumin  leads  by  gradual 
stages  to  the  formation  of  end  products,  largely  consist- 
ing of  amino  acids.  These  one  would  not  expect  to  be 
excreted  as  such  from  the  body ;  they  are  built  up  again 
into  the  body  substance  through  the  biotic  energy  of  the 
cells,  and  a  portion  also  will  be  used  up  as  fuel  for  main- 
taining that  energy  ;  consequently,  therefore,  we  do  not  find 
in  the  products  of  excretion  of  the  animal  body  just  those 
amino  acids  and  polypeptides  which  are  formed  when 
albumin  is  digested  by  pepsin  or  trypsin,  under  laboratory 
conditions. 

Some  of  these  substances,  it  is  true,  are  found  amongst  the 
products  of  excretion ;  thus  leucin  and  tyrosin  have  been 


THE    NITROGEN    CYCLE  213 

mentioned  as  occurring  under  certain  conditions  in  human 
urine,  and  ornithin  is  so  named  from  its  occurrence  in  the 
urine  of  birds.  Skatol  and  indol  are  characteristic  constituents 
of  faeces.  In  the  case  of  flesh-eating  mammals,  however,  by 
far  the  greater  proportion  of  the  nitrogen,  which  is  not  used 
up  in  adding  to,  or  maintaining,  the  body  substance,  is 
excreted  in  the  form  of  urea  contained  in  the  urine.  Urea 
is  a  comparatively  simple  substance  of  which  the  chemical 
formula  is  CO(NH2)2 ;  chemically  it  is  known  as  carbamide, 
being  the  amide  of  carbonic  acid,  CO(OH)2.  The  proportion 
of  urea  in  the  urine  is,  in  fact,  an  index  as  to  whether  proper 
physiological  equilibrium  is  being  maintained,  and  its  deter- 
mination in  the  urine  is  a  routine  test  in  medicine.  Its 
estimation  depends  on  the  fact  that  it  is  decomposed  by 
sodium  hypobromite,  with  liberation  of  nitrogen,  according  to 
the  following  equation  : — 

CO(NH2)2  +  SNaBrO  =  C02  +  N2  +  2H20  +  3NaBr 

Urea  is  also  broken  up  in  a  similar  manner  by  nitrous 
acid,  obtained  by  adding  a  mixture  of  sodium  nitrite  and 
sulphuric  acid  to  the  solution  containing  the  urea.  In 
this  case  nitrogen  is  evolved  both  from  the  nitrous  acid 
and  the  urea  in  equal  proportion,  according  to  the  following 
equation  : — 

CO(NH2)2  +  2HN02  =  C02  +  3H20  +  2N2 

This  reaction  is  of  far-reaching  importance,  as  it  probably 
represents  one  method  by  which  the  nitrogen,  originally 
consumed  as  albumin  food,  finally  reappears  as  free  nitrogen. 
In  the  case  of  animals  whose  diet  is  wholly  vegetable  the 
greater  part  of  the  nitrogen  is  excreted  as  so-called  hippuric 
acid  or  benzoyl  glycocoll,  which  has  the  formula 

C6H5CONHCH3COOH 


214      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

We  have  now  to  consider  how  these  two  main  end  products 
of  nitrogen  metabolism,  viz.,  urea  and  hippuric  acid,  are 
reabsorbed  into  the  cycle  of  nature.  They  are  not  in  them- 
selves directly  available  for  plant  food,  and  the  first  stage  in 
their  reabsorption  by  plants,  whose  nitrogen  may  serve  again 
as  food  for  animals,  consists  in  their  conversion  into  ammonia. 
That  the  conversion  of  urea  into  ammonia  was  a  fermentation 
process,  and  therefore  due  to  life  agency  in  some  form,  most 
probably  to  bacteria,  was  first  suspected  by  Pasteur,  and  also 
by  Tiegheim.  Subsequent  investigators  showed  that  numerous 
organisms  can  induce  ammoniacal  fermentation.  The  most 
active  of  these  is  a  micrococcus  known  as  Micrococcus  urece, 
and  also  a  bacillus,  Bacillus  urece.  These  organisms  are  very 
widely  distributed,  and  consequently  urine,  if  left  exposed  to 
the  air,  very  rapidly  becomes  ammoniacal,  and  the  strong  smell 
of  an  ill-kept  urinal  is  thus  accounted  for.  In  normal  health 
it  has  been  shown  that  these  organisms  are  not  present  in 
freshly  excreted  urine. 

To  demonstrate  the  ammoniacal  fermentation  of  urea, 
some  50  c.c.  of  fresh  urine  may  be  taken  and  diluted  with  an 
equal  volume  of  water  in  a  conical  flask,  thus  exposing  a  large 
surface  to  the  air  ;  the  solution  may  be  infected  with  a  drop  or 
two  of  ammoniacal  urine,  or  with  a  few  centigrams  of  garden 
soil,  and  allowed  to  stand  with  occasional  shaking  for  some 
days.  A  similar  solution  may  be  made  up  with  similarly 
infected  urine,  and  a  small  bottle  completely  filled  with  it, 
and  stoppered.  Both  flask  and  bottle  may  be  placed  in  the 
incubator  at  a  temperature  of  26°  C.  (80°  F.) ;  in  a  day  or  two 
both  solutions,  on  testing  with  litmus  paper,  will  be  found  to 
have  become  strongly  alkaline,  and  Nessler  reagent  will  reveal 
the  presence  of  considerable  quantities  of  ammonia.  It  is 
evident  from  this  experiment  that  ammoniacal  fermentation 
of  urea  can  take  place  both  under  anaerobic  and  aerobic 
conditions ;  the  organisms  of  ammoniacal  fermentation 
belong  therefore  to  the  class  known  as  facultative  aerobes. 


THE    NITROGEN   CYCLE  215 

This  circumstance  is  of  considerable  importance  in  connection 
with  the  purification  of  sewage.  If  the  fermentation  is  allowed 
to  proceed  till  approximate  completion,  and  a  drop  of  the 
solution  is  examined  under  the  high-power  microscope 
(-5^-  inch  oil  immersion),  the  micrococcus  can  be  plainly 
seen. 

Similar  results  are  obtained  if,  instead  of  urine,  an 
artificial  solution  is  made  up  in  the  following  proportions 
and  similarly  fermented  : — 

Water        1500  grams 

Urea           33      „ 

Sodium  chloride    . .         . .         . .  18      „ 

Potassium  hydrogen  phosphate  . .  5      „ 

Magnesium  sulphate         . .         . .  0'5  gram 

The  reaction  which  takes  place  in  both  these  cases  consists 
in  a  simple  hydrolytic  change  resulting  in  the  formation  of 
ammonium  carbonate,  thus  : — 

CO(NH2)2  +  H20  =  (NHJjCOj 

It  has  been  found  that  the  same  organisms  which  bring 
about  the  conversion  of  urea  into  ammonia  will  also  decompose 
uric  acid,  with  production  eventually  of  ammonia ;  and 
hippuric  acid,  with  formation  of  benzoic  acid  and  glycin 
(glycocoll  or  ammo-acetic  acid),  according  to  the  following 
equation  : — 

C6H5CONHCH2COOH  +  H00 

=  C6H5COOH  +  CH2NH2COOH 

Hippuric  acid  or  benzoyl-glycin  Aminc-acetic  acid  or  glycin 

Like  all  other  fermentations,  the  ammoniacal  fermentation 
ceases  when  a  certain  concentration  is  reached.  In  this  case 
fermentation  proceeds  until  the  ammonium  carbonate  formed 
reaches  a  concentration  of  13  per  cent. 


216      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

The  ammoniacal  fermentation  belongs  to  an  increasing 
number  of  such  changes,  which  can  ultimately  be  referred  to 
the  activity  of  a  non-living  enzyme.  In  1874  Musculus  found 
that  if  ammoniacal  urine  was  filtered  through  filter  paper, 
and  the  filter  paper  was  washed  and  dried  and  afterwards 
placed  in  a  neutral  solution  of  urea,  ammoniacal  fermentation 
took  place.  This  also  happened  if  the  filter  paper  was  washed 
with  strong  alcohol,  showing  that  the  activity  was  due  to 
something  other  than  the  living  organism.  Although  not 
absolutely  conclusive,  the  evidence  at  present  available 
indicates  that  the  micro-organisms  secrete  an  enzyme  which 
has  been  termed  urease ;  it  can  be  precipitated  by  alcohol 
and  is  destroyed  by  acids.  Sheridan  Lea  in  1885  obtained 
a  rapid  ammoniacal  fermentation  of  a  2  per  cent,  solution  of 
urea,  by  incubating  it  at  38°  C.  with  the  alcoholic  precipitate 
obtained  from  pathological  urine.  Sheridan  Lea  concluded 
that  urease  was  soluble  in  water  after  the  cells  had  been  killed 
by  alcohol,  but  that  otherwise  it  was  intracellular.  It  can 
hardly  be  said  that  Sheridan  Lea's  experiments  are  quite 
convincing ;  the  writer  has  endeavoured  to  repeat  them  with 
ordinary  urine,  so  far  with  little  success.  The  existence  of 
urease,  apart  from  the  organism,  whether  the  latter  is  in  a 
living  state  or  in  the  form  of  its  dead  cells,  is  not,  in  the  writer's 
opinion,  as  yet  fully  established,  and  it  is  possible,  therefore, 
that  the  cell  substance  itself  may  not  be  without  effect  upon 
the  reaction.  Be  this  as  it  may,  the  essential  fact  remains 
that  the  nitrogen  of  albuminoid  material  appears  in  the  course 
of  the  digestive  process  of  animals,  and  of  the  putrefactive 
changes  taking  place  in  nature,  in  the  form  of  amino  acids 
or  urea,  which  are  apparently  not  available  for  plant  food 
until  they  have  undergone  the  ammoniacal  fermentation  which 
has  just  been  described.  Nitrogen  in  the  form  of  carbonate 
of  ammonia  is  capable  of  serving  as  plant  food  ;  in  the  plant 
it  is  built  up  again  into  vegetable  albumins  which  form  the 
food  of  animals. 


THE    NITROGEN   CYCLE  217 

Nitrification. — Not  only  can  plants  absorb  their  nitro- 
gen in  the  form  of  ammonia,  but  they  can  also  make  use 
of  products  of  oxidation  of  ammonia,  viz.,  nitrites  and 
nitrates.  The  chemical  equations  showing  the  relation 
between  ammonia  and  nitrous  and  nitric  acids  are  as 
follows : — 

NH3  +  202  =  HN02  +  H20  +  0  =  HN03 

It  is  possible  in  the  laboratory  directly  to  oxidise  ammonia  to 
nitrous  acid  by  passing  electric  sparks  through  a  mixture  of 
ammonia  and  oxygen,  or  by  passing  the  mixture  over  heated 
spongy  platinum. 

It  was  Pasteur  who  first  suggested  that  the  oxidation  of 
ammonia  to  nitric  acid,  which  evidently  takes  place  in  nature, 
was  really  due  to  micro-organisms,  and  two  French  chemists, 
Schlosing  and  Muntz,  actually  proved  that  this  was  the  case. 
They  found  that  if  solutions  containing  ammonia  were  allowed 
to  percolate  through  soil,  which  was  well  aerated  at  regular 
intervals,  the  ammonia  was  mainly  converted  into  nitrate ; 
but  that  if  any  living  energy  in  the  soil  was  paralysed,  e.g. 
by  the  introduction  of  chloroform  vapour,  or  by  other  anti- 
septics, no  nitrification  took  place.  The  study  of  the  conditions 
of  nitrification  has  engaged  the  attention  of  a  great  number 
of  workers  both  in  England  and  on  the  Continent,  and  is  of 
the  very  greatest  importance  from  the  point  of  view  of  agricul- 
ture, and  the  kindred  subject  of  sewage  purification.  In  order 
to  have  a  living  idea  of  the  sequence  of  changes  which  take 
place  when  the  nitrogenous  solution  undergoes  nitrification, 
the  following  experiment  may  be  undertaken :  10  c.c.  of 
urine  may  be  added  to  a  litre  of  watef  in  a  Winchester  bottle, 
together  with  about  a  gram  of  good  garden  mould,  and  the 
solution,  which  will  occupy  rather  less  than  half  of  the  bottle, 
may  be  continually  aerated,  either  by  drawing  air  through  by 
means  of  a  Bunsen  water  pump,  or  by  attaching  the  bottle  to  a 


218      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

shaking  machine.  At  intervals  of  about  three  days  about 
20  c.c.  of  the  solution  may  be  examined  : — 

(a)  for  ammonia  by  means  of  Nessler  solution  ; 

(6)  for  nitrites  by  means  of  acetic  acid,  potassium  iodide 
and  starch,  and 

(c)  for  nitrates  by  means  of  the  Stoddart  test. 

The  following  further  details  in  regard  to  these  tests  may 
be  useful : — 

Ammonia  gives  a  reddish-brown  precipitate,  or  in  dilute 
solutions,  a  yellowish-brown  coloration,  with  an  alkaline 
solution  of  potassium  mercury  iodide,  known  as  Nessler's 
reagent.  The  depth  of  coloration  is  proportional  to  the 
amount  of  ammonia  present. 

Nitrites. — Acetic  acid  liberates  nitrous  acid  from  a  solution 
containing  nitrites ;  the  nitrous  acid,  in  its  turn,  liberates 
iodine  from  potassium  iodide,  and  the  free  iodine  gives  a 
blue  coloration  with  starch. 

Nitrates. — The  Stoddart  test  affords  a  ready  means  of 
determining  the  presence  of  nitrate  :  10  c.c.  of  the  sample, 
filtered  from  suspended  solids,  are  poured  into  a  test-tube  of 
rather  thick  glass.  About  as  much  pyrogallol  as  will  cover 
a  sixpence  is  then  dissolved  in  the  solution  and  2  c.c.  of 
strong  nitrate-free  sulphuric  acid  carefully  added  from  a 
pipette,  so  as  to  form  a  layer  in  the  lower  portion  of 
the  solution.  Dry  powdered  sodium  chloride  (salt)  about 
equal  in  quantity  to  the  pyrogallol  is  now  added,  and  if 
nitrate  is  present  a  purple  band  is  formed  immediately 
above  the  sulphuric  acid  layer.  The  intensity  of  the 
coloration  is  roughly  proportional  to  the  amount  of  nitrate 
present. 

It  will  be  found  that  a  progressive  change  takes  place  ; 
first  of  all,  formation  of  ammonia  will  be  noticed,  with  no 
nitrite  or  nitrate  ;  this  attains  a  maximum,  and  then  decreases 
with  simultaneous  appearance  of  nitrite,  but  little  or  no 
nitrate ;  finally  the  nitrites  disappear  and  there  is  left  a 


THE    NITROGEN    CYCLE  219 

solution  containing  only  nitrate.  During  the  course  of  the 
experiment  the  bottle  should  be  kept  as  far  as  possible  in 
darkness,  to  prevent  the  formation  of  green  algae  growths, 
which  combine  with  the  nitrogen  of  the  ammonia  or  the 
nitrate,  and  so  confuse  the  progress  of  the  reaction. 

Experiments  of  this  kind  were  carried  out  by  Munro  in 
1883,  who  showed  that  practically  every  form  of  nitrogenous 
organic  matter  was  capable  of  undergoing  this  series  of 
changes. 

We  are  indebted  to  the  labours  of  Warington  for  the 
exhaustive  study  of  the  conditions  under  which  nitrification 
occurs. 

He  showed  that  the  power  of  nitrification  could  be  com- 
municated to  solutions,  which  otherwise  did  not  nitrify,  by 
inoculating  them  from  solutions  in  which  nitrification  was 
taking  place. 

He  further  confirmed  the  results  of  Schlosing  and  Muntz 
by  showing  that  nitrification  could  be  inhibited  by  the  intro- 
duction of  antiseptics  such  as  chloroform  and  carbon  bisulphide. 

The  following  were  the  conditions  which  Warington  found 
to  be  essential  for  nitrification,  and  his  results  are  in  harmony 
with  those  of  other  observers,  among  whom  may  be  especially 
mentioned  Munro  and  Winogradski. 

1.  It  was  found  that  phosphates  are  the  essential  element 
of  the  food  of  the  organism  of  nitrification.     In  fact,  the  very 
interesting  observation  was  made  that  these  organisms  could 
thrive  on  purely  inorganic  material,  and  even  that  the  presence 
of  organic  matter  appears  to  have  an  inhibiting  effect.     This 
question  will  be  further  considered  in  the  light  of  more  recent 
investigations. 

2.  The  presence  of  oxygen  is  essential  to  the  activity  of 
the  nitrifying  organisms. 

3.  The  presence  of  a  base  is  also  essential  to  neutralise 
the  nitrous  and  nitric  acids  as  they  are  formed  ;  at  the  same 
time  there  must  not  be  an  excessive  alkalinity. 


220      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

4.  Like  other  organisms  the  nitrifying  organisms  have  an 
optimum  temperature  of  activity  ;  they  will  produce  effects 
at  as  low  a  temperature  as  3°  or  4°  C.  (37°  or  39°  F.), 
they  are  fairly  active  at  12°  C.  (54°  F.),  but  they  work 
best  at  37°  C.  (99°  F.).  Still  higher  temperatures  begin  to 
be  prejudicial,  and  like  other  organisms  they  are  apt  to  be 
destroyed  by  strong  sunlight.  The  latter  circumstances, 
it  may  be  mentioned,  are  believed  by  Major  Clemesha  to 
account  for  the  absence  of  nitrates  in  certain  surface  waters 
in  India. 

These  facts  have  a  very  important  bearing  on  the  processes 
of  agriculture  and  especially  also  those  of  sewage  purification. 
The  experiment  which  is  described  on  p.  217  indicates 
clearly  that  nitrification  proceeds  in  two  stages,  the  ammonia 
being  first  oxidised  to  nitrite  and  then  to  nitrate  ;  it  has  been 
found  that  these  two  reactions  are  the  work  of  separate 
organisms. 

Warington  was  not  successful  in  isolating  either  of  these, 
partly  for  the  reason  that  neither  organism  will  grow  on 
gelatine.  Winogradski  in  Russia,  and  Percy  Frankland  in 
this  country,  independently  made  use  of  gelatinous  silica  as 
a  means  of  cultivation.  The  solution  used  with  which  the 
silica  was  gelatinised  had  the  following  composition  in  the 
case  of  the  nitrous  organism  : — 

2  grams  ammonium  sulphate ; 
Q'5  gram  magnesium  sulphate  ; 

2  grams  sodium  chloride  ; 
0'4  gram  ferrous  sulphate  ; 
1000  c.c.  of  water. 

The  nitric  organism  is  more  difficult  to  isolate  even 
than  the  nitrous,  as  it  is  much  smaller.  Winogradski, 
however,  succeeded  in  1891 ;  he  made  use  of  the  following 
solution  ;— 


THE   NITROGEN    CYCLE  221 

1  gram  potassium  hydrogen  phosphate ; 
|  gram  magnesium  sulphate  ; 

trace  of  calcium  chloride ; 

2  grams  sodium  chloride  ; 
1000  c.c.  water. 

Twenty  c.c.  of  this  solution  were  placed  in  a  flat-bottomed 
flask  and  a  little  freshly  washed  magnesium  carbonate  added, 
the  flask  was  closed  with  cotton  wool  and  sterilised  ;  2  c.c.  of  a 
2  per  cent,  solution  of  ammonium  sulphate  were  then  added 
and  the  whole  inoculated  with  a  little  soil.  When  nitrate 
development  had  taken  place  subcultures  were  made  on  to 
silica  jelly.  The  researches  of  Frankland  and  Winogradski 
have  been  confirmed  by  other  investigators. 

From  the  detailed  work  of  Boullanger  and  Massol,  it  appears 
that  there  are  two  well-defined  organisms  which  convert 
ammonia  into  nitrites.  Nitrosomonas,  which  is  a  fairly  large, 
nearly  spherical  organism,  exists  in  two  varieties,  one  the 
form  usually  found  in  Europe,  and  the  other  in  certain  soils 
occurring  in  Java.  There  is  also  a  smaller  form  known  as 
nitrosococcus. 

The  nitric  organism  is  a  very  small  bacterium  whose  length 
somewhat  exceeds  its  breadth. 

These  two  organisms,  the  nitrous  and  the  nitric,  work 
together  in  nature,  and  neither  can  do  its  work  without 
the  help  of  the  other ;  the  nitric  organism  is  incapable  of 
directly  oxidising  ammonia,  and  the  nitrous  organism  cannot 
carry  the  oxidation  of  ammonia  farther  than  the  stage  of 
nitrite.  A  very  important  consequence  of  this  differential 
action  is  seen  in  the  changes  which  take  place  when  sewage 
matter  is  discharged  into  sea  water  ;  the  nitrifying  organism 
under  these  conditions  is  either  actually  destroyed  or  rendered 
inactive.  Dr.  W.  E.  Adeney  gives  the  following  figures  for  the 
results  of  spontaneous  oxidation  of  sewage,  and  comparative 
mixtures  of  sewage  and  fresh  water,  and  sewage  and  sea  water, 
respectively  : — 


222      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 


— 

PARTS  PER  100,000 

Sewage 

Sea  Water 
Mixture 

Fresh  Water 
Mixture 

At   commencement  — 

Nitrogen  as  ammonia    .  . 
Nitrogen  as  Nitrites 
Nitrogen  as  Nitrates 
Organic  Nitrogen 

0-825 

o-o 
o-o 

0-675 

0-165 

o-oo 

0-01 
0-135 

0-165 

o-oo 

0-01 
0-135 

At  conclusion  — 

Nitrogen  as  ammonia   .  . 
Nitrogen  as  nitrites 
Nitrogen  as  nitrates 
Organic  nitrogen 

0-02 

o-o 

0-92 
0-5 

0-0 
0-14 

o-o 

0-072 

o-o 
o-o 

0-142 
0-076 

The  author  has  confirmed  these  observations  in  experi- 
ments made  for  the  purpose  of  tracing  the  changes  taking  place 
when  sewage  sludge  is  discharged  into  sea  water ;  he  found, 
not  only  that  the  ultimate  product  of  oxidation  of  nitrogen 
was  nitrite  rather  than  nitrate,  but  also  that  the  actual  oxida- 
tion of  ammonia  took  place  more  slowly  in  sea  water  than 
in  fresh  water. 

He  has  also  noticed  the  same  phenomenon  of  the  pro- 
duction of  nitrite,  rather  than  of  nitrate,  in  a  case  where 
sewage  effluent  was  being  discharged  into  a  stream  containing 
large  quantities  of  calcium  chloride  from  an  ammonia  soda 
works. 

So  far  we  have  considered,  primarily,  the  oxidation  of 
solutions  containing  ammonium  salts,  with  no  admixture  of 
organic  matter,  and  with  more  or  less  pure  cultivations  of  the 
nitrous  and  nitric  organisms.  In  nature,  however,  such 
conditions  of  course  do  not  obtain  ;  we  have  there  to  do  with 
organic  matter  in  different  stages  of  decomposition,  and  with 


THE   NITROGEN   CYCLE  223 

mixtures  of  numerous  organisms.  The  conditions,  under 
which  the  final  nitrification  then  takes  place,  have  been 
worked  out  by  Adeney  in  a  series  of  very  careful  researches. 
His  method  of  research  consisted  in  exposing  solutions, 
either  of  defined  chemical  substances  such  as  urea,  asparagin, 
ammonium  tartrate,  etc.,  or  less  defined  organic  matter  such 
as  town  sewage,  or  infusions  of  peat,  to  the  prolonged  action 
of  oxygen,  in  the  presence  of  the  usual  organisms  to  be  found 
in  natural  waters.  This  was  accomplished  either  by  mixing 
the  solution  with  a  known  volume  of  aerated  tap  water,  or 
by  shaking  the  solution  periodically  with  known  volumes  of 
air.  Not  only  were  the  products  of  decomposition  and 
oxidation  determined,  such  as  ammonia  and  nitrous  and  nitric 
acid,  but  also  the  carbonic  acid  resulting  from  the  oxidation 
of  the  carbonaceous  matter  present,  as  well  as  the  resulting 
change  in  composition  of  the  dissolved  gases  present.  For 
this  purpose  Adeney  devised  a  special  form  of  gas  analysis 
apparatus,  which  enabled  him  to  analyse  the  gases  obtained 
on  boiling  out  the  solutions  in  vacuo.  He  discovered  the 
source  of  error  in  previous  determinations,  viz.,  the  fact  that 
the  carbon  dioxide  formed  by  oxidation  of  organic  matter 
is  present  largely  as  carbonate,  and  is  only  fully  recovered 
from  the  solution  if  the  latter  is  acidified  before  boiling. 
The  oversight  of  this  fact  led  Sir  Edward  Frankland  to 
conclude  that  the  rate  of  oxidation,  e.g.  of  sewage  matter, 
when  discharged  into  a  stream,  was  much  less  than  was  actually 
the  case.  As  a  result  of  prolonged  investigation,  Adeney 
arrived  at  the  following  conclusions  : — 

Oxidation  of  organic  matter  proceeds  in  two  well-defined 
stages,  which  may  be  briefly  described  as  the  carbon  oxidation 
stage,  and  the  nitrogen  oxidation  stage. 

In  the  carbon  oxidation  stage,  carbon  dioxide,  water, 
ammonia,  and  excretory  substances  are  produced ;  in  the 
second  or  nitrogen  oxidation  stage,  the  two  last-named  bodies 
are  further  fermented,  the  products  being  nitrites,  nitrates, 


224      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

and  comparatively  small  quantities  of  carbon  dioxide.  He 
confirms  the  conclusions  of  previous  observers,  by  showing 
that  in  solutions  of  organic  matter  the  nitrous  organisms 
thrive,  while  the  nitric  organisms  lose  their  vitality.  He 
also  finds  that  the  nitrous  organism  cannot  carry  oxidation 
beyond  the  stage  of  nitrite,  whereas  the  nitric  organism  only 
oxidises  nitrites  to  nitrates.  He  adds  the  further  important 
conclusion,  that  the  presence  of  peaty  or  humus  matter 
appears  to  preserve  the  vitality  of  the  nitric  organisms, 
during  the  earlier  stages  of  the  fermentation  process,  and 
establishes  conditions  whereby  it  is  possible  for  the  nitric 
organisms  to  thrive  simultaneously  with  the  nitrous.  This 
latter  conclusion  has  an  important  bearing  on  the  oxidation 
of  organic  matter  in  nature,  and  especially  under  the  controlled 
conditions  which  obtain  in  modern  processes  for  the  biological 
purification  of  sewage. 

In  all  the  researches  on  the  nitrifying  organisms  referred 
to  in  the  foregoing  pages,  the  conditions  have  been  essentially 
laboratory  conditions,  where  the  solutions  of  organic  matter 
have  been  exposed  to  air,  so  to  speak,  in  bulk,  either  by 
simple  exposure  of  a  solution  in  a  flask,  by  shaking  with  air, 
or  by  bubbling  air  through ;  the  element  of  surface  action 
has  not  been  brought  into  play.  It  is  clear  on  reflection  that 
if  the  solution  to  be  nitrified  could  be  passed  in  a  thin  film 
over  a  large  surface,  with  free  circulation  of  air,  the  conditions 
for  oxidation  would  be  very  much  more  favourable  ;  for  not 
only  would  the  presence  of  ample  oxygen  be  assured,  but  also 
the  extended  surface  would  afford  a  substratum  for  a  greatly 
increased  development  of  the  necessary  organisms.  It  is  the 
application  of  these  principles  which  has  led  to  the  modern 
developments  in  sewage  purification  processes. 

In  1869  Sir  Edward  Frankland,  acting  on  behalf  of  the 
Eoyal  Commission  on  Sewage  Disposal  then  sitting,  made  his 
classical  experiments  on  the  so-called  intermittent  filtration 
of  sewage  through  soil.  He  made  use  of  cylinders  fifteen  feet 


THE    NITROGEN    CYCLE  225 

in  height  filled  with  sand  or  earth,  and  dosed  them  with 
defined  quantities  of  sewage,  allowing  intervals  for  aeration 
between  each  dose.  By  this  method  he  was  able  to  purify 
much  greater  quantities  of  sewage  on  a  given  surface  area  of 
soil,  than  by  the  so-called  broad  irrigation  processes  formerly 
in  vogue.  At  that  time,  however,  the  true  explanation  of 
the  oxidation  change  which  took  place  was  not  properly 
understood,  and  it  was  considered  to  be  a  purely  chemical 
phenomenon.  Later  on  the  Massachusetts  State  Board  of 
Health  took  up  the  subject,  in  the  Light  of  the  researches  of 
Warington,  Winogradski,.  and  Percy  Frankland,  and  they 
worked  out  the  conditions  for  the  successful  oxidation  of 
sewage  matter  by  percolation  through  sand  filters.  They 
showed  that  the  results  depended  essentially  upon  the  presence 
of  oxygen,  and  upon  the  time  allowed  for  the  change  to  take 
place.  They  confirmed  Warington's  conclusion  that  it  was 
necessary  for  a  base  of  some  kind  to  be  present,  to  combine 
with  the  nitrous  and  nitric  acid  produced  by  the  oxidation  of 
ammonia  ;  all  other  conditions  they  considered  were  secondary 
to  these  three. 

It  was  Stoddart  who  showed  in  1893  that  the  time  factor 
could  be  gradually  decreased,  if  filters  of  more  open  material 
than  sand  were  used,  and  care  was  taken  to  distribute  the 
nitrifying  solution  in  such  a  way  that  a  thin  film  only  was 
exposed  to  the  action  of  the  air.  By  allowing  a  solution  of 
ammonium  carbonate  (1  part  N  in  10,000)  to  drip  on  to  a 
column  of  coarsely  powdered  chalk  properly  inoculated  with 
nitrifying  organisms  he  was  able  to  obtain  highly  efficient 
nitrification. 

This  experiment  of  Stoddart's  is  really  the  original  of  the 
modern  trickling  or  percolating  sewage  filter. 

Scott-Moncrieff  in  1898,  by  employing  superimposed 
trays  of  filtering  medium  for  the  final  purification  of  sewage, 
which  had  undergone  preliminary  ammoniacal  fermentation 
in  a  so-called  '  cultivation  tank/  obtained  a  high  degree 


226      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

of  nitrification.  A  very  interesting  result  was  also  demon- 
strated by  these  experiments,  viz.,  that  the  nitrification  was 
a  progressive  phenomenon,  and  its  course  was  considerably 
interfered  with  if,  after  it  had  once  been  established,  the 
sequence  of  the  trays  was  altered,  the  last  tray,  e.g.,  being 
substituted  for  the  highest,  in  which  case  the  nitrification 
was  considerably  impeded,  until  the  original  conditions  were 
re-established. 

The  bacteriological  conditions  obtaining  in  sewage  filters 
of  this  description  have  been  worked  out  in  recent  years  by 
Boullanger  and  Massol  at  the  Pasteur  Institut  at  Lille,  by 
Schulze-Schulzenstein  in  Germany,  and  by  Dr.  Harriette 
Chick  of  the  Lister  Institute.  All  these  investigators  agree 
that  the  nitrifying  organisms  found  in  ordinary  sewage  filters 
are  the  same  as  those  which  occur  in  soil.  Boullanger  and 
Massol  have  found  an  explanation  for  the  seeming  discre- 
pancy between  the  results  of  Winogradski  and  those  which 
are  obtained  on  sewage  filters.  According  to  Winogradski, 
it  will  be  remembered,  the  activity  of  the  nitrifying  organism 
is  inhibited  by  the  presence  of  ammonia  or  of  organic  matter. 
Boullanger  and  Massol  concluded  from  their  experiments,  that 
while  the  presence  of  large  quantities  of  ammonia  or  of 
organic  matter  may  impede  the  original  development  of  the 
nitric  organism,  yet  if  the  growth  of  this  is  once  established, 
its  activity  is  unaffected  by  these  conditions.  These  results 
are  in  harmony  with  Adeney's  conclusion  that  the  presence  of 
peaty  matter  is  of  assistance  in  maintaining  the  activity  of  the 
nitric  organism.  In  a  sewage  filter  the  extended  surface 
enables  an  abundant  growth  of  nitrifying  organism  to  take 
place ;  at  the  same  time  it  is  well  known  that  if  the  maxi- 
mum load,  as  it  were,  of  sewage  matter  is  put  upon  the 
filter  in  its  early  stages,  before  nitrification  is  established, 
it  is  difficult,  if  not  impossible,  for  the  right  conditions  to 
be  set  up  later.  It  is  consequently  necessary  to  '  ripen ' 
the  filter,  by  putting  on  only  comparatively  small  quantities 


THE    NITROGEN    CYCLE  227 

of  sewage  at  first,  increasing  the  quantity  as  nitrification 
becomes  established. 

Dr.  Chick  found  that  in  sewage  filters,  as' in  the  experiments 
with  solutions,  the  nitrification  took  place  in  two  well-defined 
stages,  first  nitrites  and  then  nitrates  being  formed.  The 
length  of  time  required  for  complete  nitrification  to  become 
established  depended  on  the  amount  of  ammonia  present, 
either  actually  as  ammonium  carbonate,  or  potentially  as  un- 
fermented  organic  matter  in  the  sewage  applied.  Tempera- 
ture also  has  a  marked  effect,  as  might  be  expected,  in  deter- 
mining the  time  necessary  for  nitrification  to  be  established ; 
for  this  reason  it  is  advisable  always  if  possible  to  bring  new 
sewage  filters  into  work  during  the  warmer  months  of  the 
year. 

Finally,  mention  may  be  made  of  the  importance  of  the 
character  of  the  material  used  in  the  construction  of  the  filter 
beds.  Practical  experience  has  shown  that  better  results  are 
obtained  with  a  medium  which  offers  a  maximum  of  surface ; 
thus  irregular  material,  such  as  clinkers,  gives  better  results  than 
when  a  smoother  material,  such  as  gravel,  is  used.  Experi- 
ments by  the  author  and  Percy  Gaunt  have  shown  that,  in 
addition  to  the  effect  of  surface  in  giving  an  extended  habitat 
for  bacteria,  the  majority  of  vesicular  or  porous  materials  have 
the  power,  to  a  greater  or  less  extent,  of  retaining  ammonium 
salts,  either  in  their  smaller  pores  or  in  their  larger  interstices  ; 
such  materials,  therefore,  afford  a  somewhat  longer  time  for 
the  nitrifying  action  to  take  place  when  a  solution  containing 
ammonium  salts  is  brought  in  contact  with  them. 

The  purely  physical  side  of  this  question  has  also  been 
carefully  investigated  by  W.  Clifford.  He  allowed  known 
amounts  of  water  to  trickle  through  media  of  different  kinds 
and  dimensions  at  denned  rates.  When  equilibrium  was 
established  between  the  rate  of  inflow  and  outflow,  a  known 
amount  of  sodium  chloride  solution  of  known  strength  was  run 
on  to  the  filter.  The  amount  of  chlorine  emerging  from  the 

Q2 


228      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

medium  was  determined  at  defined  intervals.  He  afterwards 
allowed  the  medium  to  drain  and  measured  the  amount  of 
drainage  water,  and  finally  dried  the  medium,  and  determined 
the  loss  of  moisture.  He  thus  measured  for  each  class  of 
medium  (a)  the  amount  of  water  passing  through  in  a  given 
time,  (b)  the  amount  of  water  held  in  the  larger  interstices, 
and  (c)  the  amount  of  water  retained  in  the  pores.  These 
experiments  showed  generally  that  the  time  of  percolation 
through  clean  filter  material  varies,  inversely  as  the  rate  of 
application  of  the  water,  and  directly  as  the  amount  of  water 
taking  part  in  the  water  movement  through  the  bed. 
This  latter  obviously  depends  on  the  size  of  particles,  and 
the  physical  character  of  the  medium. 

These  results  find  expression  in  the  following  formula  :  — 


~ 


where  c  is  a  constant,  I  the  interstitial  water  per  cubic 
yard,  B,  the  rate  of  sprinkling  per  square  yard  per  hour, 
and  T  the  average  time  of  sprinkling  through  three  feet  of 
medium. 

Unpublished  experiments  by  the  author  and  Mr.  T.  W. 
Lockett  have  shown  that  when  a  nitrifying  solution,  made  up 
after  Winogradski's  recipe,  is  allowed  to  drip  on  laboratory 
filters,  composed  respectively  of  quartz  particles  about  J  inch 
diameter,  and  of  broken  clinker  of  the  same  dimensions,  nitri- 
fication is  established  much  more  rapidly  in  the  case  of  the 
clinker  medium  than  in  the  case  of  the  quartz. 

De-nitrification.  —  De-nitrification,  as  the  name  implies, 
is  the  reverse  of  nitrification.  De-nitrification  changes  are 
concerned  either  with  :  — 

(1)  The  reduction  of  nitrates  to  nitrites,  or  ammonia  ; 
(2)  the  reduction  of  nitrates  and  nitrites  to  oxides  of  nitrogen, 


THE    NITROGEN    CYCLE  229 

NO  and  N20 ;  or  (3)  the  reduction  of  nitrates  and  nitrites 
to  nitrogen. 

The  first  characteristic  work  on  this  subject  was  done  by 
Gayon  and  Dupetit  in  1882.  They  found  that  when  a  solution 
containing  potassium  nitrate,  together  with  sewage  and  a 
little  urine,  was  allowed  to  stand  in  absence  of  air,  the  nitrate 
was  reduced.  When  using  nitrated  broth  containing  asparagin, 
they  obtained  an  evolution  of  nitric  oxide ;  they  also  noted 
the  effect  on  the  reaction  due  to  the  addition  of  carbohydrates 
and  tartrates,  etc.,  and  they  concluded  that  de-nitrification 
was  essentially  the  combustion  of  organic  matter  by  the 
oxygen  of  the  nitrates.  It  thus  naturally  proceeded  best  in 
presence  of  a  minimum  air  supply.  It  could  be  shown,  e.g., 
that  in  a  given  solution  a  greater  amount  of  de-nitrification 
took  place  in  the  lower  portion  of  the  solution  than  at  the 
surface. 

The  subject  of  de-nitrification  has  been  investigated  by 
numerous  workers,  notably  Percy  Frankland  and  Beyerinck. 

The  latter  describes  an  elegant  experiment  for  the  demon- 
stration of  the  presence  of  de-nitrifying  organisms  in  sewage. 
O'l  per  cent,  of  potassium  nitrate  and  a  little  starch  paste  is 
added  to  nutrient  gelatine,  and  the  whole  sterilised  and  poured 
into  a  Petri  dish.  A  little  sewage,  diluted,  say  twenty  times, 
with  distilled  water  is  poured  on  and  off  the  plate,  which  is 
turned  with  the  gelatine  surface  downwards  and  allowed  to 
grow  at  20°  C.  When  the  colonies  have  developed,  a  dilute 
solution  of  hydrochloric  acid  and  potassium  iodide  is  poured 
over  half  the  plate.  Wherever  nitrites  have  been  formed, 
iodine  will  be  liberated  and  will  colour  the  starch  blue.  Colonies 
on  the  other  half  of  the  plate,  similar  in  appearance  to  those 
giving  the  blue  starch-iodide  reaction,  and  which  will  not  have 
been  killed  by  the  acid,  may  be  picked  out  and  grown  separately 
in  suitable  solutions. 

For  the  study  of  the  ultimate  conversion  of  nitrate  into 
nitrogen,  the  following  solution  may  be  made  use  of  : — 


230     BACTERIOLOGICAL   AND   ENZYME    CHEMISTRY 

1  litre  of  river  water ; 

2  grams  calcium  tartrate  ; 

0*5  gram  potassium  hydrogen  phosphate ; 
O'l  gram  potassium  nitrate. 

This  is  sown  with  a  little  horse  dung,  or  straw,  and  in- 
cubated at  35°  C.  The  general  reaction  taking  place  may 
be  expressed  by  the  following  equation  : — 

4KN03  +  5C  +  2H20  =  4KHC03  +  2N2  +  C02 

It  will  be  remembered  that  when  the  decomposition  of 
cellulose  under  aerobic  conditions  was  being  considered,  a 
mixture  was  made  of  a  similar  character  to  the  solution  just 
described,  the  carbon  being  represented  by  the  carbon  of 
cellulose ;  the  importance  of  de-nitrification  as  a  natural 
phenomenon  is  thus  seen.  On  the  one  hand,  we  have  the 
nitrifying  organisms  oxidising  ammonia  to  nitrite  and  nitrate, 
while  on  the  other  hand  the  de-nitrifying  organisms  make  use 
of  the  nitrate  thus  formed,  to  oxidise  organic  matter. 

De-nitrification  is  by  no  means  so  restricted  a  phenomenon 
as  nitrification,  and  quite  a  large  number  of  organisms  have 
been  found  which  are  capable  of  bringing  about  de-nitrification 
to  a  greater  or  less  degree.  Broadly  speaking,  these  may  be 
classified  into  two  classes,  true  de-nitrifying  organisms  which 
are  capable  of  pushing  the  reaction  to  its  final  limit  and 
producing  free  nitrogen  ;  and  indirect  de-nitrifying  organisms, 
which  only  reduce  nitrates  to  nitrites,  when,  through  the  inter- 
action of  nitrites  with  amido  compounds  in  acid  solution,  as 
in  the  case  of  urea,  we  have : — 

CO(NH2)2  +  2HN02  =  2N2  +  C02  +  3H20 

Urea  Nitrous  acid 

Or  to  take  an  analogous,  but  more  complicated,  instance, 
asparagin  may  be  converted  into  malic  acid,  thus  : — 


THE    NITROGEN    CYCLE 


231 


COOH  +  2HN02    =    COOH  +  2N3  +  2H20 


CHNH3 

CH, 

CONH2 

Asparagin 


CHOH 

CH2 

COOH 

MaHc  acid 


Recently  a  somewhat  sensational  discovery  has  been 
made  by  Beyerinck  and  Minkman.  Besides  identifying  the 
de-nitrifying  organism  originally  discovered  by  Gayon  and 
Dupetit,  they  have  isolated  two  other  organisms,  which  are 
probably  the  destroyers  of  nitrates  in  the  soil.  They  describe 
the  following  experiment  :  — 

A  bottle,  with  a  well-fitting  glass  stopper,  is  filled  with 
bouillon,  containing  8  per  cent,  of  potassium  nitrate,  and 
10  to  20  grams  of  garden  soil  are  added.  After  incubation 
at  37°  C.  for  a  day  or  two,  a  considerable  froth  forms, 
which  forces  out  the  liquid  by  a  capillary  action  between  the 
stopper  and  the  neck  of  the  bottle.  The  gas  evolved  remains 
in  the  bottle,  and  on  cautiously  opening  the  bottle  at  the 
end  of  forty-eight  hours,  and  applying  a  glowing  chip,  it  will 
burst  into  flame  through  the  action  of  the  nitrous  oxide 
present.  This  has  been  found  to  amount  to  as  much  as 
90  per  cent,  of  the  gases  evolved.  They  have  also  isolated 
a  second  organism,  which  is  capable  of  causing  the  com- 
bination of  hydrogen  and  nitrous  oxide,  when  these  two  gases 
are  simultaneously  led  into  the  solution  containing  the 
organism.  From  this  combination  the  organism  appears  to 
derive  energy  which  enables  it  actually  to  decompose  carbon 
dioxide,  and  thus  utilise  the  carbon  for  building  up  its  own 
structure.  This  is  an  extremely  interesting  instance  of  the 
reabsorption  of  carbon  from  its  final  state  of  oxidation  as 
carbon  dioxide,  back  into  the  cycle  of  organic  life.  We  know, 


232      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

of  course,  that  plants  possess  this  property  through  the 
activity  of  the  chlorophyll  in  their  cells,  but  instances  of  the 
utilisation  of  the  carbon  in  carbon  dioxide  by  lower  organisms 
have  not  been  frequently  observed. 

Assimilation  of  Nitrogen.— It  will  be  seen  from  the  above 
equations,  representing  de-nitrification  changes,  that  these 
must  eventuate  in  escape  of  nitrogen  into  the  atmosphere. 
If  this  continued,  it  is  evident  that  in  time  the  stock  of  nitrogen 
available  for  life  would  become  depleted,  as  a  certain  percentage 
of  the  nitrogen  of  all  organic  matter  would  be  permanently 
lost  in  this  way.  Fortunately  a  means  exists  for  bringing 
back  this  escaped  nitrogen  once  more  into  the  cycle  of  life. 
A  certain  small  quantity  is  returned  as  nitric  acid,  through 
the  combination  of  nitrogen  and  oxygen  brought  about  by 
the  electric  discharge  of  the  lightning;  and  of  recent  years 
considerable  developments  have  taken  place  in  the  production 
of  nitric  acid  by  the  union  of  the  nitrogen  and  oxygen  of  the 
atmosphere,  by  means  of  powerful  electric  discharges  artificially 
produced.  Nitrogen  has  also  been  recovered  artificially  from 
the  atmosphere  by  the  production  of  calcium  cyanamide 
in  the  electric  furnace,  by  heating  mixtures  of  lime  or  chalk 
with  charcoal  at  a  temperature  of  2000°  C.  in  a  current  of  air. 

Calcium  carbide  is  first  formed,  which  combines  with 
nitrogen  to  form  calcium  cyanamide,  thus  : — 

CaC2  +  2N  =  CaCN2  +  C 

Calcium  cyanamide  can  be  used  as  a  source  of  nitrogen  in 
agriculture,  as  it  decomposes  readily  in  presence  of  moisture, 
yielding  calcium  carbonate  and  ammonia,  thus  : — 

CaCN2  +  3H20  =  CaC03  +  2NH3 

All  these  artificial  methods  are  dependent  upon  cheap 
electricity  for  their  economic  development,  and  the  works 


THE    NITROGEN    CYCLE  233 

for  their  production  are  therefore  situated  mainly  in  Scandi- 
navia, or  in  mountainous  districts  where  water  power  can  be 
readily  utilised.  The  amount  of  nitrogen  recovered  by  these 
artificial  processes  is,  in  the  aggregate,  of  small  account,  com- 
pared with  the  silent  but  widely  active  processes  of  nature. 
The  discovery  of  the  natural  process  by  which  the  apparent 
loss  of  nitrogen  is  made  good  is  due  to  the  researches  of  two 
German  investigators,  Hellriegel  and  Wilfarth.  It  will  be  of 
interest  at  this  point  to  follow  their  discovery  to  some 
extent  by  making  certain  actual  observations,  if  the  season 
of  the  year  permits. 

If  a  fairly  well-grown  plant  belonging  to  the  Leguminacese, 
e.g.,  an  ordinary  garden  sweet  pea,  be  carefully  pulled  up  by 
the  roots  and  the  latter  examined,  if  necessary  with  a  pocket 
lens,  a  number  of  little  nodules  will  be  observed  on  the  rootlets 
(see  Plate  II  (i) ),  which  on  pressing  will  exude  a  milky  juice. 
If  a  microscopic  preparation  is  made  of  this  juice  and  it  is 
examined  under  a  high-power  microscope,  numerous  bacteria 
will  be  found  to  be  present.  Hellriegel  and  Wilfarth  found 
that  plants,  such  as  the  sweet  pea,  were  capable  of  growing  in 
a  sterile  soil  free  from  nitrogen,  if  this  soil  were  treated  with 
an  extract  of  earth  in  which  plants  of  the  same  family  had  been 
previously  grown.  The  addition  of  this  extract  determined 
the  development  of  the  root  nodules.  They  concluded  that 
the  nodule  bacteria  in  some  way  assisted  the  plant  to  absorb 
its  nitrogen  from  the  air.  Their  conclusions  were  confirmed 
by  Breal,  who  compared  the  growth  of  rootlets  of  lupin, 
inoculated  directly  with  the  liquid  contents  of  a  root- 
nodule,  with  the  development  of  similar  rootlets  which 
had  not  been  inoculated.  The  growth,  and  the  percentage 
of  nitrogen  in  the  resulting  plant,  was  much  greater 
in  the  former  than  in  the  latter  case.  It  is  common 
knowledge  that  peas  and  beans  are  the  chief  sources 
of  nitrogen  in  a  vegetable  diet;  we  thus  see  how  by  the 
action  of  these  organisms,  in  assisting  plants  of  this  character 


234      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

to  assimilate  the  nitrogen  of  the  air,  the  nitrogen  cycle  is 
completed.  The  plants,  or  their  seeds,  furnish  food  for 
animals  and  men,  which  nitrogenous  food,  as  we  have  seen,  is 
broken  down,  first  by  the  digestive  processes  of  the  body,  and 
afterwards  by  micro-organisms,  producing  first  ammonia,  and 
finally  nitrates,  to  serve  once  more  as  food  for  plants.  It 
must  be  remembered,  besides,  that  apart  from  the  leguminous 
plants  used  in  this  way  for  food,  a  large  proportion  of  the  total 
growth  of  the  plants  of  this  order  must  suffer  decay,  and  their 
nitrogen  be  returned  directly  to  the  soil.  Indeed  this  method 
of  returning  nitrogen  to  the  soil  constitutes  one  of  the  ordinary 
processes  of  agriculture,  and  is  part  of  what  is  known  as  the 
rotation  of  crops.  After  a  crop  has  been  grown,  such  as 
wheat,  which  tends  to  exhaust  the  soil  of  its  nitrogen,  it  is 
customary  to  grow  a  crop  of  clover,  which  is  afterwards 
ploughed  into  the  soil.  The  clover  in  its  growth  absorbs 
large  quantities  of  nitrogen  from  the  air  ;  when  it  is  ploughed 
into  the  soil  it  rots,  and  once  more,  through  the  changes 
which  have  been  described,  this  nitrogen  is  converted  into 
nitrate,  which  will  again  serve  as  food  for  wheat. 

The  series  of  changes  which  has  been  discussed  in  the 
foregoing  chapters  may  be  usefully  summarised  hi  the 
following  diagram,  which  is  self-explanatory.  The  application 
of  the  knowledge,  summarised  in  this  diagram,  to  the  practical 
problems  of  agriculture  and  sewage  disposal  will  be  more 
fully  discussed  in  Chapters  XVI  and  XVII. 


THE    NITROGEN    CYCLE 


235 


Nitrogen  Fixing 
Bacteria 


The  Nitrogen  Cycle 
— >  Plants 


Nitrates, 
Nitrites, 
Ammonia, 


Free  Nitrogen 


Bacteria 


(Proteolytic 
Nitrifying  and 
De-nitrifying) 


Proteins,  fats, 
Carbohydrates,  etc. 


Animals 


Urea,  Proteins 
Amino-acids,  etc. 


CHAPTEE  XIV 

THE  SULPHUR  CYCLE 
EVERYONE  who  has  been  confronted  with  a  bad  egg  is  aware 

oo 

of  the  unpleasant  character  of  the  final  decomposition  products 
of  albumin.  The  product  most  easily  recognised  chemically 
is  sulphuretted  hydrogen  or  hydrogen  sulphide,  H2S,  whose 
presence  is  easily  demonstrated  by  holding  a  paper  soaked  in 
a  solution  of  lead  acetate  in  its  vicinity.  The  smell  of  a  rotten 
egg  is  mainly  due  to  this  gas.  Hydrogen  sulphide  is  there- 
fore often  described  as  having  a  smell  like  rotten  eggs. 

It  has  been  shown  in  Chapter  XII  that  most  varieties  of  albu- 
min contain  sulphur  in  greater  or  less  proportion,  and  they  are 
capable,  like  egg-albumin,  when  undergoing  putrefaction,  of 
liberating  this  sulphur  as  hydrogen  sulphide.  It  is  easily  seen, 
therefore,  that  decomposing  albuminous  matter  is  capable  of 
causing  considerable  nuisance  from  this  source. 

Sulphur  appears  to  be  an  essential  constituent  of  both 
animal  and  vegetable  life,  and  a  knowledge  of  its  transforma- 
tions as  it  passes  from  one  to  the  other  is  of  the  greatest  im- 
portance, especially  in  view  of  the  possibility  of  nuisance  being 
produced  during  the  process. 

The  transformations  which  sulphur  compounds  undergo 
bear  a  rough  analogy  to  the  transformations  of  nitrogen  con- 
sidered in  Chapter  XIII.  Just  as  the  plant  takes  up  nitrate 
to  furnish  the  nitrogen  for  vegetable  albumin,  which  nitrogen 
ultimately  reappears,  after  passage  through  the  animal 
organism,  as  urea  and  ammonia,  to  be  finally  again  oxidised 


THE    SULPHUR   CYCLE  237 

to  nitrates,  so  the  sulphur  supplied  to  the  plant  as  sulphates 
becomes  part  of  vegetable  and  animal  albumins,  which  again 
break  down,  yielding  hydrogen  sulphide,  and  the  latter  is 
oxidised,  either  chemically  or  biologically,  back  to  sulphate. 

Moreover,  just  as  nitrates  are  capable  of  reduction  to  form 
nitrites,  and  finally  ammonia,  so  sulphates  are  capable  of 
reduction  to  hydrogen  sulphide. 

In  the  sulphur  cycle  purely  chemical  reactions  play  a 
greater  part  than  is  apparently  the  case  with  nitrogen,  but  in 
all  cases  the  sulphur  transformations  are  capable  of  being 
facilitated  by  the  activities  of  various  organisms. 

The  chief  workers  on  this  important  question  of  the  natural 
sequence  of  combinations  entered  into  by  sulphur,  have  been 
Winogradski  in  Russia,  who  has  investigated  the  conditions 
under  which  sulphur  is  oxidised  by  certain  specific  sulphur 
organisms  ;  Beyerinck  and  van  Delden  in  Holland,  who  have 
studied  particularly  the  reduction  of  sulphates  ;  and  Letts  in 
Belfast,  who,  while  repeating  Beyerinck  and  van  Delden's 
experiments,  has,  in  conjunction  with  several  of  his  students, 
made  important  original  observations  upon  the  conditions 
under  which  sulphuretted  hydrogen  is  evolved,  in  the  actual 
circumstances  of  certain  estuaries. 

It  will  perhaps  be  simplest  to  consider  the  subject  under 
two  heads  : — 

I.  The  production  of  hydrogen  sulphide. 
II.  The  oxidation  of  hydrogen  sulphide. 

I.  Hydrogen  sulphide  can  arise  under  natural  conditions 
from  the  following  sources  : — 

(a)  The  decomposition  of  albumin,  as  already  stated  ; 

(6)  The  reduction  of  sulphates. 

Both    these    changes    are    due   to    the    action    of 
various  organisms. 


238      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

Letts  and  McKay  have  also  shown  that  carbon  dioxide, 
itself  produced  by  the  decomposition  of  organic  matter,  can 
decompose  sulphides,  such  as  ferrous  sulphide,  FeS,  yielding 
H2S.  Such  sulphides  can  also  be  decomposed  by  fatty  acids 
produced  by  other  fermentations.  The  two  sources,  a  and  b, 
of  sulphuretted  hydrogen  may  now  be  separately  considered  : — 
(a)  The  decomposition  of  albumin. — The  formation  of 
hydrogen  sulphide  by  the  decomposition  of  albumin,  through 
the  action  of  bacteria,  can  be  readily  demonstrated.  If  a  few 
drops  of  lead  acetate  solution  are  added  to  a  small  bottle  full 
of  sewage,  the  bottle  closed,  and  placed  in  an  incubator  for  a 
day  or  two,  the  solution  turns  black  from  the  presence  of  lead 
sulphide.  The  actual  organisms  capable  of  decomposing 
albumin,  with  formation  of  hydrogen  sulphide,  can  be  recog- 
nised by  an  elegant  method  suggested  by  Beyerinck  :— 

To  ordinary  nutrient  gelatine,  sufficient  white  lead  is  added 
to  obtain  a  perfectly  white  plate  ;  when  the  medium  is  poured 
into  the  Petri  dish,  a  little  sewage  diluted  with  distilled  water 
is  poured  over  the  plate.  After  it  is  set,  and  as  the  colonies 
develop,  black  dots  of  lead  sulphide  will  indicate  the  presence  of 
these  organisms,  which  are  capable  of  breaking  down  albumin 
with  production  of  hydrogen  sulphide. 

A  very  serious  case  of  nuisance  has  for  a  long  time  existed 
on  the  shores  of  Belfast  Lough.  Here  great  quantities  of  a 
seaweed,  Ulva  latissima,  flourish.  Professor  Letts  has  shown 
that  this  seaweed  contains  an  abnormally  high  albumin  con- 
tent ;  when  deprived  of  its  natural  conditions  of  growth,  the 
Ulva  is  capable  of  fermentation,  apparently  in  two  distinct 
and  successive  stages.  The  first  stage  results  in  the  produc- 
tion of  fatty  acids,  mainly  propionic,  together  with  carbon 
dioxide  and  hydrogen ;  in  the  second  stage  of  fermentation, 
in  which  a  different  species  of  micro-organism  is  concerned, 
sulphuretted  hydrogen  is  produced. 

It  is  not  at  present  certain  what  are  the  exact  sources,  in 
the  first  place  of  the  fatty  acids,  and  in  the  second  place  of  the 


THE    SULPHUR    CYCLE  239 

sulphuretted  hydrogen.  They  may  both  be  due  to  decom- 
position of  the  albumin  of  the  weed,  or  on  the  other  hand 
sulphides  may  be  produced  by  reduction  of  the  sulphates  in 
the  sea  water,  or  in  the  tissues  of  the  Ulva,  and  these  sulphides 
are  then  decomposed  by  the  fatty  acids  produced  in  the  first 
fermentation. 

The  evidence  points  to  the  hydrogen  sulphide  being  derived 
from  the  reduction  of  sulphates,  rather  than  from  the  decom- 
position of  the  albumin,  inasmuch  as  when  comparative  tests 
were  made,  by  fermenting  the  Ulva  hi  sea  water,  and  tap 
water,  respectively,  sulphuretted  hydrogen  was  much  more 
readily  evolved  from  the  sea  water  experiment  than  from  the 
tap  water.  The  reduction  of  sulphates  is  clearly,  then,  a  very 
important  source  of  hydrogen  sulphide. 

Before  considering  this  process  in  detail,  however,  it  should 
be  stated  that  the  objectionable  odour  evolved,  when  organic 
matter  is  allowed  to  putrefy,  is  not  solely  due  to  hydrogen 
sulphide.  Under  certain  conditions,  very  evil-smelling  gases 
are  evolved  in  which  no  trace  of  hydrogen  sulphide  can  be 
discovered.  These  are  probably  organic  sulphur  compounds, 
such  as  inercaptan  (C2H5SH),  also  amines,  and  substances 
such  as  skatol,  etc.,  which  are  also  products  of  albumin 
decomposition.  It  has  been  further  found  that  the  yield  of 
sulphuretted  hydrogen  can  be  increased  in  many  cases  if  a 
small  quantity  of  flowers  of  sulphur  is  added  to  the  fermenting 
mixture. 

(6)  Sulphate  reduction. — As  already  stated,  this  change 
has  been  studied  by  Beyerinck  and  van  Delden.  Beyerinck 
inoculated  suitable  solutions  containing  sulphates  with  small 
quantities  of  mud  from  the  canals  of  Delft,  and  found  that 
the  best  conditions  for  sulphate  reduction  were  as  follows  : — 

(1)  No  oxygen  must  be  present. 

(2)  No  acid  formation  must  take  place,  and  consequently 
little  or  no  sugar  should  be  present  in  the  culture  media. 

(3)  Phosphates  and  other  suitable  solids  must  be  present. 


240      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

(4)  Nitrogen  compounds  are  only  required  in  very  small 
quantities  ;  sufficient  indeed  is  contained  in  ordinary  tap  water. 

(5)  The  most  favourable  temperature  for  sulphate  re- 
duction is  about  25°  C.     Beyerinck  succeeded  in  isolating  an 
organism  which  he  termed  Spirillum  desulphuricans ;    it  is 
a  strictly  anaerobic  organism,  and  this  circumstance,  in  con- 
junction  with   its   small  need   for  nitrogenous   nutriment, 
enables  it  best  to  grow  in  solutions  which  have  been  worked 
over  by  other  organisms.    These  facts  are  of  not  a  little 
practical  interest.    Those  who  have  had  to  deal  with  samples 
of  sewage  and  effluents  will  have  noticed  that  such  samples, 
if  kept  in  stoppered  bottles,  may  become  in  time  practically 
clear,  having  only  a  small  black  sediment  at  the  bottom ; 
but  if  they  have  been  tightly  stoppered,  they  may  also  retain 
considerable    quantities   of   hydrogen    sulphide.     If   this   is 
removed  by  boiling,  very  little  residual  organic  matter  will  be 
found  to  be  present. 

Stagnant  polluted  waters,  e.g.,  the  Manchester  Ship  Canal, 
show  the  same  phenomenon.  It  is  evident,  in  both  these 
cases,  that  the  nitrogenous  organic  matter  is  broken  down  by 
ordinary  putrefactive  organisms,  and  that  final  sulphate  reduc- 
tion takes  place.  In  such  cases  sulphides,  or  hydrogen  sulphide, 
will  be  found  to  constitute  almost  all  the  oxidisable  matter  left. 

To  demonstrate  the  reduction  of  sulphates,  the  following 
solution  was  made  use  of  by  van  Delden  : — 

Tap  water 1000      grams 

Common  salt             . .         . .         . .  30          „ 

Sodium  lactate          . .         . .         . .  10          „ 

Crystallised  magnesium  sulphate   . .  8          „ 

Potassium  phosphate           . .         . .  0*5  gram 

Asparagin        . .         . .         . .         . .  0*5       „ 

This  solution  may  be  inoculated  with  a  little  sewage 
sludge,  from  which  sulphate-reducing  organisms  are  seldom, 
if  ever,  absent. 


THE    SULPHUR   CYCLE  241 

Van  Delden  isolated  an  organism  causing  the  reduction 
of  sulphates  in  sea  water,  and  found  that  it  closely  resembled 
Spirillum  desulphuricans ;  he  named  it  Microspira  estuarii. 
Both  these  organisms,  although  as  above  stated  they  do  not 
need  large  quantities  of  nitrogen,  are  not  inhibited  in  their 
growth  by  organic  matter,  if  they  are  present  in  pure  culture. 
Under  natural  conditions  the  presence  of  organic  matter 
facilitates  the  growth  of  other  organisms,  to  the  detriment  of 
the  sulphate-reducing  spirillae. 

The  reduction  of  sulphates  is  of  special  importance  in 
relation  to  the  discharge  of  sewage  into  sea  water.  There  is 
no  doubt  that,  in  absence  of  sufficient  dilution,  putrefaction 
may  set  in,  resulting,  in  the  case  of  sea  water,  in  sulphuretted 
hydrogen  production ;  so  that  the  nuisance  may  be  much 
greater  in  the  case  of  discharges  into  sea  water  than  into 
fresh  water. 

Sulphate  reduction  has  been  compared  to  de-nitrification ; 
it  will  be  remembered  that  in  the  case  of  the  reduction  of 
nitrates  the  oxygen  of  the  nitrate  with  the  assistance  of  the 
de-nitrifying  organism  combined  with  the  organic  matter 
present.  A  similar  reaction  appears  to  take  place  in  the  case 
of  sulphate  reduction ;  thus  in  the  above  described  solution, 
where  the  chief  source  at  any  rate  of  oxidisable  material  is 
sodium  lactate,  van  Delden  suggests  the  following  equation  : — 
2C3H503Na+3MgS04 

=  3MgC03  +  Na2C03  +  2C02  +  2H20  +  3H2S 

Experimental  evidence  supports  the  above  equation  fairly 
well. 

II.  Oxidation  of  Sulphur. — Unlike  ammonia,  whose  direct 
oxidation  by  purely  chemical  means  has  been  shown  to  take 
place  to  only  a  limited  extent  in  nature,  hydrogen  sulphide 
readily  oxidises  in  a  variety  of  ways.  The  simplest  is  the 
direct  oxidation  to  water  and  sulphur  according  to  the  simple 
equation : — 

H2S+  0  =  H20  +  S 


242      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

This  change  is  hastened  by  the  presence  of  certain  metallic 
oxides,  particularly  those  of  iron  and  manganese ;  thus  in 
presence  of  oxide  of  iron  the  following  changes  may  take 
place  :— 

3H2S  +  Fe203  =  2FeS  +  3H20  +  S 

In  presence  of  oxygen  and  moisture  FeS  may  readily 
oxidise  to  ferrous  sulphate,  FeS04,  thus  : — 

FeS  +  202  =  FeS04 

And  this  may  further  oxidise  with  formation  of  ferric 
sulphate,  thus  : — 

(FeSOJ3  +  0  +  H20  =  Fe2(S04)3  +  Fe(OH)2 

It  is  quite  possible  that  pyrites,  especially  when  found  in 
coal,  may  owe  its  origin  to  the  interaction  of  oxide  of  iron  and 
the  sulphides  produced  by  the  decay  of  vegetable  matter. 
When  such  '  coal  brasses/  as  this  form  of  pyrites  is  termed, 
is  exposed  to  the  air,  it  oxidises  with  formation  of  ferrous 
sulphate,  or  eventually,  it  may  be,  of  ferric  sulphate. 

When  black  sewage  mud  is  exposed  to  the  air  it  turns 
brown  and  becomes  acid,  owing  to  the  formation  of  hydrated 
oxide  of  iron  and  sulphuric  acid. 

How  far  hydrogen  sulphide  and  sulphides  are  capable  of 
being  directly  oxidised  by  solutions  of  nitrates  does  not 
appear  to  have  been  sufficiently  studied.  There  is  no  doubt 
that  nitrates  are  rapidly  reduced  in  presence  of  sulphide 
mud ;  how  far  this  is  a  purely  chemical  change  and,  if  so, 
what  is  the  exact  cause  of  the  change,  has  not  been  fully 
determined. 

Beyerinck  claims  to  have  isolated  an  organism,  B.  thioparus, 
which  brings  about  the  following  decomposition  : — 

5S  +  6KN03  +  2H20  =  K2S04  +  4KHS04  +  3N2 
The  most  frequently  occurring  and  obvious  case  of  oxida- 


THE    SULPHUR   CYCLE  243 

tion  of  hydrogen  sulphide  by  bacterial  agency  is  that  brought 
about  by  the  higher  bacteria,  classified  under  the  general  term 
of  Beggiatoa  (Fig.  3  (IA)).  These  are  the  organisms  which 
form  the  subject  of  Winogradski's  researches  above  referred 
to.  They  are  found  very  often  in  sulphur  springs  and 
wherever  putrefying  sewage  or  suchlike  organic  matter  comes 
in  contact  with  air,  as,  e.g.,  on  the  stones  of  a  stream  in  the 
neighbourhood  of  a  badly  polluting  discharge.  The  organism, 
as  a  matter  of  fact,  grows  between  wind  and  water,  but 
makes  use  of  the  sulphur  either  by  decomposition  of  the  H2S 
present,  or  by  actual  absorption  of  the  free  sulphur  formed 
by  its  spontaneous  oxidation.  If  a  strand  of  Beggiatoa  is 
examined  under  a  high-power  microscope,  very  characteristic 
granules  of  sulphur  are  seen  to  be  present  throughout  the 
organism,  as  is  shown  in  Fig.  3  (IA)  (Chapter  II).  This 
sulphur  is  the  amorphous  form  soluble  in  carbon  bisulphide. 
Beggiatoa  is  capable  of  absorbing  large  quantities  of  sulphur 
which  it  oxidises  to  sulphates  ;  for  this  purpose  it  is  necessary 
that  carbonates  should  be  present  in  the  surrounding  liquid. 
Under  its  natural  conditions  of  growth  this  will  inevitably  be 
the  case,  ammonium  carbonate,  e.g.,  being  always  present  in 
decomposing  sewage.  Beggiatoa  appears  to  use  the  sulphur 
as  a  source  of  energy  rather  than  to  increase  its  cell  substance. 
Winogradski  found  that  it  could  use  up  from  two  to  four 
times  its  weight  of  sulphur  without  increasing  in  growth. 
Under  these  circumstances,  comparatively  small  amounts 
of  organic  matter  will  suffice  to  sustain  it,  and  thus  it  can 
flourish  in  sulphur  springs,  whose  chief  constituents,  apart 
from  hydrogen  sulphide,  are  mineral  salts. 

To  summarise  the  contents  of  the  foregoing  chapter,  we 
may  conclude  that  sulphur  enters  the  cycle  of  living  nature  as 
mineral  sulphates  in  the  food  of  plants.  By  the  decom- 
position of  vegetable  albumin,  or  at  a  further  stage  from  the 
excretory  products  of  animals,  it  may  reappear  as  hydrogen 

B2 


244      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

sulphide  (sulphuretted  hydrogen).  This  may  be  re-oxidised 
to  sulphates,  either  directly  by  chemical  means,  e.g.,  oxides  of 
iron,  etc.,  or  by  the  intervention  of  bacteria.  Certain  of 
these  oxidise  it  directly  to  sulphate,  while  others  make  use 
of  the  presence  of  nitrates. 

Sulphates  are  capable  of  being  directly  reduced  to  hydrogen 
sulphide  by  certain  bacteria,  in  presence  of  small  quantities 
of  organic  matter,  but  such  changes  only  take  place  in  absence 
of  air.  These  various  changes  clearly  indicate  the  importance 
of  abundant  supplies  of  oxygen,  if  the  evolution  of  hydrogen 
sulphide,  and  the  other  less  well-defined  objectionable  gases 
which  accompany  it,  are  to  be  avoided.  The  bearing  of  this 
principle  on  the  purification  and  disposal  of  sewage  and 
other  waste  organic  matter,  will  be  further  referred  to  in 
Chapter  XVII. 


CHAPTEK  XV 

FERMENTATION  OF  INDIGO,  TEA,  COCOA,  COFFEE, 
AND  TOBACCO 

Indigo. — The  important  series  of  researches  carried  on 
during  recent  years  on  behalf  of  the  Government  of  India  on 
the  chemistry  of  natural  indigo,  and  of  the  native  processes 
of  manufacture,  is  of  especial  interest  to  the  student  of 
enzyme  chemistry. 

As  with  most  native  industries,  a  considerable  amount  of 
empirical  knowledge  and  skill  has  been  attained  in  the  manu- 
facture of  indigo,  through  centuries  of  experience,  and  the 
improvements  to  be  effected  do  not  usually  lie  on  the  surface, 
although  at  first  sight  they  may  appear  to  do  so.  Scientific 
research  of  a  high  order  is  requisite,  together  with  special 
knowledge  of  local  conditions,  if  a  real  gain  in  efficiency  is 
to  be  achieved. 

It  is  partly  for  this  reason  that  native  methods  of  manu- 
facturing indigo  have  been  practically  stationary  for  many 
years.  The  author  has  recently  seen  indigo  vats  (Plate  III  (i) ) 
near  Mirzapur,  U.P.,  whose  construction  and  methods  of  use 
do  not  greatly  differ  from  the  graphic  description  to  be  found 
in  a  volume,  '  Kural  Life  in  Bengal,'  published  in  1860. 

The  native  method  for  extracting  indigo  from  the  indigo 
plant  is  briefly  as  follows  : — 

The  plant  is  brought  to  the  factory  immediately  after 
cutting  and  placed  in  bundles  in  an  upper  series  of  stone 
or  concrete  vats.  The  bundles  are  tightly  pressed  down 


246      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

by  means  of  bamboos,  and  heavy  baulks  of  timber  levered 
down  and  fixed  in  position  by  horizontal  pins,  passing  through 
two  uprights  at  each  end  of  the  vat.  The  vats  containing  the 
pressed  bundles  are  filled  up  with  water  and  steeping  is 
continued  over  night,  the  liquor  being  allowed  to  run  off  into 
lower  vats  in  the  morning.  The  liquor  in  the  lower  vats  is 
then  thoroughly  beaten  up  by  men  who  stand  immersed  to 
the  hips  in  the  liquor  and  beat  it  with  bamboos  shaped  like 
oars,  or  artificial  beaters  of  various  kinds  are  used.  The 
object  of  this  process  is  to  bring  the  liquor  thoroughly  in  con- 
tact with  air  when  the  indigo  is  precipitated.  The  progress 
of  the  operation  is  tested  by  the  manager  by  inspection  of 
small  portions  of  the  liquor  from  time  to  time. 

On  completion  of  the  beating  process,  the  indigo  is  allowed 
to  deposit,  the  liquid  run  off  to  waste,  and  the  wet  indigo 
mud  run  on  to  draining  cloths.  When  it  has  attained  a 
suitable  consistency,  portions  are  wrapped  in  cloth  and  pressed 
like  cheeses  in  a  press ;  the  pressed  mass  is  then  cut  into 
cakes  and  finally  dried. 

The  impetus  towards  improvement  of  this  process  has 
been  due  to  the  acute  competition  during  recent  years  of 
artificial  indigo,  the  extent  of  which  may  be  gathered  from 
the  fact  that  in  1896,  out  of  a  total  weight  of  46,683  cwts.  of 
indigo  imported  into  Great  Britain,  only  7,641  cwts.  consisted 
of  the  natural  product. 

In  1902  Mr.  W.  Popple  well  Bloxam  and  his  colleagues 
began  their  researches  for  the  Government  of  Bengal.  The 
work  was  carried  on  in  India  for  two  years  and  was  afterwards 
continued  from  1905  to  1907  in  the  University  of  Leeds,  under 
the  general  supervision  of  Mr.  A.  G.  Perkin,  F.E.S. 

A  report  of  this  work  was  published  in  1908  by  the  Govern- 
ment of  India,  and  the  following  information  is  mainly  taken 
from  its  pages. 

In  the  first  place  it  should  be  explained  that  the  pure 
colouring  matter  of  indigo  is  indigotin,  which  has  the  molecular 


INDIGO  247 

formula  C16H10N202.  Careful  study  of  its  related  products 
leads  to  the  following  structural  formula  for  indigotin  which 
was  first  prepared  artificially  by  von  Baeyer  in  1878  :  — 


xoc\      xco\ 

6  4 


It  is  to  von  Baeyer  and  his  pupils  that  we  owe  the  know- 
ledge of  the  structure  of  indigo,  which  has  rendered  possible 
its  commercial  production  on  the  large  scale  from  raw 
material,  such  as  naphthalene,  found  in  coal-tar. 

Indigo  does  not  exist  as  such  in  the  indigo  plant.  Schunck 
in  1855  showed  that  the  plant  contained  a  glucoside  which  he 
termed  indican.  Schunck  regarded  this  as  a  compound  of 
indigo  with  sugar.  Kecent  investigations  of  Hoogewerff  and 
Termeulen,  which  have  been  confirmed  and  extended  by 
Perkin  and  Bloxam,  have  shown  that  indican  is  a  glucoside, 
not  of  indigo  itself,  but  of  a  substance  which  was  originally 
discovered  by  Von  Baeyer,  known  as  indoxyl  (see  note 
p.  255).  which  yields  indigo  in  contact  with  oxygen. 

In  the  steeping  process  described  above  the  indican  is 
fermented,  yielding  indoxyl  and  glucose  ;  in  the  subsequent 
beating  operation  the  indoxyl  is  oxidised.  The  equations 
representing  these  changes,  supposing  them  to  be  complete, 
are  as  follows  :  — 

C14H1?06N  +  H20  =  QgHjON  +  C^O. 

Indican  Indoxyl  Dextrose 

H2 

Indoxyl 


-  CH  /COxr-r//co 

t6H4 


Indigo 

Perkin  and  Bloxam's  researches  were  concerned  with  the 
exact  study  of  these  chemical  changes.  Before  this  was 
possible,  accurate  methods  of  analysis  had  to  be  devised  in 


248      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

order  to  determine  the  amount  of  indigotin  in  the  cake  indigo, 
and  of  indican  in  the  original  plant,  older  processes  all  giving 
conflicting  results.  The  methods  finally  devised  were  briefly 
as  follows  : — 

For  the  determination  of  indigo,  one  gram  of  indigo  was 
converted  into  a  tetrasulphonate  by  means  of  fuming  sulphuric 
acid,  and  the  tetrasulphonate  precipitated  as  potassium  salt  by 
addition  of  potassium  acetate.  The  precipitated  salt  can  be 
filtered  off,  dissolved  in  water  and  oxidised  by  potassium 
permanganate  of  known  strength. 

The  determination  of  the  indican  in  the  leaf  depends  on 
the  fact  that,  when  brought  into  contact  with  a  substance 
known  as  isatin,  a  pure  crystalline  compound  known  as 
indirubin  is  formed,  by  the  combination  of  indoxyl  and  isatin, 
according  to  the  following  equation  : — 

C8H7ON  +  C8H502N  =  C16H1002N2  +  H20 

Indoxyl  Isatin  Indirubin 

It  was  found  that  the  best  method  of  extracting  the 
indican  from  the  leaf  was  by  means  of  acetone. 

Armed  with  these  exact  methods  of  analysis,  Perkin  and 
Bloxam  have  been  able  to  show  that  the  yield  of  indigo 
obtained  in  the  native  process  by  no  means  corresponds 
with  the  theoretical  yield  which  should  be  obtained  on  the 
basis  of  the  indican  present  in  the  leaf.  Several  by-products 
are  present  in  natural  indigo,  particularly  indigo  brown,  the  in- 
vestigation of  which  indicates  that  it  is  formed  by  a  secondary 
reaction  from  indican.  Their  examination  of  a  specimen  of 
leaf  from  an  indigo-yielding  plant  from  Sumatra  has  shown 
that,  under  certain  conditions,  twice  as  much  indigo  may 
be  present,  as  in  the  best  leaf  from  Java. 

The  decomposition  of  indican  they  agreed  to  be  due  to 
the  action  of  an  enzyme  present  in  the  leaves,  rather  than 
to  the  activity  of  bacteria.  In  this  they  confirm  the  opinion 
of  other  investigators,  notably  Beyerinck,  Bergtheil,  and 
Rawson.  They  agree  with  Beyerinck  that  the  enzyme  is 


INDIGO  249 

insoluble  in  water.  The  exact  character  of  this  enzyme,  and 
especially  its  conditions  of  formation  in  the  plant,  afford 
material  for  further  study.  According  to  Beyerinck  it  is  not 
an  oxidase,  nor  has  he  been  able  to  find  this  class  of  enzyme 
in  the  indigo  plant. 

As  a  result  of  all  these  researches  improvement  in  the 
present  method  of  native  indigo  production  is  to  be  sought 
along  the  following  lines  of  investigation  : — 

1.  New  plants  such  as  the  Indigofera  sumatrana,  giving  a 
greater  yield  of  indigo,  may  be  introduced. 

2.  The  study  of  seasonal  variation  in  the  percentage  of 
indican  in  the  plant  may  result  in  an  increased  yield. 

3.  The  effects  of  manuring  may  be  further  studied,  with 
special  attention,  it  may  be,  to  the  organisms  in  the  soil.    The 
indigo  plant  is  leguminous,  and  possesses  root  nodules,  which 
also  call  for  investigation. 

4.  The  accurate  control  of  the  beating  or  blowing  operation. 
In  view  of  the  researches  of  Brown  and  Morris,  it  might 

even  be  suggested  that  the  time  of  day  at  which  the  plant 
was  gathered  would  condition,  to  some  extent,  the  pro- 
portion of  indican  present  in  the  leaves  ;  and  the  suggestion 
made  by  several  workers  that  the  leaves  rather  than  the 
whole  plant  should  be  plucked  would  seem  to  be  worth 
attention. 

It  must  always  be  remembered,  when  comparing  what  are 
generally  called  natural  processes  with  artificial  methods,  that 
prima  facie  the  advantage  lies  with  the  natural  method,  which 
depends  on  the  inexhaustible  energy  of  the  sun's  rays  ;  when 
this  advantage  is  coupled  with  cheap  labour  and  scientific 
control,  such  a  native  process  should  be  able  to  stand  con- 
siderable competition.  It  is  the  scientific  control  which  up  to 
recent  years  has  been  lacking,  and  it  may  be  hoped  that,  for 
social  and  economic  reasons,  these  researches  will  be  successful 
in  maintaining  an  industry  which  gives  healthy  and  satis- 
factory employment  to  a  large  number  of  people. 


250      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

Tea. — Tea  is  produced  in  two  forms  for  the  market,  viz., 
green  tea  and  black  tea.  In  the  manufacture  of  green  tea  the 
object  is  to  maintain  the  colour  and  to  prevent  fermentation  ; 
the  leaf  is  therefore  roasted  immediately  after  picking  and 
the  whole  process  of  manufacture  conducted  as  quickly  as 
possible.  In  the  case  of  black  tea  the  leaves  are  dried  slowly, 
and  in  the  course  of  the  process  fermentation  takes  place. 
This  fermentation  is  a  special  feature  in  the  formation  of 
Indian  black  tea,  and  has  been  the  subject  of  very  interesting 
researches  by  Dr.  H.  H.  Mann,  to  whom  the  author  is  indebted 
for  the  special  information  of  this  section. 

The  following  processes  are  involved  in  the  manufacture  of 
Indian  black  tea  : — 

1.  Withering. — Withering  of  the   leaf,  which  consists   in 
exposure  to  the  sun  on  fine  basket-work  trays. 

2.  Rotting. — Rolling  by  machine,  which  has  the  effect  of 
pressing  out  a  certain  amount  of  the  juice  of  the  leaves.     The 
soft  leaves  are  often  made  into  balls  which  are  used  to  absorb 
the  juice. 

3.  Fermentation. — These  balls  are  broken  up  and  allowed 
to  ferment  and  then  spread  out  to  dry  in  the  sun. 

4.  Firing. — This  takes  place  in  a  chest  of  shallow  firing 
drawers,  the  bottoms  of  which  are  made  of  fine  wire  gauze. 

5.  Sorting. — In  this  process  various  qualities  of  leaf  are 
sorted  by  sieving,  etc. 

Dr.  Mann's  researches  have  been  concerned  primarily  with 
the  changes  going  on  during  the  withering  and  fermentation 
processes,  and  the  relation  of  these  to  the  quality  of  the  tea. 

The  quality  of  tea  appears  to  depend  on  the  following 
factors : — 

(a)  The  flavour,  caused  principally  by  an  essential  oil. 

(b)  Pungency,  caused  in  greatest  measure  by  the  unfer- 
mented  tannin. 

(c)  Colour   of    liquor,   caused  chiefly  by   the   fermented 
tannin. 


TEA  251 

(d)  Body  of  liquor,  measured  principally  by  the  total 
soluble  matter,  of  which  a  large  part  is  tannin  both  fermented 
and  unfermented. 

It  was  found  that  the  fermentation  is  the  result  of  enzyme 
action  ;  the  presence  of  bacteria  during  the  fermentation  pro- 
cess is  distinctly  injurious,  rendering  the  tea  sour  and  unfit 
for  consumption.  In  order  to  prevent  deleterious  changes  of 
this  sort,  it  is  necessary  that  the  fermentation  should  be  carried 
on  under  aseptic  conditions,  that  is,  scrupulous  cleanliness 
must  be  maintained  throughout  the  process.  The  use  of  anti- 
septics is  injurious  to  the  enzyme  as  well  as  to  the  micro- 
organisms. If  the  temperature  also  is  kept  at  about  80°  F. 
the  change  is  found  to  be  mainly  enzymic.  The  chemical 
change  which  takes  place  during  fermentation  consists  essen- 
tially in  an  oxidation  of  the  tannin.  It  has  been  found  indeed 
that  there  are  two  enzymes  present;  one  of  these  colours 
guiachum  resin  blue  at  once,  the  other  does  so  only  in  presence 
of  hydrogen  peroxide.  The  main  ferment  is  an  oxidase,  causing 
the  darkening  of  tea  juice  and  also  of  pyrogallol  and  hydro- 
quinone.  It  has  been  found  that  the  flavour  improves  in  pro- 
portion to  the  amount  of  enzyme  in  the  leaf.  It  would  appear 
that  in  the  tea  leaf  the  tannin  is  combined  with  sugar  ;  during 
fermentation  this  compound  is  split  up  and  the  tannin  is 
oxidised  to  brown  products.  This  oxidised  tannin  combines 
with  other  substances  in  the  leaf-forming  compounds,  some  of 
which  are  insoluble  in  water  ;  there  is,  therefore,  a  decrease  in 
soluble  tannin.  It  is  possible  for  this  to  go  too  far  and  the 
pungency  of  the  tea  to  be  injuriously  affected. 

The  enzyme  increases  during  the  withering  of  the  leaf,  and 
one  of  the  most  important  results  of  Dr.  Mann's  investigations 
is  the  possibility  of  the  exact  control  of  the  withering  process. 
The  object  of  withering  is  twofold — to  soften  the  leaf  in  pre- 
paration for  rolling,  and  to  produce  the  greatest  amount  of 
enzyme.  Under  normal  conditions  these  two  changes  are 
practically  simultaneous,  but  in  very  dry  weather  the  leaf 


252      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 


may  be  physically  ready  to  roll  before  sufficient  enzyme  is 
developed ;  and  on  the  other  hand,  in  very  wet  weather,  the 
leaf  may  be  chemically  ready  for  rolling  before  it  is  properly 
withered.  It  may  be  possible,  therefore,  to  control  the  time 
of  withering,  either  retarding  it  by  heaping  up  the  leaves  or 
quickening  it,  e.g.,  by  means  of  fans,  and  so  obtaining  the 
necessary  conditions  for  the  production  of  the  best  tea. 

It  is  of  further  interest  that  the  amount  of  enzyme  in  the 
leaf  has  been  shown  to  depend  on  the  percentage  of  phosphoric 
acid  used  in  manuring  the  plants ; 
further,  much  more  enzyme  is  present 
in  leaves  plucked  at  6.30  A.M.  than'  at 
6  P.M.,  which  supports  the  suggestion 
made  with  regard  to  the  indigo  plant 
in  the  preceding  section. 

The     Fermentation    of    Cocoa. — 

Cocoa,  as  known  to  the  consumer,  is 
obtained  by  grinding  and  roasting  the 
seeds  or  beans  of  the  cocoa  fruit ;  the 
appearance  and  structure  of  the  latter 
can  be  understood  by  reference  to  Fig. 
25.  In  order  to  obtain  the  beans  free 
from  surrounding  pulp,  a  process  of 

FIG.  25,-CocoA  FKUIT  fermentation  is  resorted  to.  The  fresh 
IN  PART  SECTION.  beans,  after  separating  them  from  the 
shell,  are  piled  on  a  floor  or  filled  into 
boxes,  and  allowed  spontaneously  to  ferment.  Plate  III  (ii) 
gives  an  idea  of  the  appearance  of  these  fermenting  boxes. 
A  period  of  two  to  six  days,  according  to  circumstances,  is 
usually  allowed  for  fermentation.  A  rise  of  temperature, 
amounting  to  about  5°  C.,  takes  place  in  twenty-four  hours, 
and  in  the  course  of  four  days  the  fermenting  beans  may  have 
a  temperature  as  much  as  18°  to  20°  C.  above  the  surrounding 
atmosphere. 


PLATE    III. 


[Photo  by  Author. 

(i)  INDIGO  VATS  NEAR  MIRZAPUR,  INDIA. 


(ii)  FERMENTINCJ  BOXES  FOR  COCOA. 


COCOA  253 

The  chief  purposes  of  the  fermentation  process  are  l : — 

1.  To  arrest  the  germinating  power  of  the  seed ; 

2.  To  remove  or  contract  the  pulp  surrounding  the  seed  ; 

3.  To  loosen  the  connection  between  the  seed  and  its  testa  ; 

4.  To  develop  the  colour  of  the  bean  and  to  improve  the 
taste  of  the  cocoa. 

The  separation  of  the  pulp  is  originally  due  to  the  activity 
of  yeasts,  which  develop  in  the  sweet  juice  oozing  from  the 
pulp ;  an  alcoholic  fermentation  takes  place  in  the  inner 
portions  of  the  mass,  which  gives  place  to  an  acetic  fermenta- 
tion in  those  portions  in  contact  with  air.  These  changes 
result  in  an  elevation  of  temperature  and  a  considerable 
discharge  of  acid  juice,  which  is  sometimes  used  as  vinegar. 
At  the  same  time  the  beans  become  loosened  from  their 
surrounding  integument,  from  which  they  can  afterwards  be 
easily  separated  by  washing. 

The  bean  in  its  fresh  state  has  a  violet  colour ;  on  exposure 
to  air  the  violet  colour  changes  to  a  deep  brown.  The  change 
of  colour  from  purple  to  brown  takes  place  to  some  extent 
during  the  fermentation  process,  and  is  completed  in  the 
subsequent  drying.  It  has  been  shown  that  this  change  of 
colour  is  due  to  the  action  of  an  oxidase  in  the  cocoa  bean. 
If  the  bean  is  boiled  or  treated  with  acid,  no  change  of  colour 
can  afterwards  be  produced,  showing,  therefore,  that  it  is  due 
to  the  action  of  an  enzyme.  It  appears  that  both  an  oxidase 
and  a  peroxidase  are  present.  Thus,  if  a  freshly  cut  bean  is 
moistened  with  tincture  of  guaiacum,  a  blue  colour  is  rapidly 
produced,  indicating  the  presence  of  an  oxidase.  If  the  bean 
is  crushed  with  a  little  water  and  heated  for  five  minutes  to 
75°  C.,  no  coloration  is  given  with  guaiacum,  showing  that  the 
oxidase  is  destroyed  at  this  temperature.  On  addition  of 

1  See  The  Fermentation  of  Cacao  awl  of  Coffee,  by  Dr.  Oscar  Loew,  pub- 
lished in  the  Annual  Report  of  the  Porto  Rico  Agricultural  Experiment 
Station  for  1907,  to  which  the  author  acknowledges  his  indebtedness  in 
the  present  and  the  succeeding  sections. 


254      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

hydrogen  peroxide  to  the  unfiltered  juice,  a  blue  colour  is 
obtained,  but  is  not  developed  in  the  juice  after  filtering.  This 
indicates  the  presence  of  an  insoluble  peroxidase. 

The  flavour  of  the  cocoa  appears  to  be  improved  by  the 
fermentation  process,  probably  in  consequence  of  the  partial 
oxidation  of  a  tannin  present  in  the  bean,  but  some  difference 
of  opinion  exists  on  this  point.  The  flavour  is  chiefly 
developed  in  the  subsequent  roasting,  but  the  action  of  the 
oxidases  would  seem,  from  the  colour  produced,  to  be  a 
necessary  preliminary  to  this  process. 

The  Fermentation  of  Coffee.  —  The  coffee  fruit,  whose 
structure  is  illustrated  in  Fig.  26,  is  subjected  to  a  fermentation 
similar  to  the  one  above  described  in  connec- 
tion with  cocoa  ;  chiefly   in    order   to  loosen 
the  seeds  from  their  surrounding  integument. 
The  essential  part  of  this  process  is  a  solution, 
apparently    by  enzyme    action,   of    the    ad- 
hesive  substance  between  the  parchment  en- 
velope   and    the    slimy  layer,  so   that    after 
the    fermented    coffee  is  washed    and    dried, 
FIG.  26.        the  parchment  becomes  brittle  and  is  removed, 
COFFEE  BEAN.  e   silver  skin,  in  the  process 


of  coffee  milling.  This  last  process  is  frequently  done  in 
London,  and  not  in  the  country  where  the  coffee  is  produced. 
The  effect,  if  any,  of  the  fermentation  process  upon  the 
flavour  of  the  coffee  has  not  hitherto  been  fully  investigated. 

Tobacco.  —  The  curing  of  tobacco  is  again  a  fermentation 
process.  The  leaves  after  gathering  are  first  slightly  withered, 
then  '  sweated  '  in  moderate-sized  heaps,  and  finally  fermented 
in  large  heaps  containing  as  much  as  fifty  tons  of  tobacco. 

It  has  been  considered  that  this  fermentation  is  a  bacterial 
process,  and  pure  cultures  have  even  been  introduced  in  order 
to  impart  specific  aromas  to  the  tobacco.  More  recent 


TOBACCO  ZOO 

researches  by  Loew  and  other  chemists  of  the  United.  States 
Board  of  Agriculture,  lead  to  the  conclusion  that  the  changes  are 
essentially  due  to  enzyme  action ;  oxidases  and  peroxidases 
have  been  detected,  and  especially  a  soluble  and  insoluble 
catalase,  an  enzyme  capable  of  decomposing  hydrogen 
peroxide.  The  changes  taking  place  in  the  curing  of  tobacco 
consist,  in  the  first  place,  in  the  elimination  of  starch  and 
sugar,  by  the  continued  respiration  of  the  plant  cells  during 
drying.  The  ethereal  extract  and  the  percentage  of  tannin 
also  decrease.  During  the  fermentation  the  nicotine  also 
decreases,  and  the  colour  and  aroma  improve.  The  effect  of 
the  character  of  the  soil  on  the  quality  of  tobacco  is,  of  course, 
well  known,  and  greater  control  of  the  quality,  in  parts  of  the 
world  which  hitherto  have  not  yielded  the  finest  brands  of 
tobacco,  must  be  sought  in  investigations  similar  to  those 
which  have  been  described  in  connection  with  indigo  and 
with  tea. 

Note  on  the  Extraction  of  Indigo. — That  indoxyl  was  probably  formed 
during  the  steeping  process  appears  to  have  been  first  suggested  by  Surg. 
Lt.-Col.  G.  S.  A.  Ranking,  sometime  Professor  of  Chemistry,  Medical 
College,  Calcutta.  His  studies  (published  in  the  Journal  of  the  Asiatic 
Society  of  Bengal,  VoL  LXV.  Part  II.  No.  1,  1896)  indicated  that  there  are 
two  indigo  browns,  one  more  highly  oxidised  than  the  other  and  also  more 
soluble. 


CHAPTEE  XVI 

BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY    IN 
RELATION  TO   AGRICULTURE 

IT  is  becoming  increasingly  necessary  for  the  scientific  agri- 
culturist to  be  well  acquainted  with  the  chemical  changes 
induced  by  bacteria  and  by  enzymes.  The  economical  use  of 
farmyard  manure  is  better  understood  by  a  knowledge  of  the 
character  of  fermentation  which  it  undergoes,  both  spontane- 
ously and  in  contact  with  the  soil,  before  it  is  fitted  for  the 
food  of  plants.  The  conditions  of  fertility  of  soils,  including 
the  maintenance  of  a  sufficient  proportion  of  nitrogen,  are 
intimately  related  to  the  bacterial  life  of  the  soil. 

For  a  right  understanding  of  the  conditions  of  growth  of 
plants,  careful  study  is  required  of  the  changes  brought  about 
by  enzyme  action  in  the  various  organs  of  the  plant,  particu- 
larly in  the  seed  and  leaves. 

Important  enzyme  changes  also  occur  in  the  preparation 
of  special  fodder  or  silage  for  stock. 

Finally,  for  successful  dairy  work,  especially  the  ability  to 
maintain  a  constant  quality  in  butter  and  cheese,  a  knowledge 
of  bacteriological  chemistry  is  now  almost  essential.  In  the 
following  pages  these  aspects  of  the  subject  will  be  briefly 
dealt  with  in  order. 

Farmyard  Manure. — Stable  manure  is  of  course  a 
complex  mixture  of  substances,  and  the  possible  fermentations 
which  it  may  undergo  are  very  various.  When  the  animals 


BACTERIA    AND    ENZYMES   IN   AGRICULTURE       257 

are  kept  in  the  fields,  manure  is  returned  directly  to  the  ground, 
and  gradually  becomes  broken  down  therein.  It  is  when 
manure  is  collected  from  stables  and  stalls  that  considerable 
loss  may  occur,  if  care  is  not  taken.  Farmyard  manure  consists 
of  dung  and  urine,  mixed  with  straw  or  other  material  used  for 
bedding,  such  as  peat-moss  litter,  etc.  The  dung  will  contain 
the  undigested  portions  of  the  animals'  food,  together  with 
a  certain  amount  of  waste  material  from  the  digestive  organs. 
The  more  valuable  portion  of  the  nitrogenous  output  of  the 
animal  is  in  the  urine.  The  main  fermentations,  therefore, 
that  will  take  place  in  stable  manure  are  : — 

1.  Ammoniacal  fermentation  of  urine  and  of  hippuric  acid ; 

2.  The  breaking  down  of  albumin  derivatives ; 

3.  The  decomposition  of  carbohydrates  and  especially  of 
cellulose. 

All  of  these  have  been  referred  to  in  previous  chapters ;  it 
is  only  necessary  here  to  indicate  their  practical  bearing. 

It  is  clear,  in  the  first  place,  that  every  care  must  be  taken, 
if  the  full  value  of  the  manure  is  to  be  obtained,  that  the  urine 
is  not  allowed  to  run  to  waste ;  for  this  reason  stables  and  yards 
should  be  well  paved  and  the  manure  should  be  kept  on  an 
impervious  floor.  Another  less  obvious  cause  of  the  loss  of 
nitrogen  from  manure,  apart  from  the  actual  running  to  waste 
of  the  liquid  portions,  arises  from  the  volatilisation  of  ammonia, 
owing  to  the  dissociation  of  the  ammonium  carbonate, 
formed  by  ammoniacal  fermentation.  This  loss  is  greatest 
when  the  manure  is  fresh,  as  ammoniacal  fermentation  is 
almost  the  first  to  set  in ;  later  on  acids  are  formed  by  the 
decomposition  of  carbohydrates  and  cellulose,  which  tend  to 
fix  the  ammonia.  One  advantage  of  the  use  of  peat-moss  litter 
is  that  it  has  the  power  of  retaining  ammonia.  It  has  been 
found,  however,  by  the  experiments  of  Deherain  and  others, 
that  if  care  is  taken  to  pile  the  manure  heap  in  such  a  way  as 
to  exclude  air,  the  C02  evolved  by  various  fermentations  pre- 
vents the  dissociation  of  ammonium  carbonate  and  consequent 


258      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

loss  of  ammonia.  Following  the  ammoniacal  fermentation 
will  be  the  decomposition  of  albuminoids,  yielding  ultimately, 
as  has  been  shown,  various  amino  acids.  The  decomposition 
of  carbohydrates,  other  than  cellulose,  which  occur  in  dung, 
e.g.,  starch,  gums  and  possibly  certain  sugars,  will  also  occur 
with  some  rapidity,  yielding  acids  capable  of  uniting  with 
ammonia  and  any  other  bases  present ;  these  various  decom- 
positions take  place  with  considerable  evolution  of  carbon 
dioxide. 

The  fermentation  of  the  cellulose  is  the  longest  delayed,  and 
probably  takes  place  both  anaerobically  and  aerobically, 
according  to  the  conditions  obtaining  in  different  parts  of  the 
manure  heap.  Acids  are  also  produced  here  as  by-products ; 
probably  also  the  valuable  residual  humus  is  a  product  of  the 
fermentation  of  cellulose. 

The  quantity  of  nitrogen  in  the  manure  will  depend,  as 
already  indicated,  on  the  care  taken  to  exclude  air  in  the 
manner  of  forming  the  manure  heap.  If  the  heap  is  well 
pressed  down,  the  conditions  are  mainly  anaerobic,  and  the 
heap  can  be  kept  for  considerable  periods  without  serious 
loss  of  valuable  constituents. 

It  is  sometimes  necessary,  e.g.,  for  market  gardening,  to 
prepare  manure  quickly,  and  large  piles  may  not  then  be 
conveniently  made.  In  such  a  case  a  considerable  quantity 
of  nitrogen  passes  off  in  the  free  state,  apparently  by  direct 
oxidation  of  nitrogenous  matter. 

Well-rotted  manure  will  contain  all  the  materials  for  plant 
food,  and  the  time  which  has  elapsed  in  its  preparation  will 
be  saved  by  the  greater  availability  of  its  constituents  when 
it  is  placed  on  the  ground.  According  to  Warington  one  ton 
of  farmyard  manure  supplies  9  to  15  Ibs.  of  nitrogen,  a 
similar  amount  of  potash,  and  4  to  9  Ibs.  of  phosphoric 
acid.  It  is  thus,  of  course,  an  attenuated  manure,  and 
further  changes  have  to  take  place  after  it  is  incorporated 
with  the  soil,  before  the  plant  can  make  full  use  of  it ;  the 


BACTERIA   AND    ENZYMES    IN   AGRICULTURE        259 

physical  character  of  the  soil  is,  however,  improved  by  its 
presence.  The  resistant  portions  of  fibre  and  straw  tend  to 
make  the  soil  more  porous,  and  the  humus  which  it  contains 
increases  the  power  of  the  soil  to  retain  water  and  ammonia 
salts,  and  also  improves  the  texture  of  the  soil. 

After  the  manure  is  placed  on  the  field  the  various  amino 
compounds  will  suffer  further  decomposition,  yielding  eventu- 
ally ammonia.  It  is  a  matter  of  some  uncertainty  whether 
ammonia  is  immediately  available  for  plant  food ;  at  any 
rate,  there  is  no  doubt  that  nitrates  are  more  readily 
taken  up  by  a  plant,  and,  therefore,  a  prolonged  retention  of 
nitrogen  compounds  in  the  soil,  and  their  slow  conversion  into 
ammonia,  and  finally  into  nitrate,  is  an  advantage.  As  a 
matter  of  fact,  nitrification  of  the  ammonia  generally  takes 
place  before  it  has  been  removed  from  the  soil  by  the  plant. 
Moreover,  the  weight  of  the  dry  matter  of  the  plant  increases 
per  unit  of  nitrogen,  supplied  as  nitrate. 

The  conditions  of  nitrification  of  ammonia,  whether 
supplied  as  stable  manure,  or  in  the  various  forms  of  artificial 
manure,  especially  sulphate  of  ammonia,  have  been  con- 
sidered from  the  laboratory  point  of  view  in  Chapter  XIII ; 
the  importance  of  the  presence  of  humus  has  just  been  noted. 
It  will  be  further  clear,  upon  consideration,  that  nitrification 
will  not  take  place  satisfactorily  in  soils  which  have  become 
sour  or  acid ;  an  addition  of  lime  is  necessary  in  such  cases. 
The  beneficial  effect  which  unquestionably  attends,  in  many 
cases,  the  use  of  pressed  sludge,  which  contains  only  small 
quantities  of  nitrogen  relatively  speaking,  is  due  to  the* 
presence  of  lime,  which  has  been  added  to  facilitate  the  opera- 
tion of  pressing.  The  physical  effect  of  the  admixture  of  such 
material  with  the  soil  is  of  importance,  and  the  presence  of  a 
certain  proportion  of  matter  of  the  nature  of  humus  is  also 
beneficial. 


The  Loss  and  Recovery  of  Nitrogen  in  the  Soil.— It  is 

32 


260      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

evident  that  a  crop,  such  as  wheat,  of  a  high  nitrogen  content, 
must  diminish  the  supply  of  this  element  in  the  soil,  and  if 
such  a  crop  is  continually  grown  on  one  plot,  the  nitrogen  must 
become  exhausted  unless  replaced  in  various  ways.  Besides 
the  loss  of  nitrogen  from  cropping,  other  sources  of  loss  occur 
by  drainage  ;  the  nitrates  pass  away  in  the  subsoil  water,  and 
heavy  rainfall  on  a  porous  soil  accelerates  this  loss.  A  further 
source  of  loss  has  been  referred  to  in  Chapter  XIII,  viz.,  the 
elimination  of  nitrogen  from  nitrates  by  the  de-nitrifying 
organisms.  Against  these  sources  of  loss  of  nitrogen  have 
to  be  set  the  following  sources  of  gain,  apart  from  the  applica- 
tion of  nitrogenous  manure.  A  certain  amount  of  nitrogen 
is  added  to  the  soil  in  rain,  though,  as  already  explained,  this 
may  wash  out  more  than  it  brings.  It  is  to  the  nitrogen-fixing 
bacteria  that  we  have  largely  to  look  for  the  economic  mainten- 
ance of  the  balance  of  nitrogen  ;  and  we  have  here  an  explana- 
tion of  the  advantage  of  growing  leguminous  crops  at  intervals. 
If,  after  a  succession  of  nitrogen-exhausting  crops,  such  as 
wheat,  a  crop  of  clover  be  grown,  and  the  stubble  afterwards 
ploughed  in,  the  nitrogen  content  of  the  soil  is  greatly  in- 
creased. This  is  due,  as  has  been  explained,  to  the  action  of 
organisms,  which  find  their  habitat  in  the  root  nodules  of 
leguminous  plants,  such  as  the  clover,  which  in  some  way 
enable  the  plant  to  obtain  a  store  of  nitrogen  from  the  air. 

Experiments  on  a  small  scale  have  shown  that  it  is  possible 
greatly  to  increase  the  growth  of  such  plants,  when  grown  in 
sand,  by  inoculating  the  sand,  or  the  seeds  of  the  plants,  with 
suitable  cultures  of  nitrogen-fixing  bacteria.  Attempts  have 
been  made  to  carry  out  this  process  on  the  large  scale.  The 
best  results  have  been  obtained  with  species  of  leguminosge 
introduced  into  a  country  for  the  first  time,  e.g.,  the  soy  bean 
in  the  United  States  and  Germany,  lucerne  in  Scotland,  and 
certain  non-indigenous  plants  in  Canada.  For  crops  which 
have  already  long  been  cultivated,  e.g.,  clover  in  England,  the 
conditions  of  success  do  not  so  far  seem  to  be  fully  understood. 


BACTERIA   AND    ENZYMES   IN   AGRICULTURE      261 

Fertility  of  Soils. — Enough  has  been  said  to  show  the  great 
importance  to  the  farmer  of  the  bacterial  life  in  the  soil. 
Dr.  E.  J.  Kussell  has  carried  out  important  investigations 
showing  that  the  fertility  of  the  soil  is,  under  normal  circum- 
stances, actually  proportional  to  the  bacterial  activity  of  the 
soil.  It  is,  of  course,  obvious  that  bacterial  activity  is  a 
very  wide  term,  and  covers  the  many  classes  of  action  which 
have  been  indicated  in  the  foregoing  paragraphs,  but  Dr. 
Russell  has  found  that  the  sum  of  these  activities  can  be 
measured,  by  determining  the  rate  at  which  oxygen  is  taken 
up  by  a  given  weight  of  soil,  and  also  the  total  amount  of 
oxygen  so  taken  up.  This  he  determined  by  enclosing  the 
soil  in  a  flask,  connected  on  one  side  with  a  tube  dipping  into 
mercury,  and  on  the  other  with  a  small  receptacle  containing 
strong  potash  solution,  which  served  to  absorb  the  carbon 
dioxide  produced  by  the  oxidation  of  the  organic  matter. 
The  rise  of  the  mercury  in  the  side  tube  enabled  the  rate  and 
amount  of  oxygen  absorption  to  be  measured.  A  number 
of  these  flasks,  each  containing  soil,  whose  character  as 
regards  fertility  was  known,  was  placed  in  a  common  water- 
bath,  and  maintained  at  a  constant  temperature,  one  flask 
being  left  empty  to  serve  as  control.  It  was  found,  as  already 
stated,  that  the  absorption  of  oxygen,  and  consequently  the 
bacterial  activity,  increased  with  the  fertility. 

In  the  face  of  these  results,  it  appears  surprising  that 
experiments,  by  Russell  and  others,  should  have  shown  that 
partial  sterilisation  of  the  soil,  either  by  antiseptics,  such  as 
toluene,  or  by  heat,  should  increase  the  fertility. 

An  explanation  of  this  apparent  contradiction  is  afforded 
by  a  recent  research  by  Russell  and  Hutchinson.  They 
effected  partial  sterilisation  either  by  heating  to  98°  C.,  or 
by  addition  of  4  per  cent,  of  toluene,  which,  at  the  end  of 
three  days,  was  allowed  to  evaporate  by  spreading  out  the 
soil  in  a  thin  layer.  In  a  third  series,  the  toluene  was  left  in  ; 
in  a  fourth  series,  the  soil  was  left  untreated.  The  soils  were 


262      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

moistened,  and  kept  for  definite  periods  in  bottles,  stoppered 
with  cotton  wool,  at  the  ordinary  laboratory  temperature. 
Determinations  were  then  made  : — 

(a)  Of  the  production  of  ammonia ; 

(6)  Of  the  production  of  unstable  nitrogen  compounds ; 

(c)  Of  the  proportion  of  humus ; 

(d)  Of  the  nitrification;    and 

(e)  Of  the  total  amount  of  nitrogen. 

The  effect  of  partial  sterilisation  was  found  to  be  : — 

(1)  An  increase  in  the  amount  of  ammonia ; 

(2)  Cessation  of  nitrification. 

Besides  the  chemical  observations,  they  determined  the 
total  number  of  bacteria ;  and  they  found  that  the  increased 
ammonia  production,  due  to  partial  sterilisation,  was  accom- 
panied by  an  increased  number  of  bacteria.  The  problem 
resolves  itself  into  finding  out  why  the  bacteria  increase  so 
much  more  rapidly  in  the  partially  sterilised  than  in  the  un- 
treated soil.  They  found  that  if  untreated  soil  were  added  to 
partially  sterilised  soil,  the  rate  of  ammonia  production  was 
reduced,  but  this  was  not  the  case  if  an  extract  of  the  un- 
treated soil,  filtered,  but  still  containing  bacteria,  was  added 
to  the  partially  sterilised  soil.  This  would  indicate  that  the 
inhibiting  agent  was  something  which  affected  bacterial 
growth,  but  which  could  be  removed  by  a  coarse  filter.  Such 
an  agent  would  be  found  in  large  organisms  capable  of  feeding 
upon  bacteria.  As  a  matter  of  fact,  upon  examination, 
many  of  these  were  found  in  the  untreated  soil. 

Russell  and  Hutchinson  therefore  conclude  that  the 
large  organisms,  that  is,  protozoa  of  various  kinds,  are  an 
important  factor  in  limiting  the  bacterial  activity,  and  there- 
fore the  fertility  of  untreated  soil.  When  toluene  is  added  to 
the  soil,  or  when  the  soil  is  heated  to  98°  C.,  these  phagocytic, 
or  bacteria-consuming,  organisms  are  destroyed,  but  the 
bacterial  spores  are  not.  On  removing  the  toluene,  and 
adding  moisture,  the  spores  germinate,  and  the  other  bacteria 


BACTERIA   AND    ENZYMES   IN   AGRICULTURE       263 

multiply  with  great  rapidity,  since  they  are  now  free  from 
the  attacks  and  the  competition  of  their  enemies,  the  other 
large  organisms.  The  dead  organisms,  in  fact,  were  shown 
to  afford  food  for  the  bacteria. 

It  was  further  found  that  plant  growth  increased  in 
partially  sterilised  soil,  although  nitrification  was  inhibited; 
under  these  conditions  it  appears  that  the  plants  can  obtain 
their  nitrogen  from  a  source  other  than  nitrates. 

These  experiments  are  of  the  highest  interest,  and  show 
that  much  remains  yet  to  be  discovered  with  regard  to  the 
conditions  of  bacterial  life  in  soil,  and  its  relation  to  the 
growth  of  plants. 

Chemical  Changes  in  Plant  Cells. — When  a  plant  is  burnt, 
its  organic  constituents  disappear,  mainly  as  carbon  dioxide, 
C02,  nitrogen  and  water,  H20 ;  its  mineral  constituents 
remain  behind  in  the  ash.  The  growing  plant  builds  itself 
up  again  out  of  these  products  of  its  combustion ;  the  mineral 
constituents  and  water  it  takes  in  through  the  roots,  the 
carbon  and  oxygen  through  the  leaves,  the  nitrogen  ulti- 
mately being  supplied  from  the  sources  already  discussed. 
All  the  complex  physical  and  chemical  processes  involved 
in  building  up  a  plant  are  controlled  ultimately  by  the  vital 
energy  of  the  plant  cells,  together  with  the  energy  of  sun- 
light. The  correlation  of  all  these  processes  is  the  task  of 
physiological  botany,  and  a  knowledge  of  this  is  obviously 
inolispensable,  if  the  plant  is  to  be  grown  under  the  best  con- 
ditions and  supplied  with  its  right  food.  Enzyme  chemistry 
forms  the  foundation  knowledge  of  physiological  botany.  It 
is  clearly  necessary  to  have  some  understanding,  in  the 
first  place,  of  the  primary  chemical  changes  taking  place  in 
specific  cells,  before  establishing  their  general  relations. 

The  initial  impulse  to  plant  growth  is  to  be  found  in  the 
potential  biotic  energy  of  the  seed,  or,  more  properly  speaking, 
of  the  embryo.  This,  of  course,  like  all  forms  of  ITe  that  we 


264      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

are  acquainted  with,  has  its  origin  in  pre-existent  life,  but  the 
distinction  between  the  changes  taking  place  in  the  seed,  and 
those  which  occur  in  the  leaf,  is  that  the  former  are  not 
directly  dependent  upon  sunlight,  unless  indirectly,  it  may 
be,  for  warmth. 

The  chemical  changes  taking  place  have  been  fully  illus- 
trated in  the  study  of  the  barley  grain,  to  which  Chapters  V 
and  VI  have  been  devoted.  The  seeds  of  all  plants  contain 
in  the  endosperm  a  store  of  reserve  material,  which  has  been 
elaborated  by  the  growing  plant.  The  embryo,  as  we  have 
seen,  has  the  power  of  secreting  various  enzymes,  viz.,  cytase, 
which  breaks  down  the  cell  walls  of  the  endosperm,  and 
amylase,  which  converts  the  starch  into  sugar.  There  are 
also  present  proteolytic  enzymes,  which  break  down  the  stored 
albumin  of  the  seeds,  and,  in  the  case  of  fat-containing  seeds, 
such,  e.g.,  as  those  of  the  castor  oil  plant,  lypolytic  enzymes 
are  present,  which  break  down  the  oils  or  fats.  All  these 
changes,  it  may  be  seen,  are  essentially  concerned  with  the 
breaking  down  of  material  already  elaborated,  i.e.,  they 
are  what  is  known  as  catdbolic ;  unless  fresh  nutriment  is 
supplied,  on  the  one  hand,  and  fresh  energy  on  the  other 
hand,  growth  will  cease.  Nutriment  is  supplied  to  the 
plant,  as  already  stated,  by  the  roots,  and  by  the  leaves ; 
energy  is  supplied  by  the  leaves  only,  and  it  is  in  the  leaf  cell 
that  we  have  to  look,  to  find  what  we  may  describe  as  the 
power  house  of  the  plant. 

The  chemistry  of  the  leaf  cell  is  one  of  the  most  fascinating 
problems  which  has  occupied  the  attention  of  chemists,  but 
in  spite  of  numerous  researches  by  highly  qualified  workers, 
it  is  still  very  imperfectly  understood, 

The  simple  beginnings  and  ends  of  the  process  have  been 
known  for  a  long  time  ;  they  may  be  studied  without  difficulty, 
and  indeed  form  one  of  the  subjects  of  most '  nature  study  * 
classes.  The  following  experiments  are  easily  carried  out. 

A  portion  of  American  pond  weed,  Elodea  canadensis,  is 


BACTERIA   AND    ENZYMES   IN   AGRICULTURE      265 

placed  in  water,  in  a  cylindrical  vessel,  with  a  little  earth  at 
the  bottom  for  root  attachments,  and  the  whole  set  in  the 
sun ;  bubbles  of  gas  soon  arise  from  the  leaves  and  may  be 
readily  collected.  If  a  glowing  splinter  of  wood  be  held  in 
the  gas,  it  will  burst  into  flame,  showing  that  the  gas  con- 
sists, for  the  most  part  at  any  rate,  of  oxygen. 

On  the  other  hand,  if  the  plant  is  placed  in  darkness,  and 
air,  freed  from  C02  by  passing  through  potash  solution,  is  led 
over  the  plant,  and  then  passed  into  baryta  water,  the  latter 
will  become  turbid  from  formation  of  barium  carbonate. 

From  these  experiments,  it  is  clear  that  two  main  processes 
go  on  in  the  leaves,  the  evolution  of  oxygen  in  sunlight,  and 
of  carbon  dioxide  in  darkness.  These  two  changes,  as  a  matter 
of  fact,  take  place  at  all  times,  but  the  preponderance  of  one 
over  the  other  depends  on  the  presence  or  absence  of  sunlight. 
The  evolution  of  oxygen  is  a  building  up,  or  anabolic  process, 
arising  from  the  decomposition  of  the  carbon  dioxide  in  the  air, 
the  plant  utilising  the  carbon  and  giving  off  the  oxygen;  on 
the  other  hand,  the  evolution  of  carbon  dioxide  is  essentially 
a  process  of  respiration,  or  a  catabolic  process,  where  the 
carbonaceous  constituents  of  the  plant  are  broken  down,  with 
production  of  carbon  dioxide  and  water.  The  volume  of  the 
oxygen  given  out  in  the  assimilation  process  is  practically 
equal  to  the  volume  of  carbon  dioxide  taken  in,  sufficiently 
indicating  that  the  changes  involved  are  of  a  fairly  simple 
order.  The  problem  to  the  chemist  is  to  discover  how  the 
carbon,  taken  in  by  the  plant  as  C02,  is  built  up  into  starch  and 
cellulose,  and  by  what  stages  these  latter  are  reconverted  into 
carbon  dioxide,  and  thus  the  life  cycle  maintained.  Micro- 
scopic observation  indicates  that  starch  is  the  first  visible  pro- 
duct appearing  in  the  leaf  cell ;  but,  of  course,  between  a  simple 
substance  such  as  carbon  dioxide  and  starch,  the  chemical 
steps  must  be  very  numerous. 

In  1870  von  Baeyer  put  forward  a  very  suggestive  hypothesis 
in  regard  to  the  first  of  these  steps ;  the  simplest  carbohydrate, 


266      BACTERIOLOGICAL   AND   ENZYME   CHEMISTRY 

as  was  explained  in  Chapter  IV,  is  formaldehyde,  CH20.  Von 
Baeyer  suggested  that,  in  the  simultaneous  presence  of  light 
and  of  chlorophyll,  carbon  dioxide  and  water  may  react 
according  to  the  following  simple  equation  :  — 

C0  +  H0  =  CH0  +  0  : 


An  explanation  is  here  indicated  of  the  equivalence  alluded 
to  above  between  the  C02  decomposed  and  the  oxygen  evolved. 
It  is  easy  to  conceive  further,  that  the  formaldehyde,  by  a 
series  of  polymerisations,  can  build  up  more  complex  carbo- 
hydrates, such  as  starch.  This  hypothesis  derives  confirma- 
tion from  the  fact  that,  on  standing  in  contact  with  a  dilute 
solution  of  lime  water,  formaldehyde  does,  as  a  matter  of  fact, 
become  gradually  converted  into  a  mixture  of  hexoses.  Until 
recently,  however,  all  attempts  to  realise  the  formation  of 
formaldehyde  from  carbon  dioxide  and  water  in  the  laboratory 
were  without  success,  formic  acid  being  always  the  product 
of  the  reaction.  Nor  was  it  possible  to  detect  formaldehyde  in 
the  living  cell. 

Recently,  however,  the  subject  has  been  advanced  con- 
siderably by  the  investigations  of  Usher  and  Priestly  ;  they 
have  been  able  to  show  that  if  leaves  of  Canadian  pond  weed, 
Elodea  canadensis,  and  certain  green  seaweeds,  viz.,  Ulva  and 
Enter  omorpha,  are  first  placed  in  hot  water,  so  as  to  kill  the 
protoplasm,  and  are  then  exposed  to  moist  carbon  dioxide 
in  presence  of  light,  formaldehyde  and  hydrogen  peroxide  are 
produced,  and  can  be  detected.  If  a  suitable  catalase,  or 
hydrogen  peroxide  decomposing  enzyme,  were  introduced  into 
the  mixture,  oxygen  was  evolved. 

Under  the  conditions  of  the  experiment,  when  a  certain 
amount  of  hydrogen  peroxide  and  formaldehyde  had  been 
formed,  a  reverse  change  tended  to  be  set  up,  the  reaction 
being  expressed  as  follows  :  — 

H2C08  +  2H20  £CH20  +  2H202 


BACTERIA   AND    ENZYMES   IN    AGRICULTURE        267 

Such  a  reverse  change  would  not  take  place  in  plants,  inas- 
much as  the  products  of  the  first  reaction,  formaldehyde  and 
oxygen,  are  eliminated,  the  formaldehyde  being  utilised  for 
building  up  carbohydrates,  and  the  oxygen  passing  off. 

We  have  still  here  to  do  with  chlorophyll,  a  substance 
elaborated  by  life  processes.  Experiments  by  Fenton  in 
1907  have,  however,  shown  that  carbon  dioxide  can  be  reduced 
to  formaldehyde  in  presence  of  metallic  magnesium.  This 
experiment  is  of  interest  in  view  of  the  fact  that,  according  to 
Willstatter,  magnesium  is  an  essential  constituent  of  chloro- 
phyll, just  as  iron  is  an  essential  constituent  of  the  haemoglobin 
of  the  blood.  It  may  be  that  we  have  here  the  first  chemical 
step  in  the  series  leading  up  to  starch. 

The  conversion  of  C02  into  formaldehyde  and  oxygen  would 
thus  appear  to  be  a  purely  chemical  phenomenon,  which  under 
the  conditions  of  the  laboratory  quickly  reaches  a  limit,  but 
which  under  the  influence  of  biotic  energy  becomes  continuous, 
owing  to  the  products  of  the  reaction  being  quickly  removed. 

This  important  first  stage  in  plant  assimilation  may  be 
expressed  by  the  following  equations  : — 

i.    HO-j-COOH  HO       COOH 

Carbonic  j  acid  j     -j-    I 

HO-j-  H  HO       H 

Water  Hydrogen        Formic 

acid 


ii.     HO-j-CHO  HO     CHO 

Formic  i  acid  j   -|-     | 

HO-I-H  HO      H 

Water  Hydrogen        Formalde- 

peroxide          hyde 

The  question  still  remains,  presuming  that  the  above 
equations  bear  a  close  relation  to  actual  fact,  what  is  the  next 
stage  between  the  simple  carbohydrate,  formaldehyde  (CH20), 
and  the  more  complex  sugars  and  starches  ? 


268      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

It  might  naturally  be  assumed  that  the  first  detectable 
products  would  be  simple  sugars,  such  as  bioses  and  trioses ; 
such  trioses  have  not  been  found.  On  the  other  hand  there  is 
evidence  that  acids,  such  as  glyoxylic  acid,  CHOC02H,  and 
glycollic  acid,  CH2OHC02H,  do  occur  in  the  leaves  of  plants, 
and  the  interesting  suggestion  has  been  made  that  in  the 
process  of  reduction  of  carbonic  acid,  groups  such  as  CHO, 
CH2OH,  C02H,  CHOH,  etc.,  are  formed,  from  which  various 
combinations,  acids,  aldehydes  and  carbohydrates  may  be  built 
up.  At  the  same  time  certain  of  these  compounds  might 
combine  with  ammonia,  produced,  it  may  be,  by  reduction  of 
nitrates,  to  form  amino  acids,  the  first  products  of  albumin 
synthesis.  Still  the  fact  remains  that  these  intermediate 
products  are  not  at  all  readily  identified,  and  the  evidence  as 
to  their  presence  is  conflicting. 

The  careful  experiments  of  Brown  and  Morris,  in  their 
research  on  the  chemistry  of  foliage  leaves,  already  referred  to, 
reveal  the  somewhat  surprising  fact  that,  in  the  case,  at  any  rate, 
of  the  nasturtium  leaves,  which  constituted  the  chief  material 
of  their  research,  the  first  product  of  assimilation  is  cane  sugar. 
Their  method  of  experiment  was  to  take  leaves  which  were 
gathered  early  in  the  day,  and  dried  at  once  after  plucking, 
and  compare  their  sugar  content  with  leaves  which  were  left 
exposed  to  the  sun  for  some  hours  after  gathering,  with  leaves 
which  were  gathered  later  on  in  the  day  and  immediately  dried, 
and  with  others  placed  in  the  dark  for  some  hours  after  pluck- 
ing. The  dried  leaves  were  extracted  with  ether,  to  remove 
fat  and  chlorophyll,  and  a  weighed  portion  of  the  residue  then 
extracted  with  alcohol  to  remove  the  sugars ;  the  alcoholic 
extract  was  rendered  slightly  alkaline  with  ammonia,  to 
prevent  inversion  of  the  sugars  by  means  of  the  vegetable 
acids,  small  quantities  of  albuminous  matter  and  of  tannin 
removed  by  lead  acetate,  and  the  mixture  of  sugars,  in  the 
clear  extract  thus  obtained,  carefully  analysed  by  polarimetric 
and  copper  reduction  methods,  in  successive  stages,  viz.  : — 


BACTERIA    AND    ENZYMES   IN   AGRICULTURE      269 

(a)  At  once ; 

(b)  After  treatment  with  invertase  to  hydrolyse  the  cane 
sugar ;  and 

(c)  After  complete  inversion  by  means  of  hydrochloric  acid. 
In  this  way  cane  sugar,  dextrose,  laevulose  and  maltose 

were  determined,  and  it  was  found  in  every  case  that  the  leaves 
which  had  been  exposed  to  light,  under  conditions  where 
assimilation  processes  were  in  the  ascendant,  always  contained 
cane  sugar  in  greater  proportion  than  any  of  the  other  sugars 
present. 

Thus,  to  take  one  example,  leaves  picked  at  9  A.M.  on  a  dull 
morning  yielded  the  following  analysis  : — 

Starch  3'24 

Cane  sugar  . .         . .         . .  4*94 

Dextrose 0'81 

Laevulose  . .         . .         . .  4*78 

Maltose  1-21 

Leaves  picked  at  4  P.M.  on  the  same  day  after  seven  hours 
of  sun  gave  : — 

Starch         4'22 

Cane  sugar  . .         . .         . .  8 '02 

Dextrose O'OO 

Lsevulose     . .         . .         . .  1*57 

Maltose       3'62 

It  has  been  possible  to  synthesise  in  the  laboratory  all 
the  various  sugars  isolated  by  Brown  and  Morris ;  but  one 
important  difference  exists  between  the  products  formed  in 
the  laboratory,  and  those  produced  by  the  activity  of  the  plant 
cell.  In  all  cases  where  a  synthesis  is  effected  in  the  laboratory 
by  purely  chemical  means,  optically  inactive  derivatives 
result,  that  is,  mixtures  of  right-handed  and  left-handed  forms 
in  equal  proportions.  It  is  of  course  possible,  by  methods 


270      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

already  described,  to  separate  these  mixtures  into  their 
optically  active  constituents,  as  well  as  to  produce  optically 
active  compounds  in  the  first  instance,  if  an  optically  active 
substance  is  used  as  a  starting  point.  Thus,  e.g.,  cane 
sugar  can  be  synthesised  by  comparatively  simple  reactions 
from  glucose  and  fructose,  but  in  all  cases  where  an  opti- 
cally active  body  is  obtained,  the  agency  of  life  steps  in  at 
some  point. 

This  important  fact  was  clearly  realised  by  Pasteur,  whose 
words  on  the  subject  are  worth  quoting :  . '  To  transform 
an  inactive  compound  into  another  inactive  compound,  which 
has  the  power  of  resolving  itself  simultaneously  into  a  right- 
handed  compound  and  its  opposite,  is  in  no  way  comparable 
with  the  possibility  of  transforming  an  inactive  compound  into 
a  single  active  compound.  This  is  what  no  one  has  ever  done ; 
it  is,  on  the  other  hand,  what  living  nature  is  continually  doing 
before  our  eyes.' 

It  is  to  the  action  of  enzymes  present  in  the  living  proto- 
plasm, especially  in  chlorophyll,  that  we  must  look  for  this 
selective  synthetic  power  of  the  plant  cell.  We  have  learnt, 
through  the  work  of  Croft  Hill  and  others,  that  the  action  of 
an  enzyme  may  show  itself  in  a  building-up  or  anabolic  process, 
as  well  as  in  a  breaking-down  or  catabolic  process.  We  may 
perhaps  conceive  of  the  enzyme  as  a  kind  of  framework  into 
which  the  molecules  must  fit  themselves,  in  order  that  a  certain 
substance  may  be  produced,  either  on  the  up  or  down  grade  of 
a  chemical  change.  Thus,  in  the  case  of  maltose,  we  can  con- 
ceive the  various  atomic  groupings  setting  themselves  to  form 
maltose,  or,  on  the  other  hand,  passing  back  through  the  same 
framework  to  form  glucose,  the  hexose  which,  it  will  be 
remembered,  is  produced  when  maltase  acts  upon  maltose. 
Similarly  invertase  may  act  as  the  framework  for  the  building 
up  or  breaking  down  of  cane  sugar. 

It  is  evident  that  our  knowledge  of  this  subject  is  still  of  a 
speculative  character,  but  enough  has  been  said  to  indicate  the 


BACTERIA   AND    ENZYMES   IN   AGRICULTURE      271 

extraordinary  complexity  of  the  phenomenon  of  cell  chemistry, 
even  in  a  region  so  comparatively  simple  as  the  synthesis 
of  carbohydrates.  Apart  from  the  purely  scientific  interest  of 
researches  in  this  direction,  it  is  permissible  to  expect  that  an 
extension  of  knowledge  of  the  chemistry  of  plant  assimilation 
will  render  it  possible  more  exactly  to  adapt  the  food  supply  of 
the  plant  to  its  special  needs,  and  thus  to  conduce  to  economy 
in  plant  cultivation. 

The  Preparation  of  Silage.— In  order  to  obtain  a  store 
of  succulent  food,  for  use  when,  through  severity  of  weather, 
or  for  other  reasons,  it  is  naturally  unavailable,  it  is  a  frequent 
custom,  especially  in  America,  to  resort  to  the  operation  of 
ensilage. 

In  this  process  the  fodder,  e.g.,  hay,  beet,  cabbage  leaves, 
or  green  maize  stems,  is  packed  into  what  is  termed  a  silo, 
which  may  be  either  a  closely  pressed  heap,  protected  from 
weather  by  thatch,  or  a  large  air-tight  receptacle,  usually 
cylindrical  in  form,  into  which  the  mass  is  pressed. 

Under  these  circumstances  fermentation  sets  in,  accom- 
panied by  considerable  heating.  The  heating  is  due  to  the 
continued  respiration  of  the  still  living  cells.  In  course  of  time 
the  heat  becomes  such  that  most  bacteria  are  killed,  fermenta- 
tion ceases,  and  the  fermented  fodder  will  keep  for  a  consider- 
able time.  The  character  of  the  fermentation  depends  on 
the  nature  of  the  fodder  used,  and  on  the  amount  of  oxygen 
and  moisture  present  in  the  silo  and  in  the  original  fodder 
employed. 

Russell  has  carefully  investigated  the  changes  which  take 
place  during  the  ensilage  of  green  maizt-.  The  vital  processes 
of  the  cell  protoplasm  continue  for  some  time  after  the  maize  is 
put  in  the  silo,  the  starch  continues  to  break  down  and  the 
sugar  formed  is  partially  oxidised  to  various  acids ;  in  the 
limited  supply  of  air,  complete  combustion  to  C02  does  not 
take  place.  The  proteolytic  enzymes  of  the  cell  act  on  the 


272      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

vegetable  albumins,  forming  amino  acids,  etc. ;  these  actions, 
being  purely  enzymic,  will  go  on  after  the  cell  is  dead.  Accord- 
ing to  Russell,  bacteria  are  also  present,  yet  they  are  not  the 
chief  agents  in  the  decomposition,  though  they  probably 
attack  the  softer  cellulose,  producing  humus  and  some  fatty 
acids ;  they  also  carry  to  a  further  stage  the  decomposition 
of  certain  of  the  nitrogen  compounds.  These  changes  are 
summarised  in  the  table  on  the  opposite  page,  which  may 
be  taken  as  a  typical  statement  of  the  changes  occurring 
during  the  ensilage  of  fodder.  In  this  particular  case,  maize 
was  taken  as  the  subject  of  experiment,  and  air  was  excluded 
as  far  as  possible.  With  other  materials,  e.g.  exhausted  beet 
from  sugar  factories,  etc.,  a  greater  proportion  of  acid  may 
be  obtained,  in  which  case  sour  fodder  is  produced.  It  may 
even  be  possible  to  inoculate  silos  with  selected  ferments, 
in  order  to  obtain  the  best  results.  Incidentally  it  may  be 
mentioned  that  the  '  sauerkraut '  of  the  German  restaurant 
is  produced  by  an  analogous  species  of  fermentation. 

The  Bacteriological  Chemistry  of  Dairy  Products.— 

Starting  from  milk,  as  raw  material,  a  great  variety  of  pro- 
ducts are  obtained  in  modern  dairy  practice :  cream  of  different 
flavours,  butter  both  sweet  and  sour,  cheeses  in  great  variety, 
both  soft  and  hard.  These  different  products  are  not  in  general 
all  produced  in  one  dairy  or  even  in  one  district ;  rather  indeed 
are  we  accustomed  to  differentiate  them  according  to  the  place 
of  manufacture :  thus  Dutch  cheese  and  Swiss  cheese  differ 
from  those  produced  in  England,  and  the  different  varieties  of 
English  cheese,  as  is  well  known,  were  at  one  time  derived 
from  different  districts.  The  ultimate  reason  for  this  is  to  be 
found  in  the  bacteriological  conditions  which  long  practice  has 
established  in  various  dairying  centres.  Modern  advances  in 
dairy  practice  seek  to  render  it  possible  to  produce  any  kind  of 
dairy  product  at  will,  at  any  centre. 


BACTERIA    AND    ENZYMES   IN   AGRICULTURE       273 


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274      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

In  order  to  understand  these  developments,  the  chemical 
constituents  of  milk  must  be  first  considered.  According  to 
Warington,  cow's  milk  has  the  following  composition: — 

Water 87*0  per  cent. 

Albuminoids       . .         .  .         . .       3-7      „ 

Fat         3-9      „ 

Sugar 4-7      „ 

Ash         0-7      „ 

It  is  evident,  therefore,  that  the  possible  chemical  changes 
that  may  take  place  in  milk  are  concerned  with  the  decom- 
positions of  albumin  which  are  brought  about  by  proteolytic 
bacteria  and  enzymes,  with  the  splitting  of  fats,  and  with  the 
various  fermentations  which  lactose  or  milk  sugar  is  capable  of 
undergoing.  If  these  various  changes  are  to  be  controlled,  it 
will  be  seen  that  in  dairy  practice  the  utmost  cleanliness  is  of  the 
first  importance,  lest  normal  proteolysis  should  become  putre- 
faction, or  the  breaking  up  of  fats  and  of  sugar  should  give 
rise  to  abnormal  developments  of  butyric  acid,  and  consequent 
rancidity,  as  distinguished  from  merely  pleasant  souring. 

These  considerations  may  first  be  applied  to  the  manu- 
facture of  butter.  Butter,  as  is  well  known,  is  obtained  by  the 
churning  of  cream,  a  process  by  which  the  fat  globules,  present 
as  an  emulsion  in  milk,  are  collected  together  to  form  the  mass 
known  as  butter.  The  fat  of  milk  consists  largely  of  glycerides 
of  palmitic  and  oleic  acids,  together  with  smaller  quantities  of 
the  glycerides  of  other  fatty  acids,  notably  butyric. 

The  oldest  method  of  separating  the  cream  from  milk  is  to 
allow  the  latter  to  stand  in  wide  shallow  dishes,  when  the  fat 
particles,  being  specifically  lighter  than  the  rest  of  the  milk,  rise 
to  the  surface,  and  can  be  skimmed  off.  Such  a  process 
obviously  offers  conditions  for  contamination  of  the  cream  by 
air  infection,  especially  if  any  carelessness  is  permitted.  The 
danger  of  contamination  is  reduced  if  the  cream  is  allowed  to 
rise  in  deep  closed  vessels.  But  the  modern  process,  in  which 


BACTERIA    AND    ENZYMES   IN   AGRICULTURE       275 

the  cream  is  separated  from  the  milk  by  centrifugal  action  in 
suitable  machines,  is  the  most  rapid,  and  consequently  the 
least  liable  to  infection.  If,  at  the  same  time,  a  low  tempera- 
ture is  maintained,  the  danger  of  uncertainty  in  the  subsequent 
souring  of  the  cream  is  still  further  reduced. 

If  cream  is  churned  in  a  perfectly  fresh  state,  sweet 
butter  is  obtained,  which  is  somewhat  tasteless.  By  careful 
souring  of  the  cream  previous  to  churning,  butter  of  a  more 
defined  flavour  is  produced  ;  it  is  in  the  control  of  this  flavour 
that  bacteria  play  their  part. 

In  following  the  changes  which  they  bring  about,  it  must 
be  understood  that  the  cream,  as  used  for  churning,  will 
contain  not  only  the  fat  of  the  milk,  but  also  a  certain 
quantity  of  its  other  constituents.  The  composition  of  the 
butter  is  conditioned  therefore  by  the  method  used  for  the 
collection  of  the  cream. 

The  souring  or  '  ripening '  of  the  separated  cream  may  be 
effected  by  the  addition  of  a  small  quantity  of  sour  milk, 
which  will  contain  the  necessary  bacteria,  notably  the  lactic 
acid  bacteria.  Satisfactory  ripening  of  the  cream  can  gener- 
ally be  judged  by  practice ;  chemical  examination  of  such 
cream  should  not  discover  any  appreciable  quantity  of 
casein.  Its  presence  would  point  to  the  souring  process  having 
gone  too  far,  resulting  in  the  production  of  a  certain  amount 
of  clotting  of  the  buttermilk  present.  Working  in  this 
manner,  and  with  careful  attention  throughout  to  the  avoid- 
ance of  infection  by  deleterious  bacteria,  excellent  results  are 
obtainable  so  long  as  the  conditions  of  milk  supply  and  the 
general  control  of  the  dairy  remain  unchanged.  An  element 
of  uncertainty,  however,  still  exists,  and  the  main  reason  for 
the  greater  sale  of  Danish  butter,  compared  with  that  produced 
in  England  and  Ireland,  is  its  constancy  of  quality. 

This  constant  quality  has  been  attained  by  still  further 
development  in  the  control  of  the  souring  process. 

Such  absolute  control  is  obtained  when,  in  the  first  place, 

T2 


276      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

all  the  bacteria  present  in  the  cream  and  its  associated  milk 
are  destroyed  by  the  process  of  Pasteurisation,  and  the 
subsequent  inoculation  of  the  Pasteurised  cream  with  a  pure 
culture  of  the  necessary  bacteria. 

The  process  of  Pasteurisation  consists  in  a  rapid  heating  to 
a  temperature  sufficient  to  destroy  the  majority,  at  any  rate, 
of  the  bacteria  present,  followed  by  an  equally  rapid  cooling 
process.  By  this  method  the  composition  and  flavour  of  the 
cream  is  not  appreciably  altered.  The  pure  culture  to  be 
added  is  generally  known  as  the  '  starter.'  The  following  is  the 
method  described  by  the  Danish  bacteriologist  Weigmann  : — 

A  quantity  of  sweet  milk,  amounting  to  about  two  or  three 
per  cent,  of  the  cream  to  be  acidified,  is  heated  to  about  60°  C. 
and  quickly  cooled ;  to  it  is  added  a  pure  culture  which  is 
maintained  by  the  addition  of  sweet  Pasteurised  milk  from 
day  to  day.  A  portion  of  the  inoculated  milk  is  added  to 
the  cream,  which  is  best  prepared  by  cooling  to  a  low  tempera- 
ture and  then  quickly  warming  up  again  to  16°  or  20°  C. 
The  ripening  process  is  generally  started  in  the  evening,  and 
allowed  to  complete  itself  at  a  temperature  of  15°  to  20°  C., 
the  cream  being  ready  for  churning  on  the  following  morning. 

The  flavour  of  the  butter  produced  depends  on  the  particular 
starter  used.  It  does  not  appear  certain  whether  one  variety 
of  organism  alone  is  concerned  in  producing  specific  flavours, 
although  all  the  organisms  concerned  probably  belong  to  the 
lactic  acid  producing  species.  At  any  rate  one  organism  has 
been  found  by  Professor  Conn  in  America  which  produces  a 
very  excellent  flavour.  It  was  originally  obtained  from  a 
specimen  of  milk  from  Uruguay,  South  America,  exhibited 
at  the  World's  Fair  in  Chicago.  This  bacillus  is  known  as 
No.  41,  and  by  its  use  constant  results  have  been  maintained 
on  a  very  large  scale. 

Although,  as  has  been  stated,  the  ripening  of  cream  is 
mainly  produced  by  lactic  acid  bacteria,  the  precise  chemical 
changes,  resulting  in  the  production  of  a  different  taste  or 


BACTERIA   AND    ENZYMES   IN   AGRICULTURE       277 

aroma  in  different  cases,  is  not  very  perfectly  understood. 
Obviously  the  conditions  admit  of  the  production  of  esters, 
by  combination  of  different  organic  acids  with  various 
alcoholic  groups  in  a  great  variety  of  ways. 

The  chemistry  of  cheese  making  is  more  complex  than  the 
chemistry  of  butter  making  ;  we  have  here,  in  addition  to  the 
activity  of  lactic  organisms,  to  consider  more  especially 
proteolytic  changes  in  the  curd,  which  is  the  starting  point  of 
cheese.  Reference  has  already  been  made  to  the  chemical 
changes  which  take  place  when  the  clotting  enzyme  rennet 
is  added  to  milk.  The  first  process  in  the  manufacture  of 
cheese  consists  in  throwing  out  the  curd  or  casein  by  means  of 
rennet.  Milk  can  also  be  curdled  by  the  activity  of  acid- 
forming  organisms,  and  this  method  is  actually  employed  in 
the  preparation  of  certain  home-made  cheeses  ;  but  in  this  case 
the  curd  is  of  a  different  composition,  the  whole  of  the  albumin 
of  the  milk  being  thrown  out.  The  curd  produced  by  rennet, 
as  we  have  seen,  consists  of  casein  together  with  calcium 
phosphate  ;  the  curd  will  also  carry  down  with  it  fat  particles, 
and  will  retain,  of  course,  a  certain  proportion  of  the  whey, 
i.e.,  the  liquid  left  after  separation  of  the  curd,  and  which 
contains  the  soluble  constituents  of  the  milk.  Curd  thus 
prepared  is  tasteless,  and  in  order  to  be  converted  into 
cheese  has  to  undergo  a  ripening  process.  In  the  process 
of  cheese  making  the  curd  is  granulated,  placed  in  cloths,  and 
the  whey  pressed  out ;  the  pressed  curd  is  then  set  aside  to 
ripen. 

The  ripening  process,  in  the  case  of  cheese,  is  brought 
about,  not  only  by  bacteria,  but  also  in  certain  cases  by 
moulds.  The  former  are  chiefly  concerned  in  the  ripening  of 
hard  cheese,  which  may  take  place  at  a  fairly  high  tempera- 
ture, at  which  the  activity  of  moulds  will  be  inhibited.  The 
activity  of  moulds  is  concerned  more  with  soft  cheeses,  which 
are  allowed  to  ripen  at  a  lower  temperature. 

As  in  the  case  of  butter,  so  in  the  case  of  cheese,  if  the 


278      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

conditions  of  the  dairy  remain  constant,  a  constant  product 
may  be  obtained,  without  the  necessary  use  of  pure  cultures. 
This  constancy  of  conditions  depends,  however,  on  locality. 
Cheese  making,  e.g.,  is  carried  on  in  the  high  Alps  under 
exceptionally  favourable  conditions  in  this  respect ;  the  air 
is  pure,  the  fodder  of  the  animals  consists  mainly  of  grass  and 
hay,  from  fields  to  which  they  themselves  contribute  the  only 
manure.  In  the  lower  Alps,  on  the  other  hand,  the  fodder, 
and  consequently  the  manure,  is  subject  to  variation ;  the 
bacterial  atmosphere,  therefore,  may  change  from  time  to 
time,  and  the  variable  conditions  manifest  themselves  in  the 
dairy. 

The  chemical  changes  taking  place  in  ripening  cheese 
consist : — 

(a)  In  the  breaking  down  of  albumin ; 

(6)  The  splitting  of  fats  ; 

(c)  The  fermentation  of  sugar. 

That  these  changes  are  brought  about  by  living  organisms 
is  clear  from  the  fact  that,  if  the  curd  is  treated  with  an 
antiseptic,  no  ripening  takes  place.  Numerous  disintegration 
products  of  albumin  have  been  detected  in  ripe  cheese,  such 
as  leucin,  tyrosin,  and  even  ammonia.  The  splitting  up  of 
the  glycerides  is  carried  to  a  further  extent  than  in  the  souring 
of  cream ;  and  the  activity  of  the  lactic  organism  is  shown, 
in  many  cases,  in  the  production  of  gas,  which  causes  the 
pitting,  so  noticeable,  e.g.,  in  Gruyere  cheese. 

The  control  of  cheese  ripening  by  pure  cultures  is  not  so 
easily  carried  out  as  in  the  case  of  croam,  since  Pasteurised 
milk  will  not  curdle  with  rennet.  By  initial  care  in  the 
production  of  the  milk  and  its  maintenance  at  a  low  tempera- 
ture, a  reasonably  pure  curd  can,  however,  be  obtained. 

The  particular  organism  used  as  a  starter  will,  of  course, 
depend  on  the  character  of  cheese  to  be  produced  ;  thus  in  the 
case  of  Roquefort  cheese  the  mould  Penicillium  glaucum  is 
used,  whereas  in  the  production  of  Cheddar  cheese,  lactic 


BACTERIA    AND    ENZYMES    IN    AGRICULTURE       279 

acid  bacteria  have  been  shown  to  play  the  most  prominent 
part. 

In  addition  to  being  able  to  control  the  products  of  the 
dairy,  the  scientific  dairyman  must  also  understand  the 
causes  of  the  various  abnormal  and  deleterious  fermentations 
which  may  from  time  to  time  take  place.  These  are  mainly 
dependent  on  the  invasion  of  so-called  wild  bacteria,  whose 
nature  and  chemical  activities  have  to  be  studied.  It  would 
lead  too  far  to  consider  these  difficulties  here. 

Although  of  recent  years  some  attempts  have  been  made 
in  England  to  introduce  scientific  precision  into  dairy  work, 
very  much  yet  remains  to  be  done.  We  are  still  very  far 
from  applying  to  the  manufacture  of  dairy  products  the  same 
standard  of  scientific  thoroughness  which  has  been  so  long 
worthily  upheld  in  other  fields  of  agricultural  investigation, 
notably  at  the  seventy-year-old  experimental  statfon  at 
Rothamsted. 


CHAPTEK  XVII 

THE  CHEMISTRY  OF  SEWAGE  PURIFICATION 

OWING  to  the  general  adoption  of  the  water  carriage 
system,  together  with  the  increasing  scarcity  of  land  in  the 
vicinity  of  towns,  great  developments  have  taken  place  during 
recent  years  in  so-called  artificial  methods  for  the  purification  of 
sewage.  In  order  that  works  for  this  purpose  shall  be  designed 
with  due  regard  both  for  economy  and  efficiency,  it  is  necessary 
that  the  changes,  which  it  is  intended  to  bring  about  by  their 
means,  should  be  thoroughly  understood  by  those  concerned 
in  their  construction.  Although  the  actual  construction  of 
the  modern  sewage  works  is  largely  a  matter  of  engineering, 
the  design  depends  on  careful  adaptation  of  means  to  ends, 
and  the  bacteriological  chemist  and  the  engineer  should  here 
work  in  collaboration.  The  object  of  the  works  is  to  convert 
objectionable  waste  products,  which  if  left  to  themselves 
would  be  a  source  of  nuisance  and  danger,  into  other  sub- 
stances which  are  incapable  of  producing  such  objectionable 
developments.  In  the  course  of  the  necessary  transformation, 
at  one  point  or  another,  practically  all  the  typical  chemical 
changes,  which  have  been  considered  in  the  theoretical  chapters 
of  this  book,  are  met  with,  and  the  consideration  of  the  pro- 
cesses carried  on  in  a  modern  sewage  works  forms  therefore  an 
excellent  illustration  of  the  application  of  bacteriological  and 
enzyme  chemistry. 

It  will  be  necessary,  in  the  first  place,  carefully  to  consider 
the  composition  of  ordinary  town  sewage ;   for  this  purpose. 


SEWAGE   PURIFICATION  281 

domestic  sewage  only  will  be  referred  to,  the  question  of  the 
treatment  of  trade  effluents,  or  mixtures  of  trade  effluents  and 
sewage,  constituting  a  special  problem. 

The  main  constituents  of  domestic  sewage  may  be  described 
as  follows : — 

(i.)  MATTERS  IN  SOLUTION.     (Mainly  derived  from  urine.) 
Nitrogenous  substances,  e.g.,  urea  and  kindred  com- 
pounds. 
Mineral  salts,  chiefly  sodium  chloride  together  with 

phosphates. 

(ii.)  MATTERS  IN  SUSPENSION,  EMULSION,  OR  COLLOIDAL 
SOLUTION. — Nitrogenous  substances  of  complex  character 
containing  sulphur  (mainly  derived  from  faeces). 

Cellulose  (disintegrated  paper)  and  vegetable  debris. 
Soap  and  fat. 
(iii.)  SEDIMENTARY  MATTERS. — Silt,  clay,  sand,  etc. 

There  cannot,  of  course,  be  sharp  lines  drawn  between  these 
various  classes  of  substances.  It  will  depend,  e.g.,  on  the 
hardness  of  the  water,  how  much,  if  any,  of  the  soap  is  present 
in  solution  or  suspension  ;  substances  in  Class  ii.  will  also 
be  partially  carried  down  by  the  quickly  sedimenting  mineral 
matters  of  Class  iii. 

In  general,  about  twenty  gallons  may  be  taken  as  the  usual 
water  supply  per  head  per  day,  in  which  the  above  constituents 
are  disseminated. 

The  complete  purification  of  the  sewage,  which  is  effected 
by  bacterial  treatment,  results  finally  in  the  production  of 
some  or  all  of  the  following  substances  : — 

(i.)  GASES  : 

Methane  (Marsh  gas). 
Hydrogen. 
Nitrogen. 
Carbon  dioxide. 


282      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

(ii.)  SOLUBLE  SALTS  : 
Nitrates. 
Phosphates. 
Sulphates. 
Chlorides. 

(iii.)  Insoluble  residual  matters  conveniently  termed 
'  Humus.' l 

It  is  possible  to  transform  sewage  into  these  harmless 
products  by  direct  oxidation,  through  the  agency  of  the 
requisite  organisms,  in  presence  of  air. 

Thus,  if  a  sample  of  sewage  be  shaken  in  a  bottle  with  an 
excess  of  water  saturated  with  air,  and  allowed  to  stand  a 
sufficient  time,  under  conditions  which  allow  of  an  excess  of 
oxygen  being  always  present,  it  will  be  gradually  transformed, 
and  eventually  nothing  will  be  left  in  the  bottle  but  a  solution 
of  the  above  salts,  with  some  brown  particles  of  '  humus,' 
and  some  carbon  dioxide  in  solution. 

Although  it  can  be  shown  by  careful  analysis  that  the 
sewage  suffers  a  regular  sequence  of  changes,  yet  at  no  point 
are  offensive  gases  evolved  under  these  conditions — and 
neither  marsh  gas  nor  hydrogen  is  produced. 

In  the  above  case  the  sewage  is  purified  under  strictly 
aerobic  conditions.  In  practice  such  conditions  are  met  with 
when  sewage  is  discharged  into  a  stream  or  body  of  water,  of 
such  a  volume  that  an  excess  of  dissolved  oxygen  is  always 
present,  over  that  necessary  to  oxidise  the  sewage. 

But  it  is  rare  to  find  conditions  under  which  it  is  possible 
to  deal  with  sewage  in  this  way,  by  what  may  be  termed  the 
dilution  method.  A  favourably  situated  outfall  must  admit 

1  Strictly  speaking  the  term  '  humus  '  should  be  reserved  for  organic 
residual  matter  of  special  chemical  characteristics.  For  the  sake  of 
brevity  the  term  is  used  here  to  include  organic  matters  of  somewhat 
indefinite  composition  which  remain  undecomposed  at  the  end  of  the 
ordinary  processes  of  purification  of  sewage.  They  are  generally  associated 
with  a  fair  proportion  of  mineral  matter,  especially  phosphates,  and  lime, 
and  alumina  compounds. 


SEWAGE    PURIFICATION  283 

of  the  sewage  being  quickly  mixed  with  a  large  excess  of  water, 
so  that  at  all  times  the  oxygen  content  is  maintained  at  such 
a  point  that  offensive  products  cannot  be  produced. 

The  extensive  investigations  of  Letts  and  Adeney  on  the 
pollution  of  estuaries  and  tidal  waters  have  resulted  in  the 
suggestion  of  various  standards,  in  relation  to  the  amount  of 
oxygen  available  in  the  mixed  sewage  and  tidal  water,  to  meet 
the  different  conditions  of  discharge. 

In  the  majority  of  cases,  even  where  the  dilution  method  is 
resorted  to,  and  still  more  when  it  is  a  question  of  purification 
by  application  to  land  or  artificial  filter  beds,  some  form  of 
tank  treatment  is  required,  and  the  chemistry  of  this  process 
may  now  be  considered. 

TANK  TREATMENT  OP  SEWAGE 

When  sewage  passes  through  a  tank  of  any  description, 
deposition  of  the  heavier  matters  present  will  take  place  with 
greater  or  less  completeness  according  to  the  method  of 
construction  of  the  tank,  the  rate  at  which  the  sewage  passes 
through  and  the  addition,  or  otherwise,  of  chemical  coagulants 
to  facilitate  the  deposition  of  the  matters  in  emulsion. 

We  may  consider  tank  treatment,  according  as  it  is 
directed,  to  effect  one  or  other  of  the  following  results  : — 

(a)  Simple  sedimentation ; 

(b)  Anaerobic  decomposition ; 

(c)  Aerobic  decomposition ; 

(d)  Chemical  clarification. 

(a)  Simple  Sedimentation.— In  this  case  we  shall  expect 
only  the  heavier  matters  in  the  sewage  to  be  deposited,  that  is, 
the  mineral  substances,  sand  and  silt,  etc.,  together  with  paper, 
faeces,  grease  and  soap.  The  character  of  the  effluent  will 
depend  on  the  dilution  or  strength  of  the  original  sewage,  and 
the  distance  between  the  sewage  works  and  the  source  of  the 


284      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

sewage.  The  passage  of  sewage  through  sewers  results  in  the 
mechanical  breaking  up  and  emulsification  of  faecal  matter, 
and  the  setting  up  of  ammoniacal  fermentation  of  the  urea 
present  in  the  urine.  The  extent  to  which  these  changes  take 
place  depends  on  the  length  of  sewer  to  be  traversed,  and  also 
on  the  state  and  construction  of  the  sewer.  New  sewers 
with  smooth  surfaces  will  not  readily  allow  the  formation  of 
deposits  of  sewage  matter,  and  consequent  further  fermenta- 
tive change.  With  well-laid  sewers,  only  the  initial  stages 
of  fermentation  of  nitrogenous  matter  will  have  set  in  by  the 
time  the  sewage  reaches  the  works,  and  such  sewage  should 
therefore  be  comparatively  inoffensive.  Moreover,  in  designing 
tanks  for  simple  sedimentation,  they  should  be  of  such  a  size 
that  the  sewage  will  not  remain  in  them  sufficiently  long  for 
any  but  the  preliminary  stages  of  decomposition  to  take  place. 
The  sludge  or  deposit  from  such  a  sedimentation  tank  will, 
in  consequence  of  what  has  been  said,  also  need  to  be  very 
frequently  removed,  if  offensive  decomposition  is  not  to  take 
place,  and  not  only  must  it  be  quickly  removed,  but  it  must 
for  the  same  reason  be  quickly  disposed  of,  e.g.,  by  trenching 
into  the  ground. 

(6)  Anaerobic  Decomposition.— A  tank  designed  to  facili- 
tate anaerobic  decomposition  differs  from  a  sedimentation 
tank  chiefly  in  being  relatively  larger,  and  so  allowing  time 
for  decomposition  to  take  place  under  anaerobic  conditions. 
Such  a  tank  has  been  variously  termed  a  cesspool,  a  septic 
tank,  a  liquefying  tank,  or  a  hydrolytic  tank.  The  differences 
in  design  are  mainly  structural,  to  facilitate  deposition  and 
removal  of  the  solid  matters,  and  to  control  more  or  less  the 
character  and  extent  of  the  chemical  changes  taking  place. 
These  chemical  changes  may  at  this  point  be  usefully  con- 
sidered in  detail. 

Decomposition  of  cellulose. — The  anaerobic  decomposition 
of  cellulose  has  been  considered  in  Chapter  X,  It  was  there 


PLATE   IV. 


[Photo  by  Author, 

(i)  SEWAGE  WORKS  AT  MATUNGA,  NEAR  BOMBAY. 


[Photo  lent  by   H'.  J.  Xewton,  A.M.I.C.E. 

(ii)  PERCOLATING  FILTERS  AT  ACCRINGTON. 


SEWAGE    PURIFICATION  285 

shown  to  be  due  to  two  organisms,  one  of  which  produced 
mainly  hydrogen  and  the  other  marsh  gas.  In  both  cases 
carbon  dioxide  (C02)  and  fatty  acids  are  also  produced  as 
by-products.  The  production  of  gases  is  a  visible  indication 
that  fermentation  is  taking  place  in  the  sewage.  The  evolution 
of  nitrogen  has  often  been  regarded  as  taking  place  in  septic 
tanks  ;  the  author's  experience,  however,  would  tend  to  show 
that  such  nitrogen,  if  it  is  produced,  arises  either  from  the  air 
dissolved  in  the  incoming  sewage,  or  from  the  reduction  of 
nitrates  present  therein,  and  not  from  the  anaerobic  decom- 
position of  nitrogenous  matter.  It  may  be  taken,  therefore, 
that  the  gases  which  are  evolved  in  the  septic  tank  arise 
chiefly  from  the  decomposition  of  cellulose.  The  researches 
of  Omelianski,  described  in  Chapter  X,  showed  that  the 
optimum  temperature  for  this  fermentation  was  above 
90°  F.  For  this  reason  the  activity  of  septic  tanks  in  this 
country,  measured  solely  by  the  gas  evolved,  is  much  greater 
in  summer  than  in  winter,  and  it  never  attains  the  intensity 
observable  in  tropical  countries.  There,  where  the  tempera- 
ture seldom  is  less  than  70°  F.  and  often  of  course  much 
higher,  a  quite  extraordinary  development  of  gas  may  take 
place.  The  illustration  on  Plate  IV  (i)  is  from  a  photograph 
taken  by  the  author  at  the  installation  attached  to  the  leper 
colony  at  Matunga  near  Bombay.  Here  the  tanks  are  pro- 
vided with  gas-tight  iron  covers,  and  the  gas  is  withdrawn  from 
below  these  into  a  gas-holder.  The  carbon  dioxide  is  removed 
by  lime  purifiers,  and  the  inflammable  marsh  gas  and  hydrogen 
used  for  driving  the  engine  which  pumps  the  sewage,  and  also 
for  lighting  and  cooking  purposes.  The  gas-holder,  lime 
purifiers,  and  engine-house  are  indicated  in  the  photograph. 

Such  economic  use  of  the  gas  from  septic  tanks  has  been, 
to  a  limited  extent,  adopted  in  this  country,  but,  owing  to  the 
temperature  conditions,  it  is  hardly  likely  to  find  wide  applica- 
tion on  the  large  scale,  and  artificial  raising  of  the  temperature 
of  large  volumes  of  sewage  is  out  of  the  question.  It  is, 


286      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

however,  worthy  of  suggestion  whether  in  the  case  of  small 
manufacturing  premises,  where  much  waste  cellulose  matter 
accumulates,  e.g.,  packing  paper,  extracted  plants  from  drug 
manufactories,  waste  hops  from  breweries,  etc.,  it  might  not 
be  economical  to  produce  gas  in  this  way  in  small  tanks, 
maintained  at  the  optimum  temperature  by  waste  steam. 

Ammoniacal  fermentation. — As  already  mentioned,  the 
greater  part  of  the  nitrogen  of  sewage  is  present  as  urea,  and 
the  ammoniacal  fermentation  readily  sets  in  ;  in  many  cases 
indeed  it  may  be  almost  complete  before  the  sewage  reaches 
the  purification  works.  A  great  deal  of  discussion  as  to  the 
necessity  or  otherwise  of  preliminary  anaerobic  treatment 
of  sewage,  has  been  confused  by  the  failure  clearly  to  dis- 
tinguish between  absolutely  fresh  sewage  and  sewage  which 
has  passed,  it  may  be,  through  several  miles  of  sewers.  The 
consideration  of  the  conditions  necessary  for  nitrification; 
which  found  its  place  in  Chapter  XIII,  shows  the  necessity 
for  a  preliminary  conversion  of  urea,  and  allied  substances 
such  as  amino  acids,  into  ammonia  before  nitrification  takes 
place.  The  author  has  found  beyond  question  that  if 
absolutely  fresh  sewage  is  to  be  put  upon  a  filter,  considerably 
more  filter  space  is  requisite  to  convert  the  nitrogen  into 
nitrates,  than  if  time  is  first  aUowed,  e.g.,  by  retention  in 
tanks,  for  ammoniacal  fermentation  to  take  place.  It  is, 
however,  rarely,  in  the  case  of  an  ordinary  town  sewage  works, 
that  sewage  is  met  with  in  such  a  fresh  condition  ;  as  already 
stated,  ammoniacal  fermentation  nearly  completes  itself  in 
the  sewers,  or  at  any  rate  during  a  comparatively  short  tank 
treatment.  It  is  doubtful  whether  urea  would  ever  be  found 
in  an  ordinary  sample  of  town  sewage. 

Apart  from  the  urea,  however,  the  other  nitrogenous 
constituents  of  the  sewage  have  to  be  considered ;  these  are 
of  a  very  complex  character.  Broadly  speaking,  all  the 
various  decomposition  products  of  albumin  will  be  represented 
in  some  form  or  other,  together  with  actual  undigested  portions 


SEWAGE    PURIFICATION  287 

of  nitrogenous  food.  Apart  from  undigested  food,  it  has 
been  shown  that  the  bulk  of  faeces  consists  of  intestinal 
secretions,  epithelium  detritus,  etc.,  and  masses  of  bacteria. 
Under  the  conditions  maintained  within  the  anaerobic  tank 
these  will  all  gradually  break  down,  and  an  important  con- 
sideration here  arises,  viz.,  as  to  how  far  this  breaking  down 
should  be  carried.  We  have  seen  that  eventually,  by  the 
decomposition  of  albuminous  matter,  evil-smelling  substances 
such  as  hydrogen  sulphide,  indol,  skatol,  and  various  amines 
are  produced.  A  frequent  error  in  the  design  of  septic  tanks 
has  been  to  make  these  too  large,  so  that  the  decomposition 
of  nitrogenous  matter  is  carried  more  or  less  to  its  limit,  with 
the  production,  in  many  cases,  of  serious  nuisance.  The 
design  of  anaerobic  tanks  should  be  directed  to  the  rapid 
deposition  of  solid  matter,  and  its  retention  for  a  period 
sufficiently  long  to  enable  it  to  be  broken  down  as  far  as  is 
economically  possible,  while  the  liquid  portion  of  the  sewage 
should  be  led  away  quickly,  sufficient  time  only  being  allowed 
for  ammoniacal  fermentation  and  incipient  proteolysis  to 
take  place  therein.  The  hydrolytic  tank  of  Travis,  and  the 
Emscher-Brunnen  of  Imhofi,  have  this  object  in  view,  but 
any  design  which  distinguishes  between  the  changes  taking 
place  in  the  solid  matter  and  in  the  supernatant  liquid  is 
likely  to  be  more  or  less  successful.  It  has  been  found,  e.g., 
that  in  latrine-tanks,  where  the  greater  part  of  the  faecal 
matter  is  retained  in  a  compartment  at  the  inlet,  separated 
from  the  main  tank  by  a  pigeon-holed  wall,  that  a  very  large 
amount  of  liquefaction  of  retained  solids  takes  place  in  this 
inlet  chamber. 

Decomposition  of  fats. — There  is  evidence  that  besides 
the  decomposition  of  cellulose  and  nitrogenous  products  a 
considerable  change  takes  place  in  the  fatty  constituents  of 
the  sewage  in  the  anaerobic  tank.  Fat  is  always  present  in 
household  refuse  from  the  washing  of  plates  and  dishes  ;  all 
the  soap  which  is  used  finds  its  way  into  the  sewage,  and 


288      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

partially  digested  fatty  matter  is  often  present  in  faeces. 
Free  fat  is  readily  broken  up  by  bacterial  or  enzyme  action, 
yielding  fatty  acids  and  glycerine,  as  has  been  shown  in 
Chapter  XI.  The  higher  fatty  acids  thus  produced  may 
be  further  broken  down  into  soluble  fatty  acids  of  lower 
molecular  weight.  Soaps  also  are  capable  of  change,  but  only 
very  slowly. 

In  the  case  of  small  installations,  attached  to  institutions 
such  as  sanatoria,  asylums,  etc.,  where  a  separate  laundry 
exists,  the  author's  experience  strongly  favours  the  separate 
retention  of  the  soap,  by  treatment  with  lime  salts,  and  col- 
lection of  the  precipitated  lime  soaps  in  specially  devised 
intercepting  traps.  In  such  cases  also  it  is  desirable  to  retain 
the  grease  waste  from  the  kitchen,  which  is  quite  capable  of 
being  readily  and  economically  worked  up  into  soap  on  the 
spot.  The  retention  of  fats,  apart  from  its  economic  aspect, 
greatly  simplifies  the  operations  on  the  sewage  works,  where 
insoluble  soaps  are  liable  to  be  formed,  causing  accumulations 
in  the  tanks  and  clogging  of  the  filters  which  receive  the 
tank  effluent.  The  decomposition  of  fat  also  gives  rise  to  an 
extremely  objectionable  rancid  odour,  due  to  the  formation  of 
butyric  acid. 

To  summarise,  therefore,  the  changes  which  take  place 
in  the  anaerobic  tank,  these  are  mainly  the  decomposition  and 
gasification  of  cellulose,  the  ammoniacal  fermentation  of  urea, 
the  breaking  down  to  a  greater  or  less  extent  of  more  complex 
nitrogenous  substances,  and  the  splitting  of  fats.  These 
changes  are  almost  wholly  due  to  bacteria  and  to  enzymes, 
the  latter  in  all  probability  present  in  faeces.  The  changes 
can  be  followed  by  analysis  of  the  sewage  and  of  the  deposited 
sludge.  In  the  liquid  sewage  it  will  be  found  that  the  free 
ammonia  increases  at  the  expense  of  the  albuminoid ;  the 
oxygen  absorbed  from  permanganate,  while  possibly  not 
greatly  differing  at  the  beginning  and  end  of  the  process  in 
its  total  amount,  will  be  found  to  be  distributed  in  different 


SEWAGE    PURIFICATION  289 

proportion  between  the  easily  oxidised  matter  and  the  more 
difficultly  oxidisable  substances,  or,  in  other  words,  while  the 
oxygen  absorbed  in  four  hours  by  the  so-called  '  four  hours' 
test '  may  not  greatly  decrease,  the  oxygen  absorbed  in  three 
minutes  will  probably  increase. 

Whether  there  is  an  increase  or  decrease  in  colloidal  matter 
depends  upon  circumstances.  With  highly  concentrated 
sewages  such,  e.g.,  as  are  met  with  under  conditions  of  very 
limited  water  supply,  actual  solution  of  colloidal  matter  un- 
doubtedly takes  place,  owing  to  the  breaking  down,  e.g.,  of 
albuminoid  substances  into  amino  acids  ;  on  the  other  hand, 
with  ordinary  town  sewage,  it  may  readily  be  the  case  that 
the  colloidal  substances  in  the  effluent  from  the  tank  increase, 
owing  to  the  washing  out  of  colloids  from  the  sludge  present 
in  the  tank.  In  considering  the  changes  taking  place  in 
anaerobic  tanks,  the  exact  conditions  of  the  installation  must 
always  be  carefully  borne  in  mind. 

It  was  at  one  time  considered  essential  that  anaerobic 
tanks  should  be  closed,  in  order  to  prevent  access  of  light  and 
air ;  numerous  experiments  have  shown  that  this  condition 
is  not  necessary.  It  is  obvious  that  beneath  the  immediate 
surface  of  the  sewage  in  the  tank  the  conditions  must  be  anae- 
robic, and  the  covering  of  tanks  is  only  called  for  by  reasons 
of  sightliness,  or  to  render  more  permanent  the  scum,  which 
generally  forms  owing  to  the  throwing  up  of  solid  matter  by 
the  gases  evolved  during  the  fermentation  of  the  sludge.  A 
cover,  of  course,  is  necessary  if  these  gases  are  to  be  collected 
and  utilised  as  in  the  case  quoted,  but  is  of  little  use  for  the 
prevention  of  nuisance  unless  escaping  gases  are  collected  and 
passed  through  a  washing  tower,  e.g.,  of  coke,  down  which  a 
spray  of  water  passes. 

(c)  Aerobic  Decomposition.— In  the  early  experiments  of 
Mr.  Dibdin  at  Button  in  Surrey,  during  the  later  nineties,  the 
attempt  was  made  directly  to  treat  crude  sewage  on  a  coarse 


290      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

contact  bed,  that  is,  a  tank  filled  with  large  pieces  of  coke, 
burnt  clay  or  other  material.  Such  a  tank  is  first  filled  up 
with  the  sewage,  when  the  insoluble  and  colloidal  matters 
are,  for  the  most  part,  deposited  upon  the  surfaces  of  the 
pieces  of  material  in  the  tank,  and  the  liquid  allowed  to  run  off 
from  the  bottom  of  the  tank.  Air  enters  the  interstices  of  the 
medium  to  replace  the  liquid,  the  tank  is  allowed  to  remain 
empty  for  some  hours,  and  opportunity  is  afforded  for  the 
deposited  organic  matter  to  be  oxidised ;  the  tank  is  then 
filled  again  with  sewage  and  the  cycle  of  operations  repeated. 
A  tank  of  this  kind  may  be  termed  an  aerobic  tank,  and  the 
changes  which  go  on  in  it  are  essentially  different  from  those 
taking  place  under  anaerobic  conditions,  as  described  under  (b). 
The  process  used  at  Sutton  acted  well  in  so  far  that  the 
heavier  suspended  matter  in  the  sewage  was  largely  removed, 
and  converted  in  course  of  time  into  a  nearly  odourless 
residuum.  The  main  drawback  to  the  process  was  the  gradual 
blockage  of  the  interstices  of  the  medium  and  the  difficulty 
of  cleaning  it  without  complete  removal  from  the  tank.  This 
difficulty  Dibdin  seeks  to  avoid  in  his  recently  introduced 
slate  bed.  In  this  case,  instead  of  the  tank  being  filled  with 
irregular  lumps  of  material,  superimposed  horizontal  layers  of 
slate  are  made  use  of,  separated  by  distance  pieces  about 
two  inches  thick.  On  filling  the  tank  with  sewage,  the 
suspended  solids  deposit  themselves  on  the  slates,  and  are 
gradually  oxidised  in  the  same  manner  as  in  the  Sutton 
process.  It  is  possible  to  remove  the  deposit  from  time  to 
time  from  the  surface  of  the  slates  by  flushing  out,  and  so  to 
retain  the  water-holding  capacity  of  the  tank  undiminished. 
The  writer  has  had  occasion  to  examine  with  some  care  the 
changes  which  go  on  in  these  slate  beds,  as  they  are  called.  He 
found  that  the  oxidation  of  the  organic  matter,  and  especially 
of  the  fatty  constituents,  is  largely  due  to  masses  of  nematode 
worms,  with  infusoria,  etc.,  and,  of  course,  bacteria.  The 
deposit  on  the  slates,  in  course  of  time,  assumes  a  liver-like 


SEWAGE    PURIFICATION  291 

consistency  and  can  be  stripped  off  in  pieces  and  examined ; 
the  smell  is  not  offensive,  being  similar  to  that  of  an  exposed 
river  bank. 

If  some  of  the  material  is  placed  in  a  glass  tube  and  air  led 
over  it,  considerable  volumes  of  C02  are  given  off,  through  the 
respiration  and  other  changes  of  the  organisms  present.  If 
the  deposit  is  covered  with  water  and  air  is  excluded,  it  very 
soon  putrefies  and  becomes  offensive  ;  it  is  evident,  therefore, 
that  in  the  working  of  such  tanks  care  must  be  taken  that  the 
conditions  at  no  time  become  anaerobic.  When  the  sewage 
first  enters  the  tank  a  considerable  amount  of  air  is  dissolved 
in  it,  as  it  falls  through  the  slates,  and  a  further  quantity 
is  trapped  underneath  the  slate  surfaces ;  this  is  sufficient  to 
maintain  aerobic  conditions  for  an  hour  or  two,  which  is 
the  length  of  time  which  should  be  allowed  to  elapse  before 
the  tank  is  emptied.  On  flushing  out  the  deposit  and 
allowing  it  to  drain  and  weather  in  the  air,  it  is  gradually 
converted  into  a  brown  inoffensive  mass,  resembling  garden 
mould. 

(d)  Chemical  Clarification.— In  the  chapter  on  the 
chemistry  of  albumins  it  was  shown  that  colloidal  substances 
of  this  nature  could  be  coagulated  and  precipitated  by  addition 
to  their  solutions  of  hydrated  precipitates,  such  as  those  of  iron 
and  aluminium  hydroxides.  This  precipitation  is  made  use  of 
for  the  clarification  of  sewage.  The  chief  precipitants  used 
are  aluminium  sulphate,  ferric  sulphate,  lime  and  ferrous 
sulphate  (green  copperas)  used  in  conjunction.  The  choice 
of  precipitant  depends  on  the  relative  market  price  of  the 
particular  chemicals,  and  on  the  facilities  available  for  their 
efficient  use. 

The  right  adjustment,  e.g.,  of  lime  and  copperas  so  as  to 
keep  the  lime  always  slightly  in  excess,  requires  constant 
attention,  whereas  salts  of  alumina  are  precipitated  directly 
by  the  carbonate  of  ammonia  present  in  the  sewage.  Ferric 

u2 


292       BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

salts  have  been  found  to  be  specially  useful  in  the  case  of 
sewages  containing  an  excessive  amount  of  grease,  e.g.,  at 
Wakefield,  where  much  wool-scouring  refuse  enters  the 
sewers. 

All  processes  of  chemical  precipitation,  while  they  are 
capable  of  yielding  effluents  containing  less  suspended  matter 
than  either  of  the  processes  considered  in  the  foregoing  para- 
graphs, result  in  the  production  of  considerable  quantities  of 
sludge,  which  needs  special  care  in  its  disposal,  as  its  con- 
stituents are  still  capable  of  undergoing  offensive  decomposi- 
tion, differing  thus  from  the  residuum  left  after  well-conducted 
anaerobic  or  aerobic  treatment. 

The  choice  of  one  or  the  other  of  the  methods  of  tank 
treatment  (a),  (b),  (c)  or  (d)  depends  on  local  conditions.  In 
the  case  of  small  communities,  where  constant  attention 
cannot  be  given,  and  also  where  the  fall  is  limited,  anaerobic 
tanks  find  useful  application.  In  certain  cases  also,  notably 
at  Birmingham  and  to  some  extent  at  Manchester,  anaerobic 
treatment  has  been  found  useful,  in  the  first  case  in  order  to 
produce  an  inoffensive  sludge,  and  in  the  second  case  to 
neutralise  to  some  extent  the  effect  of  antiseptic  trade  effluents 
present  in  the  sewage,  before  the  latter  is  finally  treated  on 
filter  beds.  In  both  these  cases,  however,  the  presence  of 
considerable  quantities  of  iron  salts  in  the  sewage  diminishes 
the  chance  of  nuisance,  owing  to  the  combination  of  any 
sulphuretted  hydrogen  produced  with  the  dissolved  iron,  to 
form  black  ferrous  sulphide.  It  must  be  emphasised  that 
anaerobic  treatment,  carried  out  in  ill-designed  tanks  and 
with  imperfect  supervision,  may  be,  and  often  has  been,  a 
serious  source  of  nuisance,  and,  for  this  reason,  preliminary 
aerobic  treatment  has  often  much  to  recommend  it.  The 
slate  process  of  Dibdin  requires,  however,  an  amount  of  fall 
depending  on  the  depth  of  the  bed,  in  addition  to  that  required 
for  the  subsequent  filtration  processes.  If  this  is  available,  the 
process  can  often  find  useful  application,  it  being  understood 


SEWAGE   PURIFICATION  293 

that  some  form  of  catchment  tank  is  necessary  to  retain  the 
suspended  matter  coming  away  from  the  slates. 

It  was  at  one  time  thought  that  the  clarification  of  sewage 
by  means  of  chemicals  must  give  place  entirely  to  biological 
treatment  of  one  sort  or  another.  The  findings  of  the  Koyal 
Commission  on  Sewage  Disposal  have,  quite  rightly,  in  the 
author's  opinion,  suggested  that  many  cases  still  exist  where 
this  method  of  purification  is  to  be  preferred.  Where  the 
large  amount  of  sludge  produced  by  chemical  precipitation 
can  be  easily  and  cheaply  disposed  of,  and  where  the  space 
available  for  the  final  filtration  process  is  limited,  the  total 
expense  involved  will  probably  be  less  by  this  method  than 
by  any  other,  owing  to  the  longer  life  of  the  filter  beds  in  con- 
sequence of  the  small  amount  of  suspended  solids  passing  on 
to  them.  A  typical  case  for  the  application  of  chemicals  is 
afforded  by  the  conditions  of  the  sewage  works  at  Salford. 
Here  the  available  area  of  filters  is  necessarily  very  restricted, 
owing  to  the  site  of  the  works,  and  to  maintain  the  high  rate 
of  filtration  necessary  if  the  sewage  is  to  be  dealt  with 
thoroughly,  preliminary  treatment  is  called  for.  On  the 
other  hand,  as  the  sludge  is  sent  to  sea  in  a  steamer,  the 
standing  charges  of  which  have  always  to  be  maintained,  an 
increase  in  the  sludge  production  does  not  necessarily  mean 
a  proportional  increase  in  the  cost. 

There  are  definite  limits  to  the  economic  use  of  chemicals. 
It  has  been  shown  that  beyond  a  certain  point  an  increase  in 
the  amount  of  chemicals  added  does  not  produce  a  proportional 
reduction  in  the  amount  of  suspended  matter.  Further,  with 
very  dilute  sewages,  the  colloidal  matter  to  be  removed  is 
disseminated  through  a  large  volume  of  water,  so  that  very 
considerable  quantities  of  chemicals  have  to  be  added  in 
order  to  precipitate  it,  and  here  again  the  cost  is  out  of 
proportion  to  the  purifying  effect  obtained.  Speaking  gener- 
ally, therefore,  the  use  of  chemical  clarification  may  be 
recommended  where  the  sewage  is  concentrated,  where  the 


294      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

available  filtration  area  is  limited,  and  where   the  sludge  is 
easily  disposed  of. 

A  further  case  for  chemical  treatment  may  arise  where 
special  trade  effluents  are  present.  Thus,  at  Bilston  and 
Wolverhampton,  large  quantities  of  lime  have  to  be  added, 
to  neutralise  and  precipitate  the  acid  solution  of  ferrous 
chloride,  or  '  iron  pickle/  discharged  into  the  sewers  from 
galvanising  works. 

THE  FINAL  PURIFICATION  OF  SEWAGE 

In  general,  as  has  been  explained,  some  form  of  preliminary 
treatment  is  necessary  before  sewage  can  be  finally  mineralised 
in  biological  filter  beds.  It  is  possible,  however,  under  special 
conditions  to  treat  crude  sewage  directly  on  filters.  Where 
the  sewage  is  dilute,  and  where  considerable  fall  is  available, 
the  liquid,  after  efficient  screening  and  removal  of  the  coarser 
solids,  sand,  etc.,  in  catchpits,  may  be  directly  sprayed  upon 
coarse  percolating  filters  of  considerable  depth.  In  this  case 
the  oxidation  of  the  suspended  and  colloidal  matters  takes 
place  by  much  the  same  agencies  as  are  at  work  in  the  slate 
bed,  above  described,  and  the  resulting  granular  residue  passes 
out  at  the  bottom  of  the  filter,  and  can  be  retained  in  catchpits, 
or  on  the  surface  of  sand  strainers.  The  works  at  Rothwell 
in  the  West  Riding  of  Yorkshire  have  been  successfully 
designed  on  these  lines.  The  conditions  differ  in  such  a  filter 
from  those  obtaining  in  the  slate  bed,  in  that  the  liquid 
portion  of  the  sewage  passes  in  a  thin  film  over  the  filtering 
medium,  and  its  soluble  impurities  are  therefore  oxidised  as 
well  as  the  matters  in  suspension.  If  the  rate  of  filtration  is 
not  too  high,  it  is  even  possible  completely  to  oxidise  fairly 
strong  sewage  in  this  way.  A  periodical  renewal  of  a  portion 
of  the  filtering  medium  is,  however,  likely  to  be  called  for  in 
such  a  case.  A  good  instance  of  the  adequate  treatment  of 
strong  sewage  is  to  be  seen  at  Little  Drayton,  where  a  filter, 


SEWAGE    PURIFICATION  295 

on  the  plan  devised  by  the  late  Colonel  Ducat,  has  been  in  use 
for  many  years,  and  has  been  reported  upon  by  the  Royal 
Commission. 

In  the  majority  of  cases  it  will  probably  be  necessary,  or 
at  any  rate  preferable,  to  adopt  some  form  of  preliminary 
treatment  for  the  sewage,  before  its  final  purification  on 
filter  beds,  and  we  may  now  consider  the  changes  which  take 
place  when  such  partially  treated  sewage  is  applied  to  filters. 

The  artificial  niters  in  general  use  are  of  two  types,  which 
may  be  broadly  divided,  according  as  the  sewage  is  applied 
intermittently  or  continuously,  into — 

(a)  Contact  beds ; 

(6)  Percolating  or  trickling  filters. 

Primary 
Contact  Bed 

a™,,.- 

Valve 


Fia.   27. — CONTACT  FILTER  BED. 

(a)  Contact  Beds. — The  general  design  of  a  contact  bed 
is  seen  in  Fig.  27.  It  consists  of  a  water-tight  tank,  generally 
of  concrete,  filled  with  filtering  material  carefully  screened 
and  graded  to  a  definite  size.  The  essentials  of  such  a 
material  are  that  it  should  be  durable,  that  is,  not  likely  to 
crumble  on  use,  and  should  expose  as  large  a  surface  as 
possible.  Hard  well-fused  clinkers  fulfil  this  condition  most 
perfectly,  but  other  materials  may  also  be  used,  if  good 
clinker  is  not  available.  Effluent  from  the  preliminary  process 
is  passed  on  to  such  a  bed,  and  allowed  to  remain  in  contact, 
generally  for  about  two  hours,  and  then  run  off ;  and  if  not 
sufficiently  purified,  submitted  to  similar  treatment  on  another 
bed,  at  a  lower  level.  The  material  in  the  second  contact 


296      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

bed  must  be  of  smaller  dimensions  than  that  in  the  primary 
bed,  if  the  best  results  are  to  be  obtained.  In  special  cases  a 
third  treatment  on  still  finer  grade  material,  e.g.,  sand,  may  be 
called  for. 

The  following  are  the  principal  changes  which  take  place 
in  a  contact  bed.  The  suspended  and  colloidal  matter,  still 
present  in  the  liquid  to  be  treated,  is  mechanically  retained 
by  the  filtering  medium.  It  is  evident,  therefore,  that  the 
fineness  of  this  medium  must  increase,  as  the  amount  of 
suspended  and  colloidal  matter  decreases,  if  this  mechanical 
effect  is  to  be  obtained.  Besides  the  mere  mechanical  strain- 
ing, a  species  of  absorptive  action  also  takes  place  between  the 
surface  of  the  medium  and  the  constituents  of  the  sewage, 
which  increases  within  limits,  as  the  slimy  layer  thus  formed 
on  each  fragment  of  filtering  material  becomes  more  well 
defined.  This  slimy  layer  also  acts  as  a  sponge  retaining  an 
appreciable  proportion  of  the  liquid  applied,  together  with  its 
dissolved  constituents.  A  considerable  amount  of  purification 
will  therefore  take  place  by  purely  mechanical  and  absorptive 
action,  immediately  the  liquid  is  applied  to  the  filter.  This 
action,  however,  would  very  soon  cease,  and  the  contact  bed 
become  clogged  and  foul,  were  it  not  for  the  biological  activities 
which  are  set  up  within  it.  These  activities  are  exceedingly 
various,  and  depend  not  only  on  the  life  of  bacteria,  but  on 
many  higher  organisms,  notably  small  worms  and  many  species 
of  infusoria.  Kecent  researches,  carried  out  more  particularly 
at  the  Government  Experimental  Station  in  Berlin,  have 
emphasised  the  functions  of  these  higher  organisms,  and  it  is 
here  that  the  choice  of  the  preliminary  treatment,  whether  by 
simple  sedimentation,  by  anaerobic  or  aerobic  tanks,  or  by 
chemical  precipitation,  needs  careful  study.  If  a  sample  of 
sewage  be  collected  in  a  sterile  bottle,  and  allowed  to  stand 
freely  exposed  to  the  air,  but  protected  from  infection  by  a 
plug  of  cotton  wool,  a  film  of  organic  life  generally  makes 
its  appearance.  If  this  is  carefully  examined  under  the 


SEWAGE   PURIFICATION  297 

microscope,  after  the  lapse  of  some  days,  or  even  weeks, 
numerous  forms  of  life  are  generally  visible.  This  life, 
potentially  present  in  the  sewage,  is  probably  an  important 
initial  source  of  population  of  the  sewage  filter  beds.  The 
effect  of  the  different  methods  of  preliminary  treatment,  above 
referred  to,  upon  this  organic  life,  has  been  only  imperfectly 
studied  as  yet.  We  should  expect,  a  priori,  that  effluents 
from  simple  sedimentation,  or  from  the  aerobic  tank,  would 
be  more  favourable  to  the  existence  of  aerobic  organisms 
of  this  sort  than  either  anaerobic  treatment,  which  might 
destroy  them  owing  to  the  absence  of  oxygen,  or  chemical 
precipitation,  which  would  tend  mechanically  to  remove  them. 
It  is  not  unlikely  that  the  organic  life  of  sewage  will  vary 
according  to  the  amount  of  subsoil  and  surface  water  drainage 
entering  the  sewers.  The  author  has  indeed  found,  in  investi- 
gating the  conditions  of  purification  of  sewage  obtained  in  an 
absolutely  fresh  condition,  without  admixture  of  surface 
water,  that  decomposition  and  nitrification  take  place  with 
extreme  slowness,  when  the  sewage  is  allowed  spontaneously 
to  oxidise  in  a  bottle.  Inoculation,  by  means  of  medium 
from  a  filter,  greatly  accelerated  the  rate  of  oxidation.  He 
has  further  found  that  the  effluent  from  an  aerobic  tank 
oxidised  spontaneously  more  quickly  than  the  effluent  from 
chemical  precipitation,  containing  an  equivalent  amount  of 
oxidisable  matter. 

Whatever  the  primary  source  of  the  population  of  a  sewage 
filter  bed  may  be,  whether  derived  from  the  original  sewage 
or  from  the  bacteria  naturally  present  in  all  unsterilised 
material,  such  as  is  likely  to  be  used  for  the  construction  of 
such  filters,  there  is  no  doubt  that,  in  course  of  time,  countless 
numbers  of  bacteria,  and  other  organisms  of  the  nature 
specified  above,  establish  themselves  in  the  filter.  During 
the  period  when  the  contact  bed  is  empty,  and  when  conse- 
quently its  interstitial  spaces  are  full  of  air,  these  organisms 
act  upon  the  suspended  and  dissolved  impurities  retained 


298       BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

by  the  filtering  medium.  Unbroken  down  albuminoid  matter 
is  further  peptonised,  and  ammonia  is  oxidised  to  nitrite 
and  finally  nitrate.  Fatty  acids  and  other  carbonaceous 
matters  are  finally  oxidised  to  C02.  This  can  be  verified,  if 
the  gases  in  the  interior  of  such  a  bed  are  drawn  off  and 
analysed,  when  a  marked  increase  in  the  C02  over  that  present 
in  the  atmosphere  will  be  noticed.  Moreover,  if  a  portion 
of  the  filtering  material  is  carefully  removed  from  the  bed 
without  disturbing  its  coating  of  slime  and  is  placed  in  a  closed 
vessel  provided  with  a  manometer,  an  appreciable  rise  in  the 
mercury  may  be  observed,  owing  to  the  absorption  of  the 
oxygen  in  the  containing  vessel.  The  presence  of  nitrates  can 
be  determined  by  washing  the  material  with  water  free  from 
nitrate,  and  testing  for  the  presence  of  the  latter  in  the 
washings. 

The  changes  just  described  take  place  while  the  bed  is 
standing  empty,  and  are  characterised  by  the  predominance 
of  nitrification ;  when  the  bed  is  again  filled  with  liquid  a 
somewhat  different  set  of  conditions  arises.  Mechanical 
absorption  of  the  more  insoluble  matter  takes  place  as 
already  described,  but  oxidation  also  occurs,  through  inter- 
action of  the  nitrates  present  with  these  substances  and  with 
impurities  present  in  solution  ;  in  this  way  finely  divided 
cellulose  may  be  finally  oxidised,  as  was  explained  in 
Chapter  X.  During  these  changes,  which  may  be  grouped 
together  as  de-nitrification  changes,  loss  of  nitrogen  occurs,  as 
has  been  shown  in  Chapter  XIII,  either  as  free  nitrogen  or, 
it  may  be,  as  nitrous  oxide,  N20,  this  gas  having  actually 
been  discovered  by  Letts  in  solution  in  the  liquid  contents  of 
a  contact  bed. 

The  proper  working  of  a  contact  bed  can  be  controlled,  by 
having  regard  particularly  to  the  amount  of  nitrate  present  in 
the  effluent,  especially  in  the  first  discharge  after  a  long  period 
of  rest.  The  nitrates  present  represent  the  overplus  left  after 
de-nitrification  has  taken  place  ;  within  limits,  the  longer  the 


SEWAGE    PURIFICATION  299 

period  of  standing  empty,  the  greater  will  be  the  amount  of 
nitrate  found,  but  if  nitrates  are  present  at  all,  it  is  evident  that 
the  conditions  are  still  mainly  aerobic,  and  therefore  suited 
to  the  maintenance  of  organic  life.  If  a  contact  bed  becomes 
clogged  and  waterlogged,  not  only  will  nitrates  be  absent 
in  the  effluent,  but  very  often  crowds  of  worms  will  emerge 
at  the  surface  of  the  bed,  seeking  their  necessary  air 
supply. 

An  interesting  application  of  de-nitrification  has  been  made 
by  Letts  at  Belfast,  whose  object  was  to  produce  an  effluent 
containing  as  little  nitrogen  as  possible,  either  in  the  form  of 
ammonia  or  nitrate,  in  order  to  minimise  the  growth  of  Viva 
latissima,  which  was  found  to  derive  its  nitrogen  equally  well 
from  either  of  these  sources.  Letts  purified  a  portion  of  his 
effluent  by  means  of  trickling  filters  in  order  to  obtain  as  high 
a  yield  of  nitrate  as  possible ;  this  nitrified  effluent  was  then 
mixed  with  the  remainder  of  the  unfiltered  effluent,  and  the 
mixture  treated  on  a  de-nitrifying  bed.  The  nitrates  in  the  one 
portion  interacted  with  the  organic  matter  in  the  other,  with 
elimination  of  nitrogen,  and  production  of  a  purified  effluent, 
containing  a  minimum  of  nutriment  for  the  Viva. 

(b)  Trickling  or  Percolating  Filters.— The  operation  of  a 
trickling  filter  differs  from  that  of  a  contact  bed,  in  that  the 
liquid  is  applied  to  it  in  such  a  way  that  it  flows  over  the  frag- 
ments of  filtering  medium  in  a  thin  film,  and  the  oxidation  pro- 
cess is  consequently  continuously  proceeding.  It  is  in  this 
sense  that  the  trickling  filter  may  be  spoken  of  as  a  continuous 
filter,  as  distinguished  from  a  contact  bed,  whose  operation 
is  intermittent,  and  clearly  divisible,  as  we  have  seen,  into 
two  distinct  processes.  Mechanically  speaking,  it  is  doubtful 
whether  a  really  continuous  filter  has  yet  been  constructed. 
When  a  sewage  effluent,  e.g.,  is  sprinkled  upon  a  trickling 
filter  by  an  ordinary  rotary  distributor,  the  operation  is 
really,  of  course,  a  discontinuous  one,  each  element  of  surface 


300      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

receiving  a  dose  of  liquid  at  given  intervals  of  time,  depending 
on  the  speed  of  rotation  of  the  sprinkler. 

It  is  not  necessary  here  to  describe  in  detail  the  various 
methods  for  applying  sewage  effluent  to  trickling  filters,  an 
account  of  them  will  be  found  in  text-books  dealing  with  the 
engineering  side  of  the  problem.  It  will  only  be  briefly 
mentioned  that  distribution  may  be  effected  by  simple  inter- 
mittent discharge  on  to  a  surface  of  fine  material,  by  rotary 
distributors  such  as  are  indicated  in  Plate  IV  (ii),  by  spray 
jets  (Fig.  28),  and  by  other  mechanical  devices  of  more  or  less 
complexity. 


Supply  Pipe 


Distribution 
>/ 


Collecting 
Channel 


FIG.  28. — PERCOLATING  FILTER  WITH  SPRINKLERS. 


.  Plate  IV  (ii)  shows  a  set  of  trickling  filters  at  the  Accrington 
Sewage  Works,  which  will  sufficiently  indicate  their  general 
appearance. 

We  have  here  carefully  to  consider,  assuming  equable 
distribution  of  the  liquid  upon  the  filtering  material,  what  the 
physical  and  mechanical  conditions  are  which  result  in  the 
production  of  a  purified  effluent. 

The  efficiency  of  a  sewage  filter  depends  on  the  total  effec- 
tive surface  area  of  the  filtering  material,  together  with  a 
sufficient  air  supply.  By  effective  surface  area  is  here  meant 
the  sum  of  the  surface  areas  of  the  fragments  of  material. 
The  surface  area  of  the  filter  may  be  spoken  of  as  the  upper 
surface  area.  Thus,  if  large-sized  material  is  used,  a  greater 
quantity  of  it  is  necessary  in  order  to  obtain  the  same  total 


SEWAGE    PURIFICATION  301 

surface  area.  On  the  other  hand,  the  material  may  be  so  far 
subdivided  that  the  interstices  rapidly  fill  up  with  gelatinous 
matter,  which  in  its  turn  holds  up  water,  so  that  the  interstices 
become  reduced  and  the  circulation  of  air  is  interfered 
with. 

In  general,  therefore,  it  will  be  found  economical  to  use  the 
smallest  material  which  allows  of  free  circulation  of  air. 
The  main  direction  of  air  circulation  in  a  trickling  filter  is 
probably  from  above  downwards,  the  air  being  drawn  through 
the  filter  by  the  percolation  of  the  liquid.  An  exception  to 
this  rule  may  occur  in  cold  weather,  when  the  higher  tempera- 
ture of  the  filter,  as  compared  with  the  outside  air,  may  induce 
an  upward  current. 

It  is  obvious  that  filtering  material  must  be  avoided  which 
tends  to  weather  and  break  down,  as  the  interstices  will  then 
tend  to  be  filled  with  small  pieces  of  broken-down  material, 
and  air  circulation  will  be  impeded.  In  order  to  obtain  the 
greatest  possible  surface  area,  material  of  an  irregular  character, 
such  as  hard  furnace  clinker,  gives  the  best  results,  but  other 
available  material  can  be  used,  so  long  as  it  is  not  so  smooth  as 
to  exert  little  or  no  retaining  power,  or  retentivity,  on  the 
gelatinous  matter  deposited  upon  it. 

Further,  thorough  drainage  is  essential ;  otherwise  water 
will  tend  to  be  held  up  by  capillary  attraction  in  the  bottom 
layers  of  the  filter,  and  will  interfere  with  air  circulation.  For 
this  reason  a  concrete  bottom  for  the  filtering  medium  is  advis- 
able, the  thickness  of  which  will  depend  on  engineering  con- 
siderations. These  conditions  of  efficiency  apply  equally 
both  to  contact  beds  and  to  trickling  filters. 

It  is  obvious  that  if  the  filtering  material  is  to  be  fully 
made  use  of,  efficient  and  equable  distribution  of  the  effluent 
over  every  part  of  the  filter  is  essential ;  it  may,  however,  be 
pointed  out  that  in  certain  circumstances,  especially  in  small 
works,  it  is  well  to  have  an  ample  surface  area  of  material,  so 
that  the  efficiency  of  the  process  shall  not  be  too  dependent  on 


302      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

the  exact  operation  of  mechanical  devices  ;  in  other  words,  a 
large  factor  of  safety  should  be  provided. 

The  physical  conditions  governing  the  rate  of  passage  of 
liquid  through  trickling  filters  have  been  studied  by  W.  Clifford 
in  the  researches  referred  to  on  p.  227. 

We  are  now  in  a  position  to  follow  the  changes  which  take 
place  in  such  a  filter.  In  the  first  place,  as  in  the  contact 
bed,  a  purely  mechanical  effect  is  exerted,  and  the  suspended 
and  colloidal  matters  deposit  themselves  on  the  surface  of 
the  medium.  This  will  take  place  obviously  to  a  greater 
extent  in  the  upper  layers  of  the  filter,  and  there  is  conse- 
quently a  limit  to  the  depth  of  such  filters,  owing  to  the  con- 
centration of  deposited  matter  in  the  upper  layers,  which  will 
take  place  if  such  effluent  is  poured  upon  them  at  a  very 
high  rate.  For  this  reason  also,  trickling  filters  are  better 
adapted  to  deal  with  large  volumes  of  dilute  effluent,  rather 
than  with  a  more  concentrated  liquid,  the  application  of 
which  results  in  a  rapid  accumulation  of  undigested  organic 
matter  in  the  upper  layers  of  the  filter.  In  course  of  time 
forms  of  life  establish  themselves  in  these  filters,  worms, 
larvae,  infusoria  and  bacteria,  which  maintain  the  cycle  of 
changes.  Albuminoid  substances  are  broken  down  to  amino 
compounds,  and  finally  oxidised  to  nitrates.  The  trickling 
filter  differs  from  the  contact  bed  primarily  in  the  predomin- 
ance of  nitrification,  owing  to  the  constant  presence  of  oxygen 
in  its  interstices.  No  doubt  some  de-nitrification  takes  place 
in  the  interstices  of  the  medium,  but  speaking  generally,  a 
greater  proportion  of  the  nitrogen  is  recovered  as  nitrate  than 
in  the  case  of  a  contact  bed.  A  further  great  advantage 
possessed  by  the  trickling  filter  is  that  the  effluent  passing 
away  from  it  is  constantly  saturated  with  dissolved  oxygen, 
and  consequently  the  effluents  from  these  filters  contain  in 
general  a  greater  reserve  of  oxygen,  available  for  further 
purification  in  the  stream  into  which  the  effluent  may  flow. 
On  the  other  hand,  owing  to  their  method  of  operation,  there 


SEWAGE    PURIFICATION  303 

is  a  greater  tendency  for  incompletely  oxidised  nitrogenous 
matter  to  pass  away  from  them,  either  in  solution,  in  the 
colloidal  state,  as  granular  residual  '  humus,'  or  as  debris  of 
growths  formed  in  the  filter.  In  the  contact  bed,  as  has  been 
shown,  the  oxygen  of  the  nitrate  interacts  with  the  undecom- 
posed  oxidisable  matter  during  the  period  of  standing  full. 

It  is  necessary,  in  the  case  of  trickling  filters,  that  means 
should  in  all  cases  be  provided  for  arresting  suspended  matter 
which  continuously  passes  away  from  them.  For  this  purpose 
either  so-called  '  humus  tanks  '  or  sand  strainers  may  be 
employed.  Especially  are  these  necessary  after  the  filter 
has  had  a  period  of  rest.  The  colloidal  matter  deposited  on 
the  filtering  medium  suffers  oxidation  during  such  times,  and 
is  rendered  granular,  and  readily  detaches  itself  in  conse- 
quence from  the  filtering  material.  At  such  times,  therefore, 
the  effluent  will  contain  abnormal  quantities  of  solid  matter. 
This  is  also  the  case  in  spring  time,  when  the  organic  life  in 
the  filter  is  particularly  active.  The  material  which  has 
been  stored  during  the  previous  months  is  then  to  a  large 
extent  ejected  from  the  filter. 

This  phenomenon  is  further  instructive,  as  showing  that 
the  changes  taking  place  in  these  filters  are  by  no  means 
instantaneous,  but  take  place  over  a  prolonged  period  of  time. 

CONDITIONS  GOVERNING  CHOICE  OP  FILTRATION  METHODS 

In  designing  works  for  the  purification  of  sewage,  the 
choice  of  method  for  the  final  purification  of  the  effluent 
must  depend  on  a  number  of  factors,  more  particularly  the 
following  :  (a)  area  of  land  and  fall  available  ;  (6)  strength 
of  sewage  ;  (c)  methods  of  preliminary  treatment.  The  full 
discussion  of  this  aspect  of  the  question  involves  engineering 
considerations,  which  are  outside  the  scope  of  this  work,  but 
it  is  obvious  that  deep  trickling  filters  can  only  be  made  use 
of  economically  when  there  is  sufficient  fall  between  the 


304      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

outlet  of  the  tanks  and  the  point  of  final  discharge.  More- 
over, even  if  the  total  amount  of  fall  be  adequate,  yet  if  the 
ground  slopes  gradually  away,  it  may  be  more  economical  to 
put  down  primary  and  secondary  contact  beds  and  so  avoid 
the  excavation  necessary  for  deep  trickling  filters.  The 
amount  of  solid  matter  discharged  from  contact  beds,  especi- 
ally from  secondary  beds  of  fine  material,  is  not  so  great  as 
from  open  percolating  filters,  and  consequently,  in  such  a 
case,  the  final  provision  of  humus  tanks  may  be  on  a  less 
extensive  scale.  This  is  sometimes  a  factor  in  the  choice  of 
method.  On  the  other  hand,  the  material  in  contact  beds 
generally  needs  to  be  removed  and  washed  more  often  than 
the  medium  in  trickling  filters. 

As  already  indicated,  a  concentrated  sewage  lends  itself 
for  various  reasons  to  treatment  by  contact  beds,  at  any  rate 
as  a  preliminary  step.  Probably  the  most  satisfactory  method 
in  such  a  case  is  preliminary  treatment  in  contact  beds, 
followed  by  final  purification  on  trickling  filters.  For  weak 
sewages,  it  is  probable  that  trickling  filters  are  always  to  be 
preferred.  The  method  of  preliminary  treatment  conditions 
primarily  the  grade  of  material  to  be  used  on  the  filter  bed. 
The  freer  the  effluent  from  suspended  or  colloidal  matter, 
the  finer  the  grade  of  material  that  can  be  economically  used. 
Where  there  is  little  depth  available  for  filter  beds,  the  thorough 
clarification  of  the  sewage  may  be  desirable,  so  that  fine-grade 
material  can  be  used,  and  the  lack  of  depth  made  up  for  by 
the  extended  surface  area  of  the  particles. 

THE    INTERPRETATION    OF    SEWAGE    AND    WATER    ANALYSES 

The  methods  used  in  the  analysis  of  sewage  and  sewage 
effluents  must  be  looked  for  in  the  books  specially  devoted 
to  the  subject.  A  short  space  may  be  usefully  given  here  to 
the  interpretation  to  be  placed  on  the  results  of  these  analyses, 
when  viewed  in  the  light  of  the  information  given  in  the  fore- 
going pages. 


SEWAGE    PURIFICATION  305 

The  methods  of  sewage  and  of  water  analysis  are  closely 
allied,  the  chief  difference  being  in  matters  of  detail,  necessi- 
tated by  the  different  quantities  of  oxidisable  or  organic 
matter  which  have  to  be  determined  in  a  given  volume  of  the 
respective  liquids.  As  a  matter  of  fact,  a  sewage  effluent  of 
high  quality  may  contain  no  more  organic  matter  than  a  low- 
grade  drinking  water. 

The  main  difference  between  the  two  branches  of  analysis 
lies  in  the  significance  of  the  presence  of  nitrate  in  the  two 
cases,  and  the  importance  attaching  to  the  determination  of 
the  number  and  character  of  the  bacteria  present. 

A  good  sewage  effluent,  as  we  have  seen,  is  generally 
characterised  by  the  presence  of  an  abundant  proportion  of 
nitrates.  The  presence  of  nitrates  in  a  water  supply  may  often, 
on  the  other  hand,  give  rise  to  suspicion,  as  pointing  to  the 
oxidation  of  previously  present  organic  matter.  An  excep- 
tion to  this  rule  is  met  with  in  the  case  of  deep  well  waters, 
where  the  nitrates  may  arise  from  long  past  deposits.  In  such 
cases,  as  a  rule,  the  nitrates  will  be  unaccompanied  by  nitrites  ; 
the  presence  of  the  latter,  which  are  unstable  intermediate  sub- 
stances, point  to  an  oxidation  process  in  actual  operation,  or 
possibly,  of  course,  de-nitrification  changes  which  may  be 
equally  due  to  organic  matter. 

In  regard  to  the  presence  of  bacteria,  these  are  of  compara- 
tively little  significance  in  the  case  of  an  ordinary  sewage 
effluent,  as  none  of  the  processes  of  sewage  purification  in 
common  use,  short  of  sterilisation  or  slow  sand  filtration,  do 
more  than  reduce  the  number  of  organisms  present.  For  this 
reason  the  detection  of  Bacillus  coli  in  a  drinking  water  is 
presumptive  evidence  of  sewage  pollution.  This  test  is  one  of 
extreme  delicacy  and  it  is,  therefore,  quite  possible  for  a  sample 
of  water  to  pass  the  usual  chemical  tests,  and  yet  to  be  placed 
under  suspicion,  when  examined  bacteriologically. 

The  Analysis  01  Sewage.— Bearing  in  mind  the  importance 

z 


306      BACTERIOLOGICAL   AND    ENZYME    CHEMISTRY 

of  chemical  evidence  in  the  case  of  sewage  or  sewage  effluents, 
the  factors  generally  determined  in  a  sewage  analysis  are  : — 

(a)  Total  oxidisable  matter  as  measured  by  the  oxygen 
absorbed  from  acid  permanganate  in  four  hours  and  in  three 
minutes ; 

(6)  Nitrogen,  either  ammoniacal,  albuminoid,  nitrous  or 
nitric ; 

(c)  Chlorine; 

(d)  Suspended  matter  / 

(e)  Putrescibility ; 

(f)  Consumption  of  dissolved  oxygen. 

The  objects  of  sewage  analysis  may  be  defined  as  follows  : — 

(1)  To  determine  the  character  of  the  sewage  to  be  treated. 

(2)  To  determine  the  efficiency  of  purification  works. 

(3)  To  determine  the  effect  of  the  discharge  of  sewage  or 

effluents  into  various  bodies  of  water,  either  river, 
lake  or  sea. 

1.  Taking  these  objects  in  order,  it  is  of  great  importance, 
when  designing  works  for  sewage  purification,  to  ascertain  the 
concentration  or  strength  of  the  sewage  to  be  treated,  as  the 
amount  of  filter  space  provided  must  necessarily  depend  on 
the  amount  of  organic  matter  to  be  transformed. 

The  Local  Government  Board  has  recently  issued  a  memo- 
randum, based  on  the  Fifth  Report  of  the  Royal  Commission 
on  Sewage  Disposal,  which  defines  roughly  what  is  meant  by 
'  strong/  '  average/  or  '  weak  '  sewage.  Using  permanganate, 
1  c.c.  of  which  equals  one  milligram  of  oxygen  (which  is  ten 
times  the  strength  frequently  used),  and  assuming  that  the 
determination  is  made  at  80°  F.,  the  amount  of  oxygen 
absorbed  by  the  different  strengths  of  sewage  is  taken  as 
follows : — 

'  Strong  '  sewage  17  to  25  parts  per  100,000 

*  Average  '  sewage  10  to  12  parts  per  100,000 

'  Weak  '  sewage   7  to    8  parts  per  100,000 


SEWAGE    PURIFICATION  307 

The  other  analytical  figures,  in  the  absence  of  trade 
effluents,  will  probably  vary  in  proportion. 

It  is,  of  course,  necessary  in  determining  the  strength  of 
sewage  that  an  average  be  taken  if  possible  over  several  days, 
samples  being  taken  hourly  and  mixed  in  proportion  to  the  rate 
of  flow. 

2.  In  determining  the  efficiency  of  purification  works,  the 
analysis   will  show  the   progressive   reduction   in   impurity 
attained  in  the  various  stages  of  the  process.     The  oxygen 
absorption,  the  ammoniacal   and   albuminoid  nitrogen  and 
the  suspended    matters   should    decrease.      A   considerable 
proportion  of  the  nitrogen  should  reappear  as  nitrate.     The 
resultant  effluent   should  have   lost  its  putrescibility,  that 
is  to  say,  when  kept  in  a  closed  and  full  bottle  for  a  few 
days,  at    a   temperature,    say,   of    80°   F.,   it    should    not 
become  offensive. 

The  chlorine  figure,  which  is  due  to  the  sodium  chloride 
present  in  the  sewage,  is  unaltered  by  the  purification  process, 
and  therefore  serves  as  a  useful  index  to  show  whether  the 
effluent  really  represents  the  sewage  from  which  it  is  produced. 
In  a  true  comparison  the  chlorine  number  should  be  the  same 
in  both  cases. 

3.  The  effect  of  an  effluent  upon  a  body  of  water  depends 
essentially,  as  we  have   seen,   on  the  amount  of    dissolved 
oxygen  it  is  capable  of  abstracting  from  a  body  of  water, 
and  the  Royal  Commission  have  therefore  summarised,  as 
it  were,  the  various  methods   of  sewage  analysis,  and  have 
sought  to  define  a  good  effluent  in  terms  of  its  power  of  con- 
suming dissolved  oxygen.     The  importance  of  the  absence 
of  suspended  solids,  which  may  form  troublesome  deposits, 
is  also  recognised,  and  they  suggest  that  an  effluent  would 
generally  be  satisfactory  if  it  complied  with  the  following 
conditions  : — 

(1)  '  That  it  should  not  contain  more  than  three  parts 
per  100,000  of  suspended  matter;  and 

x2 


308      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

(2)  That  after  being  filtered  through  filter  paper  it  should 
not  absorb  more  than  : — 

(a)  0*5  part  by  weight  per  100,000  of  dissolved  or  atmo- 
spheric oxygen  in  twenty-four  hours ; 

(b)  I/O  part  by  weight  per  100,000  of  dissolved  or  atmo- 
spheric oxygen  in  forty-eight  hours  ;   or, 

(c)  1*5  parts  by  weight  per  100,000  of  dissolved  or  atmo- 
spheric oxygen  in  5  days/ 

Although  these  tests  are  open  to  some  criticism  in  matters 
of  detail,  they  do  broadly  serve  to  determine  whether  an 
effluent  is  likely  to  give  rise  to  nuisance  or  not.  They  may  be 
hardly  stringent  enough  for  special  cases,  e.g.,  if  the  effluent 
enters  a  stream  used  for  water  supply  ;  or  on  the  other  hand 
may  be  unnecessarily  severe,  when  ample  dilution  takes  place, 
and  the  water  into  which  the  effluent  is  discharged  is  not  used 
for  drinking. 

The  adequacy  or  otherwise  of  the  purification  effected 
under  given  conditions  can  generally  be  judged  from  a  careful 
examination  of  the  conditions  obtaining  at  the  point  of  dis- 
charge, especially  the  various  forms  of  living  growth  which  can 
be  there  observed.  The  presence  of  Beggiatoa,  for  instance, 
would  indicate  that  unoxidised  sulphides  are  still  present, 
and,  consequently,  that  the  purification  was  imperfect.  Such 
a  state  of  things  is  almost  certain  to  give  rise  to  nuisance. 
Other  forms  of  sewage  fungus,  such  as  Sphaerotilus  natans  or 
Leptomitus  lacteus,  are  also  characteristic  of  imperfect  purifica- 
tion. Certain  protozoa,  such  as  carchesium,  indicate  a  more 
satisfactory  purification,  still  stopping  short,  however,  of 
complete  mineralisation.  A  first-class  effluent  can  generally 
be  recognised  by  an  increased  development  of  healthy  aquatic 
vegetation  in  its  vicinity,  owing,  doubtless,  to  the  nitrates 
present. 

The  Analysis  Of  Water. — Turning  now  to  the  subject  of 
the  analysis  of  water,  while  it  is  true  that  water  may  contain 


SEWAGE    PURIFICATION  309 

very  little  organic  impurity — so  that,  on  the  results  of  chemical 
analysis  alone,  it  might  be  passed  as  satisfactory,  and  yet 
reveal  the  presence  of  B.  coli  when  examined  bacteriologically 
— yet  in  the  author's  experience,  if  a  series  of  comparative 
samples  are  taken,  and  the  analysis  carried  out  with  special 
care,  the  chemical  and  bacteriological  indications  are  usually 
of  the  same  character,  and  the  conditions  which  tend  to  improve 
the  chemical  composition  of  the  water,  tend  also  to  the  removal 
or  diminution  of  dangerous  organisms. 

Thus,  e.g.,  Houston  has  shown  that  prolonged  storage 
tends  gradually  to  decrease  the  number  of  organisms  present 
in  a  water  supply,  and  especially  the  less  resistant  organisms 
such  as  the  typhoid  bacillus.  There  is  no  doubt  that  the 
number  of  bacteria  decreases  as  the  amount  of  pabulum 
diminishes,  and  vice  versa.  Kecent  experiments  in  France  by 
Miquel  and  Mouchet  have  shown  that  the  impurities  in  water 
can  be  oxidised  by  spraying  over  filters  worked  on  similar 
principles  to  the  sewage  trickling  filter,  but  of  course  with 
material  of  smaller  dimensions.  With  the  chemical  improve- 
ment of  the  water,  there  is  again  diminution  in  its  bacterial 
content,  but  an  extraordinary  increase  in  the  number  of 
organisms  takes  place  if  the  filters  are  dosed  with  a  solution  of 
peptone. 

Besides  the  gradual  destruction  of  their  pabulum  which 
takes  place  on  storage,  the  effect  of  sunlight  is  of  great  import- 
ance in  diminishing  the  number  of  bacteria  present,  especially 
certain  kinds,  and  those  the  more  dangerous.  This  aspect  of 
the  matter  has  been  dealt  with  by  Major  W.  W.  Clemesha,  in 
his  extensive  study  of  the  bacteriology  of  drinking  water 
supplies  in  tropical  countries,  undertaken  particularly  in 
reference  to  the  water  supplies  of  Madras.  He  endeavoured 
in  his  researches  to  differentiate  the  various  organisms 
allied  to  Bacillus  coli,  by  an  extension  of  the  method  sug- 
gested by  MacConkey,  who  divided  fsecal  bacilli  into  four 
groups : — 


310      BACTERIOLOGICAL    AND    ENZYME    CHEMISTRY 

Group    I.    Ferments  neither  saccharose  nor  dulcite  ; 

Group  II.  Ferments  dulcite  but  not  saccharose  ; 

Group  III.  Ferments  dulcite  and  saccharose  ; 

Group  IV.  Ferments  saccharose  but  not  dulcite. 

To  these  Major  Clemesha  added  sundry  other  fermentative 
tests,  whereby  he  was  able  to  some  extent  to  classify  numerous 
varieties  of  coli-like  organisms  present,  all  of  which  are 
capable  of  fermenting  lactose.  He  found  that  certain  of 
these  were  characteristic  of  water  which  was  obviously 
recently  polluted ;  others,  on  the  other  hand,  alone  survived 
when  the  water  had  been  exposed  to  the  sun  for  some  time, 
or  was  drawn  from  a  well  after  long  drought,  etc. 

As  nearly  all  the  chief  water  supplies  in  tropical  coun- 
tries are,  so  to  speak,  of  natural  origin,  that  is,  from  wells, 
rivers  or  lakes,  and  are  subject  to  occasional  pollution,  and, 
therefore,  according  to  English  standards  would  be  classi- 
fied as  dangerous,  it  is  obviously  of  importance  to  be  able 
to  differentiate  between  the  residue  of  pollution  and  the 
presence  of  deleterious  matter  of  recent  introduction.  While, 
no  doubt,  further  research  and  many  more  data  are  requisite 
before  it  is  possible,  under  all  circumstances,  to  distinguish 
between  harmless  arid  potentially  dangerous  supplies  by  the 
characteristics  of  the  organisms  present,  or  *by  the  chemical 
reactions  which  they  produce  under  given  conditions,  Major 
Clemesha 's  researches  are  a  very  interesting  application  of 
bacteriological  chemistry  to  the  classification  of  water  supplies. 

The    Biological    Purification    of    Trade    Effluents.— 

Numerous  effluents  from  manufacturing  processes  are  highly 
charged  with  organic  matter,  and  are  capable  of  bacteriological 
purification  by  methods  analogous  to  those  used  in  the 
purification  of  sewage ;  such  effluents  are,  e.g.,  those  from 
breweries  and  distilleries,  from  tanneries  and  hide-dressing 
works,  from  beetroot  sugar  factories,  starch  works,  wool- 
scouring  works,  bone  manure  and  glue  factories. 


SEWAGE    PURIFICATION  311 

Special  methods  have  to  be  used  in  each  case,  according  to 
the  character  of  the  effluent  to  be  treated,  and  dilution  is  fre- 
quently necessary,  e.g.,  in  the  case  of  pot  ale  from  distilleries, 
before  purification  can  be  effected.  In  the  case  of  effluents 
containing  sugar  or  starch,  care  has  to  be  taken  lest  acid 
fermentation  should  set  in,  especially  formation  of  butyric 
acid,  which  is  liable  to  create  serious  nuisance.  For  this 
reason  it  is  generally  found  necessary  to  avoid  anaerobic 
treatment  in  the  case  of  these  effluents,  and  a  preliminary 
addition  of  lime  is  often  advantageous. 


BIBLIOGRAPHY 


General  Text  Books 
Biochemie  der  Pflanzen.     CZAPEK. 

Enzymes  and  their  Applications.     EFFRONT  (translated  by  Prescott). 
Fermentation.     REYNOLDS  GREEN. 
Ferments  and  their  Actions.     OPPENHEIMER. 
Laboratory  Studies  for  Brewing  Students.     ADRIAN  J.  BROWN. 
Monographs  on  Bio- Chemistry.     Edited  by  PLIMMER  and  HOPKINS. 
Technical  Mycology.     LAFAR. 
Traite  de  Micro-biologie.     DUCLAUX. 

Zerzetzung    Stickstofffreier    Organischer     Substanzen    durch    Bakterien. 
EMMERLINQ. 


CHAPTER  I 

Text  Books 

Acht  Vortrage  iiber  Physikalische  Chemie.     VAN'T  HOFF. 
Cell  as  the  Unit  of  Life.     MACFADYAN. 
Chemical  Letters.     LIEBIG. 

Colloids  and  the  Ultra  Microscope.     ZSIGMONDY  (translated  by  Alexander). 
Physical  Chemistry :  its  bearing  on  Biology  and  Medicine.     J.  C.  PHILIP. 
Recent  Advances  in  Physiology  and  Bio-chemistry.     Edited  by  LEONARD 
HILL. 

CHAPTER  II 

Text  Books 

Applied  Bacteriology.     PEARMAIN  and  MOORE. 

Bacteria  and  Public  Health.     NEWMAN. 

Course  of  Elementary  Practical  Bacteriology.     KANTHACK  and  DRYSDALE. 

Fermentation  Organisms.     KLOCKER. 

Manual  of  Bacteriology.     HEWLETT, 


BIBLIOGRAPHY  313 

CHAPTERS  III— IV 
Text  Books 


Modern  Organic  Chemistry.     KEANE. 

On  Light.    TYNDALL. 

Organic  Chemistry.     REMSEN. 

Organic  Chemistry.     COHEN. 

Outlines  of  Organic  Chemistry.     MOORE. 

Physikalische  Krystallographie.     GROTH. 

Practical  Physical  Chemistry.     FINDLAY. 


CHAPTER  V 

Text  Book 
Principles  and  Practice  of  Brewing.     SYKES  and  LINO. 

Scientific  Papers 

BROWN,  MORRIS  and  MILLAR.     Experimental  Methods  employed  in  the 

Determination  of  the  Products  of  Starch   Hydrolysis   by  Diastase. 

Journ.  Chem.  Soc.  Trans.,  1897,  p.  72. 
BROWN  and  MILLAR.     The  Stable  Dextrin  of  Starch  Transformations,  and 

its  Relation  to  the  Maltodextrins  and  Soluble  Starch.    Journ.  Chem. 

Soc.  Trans.,  1899,  p.  315. 
Report  of  Malt-Analysis   Committee  to  the  Council  of  the  Institute  of 

Brewing.     Journal  of  the  Inst.  of  Brewing,  Vol.  XII,  No.  1,  1906. 


CHAPTER  VI 

Scientific  Papers 

B.  H.  BUXTON.     Mycotic  Enzymes.    American  Medicine,  Vol.  VI,  No.  4, 

1903,  pp.  137-142. 
BROWN  and  MORRIS.     Contribution  to  the  Chemistry  and  Physiology  of 

Foliage  Leaves.    Journ.  Chem.  Soc.  Trans.,  1893,  p.  629. 
On  the  Germination  of  some  of  the  Graminae.   Journ.  Chem.  Soc.  Trans., 

1890,  p.  458. 

DELEPINE.     Case  of  Melanomycosis  of  the  Skin.     Trans.  Path.  Soc.  Lond., 
1891. 


314  BIBLIOGEAPHY 


CHAPTER  VII 

A.  CROFT  HILL.     Reversible  Zymohydrolysis.     Journ.  Chew.  Soc.  Trans. 

1898,  p.  634. 
0' SULLIVAN   and   TOMPSON.     On   Invertase.    Journ.     Chem.  Soc.  Trans. 

1890,  p.  926. 


CHAPTER  VIII 

Text  Books 

Die  Zymase-Gahrung.     BUCHNER. 
Etudes  sur  la  Biere.     PASTEUR. 
Principles  and  Practice  of  Brewing.     SYKES.  and  LING. 

Scientific  Papers 

ALBERT.     Einfacher  Versuch  zur  Veranschaulichung  der  Zymase  Wirkuns. 

Ber.  d.  Deut.  Chem.  Ges.,  1900,  XXXIII,  3775. 
BUCHNER.     Alcoholische  Gahrung  ohne  Hefezellen,  Ber.  d.  Deut.  Chem. 

Ges.,  30  (1897),  117  ;   30  (1897),  1110. 
HARDEN  and  YOUNG.     The  Alcoholic  Ferment  of  Yeast-juice.     Roy.  Soc. 

Proc.,  B.,  Vol.  77,  1906,  405 ;   Vol.  78,  1906,  369 ;   Vol.  80,  1908,  299  ; 

Vol.  81,  1909,  336. 
HARDEN   and   NORRIS.     The   Fermentation   of   Galactose   by   Yeast   and 

Yeast-juice.    Roy.  Soc.  Proc.,  B.,  Vol.  82,  1910,  645. 
SLATOR.     Studies  in  Fermentation.    Journ.  Chem.  Soc.  Trans.,  89,  1906, 

128 ;   94,  1908,  217 ;   97,  1910,  922. 


CHAPTER  IX 

FRANKLAND  and  MACGREGOR.  Sarcolactic  Acid  obtained  by  the  Fermenta- 
tion of  Inactive  Lactic  Acid.  Journ.  Chem.  Soc.  Trans.,  63,  1893,  1028. 

HARDEN.  The  Chemical  Action  of  Bacillus  Coli  Communis  on  Carbo- 
hydrates and  Allied  Compounds.  Journ.  Chem.  Soc.  Trans.,  79,  1901, 
610.  (See  also  Trans.  Jenner  Inst.,  2,  1899,  126.) 

HARDEN  and  WALPOLE.  Chemical  Action  of  Bacillus  lactis  aerogenes  on 
Glucose  and  Mannitol.  Roy.  Soc.  Proc.,  B.,  Vol.  77,  1906,  399. 

PROCTER.  Problems  of  the  Leather  Industry.  Journ.  Soc.  Chem.  Ind., 
Vol.  29,  1910,  329. 

WOOD.  Bacteriology  of  the  Leather  Industry.  Journ.  Soc.  Chem.  Ind., 
Vol.  29,  1910,  666. 


BIBLIOGRAPHY  315 

CHAPTER  X 

Text  Books 

Cellulose.  Researches  on  Cellulose,  1895-1900,  1900-1905,  CROSS  and 
BE  VAN. 

Scientific  Papers 

HORACE  T.  BROWN.  On  the  Search  for  a  Cytolytic  Enzyme  in  the 
Digestive  Tract  of  Certain  Grain-feeding  Animals.  Journ.  Chem.  Soc. 
Trans.,  1892,  p.  352. 

C.  VAN  ITERSON.  The  Decomposition  of  Cellulose  by  Aerobic  Organisms. 
Centralblatt  fur  Bakt.,  XI,  No.  23. 

MANGIN.     Composes  Pectiques.     Journ.  de  Botanique,  1891-3. 

OMELIANSKI.     Centralblatt  fur  Bakt.,  II,  1902,  pp.  193  et  seq. 

CHAPTER  XI 

Scientific  Papers 

ARMSTRONG  and  ORMEROD.     Studies  in  Enzyme  Action.     Roy.  Soc.  Proc., 

B.,  Vol.  76,  606 ;  B.,  Vol.  78,  526. 
BERTRAND.     Sur  le  latex  de  1'arbre  a  laque.     Compt.  Rend.,  118,  1894, 

p.  1215. 
GORTNER.     A  Contribution  to  the  Study  of  the  Oxydases.    Journ.  Chem. 

Soc.  Trans.,  Vol.  97,  1910,  p.  110. 

KASTLE  and  LOEVENHART.     Amer.  Chem.  Jour.,  XXIV,  1900,  491. 
MANN.     The  Ferment  of  the  Tea- Leaf.    Part  I,  p.  5,  Indian  Tea  Association. 
YOSHIDA.     Chemistry  of  Lacquer.    Journ.  Chem.  Soc.  Trans.,  43,  1883,  472. 

CHAPTER  XII 

Text  Books 

Chemistry  of  the  Albumins.     SCHRYVER. 

Chemistry  of  the  Proteids.     MANN. 

Untersuchungen  iiber  Amino-sauren  Polypeptide  und  Proteine.     FISCHER, 

Scientific  Papers 

B.  II.  BUXTON.     Construction  of  the  Proteid  Molecule.    American  Medicine, 

Vol.  VI,  No.  15,  pp.  581-3. 
CLARK  and  GAQE.     A  Review  of  Twenty-one  Years'  Experiments  on  the 

Purification  of  Sewage  at  the  Lawrence  Experiment  Station,  pp.  283-5. 
PROCTER.     Problems  of  the  Leather  Industry.     Journ.  Soc.  Chem.  Ind., 

Vol.  29,  1910,  329. 
WOOD.     Bacteriology  of  the  Leather  Industry.     Journ.  Soc.  Chem.  Ind., 

Vol.  29,  1910,  666. 


316  BIBLIOGRAPHY 

CHAPTER  XIH 

Text  Books 

Trait6  de  Chemie  Agricole.     DEHERAIN. 
Traite  de  Chemie  Agricole.     KAYSER. 

Scientific  Papers 

ADENEY.     Appendix  VI.  to  Fifth  Report  of  Royal  Commission  on  Sewage 

Disposal,  pp.  5-111. 

BEYERINCK  and  MINKMAN.     Cent.  /.  BaJct.,  25,  30  Abt.,  II,  1043. 
BOULANGER   and   MASSOL.     '  Recherches   sur   FEpuration   Biologique   et 

Chimique  des  Eaux  d'Egout,'  Calmette  et  confreres,  Vol.  I,  chap.  vi. 

p.  89. 
CLIFFORD.     On  Percolating  Filters.     Proc.  Inst.  Civil  Eng.,  CLXXII,  1908, 

283. 

FRANKLAND,  P.  F.  and  G.  C.     The  Nitrifying  Process  and  its  Specific  Fer- 
ment.    Phil  Trans.  Roy.  Soc.,  B.,  1890,  1107. 
GAYON  and  DUPETIT.    Sur  la  fermentation  des  nitrates.  Compt.  Bend.,  1882, 

pp.  644,  1365. 

MUNRO.     The  Formation  and  Decomposition  of  Nitrates  in  Artificial  Solu- 
tions, and  in  River  and  Spring  Water.      Journ.  Chem.  Soc.  Trans.,  49, 

1886,    632. 

v.  MUSCULTJS.     Sur  le  ferment  de  1'uree.     Compt.  Bend.,  82,  1876. 
SCHLOESING  and  MTINTZ.     Recherches  sur  la  nitrification  par  les  ferments 

organises.     Compt.  Bend.,  V.  84,  1877,  p.  301 ;  V.  85,  1877,  p.  1018  ; 

V.  86,  1878,  p.  892  ;   V.  89,  1879,  pp.  891,  1074. 
SHERIDAN  LEA.     Some  Notes  on  the  Isolation  of  a  Soluble  Urea  Ferment 

from  the  Torula  ureae.     Journal  of  Physiology,  XI,  1890,  226. 
WARINGTON.     On  Nitrification.     Journ.  Chem.  Soc.  Trans.,  1878,  p.  44 ; 

1879,  p.  429 ;    1884,  p.  637  ;    1890,  p.  484. 
WIENOGRADSKI.     Recherches  sur  les  organismes  de  la  nitrification.    Ann.  de 

Vlnstitut  Pasteur,  4  (1890),  213,  257,  760 ;   5  (1891),  92,  577. 


CHAPTER  XIV 

Scientific  Papers 

BEYERINCK,  M.  W.  Uber  Spirillum  desulphuricans  als  Ursache  von  Sulfat- 
reduktion.  Cent.  f.  Bakt.,  2  Abt.,  1895,  I,  p.  1 ;  II,  p.  169. 

A.  VAN  DELDEN.  Beitrag  zur  Kenntniss  der  Sulfatreduction  durch 
Bakterien.  Cent.  f.  Bakt.,  2  Abt.,  1903,  Vol.  XI,  pp.  81  and  113. 

LETTS.  Appendix  VI.  to  Fifth  Report  of  Roval  Commission  on  Sewage 
Disposal,  pp.  111-169. 


BIBLIOGRAPHY  317 

CHAPTER  XV 
Scientific  Papers 

Report  to  the  Government  of  India  containing  an  Account  of  the  Research 
Work  on  Indigo  performed  in  the  University  of  Leeds,  1905-1907,  by 
W.  Popplewell  Bloxam.     1908. 
MANN.    The  Fermentation  of  Tea.     Indian  Tea  Association,  1906. 

CHAPTER  XVI 

Text  Books 

Chemistry  of  the  Farm.     WARINGTON. 
Traite  de  Chemie  Agricole.     DEHERAIN. 
Traite  de  Chemie  Agricole.     KAYSER. 

Scientific  Papers 
E.  J.  RUSSELL.     The  Chemical  Changes  taking  place  during  the  Ensilage 

of  Maize.     Journal  of  Agricultural  Science,  Vol.  II,  Part  4,  July  1908. 
Oxidation  in  Soils  and  its  Connexion  with  Fertility.     Journal  of  Agri- 
cultural Science,  Vol.  I,  Part  3,  October  1905. 
E.  J.  RUSSELL  and  H.  B.  HUTCHINSON.     Effect  of  Partial  Sterilisation  of 

Soil  on  the  Production  of  Plant  Food.     Journal  of  Agricultural  Science, 

VoL  HI,  Part  2,  October  1909. 
MELDOLA.     The  Living  Organism  as  a  Chemical  Agency.    Journ.  Chem.  Soc. 

Trans.,  1906,  Vol.  89,  p.  749. 
WEIGMANN.      Uber  den  jetzigen  Stand  der  bakteriologischen  Forschung 

auf  dem  Gebiete  des    Kasereifungs-prozesses.     Cent.  /.  Bakt.,  2  Abt., 

1896,  H,  150. 

CHAPTER  XVII 

Text  Books 

Examination  of  Water  and  Water  Supplies.     THRESH. 
Filtration  of  Public  Water  Supplies.     HAZEN. 
Micro-organisms  in  Water.     P.  F.  and  G.  C.  FRANKLAND. 
Modern  Methods  of  Water  Purification.     DON  and  CHISHOLM. 
Principles  of  Sewage  Treatment.     DUNBAR  and  CALVERT. 
Sewage  Disposal.     KINNICUTT,  WINSLOW  and  PRATT. 
Sewage  Disposal  Works.     RAIKES. 
Sewage  Works  Analyses.     FOWLER. 
Volumetric  Analysis.     SUTTON. 

Reports 

CLARK  and  GAGE.     A  Review  of  Twenty-one  Years*  Experiments  on  the 

Purification  of  Sewage  at  the  Lawrence  Experiment  Station. 
CLEMESHA.     Report  on  the  Water  Supplies  of  the  Madras  Presidency. 
ROYAL  COMMISSION  ON  SEWAGE  DISPOSAL.     Fifth  Report. 


INDEX 


a-AMlNO-GLUTAEic  acid,  198 

Absorption,  11 

Accrington  Sewage  Works,  300 

Acetaldehyde,  61 

Acetamide,  56,  59 

Acetic  acid,  148,  149,  154,  157 

Acetic  acid  anhydride,  160 
bacteria,  157 
fermentation  of  alcohol,  13 

Acetylene  hydrocarbons,  51 

Acid  albumin,  86 

Acid  amide,  56,  59 

Acrospire,  101 

Adeney,  W.  E.,  221,  223,  226,  283 

Adenin,  200 

Adonite,  93 

Adsorption,  186 

Aerobic  conditions,  162 
tank  treatment,  289 

Agar  medium,  24 

Alanin,  195 

Albert,  138 

Albumin— classification  of,  207-210 ; 
constitution  of,  203-204 ;  in 
living  cell,  12 ;  in  protoplasm, 
182;  in  zymase,  138,  140; 
precipitation  by  metallic  hydr- 
oxides, 186-187  ;  preparation  of 
form  soluble  in  alcohol,  187- 
188  ;  preparation  of  crystalline, 
185  ;  primary  disintegration  pro- 
ducts of,  192-201  ;  products  of 
enzymic  action  on,  191-192 ; 
separation  and  extraction  of, 
204-207  ;  synthesis  of  disintegra- 
tion products  of,  201-202 ;  ultimate 
analysis  of,  188 ;  ultimate  de- 
composition of,  238-239 

Albuminoid  ammonia,  187 


Albuminoids,  209,  257 

Albumoids,  209 

Albumoses,  191 

Alcohol,  52,  131,  132,  155-157 

Alcohol  vapour,  3 

Alcoholic  fermentation  of  sugar,  13, 

132 
Aldehydes,  53,  59,  85,  86,  88,  98, 

145,  146,  148 
Aldohexose,  86 
Aldopentose,  86 
Aldotetrose,  86 
Aldoses,  85-91 
Aleurone,  119 

Aliphatic  compounds,  51,  52 
Alizarin,  1 
Alkali  albumin,  85 
Alkaline  tartrate  solution,   112 
Almond  nitril  glucoside,  96 
Alyl  mustard  oil,  97 
Amine,  59,  239 

Amino-acetic  acid,  56,  59,  192,  194 
Amino  acids,  192,  193 
Amino  compounds,  53,  56,  59 
Amino-di-carboxylic  acid,  198 
Amino-propionic   acid,    195 
Amino-succinic  acid,  198 
Amino-valerianic  acid,   196 
Ammonia,  56,  218 
Ammoniacal  fermentation  of  urine, 

257,  286 

Ammonium  carbonate,  14 
phosphate,  27 
sulphate,  25,  27 
Amoeba,  181 

Amphoteric  substances,  185 
Amygdalin,  96,  136 
Amylase,  25,  95,  113,  114,  118,  120,. 

264  ;    action  on  starch,  100-117  ; 


INDEX 


319 


Amylase  in    living    cell,    118-125; 

preparation  of,  104,  106 
Amyloid,  209 
Amylum,  100 
Anabolic  process,  265 
Anaerobic  conditions,  162 
Analyser,  71,  109 
Aniline,  56,  59 
Animal  fats,  170 
Anthracene,  51 
Apple,  161,  175 
Apricots,  96 
Arabinose,  91,  154 
cyanhydrin,  91 
Arbutin,  97 
Arginin,  196,  197,  198 
Armstrong,  E.  F.,  99,  138,  172 
Asparagin,  116 
Aspartic  acid,  198 
Aspergillinae,  19 

Aspergillus  niger,  18,  123,  129,  174 
Asymmetric  carbon  atom,  76,  90 
Atoms,  35,  36,  40 
Avogadro,  37,  38 


BACILLUS  ANTHBACIS,  122 

B.  coli  communis,  26,  122,  152,  154, 
155,  196,  305,  309 

B.  lactis  aerogenes,  122 

B.  megatherium,  122 

B.  No.  41.. 276 

B.  thioparus,  242 

B.  ureae,  214 

Bacteria,  13,  16,  147-149;  decom- 
position of  cellulose  by,  162-166  ; 
microscopic  examination  of,  30, 
31  ;  Motile,  31 

Bacteriaceae,  18 

Bacterial  filter  beds,  167 

Barley,  101,  118-121,  136,  162,  163, 
171 

Bating  or  puering,  157,  211 

Becker,  132,  133 

Becker,  211 

Beetroot,  94 

Beet  sugar,  84 

Beggiatoa,  17,  243,  308 

Belfast  Lough,  nuisance  on,  238 

Benzaldehyde,  96 

Benzene,  48,  51,  60 

Bergtheil,  248 

Berkefeld  filter,  137 


Bernard,  Claude,  170 

Berthelot,  127,  136 

Bertrand,  176 

Berzelius,  36 

Beyerinck,  229,  231,  237,  238,  239, 

240,  242,  248,  249 
Bilston,  sewage  of,  294 
Biot,  75 

Biotic  energy,  12 
Bitter  almonds,  96,  97,  135 
Biuret,  105,  184,  191 
Blood-corpuscle,  9 
Blood  serum,  139 
Bloxam.   W.   Popplewell,   246,   247, 

248 

Botcher's  Chamber,  32,  33 
Bouillon,  22 

Boulanger  and  Massol,  221,  223 
Boyle,  Robert,  36,  37 
Boyle's  law,  37 
Breal,  233 

Bromine,  action  on  benzene,  etc.,  60 
Brown,  A.  J.,  128 
Brown,  Horace  T.,  163 
Brown  and  Morris,   120,   121,  124, 

125,  249,  268,  269 
Buchner,  13,  136,  137,  138 
Budding  of  yeast,  18 
Butter,  manufacture  of,  274-277 
Butter  fat,  170,  171 
Butylene  glycol,  156 
Butyric  acid,  150,  157,  170 


CADAVEKIN,  201 

Calcium  lactate,  150,  151 
pectate,  161,  168 

Calcspar,  69 

Caldwett,  96 

Cano  sugar,  acted  on  by  acid,  5, 
by  invertase,  126-127,  by  yeast 
juice,  137,  by  zymin,  138 ;  a 
di-saccharose,  84 ;  in  Raulin's 
solution,  27 ;  constitution  of, 
99 ;  first  assimilation  product  of 
nasturtium  leaves,  268  ;  occur- 
rence and  manufacture  of,  94-95  ; 
preparation  of  alcohol  from, 
131-132 

Carbohydrates,  83,  84 

Carbonyl  group,  46,  53,  57 

Carboxyl  group,  54,  59,  145 

Carchesium,  308 


320 


INDEX 


Casein,  95,  170,  179 

Caseinogen,  179 

Castor  oil  seeds,  171,  172,  178 

Catabolic  changes,  264,  265,  270 

Catalase,  255,  256 

Catalysis,  3-5,  12,  134 

Catalyst,  143 

Catalytic  substance,  13,  135 

Cavendish,  133 

Celery,  92 

Cell,  6,  7,  9,  11,  12 
globulin,  208 

Cellulose,  action  of  anaerobic  bac- 
teria in  cellulose,  163-165,  of 
enzyme  cytase  on,  162-163,  of 
Schweitzer's  reagent,  162  ;  aero- 
bic destruction  of  cellulose,  165- 
167  ;  a  polysaccharose,  84,  96  ; 
classification  of,  160-161 ;  decom- 
position in  farmyard  manure,  257- 
258 ;  in  nature,  162 ;  in  septic 
tank,  284-286  ;  fermentation  of, 
2  ;  in  barley  grain,  120  ;  prepara- 
tion of,  159 

Chamberland  filter  candle,  139 

Cheese,  19,  95,  272,  275  ;  making, 
277  ;  ripening  of,  278 

Chick,  Dr.  Harriette,  226,  227 

Chloroform,  117,  126,  129 

Chlorophyll,  18,  266 

Cholera  organism,  196 

Chromatogenic  group,  209 

Chromogenic  bacteria,  167 

Chymosin,  176 

Cladotricheae,  18 

Clarification  test,  186 

Clark,  H.   W.  (see  Gage),  210 

Clemesha,  Major  W.  W.,  220,  309, 310 

Clifford,  W.,  227,  302 

Clotting  enzymes,  178 

Clove  oil,  119 

Clover,  260 

Co-ferment,  141 

Coal  brasses,  242 

Coccaceae,  18 

Cocoa,  fermentation  of,  252,  253 

Coffee,  fermentation  of,  254 

Collagin,  209 

Colloidal  gold,  10 

matter  in  anaerobic  tank,  289 

Colloids,  7-11,  139,  140,  184,  186 

Colouring  matters  from  albumins, 
209 


Combustion  analysis,  42 

Conidia,  18 

Coniferin,  97 

Conif eryl  alcohol,  97 

Conn,  Prof.,  276 

Constant  temperature  incubator,  102 

water  bath,  102,  103 
Constitutional  formula,  49,  58,  60 
Contact  beds,  295-299 
Cotton  fibre,  150 
Courtauld,  96 

Cream,  souring  of,  275-277 
Croft  Hill,  128,  129,  174,  270 
Cross  and  Bevan,  159,  160 
Crystalline  albumin,   185 
Crystallisation,  49 
Crystalloids,  7,  8,  11,  12,  140 
Culture  media  for  bacteria,  22 

moulds,  27 

yeasts,  27 
Cupric  oxide  reducing  power,  107, 

111,  125 

Cuprous  oxide,  90 
Curd,  277 
Cyanhydrin,  59 
Cyanides,  57,  61 
Cyanogen  group,  59 
Cystin  a  and  /3,  199 
Cytase,  162,  163,  264 


DAIRY  products,  bacteriological 
chemistry  of,  272-279 

Dalton,  36,  37 

Danish  butter,  275 

Dauerhefe,  138 

Delepine,  Sheridan,  123 

Denitrification,  228 ;  in  contact  beds, 
298-299 

Dextrines,  determination  of,  in 
digest  from  leaves,  124-125 ; 
products  of  starch  hydrolysis, 
104 ;  solution  factor  of,  108 ; 
specific  rotatory  power  of,  111  ; 
formation  of,  from  starch,  114-115 

Dextro-mannit,  91 

Dextro-mannose,  91 

Dextro-rotatory,  75 

Dextrose,  glucose  or  grape  sugar — 
a  mono-saccharose,  84 ;  acid 
fermentation  of,  154-156  ;  action 
of  maltose  upon,  14;  action  on 


INDEX 


321 


silver  or  copper  solutions,  90 ; 
alcoholic  fermentation  of,  131- 
132,  136 ;  constituent  of  gluco- 
sides,  96-99;  constituent  of 
nutrient  medium,  26 ;  constitution 
of,  94 ;  cupric  oxide  reducing, 
power  of,  111 ;  decomposition 
product  of  indican,  247 ;  fer- 
mentation by  yeast,  2,  by  yeast 
juice,  Buchner,  131,  137,  Harden 
and  Young,  139-142;  formed 
by  acid  on  cane  sugar,  5  ;  growth 
of  yeast  in  mixture  of  galactose 
and  dextrose,  143-144;  prepara- 
tion of,  92,  from  maltase,  128- 
129,  from  oil  of  bitter  almonds, 
135;  relation  to  plant  assimila- 
tion, 269  ;  synthesis  of  isolactose 
from  mixture  with  galactose,  130  ; 
use  in  standardising  Fehling 
solution,  113 

Di-aci-piperazin,  202 

Di-amino-acids,  197 

Di-hexose,  94 

Di-oxy-acetone,  85 

Di-saccharoses,  83,  84,  94,  99,  129 

Dialysable  matter,  141,  1*2 

Dialysed  silicic  acid,  25 

Dialyser,  8,  25,  139 

Dialysis,  8,  10,  139,  186 

Diastase,  104 

Diastatic  activity  of  malt,  111 

Dibdin,  289,  290,  292 

Dilution  method  of  sewage  purifica- 
tion, 282 

Diose,  86 

Dipropinyl,  60 

Dissolved  oxygen,  307 

Distillation,  49,  50 

Dobereiner,  136 

Double  refraction,  69 

Drop  culture,  30,  34 

Dubrunfaut,  136 

Ducat  sewage  filter,  295 

Duclaux,  128 

Dulcite  or  dulcitol,  92,  93 

Dutch  cheese,  272 


EDESTTN,  207 
Eflront,  114 
Egg,  236 


Egg-albumin,  183,  184,  188,  208 

Elastin,  209 

Electrons,  35 

Element,  36 

Elodia  Canadensis,  264,  266 

Embryo,  118,  120,  121,  124,  263,  264 

Empirical  formulae,  43 

Emscher-Brunnen,  287 

Emulsin,  96,  97,  136 

Enantiomorphous,  78 

Endosperm,  120,  162,  163,  264 

Ensilage,  changes  during,  273 

Enteromorpha,  266 

Enzymes,  action  on  disaccharoses, 
94,  on  cellulose  of,  161-163,  on 
glucosides,  96-99  ;  and  acid  fer- 
mentation, 147  ;  and  ammoniacal 
fermentation,  216 ;  and  stereo- 
isomerism,  82  ;  and  tea  fermenta- 
tion, 251 ;  as  catalysts,  5 ;  as 
colloids,  10;  clotting,  178-180; 
coagulation  of  casein  by,  78-79, 
95-97 ;  comparison  with  micro- 
organisms, 117 ;  conditions  of 
action  of  (illustrated  by  amylase), 
115-117 ;  decomposition  of  in- 
dican by,  248-249 ;  fat-splitting, 
169-174;  history  of,  135-136; 
in  anaerobic  tank,  288 ;  in 
cheese  making,  277 ;  in  coffee 
bean,  254 ;  in  cocoa  bean,  253  ;  in 
embryo  of  barley  grain,  120-123  ; 
in  plant  assimilation,  264-271  ; 
in  preparation  of  silage,  271,  273  ; 
in  agriculture,  256-279 ;  in 
tobacco  curing,  253 ;  in  yeast, 
138-139,  142-143;  isolation  of, 
13  ;  Liebig's  views  on  action  of, 
134-135 ;  oxidising  (oxidase), 
175-178;  proteolytic,  189-192; 
proteolytic  action  on  white  of 
eggs,  190-191  ;  proteolytic  bac- 
teria, 210  ;  proteolytic,  in  gastric 
juice  (pepsin),  189,  in  pan- 
creatic juice  (trypsin),  189-190, 
in  tannery,  211 ;  reactions  and 
methods  of  preparation  of  (illus- 
trated by  amylase),  101-106; 
reactions  reversible,  6,  14,  129, 
130  ;  resolution  of  inactive  com- 
pounds by,  91  ;  stoppage  of 
action  of,  by  caustic  soda,  113 

Erodin,  211 


322 


INDEX 


Esters,  5,  6,  54,  59  ;  decomposition 

of,  172,  173,  174 
Ethereal  salts,  5 
Ethers,  55 
Ethyl  acetate,  5 

alcohol,  154,  155 

esters,  192,  193 
Extra-cellular  enzyme,  127 
Extraordinary  ray,  69 


FARMYARD  MANURE,  256,  257,  258, 
259 

Fat  digestion,  2 

Fat-splitting  enzyme,  169 

Fats,  187  ;   decomposition  of,  287 

Fatty  acid,  2 

Fehling  solution,  preparation  of, 
112 ;  test,  for  cupric  oxide  re- 
ducing power,  111,  for  maltose,  95, 
for  progress  of  saccharification, 
103,  for  reducing  sugar,  90 ;  use 
in  determining  invert  sugar,  126, 
128,  for  detection  of  amylase  in 
saliva,  124,  for  titration  in  amy- 
lase reaction,  116-117 

Fenton,  267 

Fibrin,  179 

ferment,  179 

Fibrinogen,  179,  208 

Fibroin,  209 

Fischer,  Emil,  on  alanin,  195  ;  on 
amino-acids  and  polypeptides, 
192  ;  on  glucosides,  96-99 ;  on 
serin,  197  ;  on  sugar  chemistry, 
80,  90  ;  on  syntheses  by  enzyme 
action,  14,  129 

Fischer  and  Armstrong,  129 

Five-carbon  alcohols,  93 
sugars,  93 

Flagellae,  31 

Flax,  retting  of,  168 

Formaldehyde,  83,  84,  145,  266, 
267 

Formalin,  145 

Formic  acid,  154 

Fractional  crystallisation,  49,  50,  91 

Frankland,  Sir  Edward,  223,  224 

Frankland,  Percy,  on  bacteria,  20  ; 
on  denitrification,  229 ;  on  silica 
jelly,  25,  220-221 
and  Macgregor,   fermentation   of 
calcium  lactate,   150-157 


Freudenreich  flask,  32,  33 

Fructose  fruit  sugar  or  Isevulose — 
acid  fermentation  of,  154  ;  fer- 
mentation by  yeast  juice,  137, 
in  presence  of  phosphates,  143  ; 
preparation  of,  92  ;  product  of 
inversion  of  cane  sugar,  94,  126, 
131,  of  plant  assimilation,  269 


G.P.B.,  23,  27 

Galactose,  92,  94,  130,  143,  144,  154 

Gage  (see  Clark),  210 

Gaunt,  P.,  227 

Gay  Lussac,  37,  133,  134 

Gay  on  and  Dupetit,  229,  231 

Gelatine   culture   medium,    20,    23, 

26,  30 

Germ,  101,  120 
Glacial  acetic  acid,  1 
Globulin,  179,  183 
Glucase,  128 
Glucosamin,  201 
Glucose  (see  Dextrine) 
Glucose- osazone,  89 
Glucose-osone,  89 

Glucosides,  96,  97,  98,  99,  136,  247 
Glutaminic  acid,  198 
Glycerine  or  Glycerol,  2,  8,  34,  85, 

86,  169,  170 
Glycerolaldehyde,  85 
Glycerol  ester,  169 
Glycerose,  86 
Glycocol  or  glycin,  56,  86,  192,  193, 

194 

Glycol,  85,  86 
Glycolaldehyde,  85 
Glycollic  acid,  87 
Glycoproteids,  209 
Glycyl-glycin,  202 
Glyoxylic  acid,  268 
Gortner,  176 
Granulose,  100 
Grape  sugar  (see  Dextrose) 
Grapes,  132,  133 
Green  tea,  250 
Gruyere  cheese,  278 
Guanidin,  198 
Guanin,  200 
Guaiacum  resin,  104,  105 

HAEMOGLOBIN,  208 

Hammer  sten,  179 


INDEX 


323 


Hanscn,  32,  143,  149 

Harden,  A.   (see  also  Harden    and 

Young),  effect  of  blood  serum  on 

yeast  juice,    139,    of    phosphates 

on  fermentation,  116  ;    researches 

on  acid  fermentation,    152,    156, 

on  zymase,  13 
Harden  and  Walpole,  action  of   B. 

lactis  aerogenes  on  glucose  and 

mannit,   156 
Harden  and  Young,  on  fermentation 

by  yeast  juice,  139-143 
Harnack,  187 
Hearson  incubator,  29 
Helicin,  97 

Hellriegel  and  Wilfarth,  233 
Hempel  gas  burette,  163,  166 
Heterocyclic  compounds,  51 
Hexite,  86 

Hexone  bases,  197,  204 
Hexose  phosphate,  142 
Hexoses,  83,  90,  91,  94,  99,  270 
Hexyl  iodide,  92 
Hippuric  acid,  213.  215,  257 
Histidin,  197,  198 
Histones,  204,  208 
Hoffmeister,  203 
Homologous  series,  47 
Honey,  92 
Hoogewerff,  247 
Humus,  167,  258,  259,  282 

tanks,  303 

Hutchinson,  261,  262 
Hydrazine,  57,  88 
Hydrazone,  87 
Hydrides,  4 
Hydriodic  acid,  92 
Hydrocyanic  acid,  59,  61 
Hydrogel,  10 
Hydrogen  cyanide,  96 

sulphide,    oxidation    of,    237 ; 

production  of,  237 
Hydrolysis,  6-57  ;  of  cellulose,  160  ; 

of  fata,  170  ;    of  glucosides,  97  ; 

of  starch,  101 
Hydrolytic  tank,  287 
Hydroquinone,  97 
Hydrosol,  10 
Hydroxides,      gelatinous      mineral, 

10 

Hydroxy-amino  acids,  197 
Hydroxyl  group,  52,  146 
Hyphae,  18,  19,  33,  34 


Hyphomycetes,  18 
Hypoxanthin,  200 


Imhoff,  287 
Imino  group,  196 
Incubator,  29 
Indian  black  tea,  250 
Indican,  97,  247,  249 
Indigo,  2,  245-249 

brown,  248 

Indigofera  Sumatrana,  249 
Indigotin,  246,  248 
Indirubin,  248 
Indol,  194,  195,  196 
Indol-amino-propionic  acid,  195 
Indoxyl,  97,  247,  248 
Infusoria,  34 
Inosite,  92,  150 
Inversion,  94 
Invert  sugar,  92,  126 
Invertase,  117,  126,  128,  131,  136 
Ions,  11 

Irreversible  action,  10 
Isatin,  248 

Iso-butyl-a-amino-acetic  acid,  196 
Isocyanide,  6i 
Isocyclic  compounds,  51 
Isolactase,  129 
Isomaltose,  14 

Isomeric  compounds,  60,  100 
Isomerisra,  58,  60 
Isotonic  solutions,  12 


JAPANESE  LACQUER,  175 

Java,  nitrous  organism  from,  221 


Kastel,  172,  174 
Kekule,  47,  48 
Keratin,  209 
Ketohexose,  86,  90,  92 
Ketopentose,  86 
Ketotetrose,  86 
Ketone  aldehyde,  89 
Ketones,  53,  59,  85,  145,  148 
Ketonic  acid,  62 
Ketoses,  85,  86 
Kieselguhr,   137 
Kiliani,  89,  91 
Kipping,  82 
Koch,  20 


324 


INDEX 


Koch's  cholera  bacillus,  122 
Kossel,  197 
Kuhne,  25 


LAB,  178 

Laccase,  175,  176 

Laccol,  176 

Lactalbumin,  183,  208 

Lactase,  130 

Lactic  acid  fermentation  of  sugar,  13 
acids,  61,  149,  151,  155,  157 

Lacto-globulin,  208 

Lactose,  a  disaccharose,  84 ;  acid 
fermentation  of,  145-149 ;  for- 
mation of  lactic  acid  from,  61  ; 
inversion  of,  by  acids,  etc.,  92-94  ; 
unaffected  by  yeast  juice,  137 ; 
preparation  of,  95 

Lsevo-lactic  acid,  154 

Laevo-rotatory,  75 ;  zinc  lactate,  151 

Lsevulose  (see,  Fruit  sugar) 

Latour,  Gagniard  de,  134 

Latrine  tanks,  287 

Lavoisier,  132 

Le  Bel,  76 

Leather,  19 

Leguminosae,  125,  260 

Leptomitus  lacteus,  308 

Leptotricheae,  18 

Letts,  on  application  of  denitrifica- 
tion  in  sewage  purification,  299  ; 
on  fermentation  of  Ulv a  latissimu, 
238 ;  on  pollution  of  estuaries, 
283 ;  on  production  of  nitrous 
oxide  in  contact  beds,  298 ;  on 
reduction  of  sulphates,  237 

Leucin,  195,  196 

Libavius,  132 

Liebig,  96,  134,  135 

Liebig's  meat  extn  ct,  23 

Lignine,  22 

Lignite,  167 

Lime,  259 

Lipase,  170,  171,  172,  1"3 

Lippich,  108,  109 

Liquor  pancreaticus,  170 
pepticus,  189 

Lister,  20 

Little  Drayton  sewage  filter,  294 

Lockett,  228 

Loevenhart,  172,  174 

Loew,  254 


Lucerne,  260 
Lypolytic  enzyme,  204 
Lysin,  197,  198 


MacConkey,  309 
MacGregor,  150 
Madagascar  manna,  92 
Madder  plant,  1 
Madras,  water  supplies  of,  309 
Maize,  100 
Malonic  acid,  63 

Malt,    characteristics    and    manu- 
facture of,  101  ;  distribution 
of  amylase  in,  120  ;  prepara- 
tion of  amylase  from,  104 
extract,  action  on  leaf  extract, 
125;     on    starch,    103-104, 
117  ;   preparation  of,  102 
sugar,  94 

Maltase,  14,  126,  129,  270 
Maltose,  cupric  oxide  reducing  power 
of,  111  ;  detection  of,  113  ;  hydro- 
lysis by  maltase,  128-129 ;  in- 
version  of,  94 ;  occurrence  in  plant 
assimilation,  269-270;  prepara- 
tion of,  95  ;  product  of  sacchari- 
fication  of  starch,  104 ;  produc- 
tion from  starch  by  Aspergillus 
niger,  123 ;  in  leaves  of  tro- 
paeolum  majus,  125 ;  solution 
factor  of,  108 

Manchester  Ship  Canal,  240 
Mangin,  161 

Mann,  H.  H.,  177,  250,  251 
Manna,  91,  92 
Mannite  or  mannitol,  91,  137,  154, 

155,  156 

Mannose,  91,  142,  143 
Mariotte,  37 
Marsh  gas,  2,  147 
Martin,  Dr.,  140 

filter,  140 
Massachusetts     State     Board      of 

Health,  225 

Matunga  installation,  285 
McKay,  238 
Melanin,  209 
Mercaptan,  239 
Methyl  alcohol,  84,  98 
esters,  98 
glucosidee,  98 
Methyl-amine,  56,  59 


INDEX 


325 


Methylene,  86 

blue,  33 
Micro-organisms,    14,    16,    19,    21  ; 

comparison   with   enzymes,  117  ; 

isolation  of  enzyme  from,   136 ; 

secretion  of  enzyme  by,  122-124 
Microbe,  13 

Micrococcus  ureae,  136,  214 
Micros pira  estuarii,  241 
Milk,  95,  145,   149,   178;   chemical 

constituents   of,    274 
sugar  (see  Lactose) 
Millon's  reagent,  105,  184 
Minkman,  231 
Mitscherlich,  75,  136 
Moist  chamber,  32 
Molasses,  95 
Molecular  formula,  41 

weight,  11 

Molecules,  36,  37,  38,  40 
Mono-amino  acids,  194 
Mono-saccharoses,  83,  84 
Monochlorbenzene,  48,  60 
Moulds,  16,  17,  18,  19,  33,  34,  162 ; 

examination  of,  33 
Mucins,  209 
Mucorineae,  19 
Mucus,  209 
J/unro,  219 
Musculus,  136,  215 
Mushrooms,  175,  176 
Mycelia,  18,  34 
Mycoderma  aceti,  149 
Myosin,  208 


NAPHTHALENE,  51 

Nascent  state,  39 

Nasturtium  leaves,  124,  268 

Nessler's  reagent,  218 

Nicol  prism,  70,  109 

Nicotine,  255 

Nitrates,  Stoddart  test,  218 

Nitric  acid,  87,  105 
organism,  220,  221 

Nitrification,  217-228 

Nitrites,  test  for,  218 

Nitrogen,  14,  20,  23,  59,  82 ;  assimi- 
lation of,  232;  cycle,  212-235; 
groups  containing,  56 ;  in  soil, 
259-260 

Nitroso-coccus,  221 

Nitroso-indol,  196 


Nitroso-monas,  221 
Nitrous  organism,  220,  221 
Nucleo-proteids,  209 


OATS,  163 

Octadecapeptide,  202 

Oidaceae,  19 

Oil  immersion  lens,  30 

Olefine  hydrocarbons,  48,  51 

Omelianski,  163,  167,  285 

Optical  activity,  97,  107,  108,  124, 

125 

Ordinary  ray,  69 
Ormerod,   172.     See   Armstrong 
Ornithin,  190 

Osazone,  88,  113,  129,  160 
Osmotic  pressure,  11,  12,  44 
0' Sullivan  and  Tompson,  127 
Oxidases,   40,   175,   249,   251,   253, 

255 

Oxidation,  40 
Oximes,  57 
Oxiurushic  acid,  176 
Oxycelluloses,  160 
Ozone,  39 


PALM,  1 

Pancreatic  extract,  124,  170,  171 

Paraffin,  118,  119 

Paraffin  hydrocarbons,  47,  51 

Pasteur,  on  acetic  acid  fermenta- 
tion, 149 ;  on  conversion  of 
urea  into  ammonia,  214;  on 
dilution  method  of  culture,  20; 
on  oxidation  of  ammonia,  217; 
on  production  of  optically  active 
compounds,  270 ;  on  spontaneous 
generation,  134-135 

Pasteurisation,  276 

Pathogenic  organisms,  19,  123 

Pay  en,  136 

Peaches,  96 

Pear,  151 

Peat,  167 

Pectase,  161,  179 

Pectic  acid,  161 

Pectin,  161,  168,  179 

Pectose,  161,  162 

bodies,  decomposition  of,  167 

Penicilliaceae,  19 

Penicillium  glaucum,  278 


326 


INDEX 


Pentite,  86 
Pepsin,  189,  190 

action  on  albumin,  190,  191 
Peptides,  208 

Peptones,  191,  192,  206,  208 
Percolating  filters,  299 
Perkin,  A.  0.,  246,  247,  248 
Permanent  yeast,  138 
Person,  149 
Persoz,  136 
Petri  dish,  20 
Phenolic  compounds,  55 
Phenols,  55 
Phenyl  alanin,  195 

hydrazine,   50,   54,  59,   87,  88, 

95,  96,  113,  160 
Phenylamine    or    amino    benzene, 

56,  59 

Philosopher's  Stone,  132 
Phlogiston,  153 
Phosphate,  constituent  of  bacterial 

food,  149;    in  yeast  juice,  142- 

143 
Phosphorus    containing    albumins, 

208 

PiotrowsJci,  183 
Plant   cells,    chemical   changes   in, 

263-271 

globulins   and  vitellins,   208 
Plasmolysis,  12 
Plate  culture,  20,  30 
Platinum  black,  145,  146 

spongy,  3,  4,  145,  146 
Plums,  96 
Polarimeter,       determination        of 

optical    activity     by,     108-111 ; 

examination    of    action    of    in- 

vertase  by,   126,  of  maltose  by, 

129,  of  zinc  lactate  solution  by, 

151,     of     glucose     solution     in, 

97 ;  theory  of,  65-75 
Polarisation,  65 

Polariscope,   108.     See  Polarimeter 
Polariser,  71,  109 
Polymethylene  hydrocarbons,  51 
Polypeptides,  192,  194,  201 
Polysaccharoses,  83,  84,  96,  99 
Pope,  82 
Popp,  211 
Potatoes,  100,  163 
Precipitants  for  sewage,  291 
Precipitins,  11 
Priestly,  266 


Primary  alcohols,  52,  59 

amine,  59 
Prolin,  199 

Prosthetic  group,  208-209 
Protamines,  204,  208 
Proteids,  208 
Proteins,  182 
Proteolysis,  210 
Proteolytic  enzyme,  138,  140,  142, 

157,  189,  264,  271,  277 
Protoplasm,  7,  12,  15,  181,  182 
Ptomaines,  201 
Puering    process     or    bating,    157, 

211 
Pure  culture,  20 

of  bacteria,  27 
Purin,  200 
Purin  bases,  200 
Putrescin,  201 
Pyrollidin-carboxylic  acid,  199 


QUINOLINE,  52 


RACEMIC  ACID,  80 

Radio-activity,  106 

Raoult,  44 

Raulin's  solution,  27,  128 

Rawson,  248 

Reamur,  136 

Reduction,  39 

Rennet,  95,  178,  179 

Respiratory  fermentation,  14 

Reversible  enzyme  action,  129 
reaction,  6,  10,  55 

Ribose,  93 

Rice,  100 

Ring  hydrocarbons,   48 

Roquefort  cheese,   278 

Rothamsted,   279 

Rothwell  Sewage  Works,  294 

Royal  Commission  on  Sewage  Dis- 
posal— findings  re  chemical  clari- 
fication of  sewage,  293-295 ; 
standards  of  purity  for  effluents, 
307,  308 

Russell,  E.  J.,  261,  262,  271,  272 


SACCHABIFICATION,  122,  123 
Saccharomycetes,  18 
Saccharose  or  sucrose,  94,  99 


INDEX 


327 


Salicin,  97 

Salicyl  alcohol,  97 

Saliva,  124 

Saponification  of  a  fat,  176 

Sauerkraut,  272 

Schiff,  191 

Schizomycetes,  18 

Schlosing  and  Muntz,  217,  219 

Schryver,  185 

Schulze-Schulzenstein,  226 

Schunck,  247 

Schwann,  134 

Schweitzer's  reagent,  161,  162 

Scott-Moncrieff,  225 

Scutellar  epithelium,  120,  121 

Secondary  alcohols,  52,  59 

Septum,  semi- permeable,  11 

Serin,  193,  197 

Serum,  179 

albumin,  183,  208 
globulin,  208 
therapy,  11 

Sewage,  aerobic  tank  treatment  of, 
289-291  ;  analyses,  305-308 ; 
interpretation  of,  304-305 ;  an- 
aerobic decomposition,  284-289  ; 
chemical  clarification,  291-294 ; 
choice  of  filtration  methods,  303- 
304 ;  direct  treatment  on  niters, 
294-295;  final  purification  of, 
294-304 ;  simple  sedimentation, 
283 ;  standards  for  purity  of 
effluents,  307-308;  strength  of, 
306-307;  tank  treatment  of, 
283  ;  treatment  on  trickling  and 
percolating  filters,  299-303 
Sewage  mud,  242 
Sheridan  Lea,  216 
Siedentopf,  9 

Silage,  preparation  of,  271 
Silica  jelly,  25 
Silo,  271,  272 
Sinigrin,  97 
Six-carbon  alcohols,  93 

sugars,  94 

Skatol,  194,  195,  239 
Skatol-amino-aeetic  acid,  195 
Slate  bed,  290 
Slator,  143 
Sludge,  284 
Soap,   169;  precipitation   of,   from 

sewage,  288 
Sohxlet  apparatus,  171 


Soils,  fertility  of,  261-263 ;  inocula- 
tion of,  260 
Solution  factor,  108 
Sorbite,  93 
Soy  bean,  260 
Spallanzani,  136 
Specific  gravity,  107,  108 

rotatory  power,  110,  111 
Spermatozoa  of  fishes,  208 
Sphaerotilus  natans,  308 
Spirillum  desulphuricans,  240,  241 
Spongin,  209 
Spore  formation,  17 
Stab  culture,  29 
Stahl,  132 

Staining  culture  medium,  28,  31 
Starch,  84,  96,  100,  107,  111,  119, 

120,  122,  124,  125,   161,  16i>,  269 
Steapsin,  170 
Stearic  acid,  169 
Stearin,  169 

Stereo-chemical  formulae,  78 
Stereo-isomerism,  90 
Steriliser,  21 
Sterility,  21 
Stoddart,  225 
Subculture,  30 
Succinic  acid,  154,  155 
Sucrase,  126 

Sugar,  13, 82, 84, 92, 95, 103, 108,  111, 

113,  121,  122,  128,  133,  138, 

145,148, 155,160, 167,268,  269 

cane,  94 

Sulphates,    reduction  of,  237,  239, 

240,  241,  244 

Sulphur,   3,   236,   237,  243 ;  oxida- 
tion of,  241 
springs,  243 
Sulphuretted   hydrogen,    236,    237, 

238,  239,  241,  242,  244 
Sutton,  289,  290 


TANK  treatment  of  sewage,  283 

Tannery,  bating   and  puering  pro- 
cess in,  157,  211 

Tannin,  177,  251 

Tartaric  acid,  27,  29,  87 

Tea,     manufacture     of,     250-251 ; 
quality  of,  250 

Termeukn,  247 

Tertiary  alcohols,  52,  59 
amine,  59 


328 


INDEX 


Tetrite,  86 

Thalli,  18 

Thrombase,  179,  180 

Thymol,  117,  123 

Tiegheim,  214 

Tobacco,  fermentation  of,  254 

Toluene,  117,  129 

Tourmaline,  68 

Toxins,  11,  123 

'Trade  effluents,  biological  purifica- 
tion of,  310,  311 

Travis,  287 

Treacle,  95 

Trickling  filters,  299-303 

Trimethylene,  51 

Triose,  83 

Tropaeolum  majus,  124 

Trypsin,  189,  190,  195;  action  on 
albumin  of,  190,  191,  192 

Tryptophane,   195 

Tyndall,  134,  135 

Tyndall  phenomenon,  8 

Tyrosin,  176,  177,  195 

Tyrosinase,  176,  177 

ULTRAMICROSCOPE,  8,  9 

Ulva  latissima,  238,  239,  266,  299 

Urea,  14,  40,  56,  105,  136,  213,  215 

Urease,  136,  216 

Uric  acid,  2 

Urushic  acid,  175 

Usher,  266 

VALENCY,  41,  46 
Valentine,  Basil,  133 
Van  Delden.  237,  239,  240,  241 
Van  Helmont,  132 
Van  Iterson,  165,  166,  167 
Van't  Hoff,  44,  76 
'Vegetable  fats,  17 
Vinegar,  19,  157 
Vital  action,  12 
Von  Baeyer,  247,  265,  266 

WAKEFIELD  sewage,  precipitation 
of,  292 


Walpole,  156 

Warington,  219,  220,  258,  274 

Water,  analyses  of,  308-310  ;  inter- 
pretation of,  304-305 

Water-bath,  21 

Wave  length,  66 
motion,  66 

Weigmann,  276 

Wheat,  100,  121,  160,  260 

Whey,  277 

Winogradski,  decomposition  of  pec- 
tose  bodies,  167  ;  on  nitrification, 
219-221,  226  ;  on  sulphur  oxida- 
tion, 237-243 

Willstdtter,  267 

Wine,  148,  157,  175 

Witte's  peptone,  23,  154 

Wohler,  40,  96 

Wolverhampton,  sewage  of,  294 

Wood,  J.  T.,  211    " 

Worms,  174 

Wort,  143 

gelatine,  25,  32,  33 

XANTHIN,  200 

Xanthoproteic  reaction,  105,  184 

Xylite,  93 

Xylol,  119 

Xylose,  93 

YEAST,  action  on  grape  sugar  of,  2  ; 
characteristics  of,  16-19 ;  de- 
tection of  maltose  in,  128-129  ; 
extraction  of  invertase  from,  126- 
127  ;  fermentation  of  grape  sugar 
by,  131-144 ;  no  action  on 
maltose  and  milk  sugar,  95-96 ; 
oxidation  of  acetic  acid  by,  149 ; 
use  of,  in  purifying  amylase, 
106 ;  variety  of  functions  of, 
123  ;  zymase  in  cells  of,  13 

Yoshida,  175 

Young,  139,  142 

Zsigmondy,  9 

Zymase,  13,  127,  137,  138,  139 

Zymin,  138,  139 


Spottificoode  &  Co.  Ltd.,  Printers.  Colchester,  London  and  Eton. 


Engineering 
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