RATES OF ACETIC ACID FORMATION FROM ETHANOL BY

ACETOBACTER SUBOXYDANS

A THESIS

BY

LACEY WALIŒ

Submitted to the Faeulty of Graduate Studies and

Researeh, MeGill University, in partial fulfillment

of the requirements for the degree of:

MASTER OF SCIENCE

IN

AGRICULTURAL BACTERIOLOGY

Department of Agricultural Baeteriology Macdonald College of MeGill University, Canada.

August, 1958.

i

ACKNOWLEDGEMENTS

The autbor wishes to make the following acknowledgements:

To bis Director of Researcb, Dr. A. c. Blackwood, Professer ot Bacteriology and Chairman of the Department ot Bacteriology, Macdonald Collage, for advice given during the course of this investigation and for guidance in the preparation of the manuscript.

To Dr. w. F. Oliver, Associate Professer of Agricultural Physics, Macdonald College, for assistance in building the d.c. rectifier used with the constant-volume dispensing apparatus.

To the National Research Council, Ottawa, for the award of a National Research Scholarship whicb defrayed the expenses involved in this investigation.

I

II

III

TABLE OF CONTENTS

ACKNOWLEDGEMENTS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

INTRODUCTION ••••••••••••••••••••••••••••••••••

LITERATURE REVIEW •••••••••••••••••••••••••••••

Vinegar Production •••••••••••••••••••••••••• Mechanism of Acetic Acid Formation •••••••••• Methods of Production •••••••••••••••••••••••

The Orleans or French Method •••••••••••••• The Quick Vinegar Process ••••••.•••••••••• The Frings Generator and Similar Methods •• Submerged Fermentation ••••••••••••••••••••

Sorne Factors Affecting Vinegar Production ••• Basal Medium ••.•......•..•••..•.•••••••••• Ethanol Concentration ••••••••••••••••••••• I>l1 •••••••••••••••••••••••••••••••••••••••• Temperature •••••••••••••••••••••••••••••••

The Genus Acetobacter ••••••••••••••••••••••• Classification •••••••••••••••••••••••••••• The Organism- Acetobacter suboxydans •••••

Carbohydrate and Nitrogen Nutrition ••••• Growth Factors •••••••••••••••••••••••••• Mineral Nutrition •••••••••••••••••••••••

MATERIALS AND METHODS •••••••••••••••••••••••••

ii

1

1

3

~ ~ 9

10 11 12 12 13 14 15 16 16 20 21 22 23 24

Organism • • . • . • • . • • . . • • • • • • . • . • . • . • • • . • • . • . • • 24 Inocultl.Dl • • • • • • • • • . • • • • • • • • . • • • • • • • • • . • . . • . • • 24

Batch Fermentation •••••••••••••••••••••••• 24 Continuous Fermentation................... 24

Media • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 25 Batch Fermentation •••••••••••••••••••••••• 25 Continuous Fermentation ••••••••••••••••••• 26

Growth Conditions ••••••••••••••••••••••••••• 27 Batch Fermentation........................ 27 Continuous Bubmerged Fermentation......... 28

iii

~alytical Techniques •••••••.••••••••••••••• 32 Determination of Acetate •••••••••••••••••• 32

1

Determination of Ethanol •••••••••••••••••• 33 Determination of pH....................... 3

365

Determination of Turbidity ••••••••••••••••

IV RESULTS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 37

Batch Fermentation •••••••••••••••••••••••••• 37 Effect of Ethanol Concentration ••••••••••• 37 Effect of Tryptone Concentration •••••••••• 41

Continuous Fermentation..................... 44 Effect of pH.............................. 44 Effect of Initial Ethanol Concentration ••• 51 Effect of the Concentration of Corn Steep

Liquor ••••• 54 Effect of Addition of Large Volumes of

Fresh Medium •• 57

v DISCUSSION • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 61

VI SU'J14I'w1ARY • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 68

VII REFERENCES • • • • • • • • • • • • • • • • • • • • • • • • • • • • • . • . • • • • 69

VIII APPENDIX TABLES ••••••••••••••••••••••••••••••• 74

I

INTRODUCTION

Vinegar is the condiment made from sugary or enzyma­

tically hydrolysed starchy materials by an alcoholic fermen­

tation followed by oxidation to acetic acid. It is used in

the home as a condiment and in the commercial manufacture or

mayonnaise, french dressing, pickles, relishes, catsup,

prepared mustards and horse radish; it is also used as a

fungistatic and antiseptic agent and in the photographie and

the plastic industries.

The Acetobacter ~· commonly referred to as the

acetic acid bacteria, have been used, knowingly or not, from

ancient times, to produce vinegar. During that period, vine­

gar production was mostly an art, but the studies of Kutzing

and Pasteur on the fermentation created an upsurge of

scientific thought with the result that the art of vinegar

production gradually became scientific in nature.

Classification or the group of organisms producing

acetic acid into genus and species, though far from settled,

allowed rapid progress to be made, until today, there are a

few species which are well known for their ability to oxidize

ethanol to acetic acid. Among this group of organisms is

Acetobacter suboxYdans, the organism used in this study.

Bacteriologists who first worked with the genus

2

Acetobacter omitted details concerning their scientific app­

roaeh to the subject of vinegar production; quite often, the

composition of the medium, the pH and even at times the tem­

perature of incubation were omitted. Omission of such vital

data caused rapid scientific progress to be a wish rather

than a reality, but, with the progress of time, such details

became more widely known.

Tbe aim of this project is to study the rates of

acetic acid formation from ethanol by A· suboxydans under

varions physical and chemical conditions using the bateh and

continuons submerged fermentation methods.

3

II

LITERATURE REVIEW

Vinegar Production

Ordinary vinegar is dilute acetic acid obtained from

the oxidation of alcoholic liquids, and it was probably in this

form that it was known from the earliest times. Vinegar was

mentioned in three books of the Old Testament and Hippocrates

is reported to have prescribed it as a medicine for his pa­

tients. Livy, the Latin prose writer, reported in his book

No. ~ that Hannibal overcame the difficulty offered to the

passage of his army in crossing the rocks or the Alps by dis­

solving the rocks with vinegar so that a pathway could be made.

Thus, vinegar was known for a long time. In ancient literature,

however, there seemed to be a lack of knowledge concerning

vinegar production and, as a consequence, we are indebted to

the much abused alchemists of more recent times for the first

knowledge concerning the purification and concentration of the

acid by distillation.

Gerber, Stahl, Loewitz, Durande, Vanquelin and

Thenard (Brannt 1900) provided information concerning the

purification and concentration of vinegar. In 1814, Berzelius

(Brannt 1900) determined the exact chemical composition of

acetic acid while Dobereiner (Brannt 1900) studied the nature

4

,of the acid and proved that alcohol was oxidized at the

expense of atmospheric oxygen to acetic acid and water.

Dobereiner (Brannt 1900) also proved that no carbonic acid

was formed as a result of the oxidation. Schutzenbach in 1823

(Brannt 1900), approximately one year after Dobereiner's dis­

covery, introduced a quick vinegar process.

Although sorne chemical knowledge was known about the

production of vinegar up to that time, nothing was known about

its microbiological origin. In 1822, however, Persoon (Smyth

and Obold 1930) observed that whenever any alcoholic liquid was

exposed to air, a tough membrane formed on the surface. He

called this skin Mycoderma, currently called "the mother of

vinegar". Kutzing (Smyth and Obold 1930) proved that this

Mycoderma consisted of bacteria and he classified these

bacteria as algae and named them Ulvina aceti. Liebig (Smyth

and Obold 1930), a German chemist, stated that the process, as

described by Kutzing, was devoid of life. Pasteur, in 1864

showed that the process was a physiological one, and that it

was due to the presence of a minute organism which he named

Mycoderma aceti. This discovery confirmed Kutzing's findings,

and so the foundation for the sci.entific study of vinegar pro­

duction was laid. Hansen (1935) demonstrated that two species

of bacteria might be associated with the process and he named

these, Mycoderma aceti and Mycoderma pasteurianum.

Acetic acid is formed by more than one species of

Acetobacter and workers such as Henneberg and Beijerinck

(Smyth and Obold 1930) have described other species. Since

that time, a number of pellicle and non-pellicle forming

acetifying organisms were reported and over twenty five species

are now known. These organisms are classified under the genus

Acetobacter in Bergey•s Manual of Determinative Bacteriology

(Breed et al 1957).

As defined from the French vinaigre, the word vine­

gar means sour or sharp wine. Species of Acetobacter vary in

their ability to oxidize or dehydrogenate substrates; some

organisms such as A· xYlinum and A· aceti can overoxidize the

acetic acid to carbon dioxide and water; others such as A· suboxydans effect incomplete oxidation of a substrate such as

ethanol thereby forming acetic acid.

The Food and Drugs Act in Canada requires that the

vinegar must contain not less than 4.1% nor more than 12.3%

acet1c ac1d.

Mechanism of Acetic Acid Formation

It was known for a long time that platinum black

oxidized alcohol to a chemical end product. Davy (Brannt

1900) synthesized acetate by using platinum black and wine.

Dobereiner (Brannt 1900) proved that atmospheric oxygen was

involved in the oxidation of alcohol while Liebig (Smyth and

6

Obold 1930) studied an intermediate compound during such an

oxidation and named it aldehyde; Royer (Prescott and Dunn 1949)

established acetaldehyde as an intermediate product of the

reaction.

A number of intermediary pathways were described to

explain the mechanics of oxidation of alcohol to acetic acid.

Neuberg and Windisch (Prescott and Dunn 1949) proposed that

ethanol is aerobically converted to acetaldehyde which isthen

dismutated, according to the reactions listed below, to equi­

molar quantities of ethanol and acetic acid •

1,1 CH3. C=O + HOH

.... H ~ 2CH3• C=O

Acetaldehyde

,OH ---;. CH3• C-H

'oH

+

Acetaldehyde Hydrated acetaldehyde

PH H PH CH3• Cb~ + CH3• C=O ~ CH

3• C=O +

Hydrated aldehyde Acetaldehyde Acetic acid

2HOH

H CH • è-OH

3 li Ethanol

All of the ethanol is converted to acetic acid by

alternate oxidation and dismutation.

Another proposed mechanism for the oxidation of

ethanol to acetic acid by these organisms, is that the bacteria

dehydrogenate ethanol to acetaldehyde and this aldehyde, which

is then hydrated, is dehydrogenated to form acetic acid with

molecular oxygen acting as the hydrogen acceptor.

7

.The following equations show the postulated reactions.

(Underkofler and Hickey 19,4).

lj CH3• ~-OH + . ~0" Alcohol dehYdrogenase) . CH

3• ~:0 + ·2H +2e

Acetaldehyde Ethanol

CH3• ~=0 + HOH

Acetaldehyde

,OH CH 3 • C,- H + no "

OH Hydrated aldehyde

OH CH3• é- H

OH Hydrated aldehyde

,&lde!llde de!lydrogenase• cu3

• 8-0H•2H • 2e

Acetic acid

Cytochrome system 2 HOH

Products other than acetic acid are formed during the

incomplete oxidation; these products, among others, include

aldehydes, esters, acetoin, glycerol and lactic, malic and

tartaric acids. In very acid media or with concentrated sus­

pensions, acetaldehyde may accumulate because aldehyde de­

hydrogenase functions best at an alkaline pH.

Methods of Production

Vinegar can be made from those products, which, when

fermented by yeast, can yield alcohol. Raw materials such as

apples, grapes, peaches, plums, figs and oranges, most enzy­

matically hydrolysed starchy substances, beer and wine can be

used. In the United States of America, wine, apple juice and

eider are used; in France, Italy, Spain and Greece, wine is

8

used, while in England malt and even honey are used as natural

substances.

The quality of the vinegar manufactured is dependent

on the quality of the raw material being used. Obviously,

materials such as fruits should be clean and at the correct

stage of maturity, while with wine or ether alcoholic media,

no preservatives should be present. The quality of the vinegar

will be increased if the usual plant sanitation precautions

are observed. The actual acetifying process should be pre­

ceded by an alcoholic fermentation irrespective of the nature

of the raw materials used. Adjustment of the concentration

after alcoholic fermentation may be necessary to ensure that

a proper fermentation occurs.

The Orleans or French Method.

This method is the oldest, and, according to Prescott

and Dunn (1949), it is the best method for the production of

table vinegar. The containers used are barrels of about 200 L

capacity; each barrel is filled about one-third with vinegar

acting as a starter, and at the same time, about ten to fifteen

litres of wine are added. The same volume of wine is added to

the barrel at weekly intervals for four weeks, and after the

fifth week, about ten to fifteen litres of vinegar are with­

drawn and the same volume of wine is added to the barrel. Air

is admitted to the barrels through screened holes, one at

éach end of the barrel, above the vinegar level, and the

9

Acetobacter form a zoogleal mat which contains a large n~

ber of bacteria; the mat if it is not supported, will sink and

the organisms will fail to produce acetic acid; this method

is a "slow process" that involves much attention. The oxygen

supply is a limiting factor in the rate of fermentation when

this process is used.

The Quick Vinegar Process

This method, also termed the "German Process",

originated with the discovery by Boerhaave (Prescott and Dunn

1949) that if wine was allowed to trickle through a tall re­

ceptacle containing loosely packed pomace, vinegar could be

produced. Schutzenbach in 1823 (Prescott and Dunn 1949) modi­

fied the Boerhaave method by using other types of porous mater­

ials to expose the organisms to the air as much as was possible.

The generator is designed so that the maximum surface exposure

for a definite volume of vinegar is obtained; this ensures an

adequate surface area on which the air can act. The oxidation

of the alcohol is started by the droplet dispersion of the

vinegar stock which is allowed to come into contact with beach­

wood shavings on the shelves of the generator; this vinegar

stock is trickled through the beechwood packing and the ethanol

is oxidized by the Acetobacter to acetic acid at a rate faster

than in the Orleans process; air is allowed to enter the gene­

rator through the vents in the bottom shelf of the generator and

the temperature is regulated by the rate of flow of the vinegar

10

through the generator.

The Frings Generator and Similar Methods.

This process is similar to the "German Process". The

generator is usually an airtight wooden tank plaeed on concrete

beams. Near the bottom, on the inside of the tank, is a grat­

ing which supports beechwood shavings piled to about one foot

from the top of the generator; below the grating is a ohamber

which has a fattcet for removal of the product, cooling coils

to control the temperature and an air sparger through whicb

air is introduced. The liquor is pumped to the top of the

generator and distributed by a rotating sparger arm which

sprays the liquor evenly over the shavings; air introduced at

the bottom passes up through the shavings and througb an ex­

hause vent at the top; the rate of air flow and temperature

are usually controlled with automatic control equipment.

A small scale Frings type generator was used by

Allgeier, Wisthoff and Hildebrandt (1953). Its operation con­

sisted of a cycle starting with the introduction of the charge

followed by a period of fermentation long enough to allow com­

plete oxidation of alcohol. Some of the product is then with­

drawn and a fresh charge is added to renew the cycle. A seven

day cycle is used and the generators are placed in a thermo­

statically controlled room at 30 ± 0.5°c.

Several otber processes for vinegar manufacture bave

been described; Rosentiel (Perlman et al 1953) sbowed that the

11

Luxembourg or Michaelis methods using rotating drums appear

to be less efficient than the modified Frings generator

process.

Submerged Fermentation.

Oxidation of alcohol to acetic acid by submerged

fermentation has received attention in recent years.

Hromatka and Ebner (1950) compared rates in a generator ox­

idation with submerged culture oxidation and found the oxygen

consumption of the former was a linear function of time, while

the latter was an exponential function of the time. In the

submerged fermentation they found that the acid formation was

thirty-two times as fast as in the generator process for the

oxidation of the same amount of alcohol. The Acetobacter

culture used in the submerged process had a Q02 of 7750 milli­

litres of oxygen per gram dry weight of cells per hour.

Again, Hromatka and Ebner (1954) have described, in part, a

submerged process, which gave 0.5% increase in acid formation

per hour which is at least ten times as fast as the original

Frings generator. The workers also stated that about twenty

times the amount of acid could be produced compared with the

Frings generator if the space in the apparatus was completely

used for the submerged process.

Maxon (1955) reviewed the method of continuous fer­

mentation for different processes but no r ef erence was made

about the method being used for the production of acetic acid,

12

except the paper by Vaughn. (Underkofler and Hickey 1954)

One of the disadvantages of the continuons method, however,

is that contamination is a real hazard, but this presents

no real problem in the vinegar fermentation. There can be

no doubt that, with the advance of the technology of con­

tinuons submerged fermentation, manufacturers will inevitably

turn to this type of operation for production of desired

products such as acetic acid.

§orne Factors Affecting Vinegar Production

The production of acetic acid from ethanol by

Acetobacter~ is affected by several factors, e.g., selec­

tion of the micro-organisms, nature of the raw materiels,

initial concentration of ethanol, pH, oxygen supply, tempera­

ture and nutrients. It should be emphasized that the basic

requirement for efficient oxidation of alcohol to acetic acid

is an adequate supply of air.

Basal Medium.

Shimwell (1948) stated that in the manufacture of

spirit vinegar, industrial alcohol is diluted to the desired

concentration and nutrients are added to supply the nitrogen

requirements of the organisms. Corn steep liquor is used

mostly for its nitrogen content. Rao and Stokes (1953) showed

that A· suboxYdans and A· melanogenum cannot grow in chemi­

cally defined media with ethanol as the sole carbon and energy

13

source but that growth occurred when yeast autolysate or

other complex biological material was present. Evidence is

also presented to show that the growth-promoting substance

in the biological materials is due to reducing sugars and

their derivatives. They concluded that a two substrate

phase is required for some Acetobacter ~· and also that

carbohydrates are necessary to initiate growth of the or­

ganisms~ When growth occurred, the ethanol was used as an

additional source of carbon and energy and thereby oxidized

to acetic acid. Low levels of sugar in the presence of com­

plex biological materials such as liver extract should there­

fore cause more complete oxidation of the alcohol. Rainbow

and Mitson (1953) presented evidence to show that A· suboxydans,

A· mobile and A· acidum utilized ammonium sulfate as the sole

source of nitrogen only when lactate was present. An induction

period suggested that adaptive enzymes are involved. Rao (1957)

stated that the utilization of ammonium nitrogen is profoundly

influenced by the carbon source. Mineral nutrition studies on

the Acetobacter ~· are lacking, the only paper being that or

Mulder (Rao 1957), who showed that copper was essential for

the growth of A· aceti.

Ethanol Concentration.

The ethanol concentration of the medium is very

important. Prescott and Dunn (1949) stated that 10 to 13%

alcohol concentration is readily fermented and they warned

14

against using too low a concentration of alcohol since the

acetic acid formed may be, in turn, oxidized to carbon dioxide

and water. Perlman et al (1953), however, stated that the

strains of Acetobacter ~· which tolerated high alcohol con­

centrations were not the most desirable ones for oxidation of

ethanol to acetic acid; the rapid oxidizers of ethanol were

usually unable to tolerate and grow in a 4% ethanol con­

centration. Steel and Walker (1957) showed that !• acetigenum

and A· xYlinum var. africanum tolerated a 4% ethanol concentra­

tion while severa! cellulose-less auxotrophic mutants tolerated

as high as 10 to 12% ethanol concentration. Smart (1956)

showed that with a 3% ethanol concentration in a basal yeast

extract medium, 86% of the theoretical yield was obtained, while

at a 6% concentration, a 69% yield was obtained; higher con­

centrations produced insignificant yields. In general,

Acetobacter~· differ quite markedly in their tolerance of,

and ability to oxidize, different alcohol levels; an alcohol

concentration of 3 to 4% appears to be satisfactory because

most of the rapid oxidizers can hardly tolerate any higher

concentrations.

lili· The initial pH of the liquid medium to be oxidized

also deserves attention. Shimwell (1939) stated that severa!

Acetobacter ~· have a pH range below 3.5 for maximum growth.

Steel and Walker (1957) showed t hat !· acet i genum and !· xYlinum

15

h.ad optimum pH growth ranges from pH 5 to 6 while selected

auxotrophs varied from pH 7.5- 8.5. Tosic and Walker

(1946 Part I) studied severa! Acetobacter organisms with

relation to pH, on standard malt wort medium over the range

7.05 to 2.50, growth being observed at 12 hour intervals.

Results from these studies indicated that A· aceti,

A. acetigenum, A· suboxYdans, A· gluconicum, A· pasteurianum,

A· capsulatum, A· kutzingianus and A· xYlinum grew best be­

tween the pH range 6.0 to 4.5; A· acetosum and A. ascendens

grew best at the range 5.0 to 4.0. Growth of the majority of

the species used was retarded to a certain extent at pH 7.05

and more so at pH 3.5. None of the organisms grew at pH 2.5.

Temperature

The temperature at which the oxidation of alcohol

occurs is very critical. Slow growth attendant with a morpho­

logical change is observed with organisms grown below 15°C.

Normal development is observed between 15 and 34°C and sometimes

a zoogleal mat is formed; at higher temperatures, long thread­

like coenocytic filaments with irregular bulging and occasional

branching are formed. This latter condition, if induced by

prolonged high temperatures, will result in malfunctioning of

the organisms, but if the temperature is lowered to about 30°C

after such a period of high temperature, sorne cells regain

their normal functioning. Evidently, the exact optimum tem•

pe~âture to be used will depend on the organism and the process

16

.being used. A slow fermentation is obtained when too low a

temperature is used while alcohol evaporation and acetic

acid losses are effected if too high a temperature is used.

Steel and Walker (1957) used A· acetigenum and A· xylinum

var. africanum and selected auxotrophs at a temperature of 0 0 40 c; the mutants grew well at 40 C in contrast to the parent

type. Hromatka and Ebner (1954), in their submerged methods,

used temperatures ranging from 28 to 33°C and obtained satis­

factory growth. Tosic and Walker (1946 Part II) studied ten

Acetobacter organisms on standard malt wort medium at tempera­

tures of 14, 20, 25, 28, 30, 32, 37, and 42°C. Eight of the 0 ten species had an optimum temperature for growth at 30 c;

A· gluconicum and A· xYlinum grew fastest between 25 to 28°C 0 but A. xylinum could not grow at 30 C; none of the species

0 grew at 42 C. Smart (1956) reported that satisfactory growth

was obtained at 25°C on a yeast extract medium with the organ-

isms used.

It can therefore be concluded that irrespective of

the method being used, a temperature range between 25 and 34°C

appears to be satisfactory for vinegar production.

The Genus Acetobacter

Classification.

The correct generic term for the bacteria that pro­

duce acetic acid from alcohol is Acetobacter. Words synonymous

17

with Acetobacter have been used, e.g., Bacterium, Bacillus,

Ulvina, Umbina, Acetobacterium and Termobacterium. Vaughn

(1942) stated that acetic acid bacteria are related to the

family Pseudomonadaceae due to their morphological character­

istics. Breed et al (1957) placed the Acetobacter in the

family Pseudomonadaceae, tribe Pseudomonadeae which contains

the genus Pseudomonas. Breed et al (1957) described the

organisms as having the following characteristics:

Individual cells are ellipsoidal to long and are

rod-shaped, occurring singly or in pairs or in short and long

chains; they are motile with polar flagella or they are non­

motile; involution forms may be elongated, spherical, fila­

mentons, club-shaped, swollen, curved or even branched; young

cells are Gram negative but older ones may be Gram variable.

The bacteria may be obligate aerobes and are usually catalase

positive; varions organic compounds are oxidized to organic

acids and other end products that may be furtber oxidized.

Products of oxidation may include the ~ollowing: acetic acid

from alcohol, gluconic and ketogluconic from glucose, dihY­

droxy-acetone from glycerol and sorbose from sorbitol.

Their mode of nutrition is complex but best growth is obtained

in yeast infusion or autolysed yeast media with ethanol or

some other oxidizable substrate (s) included. The organisms

do not form spores; the optimum temperature for growth varies

quite widely.

18

Vaughn (1942) stated that Acetobacter ~· should be

elassified according to their ability to oxidize a substrate

such as alcohol or glucose. Shimwell (1948), however, stated

that acid production from different sugars and alcohols eannot

be established as a reliable criterion on which Acetobacter ~·

should be classified.

Tosic and Walker (1946 Part I) presented cultural media

and procedures which they considered suitable for the character­

ization of ten cultures of Acetobacter ~· from the National

Collection of Type Cultures, The Lister Institute, England.

They investigated the morphological, cultural and biochemical

characteristics of the organisms. Morpbologieal studies in­

cluded shape, size and arrangement of cells and involution forms;

biochemical characteristics included gas formation, acid produc­

tion, utilization of acetic acid, the Voges Proskauer test and

the catalase test; physiological characteristics included tem­

perature, aerobiosis, pH and alcohol tolerance. These investi­

gators claimed that the eharacteristics agreed with those they

previously published, and that this was the.first attempt made

towards standardizing the procedure for such characterization

of Acetobacter species.

Walker and Kulka (1949) presented a summary of the

important cultural, physiological and biochemical character­

istics which they suggested should be used for identification

of these organisms. The authors stated that of the morphological

19

cbaracteristics, that of motility was the most important;

growth in wort and beer, the behaviour and appearance on wort

agar and the type of giant colonies produced on semi-solid

wort agar were the most important cultural cbaracteristics.

Incubation temperature and oxygen requirements formed the most

important physiological characteristics, while acid production

from carbohydrates, the utilization of ammonium salts as the

sole nitrogen source and the ketogenic activity of the organisms

were the most important biochemical characteristics.

Liefson (1954) suggested that the genus Acetobacter

be divided on the basis of flagellation and acetate oxidation;

e.g. Acetobacter~., those which can oxidize acetate and have

peritrichous flagella, and Acetomonas, consisting of those that

cannot oxidize acetate and are polarly flagellated. Rainbow

and Mitson (1953) suggested lactophilié · and glycophilic

groups which corresponded to Liefson's Acetobacter and Acetomonas;

Frateur (1950) proposed a scheme of classification of Acetobacter

based on the following characteristics: catalase character,

ketogenic capacity and abil1ty to oxidize acetate to carbon

dioxide and water. He used these characteristics to group the

organisms into four groups, each subdivided into species based

on the ability of the cultures to oxidize glucose, to use ammo­

nium nitrogen as the sole source of nitrogen in Hoyer's medium

and to form pigments and cellulose.

There is no widely accepted classification for the

20

group of organisms which Breed et al (1957) term Acetobacter.

Rao (1957), however, considers Frateur•s (1950) scheme of

classification to be the best available. Indeed, the words

of Stanier and van Niel (1941) are so appropriate in the classi­

fication of these organisms that their repetition here is

justified:

"in any attempt to subdivide the organisms belonging

to one natural group of bacteria into species, one would have

to create as many species as there are organisms which differ

in sufficiently fundamental characteristics, regardless of the

existence of the intermediate types. It depends entirely on

the scientific tact of the investigators to decide which charac­

ter shall be deemed worthy of the designation 'sufficiently

fundamental'."

The Organism - Acetobacter suboxYdans.

According to Breed et al (1957), this organism has the

following characteristics and description: it can be isolated

from spoiled beer, souring fruits and wine; occurs as short

rods, single or in chains and is non-motile; a hardly visible

pellicle is formed on liquid media; pantothenic acid, nicotinic

acid, para-aminobenzoic acid, valine, alanine, iso-leucine

histidine, cystine and proline acids, mineral salts and a two

phase ox1dizable system with alcohol and glucose all comprise

the minimum nutritional requirements for this organism; acid

is produced from ethanol, propanol , glycols, glucose, glycerol

21

and sorbitol; the optimum temperature for growth is 30°c;

partial oxidation of substrates is indicated by the formation

of calcium-5-gluconate crystals on the surface of the agar

slants containing glucose and calcium carbonate. No capsule

appears evident and, according to Liefson (1954), peritrichous

flagella are present; this seems to be contradictory to the

"non-motility" mentioned by Breed et al (1957). The organism

is catalase positive and forms no visible cellulosic pellicle

in liquid media.

Carbohydrate and Nitrogen nutrition.

The general nutrition of this organism is not well

established. Rao and Stokes (1953) showed that it could not

be grown on chemically defined media with ethanol as the sole

carbon and energy source. When autolysed yeast or ether com­

plex biological material such as liver extract was included,

however, the organism grew quite well. These workers also

showed that in a two substrate system, with glucose and ethanol

as carbon and energy sources, growth is accompanied by assimi­

lation of some of the carbon into cell material. Hall et al

(1952), Rao and Stokes (1953) demonstrated that the pH and

carbohydrate present in the medium influenced the growth of

A· suboxydans; above pH 5.3, with glucose present, alanine was

not essential, while niacin and valine were not required; if

glycerol or sorbitol was substituted for glucose at pH 5.0 to

5.3, all three acids were required.

22

Fulmer et al (1944) used an acid alfalfa extract as

a nitrogen source but it was inferior to autolysed yeast in

this respect. Liggett and Koffler (1948) suggested that corn

steep liquor could be used as a nitrogen source for A· suboxydans. Rainbow and Mitson (1953) found casein hydrolysate

to be a good source of nitrogen while ammonium was used as a

nitrogen source when lactate was present.

Stokes and Larsen (1945) showed that A· suboxydans

A.T.c.c. 621 required six amino acids, two being accessory for

its maximum oxidative capacity. Several strains of A· suboxydans

could not use lactate or ethanol as the sole source of carbon

and energy even when all the necessary growth factors were pre­

sent. Rainbow and Mitson (1953) used a group of organisms, one

of which was A· suboxydans N.T.C. 7069,on a medium at pH 5.8 in

which the carbohydrate, nitrogen and growth factor sources were

varied; the workers reported that A· suboxydans grew poorly in

media containing amine acids and glucose but grew well when

lactate replaced glucose. Resting cells of the organism can

deaminate, by oxidation, a number of amine acids, whose products,

so far, cannot be identified. Leucine, isoleucine and valine,

essential for growth, are not attacked.

Growth Factors.

Rainbow and Mitson (1953), Lampen et al (1942) and

Underkofler et al (1943) showed that p-aminobenzoic acid,

pantothenic acid and nicotinic acid are required by A· suboxydans

23

The organism is also used for the assay of p-aminobenzoic

acid; in the presence of purines, smaller amounts of the acid

are required. Baddiley et al (1953) identified pantetheine-4-

phosphate, one of the fragments obtained from the action of

potato pyrophosphatase as a factor that stimulated the organism

quite markedly - the so called "Acetobacter Stimulatory Factor".

Karabinoos and Dicken (1944) showed that the growth promoting

activity of biotin is due to a nicotinic acid contaminant.

Mineral Nutrition.

There is no specifie work done on the mineral nutrition

of this organism. Rao (1957) stated that work on mineral

nutrition is only known for A· aceti.

Qrganism

II~

MATERIALS AND METHODS

24

The organism used in this study was Acetobacter

suboXfdans A.T.c.c. 621, obtained in a lyophilized state

from the Prairie Regional Laboratory of the National Research

Council, Saskatoon, and numbered as Gl.

Inoculum

Batch Fermentation.

The lyophilized culture was trans!erred aseptically

to a medium containing tryptone (0.1%), liver extract ~.0% v/v)

and ethanol C% v/v). When growth occurred, slants of a simi-

lar medium, but with agar (1%) and calcium carbonate (5%) added,were inoculated from the grow~ng culture; after a long

period, growth was observed by the clear zones that were calcium

carbonate free; crystals of calcium gluconate formed on the sides

of the test tube opposite these clear zones. In every experimen~

a fresh inoculation of 5 ml was made from the slants into a

flask containing the medium to be used and the culture was then

aerated on a reciprocal shaker !or 24 hours at 30 ± 2°C, prior

to the inoculation into the test flasks.

Cont1nuous Fermentation.

Inoculation was made directly, from the slants pre­

viously described, into a medium conta1ning malt extract (0.1%),

25

autolysed yeast (0.1%), corn steep liquor (3.0% v/v) and

mannitol (0.1%). The flasks were incubated in 500 ml

Erlenmeyer flasks on a New Brunswick gyrorotary shaker (model

G.lO), having an eccentricity of , inch; the duration of in­

cubation was 96 hours at 30 ~ 0.5°C. The fructose produced was

identified by reference spots on paper chromatograms after the

method of Trevellyan et al (1950). A sub-culture was then made

into a flask containing a similar medium except that a 0.001%

mannitol was used and a (3% v/v) ethanol. After a 7 day period

of adaptation, acetic acid was produced. The culture was main­

tained in this condition by continuons withdrawal of the product

with subsequent addition of fresb medium and (3% v/v) ethanol.

The inoculum for the first continuons experiment was taken from

the sub-culture described, while the inoculum for subsequent

continuons experiments was taken from each immediate previous

experiment; in the latter instance the culture giving the

highest percentage efficiency on substrate added in any test

flask, was used as the inoculum for the next experiment.

Media

Batch Fermentation.

The medium contained:

Tryptone

Glucose

Ethanol

Caco3 Liver extract

Nicotinic acid

Pantothenic acid

0.1 gm

0.01 n

3.0 ml

5.0 gni

4.0 ml

20.0 ug

20.0 "

p-aminobenzoic acid 20.0 "

Inoculum 5.0 ml

Distilled water to make 100.0 ml

The calcium carbonate was sterilized separately and

added aseptically at the time of inoculation. The liver ex­

tract was prepared by shredding 1 lb. of beef liver into 2 L

of water; this was steamed and the fat was removed by skimming; 0 the filtered liquor was then stored at 5 c.

Continuons fermentation.

The medium contained:

Malt extract

Autolysed yeast

Ethanol

Corn steep liquor

Nicotinic acid

Pantothenic acid

0.1 gm

0.1 tl

3.0 ml

3.0 "

20.0 ug

20.0 "

p-aminobenzoic acid 20.0 t1

Inoculum 5.0 ml

Distilled water to make 100.0 ml

27

The corn steep liquor was prepared after the method

of Liggett and Koffler (1948). After the experiment to

determine the concentration of corn steep liquor to be used,

the following original procedure was developed: The raw

corn steep liquor was adjusted witb distilled water to 20%

soluble solids by use of a Balling's saccharimeter. The

liquid was then centrifuged in a Lourdes centrifuge Model

L.R.at 5°C, for 25 minutes at 10,400 g. The supernatant was

decanted, sterilized using a Millipore filter and stored at

5°C.

Growth Conditions

Batch Fermentation.

After inoculation into medium, the cultures were

incubated in 500 ml Erlenmeyer flasks at a temperature range

of 27 to 33°C. on a reciprocal shaker. Samples were with­

drawn at eacb 24 hour interval up to the 96th hour. Whenever

ethanol determinations were made, the samples were deprotein­

ized prior to the analysis (Neish 1952). Zero time samples

were always taken and analysed for pH, alcohol and acetate

concentration. The initial pH was always 4.), any necessary

adjustment being made with 0.5 M. sodium acetate buffer.

Each experiment was continued for a further period of 168

hours to find out if the organism bad reached a steady maxi­

mum rate of oxidation with continuous withdrawal of product

28

and addition of fresh medium~

Continuous Submerged Fermentation.

An apparatus (Fig. 1) for delivering a constant volume

of fresh medium was made following the design of Soltero and

Johnson (1954). Six electrolytic cells containing N/10

H2so4 were made and coupled in series to a d-e rectifier

built by Dr. W. F. Oliver, Department of Physics, following

the design of Soltero and Johnson (1954); the electrodes were

made of platinum spot welded to copper and fused through glass

tubing. Each electrolytic cell was coupled to a burette by

a glass tubing. A unit consisted of an electrolytic cell and

sterile clamped burette with its tip connected by a rubber

tubing to a capillary tubing (0.4 mm diameter) which passed

through absorbent cotton covered with gauze and touched the

bottom of the 500 ml modified Erlenmeyer flask on a New rcrn

6 Brunswick gyrorotary shaker (Model G 10) at 2 4 r.p.m. Each Il

flask was modified by making a hole about 3.5 inches from the

base and inserting a serum rubber stopper which made sampling

with a hypodermic syringe possible. The capillary tubing was

bent to a 90° angle, 2 cm from the lower tip, to which was

attached a 6 cm length rubber tubing that extended the tip to

the side of the flask where the greatest volume of broth was

whirled. This arrangement prevented the 11suck-back 11 of air.

The complete apparatus was then calibrated as follows:

29

g!g89 tybjog .

. ;~. rubber connection .·· rybber stopper:

sterile cotton pluo

rubber stopper

burette e

~ .... -~1~2~5!..!m!!Ll .!iE!.!r '!.!!!.!JRl!JmlJel)yt§t!L.r

rubbtr connectlon

caplllary tubint

çott..on- gauze plug

500 ml Erlenmeyer

serum rubber pluo

le

rubber tubtn·

- Fig.l Schemati-c diagram of continuous-feed apparatus.

(Not drawn to scale)

30

Each unit was sterilized and assembled. The burette and

flask were filled with medium and allowed to equilibrate

for 24 hours to detect leakage. The rectifier was then

turned on and the rate of delivery of each unit, coupled

in series, was determined twice for the range 0 to 5 milliamps and tbree times for the working range 0 to 2

milliamps. (Fig. 2 )

When an experiment was to be performed, the pro­

cedure used for the calibration was followed except that

six un-modified Erlenmeyer flasks containing distilled water

were used for the 24 hour equilibration. During this period,

the modified-type Erlenmeyer flasks were inoculated and

allowed to undergo a 24 hour fermentation after taking zero

time samples. If no leakage occurred, the rubber tubing

connecting the burette tip to the capillary tubing in the

flask was clamped and the cotton stopper with the capillary

tubing was transferred aseptically to the modified Erlenmeyers

that bad already undergone a 24 hour period of fermentation

on the shaker. The cotton stopper was then fastened and

samples were taken for the end of the 24 hour period. The

rectifier was started at a predetermined setting so as to

deliver between 10 to 12 ml of medium per 24 hours. The

clamps on the rubber tubing were then unfastened. Samples

were withdrawn up to the 96th hour and a portion of each was

analysed for pH and turbidity; the remainder was centrifuged

1.

A 31 ·05

·03 k = • 01

•01

., -::s 0 c E . 1 3. 5 ... ., o.

"D ., ... ., > ·016 -., "D

8 • ·014 ., ... -§

E ·012

•010 k = • o ·l

•008

·006

·004

·002

0

·2 ·6 1·0 1·4 1·8

milliampa

Fig . 2 . Cal i bration curves of t he continuous-feed apparatus. A - duplicate determinations. B - triplicata determi nations.

32

and analysed for residual ethanol and for acetate. The

initial pH, except stated to the contrary, was always 4.3.

Ethanol was delivered in the medium of the burette at a

concentration of (3% v/v) to keep the concentration in each

flask as close to (3% v/v) as possible. Each experiment was

continued for a further 168 hours to find out if the organism

had reached a steady maximum rate of oxidation with continuous

withdrawal of product and addition of fresh medium.

Analytical Techniques

Determination of Acetate.

Preliminary experiments using the steam distillation

method for the determination of volatile organic acids (Neish

1952) indicated that direct titration using standardized N/10

NaOH was adequate for the determination of total acidity,

expressed as acetic acid. Sodium hydroxide, free of carbon

dioxide, was standardized with potassium acid phthalate with

subsequent counter-standardization with standard HCl. The

alkali was stored using a Caroxite breather to absorb carbon

dioxide from the air. For titrating, a Fischer automatic

electric titrimeter was used with pH 7 as the end point, but

when titrating with a burette, phenolphthalein indicator was

used. The acetate actually produced in any interval, e.g.,

the 24-48 hour, was found by deducting the total acetate

formed at the 0-24 hour interval from the total acetate value

at the end of the 24-48 hour interval. The total acetate in-

33

cluded the initial acidity.

Whenever readjustment was made with alka1i, as

with the pH experiments, eaeh interva1 was eorrected for

"the buffered acetate" which was determined after the ad­

justment of the pH with alka1i and prior to the start of the

new interva1. The number of mi11i-equivalents or milligrams,

total or corrected, was calculated per 100 ml of broth.

Determination of ·Ethanol.

Residual ethanol was determined by the micro­

diffusion method of Conway (1947) as modified by Neish (1952)

using porcelain No. 2 Conway diffusion plates. The centri­

fuged samples were diluted with distilled water so that their

ethanol concentrations were brought within the effective range

of the method (0.1 to 0.4 mgm of ethanol) according to the

theoretical amount of ethanol used as calculated from the amount

of acid formed. The reagents used were prepared as described

by Neish (1952). After introduction of the sample and the

oxidizing mixture, each plate was sealed with an etched glass

eover smeared with 60:40 mixture of vasel1ne-paraff1n and in­

eubated at room temperature for 36 hours. A 5.00 ml micro­

burette or a calibrated micrometer syringe (Neish 1952) was

used in titrating with standardized N/10 sodium thiosulfate.

A blank was run for each set of determinations and the results

are expressed as milligrams of ethanol per 100 ml of broth.

The following is an example of the calculations to

34

determine the amount of ethanol present in each flask at the

end of each interval.

(Titre of blank - Titre of sample) x

Normality of sodium thiosulfate

= meq. of dichromate reduced.

46 mgm of ethanol are reduced by 4 meq. of dichromate,

.~ the meq. of dichromate reduced by the sample x 11.5

= the mgm of ethanol present per volume of sample

used.

This latter value x dilution factor x 100

= mgm of ethanol present per 100 ml of broth.

The amount of ethanol supplied and the amount fermented

were calculated for each interval along with the amount of acetate

produced. From these data, fermentation efficiencies were cal­

culated based either on the substrate added or on the substrate

fermented. These calculations were made on the assumption that

the organism converted 1 mole of ethanol (46 gms) to 1 mole of

acetate (64 gros); thus from 1 gm of ethanol, 1.304 gms of

acetate were expected.

An example of the calculations is given for an experi­

ment, at the 24-48th hour interval.

35

Ethanol

Amount in flask at the 24th hour ------------ 2800 gms

tt added between 24-48th Il ------------ 359 tt

tt in flask at the 48th " ------------ 2270 "

" fermented between 24-48th Il ------------ 889 tl

Acetate

Amount in f1ask at the 24th hour ------------ 84 gms

Il tl Il Il " 48th tl

" produced between 24-48th "

Ca1cu1ations

Fermentation efficiency based on

Efficiency = 948 x 100 3159 x 1.304

------------ 1032

------------ 948

substrate added.

23%

Fermentation efficiency based on substrate fermented.

Efficiency :

Determination of pH.

~48 x 100 89 x 1.304

When the Fischer automatic titrimeter was used, the

pH was determined before each samp1e was titrated. The instru­

ment was standarized with pH 4.0 buffer and the samp1e pH was

read by reference to the nu11 point of the e1ectronic eye.

When the automatic titrimeter was not used for the titration,

pH measurements were made with the Beckmann Mode1 N pH meter,

"

"

standarized with pH 4.0 buffer. Simultaneous comparative

determinations with the two instruments agreed.

Determination of TurbiditY.

Turbidimetric measurements were made with the Model

9 Coleman Nepho-colorimeter D 202. No filter was used.

Immediately before each set of daily readings the instrument

was calibrated, e.g., one division on the optical density scale

was made equivalent to 4 nephelos, using the 76 nephelos

standard unit. If growth was too dense, the sample was then

diluted with distilled water. All results are reported as

nephelos per millilitre of broth.

IV 37

RESULTS

Batch Fermentation

The affects of variations in the initial ethanol

concentration and in the tryptone concentration were studied

in the batch fermentation. A concentration of 4% liver

extract was used as a supplementary nutrient source necessary

for the growth of the organism.

Effect of Ethanol Concentration.

The results (Fig. 3, Appendix Table I) indicated

that 3% ethanol and 0.1% tryptone were adequate for the forma­

tion of the maximum amount of acetate obtained. No appreciable

amount of ethanol was oxidized with 0.05% tryptone and evidently

maximum acetate formation does not occur when the tryptone con­

centration is too low. The percentage efficiency, calculated

on the basis of the substrate fermented (Fig. 4a, Appendix

Table II) showed that 84% of the theoretical yield was obtained;

for the percentage efficiency calculated on the basis of

substrate added (Fig. 4b, Appendix Table II) 80% of the theo­

retical yield was obtained.

ë 0 0

45

15

... 45 •• c. ' C7 • E .. ., -.E 1 5 • "<>

0

45

15

Fig. 3.

• 05% trypton8

·1 % tryptone

· 3 % tryptone

n 3 5 7 9

ethanol , mgm percent

Effect of initial ethanol and tryptone concentra­tions on acetate formation at 72 hours in a batch fermentation with A· suboxydans.

39

90 • 05 % tryptone

70

50

. 30

'10

"ô ~ _.

90 • 1% tryptone ~ () ...-c CD () 70

:0:: -• Il:

. 50 0 ·--D -c 30 Q)

E .... Q) , - 10

-

90 • 3 °.4 tryptone

70

50

10

3 5 7 9

ethanol , mgm percent

Fig. 4(a). Percentage efficiency of acetate formation at 72 hours based on the substrate utilized in a batch fermentat i on with A· suboxydans supplied with dif fe rent concent r attons of ethanol~

40 • 05 %tryptone

90

70

50

30

10

'7 ~ - • 1 % tryptone 90

""' u r--c: ., 'ü 70 ;;: .... ., c: 50 0

~ -c: • E ... ., .... 10

~

90 • 3% tryptone

70

50

r--

30

10

3 5 7 9 ethanol , mom percent

Fig. 4(b). Percentage efficiency of Acetate formation at 72 hours based on the substrate added in a batch fermentation with A· suboxydans supplied with different concentrations of ethanol.

41

·Effect of Tryptone Concentration.

A tryptone concentration of 0.1% gave greater yield

of acetate than any of the other concentration studied.

(Fig. 5, Appendix Table III) The results given in Fig. 5

include only 0.01% and 0.1% concentrations of tryptone, since

these values represent the extremes of acetate production

except for the 4% tryptone concentration which gave the lowest

amount of acetate. Apparently there appears to be a maximum

and a minimum limit at which reasonable acetate formation

occurs.

The results also show that maximum acetate production

is accompanied by an increase in turbidity and a decrease in

pH and ethanol concentration. The percentage efficiency of

acetate formation calculated, at 96 hours, on the basis of the

substrate fermented (Fig. 6A, Appendix Table IV) showed that

the percentage of the theoretical yield was 79.3% for .Ol%

tryptone and 89.1% for 0.1% tryptone; for the percentage

efficiency calculated on the basis of the substrate added

at 96 hours, (Fig. 6B, Appendix Table IV) 51.6% of the theoreti­

cal yield occurred at 0.01% tryptone while 76.1% occurred at

0.1% tryptone.

42

• 0 1 % tryptone

--E E oe 0 ,... .... ~8 Q)Q -«» :;;a. --o- «».._ .... ii ut :I: Q) Q) Cl) PH ga. ... o Q. ë;O.

::::~- -=-- --Q) ace tata -6e 0" .s:: Q) . Q. .,.-- 'iii lOt E CD . !E c et~onol 15 150 turbidity 1·5 80

0

•1 % tr tone

45 450~ acetate

PH

"'-.... thanol ~anot 1 · 5 800 15 150 · • furbldifi--~-::::...__,..---1

~~------ ---0 24 72

time in hours

Fig. 5. Effect of tryptone concentration on acetate formation in a batch fermentation of 3% ethanol with A· suboxydans.

90

70

50

30

"'7l 10 ~ ->.. () c .!! () --Q)

c 0 -0 -c Q)

E ... • -90

70

50

. 30

10

Fig. 6.

43

A

,--

-r-- . ,--

0 -r-- . .----0 -- 0 0 0 . .

0 0 0

8

,

r--

-. 0

r--r-

- r-- -r-- - 0 - 0 0 0 . 0 . 0 0

24 72

tlme ln hours

Percentage efficiency of acetate formation from the substrate fermented (A) and substrate added (B) in a batch fermentation with A. suboxydans supp1ied with 0.01 or 0.1% tryptone.

-. 0

)

r-

-. 0

44

Qontinuous Fermentation

Effect of pH.

The effect of various factors affecting the rate of

acetate formation in a continuous fermentation were studied;

these factors included pH, initial ethanol concentration,

nitrogen nutrition, and addition of large volumes of fresh

medium.

The pH values first studied, were 1, 2, 3, 4, 5, and 6. No growth occurred at pH's 1, 2 or 6 and thus the

data for the pH values of 3, 4 and 5 are given in Fig. 7,

Appendix Table v. The results indicated that acetate forma­

tion is accompanied by an increase in turbidity followed by a

decrease in pH and ethanol concentration. A delay in acetate

formation occurred at pH 3 until 96 hours, while at pH 4 and

5, maximum acetate formation occurred after 48 hours.

The percentage efficiencies, for pH 4 and 5, cal­

culated on the basis of the substrate fermented (Fig. 8A,

Appendix Table VI) were over 90% of the theoretical yield;

for the substrate added (Fig. 8B, Appendix Table VI), over

50% of the theoretical yield occurred. Most of the ethanol

that disappeared was fermented by the organism. (Fig. 8A,

Appendix Table VI) The calculations of the percentage

efficiencies are based on the actual amount of acetate formed

at each 24 hour interval but no constant rate of oxidation was

reached.

The pH values previously studied showed that the

optimum pH at which the organism oxidized ethanol to acetate

appeared to be between pH 3.0 and 5.0. Consequently, it

was decided to study the intermediate values 3.5, 3.75, 4.0,

4.25, 4.5 and 5.0 to find the optimum pH value. The results

(Fig. 9a, 9b, Appendix Table VII) showed that growth occurred

at each pH value and the optimum pH for acetate production by

the organism was 4.25. At this pH value, the yield of acetate

was lOO% of the theoretical value. Relatively uniform fermen­

tation efficiencies were found for the substrate fermented

(Fig. lOA, Appendix Table VIII) and the substrate added.

(Fig. lOB, Appendix Table VIII) No steady rate of oxidation

was reached.

46

pH 3

Ë -E - 00 E

-·----------·~ a.O ~0 -o ,... ... =--a. o- :.oa. .... ~ ... ·- • u. :a., l: - Q. 0 Q. '-0 Q. 0

::J-.Se -at

E .c 0 Q. •• 0

E at ~E c

30 300 . . ·~· 3·0 160<

~-1·5 800 15 150 ·7 . turbidity .

acetate 0 . . •

PH4

. 4 5 45n acetate · If.· 5 ~40C

IV 0 •:::::::::::::::::===1

------.·----......:-- -·-----· PH

~ -~= t.urbidlty

15150 7~------· 1·5 800

0 .

PH 5

-----· pH - 4·5 ~400 45 450 •--..; ----· ~. acetate

1· 5 800

/. · -----: ttnonol -L . ____.- . turbidity

----------·7· L-~~0~----------·--------~~--------~--------~--L-~ ..

15 150

24 72

tlme ln hours

Fig. 7. Effect of initial pH on a continuous fermentation with A· suboxydans.

>­() c • :2 --• c: 0

A

90

70

50

10

; 8 0 -r: • E 90 .. • -

70

50

30

lO

r--5

..--4

3 3 l 1

3 3 J._ ~

24

tl me in

47

r--r-- 5

4 r--L

3 .

3 4 5 4 5 l • L ' L

r--3

3 4 5 45 + ! ! l L 72

hours

Fig. 8. Pe~centage efficiency of acetate formation per 24 hour interval from thes1bstrate fermented (A) and the substrate added (B) in a continuous fermentation with A. suboxydans with the initial pH set at J.O, 4.0-or 5.0.

...

48

pH 3·5

--·~ ethanol

o_ e - c e >-e 0 -0 .co

~0 _._ -o _., ·---- .,_

o- ~o. ... r; ... .QCIJ

• ·~· pH :1: - . u• ... 0 Q. g Q.

c -o. :;,--· • turbidlty ~ e E .c Q. . • 0

0 • -;:7' .

ace tate ~ E E c 15 150 1·5 aoc ------· 0

.

pH3•75

45 450 ~. . pH 4·5 ~40(

-~ ocefafe

---------~·--- ~ ' ~ ..-=::: /. _......---;:= turbidity

1· 5 800 15 150 .

0 .,

pH4·0

45 450 ~ .. . ~ pH_ 4·5 ~40C

------=:·" ~~~- acetate

"7 ""' • turbidity • --• ~ . ;;;;;;;;>

15 150 /. . -1ffiOnOÎ t-5 800

0 24 72

time in hours

Fig. 9(a). Effect of initial pH on a continuons fe rmentat ion with ·A· suboxydans.

49

pH 4•25

~i ~~~~·___:::;--== -·:1: u Q. ... 0 :- • Q. c :::~- ' t b' . e - ~ . ....._ . . ur 1d1ty

0 a. ...... E !

15 150 1·5 800

0 L-------~~-------4---------+--------~

pH4·5

45 450 ~. . /.- .:~:: ~.z·/ . '-· ~

'::::: -: turbidity ,

151:0~' .. -

4·5 240C

1·5 soc

PH5·0

P!!.-----

45450~ .~ethanol 4·5240C

~:~. -·~

1·5 800

24 72

time ln· hours

Fig. 9(b). Effect of initial pH'on a continuous fermentation with A· suboxydans.

90

70

50

30

~ · 10 ~

~ ()

. c: .!? () --1)

c: 0 -Cl ... c: CD e L.. CD - 90

70

50

30

10

A

r--lt) . ft)

8

lt)

l'f)

50

-Ill 0

lt) 0 lt) lt) 0 lt) 0 ~

lt) IC) r:- N ~ ,.._

~ v ~ lt)

v ~ Ill f() rt) rt)

)

~ ..

r-~ ' r-o ...

10 · ,.._v 1---

r-- r--rf> . lt)

lt) lt) ~

(\1 rt)

-~---IC)ro oit r-,.._ . ~0 0 . v v lb rt) U)

24 72

time in hours

Fig. 10. Percentage efficiency of acetate formation per 24 hour interval from the substrate fermented (A) and the substrate added (B) in a continuous fermentation with A. suboxydans with the initial pH set at 3.5 to 5:0 as indicated.

51

Effect of Initial Ethanol Concentration.

The batch fermentation on initial ethanol concentra­

tion indicated that 1, 3 and 7% were suitable for study. The

results of such an experiment (Fig. 11, Appendix Table IX)

showed that the peak of acetate formation occurred with 1 and

3% ethanol at 72 hours. Acetate formation was accompanied by

an increase in turbidity, followed by a decrease in pH and

ethanol concentration.

The percentage efficiency on the substrate fermented

(Fig. 12A, Appendix Table X) and the substrate added (Fig. 12B,

Appendix Table X) showed that at the 1% ethanol concentration,

approximately lOO% of the theoretical yield was obtained.

The 3% ethanol concentration reached 99% of the theoretical

yield calculated on the basis of the substrate fermented and

47.8% for the substrate added. No steady maximum rate of

oxidation was reached.

1%

! E

.......

ër-----·

H ----·'\-- pH ~ l \ ·-----:-.-u-rb-id_i_ty __

• ~·- acetate

15'0 A. :~~ 1-------: ~~

0

3%

7% -~ - -· ethanol ~. ·-

45 450 pH . . .

15 150

0 ---- -·-----·-----· turbidlty

acetate

24 72

time in hours

52

-0 E co 00 =-CD ~

CD ::r: 0 a. Q. :::1 ., e •• 0

• e ...... 1·5 300

4·5 240<

800

4·5 15100

1·5 1700

Big.ll. Effect of initial ethanol concentration on a continuous fermentation with A· suboxydans.

90

70

50

30

il 10 ->o 0 c • ·s --• c 0 :;: 0 -c • Ë 90 • -

70

50

3C

10

A

8

-,.- 1

1

.

1--3

7 J,

r--

I

,__ 3

1--3

7

!

7 J.

1 i

1 .~

,.-

3

24 72

time ln hours

7

l

7 L

53

Fig. 12. Percentage efficiency of acetate formation per 24 hour interval from the substrate fermented (A) and the substrate added (B) in a continuous fermentation with A. suhoxydans with the initial ethanol concentration set at 1, 3 or 7%.

Effect of the Concentration of Corn Steep Liguor.

The concentrations of corn steep liquor studied were

·1, 3 and 7%. The results (Fig. 13, Appendix Table XI) in­

dicated that low concentrations of corn steep liquor do not

faveur large yields of acetate. This fact, also evident at

low concentrations of tryptone (Appendix Table III) indicates

that low concentrations of the nitrogen source do not faveur

high acetate yields calculated on the basis of the substrate

added. Acetate formation is also accompanied by an increase

in turbidity and a decrease in pH and ethanol concentrations.

A slightly higher acetate yield was obtained at the 7% con­

centration than at either the 1 or 3%.

The percentage efficiency calculated on the basis

of substrate fermented (Fig. 14A, Appendix Table XII) in­

dicated that approximately 80% of the theoretical yield was

obtained for 3% corn steep liquor; the percentage efficiency

calculated on the basis of the substrate added (Fig. 14B,

Appendix Table XII) indicated that all concentrations of corn

steep liquor gave yields that were below 50% of the theoreti­

cal yield. No steady rate of oxidation was reached.

1 (%)

e ·----·- ethanol _ -0 >.._ JL-------·----- -· -o- ·---- pH - ·-G) ·~ ;:~: ~ g ~ ~~ ------· turbidity - ~ .

; ; .~ E· c

E

:c o.

55

-oe 5o .r=o --G) .. - G) 0 o. ~

~ E ln 0 f E

15 150

0

~1·5

·-------· ~========~··==========~--------+-------~ '

800

3(%)

-· 45 450 _____ :~~ t::bidity

/~=-./ acetate --------- -·

4·5 240(

15 150 1·5 80(

0

15 150

turbidity _

45 450 -----·~/ /. • ·?'-·- . PH .

------· ·~;~ - / ' acetate

0

4· 5l240C

1·5 800

24 72

time in hours

Fig. 13. Effect of the concentration of corn steep liquor on a continuous fermentation with A· suboxydans.

90

70

·50

30

"ô 10 00::: -

c 0 -0 -c .,

. e 9o L. • ..

70

5

30

10

A

1 1

B

'

....-- ,-3 ,__ 7

r--7

.--~

1 3

...--3

,.... 1

ri, 3 7 L L

3

7 3 7

1 J. ~

1 ~ 3 7

24 72

time in hours

Fig. 14. Percentage of . efficiency _of acetate formation per 24 hour interval from the substrate fermen- . ted (A) and the substrate added (B) in a continuous fermentation with A. suboxydans with the concentration of corn steëp liquor set at 1,- 3 or 7%.

57

Effect of Addition of Large Volumes of Fresh Medium.

This experiment was organized to combine all of the

factors that were previously studied. The experiment was

separated into two parts, A and B, three flasks per experiment.

The results for acetate formation are presented in Fig. 15-1

Appendix Table XIII. After inoculation, the culture reached

its peak of oxidation at 72 hours and then entered a stationary

phase for 96 hours. At the end of this time (168 hours in­

cubation time), 50 ml of broth were withdrawn and replaced by

fresh medium in Experiment A and 10 ml in Experiment B. After

72 hours, the peak of oxidation occurred (240 hours incubation

time) and 75.5% of the theoretical yield of acetic acid cal­

culated on the basis of the substrate added (Fig. 16-2 Appendix

Table XIV) was obtained for A while 11.3% was obtained for B.

Experiments A and B were now treated in the same

manner; 48 hours after reaching the peak of oxidation (288

hours incubation time) 50 ml of broth were withdrawn and re­

placed by 50 ml of fresh medium. The peak of oxidation oc­

curred 72 hours after the refortification. (360 hours incuba­

tion time) The percentage efficiency calculated on the basis

of the substrate added was 48.5% for A and 50.5 for B.

(Fig. 16-2, Appendix Table XIV)

One-half hour after the peak of oxidation 50 ml of

the broth were withdrawn and replaced by 50 ml of fresh medium

58

so that the stationary phase was extremely brief. The peak

of oxidation occurred 72 hours after the refortification.

(432 hours incubation time) The percentage efficiency cal­

culated on the basis of the substrate added was 47.0 for A

and 40.5 for B. (Fig. 16-2, Appendix Table XIV)

The over-all percentage efficiency calculated on

the basis of the substrate added indicated that 76.1% was

obtained for Experiment A while 68.0% was obtained for Experi­

ment B. The withdrawal of 50 ml of broth and replacement by

50 ml of fresh medium in both experiments gave uniform yields

of acetate. The highest rate (1.0 mgm/ml/hr)of oxidation

of ethanol to acetate occurred at 240 hours. The corresponding

decrease in ethanol concentration during oxidation to acetate

is shown in Fig. 15-2, Appendix Table XIII.

E 0 0

... G)

Q.

C7' 50 G)

E

; 30 -0 -G)

refortificotion with fresh medium

A- 50 ml

B- IOml

A- 50ml

B-50ml

A- 50ml

B -50 ml

J j . 1 ~·-• B • !__....-: ~ 1 ~

u 0

/• __ A,'--8 __ 1 J/A !/'

0 1 ~ 1 • 1

E

L. G)

Cl.

E 2100 a> E

_"t500 0 c 0 .c -; 900

~ ~

2

ethanol reforti flcation to (3 %)

71~ ~~ i ·-

•

A,B

72 168 240 360 432

time in hours

59

Fig. 15 Effect of large amounts of fresh medium added at varying intervals on acetate formation (1) and ethanol utilization (2) in a fermentation with f!. suhoxydans.

90

70

50

30

~ 10

--Cll

C" 0 -0

c c;:

E

-

' ' 1

r '

l.

..---A 8

-72

60

A 8

---- -- ____ _._ __ ......

2

r--

A

.---T 1 A B

1 r--

1 B

1

~ 240 3()0 432

time in hours

Fig . 16. Percentage efficiency of acetate format.ion as affefted by addition of fresh medium (as described in Fig . 1 5) in a fermentation with ~· suboxvdans.

1 - Calculated on basis of suhstrate f ermented. 2 - Calculated en basis of substrate added .

v

DISCUSSION

61

The oxidation of ethanol to vinegar, with its active

chemical ingredient as acetate, is of great importance be­

cause edible vinegar, by law, must be of biological origin.

The main reason for this is that the characteristic flaveur

of vinegar develops during the fermentation and it is diffi­

cult to create an artificial flaveur that is satisfactory as

would be required in the synthetic process. There is, there­

fore, no competition from the chemical industry though acetate

can be manufactured synthetically from acetylene or by the

catalytie oxidation of ethanol. One advantage of all syn­

thetic processes is that there is no difficulty in coneentrat­

ing the acetate to any strength while ordinary vinegar must

undergo careful chemical processing before a eoncentrated pro­

duct can be obtained. Synthetic acetate is used, not for

edible purposes, but for the manufacture of cellulose acetate,

white lead, for precipitating casein from milk, for precipi­

tating the latex from natural rubber and for numerous other

purposes.

Vinegar production, however, is essentially an aerobic

fermentation. Two mechanisms of production have been proposed

in the literature, and the process whereby molecular

oxygen acts as the hydrogen accepter appears to be the more

tenable one. All the mechanisms proposed show that 1 mole of

ethanol is converted to 1 mole of acetate.

CH3 - CH2 OH + o,. (Mol. wt. • 46)

CH COOH + H 0 3 2

(mol. wt. = 60)

Thus 1.304 grams of acetate can be obtained from

1 gram of ethanol if the biological conversion is complete.

The work presented deals with the factors of ethanol

concentration, nitrogen nutrition, pH and the effect of the

addition of large volumes of fresh medium. One result round

in all the experiments, though obvious, is that acetate forma­

tion occurs with an increase in turbidity and a decrease in

pH and ethanol concentration. The situation is analogous to

the production of 5 ketogluconic acid from glucose and many

other similar fermentations.

The ethanol studies for both the batch and continuous

submerged fermentations indicate that !· suboxydans cannot

oxidize the 5, 7 or 9% ethanol concentrations; the organism

cannot grow at an initial ethanol concentration much greater

than 3%. At 1% concentration the organism completely oxidized

the ethanol to acetate. The results confirm the statements of

Perlman et al (1953), Steel and Walker (1957) and Smart (1956),

who showed in several different ways, that Acetobacter ~·

that oxidize ethanol can hardly grow in concentrations greater

than 4%. Although A· suboxydans does not oxidize initial

ethanol concentrations much greater than 3%, it is possible

that the organism can tolerate higher concentrations and

oxidize such concentrations after a delayed lag phase. Since

the Food and Drug Act in Canada requires that the concentra­

tion of acetate present in vinegar should not be less than

4.1%, an ethanol concentration of least 3.1% should be used

to obtain the minimum desired strength.

The nitrogen nutrition of this organism appears to

be of paramount importance. The results indicate that low

concentrations of the nitrogen source do not favour high

acetate yields; thus it appears that the available nitrogen

might be a limiting factor. Corn steep liquor and tryptone

botb gave good growth and high yields of acetate; the liquor

has the advantage that it is relatively cheap and the addition

of synthetic growtb factors are unnecessary; tryptone, on the

contrary, is an expansive product, and requires several growth

factors for adequate growth of the organism. In general 3%

corn steep liquor or 0.1% tryptone concentration appears to

be satisfactory for supporting the growth of A· suboxYdans in

fermenting 3% ethanol. Rao and Stokes (1953) showed that the

available carbon source greatly influences the nitrogen require­

ment of Acetobacter species; thus future studies of the inter­

action between the types of carbon and nitrogen sources appear

to be justified. The supplementary sources of nutrient -

autolysed yeast and malt extract - supported the growth of

A· suboXfdans quite satisfactorily. Allegeier et al (1954)

used media containing either antolysed yeast and corn steep

liquor or autolysed yeast and Diamalt and obtained good

results.

64

The pH studies clearly indicate that A. suboxydans

grows from pH 3.0 to pH 5.0. At pH 3.0, however, growth of

the organism did not occur until 96 hours showing that low

pH induces a prolongation of the lag phase. Tosic and

Walker (1946 Part II) also reported that species of

Acetobacter, one of which was A· suboxydans, show marked

retardation of growth at pH 3.0 and no growth at pH 2.5.

These workers also reported that A. suboxydans had an optimum

growth range between 4.5 and 6.0. The present studies do not

confirm this latter report because the optimum pH for this

organism was found to be 4.25. The pH values 4.0 and 5.0 show

uniform acetate yields based on the substrate fermented and the

substrate added. At these latter pH values no further acetate

was formed after 48 hours. These results indicate that the pH

value 4.25 is best suited for the oxidation of ethanol to

acetate with A. suboxYdans.

The previous experiments show that if 5 ml, from a

submerged culture, that had already reached its stationary

phase, are inoculated into fresh medium, as at the start of

a new experiment, the inoculated culture reaches its peak of

oxidation in either 48 or 72 hours. Evidently the number of

cells in an old culture is not as important as the volume of

'fresh medium added; thus fresh nutrient has a revitalizing

, effect on the fermentation and therefore causes a rapid

formation of young cells. Consequently, two simultaneous

experiments were organised, A and B, three flasks per expert­

ment. The difference between the values obtained for the

theoretical yield, based on the substrate added (240 hours in­

cubation time) was enormous - 75.5% for Experiment A and 11.3%

for Experiment B. Subsequent withdrawals of 50 ml of broth and

addition of 50 ml of fresh medium to both Experiments A and B

indicate that uniform rates of oxidation occurred. The per­

centage efficiencies based on substrate fermented showed that

almost all the ethanol was converted to acetate, 90% or over in

all trials. This experiment, using addition of large volumes of

fresh medium to a culture in the stationary phase, could be

criticized because of the number of variables involved. What

is important, however, is the fact that the stationary phase

can be decreased and a point reached whereby continuons with­

drawal of the product and addition of fresh nutrient would

create a steady rate of oxidation by the organism. This is in­

deed, the main object of this investigation - to induce the or­

ganism to attain a maximum rate of oxidation of ethanol to

acetate so that biological commercial production could be in­

creased with a decrease in the fermentation time. In this latter

respect, the continuous submerged fermentation is of greater im­

portance than the batch method. One great disadvantage of the

66

pontinuous submerged fermentation method is that contamination

could be hazardous, but this does not present a difficult

problem in the vinegar fermentation since most ether species

of bacteria cannot grow at such low pH values. The advantages

of continuons submerged fermentation greatly outweigh its dis­

advantages; for instance, there is a marked reduction in fer­

mentation time as is clearly illustrated by the 96 hours re­

quired for the peak of oxidation in the batch fermentation

studies compared to 48 or 72 hours for the continuous fermen­

tation studies; also, the factors such as pH and nutrient can

be studied without changing the conditions as is characteristic

of the batch fermentation; finally,the fact that a steady rate

of oxidation is possible makes the continuous submerged fermen­

tation method superior to the batch fermentation method, to the

extent that manufacturers of vinegar might turn to this method

of production for higher yields.

Severa! investigators report rates of acetate forma­

tion from ethanol in different ways. The following data compare

the rates of oxidation found in sorne of the work of these in­

vestigators to the rate found in t his present work.

Rate Author

Fermentation type mgm of acetate/ml/hr

Allegeier et al (1953) Frings type 1.16

Hromatka (1954) Submerged 4.8 (Approx.)

Frings (Prescott & Dunn, Frings 0.48 1949)

Walke (1958) Continuous submerged 1 - Overall range 0.31 to 0.41

2 - Best conditions 1.00

The best conditions for oxidation in this investigation occur

with the experiment where additions of large volumes of fresh

medium were made to the submerged fermentation in its stationary

phase. This rate of 1.0 mgm/ml/hr that was found is a rather

reasonable rate of oxidation and compares quite favourably with

those of other workers with the exception of Hromatka (1954).

However, under no circumstance, in the present investigation,

except in the latter conditions described, was a steady rate

of oxidation approached.

Finally, the use of pure cultures of Acetobacter

~· for oxidation of ethanol to acetate, as done in this in­

vestigation, is a commercial possibility, although the cost

involved in such an aseptic operation could make the process

uneconomical. Possibly, a mutant strain of A· suboxydans could

be selected that would produce vinegar at rates presently un-

known in the commercial processes.

VI

SUMMARY

A· suboxydans can oxidize ethanol to acetate quite

satisfactorily, using the submerged fermentation method,

when autolysed yeast and malt extract are used as supple­

mentary sources of nutrients.

A· suboxydans in beth the batch and continuous fermen­

tation methods, gives high acetate yields at 1 or 3% ethanol

concentration but not at the 5 or 7% levels.

A· suboxYdans oxidizes 3% ethanol to acetate in medium

containing 0.1% tyrptone or 3% corn steep liquor. These

concentrations of the nitrogen source appear to be minimal

values since lower concentrations do not faveur high acetate

yields.

A· suboxydans grows at pH values between 3.0 and 5.0.

No growth occurs below pH 3.0 or above pH 6.0. The optimum

pH value is 4.25.

Addition of large volumes of fresh medium containing

ethanol faveur rapid oxidation of the ethanol thereby giving

high yields of acetate.

68

The continuous submerged method of fermentation is

superior to the batch fermentation method because higher uni­

form rates of oxidation of ethanol to acetate were found.

This accompanied higher fermentation efficiencies based on

either the amount of ethanol fermented or the amount added.

VII

REFERENCES

ALLEGEIER, R.J., WISTHOFF, R.T. and HILDEBRANDT, F.M.

1953 Pilot plant vinegar generatcrs. Ind. and Eng. Chem. 45: 489-494

----------·-· 1954 Packing for vinegar generators.

Ind. and Eng. Chem. 45: 2023-2026

BADDILEY, J., TRAIN, E.M., NOVELLI, G.D. and LIPWÂNN, F.

1953

BRANNT, W.T.

Structure of Co-Enzyme A. Nature 171: 76

1900 A practical treatise on the manufacture of vinegar and acetates, eider, fruit and wines and the preservation of fruits, vegetables, meat, fish and eggs. (2nd Edition) Henry Carey Band & Co., Phila., Pa.

BFEED, R.S., MURRAY, E.G.D. and SMITH, A.P.

1957

CONWAY, E.J.

Bergey's Manual of Determinative Bacteriology. (7th Edition) The Williams and Wilkins Co., Baltimore.

1947 Micro-diffusion analysis and volumetrie errer. Crosby, Lockwood and Son, London, E.C. 4.

FRATEUR, J.

1950 Essai sur la systematique des Acetobacters. La Cellule, 53.

Ft~MEP, E.I., BANTZ, A.C. and UNDERKOFLER, L.A.

1944 The use of alfalfa extract to supply nutrients for the growth of Acetobacter suboxydans. Iowa State Coll. Journ. Sei. 18: No. 4 369-376

70

HALL, A.N., TIWARI, K.S. and WALKER, T.K.

1952 The influence of pH value and of carbon source on the Nutritional Requirements of Acetobacter suboxydans. Biochem. J. (London) 51: 36

HANSEN, A.E.

1935 Making vinegar by the Frings process. Food Ind. 7: 277-312

HROMATKA, O. and EBNER, H.

1950

1954

Researches in vinegar fermentation. Biol. Abs. Vol. 24: No. 11 (33561)

Procedure for the production of carboxy11c acids. Austrian patent No. 179283.

KARABINOOS, J.V. and DICKEN, M.

1944 The Isolation of Nicotinic Acid from Mi1k and its Ro1e as an Essential Growth Factor for A. suboxydans. Arch. Eiochem. 4: 211 -

LAMPEN, J.o., UNDPRKOFLER, L.A. and PETERSON, W.H.

1942 p-Aminobenzoic acid. A growth factor for Acetobacter suboxYdans. J. Biol. Chem. 146: 277-278

LIEFSON, E.

1954 The flagellation and taxonomy of species of Acetobacter. Antonie van Leeuwenhoek. J. M1crobio1. Serol. 20: 102-110

LIGGETT, R.W. and KOFFLER, H.

1948 Corn steep liquor in microbiology. Bact. Rev. 12: 297-311

MAXON, WILLIAM D.

1955 Continuous fermentation. App. Micro. 3: 110-122

71

NEISH, A.C.

1952 Ana1ytica1 methods for bacterial fermentations. National Research Council No. 2952, Ottawa.

PERLMAN, D., TEMPEL Jr. A.E. and BROWN, W.E.

1953 Fermentation. Ind. and Eng. Chem. 45: 1944-1969.

PRESCOTT, S.C. and DUNN, C.G.

1949 Industrial Microbio1ogy. (2nd Edition) McGraw Hill Book Co. Inc., N.Y.

RAO, M.R.R. and STOKES, J.L.

1953

RAO, M.R.R.

1957

Utilization of ethanol by acetic acid bacteria. J. Bact. 66: 634-638.

Acetic acid bacteria. Ann. Rev. Micro. 11: 317-338.

RAINBOW, C.

1953

and MITSON, G.W.

SHIMWELL, J.L.

1939

Nutritional requirements of acetic acid bacteria. J. Gen. Microbiol. 9: 371-375.

Some facts and fallacies in brewing bacteriology. Wallerstein Labs. Comm. 2: 55.

1948 Brewing Bacterio1ogy IV. The acetic acid bacteria. Wallerstein Labs. Comm. 11: 27-39.

SMART, C.E.

1956 Rates of acetic acid formation. PhD. Thesis. McGill University.

SMYTH, H.F. and OBOLD, W.L.

1930 The utilization of Bacteria, Yeast and Molds in industrial processes. The Williams and Wilkins Co., Baltimore.

SOLTERO, F.V. and JOIDfSON, M.J.

72

1954 Continuous addition of glucose for evaluation of penicillin producing cultures. App. Micro. 2: 41-44

STANIER, R.Y. and van NIEL, C.B.

1941 The main outlines of bacterial classification. J. Bact. 42: 437-466

STEEL, R.S. and WALKER, T.K.

1957 Â comparative study of cellu1ose-producing cultures and ce1lulose-1ess mutants of certain Acetobacter ~· J. Gen. Micro. 17: 445-452

STOKES, J.L. and LARSEN, A.

1945 Amine acid requirements of Acetobacter suboxydans J. Bact. 49: 495-501

TOSIC, J. and WALKER, T.K.

1946 Part I - A procedure for the characterization of the acetic acid bacteria. J. Soc. Chem. Indus. 65: 104-107

1946 Part II - A procedure for the characterization of the acetic acid bacteria. J. Soc. Chem. Indus. 65: 180-184

TREVELLYAN, W.E., PROCTER, D.P. and HARRISON, J.S.

1950 Detection of sugars on paper chromatography. Nature 166: 444

UNDERKOFLER, L.A. and HICKEY, R.J.

1954 Industria1 fermentation. Chem. Pub. Co. Inc., N.Y.

UNDERKOFLER, L.A., BANTZ, A.C. and PETERSON, W.H.

1943

VAUGHN, R.H.

1942

Growth factors for bacteria. J. Bact. 45: 183-190.

The acetic acid bacteria. Wa11erstein Labs. Comm. 5: 5-26.

WALKER, T.K. and TOSIC, J.

1946 The characterization and identification of acetic acid bacteria. Journ. Inst. Brew. 52: 238-249.

WALKER, T .K. and KULKA, D.

73

1949 Comparative studies of Acetobacter ~· with particular reference to beer contaminants. Wa1lerstein Labs. Comm. 12: 7-28.

VIII

APPENDIX TABLES

TABLE I

Effect of initial ethanol and tryptone concentrations on

acetate formation at 72 hours in a batch fermentation with

A· suboxydans.

Ti me

74

% % in Acetate Residual ethanol Tryptone Ethanol hours pH meq/100 ml · mgm/100 ml

0.05 1 0 4.5 1.1 950 24 4.4 1.62 ~20 48 4.3 1.7 62 72 4.2 2.24 575

3 0 4.5 1.1 2850 24 4.5 1.63 1495. 48 4.3 ·1.7 1036 72 4.0 2.5 805

5 0 4.5 1.1 4~50 24 4.5 1.47 1 40 48 4.4 1.5 1380 72 4.4 2.4 1380

7 0 4.5 1.1 6650 24 4.5 1. 5.6 2645 48 4.4 1.6 2415 72 4.5 1.7 2185

9 0 4.5 1.1 8550 24 4.5 1.1 4140 48 4.4 1.1 3680 72 4.4 1.4 2530

75

TABLE I Continued

Time % % in Acetate Residual ethanol

Tryptone Ethanol hours pH meq/100 ml mgm/100 ml

0.1 1 0 4.5 1.1 905 24 4.1 3.96 805 48 3.1 12.0 575 72 2.9 13.9 230

3 0 4.5 1.1 2850 24 4.1 3.9 862 48 3.0 33.2 230 72 2.7 5o.o 115

0 4.5 1.1 4~50 24 4.5 1.~ 1 40 48 4.2 1. 14~5 72 4.1 2.5 13 0

7 0 4.5 1.1 6650 24 4.5 1.56 1840 48 4.5 1.6 1380 72 . 4.5 1.9 1265

9 0 4.5 1.1 8550 24 4.5 1.68 2645 48 4.5 1.67 2415 72 4.5 1.95 2185

.TABLE I Cont inued

Time %

Tryptone % in Acetate Residual ethanol

Ethanol ,hours pH meq/100 ml mgm/100 ml

0.3 1 0 4.5 1.1 é50 24 4.5 1.65 62 48 4.4 1.7 575 72 3-7 7.2 515

3 0 4.5 1.1 2850 24 4.5 1.~ 14é5 48 4.4 1. 13 0 72 3-7 12.5 1265

5 0 4.5 1.1 4750 24 4.5 1.82 1980 48 4.4 1.86 1495 72 4.5 2.5 1265

7 0 4.5 1.1 6650 24 4.5 3.3 2530 48 4.4 5.1 2300 72 4.3 5.0 2185

9 0 4.5 1.1 8550 24 4.5 1.2 3105 48 4.3 1.5 1980 72 4.2 1.7 1840

77

1'ABLE II

The percentage etficiency of acetate formation calculated

tor 72 hours on the basis of substrate fermented and

substrate added in a batch fermentation with !• suboxYdans

at 72 hours suppl1ed with different ethanol concentrations.

% . % Percentage efficiencY Tryptone Ethanol Substrate fermented Substrate added

0.05 1 27.4 10.8 3 5.6 4.0 5 3.2 2.3 7 0 0 9 0 0

0.1 1 ~1.7 66·~ 3 4.1 8 r,

5 3.4 2.4 7 0 0 9 0 0

0.3 1 86.7 3é·7 3 33.3 1 .5 5 3.4 2.4 7 0 0 9 0 0

78

TABLE III

Effect of tryptone concentration on acetate formation in

a batch fermentation of 3% ethanol with A· suboxYdans.

Time % in Acetate Turbidity Residual ethanol

Tryptone hours pH meq/100 ml nehpelos/ml mgm/100 ml

0.01 48 3.4 26.7 68 1265 72 3.1 29.6 84.0 1070 96 3.0 32.6 84.1 993

0.1 48 3.2 32.6 100 1135 72 2.9 45' .1 104 750 96 2.9 47.0 106 425

0.3 48 3.2 34.2 99 875 72 3.1 38.6 102 590 96 3.1 45.0 108 400

3.0 48 3.6 27.6 92 1137 72 3.2 43.0 lOi 650 96 3.2 45.0 10 420

4.0 48 4.4 9.0 20 400 72 4.2 9.6 22 2100 96 3.9 22.0 68 1561

79

TABLE IV

Percentage efficiency of acetate formation calculated

on the basis of substrate fermented and substrate added

in a batch fermentation with A· suboxydans supplied with

0.01 or 0.1% tryptone.

% Tryptone

o.o1

0.1

Time in hours

48

72

96

48

72

96

Perpentage efficiencY Substrate fermented Substrate added

77.5 43.1

76.5 47.7

79.3 51.6

87.4 52.6

98.4 72.8

89.1 76.1

80

TABLE V

~ffect of initial pH on a continuous fermentation with

A· suboxYdans.

Turbidity Residual Time in Initial Sample Acetate nephelos ethano~, Hours pH pH meq/100 ml per ml mgm/100 ml

0 1 1.2 12.0 70 2850 2 2.2 6.0 70 2850 . 3 3.1 4.5 70 2850 4 4.0 3.6 70 2850 g 5.0 2.1 70 2850

6.1 1.0 70 2850

24 1 1.2 12.0 70 2810 2 2.1 6.0 ~g 2800 3 3.1 4.5 2800 4 3.8 3.7 140 2800 g 4.8 2.2 140 2812

6.0 1.0 70 2804

48 1 1.2 11.9 80 3000 2 2.1 5.9 80 3008 3 3.1 4.5 80 3004 4 3.6 44.1 190 1060 g 4.0 37.6 2~g 1410

8.0 1.0 3050

72 1 1.3 11.9 80 3380 2 2.0 5.9 80 3392 3 3.0 4.5 140 3420 4 4.1 44.1 210 1360 g 4.8 37.5 236 1742

9.2 1.0 80 3420

Time in hours

Initial pH

1 2 3 4

l

TABLE V Continued

Sample pH

1.3 1.9 2.5 4.0 4.9

10.0

Turbidity Acetate nephelos

meq/100 ml per ml

11.8 5.7

30.0 44.0 37.5 1.0

80 80

190 210 2~g

81

Residual ethanol,

mgm/100 ml

3600 3610 2310 1o1o 1990 3647

TABLE VI

Percentage efficiency of acetate formation calculated per

24 hour interval on the basis of substrate fermented and

substrate added in a continuous fermentation with

A· suboxydans, with the initial pH set at 3, 4 or 5.

pH

3.0

4.0

5.0

"Time in hours

24 48 72 96

24 48 72 96

24 48 72 96

Percentage efficiencY Substrate fermented Substrate added

0 0 0 0 0 0

86.3 21

61.5 1 90.4 59.6

0 0 0 0

81.6 1 94.5 52.1

0 0 0 0

' ~

. TABLE VII

· Effect of initial pH on a continuous fermentation with

A· suboxYdans.

Time in hours

0

24

48

72

Initial pH

3.5 3.75 4.0 4.25 4.5 5.0

3.5 3.75 4.0 4.25 4.5 5.0

3.5 3.75 4.0 4.25 4.5 5.0

3.5 3.75 4.0 4.25 4.5 5.0

Sample pH

3.5 3.75 4.0 4.25 4.5 5.0

3.2 2.4 3.2 3.3 3.5 3.6

3.0 3.2 3.4 3·3 3.5 4.4

3.6 3.7 4.3 4.4 4.8 5.6

Acetate meq/100 ml

5.0 4.7 3.2 3.0 3.7 1.6

5.4 22.6 20.0 26.0 17.0 17.0

27.6 36.0 42.0 44.2 37.0 27.0

27.6 36.0 42.0 44.2 37.0 27.0

Turbidity, nephelos

per ml

70 70 70 70 70 70

140 180 180 1é0 1 0 180

230 220 220 260 230 190

240 245 250 250 2~0 1 0

Residual ethanol,

mgm/100 ml

2850 2850 2850 2850 2850 2850

2820 2010 2062 1850 2030 2152

1870 1064 1000

650 1263 1834

2020 1260 1271

930 1492 2138

Time in hours

96

Initial pH

3.5 3.75 4.0 4.25 4.5 5.0

T4BLE VII Continued

Sample pH

3.7 4.0 4.5 4.6 4.9 6.0

Turbidity, Acetate nephelos

meq/100 ml per ml

27.0 240 35.8 240 42.0 240 44.0 250 36.8 230 27.0 190

84

Residual ethanol,

mgm/100 ml

2200 1510 1520 1221 1740 2328

TABLE VIII

Percentage efficiency of acetate formation ca1cu1ated per

24 hours interva1 on the basis of substrate fermented and

substrate added in a continuous fermentation with

A· suboxYdans, with the pH set as indicated.

pH

3.5

3·75'

4.0

4.25'

4.5'

5.0

Time in hours

24 48 72 96

24 48 72 96

24 48 72 96

24 48 72 96

24 48 72 96

24 48 72 96

Percentage efficiencY Substrate fermented Substrate added

61.5 o.6 99.0 40.3 0 0 0 0

98.0 28.9 100.0 54.2

0 0 0 0

98.1 2~.2 99.8 5 .2 0 0 0 0

100.0 i7.2 99.4 9.8 0 0 0 0

98.9 28.4 99.4 46.3 0 0 0 0

9é·' 24.4 9 .5' 25.8 0 0 Q 0

86

TABLE IX

Effect of initial ethanol concentration on a continuous

fermentation with A. suboxYdans.

% Time in Acetate TurbiditY Residual ethanol Ethanol _hours pH .meq/100 ml .nephelos/ml . mgm/100 ml

1 0 4.~ 1.0 45 950 24 3. 0.4 50 930 48 3.1 20.1 190 5 72 3.1 20.0 220 260 96 3.1 20.1 220 490

3 0 4.3 1.0 45 2850 24 3.9 2.4 47 25é6 48 3.2 14.6 140 19 0 72 2.9 36.6 250 1100 96 2.9 36.0 250 1392

7 0 4.3 1.0 45 6650 24 4.0 0.2 46 6,300 48 3.9 0.3 47 6280 72 3.9 0.3 47 6400 96 3-9 0.3 47 6620

.TABLE X

.Percentage efficiency of acetate formation calculated per

24 hour interval on the basis of substrate fermented and

substrate added in a continuons fermentation with

A· suboxYdans with the initial ethanol concentration set

at 1, 3 or 7%.

% Ethanol

1

3

7

Time in hours

24 48 72 96

24 48 72 96

24 48 72 96

Percentage efficiencr Substrate fermented Substrate added

92.3 1.9 99.9 100.0

0 0 0 0

~7.5 3.0 3.4 21.1

99.5 47.8 0 0

0 0 0 0 0 0 0 0

88

TABLE XI

Effect of the concentration of corn steep liquor on a

continuous fermentation with A· suboX1dans.

Corn steep Time in Acetate Turbidity Residual ethanol . liquor hours . pH . meq/100 ml nephelos/ml · mgm/100 ml

1 0 4.4 1.0 190 285'0 24 4.4 1.0 190 2800 48 4.3 3.0 266 2630 72 3.9 9.2 360 245'0 96 3.7 14.4 440 25'00

3 0 4.4 1.0 190 285'0 24 4.~ 1.4 266 2802 48 3. 17.2 422 2270 72 3.5 25.0 512 1900 96 3.5 25'.0 5'20 2200

7 0 4.4 1.0 190 2850 24 4.4 1.8 210 2790 48 3.6 25.6 520 1704 72 3.4 27.4 540 135'0 96 3.4 27.3 542 1704

TABLE XII

Percentage efficiency of acetate formation ca1cu1ated per

24 hour interva1 on the basis of substrate fermented and

substrate added in a continuous fermentation with

A· suboxYdans with the concentration of corn steep 1iquor

set at 1, 3 or 7%.

% Corn steep

11quor

1

3

7

Time in hours

24 48 72 96

24 48 72 96

24 48 72 96

Percentage efficiency Substrate fermented Substrate added

0 0 17.3 2.9 g3.9 é·6 2.1 .5

~7.0 o.6 1.7 23.0

51.7 13.9 0 0

61.5 1.2 76.6 36.8 2.6 0.9 0 0

90

TABLE XIII

Effect of large amounts of fresh medium added at varying

intervals on acetate formation and the ethanol utilization

in a fermentation with A· suboxydans.

Time Residual Medium in Acetate Turbidity ethanol,

added, ml hours pH meq/100 ml nephelos/ml mgm/100 ml

# ·EXJ2 e riment A:

0 0 4.2 3.0 110 2850 50 72 3.6 40.7 280 1100 50 240 3.2 50.2 268 630 50 360 3.2 46.6 290 1398

432 2.9 52.4 310 1500

# ~:Qer;l.ment B:

0 0 4.2 3.0 110 2850 10 72 3.6 40.7 280 1100 50 240 4.0 27.0 260 2500 50 360 3.2 45.0 305 î~é6 43~ 3.0 49.1 300

# Triplicata determinations.

91

'l'ABLE XIV

Percentage efficiency of acetate formation as affected by

addition of fresh medium (as described in Fig. 15) in a

fermentation with A· suboxydans.

Medium added, ml

Ex:eeriment A:

50 50 50

~nerim~nt ;e:

10 50 50

Time in hours

72 240 360 432

72 240 360 432

Percentage efficiency Substrate fermented Substrate added

98.8 60.7 96.9 75.5 95.2 48.5 99.0 47.0

98.8 60.7 92.1 11.3 9é.l 50.5 9 .8 40.5