UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN

AGRICULTURAL SCIENCES

Vol. 4, No. 1, pp. 1-66, pis. 1-2, 15 text-figures December 31, 1918

THE FERMENTATION ORGANISMS OF
CALIFORNIA GRAPES1

BY

W. V. CEUESS

CONTENTS

PAGE

Introduction 3

I. General discussion of grape organisms 3

Molds 4

Penicillium 4

Aspergillus niger 4

Oidium 4

Borytis cinerea 6

Mucor : 6

Monilia 6

True Yeasts 6

S. ellipsoideus 6

S. cerevisiae 8

S. malei 8

S. pastorianus 8

S. anomalus 8

S. ludiwigii 9

S. marxianus 9

Psuedo Yeasts 9

Apiculatus 9

Mycoderma 9

Torula 10

1 1 . Properties of molds and bacteria from California grapes 10

Sources of cultures 10

Molds 12

Penicillium 12

Olive color penicillium species 12

Aspergillus 12

Mucor 14

Dematium 14

Monilia 14

Green molds (unidentified) 14

Bacteria 14

i The writer wishes to thank Professor F. T. Bioletti for his helpful sug-
gestions for carrying out these investigations and preparation of the manuscript.

University of California Publications in Agricultural Sciences [Vol.4

PAGE

III. Characteristics of yeasts from California grapes 16

Methods of study 16

Morphology 16

Fermentation tests 16

Yeasts studied 18

Apiculatus yeasts 18

Organism 35 (Apiculatus) morphology 18

Organism 72 (Apiculatus) morphology 19

Organisms 35 and 37, rates of fermentation 19

Mycoderma forms 22

Organism 65, morphology 22

Organism 68, morphology 23

Organism 70, morphology 23

Organism 71, morphology 24

Organism 73, morphology 24

Organism 76, morphology 25

Organism 78, morphology 25

Fermentation tests of Mycoderma forms 26

Torula forms 30

Organism 37, morphology 30

Organism 77, morphology 31

Fermentation tests of torula yeast 32

S. Pastorianus and Willia yeast 34

Organism 36, (S. Pastorianus) morphology 34

Organism 69 (Willia species) morphology 35

Fermentation tests of S. Pastorianus and Willia 35

True wine yeasts, S. Ellipsoideus 38

Organism 64, morphology 38

Organism 66, morphology 39

Organism 67, morphology 39

Organism 74, morphology 40

Organism 75, morphology 40

Organism 79, morphology 41

Fermentation records of true wine yeasts at 33° C 41

Attenuation and alcohol formation at 33° C 44

Comparison of fermentations at 33°C and 24°C (all varieties).... 46

IV. Influence of locality on the character of the micro-organisms on grapes.. 50

Methods of taking samples 51

Methods of counting living cells on grapes 51

Results of countings 52

Micro-organisms on grapes from Davis 52

Micro-organisms on grapes from El Centro 52

Micro-organisms on grapes from Fresno 52

Micro-organisms on grapes from Martinez 53

Micro-organisms on grapes from Ripon 53

Yeasts from the Tulare experiment station 53

Discussion of results 53

V. Influence of the stage, of ripeness on the character of the micro-organisms

on grapes 54

Sampling 54

Results 55

1918] Cruess: Fermentation Organisms of California Grapes 3

PAGE

VI. Changes in the numbers and character of the micro-organisms on grapes

during shipment from vineyard to cellar 56

VII. Character and number of micro-organisms on grapes as received at the

winery during the seasons of 1911 and 1912 57

VIII. Experiments upon the control of micro-organisms on grapes for wine

making 59

Before shipment 59

After arrival at cellar 62

Summary of parts II - VIII (inclusive) 63

INTRODUCTION

The manufacture of wine, grape-juice, and raisins and the shipping
of grapes depend very largely upon the control of micro-organisms,
particularly of those occurring naturally upon the fruit. This eco-
nomic fact and the scientific interest of the subject led to a study of
the micro-organisms occurring on the grapes of California. The
investigation was qualitative and quantitative, covering the effect on
type and number of micro-organisms of (a) locality, (6) degree of
ripeness, (c) shipment from vineyard to winery. It included studies
of (d) the micro-organisms normally found on grapes as received at
the winery, (e) their control in fermentation, and (/) their morpho-
logical and physiological characteristics.

I. GENERAL DISCUSSION OF GRAPE MICRO-ORGANISMS

Several groups of micro-organisms are normally found on grapes.
These may be classified under the general terms : (1) Budding fungi,
including molds, true yeasts which form spores, and pseudo-yeasts,
which do not form spores; and (2) fission fungi, including bacteria
(non-motile rods), bacilli (motile rods), the various forms of Coc-
caceae, and the fission yeasts.

Representatives of all the above groups except the bacilli, Coc-
caceae, and fission yeasts were found in California grapes. Most
attention has been given to the fermentation organisms: i.e., yeasts.
The molds and bacteria have in most cases been merely listed.

The more important forms of organisms occurring on grapes are
the following:

University of California Publications in Agricultural Sciences [Vol. 4

Molds

Penicillium. — This group includes a large number of varieties,
which are the most widely distributed and common of all the molds.
They are characterized by the method of formation and grouping of
conidia shown in a typical manner in figure 1.

The most common mold of this group met with on grapes is Peni-
cillium glaucum or Penicillium expansum. It is the common green
mold and is the cause of moldy flavors in grapes, barrels, etc.

During the first stages of growth it appears as a cottony, white
mass of mycelial threads. These white threads soon develop fructifi-
cations bearing large numbers of conidia that are green in young
cultures and brown in old. These give a powdery appearance to the
culture. The individual conidiophores are branching and present a
broomlike appearance under the microscope.

It does not carry on alcoholic fermentation, but may destroy sugar
by oxidation to C02 and H20 or the formation of penicillic acid.

Aspergillus niger. — This is a black mold of very common occur-
rence on California grapes (see fig. 1). On grapes and must it forms
a white mycelium from which spring short rods bearing aggregations
of black cells. These conidiophores are not branching, thus differing
from penicillium. These groups of cells are easily discernible with
the unaided eye. On the grapes of some districts the spores or conidia
may be broken away from the main growth of the mold during pick-
ing and rise as a black dust. At present it is not held to be very
harmful in wine making.

Oidium or Powdery Mildew of the Vine. — This fungus is of more
concern to the grape grower than it is to the wine maker and will be
found fully described in Bulletin 1862 of the University of California
Agricultural Experiment Station (see fig. 1). This mold may pre-
vent the grapes reaching maturity or may cause them to crack and
thus to be liable to attack by penicillium. In both cases, the grapes
affected may become unfit for wine making. The microscopical appear-
ance of the summer form of the powdery mildew is shown in figure 1.
It occurs most commonly as a downy white growth on the leaves, canes,
and grapes during moist or foggy weather and is most prevalent dur-
ing early summer. It forms winter spores or perithecia on the canes,
in which form the organism lives through the winter.

zBioletti, V. T. (Oidium or Powdery Mildew of the Vine), Univ. Calif. Exp.
Si;,.. Bull. ISO, pp. 317-327, 1907.

1918] Cruess: Fermentation Organisms of California Grapes

/ FZNICILLIUM k80O Z.ftSPEiRGt LL 1/5*500

3-BOTFtVTUSxdOO -f MUCOR* /SO-

SOIDI UM x dOO €>E>FKO VV/V MOL 0*800

Figure 1
Molds from California grapes:

1. Penicillium variety from California grapes, magnified 800 diameters.

2. Aspergillus variety from California grapes; X 800.

3. Botrytis cinerea from California grapes; X 800.

4. Mucor variety from California grapes; X 150.

5. Oidium or Powdery Mildew from California vine, summer form; X 800.

6. Brown mold, parasitic fungus from California grapes; X 800. Species

and variety not determined.

6 University of California Publications in Agricultural Sciences [Vol. 4

Botrytis cinerea. — This mold is a parasite or facultative sapro-
phyte occurring on grapes in moist climates (see fig. 1). It rarely
develops on California grapes during the wine making season but is
often found on grapes left on the vine during the winter. It appears
as a gray, matted growth on the surface of the grapes. The indi-
vidual fructifications may be seen with the naked eye on short upright
conidiophores. Under the microscope, the conidia may be seen in
grapelike clusters.

The mold did not grow readily in culture media, but will develop
profusely on infected grapes in a moist jar.

In Europe, the Botrytis is considered beneficial in the Sauternes
district, because it causes a concentration of the sugar content of the
grapes by favoring evaporation of the moisture. It produces also an
oxydase that acts vigorously upon the color of the grapes or wine.

Mucor. — Mucor mold will nearly always be found in an examina-
tion of California grapes (see fig. 1). Since it develops very slowly
in comparison with the commoner molds and yeasts found on grapes
it is not probable that it does very much harm in wine making.

In pure cultures, it produces a gray filamentous mass of mycelial
threads, from which upright rods are given off, bearing at their upper-
most ends spherical sporangia filled with large numbers of spores.
These sporangia are easily seen with the unaided eye. Most of the
mucor molds are capable of forming yeastlike cells and carrying on a
feeble alcoholic fermentation in sugary liquids.

Monilia. — The monilia molds and especially Monilia Candida occur
very commonly on fruits of all kinds (see fig. 2). Most of the grapes
examined in 1912 bore considerable numbers of the cells of this
organism.

In young cultures, it forms colorless yeastlike cells and gives a
feeble fermentation. The fermentation is followed by a moldlike
growth on the surface of the grape must, etc., which in old cultures
becomes olive green in spots.

True Yeasts

Culture Yeasts. — (1) Saccharomyces ellipsoideus (True Wine
Yeast). In general, the ellipsoideus yeasts are characterized by rapid
growth in grape must with the production of a strong fermentation,
yielding 10% to 16% of alcohol. They differ from the beer yeasts,
8. cerevisiae, principally in their higher alcohol-forming power and

1918]

Cruess: Fermentation Organisms of California Grapes

ill the flavor of the fermented liquids, the wine yeast giving a vinous
flavor to fermented liquids and the beer yeast a beer flavor. In grape
must, the S. ellipsoideus forms a cloudy growth and a pasty or gran-
ular sediment in the bottom of the container during the main fer-
mentation. After fermentation is complete the suspended cells settle

I.DEMflTIUMxSOa % MONILW x 800

3. VI A/e6/7/=f BftCT£/?//9x/OCO ^ TOURNC BnCTOf/ffx/OOO.

Figure 2
Molds and yeasts from California grapes:

1. Dematium variety from California grapes; X 800.

2. Monilia variety from California grapes; X 800.

3. Vinegar bacteria from California grapes; X 1000.

4. Tourne bacteria from California wine; X 1000.

out, giving a clear liquid and compact sediment. Under suitable con-
ditions spores are formed. The usual shape of the cells is shortly
ellipsoidal, although this will vary from spherical to elongate, with
the different varieties. The shape of the spores is spherical. The
appearance of several specimens of S. ellipsoideus from California
grapes and of cells containing spores will be found in figures 5 and 6.

8 University of California Publications in Agricultural Sciences [Vol. 4

The main part of all wine fermentations is carried on by this yeast.
There are a great many different varieties in this group and these vary
considerably in their suitability for wine-making purposes. Modern
methods of wine making aim to make use of the most desirable
varieties.

2. Saccharomyces cerevisiae (Beer Yeast). — It is possible that
these yeasts occur on grapes to a more or less limited extent, depend-
ing upon the proximity to vineyards of breweries or distilleries using
these yeasts. No reference has been found in the literature describing
their occurrence on grapes and none have been found on California
grapes. There is probably little likelihood of their being present on
grapes in sufficient numbers to influence in any way the fermenta-
tions. This yeast is worthy of mention in connection with wine mak-
ing, however, because it has been used in the past to start wine fer-
mentations. "Where brewery yeast is so used, it produces a wine of
beerlike flavor and low alcohol content and with an excess of unfer-
mented sugar. Such a wine is not palatable and is very liable to be
attacked by bacteria and to be lost through bacterial decomposition.

3. Saccharomyces malei. — This yeast occurs on apples and is
similar to 8. ellipsoideus, but usually forms less alcohol. It was not
found on any samples of grapes examined, but probably occurs occa-
sionally on grapes.

Wild Yeasts. — (1) Saccharomyces pastorianus. — This group of
yeasts is characterized by its elongate or sausage-shaped appearance
and its ability to form spores. Members of this group of yeasts have
been found on two samples of California grapes. They form small
amounts of alcohol in grape must and at the same time usually pro-
duce undesirable flavors and odors — usually a bitter flavor. The mic-
roscopical appearance of a culture of S. pastorianus yeast from Cali-
fornia grapes is shown in figures 3 and 6.

2. Saccharomyces anomalus (Willia Yeasts). — The yeasts of this
group are characterized by the production of hat-shaped spores. The
appearance of such spores is shown in figure 7. The members of this
group grow rapidly in must and form a wrinkled film. They carry
on a weak fermentation with the production of small amounts of
alcohol. Liquids fermented by these yeasts are high in aromatic com-
pounds of various sorts and for this reason their use has been sug-
gested as ;i means of flavoring various fermented beverages. Cells
from a ei i H me isolated from California grapes are shown magnified
1000 diameters in figures 3 and 6.

1918] Cruess: Fermentation Organisms of California Grapes 9

3. Saccharomyces ludwigii. — This yeast has been found on grapes
grown in Europe. In microscopical appearance it resembles the
S. apiculatus yeast, but is much larger. It was not found on Cali-
fornia grapes.

4. Saccharomyces marxianus. — This yeast has been reported as
being present on grapes. It is recognized by its kidney-shaped spores.
It was not found on the samples of California grapes examined.

Pseudo-Yeasts

Apiculatus Yeast. — The apiculatus yeast (Saccharomyces apicu-
latus, Hansenia apiculata) is recognized by the peculiarly pointed ap-
pearance of many of its cells (fig. 3) . According to most authorities it
does not form spores and so is placed in the group of pseudo-yeasts.
According to Lindner, however, it forms spores in drop cultures, one
spore per cell. It carries on a feeble bottom fermentation in grape
must but does not have the power to ferment saccharose, maltose,
or lactose. In must it gives from 0% to 6% alcohol and at the same
time produces fruity flavors and odors. It settles more slowly and
less completely than 8. ellipsoideus. Apiculatus yeast probably does
more harm in wine making than all other varieties of wild yeasts com-
bined, because of its large numbers on grapes and its very rapid
development after the grapes are crushed. It develops so rapidly that
the first stages of most natural wine fermentations are carried on by
this yeast and the preliminary fermentation of grapes is often spoken
of as the "apiculate" stage. During this preliminary fermentation
it produces undesirable flavors and aromas, destroys yeast food that
should have gone to the true wine yeast, forms compounds deleterious
to the vigorous development of the true wine yeast and gives a great
many cells of low specific gravity that settle out slowly after the main
fermentation is over. After the S. ellipsoideus yeast has formed 8 to
10 per cent alcohol the Apiculatus yeast is killed and will not be found
in the living state in the finished wine.

Mycoderma Types. — The Mycoderma forms are known to all wine
makers as "wine flowers" (fig. 4). They are present in considerable
numbers on uncrushed grapes and have been found on nearly all
samples of California grapes so far examined. In pure cultures in
liquids, they appear as white films, usually more or less deeply
wrinkled. From time to time portions of the pellicle are detached
and fall to the bottom giving in time a large amount of sediment. A

10 University of California Publications in Agricultural Sciences [Vol.4

feeble fermentation is carried on at the surface of the liquid, pro-
vided that it contains fermentable sugar. Under the microscope the
yeast appears as cells of irregular shape and size, the usual form, how-
ever, being elongate or sausage-shaped. The cells tend to adhere
together in groups and chains. It is thought that the cells are main-
tained at the surface of the liquid by means of small bubbles of air
occluded between them.

In pure cultures in grape must, the Mycoderma yeasts are capable
of destroying varying amounts of sugar with the production of small
amounts of alcohol, in most cases, and obnoxious flavors and aromas.
It is probable that in many natural wine fermentations they are more
or less active at the beginning of the fermentation. They undoubtedly
cause trouble in tanks of wine that are not kept well filled during
storage. They are aerobic.

Torula Yeasts. — Under the heading of Torula are placed most of
the yeasts that do not fit into other groups. Consequently, this name
covers yeasts of wide variation in properties. As a group they do not
form spores; they form small amounts of alcohol, do not normally
develop as a film growth, and the form considered as typical is spher-
ical, although there is considerable variation from the typical form.
They are found on grapes fairly commonly, though less abundantly
than 8. apiculatus and Mycoderma. Two forms from California
grapes are shown in figure 3.

II. PROPERTIES OF MOLDS AND BACTERIA FROM
CALIFORNIA GRAPES

Sources of Cultures

In 1911 micro-organisms were isolated from grapes received at the
winery of J. E. Colton, Martinez, California, and from grapes picked
at the University Farm, Davis. Samples were obtained in 1912 from
Fresno and El Centro. The properties of these micro-organisms were
studied more or less in detail. Most attention was paid to the or-
ganisms and characteristics that were of importance in wine making.

Method of Separation. — The materials used in the separation of
the various types of organisms present on the grapes were sterile
grape must agar, sterile grape must, petri dishes, and a small platinum
inoculating rod. The grape must agar was made by dissolving 20
grams of agar agar in 1000 c.c. of boiling water to which was added
60 c.c. of grape must after the agar had dissolved. It is necessary

1918] Cruess: Fermentation Organisms of California Grapes

11

Figure 3
Yeasts from California grapes magnified 1000 diameters:

35. S. apiculatus from Davis grapes.
72. S. apiculatus from Acampo grapes.
37. Torula from Davis grapes.

77. Torula from Contra Costa County grapes.

36. S. pastorianus from Davis grapes.
69. Willia species -from Acampo grapes.

12 University of California Publications in Agricultural Sciences [Vol. 4

to use this high diluation of must in the agar in order that the agar
will not be hydrolyzed by the acid of the must. At this dilution the
agar contains about .05 per cent acid as tartaric. This should permit
the growth of all forms. The hot agar solution was filtered through
cotton wool and filled in 10 c.c. portions into test tubes plugged with
cotton wool. It was sterilized at 15 pounds pressure in an autoclave.
The grape must was clarified by boiling with the white of egg and
filtration. The clear must was filled into plugged test tubes and
sterilized at 100° C. The Petri dishes were sterilized in packages of
three by dry heat. The type of dish used is shown in figure 3.

Samples of the grapes received at the winery were taken from the
center of the boxes and crushed into sterile containers. A small
amount of the must was in each case transferred by a sterile platinum
wire to a tube of melted agar kept at 40° C to 45° C, and further
dilutions were made by transfer to other tubes of agar.

The organisms were separated by growth on agar must. They were
purified by replating on the same medium. Subcultures were then
made in sterile must, on agar slants and permanent stock cultures were
made in sterile 10 per cent cane sugar in Freudenreich flasks.

The pure cultures obtained in this way are discussed in groups,
the members of each group having certain characteristics in common.
No attempt has been made so far to study the molds and bacteria in
detail and for this reason they have simply been listed with a few
words of explanation.

Molds

Penicillium Species (probably Penicillium expansum) . — Found on
nearly all samples examined ; produces fructifications bearing numer-
ous round conidia as shown in figure 1 ; forms sclerotia, resistant
vegetative forms, in old cultures; no fermentation in must, but grows
vigorously in all culture media tested; giving a characteristic moldy
odor.

Penicillium Species of Olive Green Color. — Forms ellipsoidal coni-
dia ; very common on California grapes and also found in samples of
pasteurized unfermented grape juice that had molded after pasteuri-
zation.

Aspergillus. — Two forms: (a) produces small, smooth, black coni-
<li;i ; (b) produces large black conidia with spikelike projections. The
Jippearance of the one yielding the smooth conidia is shown in figure 1.

Botrytis (probably H. cinerea). — Found on many samples of grapes

1918]

Cruess: Fermentation Organisms of California Grapes

13

Figure 4
Yeasts from California grapes:

65. Mycoderma yeast from Acampo grapes.
68. Mycoderma yeast from Acampo grapes.

70. Mycoderma yeast from Acampo grapes.

71. Mycoderma yeast from Contra Costa Grapes.
73. Mycoderma yeast from Acampo grapes.

76. Mycoderma yeast from Acampo grapes.

14 University of California Publications in Agricultural Sciences [Vol. 4

after the early rains of 1912. It gives the characteristic Botrytis
growth on the grapes but is very reluctant to grow on any of the
ordinary culture media, the best artificial medium found so far being
grape must gelatin made up by dissolving gelatin in undiluted grape
must. The appearance of this mold from grapes grown at the vine-
yard of J. Swett and Son, Martinez, is shown in figure 1.

Mucor. — Color, gray; spores, shortly ellipsoidal. Does not pro-
duce fermentation in grape must. Its microscopical appearance is
given in figure 1.

Dematium Variety. — Grows vigorously in must giving a slimy mass
of colorless cells and a surface growth of black cells arranged in long
chains. Found in large numbers on grapes examined in 1912. Does
not cause fermentation in must. Microscopical appearance is given
in figure 2.

Monilia Species. — Colonies on agar resemble yeast colonies with
rootlike projections into the depths of the medium. Gives a feeble
fermentation in grape must, followed by a mycelial growth on the
surface of the liquid ; in old cultures, part of the mycelium becomes
olive green (fig. 2).

Dark Green Mold (unidentified). — Found growing profusely as a
parasitic fungus on skins of grapes after the early rains in fall of
1912 ; grows equally well on artificial media in the laboratory, so ma}7
be termed a faculatative parasite, It causes no visible fermentation
in must and is odorless. Its microscopical appearance is given in
figure 1. It may possibly be a Cladosporium form.

Bacteria

Vinegar Bacteria. — The only forms of bacteria so far found on
grapes examined at the laboratory or winery have been vinegar
bacteria. They were observed on two different lots of grapes, both of
which had been shipped long distances and were in a badly broken
and moldy condition. Both cultures grew rapidly in grape must
giving a heavy tough film. One culture gave bacteria of medium
length that tended to form in chains, while the other developed in
the form of very short rods usually grouped in pairs (fig. 2).

"Tourne" Bacteria. — The form shown in figure 2 is found in wines
quite often, but has not been met with on California grapes. It grows
in the absence of air and gives a "mousey" taste to badly affected
wines (fig. 2). This organism is otherwise known as Bacterium
mannito-poeum. It occurs in vinegar and fermented fruit juices.

1918] Critess: Fermentation Organisms of California Grapes

15

Figure 5
Yeasts from California grapes:

78. Mycoderma yeast from Contra Costa County grapes.
64. S. ellipsoideus from Acampo grapes.

66. S. ellipsoideus from Davis grapes.

67. S. ellipsoideus from Contra Costa County grapes.

74. S. ellipsoideus from Acampo grapes.

75. S. ellipsoideus from Contra Costa County grapes.

16 University of California Publications in Agricultural Sciences [Vol. 4

III. CHARACTERISTICS OF YEASTS FROM CALIFORNIA

GRAPES

Methods of Study

The yeasts isolated from grapes in 1911 according to the methods
given on preceding pages were studied in accordance with the follow-
ing outline. From the tests made, it was possible to determine the
specific character of the yeasts and to obtain a fair idea as to their
suitability or unsuitability for wine making purposes.

Morphology. — (1) Macroscopical Appearance. — In must, beer wort,
nutrient dextrose, saccharose, and lactose solutions ; on agar must and
on gelatin must.

(2) Microscopical Appearance. — Size, form, and general appear-
ance from five days' growth in grape must. Microscopical appearance
of agar colonies.

(3) Spore Formation. — Observed by placing the sediment from
vigorous cultures on gypsum blocks partly immersed in water in wide
mouthed bottles plugged with cotton wool. This arrangement gives
the necessary conditions of aeration, poor food supply, and moisture.
Tests were made of spore formation at 22° C and 28° C.

Fermentation Tests. — (1) Rates of Fermentation. — The rates of
fermentation in grape musts of 23.01% and 29.75% Balling, in beer
wort, nutrient3 dextrose solution, nutrient saccharose solution and
nutrient lactose solution were determined on 100 c.c. portions of the
above liquids by noting the loss in weight during fermentation after
inoculation with pure cultures of the organism under observation.
The tests were carried out at 33° C. The loss in weight is due to the
formation of carbon dioxide during fermentation. The escape of this
gas causes the change in weight by which the fermentation may be
followed. The main chemical reaction involved is the following :

C H O = 2CO + 2C H OH

6 12 6 2 ' 2 5

A check flask, which was not inoculated, was used as a means of
obtaining the loss in weight due to evaporation. The figures given in
the tables are corrected for this loss. Points representing the time in
hours reckoned from the time of inoculation and also representing the

— i

8 The nutrient solutions used for the dextrose, saccharose, and lactose con-
sisted of .01 grams magnesium sulfate, .5 grams of dipotassium phosphate,
10 grams Witte 's pepton dissolved in 1000 c.c. of water. To a portion of this
solution was added 15% dextrose, to a second part 15% saccharose, and to the
third, 15% lactose.

1918]

Cruess: Fermentation Organisms of California Grapes

17

66

W/ TH SPOftCS

79

WITH SRORES-

WITH

69

S/=OF?£S.

Figure 6
Yeasts from California grapes showing spore formation:
79. S. ellipsoideus from Contra Costa County, California.
66. S. ellipsoideus from Davis grapes showing spores.
75. S. ellipsoideus from Contra Costa County grapes, showing spores.
79. S. ellipsoideus from Contra Costa County grapes, showing spores.
36. S. pastorianus from Davis grapes, showing spores.
69. Willia from Acampo grapes, showing spores.

18 University of California Publications in Agricultural Sciences [Vol.4

loss in weight have been plotted on diagrams and the individual points
connected to give "fermentation curves." These curves show at a
glance the character of the fermentations and serve as convenient
means of comparison.

(1) Products of Fermentation. — The products of fermentation in
the solutions given above were determined after the rates of fermenta-
tion were ascertained. Similar tests were made in grape must of 30° 4
Balling fermented by the various yeasts at 24° C.

Yeasts Studied

The yeasts have been taken up in the following order : (1) S. apicu-
latus yeasts, (2) Mycoderma forms, (3) Torula yeasts, (4) S. pastor-
ianus and Willia yeasts, and (5) S. ellipsoideus yeasts. The char-
acteristics of each have been discussed according to the outline given
above.

APICULATUS YEASTS FROM CALIFORNIA GRAPES

Two apiculatus yeasts were studied. One of these was isolated
from grapes grown at Davis and the other from grapes grown at
Acampo. The morphology and biological characteristics were studied
as previously outlined.

Microscopical appearance is based on cells from five days' growth
in grape must of 15% Balling. The agar used for plate cultures con-
sisted of 2 grams of agar agar, 60 c.c. of must, and 1000 c.c. of water.
The gelatin medium consisted of 15 grams of gelatin to each 100 c.c.
of grape must.

Organism 35. Apiculatus from Grapes Grown at Davis

Morphology. — Microscopical Appearance. — Majority of cells lemon-
shape. Cells broader and larger per cent apiculate-shape than in case
of yeast no. 72.

Size. — Average, 4.5/* x 2.4//,. Maximum, 7.5/a x4.5^. Minimum,
3/x x 1.5/x.

Spore Formation. — No spores at 22° C or 28° C.

Colonics on Agar Agar. — Flat. Elevation uniform. Smaller than
8. elUpsoideus colonics. Semitranslucent. Edges smooth and entire.
Under octfcroscope the edges of the colonics are entire.

4 Degrees Balling indicates total dissolved solids in grams per 100 grams,
as cane Bugar.

1918] Cruess: Fermentation Organisms of California Grapes 19

Colonies on Gelatin. — Small. Gelatin is slowly liquefied, giving
craterlike depressions. Edges entire.

Growth in Liquid Media. — In grape must, beer wort, nutrient
dextrose, and saccharose solutions, rapid but fermentation feeble.
Growth in lactose slight and no fermentation. Sediment and growth
in liquid, fine grained. Cells settle slowly. Liquid becomes fruity
in flavor and odor.

Organism 72. Apicnlatus from Grapes Grown at Acampo

Morphology. — Microscopical Appearance. — Majority of cells sau-
sage-shaped or spherical. Only a few typical apiculate cells. De-
cidedly different from no. 35.

Size. — Average, 4.5/x x 2.7/*. Maximum, 6/x x 2.7/x. Minimum
2.2/XX1.5/*.

Spore Formation. — No spores at 28° C or 22° C.

Colonies on Agar Agar. — Small. Edges undulate or indented.
Centers slightly elevated. Opaque and porcelainous. Under low
power, edges entire. Different from no. 35.

Colonies on Gelatin. — Small. Edges branchlike. Waxy luster.
Edges under microscope lacerate, i.e., saw-tooth appearance. Gelatin
slowly liquefied to form craterlike depressions around colonies ; whole
plate liquefied in few days.

Growth in Liquid Media. — In grape must, beer wort, and nutrient
dextrose, growth is rapid. Almost no growth in saccharose and lactose
nutrient solutions. Deposit and growth in liquid fine grained. Settles
very slowly. Fruity odor and flavor in fermented liquid.

Rates of Fermentation, Various Media.
The following solutions were used :
{a) Grape must, 23.01° Balling.

(b) Grape must, 29.75° Balling.

(c) Sweet beer wort, 14° Balling.
(d)5 Nutrient dextrose, 15% dextrose,
(e) Nutrient saccharose, 15% saccharose.
(/) Nutrient lactose, 15% saccharose.

5 The nutrient solutions (d), (e) , (/), contained .01 grams MgS(X .5 grams
K2HP04, 10 grams Witte's pepton, per 1000 c.c. water, and 150 grams of the
respective sugars per 1000 c.c.

20 University of California Publications in Agricultural Sciences [Vol.4

One hundred cubic centimeters of the above solutions were steril-
ized in small plugged flasks. The solutions were inoculated in each
instance with one platinum loopful of a vigorous culture. Non-
inoculated checks were used to correct for loss due to evaporation.
The inoculated flasks were placed in an incubator at 33° C.

The flasks were weighed at intervals during fermentation. The
loss in weight corrected for loss due to evaporation was taken as a
measure of the rate of fermentation.

TABLE 1

Data on Eates of Fermentation of Yeasts Numbers 35 and 72 (S. apiculatus)
in Various Sugar Solutions

Must of

Yeast
lumber

35

Time in
hours

46

23° Balling, Concentrated
loss in must, loss
grams in grams

1.85 .33

Wort loss,
in grams

.35

Dextrose
loss, in
grams

.45

Saccharose
loss, in
grams

.03

Lactose
loss, in
grams

.00

35

73

2.84 1.10

.45

.65

.02

.00

35

96

3.26 2.05

.47

.79

.01

.00

35

118

3.42 2.53

.47

.93

.01

.00

35

167

3.54 2.90

.49

1.13

.00

.00

35

215

3.54 2.95

.57

1.35

.03

.00

35

239

3.57 2.98

.58

1.40

.00

.00

72

27

.18 .01

.35

.10

.00

.00

72

52

1.09 .19

.56

.33

.00

.00

72

77

1.92 .42

.59

.83

.00

.00

72

125

2.75 1.15

.69

.98

.00

.00

72

167

2.84 1.32

.96

.00

.00

72

244

3.18 2.05

.97

1.21

.00

.00

72

341

3.50 2.47

1.10

1.42

.00

.00

72

421

3.55 2.53

1.13

1.44

.00

.00

From an examination of the data given in table 1 and of the
fermentation curves in the accompanying diagram, it is evident that
both yeasts have very low fermenting power and slow rates of fer-
mentation. In grape must at 33° C, yeast no. 35 produces a more
rapid fermentation than yeast no. 72, but in beer wort and dextrose
solutions the two yeasts are similar. The concentrated must of 29.75°
Balling has a greater retarding effect on yeast no. 72 than on yeast
no. 35. Neither yeast fermented saccharose or lactose. Because of
their difference of morphology and in the character of their fermenta-
1 ions, it is justifiable to designate them as two different varieties of
apiculatus yeast.

Attenuation and Alcohol Formation. — The liquids listed in table 2
were analyzed after fermentation. The term "attenuation" in the
accompanying table refers to the loss in sugar during fermentation.
The alcohol, theoretical and observed, is reported as volume per cent.

1918]

Cruess: Fermentation Organisms of California Grapes

21

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TABLE 2

Yeasts Numbers 35 and 72 (S. apiculatus). Attenuation and Yields
Alcohol in Must, Beer Wort, Dextrose, Saccharose, and
Lactose Solutions

Yeast
Xo.

72

72

72

72

72

72

Attenuation
in grams
per 100
c.c.

Medium

Must of 23° Balling 3.81

Must of 29?75 Balling 1.20

Beer wort of 14?56 Balling 1.61

Dextrose solution, 14?9 Ball. 2.73

Saccharose sol., 13?73 Ball. .78

Lactose solution, 15?16 Ball. .31

Theoretical
yield of
alcohol,
per cent

2.50

.79
1.11
1.79

.53

.20

Observed
yield of
alcohol,
per cent

1.00

.30

.30

.65

.00

.00

35 Must of 23° Balling 8.50

35 Must of 29?75 Balling 6.15

35 Beer wort of 14?56 Balling 1.35

35 Dextrose solution, 14?9 Ball. 3.25

35 Saccharose sol., 13? 73 Ball. .00

35 Lactose solution, 15?16 Ball. .00

5.58

4.03

.93

2.13

.00

.00

3.05
2.35

.65
.00
.00

Per cent of
theoretical

yield ob-
yield

obtained

40.0

37.9

27.6

36.3
.00
.00

54.5
58.3

30.5

22 University of California Publications in Agricultural Sciences [Vol. 4

The results show that in most cases, less than 50% of the alcohol,
theoretically obtainable from the sugar fermented, was actually formed
by the two yeasts. For example, yeast no. 35 destroyed 3.81% sugar
and should have produced 2.5% alcohol in must of 23° Balling, while
the actual yield was only 1% or 40% of the amount that was theo-
retically possible. True wine yeasts of the 8. ellipsoideus type pro-
duce at least 90% of the theoretical yield in grape must.

Since the yields of alcohol are so low and the flavor of the fer-
mented liquids is not desirable, the two yeasts may be considered
of little use for industrial purposes. Their importance lies in their
power to cause trouble in wine making by growing in natural fer-
mentations before the true wine yeast has developed. In this way they
may injure the flavor and clearing quality of the wine and favor
"stuck" tanks by the production of compounds that are injurious
to the 8. ellipsoideus yeast. Because of their very common occurrence
in large numbers on California grapes, measures should always be
taken to discourage their growth.

MYCODERMA FORMS FROM CALIFORNIA GRAPES

Under Mycoderma forms, seven film forming organisms have been
described in the following pages. Some of these organisms are yeast-
like in appearance and in their fermentation properties ; other re-
semble molds more closely than they do yeasts. All have the common
properties of forming heavy films on nutrient liquids and of not
forming spores.

Organism 65. Mycoderma from Acampo Grapes.

Morphology. — Microscopical Appearance. — Sausage-shape to fila-
mentous. Tends to grow in mycelial threads (fig. 4).

Size. — Average, 9^ x 2.5/x. Maximum, 40/x x 2.5/x. Minimum,
2/x x 1.5/x.

Spore Formation. — No spores at 22° C and 28° C.

Colonies on Agar Agar. — Small and growth slow. Powdery ap-
pearance. Color, white. Flat. Growth at surface only. Edges made
up of mycelial threads. Under low power, colonies are made up of
chains of long cells. These spread over the surface of the agar.

Colonies on Gelatin. — More vigorous than on agar. Concentric
ring growth. Powdery white. Flat. Under microscope, same as on
agar. Gelatin slowly liquefied.

1918] Cruess: Fermentation Organisms of California Grapes 23

Growth on Liquid Media. — Grows profusely on surface of grape
must, beer wort, dextrose, saccharose and lactose solutions, but does
not produce visible fermentation in any of these liquids. Film remains
permanently on the surface. Does not sink. Film is very tough in
texture.

Organism 68. Mycoderma from Grapes Grown at Acampo, California

Morphology. — Microscopical Appearance. — Predominating form
sausage-shaped. Cells vary from spherical to long-rod shaped cells
(fig. 4).

Size. — Average, 7.5/x x 3.75/x. Maximum, 15/* x 3.75/x. Minimum,
3/x x S/x.

Spore Formation. — No spores formed. at 22° C or 28° C.

Colonies on Agar Agar. — Large. Edges entire. Waxy luster.
Centers of colonies slightly elevated. Edges entire. Opaque. Under
low power of microscope, internal structure granular and edges are
surrounded by long chains of cells of ellipsoidal shape.

Colonies on Gelatin. Filmy, translucent colonies, that rapidly
cover the whole surface. Under microscope the colonies are made up
of chains of cells.

Growth in Liquid Media. — Heavy growth in grape must, beer wort,
and dextrose with formation of large sediment, the volume of which
may be equal to 10 per cent of the total volume of the liquid. Growth
slow in lactose and saccharose solutions. Visible fermentation very
slow in grape must, beer wort and dextrose solution at first, but finally
becomes vigorous in must and dextrose solutions. No visible fermenta-
tion in lactose and saccharose.

Organism 70. Mycoderma from Acampo Grapes

Morphology. — Microscopical Appearance. — Usual form, sausage-
shape, but vary from spherical to sausage-shaped. Cells are usually
grouped in branching chains (fig. 4).

Size. — Average, 7/* x 2/x. Maximum, 18/x. x 2/x. Minimum, 1.5/x x 1.5^.

Spore Formation. — No spores formed at 22° C and 28° C.

Colonies on Agar Agar. Large. Center elevated. Surface cor-
rugated by ridges radiating from centers of colonies. Centers of
colonies brown ; edges yellowish white. Edges indented. Low power
of microscope shows chains of cells growing from edges of colonies;
this growth is beneath the surface of the agar (see plate 2).

24 University of California Publications in Agricultural Sciences [Vol.4

Colonies on Gelatin. — Large. Centers convex. Colonies flatten
near edges and become filmy in appearance. Edges entire under
microscope. Gelatin slowly liquefied.

Growth in Liquid Media. — Heavy, wrinkled surface growth wrhich
frequently falls to bottom forming voluminous sediment. Fermenta-
tion slow. No fermentation in lactose.

Organism 71. Mycoderma from Contra Costa County Grapes

Morphology. — Microscopical Appearance. — Predominating form
sausage-shaped. Varies from spherical to sausage-shape (fig. 4).

Size. — Average, 6.9/x x 2/x. Maximum, 18/x x 2/x. Minimum,

1.5/X X 1.5/X.

Spore Formation.— No spores at 22° C or 28° C.

Colonies on Agar Agar. — Large. Flat. Spreading. Edges lobate.
Edges entire under microscope (see plate 2).

Colonies on Gelatin. — Flat. Filmy. Translucent. Rapidly cover
entire surface of the gelatin. Slow liquefaction (see plate 2).

Growth in Liquid Media. — A wrinkled, loosely coherent film on all
liquids tested. Films frequently sink and are quickly replaced by new
growth. Fermentation in must, beer wort, and dextrose fairly vigor-
ous, but extends over long period. No perceptible fermentation in sac-
charose and lactose.

Organism 73. Mycoderma from Grapes Grown at Acampo

Morphology. — Microscopical Appearance. — Usual form sausage-
shaped. Varies from spherical to sausage-shaped (fig. 4).

Size. — Average, 6.7/*. x 2.5/x. Maximum, 18/x x 3/x. Minimum,
3/x x 1.5/*.

Spore Formation. — No spore formation at 22° C and 28° C.

Colonies on Agar Agar. — Centers elevated. Edges flat and ser-
rated. Shape irregular. Under the microscope chains of cells are
seen to radiate from the edges of the colonies. The chains are made
up of long cells with pairs of short ellipsoidal cells at the junctures
of the long cells.

Colonies on Gelatin. — Flat. Smooth. Filmy. Translucent. Gela-
liti rapidly covered by a filamentous growth. Gelatin is slowly
softened to a syrupy consistency.

Growth in Liquid Media. — Growth vigorous in all liquid media
tested. Vigorous fermentation in grape must and dextrose solution.

1918] Cruess: Fermentation Organisms of California Grapes 25

During fermentation a heavy wrinkled film is formed with a moderate
amount of sediment growth. After fermentation most of the film
growth settles to the bottom. Fermentation is more vigorous and film
formation is not so well developed as in no. 70.

Organism 76. Mycoderma from Grapes Grown at Acampo

Morphology. — Microscopical Appearance. — Sausage-shaped to fila-
mentous (fig. 4).

Size. — Average, 10.5/x x 2.1/x. Maximum, 75/*. x 2.5/*. Minimum,
3/x x 2>fx.

Spore Formation. — No spores formed at 22° C or 28° C.

Colonies on Agar Agar. — Large. Flat. Surface smooth. Centers
slightly elevated. Edges smooth and entire under microscope.

Colonies on Gelatin. — Large. Resinous. Translucent. Surface of
colonies deeply corrugated. Mycelial projections from edges visible
to naked eye. Surface glistening and slimy. Under microscope edges
are fringed by branching chains of very long cells.

Growth in Liquid Media. — Growth in all media tested consists first
of powdery white colonies. These coalesce to form a resinous heavy
film resembling a growth of vinegar bacteria. The growth sinks after
several weeks and is replaced by a second film. No visible fermenta-
tion in any liquids tested.

Organism 78. Mycoderma from Contra Costa County Grapes

Morphology. — Microscopical Appearance. — Sausage-shaped cells
predominate. Very long filamentous cells not uncommon (fig. 4) .

Sige. — Average, 10.5/x x 3/x. Maximum, 37.5/a x 3/x. Minimum,
4.5/x x 2{x.

Spore Formation.— No spores at 22° C and 28° C.

Colonies on Agar Agar. — Large and flat. Centers slightly elevated.
Waxy luster. Edges show hazy growth of mycelium. Under micro-
scope colonies are seen to be surrounded by radiating mycelial threads.

Colonies on Gelatin — Large. Flat. Smooth. Translucent. Rapidly
cover the entire surface of the gelatin. Gelatin rapidly softened to a
syrupy consistency (see plate 2).

Growth in Liquid Media. — In grape must, beer wort, and dextrose,
solutions, growth is rapid and fermentation vigorous; in saccharose
growth vigorous and fermentation slow ; little growth in lactose. Con-
sists chiefly of surface growth. Small sediment.

26 University of California Publications in Agricultural Sciences [Vol. 4

Fermentation Tests of Mycoderma Forms from California Grapes.

The data obtained by noting the loss in weight at 33° C of flasks
containing 100 c.c. of grape must 23.01° Balling, grape must 29.8°
Balling, beer wort 14° Balling, dextrose 15° Balling, saccharose 15°
Balling, and lactose 15° Balling solutions, respectively, after inocula-
tion with pure cultures of the above yeasts appear in table 3. The
attenuation, representing the sugar destroyed, together with the yields
of alcohol in the various sugar solutions tested are given in table 3.
After each table the results of the tests are discussed. The nutrient
solutions used were the same as those described for yeasts 35 and 72.
See description of apiculatus yeasts.

TABLE 3
Fermentation Tests of Mycoderma Forms from California Grapes

Dextrose Saccharose Lactose

Yeast
number

Time in
hours

Must of

23° Balling,

loss in

grams

Must of

29° Balling,

loss, in

grams

Wort loss,
in grams

14 per cent
solution

loss, in

grams

15 per cent

solution

loss, in

grams

15 per cent

solution

loss, in

grams

15 per cent

solution

68

18

.00

.00

.04

.07

.00

.00

68

42

.70

.01

.21

.31

.10

.00

68

66

1.51

.24

.38

.93

.10

.00

68

92

2.45

.62

.37

2.10

.10

.00

68

123

3.35

1.32

.38

2.90

.10

.00

68

165

5.80

2.00

.40

5.89

.10

.03

68

236

8.45

3.30

.42

6.40

.10

.03

68

284

8.48

3.80

.50

6.40

.10

.03

68

314

8.48

3.80

.51

.10

.03

65

18

.00

.00

.00

.00

.00

.00

65

42

.03

.00

.00

.00

.01

.00

65

66

.03

.00

.03

.00

.01

.00

65

92

.03

.00

.08

.04

.01

.00

65

123

.03

.04

.08

.09

.07

.03

65

165

.03

.04

.12

.09

.07

.03

65

236

.03

.04

.17

.09

.07

.03

70

18

.02

.03

.00

.10

.00

.00

70

42

.10

.05

.06

.18

.20

.00

70

66

.38

.08

.33

.41

.20

.06

70

92

.80

.38

.42

.25

.05

70

123

1.90

.08

.80

1.30

.60

.20

70

165

2.47

.83

1.00

1.65

.90

.20

70

236

2.57

.95

1.00

1.65

.90

.30

71

46

.85

.35

.25

.00

.00

71

73

1.85

1.17

.34

.46

.00

71

96

2.70

2.19

.41

.00

71

118

3.74

.■',.19

.42

2.31

.01

71

167

5.47

3.89

.42

4.20

.00

.00

71

215

6.40

5.90

.44

.04

71

239

6.85

6.40

.45

.04

1918]

Cruess: Fermentation Organisms of California Grapes

27

TABLE 3-

-(Continued)

Yeast
lumber

Time in
hours

Must of
23° Ballin
loss in
grams

Must of
g, 29° Balling
loss, in
grams

Wort loss,
in grams

14 per cent
solution

Dextrose

loss, in

grams

15 per cent

solution

Saccharose
loss, in
grams

15 per cent
solution

Lactose

loss, in

grams

15 per cent

solution

73

27

.20

.06

.18

.66

.10

.00

73

52

1.09

.61

.34

1.79

.28

.00

73

77

2.26

1.56

.49

3.21

.58

.00

73

125

4.14

3.34

.59

4.11

.93

.00

73

167

5.69

4.59

.61

5.72

1.19

.00

73

244

9.25

6.62

.72

7.07

1.41

.00

73

341

9.32

8.24

.87

7.35

1.52

.00

73

421

9.52

9.65

.92

.00

76

27

.00

.04

.00

.00

.00

.00

76

52

.04

.08

.04

.07

.00

76

77

.00

76

125

.00

76

167

.31

.13

.24

.20

.00

76

244

.42

.39

.46

.41

.00

76

341

.62

.69

.62

.50

.00

76

421

.64

.69

.70

.54

.00

78

27

.23

.16

.23

.38

.23

.00

78

52

1.12

.44

.54

.80

.54

.00

78

77

2.37

1.64

1.75

2.05

1.37

.00

78

125

4.34

3.29

3.24

3.38

1.74

.00

78

167

6.05

4.60

4.52

1.95

.00

78

244

7.55

6.20

5.25

6.16

2.26

.00

78

341

9.50

7.50

6.98

2.50

.00

78

421

9.85

8.05

7.29

2.70

.00

The seven different varieties of mycoderma yeasts, whose rates of
fermentation in normal must, concentrated must, beer wort, dextrose,
saccharose and lactose solutions were studied gave great variation in
the rate of fermentation and the amounts of sugar fermented. Organ-
ism 65 did not cause fermentation in any of the liquids tested, though
it gave a vigorous growth. On the other hand, yeasts 68, 73, and 78,
developed fairly strong fermentations in must and beer wort, destroy-
ing nearly as much sugar as the true wine yeasts. Organism 76,
caused a very feeble fermentation in grape must but did not ferment
any of the other liquids tested. The mycoderma forms did not ferment
so rapidly in dextrose solution as in grape must. Contrasted with the
mycoderma forms the true wine yeasts did not exhibit this difference.
Lactose was not fermented by any of the mycoderma forms. Sac-
charose was attacked to a slight degree by forms 70, 73, and 78, the
amount of sugar actually fermented in each case being small. In
general, the fermentation curves of the forms shown in the diagrams

28

University of California Publications in Agricultural Sciences [Vol. 4

differ from those of the true wine yeasts shown in figures 12 to 15, by
their low and gentle slope throughout. In most cases they show a
slower start of fermentation than do the 8. ellipsoideus yeasts. Be-

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50 3

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60 4

00 -*

40

Fig. 8. — Fermentation curves of Mycoderma forms in must of 2.3° and 29 ?!
Balling and beer wort.

cause of their slow and incomplete fermentations the mycoderma forms
discussed above are not desirable types to have in wine fermentations.
Attenuation and Alcohol Formation. — Analyses were made of the
Liquids fermented by the mycoderma forms (table 4).

1918]

Cruess: Fermentation Organisms of California Grapes

29

TABLE 4

Yeasts Numbers 65, 68, 70, 71, 73, 76, and 78. Attenuation and Yields of

Alcohol in Must, Beer Wort, Dextrose, Saccharose, and

Lactose Solutions

feast

No.

Medium

Attenuation

in grams

per 100

c.c

Theoretical
yield of
alcohol

Observed
yield of
alcohol

Per cent of

theoretical

yield

obtained

64

Must of 23? Balling

0.00

65

Must of 29!8 Balling

0.00
0.00

65

Beer wort of 14° Balling ...

65

Dextrose solution, 15%

0.00

65

Saccharose solution, 15% .

0.00

65

Lactose solution, 15%

0.00

68

Must of 23° Balling

... 19.88

13.08

7.45

56.9

68

Must of 32° Balling

... 7.84

5.15

4.50

89.3

68

Beer wort of 14° Balling .

... .86

.62

.60

96.8

68

Dextrose solution, 15%

.... 12.68

8.30

6.40

77.1

68

Saccharose solution, 15% .

.03

.02

0.00

00.0

68

Lactose solution, 15%

... .27

.19

0.00

00.0

70

Must of 23° Balling

... 6.66

4.38

2.55

58.2

70

Must of 29?8 Balling

.... 2.05

1.35

1.00

74.5

70

Beer wort of 14° Balling ...

... 1.43

.96

.80

83.3

70

Dextrose solution, 15%

... 4.51

2.96

1.70

57.4

70

Saccharose solution, 15% .

70

Lactose solution, 15%

... 0.00

0.00

0.00

00.0

71

Must of 23° Balling

... 17.61

11.58

8.80

75.9

71

Must of 29?8 Balling

7.90

71

Beer wort of 14° Balling ...

... 2.91

2.03

0.00

00.0

71

Dextrose solution, 15% ....

.... 12.20

8.02

4.10

51.1

71

Saccharose solution, 15% .

08

.05

0.00

00.0

71

Lactose solution, 15%

16

.11

0.00

00.0

73

Must of 23° Balling

... 12.01

7.90

7.50

94.9

73

Must of 29?8 Balling

... 14.75

9.70

6.80

70.1

73

Beer wort of 14° Balling ...

.... 1.61

1.16

.52

44.8

73

Dextrose solution, 15% ....

.... 12.60

8.29

5.60

67.5

73

Saccharose solution, 15% .

.... 2.08

1.50

1.50

100

73

Lactose solution, 15%

35

.25

0.00

00.0

76

Must of 23° Balling

0.00
0.00

76

Must of 29?8 Balling

76

Beer wort of 14° Balling ...

0.00

76

Dextrose solution, 15% ....

0.00

76

Saccharose solution, 15% .

0.00

76

Lactose solution, 15%

0.00

78

Must of 23° Balling

.... 18.87

12.41

10.30

83.1

78

Must of 29?8 Balling

.... 14.13

9.29

9.00

97.9

78

Beer wort of 14° Balling ...

.... 12.15

7.90

7.20

91.1

78

Dextrose solution, 15% ....

.... 13.22

8.69

6.20

71.3

78

Saccharose solution, 15%

.... 3.93

2.86

1.40

48.9

78

Lactose solution, 15%

36

.26

0.00

00.0

30 University of California Publications in Agricultural Sciences [Vol.4

An examination of the data given in table 4 shows that in grape
must, which was the most favorable medium for the activity of the
yeasts under observation, amounts of alcohol varying* from .00% to
10.3% were formed. Some of the organisms, namely, 73 and 78, gave
good yields of alcohol for the sugar fermented in grape must, while
others e.g., 68, 70, 71, gave low yields. Yeasts 65 and 76 gave no alcohol
in any of the liquids in which they were grown, although they destroyed
small amounts of sugar in some of the solutions. None of the organisms
of this group fermented beer wort to any notable degree and the fer-
mentations that did take place in this liquid probably represented the
destruction of dextrose present, only, while it is probable that the mal-
tose was not attacked. Saccharose likewise proved very resistant to fer-
mentation by the above forms. Organisms 68, 71, 73, and 78 formed
considerable amounts of alcohol in dextrose solution, indicating that
this sugar is more easily attacked than cane sugar or maltose. These
facts indicate that the yeasts under discussion are deficient in invertase
and maltase, enzymes that are necessary to the fermentation of saccha-
rose and maltose.

Since the mycoderma forms discussed above do not ferment beer
wort or cane sugar, they would be useless in the manufacture of beer
or of alcohol from materials containing cane sugar. The amounts of
alcohol formed in grape must by several of the organisms might be
sufficient for some purposes, but their use for the production of wine
is excluded by the fact that all of them produce disagreeable tastes and
odors in grape must, resulting in the formation of an undririkable
fermented liquid in all cases. Therefore, it is to the winemaker's
interest to prevent the growth of these organisms.

TOEULA FORMS FROM CALIFORNIA GRAPES

Two yeasts of the torula type were found on grapes examined
in 1911, one from Davis grapes and the other from grapes grown in
Contra Costa County, California.

Organism 37. Torula Yeast from Grapes Grown at Davis

Morphology. — Microscopical Appearance. — Varies from spherical
to shortly ellipsoidal in form; latter form predominating. Cells tend
to form chains of cells of various sizes (see fig. 3).

Size. — Average, 4.6/x x 3.7/x. Maximum, 15/x x 6fi. Minimum, 3/x x 3/^.

Spore Formation. — No spores formed at 22° C or 28° C.

1918]

Cruess: Fermentation Organisms of California Grapes

31

Colonies on Agar Agar. — Almost flat, but centers, slightly elevated.
White. Waxy luster. Edges entire. Colonies in the agar, spherical.
Under microscope, edges of colonies entire.

Colonies on Gelatin. — Small. White. Waxy luster. Gelatin quickly
liquefied.

Growth in Liquid Media. — Growth in grape must, dextrose, and beer
wort, rapid, with formation of considerable sediment, and develop-
ment of a feeble bottom fermentation. No film formation. No fer-
mentation in lactose solutions, but good growth. Feeble fermenta-
tion and good growth in saccharose. Rancid taste and odor developed
in fermented liquids.

40 no izo if>o znn 2.-40 2.?>n ^zo ?*>f>c> ioo 4*0

-s

<o

HMF

IN Hi

1UHS

-78

4

/

s

fl

w rt

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NTH'

TION.

5 RT

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5

mt

in n

rxr^

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^Ol

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—73

0

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71

n

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ION

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76

•

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75

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timl

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IN h

OURZ

\ *— »

^

itr~h

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bo h

bO ft

00 z-

W X

vo o>

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bO *l

00 -5^C

Fig. 9. — Fermentation curves of Mycoderma forms in dextrose and saccha-
rose solutions.

Organism 77. Torula from Contra Costa County Grapes

Morphology. — Microscopical Appearance. — Vary from ellipsoidal
to sausage-shaped with ellipsoidal form predominating (see fig. 3).
Tends to form short chains.

Size. — Average, 4/x, x 2.1 fx. Maximum, 8p x 4.5^. Minimum,
2.2/*xl.5/x.

Colonies on Agar Agar. — Small. White. Convex. Waxy luster.
Spherical colonies in the agar. Under the microscope colonies are
found to be surrounded by straight chains of long cells. Most other
yeasts examined that formed side chains in this manner produced
branching cells.

Colonies on Gelatin. — Contoured surface with highest elevation at
centers of colonies. Centers of colonies colorless. Chalklike at edges.

32 University of California Publications in Agricultural Sciences [Vol. 4

Undermicroscope, colonies are fringed with branching chains of long
cells.

Growth in Liquid Media. — Growth in all liquids slow. Feeble fer-
mentation in grape must, beer wort, and dextose. No surface growth.
No fermentation in lactose and saccharose.

Rates of Fermentation of Torulas 37 and 77 in Various Media.

Kates of fermentation in grape must of 23° Balling, and 29.8° Balling,
beer wort of 14° Balling, and 15% solutions of dextrose, saccharose,
and lactose were tested as previously described for apiculatus and
mycoderma forms. The results are given in table 5 and figure 10.

TABLE 5

Fermentation Tests at 33° C op Torula Forms from California Yfasts

Must of Dextrose Saccharose Lactose

Must of of29°8 Wort loss, loss in loss, in loss, in

13° Balling, Balling, in grams grams grams grams

Yeast
number

Time in
hours

loss in
grams

loss, in
grams

14 per cent
solution

15 per cent
solution

15 per cent
solution

15 per ci
solutio

37

18

.01

,00

.04

.12

.07

.02

37

42

.19

.07

.31

.54

.29

.07

37

66

.51

.45

.98

.38

37

92

.63

.50

1.40

.43

.08

37

165

.87

.51

2.74

.54

.08

37

236

.87

.13

.51

3.64

.54

.09

77

46

.17

.01

.23

.23

.01

.03

77

73

.84

.15

.48

.48

77

96

1.14

.42

.58

.58

.10

77

118

1.30

.81

.68

.68

77

167

1.37

.86

.78

.78

.10

77

215

1.44

1.03

.83

.83

77

239

1.48

1.08

.95

.95

.03

Torulas 37 and 77 were able to produce only very feeble fermenta-
tions in the liquids tested. Torula yeast 77 was not able to ferment
must of 30° Balling, or saccharose solution. In must of 23° Balling,
and in beer wort the fermentations of the two yeasts were about equal.
In must, beer wort, and saccharose the fermentations were completed
in a short time, about 120 hours. In comparison with the fermenta-
tion curves of the 8. elUpsoideus yeasts (figs. 12 to 15), the yeasts
above gave very weak and incomplete fermentations. The flavors
of the fermented liquids were disagreeable. Therefore, the yeasts
seen i to have no practical value but may cause defective flavors in
wine where their growth is not prevented.

Attenuation and Yields of Alcohol. — The fermented liquids were
analyzed for alcohol with the results reported in table 6.

1918]

Cruess: Fermentation Organisms of California Grapes

33

0 40 A

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60 2

on &io 2,no 7>2,o 7S<bn ^no

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Fig. 10. — Fermentation curves of yeasts 37 and 77 in grape must, beer wort,
dextrose and saccharose solutions.

34 University of California Publications in Agricultural Sciences [Vol.4

TABLE 6

Yeasts 37 and 77. Attenuation and Yields of Alcohol in Must, Beer Wort,
Dextrose, Saccharose, and Lactose Solutions

Attenuation Per cent of

in grams Theoretical Observed theoretical

Yeast per 100 yield of yield of yield

No. Medium c.c. alcohol alcohol obtained

37 Must of 23° Balling 5.21 3.42 .60 17.6

37 Must of 29?8 Balling 05 .03 .00 .0

37 Beer wort of 14° Balling .20

37 Dextrose solution, 15% 7.96 5.22 3.50 67.3

37 Saccharose solution, 15% 64 .43 .40 93.02

37 Lactose solution, 15% 06 .04 .00 .0

77 Must of 23° Balling 4.58 2.99 1.31 43.7

77 Must of 29?8 Balling 3.85 2.53 .65 25.7

77 Beer wort of 14° Balling .... 2.91 2.00 .30 15.0

77 Dextrose solution, 15% 4.55 2.99 .65 21.7

77 Saccharose solution, 15% .00 .0

77 Lactose solution, 15% 06 .04 .00 .0

Analyses of the fermented liquids from fermentation of Torulas
37 and 77 in grape must, etc., showed that the absolute amounts of
alcohol formed were small and that the yields for each per cent of
sugar destroyed were in all cases, except for yeast 37 in dextrose, very
low. In the majority of the fermentations, the yields were less than
50 per cent of the theoretical. These yeasts have the defects of giving
slow incomplete fermentations, low yields of alcohol for sugar fer-
mented and of producing liquids of disagreeable odor and flavor.

PASTORIANUS AND WILLI A YEAST FROM CALIFORNIA GRAPES

One S. pastorianus and one Willia yeast were obtained from grapes
in 1911. Both are true yeasts and form spores.

Organism 36. S. pastorianus Yeast from Grapes Grown at Davis

Morphology. — Microscopical Appearance. — Form varies from
spherical to sausage-shaped. The ellipsoidal form predominating.
Much smaller than true ellipsoideus yeast (fig. 3).

Size. — Average, 4.5/* x 3/x. Maximum, 7.5w x 3/x. Minimum,
1.5/x x 1.5ft.

Spore. — Produced in great abundance in 24 hours on gypsum spore
blocks. Spores also form in grape must cultures. Spores spherical in
form. Number per cell, 2 to 4. Size of spores, 2/x x 2/x, (fig. 7) .

Colonies on Agar Agar. — Round. Slightly convex. White. "Waxy
Luster. Colonies in agar spherical. Under the low power, the colonies

1918] Cruess : Fermentation Organisms of California Grapes 35

are surrounded by chains of cells made up of long central cells with
pairs of short ellipsoidal cells at the junctures of the larger cells.

Colonies on Gelatin. — Flat. Concentric rings of growth. Outer
edges of colonies sharply indented. Gelatin slowly softened but not
completely liquefied.

Growth in Liquid Media. — Heavy wrinkled surface growth which
sinks to bottom frequently. No visible fermentation. Taste of liquids
bitter and disagreeable.

Organism 69. Willia Species (Saccharomyces anomalus) from Grapes
Grown at Acampo, California

Morphology. — Microscopical Appearance. — Size and shape very
irregular. The larger cells usually sausage-shaped or ellipsoidal and
the smaller ones spherical. Larger than typical S. anomalus in the
University collection (fig. 3).

Size. — Average, 6/a x 3^. Maximum, 14^ x 2.5//,. Minimum, 3/x x 3/x.

Spore Formation. — Spores are formed abundantly on gysum blocks
at 28° C and 22° C, in three days. Spores are of typical Willia form,
i.e., hat-shaped (fig. 7). Number of spores per cell varies from 2 to 8.
Size of spores, 4//, x 1.5/x.

Colonies on Agar Agar. — Medium size. Flat. White. Waxy
luster. Edges entire. Colonies on the agar are spherical. LTnder the
microscope the edges of the colonies are entire.

Colonies on Gelatin. — Large. Flat. White. Chalklike. Colonies
spread rapidly and soon cover entire surface and the gelatin is rapidly
liquefied.

Growth in Liquid Media. — A chalky white film develops on all
liquids tested, with flocculent growth at bottom of flask. Prolonged
top fermentation takes place in all liquids except lactose. Strong
odor of ethyl acetate is developed. The fermented liquids possess a
burning taste.

Rates of Fermentation of Yeasts 36 and 69. — These yeasts were
tested for their rates of fermentation in various media as previously
described for other organisms. The results are given in table 7 and
figure 11.

From the data given in table 7, yeast 36 is seen to give scarcely any
fermentation in any of the liquids tested, the maximum loss in weight
per 100 c.c. being .68 grams in must of 29.8° Balling. On the other
hand, yeast 69 gave fairly strong fermentations in must, beer wort,
dextrose, and saccharose solutions. Neither yeast fermented lactose,

36 University of California Publications in Agricultural Sciences ' [Vol. 4

TABLE 7
Data of Eates of Fermentation of Yeasts Numbers 36 and 69 in Various

Sugar Solutions

Yeast
number

Time in
hours

Must of

23° Balling,

loss in

grams

Concentrated Wort loss,
must, loss in grams
in grams 14 per cent
29? 8 Ball. Balling.

loss, in

grams

15 per cent

solution

loss, in

grams

15 per cent

solution

loss, in

grams

15 per cer

solution

36

46

.03

.03

.03

.03

.00

.00

36

73

.04

.04

.03

.06

.00

.00

36

96

.09

.11

.03

.08

.00

.01

36

118

.24

.16

.13

.13

.00

.01

36

167

.33

.43

.13

.19

.00

.02

36

215

.45

.63

.13

.26

.00

.02

36

239

.46

.68

.28

.00

.02

69

18

.01

.10

.13

.01

.10

.03

69

42

.58

.18

.46

.13

.28

.14

69

66

1.10

.23

.56

.77

.86

.14

69

92

1.79

.80

1.71

1.77

1.90

69

123

2.49

2.20

1.75

2.87

2.88

69

165

3.22

2.50

1.81

3.99

3.78

69

236

4.02

4.88

1.92

5.31

4.62

.14

69

283

4.49

5.55

1.93

5.77

4.98

69

314

4.62

5.75

5.79

5.03

while yeast 36 did not attack saccharose. Although yeast 69 gives
more vigorous fermentations than does yeast 36, still it does not ap-
proach the S. ellipsoideus yeasts from California grapes in rate of
fermentation or fermentative power.

Attenuation and Alcohol Yields of Yeasts 36 and 69. — The fer-
mented liquids were analyzed with the results given in the accom-
panying table.

TABLE 8

Yeasts Numbers 36 and 69. Attenuation and Yields of Alcohol in Must,

Beer Wort, Dextrose, Saccharose, and Lactose Solutions

Attenuation Per cent of

in grams Theoretical Ons€rved theoretical

Yeast per 100 yield of yield of yield

No. Medium c.c alcohol alcohol obtained

36 Must of 23° Balling 2.49 1.95 .30 15.3

36 Must of 29?8 Balling 1.65 1.08 .00 .0

36 Beer wort of 14° Balling 31 .20 .00 .0

36 Dextrose solution, 15% 1.95 1.28 .24 18.9

36 Saccharose solution, 15% 02 .67 .00

36 Lactose solution, 15% 11 .07 .00

69 Must of 23° Balling 11.12 7.30 1.25 17.1

69 Must of 29?8 Balling 2.92 1.90 1.00 52.6

69 Beer wort of 14° Balling .... 1.40 .92 .50 54.3

69 Dextrose solution, 15% 11.03 7.25 3.10 42.6

69 Saccharose solution, 15% 7.73 5.09 2.60 51.2

69 Lactose solution, 15% 1.25 .80 .00 .0

1918]

Crusss: Fermentation Organisms of California Grapes

37

4Q SO 120 I6Q 2QQ 2*Q 280 7*>20 ^0^00

time:

>N HOURfS

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AT 3

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69

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s

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<T)

SfiCCHffFTO

SF SOLUTION

TIME IN HOURS

4o ro zk? 7eo WO ~^D %$0 d^O Ot>U <W0

Fig. 11. — Fermentation curves for yeasts 36 and 69 in grape must, beer
wort, dextrose, and saccharose solution at 33 °C.

38 University of California Publications in Agricultural Sciences [Vol. 4

Yeast 69 destroyed larger amounts of sugar than did yeast 36 and
formed greater amounts of alcohol, but both yeasts gave very low
yields of alcohol compared to the amount of sugar fermented. In
most cases the yield was below 50% of the theoretical, based on the
sugar destroyed. Yeast 69 formed 3.1% alcohol in dextrose solution,
this being the largest amount of alcohol produced by either yeast.

Because of its disagreeable flavor and low alcohol formation, yeast
36 is worthless for fermentation purposes. Yeast 69 produces a very
aromatic odor and flavor in liquids in which it grows. These fer-
mented materials are not pleasing to drink by themselves but when
added in small amounts to new wine they have the property of
"aging" the new wine so treated. Therefore it may be possible to use
this yeast as an aid to aging wine either by adding some of the liquid
fermented by it to new wine or by growing it directly in new wine,
and in this latter case, allowing the desired flavors to be developed in
the wine itself. Yeasts of this same group are used to some extent
in the aging of "sake" (Japanese beer).

TEUE WINE YEASTS, SACCHAROMYCES ELLIPSOIDEUS FROM
CALIFORNIA GRAPES

Six kinds of true wine yeast, S. ellipsoideus, were isolated from
grapes in 1911 and have been discussed as a group in the following
pages.

Organism 64. Saccharomyces ellipsoideus from Grapes
Grown at Acampo

Morphology. — Microscopical Appearance. — Form varies from
shortly ellipsoidal to spherical. Cells shorter than those of no. 74
(fig. 5).

Size. — Average, 7.5/x, x 6/x. Maximum, 12//, x 3/x. Minimum, 3/x x 3/x.

Spore Formation. — Abundant spore formation at 22° C and 28° C.
Usually 3 spores per cell. Shape spherical. Size, 3,/x x 3/x.

Colonies on Agar Agar. — Medium size. Round. Moderately con-
vex. White. Waxy luster. Edges entire. Under microscope edges
are entire.

Colonies on Gelatin. — Large. Centers elevated. Radiating ridges
from center. Centers slightly elevated. Edges of colonies are un-
dulate in appearance. Gelatin slowly softened but not liquefied.
Edges of colonies entire under microscope.

1918] Cruess: Fermentation Organisms of California Grapes 39

Growth in Liquid Media. — Rapid in grape must, beer wort, dex-
trose, and saccharose solutions with vigorous fermentation and forma-
tion of fine grained sediment. No fermentation in lactose, but fair
growth. Characteristic vinous odor and flavor developed.

Organism 66. Saccharomyces ellipsoideus from Grapes
Grown at Davis

Morphology. — Microscopical Appearance. — Predominating form
oblong with tendency to be pointed at one end. Resembles S. cerevisiae
in this regard (fig. 5).

Size. — Average, 9/x, x 5.7/x. Maximum, 13.5//, x 4/*. Minimum,
4/x x 3/x. Considerably larger than most varieties of S. ellipsoideus
from California grapes.

Spore Formation. — Spores are formed abundantly at 22° C and
28° C but more rapidly at the lower temperature. Number per cell
2 to 3. Size, 3/* x 3/*.

Colonies on Agar Agar. — Size, medium. Convex. Circular.
White. Waxy luster. Edges entire macroscopically and microsco-
pically. Colonies in the agar are usually lens-shaped.

Colonies on Gelatin. — Colonies more flat than on agar. Smooth.
Under microscope edges of colonies appear bordered with branching
chains of ellipsoidal cells. Gelatin slowly softened but not liquefied.

Growth in Liquid Media. — Rapid with vigorous fermentation in all
solutions tested except lactose. Grows in lactose. Sediment fine
grained. Settles quickly after fermentation. Typical vinous flavor
developed in fermented liquids.

Organism 67. Saccharomyces ellipsoideus from Grapes Grown
in Contra Costa County

Morphology. — Microscopical Appearance. — Ellipsoidal to spherical,
former type predominating. Resembles yeast 74 in general appear-
ance (fig. 5).

Size. — Average, 6.75/x x 5.25/x. Maximum, 9jx x 6.75/x. Minimum,
3/x x 3/x.

Spore Formation. — Large numbers of spores are formed at 22° C
and 28° C in 24 hours on spore blocks. Number per cell, 2 or 3. Size,
3/x x 3/x.

Colonies on Agar Agar. — Small. Convex. White. Waxy luster.
Colonies in agar are spherical. Edges of colonies entire, both macro-
scopically and microscopically.

40 University of California Publications in Agricultural Sciences [Vol. 4

Colonies on Gelatin. — Large. Centers of colonies slightly de-
pressed. Edges are undulate. Under microscope, colonies are fringed
with short straight chains of ellipsoidal cells. The gelatin is not
liquefied or softened.

Growth in Liquid Media. — Growth rapid and fermentation vigor-
ous in grape must, beer wort, dextrose, and saccharose. Growth slight
and fermentation absent in lactose. Fermentation more rapid at 20° C
than at 33° C. Sediment fine grained. Typical vinous flavor and

Organism 74. Saccharomyces ellipsoideus from Grapes Grown

at Acampo

Morphology. — Microscopical Appearance. — Usual form oblong
rather than shortly ellipsoidal. Decidedly different in form than
yeast 64 from the same grapes (fig. 5).

Size. — Average, 6.6/x x 4.6^. Maximum, 9/x, x 4.5/x. Minimum,
Sfi x 3/x. Smaller than yeasts 66 and 79, which it resembles in shape.

Spore Formation. — Few spores only at 28° C in 48 hours and none
in 24 hours. More formed at 22° C in 48 hours. Number per cell, 2.
Size, Sjx x 3/x (fig. 6).

Colonies on Agar Agar. — Large. Round. Centers elevated.
Colonies in agar lens-shaped. Edges of colonies entire both macrosco-
pically and microscopically.

Colonies on Gelatin. — Large. Low elevation. Colonies in the
gelatin spherical. Edges of colonies entire both macroscopically and
microscopically. No liquefaction.

Growth in Liquid Media. — Rapid with vigorous fermentation in
must, beer wort, dextrose, and saccharose solutions. Small growth and
no fermentation in lactose. Typical vinous taste and odor.

Organism 75. Saccharomyces ellipsoideus from Grapes Grown in
Contra Costa Cownty

Morphology. — Microscopical Appearance. — Spherical to shortly
ellipsoidal (fig. 5).

Size. — Average, 6.3//. x 5.5/x. Maximum, 8^ x 8/x. Minimum, 3^ x 3fx.

Spore Formation. — No spores formed at 28° C at 72 hours, but
abundantly after 144 hours. This differs from yeast 67 from the same
grapes; 67 forms spores in 24 hours at 28° C. Numbers of spores per
cell, 2. Size, 3^x3^ (fig. 6).

1918] Cruess: Fermentation Organisms of California Grapes 41

Colonies on Agar Agar. — Medium size. Round. Convex. White.
Waxy luster. Colonies in the agar are lens-shaped. Edges of colonies
entire, both macroseopieally and microscopically.

Colonies on Gelatin. — Large. Low and of even elevation. Sur-
face radiately ridged. Edges undulate. Under microscope colonies
are bordered by short straight chains of ellipsoidal cells.

Growth in Liquid Media. — Rapid and fermentation vigorous in
grape must, beer wort, dextrose, and saccharose. Slight and no fer-
mentation in lactose. Typical vinous flavor and odor in fermented
liquids.

Organism 79. Saccharomyces ellipsoideus from Grapes Grown in
Contra Costa County

Morphology. — Microscopical Appearance. — Cells large and oblong.
Not strictly ellipsoidal in form (fig. 6).

Size. — Average, 8.25/x x 5/x. Maximum, 12/* x 6/x. Minimum,
4.5/a x 3/a.

Spore Formation. — Spores are formed abundantly in 24 hours at
28° C. Number per cell, 2 to 4. Size, 2/i x 2/* and 3/x x 3/x (fig. 6) .

Colonies on Agar Agar. — Medium size. Spherical. Convex. White.
Waxy luster. Colonies in agar lens-shaped. Edges entire both macro-
seopieally and microscopically.

Colonies on Gelatin. Flat. Edges hazy. White. Waxy luster.
Under microscope edges are bordered by branching chains of el-
lipsoidal cells.

Growth in Liquid Media. — In grape must, beer wort, dextrose, and
saccharose, rapid and fermentation vigorous. In lactose, slow and no
fermentation. Typical vinous taste and odor developed.

Fermentation Records at 33° C. — Fermentations were carried out
in 100 c.c. portions of various liquids at 33° C and records of the rates
of fermentation were kept as previously explained.

All of the yeasts with the exception of yeast 67 show rapid fer-
mentations in must of 23° Balling, and 30° Balling, beer wort, dextrose
and saccharose solutions, there being little to choose between them
in the rapidity of fermentation and the amount of sugar destroyed.
The yeasts show considerable variation in the fermentation of beer
wort. Apparently some of the yeasts ferment only the dextrose of
the beer wort and not the maltose. Yeast 67, however, is very strongly
inhibited in its activity by the moderate temperature of 33° C, because
it causes only very feeble and incomplete fermentations at this tern-

42

University of California Publications in Agricultural Sciences [Vol. 4

TABLE 9

Data on Eates of Fermentation of Yeasts Numbers 64, 66, 67, 74, 75, and 79

at 33° C in Various Sugar Solutions

Yeast
number

64

64

64

64

64

64

64

79
79
79
79
79
79
79

Time in
hours

18

42

66

92
123
165
236

46
73
96
118
167
215
239

Must of

23° Balling,

loss in

grams

1.42

4.18
6.72
8.09
8.26
8.33
8.88

Must of
of 29?8
Balling
loss in
grams

.00

.66
4.33
7.90
8.70
9.70
10.00

Wort loss,
in grams
14° Ball.

.87

2.57

2.75

2.94

2.94

3.04

3.16

Dextrose Saccharose
loss, in loss in

grams grams

15 per cent 15 per cent
solution

.49
1.95
2.32

4.74
5.84
6.49
6.77

4.96

7.77
8.40
8.74
8.88
9.07
9.10

3.21
6.97
8.84
10.14
10.83
11.27
11.40

1.81
2.42
2.63

2.83
2.83
3.07
3.07

1.79
3.37
4.52
5.66
6.38
6.66
6.68

solution

.68
2.43
3.96
5.35
6.30
6.88
7.30

1.80
3.66
4.77
5.71
6.40
6.55
6.60

Lactose

loss, in

grams

15 per cent

solution

.03

.07
.07

66

27

1.83

.05

.06

.44

.60

.00

66

52

5.92

.69

.55

2.13

1.89

.00

66

77

8.49

4.11

2.49

5.23

2.91

.00

66

125

10.09

9.31

6.64

6.81

4.59

.00

66

167

10.44

11.71

8.69

5.56

.00

66

244

10.98

11.85

7.54

7.38

.00

66

341

11.68

12.59

8.35

.00

66

421

12.82

8.55

.00

67

27

.14

.04

.33

.66

.31

.00

67

52

.89

.14

.53

1.80

.42

.00

67

77

1.99

.92

.64

3.21

.64

.00

67

125

2.79

1.57

.67

4.11

.83

.00

67

244

2.99

1.84

.72

5.72

.84

.00

67

341

3.33

2.40

.75

7.07

1.13

.00

67

421

3.50

2.85

.75

7.35

1.30

.00

67

4.59

2.98

1.39

.00

74

27

2.69

.16

1.76

.83

.93

.00

74

52

5.71

2.89

2.54

3.52

2.74

.00

74

77

7.81

6.15

3.99

5.52

4.42

.00

74

125

9.14

9.77

4.27

6.59

.00

74

167

9.31

11.87

4.32

7.23

7.09

.00

74

244

10.87

12.38

4.65

7.62

.00

74

341

11.50

12.53

5.00

7.87

.00

74

421

12.65

5.15

7.97

.00

75

27

2.08

.08

.66

.38

.61

.00

75

52

4.72

.67

1.29

.80

1.57

.00

75

77

6.74

4.05

1.89

2.05

2.47

.00

75

125

8.06

6.89

2.64

3.38

4.04

.00

75

167

8.74

7.94

3.12

6.17

5.17

.00

75

244

9.85

8.88

3.40

6.98

6.78

.00

75

341

10.97

10.66

3.55

7.29

7.60

.00

75

421

11.35

10.66

3.65

7.90

.00
.00
.00
.00
.00
.00
.00

1918]

Cruess: Fermentation Organisms of California Grapes

43

perature. The presence of such yeast as this in natural wine fermen-
tations would render such fermentations very liable to "sticking" if
the temperature arose much above 33° C. It is possible that the

^o an 12,0 if>n zoo £40 %

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Fig. 12. — Fermentation curves of true wine yeasts from California grapes.

reason that some wines will ferment completely even at temperatures
above 38° C, and others stick with unfermented sugar is because of
the difference in the character of the natural wine yeast present. The
possibility of a yeast similar to yeast 67 being the only wine yeast
present in a natural wine fermentation makes it very desirable to

44 University of California Publications in Agricultural Sciences [Vol. 4

insure good fermentations by the use of pure and selected varieties
of wine yeast.

Lactose supported the growth of the 8. ellipsoideus yeasts discussed
above, but was not fermented by any of them.

Attenuation and Alcohol Formation at 33° C. — The fermented
liquids were analyzed with the results in table 10.

TABLE 10

Yeasts Numbers 64, 66, 67, 74, 75, 79. Attenuation and Yields of Alcohol
in Must, Beer Wort, Dextrose, Saccharose, and Lactose Solutions

Sfeasl
No.

Medium

Attenuation
in grams
per 100
c.c

Theoretical
yield of
alcohol

Observed
yield of
alcohol

Per cent of

theoretical

yield

obtained

64

Must of 23° Balling

... 19.70

12.80

11.75

93.4

64

Must of 29?8 Balling

... 21.40

13.90

12.80

92.1

64

Beer wort of 14° Balling ...

... 6.16

4.39

3.40

77.4

64

Dextrose solution, 15%

... 11.69

7.60

6.00

79.0

64

Saccharose solution, 15% ...

... 12.23

8.80

7.15

81.4

64

Lactose solution, 15% ...

.00

.0

66

Must of 23° Balling

... 19.41

12.70
12.90

11.40
11.40

89.7

66

Must of 29?8 Balling

... 19.75

88.4

66

Beer wort, of 14° Balling .

5.70

66

Dextrose solution, 15%

... 13.20

8.60

7.95

92.4

66

Saccharose solution, 15% ...

... 12.03

8.30

7.55

90.9

66

Lactose solution, 15%

.00

67

Must of 23° Balling

... 5.41

3.55

2.00

56.4

67

Must of 29?8 Balling

... 3.85

2.53

1.30

51.3

67

Beer wort of 14° Balling .

... 1.61

1.11

.65

58.6

67

Dextrose solution, 15%

... 12.75

8.38

7.95

94.7

67

Saccharose solution, 15% ...

... 2.82

1.94

1.00

51.5

67

Lactose solution, 15%

... .36

.24

.00

.0

74

Must of 23° Balling

... 19.31

12.70

11.40

89.7

74

Must of 29?8 Balling

... 20.95

13.70

11.40

83.2

74

Beer wort, of 14° Balling .

... 7.96

5.30

4.10

75.3

74

Dextrose solution, 15%

... 11.64

7.60

5.70

75.0

74

Saccharose solution, 15% ...

... 11.83

8.18

7.95

97.2

74

Lactose solution, 15%

.00

75

Must of 23° Balling

... 18.31

12.03

8.01

66.6

75

Must of 29?8 Balling

... 22.10

14.53

75

Beer wort of 14° Balling .

... 6.07

4.17

2.34

57.3

75

Dextrose solution, 15%

... 12.55

8.60

6.40

74.4

75

Saccharose solution, 15% ...

... 12.62

8.70

8.60

98.8

75

Lactose solution, 15%

... .36

.25

.00

.0

79

Must of 23° Balling

... 18.51

12.17

79

Must of 29?8 Balling

... 18.54

12.20

11.45

93.8

79

Beer wort of 14° Balling .

... 6.36

4.38

2.35

53.7

79

Dextrose solution, 15%

... 12.60

8.20

6.00

72.9

79

Saccharose solution, 15% ...

... 11.43

7.87

6.00

76.2

79

Lactose solution, 15%

... .06

.04

.00

.0

1918]

Cruess : Fermentation Organisms of California Grapes

45

^ ^0 im tm *on z^n &m 3$a -y? ^pn ^y

^F7D

Fig. 13. — Fermentation curves of true wine yeasts from California grapes.

The effect of a moderately high temperature on yeast 67 is shown
by the analyses of the fermented liquids, from which it is seen that
the absolute amounts of alcohol formed by this yeast are small and
that the per cents of the theoretical yields obtained were very small.
Thus at 33° C it not only gives very incomplete fermentations, but
is also very wasteful of the sugar that it ferments. Yeast 75 gave
the highest yield of alcohol, 14.53 per cent. None of the yeasts gave as
much alcohol in must of 30° Balling, at 33° C as they did at 24° C.
Contrasted with the yeasts discussed in the previous pages the S.
ellipsoideus yeasts gave larger yields of alcohol and in general a larger
per cent of the sugar destroyed was transformed into alcohol.

46

University of California Publications in Agricultural Sciences [Vol.4

Comparison of Fermentations at 33° C and 24° C (all Varieties)

The nineteen fermentation organisms discussed in the preceding
pages were transferred to grape must of 29.8° Balling, and the pro-
ducts of fermentation at 24° C and 33° C studied and compared. The
results appear in table 11.

TABLE 11

Comparison of Products of Fermentation of California Yeasts in Grape

Must of 30° Balling Fermented at 24° C and 33° C

Attenuation
in grams
per 100
Organism c.c.

35. S. apiculatus 8.31

72. S. apiculatus 6.80

65. Mycoderma species .... 2.70

68. Mycoderma species ... . 4.20

70. Mycoderma species .... 3.40

71. Mycoderma species .... 8.20

73. Mycoderma species .... 23.90

76. Mycoderma species .... 3.20

78. Mycoderma species .... 10.10
37. Torula species 3.70

77. Torula species 6.47

36. S. pastorianus 2.80

69. Willia anomala 5.11

64. S. ellipsoideus 24.60

66. S. ellipsoideus 22.00

67. S. ellipsoideus 24.01

74. S. ellipsoideus 22.50

75. S. ellipsoideus 22.70

79. S. ellipsoideus 25.30

35. S. apiculatus 6.15

72. S. apiculatus 1.20

65. Mycoderma vini 0.00

68. Mycoderma vini 7.84

70. Mycoderma vini 2.05

71. Mycoderma vini

73. Mycoderma vini 14.75

76. Mycoderma vini 00.00

78. Mycoderma vini 14.13

37. Torula species 05

77. Torula species 3.85

36. S. pastorianus 1.65

69. Willia Anomala 2.92

64. S. ellipsoideus 21.40

00. S. ellipsoideus 19.75

07. S. ellipsoideus 3.85

74. S. ellipsoideus 20.95

7;1. S. Hlipsoi<l<Mis 22.10

79. S. cllipso'i-lcus 18.54

Theoretical
yield of
alcohol,
volume,
per cent

5.46

Alcohol
obtained,
volume,
per cent

2.60

Per cent of
theoretical

yield
obtained

47.6

Temperature
24° C

4.47

3.20

72.7

24° C

1.70

0.00

00.0

24° C

2.70

1.20

44.4

24° C

2.20

.60

27.3

24° C

5.40

3.20

59.2

24° C

15.70

13.50

86.0

24° C

2.10

.65

30.9

24° C

6.64

4.05

60.9

24° C

2.43

.30

12.3

24° C

4.25

1.05

24.7

24° C

1.85

0.00

00.0

24° C

3.35

1.05

31.3

24° C

16.20

15.60

96.3

24° C

14.40

14.10

97.9

24° C

15.80

15.30

96.8

24° C

14.80

14.20

95.2

24° C

14.90

14.40

96.6

24° C

16.60

15.55

93.7

24° C

.79

.30

37.9

33° C

4.03

2.35

58.30

33° C

0.00

0.00

00.0

33° C

5.15

4.50

89.30

33° C

1.35

1.00

74.50

33° C

7.90

33° C

9.70

6.80

70.10

33° C

0.00

0.00

00.00

33° C

9.29

9.00

97.90

33° C

.03

0.00

00.00

33° C

2.53

.65

25.70

33° C

1.08

.00

00.00

33° C

1.90

1.00

52.60

33° C

13.90

12.80

92.10

33° C

12.90

11.40

88.40

33° C

2.53

1.30

51.30

33° C

13.70

11.40

83.20

33° C

14.53

33° C

12.20

11.45

93.80

33° C

1918] Cruess: Fermentation Organisms of California Grapes 47

TABLE 11— (Continued)

Loss in fixed
acid during
Volatile acid, grams fermentation,
Organism per 100 c.c. grams per 100 c.c. Temperature

35. S. apiculatus 110 —.10 24° C

77. S. apiculatus 100 .05 24° C

65. Mycoderma vini 020 .06 24° C

68. Mycoderma vini 050 —.19 24° C

70. Mycodrema vini 030 — .10 24° C

71. Mycoderma vini 043 — .07 24° C

73. Mycoderma vini 097 .02 24° C

76. Mycoderma vini 020 .04 24° C

78. Mycoderma vini ' .056 — .08 24° C

37. Torula species 084 .06 24° C

77. Torula species 020 — .02 24° C

36. S. pastorianus 030 —.04 24° C

69. Willia anomala 080 —.06 24° C

64. S. ellipsoideus 060 .06 24° C

66. S. ellipsoideus 050 .10 24° C

67. S. ellipsoideus 080 .12 24° C

74. S. ellipsoideus 078 .11 24° C

75. 8. ellipsoideus 070 .06 24° C

79. S. ellipsoideus 090 .02 24° C

Discussion of Results in Table 11. — Organisms 35, 72, 73, 76, 37,
64, 66, 67, and 74 gave higher percentages of the theoretical yield at
24° C than at 33° C. Organisms 68, 70, 77, and 69 gave higher per-
centages of the theoretical at 33° C than at 24° C and organism 79
gave approximately the same percentages at 33° C and 24° C, while
organisms 65 and 36 gave no alcohol at either temperature.

In regard to the absolute amounts of alcohol formed, the relations
are somewhat different. Yeasts 68, 70, 71, and 78 gave higher yields
of alcohol at 33° C than at 24° C. All of these are Mycoderma forms.
This might indicate that these forms have a higher optimum than the
other forms tested. All other forms tested gave higher yields of
alcohol at 24° C than at 33° C. The results are especially striking
with 8. ellipsoideus, number 67, in this respect. It gave 1.3% alcohol
at 33° C and 15.3% at 24° C.

The wild yeasts, i.e., the 8. apiculatus, Mycoderma, Torula, S. pas-
torianus, and Willia yeasts, with the exception of organism 73, all
gave small amounts of alcohol and were very wasteful of the sugar
fermented. The percentage of the theoretical yield varied from 0%
to 72%. On the other hand, the 8. ellipsoideus yeasts gave fairly
high amounts of alcohol, varying from 14.1% to 15.6% at 24° C, and
all gave over 93% of the theoretical yield of alcohol from the sugar
destroyed at 24° C. These facts emphasize the necessity of favoring

48 University of California Publications in Agricultural Sciences [Vol. 4

the growth of the true wine yeasts and discouraging the development
of the wild yeasts.

It is commonly held by enologists in general that wild yeasts as a
rule destroy large amounts of tartaric acid and form high amounts of
volatile acid during fermentation. To test this belief with the Cali-
fornia wild yeasts under observation, analyses of the original unfer-
mented must used in the above tests and of the fermented liquids were
made. Contrary to expectations they did not destroy abnormally large
amounts of fixed acid during fermentation, so that it is possible that
the low yield of fixed acid in naturally fermented wines compared to
those fermented with pure yeast and sulfur ous acid may be due to other
causes than the development of the wild yeasts. For example, it may
be due to bacteria, or the higher yield of fixed acid in wines in which
sulfurous acid has been used may be due to the chemical effect of the
sulfurous acid on the cream of tartar which it may protect from preci-
pitation by the alcohol of the wine. The fact that the use of sulfurous
acid in wine making gives a higher yield of fixed acid in the finished
wine made from the same grapes by natural fermentation, and the
results of the above tests, simply cast doubt on the theory held that
the low yield of fixed acid in naturally fermented wines is due to
the activity of the wild yeasts. They do not necessarily prove the
contrary view, because of the small number of yeasts under obser-
vation.

The largest amount of volatile acid was formed by the S. apicu-
latus yeast, which gave 0.11%. This, in itself, is considerably below
the legal limit of 0.140% for red wines and 0.120% for white wines.
The real point to note, however, is that the apiculatus yeast develops
in natural wine fermentations before the true wine yeast has had a
chance to carry on any great amount of fermentation, so that the
volatile acid formed by the apiculatus yeast is added to that later
formed by the true wine yeast and other yeasts. In this way the
volatile acid may rise dangerously high. Added to this, is the fact
that the apiculatus yeast produces compounds during fermentation
that are prejudicial to the activity of the true wine yeast and so cause
it to behave abnormally and produce larger amounts of volatile acid
than it otherwise would. It may be stated that wine ceases to be com-
mercially salable when it contains more than 0.14% volatile acid.

From the data and discussions given in the foregoing pages on the
properties of California yeasts, it may be said that the yeasts of the
8. (Uipsmdeus or true wine yeast type are the only ones so far found

1918]

Cruess: Fermentation Organisms of California Grapes

49

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that are suitable for the production of wine. Although several of the
wild yeasts produce fairly large amounts of alcohol and low volatile
acid during fermentation, they can not be used in wine making, be-
cause they produce undesirable flavors and odors. An objection to the
California 8. ellipsoideus yeasts is the fine grained character of their
sediments, giving slow clearing after fermentation and difficulty in
racking. Another defect is that some of them seem to be weakened
very perceptibily by such a moderate temperature as 90° F (32° C).

50

University of California Publications in Agricultural Sciences [Vol. 4

2Q 12Q. IW. &na &3Q. ZiW TifiQ 7>W dQQ "HP

366) 1UO 44b

Fig. 15. — Fermentation curves of true wine yeasts from California grapes.

Several varieties of 8. ellipsoideus isolated in France and cultures
of which are in the University collection give much better results than
any of the yeasts isolated from California grapes in this laboratory.
Consequently i1 seems desirable to use one of the French varieties
rather than any of the above California yeasts for commercial fer-
mentation of grapes for wine.

1918] Cruess : Fermentation Organisms of California Grapes 51

IV. INFLUENCE OF LOCALITY ON THE CHARACTER OF

THE MICRO-ORGANSIMS ON GRAPES

Methods of Taking Samples6

Samples of grapes were obtained from Davis, Fresno, and El
Centro, all localities in California. Two Mason fruit jars of one quart
capacity each were plugged with cotton wool, packed into a small box
with excelsior, and the box and contents were sterilized at 150° C in
a dry heat sterilizer. Two such jars were sent to each of the above
localities. Several small bunches of grapes were cut and allowed to
fall into the open jars. The cotton plugs were then replaced. The
grapes were sent to Berkeley where the numbers of the different
groups of yeasts present were determined. Samples from Contra
Costa County, California, were taken in sterile paper bags. The organ-
isms on grapes from Ripon, San Joaquin County, California, were
determined at the vineyard.

Method of Counting Living Cells on Grapes
The same sort of grape must agar, and sterile petri dishes pre-
viously described were employed in the counting tests. In addition
to the above apparatus a number of sterile one c.c. pipettes, and test
tubes, each containing 9 c.c. of sterile water were necessary. On
arrival at the laboratory, the grapes were crushed in the jars by means
of a sterile pestle. One cubic centimeter of the fresh juice was re-
moved by means of a sterile 1 c.c. pipette and transferred to a tube
of 9 c.c. sterile water. With another sterile 1 c.c. pipette one cubic
centimeter of the liquid in the first tube of water was transferred to
a second tube. This process was carried progressively to a third,
fourth, and occasionally a fifth tube, depending on the condition of the
grapes. This gave a dilution of 1 :10 in the first tube, 1 :100 in the
second tube, 1 :1000 in the third, and 1 :10,000 in the fourth. From
each of these tubes one cubic centimeter of the liquid was removed to
separate petri dishes. Melted agar must at 40° C to 45° C was poured
into each plate and the plates set away to allow development of the
individual cells into colonies. Counts of the number of colonies and
notes of the numbers of each type of organism present were made.
From the dilutions used it was possible to calculate the number of
active cells of each kind of micro-organism present in a unit volume
of the original liquid (see plate 1).

6 The writer wishes to express his appreciation of the aid given him by
Messrs. Flossfeder, Way, and Packard in taking samples.

52

University of California Publications in Agricultural Sciences [Vol. 4

Results op Counting
The results of the countings made, appear in the following tables

TABLE 12

Micro-Organisims on Grapes from Davis

Sample number 1, Sample number 2,

first crop, second crop,

Muscat grapes, Muscat grapes,

Organism cells per c.c. cells per c.c.

Penicillium, green mold (probably glaucum none none

Aspergillus mold (probably niger) none 1,000

Mucor mold 20,000 none

Dematium mold 130,000 105,000

Powdery white mold none 3,000

S. apiculatus (a wild yeast) less. than 100* less than 100

Other wild yeasts 200,000

True wine yeast (S. ellipsoideus) none none

* The dilutions below 1:100 were so badly overgrown with the dematium mold that it
was not possible to differentiate the yeasts present, so that "less than 100 per c.c." may
mean from none to 100 per c.c. It signifies that no S. apiculatus or true wine yeasts appeared
on the dilution of 1:100. There were no true wine yeast cells present, however, because
all of the samples were plated out again during fermentation and in this way true wine yeast
cells were proven to be absent. Added proof of the absence of true wine yeast is given
by the fact that the crushed grapes did not undergo true wine yeast fermentation but after
a feeble wild yeast fermentation became moldy and rotted.

TABLE 13
Micro-Organisms on Grapes from El Centro

Sample number 1, Sample number 2,

Organism cells per c.c. cells per c.c.

Penicillium (olive green mold) 2,400,000 20,000,000

Penicillium (blue green mold) none 50,000

Fusarium-like mold 30,000 none

Aspergillus mold 40,000 100,000

S. apiculatus less than 100 less than 100

Wild yeasts, Mycoderma vini type .... 400,000 8,000,000

True wine yeasts (S. ellipsoideus) none none

There were no true wine yeasts present because the grapes did
not undergo a wine yeast fermentation but simply became very moldy
and finally rotted.

TABLE 14
Micro-Organisms from Grapes Grown at Fresno

White grapes, Red grapes,

Organism cells per c.c. cells per c.c.

Penicillium (olive green mold) 10,000 15,000

Aspergillus mold : 10,000 1,000

Fusarium-like mold 10,000 4,000

Dematium mold 7,500 560,000

Mucor mold 200 less than 100

S. apiculatus less than 100 less than 100

True wine yeast (S. ellipsoideus less than 100 less than 100

These grapes underwent true wine yeast fermentation, proving
the presence of a few 8. ellipsoideus.

1918] Cruess: Fermentation Organisms of California Grapes 53

TABLE 15
Micro-Organisms on Grapes from Vineyard of J. Swett & Son, Martinez

Alicante Sauvignon Isabella

Bouschet grapes, verte grapes, grapes,

Organism cells per c.c. cells per c.c. cells per c.c.

Penicillium (green mold) 5,000 3,000 15

Mucor (gray mold) 2,000 none none

Dematium (tree mold) 17,000 22,000 18,000

Aspergillus mold none none 200

Monilia mold 12,000 30,000 10,000

Wild yeasts 14,000 36,000 100

True wine yeast (S. ellipsoideus a few a few a few

These grapes underwent normal alcoholic fermentation, proving
the presence of S. ellipsoideus.

TABLE 16
Micro-Organisms from Grapes Grown at Kipon*

Organism Cells per c.c.

Penicillium (blue green mold) 1,700

Mucor mold 100

Aspergillus mold less than 100

Wild yeasts 2,600

True wine yeasts less than 100 but more than 0

* The above counts were made by plating the grapes in the vineyard.

Yeasts From the Tulare Experiment Station

In 1906 grapes were gathered at the Tulare substation by Pro-
fessor Bioletti7 under conditions that precluded contamination by
organisms not present on the grapes. The yeasts present on them
were isolated by H. C. Holm and their properties studied. All of
them were found to be wild yeasts of very low fermenting power and
all gave undrinkable fermented musts. No true wine yeast was found.

Discussion of Tables 12 to 16, Inclusive

The grapes from Davis, Fresno, El Centro, Ripon, and Martinez
were all allowed to ferment after crushing and plating tests were made
during fermentation in order to obtain the true wine yeast if present.
The Davis grapes and the El Centro samples did not undergo true
yeast fermentations, but after a short wild-yeast fermentation, became
very moldy and finally rotted. The grapes from the other localities
underwent normal true wine yeast fermentations and the yeasts of
this type present were found on dilutions in agar must made during
fermentation.

7 Bioletti, F. T., and Holm, H. C, Calif. Exp. Sta. Bull. 197, pp. 169, 175, 1908.

54 University of California Publications in Agricultural Sciences [Vol. 4

Wine is not made in the regions in the immediate vicinity of Tulare
and El Centro, and none is made at the University Farm, Davis. The
grapes from Fresno, Ripon, and Martinez came from places near
wineries and on all of these samples true wine yeasts were found.
These tests indicate that grapes from regions remote from wineries
have smaller numbers of true wine yeast cells on their surfaces than
grapes from wine making regions. Reasoning from these facts it is
probable that the grapes gathered during the beginning of the season
will have fewer true wine yeast cells than those picked later in the
crushing season. Thus the use of pure yeast is more necessary on
grapes from regions distant from wineries and on grapes gathered
during the forepart of the season that it is on those picked in wine
making regions after the season has progressed for several weeks.
The figures given in Part IV indicate the influence of the time of
picking on the numbers of yeasts, etc., present.

All the samples tested above gave a great preponderance of molds
and wild yeasts over the true wine yeasts. It can easily be seen that
if there are several hundred thousand wild yeasts and a million or
two mold cells for each one to a hundred true yeast cells, as was
actually the case in several instances, the true wine yeasts will have
overwhelming odds against which to develop. Where such a condi-
tion of affairs exists the wild yeasts must of necessity cause consider-
able damage. It is not probable that the molds do a great deal of
damage after crushing because the conditions are not very favorable
for their growth in the crushed grapes, but they undoubtedly de-
preciate the value of grapes for wine making during long shipments.

In summarizing the above data it may be stated that grapes, both
from wine making regions and from those in which wine is not made,
are deficient in true wine yeasts and carry a large number of molds
and wild yeasts prejudicial to the making of the best wines. Grapes
from regions remote from wineries seem to bear smaller numbers of
the true wine yeasts than those from wine making localities.

V. INFLUENCE OF THE STAGE OF RIPENESS ON THE
CHARACTER OF THE MICRO-ORGANISMS ON GRAPES

Sampling

Grapes were gathered in sterile paper bags from a Zinfandel vine
in a large vineyard at Muir Station in Contra Costa County, Cali-
fornia, in 1912. One sample was taken when the grapes were green,
a second when they had begun to turn, and the third in the middle of

1918]

Cruess : Fermentation Organisms of California Grapes

55

September when the grapes were ripe. Since the samples were all
from the same vine, they may be taken as being more or less com-
parative. A sample was taken from an Alicante Bouschet vine in
the same vineyard as the Zinfandel when the grapes were green and
a second sample in the middle of September from another vine of the
same variety in the vicinity of the first one.

The grapes were crushed in the laboratory, using precautions
against outside infection, and counts of the active cells present were
made as in the tests recorded in Part III.

Eesults
The results of the tests appear in the accompanying tables .

TABLE 17
Numbers of Micro-Organisms on Grapes at Different Stages of Ripeness

Ripeness of
grapes at
sampling

Type of

organisms

found

Cells per c.c.

of must,

Zinfandel grapes

Cells per c.c.
of must, Alicante
Bouschet grapes

r

Molds

1,040,000

100

Hard, greenJ

"Wild yeasts

less than 10

less than 10

I

S. ellipsoideus

none

none

r

Molds

1,000,000

ginning to eolorJ

Wild yeasts

175,000

1

S. ellipsoideus

none

f

Molds

190

22,000

RipeJ

Wild yeasts

3,360

26,000

1

S. ellipsoideus

less than ls
but present

a few

The crushed samples were allowed to stand in sterile jars plugged
with cotton. Samples taken when the grapes were hard green and
beginning to color, molded and putrefied, but did not ferment, indi-
cating absence of 8. ellipsoideus. The samples of ripe grapes both
fermented, showing presence of 8. ellipsoideus.

On the green grapes molds were about the only kind of organisms
present. When the grapes began to turn in color the wild yeasts had
made their appearance in large numbers, but no true yeasts were in
evidence. On the ripe grapes were found chiefly wild yeasts and
molds and in addition to these a few 8. ellipsoideus cells, not enough
to develop on the plates of dilution 1 :10, but still enough to cause
the grapes to undergo a true yeast fermentation after standing several
days in a sterile flask plugged with cotton wool.

56 University of California Publications in Agricultural Sciences [Vol. 4

The surfaces of the hard green grapes are very poor places for the
development of micro-organisms; and molds predominate probably
because they are merely present in the dust on the surface of the
grapes. As the grapes soften during ripening some of them are broken
by birds and insects and the yeasts, etc., develop in the broken berries.
From these the cells are carried to the surfaces of other grapes by
insects, etc., so that as the season advances the numbers of yeasts
increase. Even on the ripe grapes, however, the numbers of the true
wine yeast cells, 8. ellipsoideus, were small and were greatly exceeded
by those of the molds and wild yeasts. The data of this table do not
confirm the statement often made that the numbers of micro-organisms
increase during ripening.

VI. CHANGES IN THE NUMBERS AND CHARACTER OF THE

MICRO-ORGANISMS ON GRAPES DURING SHIPMENT

FROM VINEYARD TO CELLAR

Burger grapes were picked at the vineyard of J. Swett & Son,
Ripon, California, and dilution plates on agar were made at the vine-
yard on the freshly picked grapes. Counts were also made on grapes
from the same block of the vineyard after they had stood about eigh-
teen hours in boxes. The sample in this latter case was taken from a
lot of two boxes that were crushed into a sterile barrel. After the
grapes arrived at the winery in Martinez three days later an average
sample was taken and counts of the micro-organisms present again
made.

Alicante Bouschet grapes were plated at the vineyard and on
arrival three days later at the winery. The results of the above tests
appear in tables 18 and 19.

TABLE 18

Change in Micro-Organisms on Burger Grapes After Picking

Grapes after Grapes after
Grapes in 18 hours 3 days on

vineyard, in boxes, board cars,

cells per c.c. cells per c.c. cells per c.c.

Organism of must of must of must

1. Penicillium mold 1,700 145,000 ]

2. Mucor mold 100 50,000 (

3. Aspergillus mold none none ^9,200,000

4. Dematium mold less than 1 72,000

5. Myeodera forms and Torula

yeasts 2,500 160,000 4,700,000

0. S. apiculatus yeast less than 1 less than 10 less than 1,000

7. True wine yeast (S. ellipsoideus) less than 1 less than 100 500,000

8. Vinegar bacteria none none 800,000

1918] Cruess: Fermentation Organisms of California Grapes 57

TABLE 19
Change in Micro-Organisms on Alicante Bouschet Grapes During Shipment

Grapes three days
Grrapes in vineyard, later at cellar,

cells per c.c. cells per c.c.

Organism of must of must

1. Penicillium mold 320,000 2,500,000

2. Mycoderma forms 125 5,400,000

3. Other wild yeasts none less than 1,000

4. True wine yeast (S. ellipsoideus) 25 440,000

5. Vinegar bacteria none less than 100

The effect of standing over night in the boxes was to greatly
increase the numbers of wild yeasts and molds present without mate-
rially affecting the quantity of true wine yeast present. No doubt a
great deal of the mold and wild yeast cells came from the sides and
bottoms of the boxes, where they had collected from broken and moldy
berries during previous shipments. Thus, it may be seen how grapes
almost free from micro-organisms could acquire large numbers by
standing in boxes a few hours. Some of the increase is, of course,
due to normal increase of the cells present on the surface of the grapes
and to increase of organisms in the grapes broken during picking.

The grapes were three days on the road from the vineyard to the
winery and arrived in a moldy condition, but in no worse state than
other Burger grapes shipped under like conditions. The plating tests
bore out what the appearance indicated, namely, the presence of great
numbers of molds, 9,000,000 per cubic centimeter; wild yeasts,
4,700,000, and even large numbers of vinegar bacteria, 800,000 per
cubic centimeter. The wine yeast was present in less numbers than
any of the other organisms, there being only 500,000 of them per
cubic centimeter.

The Alicante Bouschet grapes gave similar results, but being firmer
grapes they arrived in better condition than the Burgers, as was
shown by their appearance and by counting tests.

A method of controlling the micro-organisms on grapes during
shipment is discussed under Part VII.

VII. CHARACTER AND NUMBERS OF MICRO-ORGANISMS

ON GRAPES AS RECEIVED AT THE WINERY DURING

THE SEASONS OF 1911 AND 1912

Samples of the freshly crushed grapes from two wineries in Contra
Costa County were taken in 1911 and 1912 and counts of the active
cells present were made with the results given in the following table :

58

University of California Publications in Agricultural Sciences [Vol. 4

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1918] Cruess: Fermentation Organisms of California Grapes 59

These tests justify the statement that grapes ordinarily arrive at
the winery with a preponderance of the undesirable organisms as com-
pared with the desirable true wine yeast, 8. ellipsoideus. The actual
numbers of all molds, wild yeasts, etc., are less on grapes gathered
near the winery than on those shipped considerable distances before
crushing; but the ratio of molds and wild yeasts to true yeasts is
greater on clean grapes than on moldy ones judging from the above
tests. Therefore, there is just as much, or even a greater need for
the use of pure yeast with clean grapes than with grapes in poor
condition, although it is extremely desirable to check the growth of the
undesirable and promote the development of the desirable types in
both cases.

VIII. EXPERIMENTS UPON THE CONTROL OF MICRO-
ORGANISMS ON GRAPES FOR WINE MAKING

Before Shipment

Burger and Alicante Bouschet grapes from the vineyard of J.
Swett & Son were used in the tests discussed below.

Several boxes of Burgers were crushed into a barrel that had been
sterilized with strong potassium metabisulfite solution and rinsed out
with clean water to remove the metabisulfite. To the crushed grapes
was added potassium metabisulfite at the rate of 12 ounces8 per ton
of grapes. This was added in the form of a 10 per cent solution. Four
clean Mason fruit jars were filled with the crushed grapes and sul-
fited at the rates of 6, 8, 9, and 14 ounces per ton. The metabisulfite
is decomposed by the tartaric acid of the grapes to give sulfur dioxide
and cream of tartar.

Several bunches of Alicante Bouschet grapes were crushed into a
Mason jar and were treated at the rate of about eight ounces of meta-
bisulfite per ton. The treated Burgers and Alicante Bouschet were
shipped from Ripon to Martinez in the same car with untreated
grapes. Counts of the micro-organisms on the grapes before treatment
and on the treated and untreated grapes at the winery upon their
arrival three days later were made.

s Twelve ounces of K2 S2 03 (potassium metabisulfite) per ton corresponds
to approximately 190 milligrams S02 (sulfur dioxide) per kilogram of grapes.

60

University of California Publications in Agricultural Sciences [Vol. 4

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1918] Cruess: Fermentation Organisms of California Grapes 61

The Burger grapes in the jars all arrived in good condition, i.e.,
with no visible mold growth and no fermentation ; in fact, none of the
treated grapes were moldy or fermenting. The untreated grapes of
both varieties shipped in the ordinary way were visibly moldy and
in some bunches the odors of fermentation and acetic acid were
noticeable.

The amounts of sulfurous acid used were small and yery much
below the legal amount of 350 milligrams per liter allowed in finished
wine in France. Analyses of the samples for sulfur dioxide on arrival
at the cellar are tabulated in table 24. The grapes in the barrel were
sulfited twelve ounces per ton, which is the equivalent to 190 milli-
grams of sulfurous acid per kilogram. On arrival at the cellar they
contained only 110 milligrams of total sulfurous acid per liter. In
other words almost one-half of the sulfurous acid had disappeared
during shipment.

TABLE 22
Sulfurous Acid in Treated Grapes on Arrival at the Cellar

Total sulfurous Free sulfurous
acid in milligrams acid in milligrams
Sample per liter per liter

1. Barrel of sulfited Burgers 110 45

2. Jar number 1, Burger 48 16

3. Jar number 2, Burger 60 20

4. Jar number 3, Burger 68 • 32

5. Jar number 4, Burger 160 64

It was thought possible that the sprinkling of a little dilute potas-
sium metabisulfite solution over the grapes in the boxes before ship-
ment might protect them against mold growth, etc. Accordingly,
about 150 c.c. of a 5 per cent solution of potassium metabisulfite was
sprinkled throughout the bottom, center, and top of each of several
boxes of Burger grapes and these were shipped with the untreated
grapes. Contrary to expectations, they arrived in about as poor con-
dition as the untreated grapes. The cause for the ineffectiveness of
the treatment probably was that the metabisulfite solution dried out
rapidly during shipment and thus became of little use in checking
mold growth.

Discussion of Tables 19, 20, and 21. — From the results of these
tables it can be seen that the wild yeasts and molds increase enor-
mously during shipment under the conditions of the above experi-
ments, which represent a fair average of the present commercial
methods of shipping. A moderate amount of sulfurous acid held the
undesirable organisms in check so well that none of these could be
found in one of the samples of treated grapes and only a few in the

62 University of California Publications in Agricultural Sciences [Vol.4

other. The true wine yeasts were not entirely suppressed but actually
increased in numbers, though fermentation had not started.

This method could be applied commercially by crushing the grapes
into wooden or iron tank cars at the vineyard and at the same time
adding sulfur ous acid in the form of 6% sulfurous acid solution or
potassium metabisulfite. For normal conditions and where it is desired
to prevent all fermentation and growth of mold, etc., during shipping,
it would be advisable to use from twelve to sixteen ounces of the
metabisulfite or three-quarters to one gallon of a 6% sulfurous acid
per ton of crushed grapes. If it should be desired to prevent only the
growth of molds and wild yeasts, twelve ounces of the metabisulfite
or three-quarters of a gallon of the sulfurous acid 6% solution per
ton would be sufficient in all cases. If the grapes should undergo
wine yeast fermentation no great harm would be done. In fact, one
step further could be made and pure yeast as well as metabisulfite
could be added, in this way making use of the cars as fermenters and
insuring good fermentations. It is possible that too much tannin
would be extracted in the shipment of white grapes by the crushing
and sulfiting method, but if no fermentation should take place it is not
likely that this would happen.

After Arrival at Cellar

From the tests recorded in Part VII, it may be seen that the grapes
as ordinarily received at the cellar have on their surfaces large num-
bers of fermentation organisms, most of which are of no value in wine
making, or are of no direct harm to the production of good wine. As
the season progresses, the numbers of the true wine yeast may increase
in dirty conveyors, in the boxes, etc. ; but this is an uncertain method
because the kind and the numbers of the yeasts present are not under
control. After heavy rains such as in 1912, the numbers of all yeasts
on the grapes are diminished by being washed off. Such grapes may
undergo all manner of mold and wild yeast fermentations because of
the lack of true wine yeast. This was actually the case in wineries in
Sonoma County, California, in 1912.

For these reasons, it becomes advisable to apply methods of wine
making that discourage the growth of the undesirable types and favor
the development of the true wine yeast. Experiments carried out in
1911, 1913, and 1914 and fully discussed in Bulletin 230 and Circular
140 of the California Agricultural Experiment Station demonstrated
that the use of moderate amounts of sulfurous acid and the subse-
quent application of selected pure yeast to the crushed grapes or
defecated must gave uniformly good fermentations and sound wines.

1918] Cruess: Fermentation Organisms of California Grapes 63

SUMMARY OF PARTS II TO VIII

1. Nineteen different organisms from California grapes have been
studied and their properties described. The majority of these were
found to be types that are harmful in wine making. The true wine
yeasts were not of the best varieties. If the yeasts so far studied are
to be taken as representative of California yeasts in general, wine
makers should not rely on natural fermentations for the production
of their wines.

2. Grapes were gathered in vineyards at Davis, El Centro, Fresno,
Ripon, and Martinez under conditions excluding, as far as could be,
the possibility of outside contamination. The numbers and character
of the micro-organisms present in a unit volume of the crushed grapes
from each locality were determined. In all cases the undesirable
organisms enormously outnumbered the true wine yeasts. Grapes
from Tulare in 1906, Davis in 1912, and El Centro in 1912 contained
no true wine yeasts, indicating that such yeasts are very rare in regions
where wine making is not being carried on.

3. On the surfaces of the green grapes examined were found mostly
molds with very few or no yeasts of any kind. As the grapes ripened
wild yeasts made their appearance. The true wine yeasts were the
last to appear.

4. As the grapes stand in boxes after picking in the vineyard, or
on cars during shipment to the winery, the organisms on their surfaces
greatly increase. During this increase in numbers of cells the molds
and wild yeasts still remain greatly in excess of the true wine yeasts.

5. Grapes as received at two wineries in 1911 and 1912 under
normal working conditions contained large numbers of molds and wild
yeast cells and in one case vinegar bacteria, but there were very few
wine yeasts (S. ellipsoideus) in all cases, compared to the numbers of
other organisms present.

6. The molds, wild yeasts, and bacteria may be eliminated from
the grapes during shipment by crushing the grapes at the vineyard
and adding moderate amounts of sulfurous acid. The amounts of
sulfurous acid necessary are far below the limits allowed by law in
wine making countries and a great deal of this disappears during
shipment and fermentation.

7. After the grapes have arrived at the cellar, the development of
the undesirable types of fermentation organisms may be checked by
the use of sulfurous acid and good fermentations assured by the use
of pure selected yeast.

PLATE 1

Apparatus used in isolating micro-organisms from grapes: Mason jar
plugged with cotton and sterilized. Sterile petri dishes. Sterile 1 c.c. pipettes
Sterile agar and sterile must in tubes.

[64]

PLATE 2

Colonies of yeasts 70, 71, and 78, on agar agar and gelatin. Upper row
of plates agar agar; lower, gelatin. Reading from left to right colonies repre-
sent numbers 70, 71, and 78.

[66]