334?“: This is to certify that the thesis entitled A STUDY OF 30% FACTORS AFFECTING GROWTH AND OXIDATIONS OF ACETOBACTER ACETI presented by Henry Paul Meloche has been accepted towards fulfillment of the requirements for Ph .D. degree in Miorobioloy and Public Health WWW Miajor professor A STUDY 01' SOME FAGNRS AFFECTING GROW AND OXIDAIIOUS Ol' AGEI'OBACTER ACETI 3! Henry Peal lleloche, Jr. A! ABSTRACT Sub-titted to the School for edmced Grahame Studies of lichigen State University of Agriculture end Applied Science in partial fulfillment of the require-eats for the degree of moron 01' PHILOSOPHY Depart-eat of Iicrebiology end Public Health You: 1956 ‘3 I. I B R A. 3?. Y" Pv‘licl‘iigan State Unék’ct flit)" v- 1-— 1“." -‘ V U ABSTRACT A study was made of factors affecting the growth and oxidations of Acetobacter aceti in.mineral medium containing ethanol. It was found that the organism would not grow unless dextrose was: included in the mineral medium. In.a mineral salts dextrose medium, growth was stimulated by the presence of pantothenic acid, folic acid, thiamine and biotin. However, the acid production of only one of the two strains studied was stimulated in the presence of the above vitamins. There was no stimulation of the organism by amino acids. Cell suspensions of both strains of A, aggti_oxidized.ethanol to acetate, but would not oxidize dextrose, gluconate, pyruvate, or acetate. The addition of the vitamins listed above did not "spark" the oxidan tion of either dextrose or acetate. However, evidence was presented which indicated that cell suspensions grown in a medium containing dex- trose and yeast extract would completely oxidize ethanol and acetate, and partially oxidize dextrose. Experimental pilot plant vinegar generators were inoculated with A, aggti, started and maintained on a mineral medium.containing dex- trose and ethanol. This study indicated that the major factors to be controlled in the generators were the concentrations of ethanol and dextrose, and the air supply. Vinegar eels did not affect the rate of vinegar production in normally functioning generators. I .l I'll.- III llllllll . III II I ll lull ll 1 -.I.nlo......:h§e\nnllhfs.ljflfl\ ‘3 .. . , ,, . .;.I... 1401 A STUDY OF 803 IAGTOBS ”TIMING GORE AND OXIDATIONS 01‘ AQTOBACTE AMI By Henry Paul lleloche, Jr. A E1318 Sumtted to the School for advanced Gretna-u Studies ef lichim State University of Agriculture end Applied Science in partial fulfillment of the require-eats for the degree of DOCTOR or PHILOSOPHY Department of Iicrebiolegy end Public leelth 1955 fl /.— 7-2153 (,s 0129-7 Afims i'he author is perticulerly grateful to Dr. R. I. Gestilew of the Department of licrobiolog and Public Keelth, without whose guidance end more). support this work would have been impossible. he author also wishes to that Dr. H. L. Sadat! of the Department of licrobiol. egy and Public Health, end Dr. 6. A. Hoppert of the Department of ministry, for their helpful suggestions, critiei-s, end edviee. um 01' some mas I.ImowcrIONe................o... 1 11¢meITW.......o......... 3 Nutrition of acetic acid bacteria . . . . . . . . . . 3 '44 Glucose aetaholisa of the chudononadacgg with special reference to getobacteg . . . . . . . . 5 0perationofvine¢argeuerators........... 8 lffect of the flaw eel on acetic fermentation. . . 9 III.mummmons................. 11 he effect of carbon source on the growth of m- bacteracetiinndneralaediua ......... 11 The vitaain and aaino acid nutrition of atebaotgr acetiinnineralaediua............. l3 Ilene-atria studies of the oxidation of various carbon sources by gcetobacter ac_e_ti . . . . . . . . . . 13 Studies of pilot-plant viuear generators . . . . . . 1M IV.RESULTS...................'.... 17 he effect of carbon source on the growth of mtg- !cteracetiinainerallediua ......... 17 the effect of vituins and alias acids on the rate of growth and acid production by Acetobacter geti ill-inorallodiul................ 19 |IEhe oxidation of various carbon sources by £9.23: hot” §.t1 O O O O O O O 0 O O O O O O O O O O 23 Studies of pilot—plant vinegar generators . . . . . . £3 '0 D1 80113810! 0 0 e e e e e e e e e e e e e e e e e e e e -n 0 mm m mummlons e 0 e e e e e e e e e 0 e e e e % Bmzom e e e e e e e e e e e e e e e e e e e o o “7 LIST OF TABLES TABLE PAGE 1 Growth of A. aceti in mm with various carbon sources . . 18 2 Growth of A. ac___e_____ti in HIM plus 0.5 percent acetic acid (pH 6.0) with vitamin and amino acid supplements. . . . 18 3 Time required for film formation of A. aceti in mm plus 2 percent dextrose with vitamin and amino acid supplements......................20 l4 Effect of the omission of various vitamins from the vitamin supplement on the time required for film formation by a. aceti in IBM plus 2 percent dextrose. . 20 5 Effect of vitamins on the rate of film formation and acid production by A. aceti in HMM plus 2 percent dextrosee e e e e e e e e e e e e e e e e e e e e e e e 22 6 Oxygen uptake by _A_. aceti with various carbon sources . . 21+ 7 Oxygen uptake by A. aceti strain 581 with dextrose and ethanol in the presence of vitamins . . . . . . . . . . 21+ 8 Oxygen uptake with various carbon sources by _A_. aceti strain 578 grown in enriched medium . . . . . . . . . . 27 9 Changes occurring in generators with.high initial dextrose concentrations . . . . . . . . . . . . . . . . 30 10 Changes occurring in generators with an air flow of 125 m Per minute . O C O O O O O O O O O O O O O O O O 33 11 Changes occurring in generators with an air flow of l+OOleperminute.................... 35 12 The effect of vinegar eels on the operation of normally functioning vinegar generators . . . . . . . . . . . . 38 A STUDY OF SOME IAO’I‘ORS “EWING EDITH AID OXIDATIONS 01' ACROBACTER ACME INTRONCIION fhe acetic acid fermentation is one of the acre cemenly known biological phenomena. It serves an important role in nature's censor- vation of carbon, and from earliest recorded history vinegar has been i-pertant to ma as a condiment, In recent years, vinegar has become a very important econoaical itea to the food industry. Also, this fermentation is often responsible for spoilage of alcoholic foodstuffs. Most of the vinegar produced in the Qnited States is aanufactured by the generator process, also known as the quick or Ger-an process. fhe equipment used consists essentially of a cylindrical tower filled with a material such as corn cobs, ceha, beechwocd shavings, e_t_9_.. his material presents a large surface area on diich the bacteria grow and provides for maxi-ml cxidative conditions. It is desired that the supporting material impart no flavor or color to the vinear. In addition, the tower has provisions for the distribution of the sub- strate to be fermented, the collection of vinegar, and aeration of the system. Vinegar can be nde in generators either by the "one run' or tande- processes. In the for-er the alcoholic stock is completely con- verted in one generator, and in the latter the stock is passed through a umber of generators until the desired acidity is reached. llost producers axing grain or distilled vinegar incorporate a ”bacteria food“ with their alcoholic substrate in order to supply 2 nutritional nterials which their medium lacks. However, the untri- tioml value of these 'bacteria foods” in relation to the acetic acid bacteria is undetermined, and thus their importance is questioned. In addition, they are expensive. It is known that some species of the acetic acid bacteria, Agog;- m 9391; in particular, can thrive in mineral media. Therefore, it would appear to be an ideal organism to use in the production of grain or distilled vinegar. Howevor, A. mt; along with isotobacter mm- is considered undesirable in a generator because both reportedly will completely oxidise any carbon source which is available to them. However, if this overoxidation could be controlled, it might be pos- sible to start and maintain a generator with a mineral—alcohol media inoculated with A. M. This would amount to a considerable econom— ic saving by eliminating unnecessary additives. Pimlly, generators operated on a mineral medium would provide basic information on the mechani- cf the acetic fermentation. Consequently, this stow was undertaken to determine factors in- volved in starting and nintaining generators with A. m}; in mineral media; and the major factors which influenced the operation of gener- ators. In addition studies were lmde to determine some of the factors affecting the oxidation of various carbon sources by A. 9_c_e_t_i_ gown in mineral media. REVIEW OF LITERATURE NUTRIEION OF ACETIC ACID BACTERIA Pasteur (1868) noted that gooderma a_c_gt_i_._ (Acetobacter a_3_e_t_i) could grow in a mineral salts medium utilising the monium ion as the sole nitrogen source only if glucose or acetate were added in conJunction with ethanol as a carbon source. Hoyer (1898) and DeiJerinck (1898) confirmed these observations. However, Prateur (1950) reported that a phosphate buffer would replace the acetate or glucose requironent so that A. M could utilise ethanol as the sole carbon source. nu. nadir. is known as modified Hoyer's mineral medium. The same author noted that Acetcvbacter pegsxidags and Aggtcbacter lg‘vanisnse would, also, grow in this medium. Rae and Stokes (1953a) found that the ability to utilise inorganic nitrogen is not as rare among species of 1Eetcbacter as was previously considered. Many strains of Acetoba'cter cuboid“; and Acotobactor melanoggnum possessed this ability under two conditions: (a) the pres- ence of growth factors and (b) the presence of appropriate sources of carbon and energy. A. subogdans required pantothenic, nicotinic, and p-aIincbensoic acids (PARA), while A. melancgguum required the above growth factors and thiamine. me organisms could utilise either glucose, arabinose, unnitol, or scrbitol served as sources of carbon. Ethanol, pyruvate and lactate could be used as carbon sources only if combined'with hydrolysed casein and an unknown growth factor( s) found in yeast auto- lysate, tryptone and other complex biological materials. Ihey (1953b) u later reported the unknown growth factor( s) to be reducing sugars or substances related to them. Studying seven strains of acetic acid bacteria, Rainbow and Mitson (1953) found that dcetobacter mmcosum, .Acetobacter mobile, and d- subozvdans (could utilize ammonium nitrogen as the sole nitrogen source when glucose and lactate were present as carbon sources. Iaushal and Walker (1951) found that écetobacter pasteurianumg Acetojsjaccter 1932; w and Acetobacter acetiggnum would utilise amonium sulfate as the nitrogen source with ethanol and glucose as carbon sources. Iratenn (1950) considered the first two organisms mentioned above as varieties of icetobacter m which he found “was unable to grow under such conditions. In all, ten species of gatobacter were listed in the literature as havingbeen able to utilise inorganic nitrogen under proper condi— tions. Vitamin and Agino acid requirements 21; various Afcetobacter species - A. suboqdans has been suggested for the assay of 13m by Lampen, Unterbofler, and Peterson (1912). This organism required :FA‘BA for growth in a basal medium. Karabinos and Dicken (19%) reported that nicotinic acid isolated from milk was essential for the growth of this organism. Unterlnofler, Bants, and Peterson (1918) found that this organism could synthesise riboflavin. In addition, they noted that pantothsnic acid or one of its derivatives, alpha—hydrory-beta-dimethyl- gamm-butyrolactone, 3mm. and nicotinic acid were required for the growth of A. subcgdans. According to Stokes and Larson (1945), A. suboxydans required valine, isoleucine, alanine, histidine and cystine or methionine for growth. Growth occurred at suboptinim levels of these amino acids with the addition of amonium sulfate. Litsky, lsslen, i‘epper, and Miller (1953) reported that run was the only vitamin required for the growth of Aggtobacter glinum. Tep- per and Litsky (1953) were able to mintain five serial subcultures of two strains of this organism in a medium containing mineral salts as essential vitamins, and a combination of isoleucine, valine and alanine. Pods and Vaughn (1953) found six cultures of A. melanogenum to require pantothsnic acid, rm, nicotinic acid, and thiamine; but they did not require amino acids. One culture each of .gfcetobacter ogdans, and 5. reasons required pantothsnic acid, rm. nicotinic acid, and valine, isoleucine, alanine, cystine, histidine, and proline, In ad- dition, A. rancens required either glutsmio or aspartic acids for best growth. I Rainbow and Mitson (1953)) found that ;. subogdans, Aoetobacter capsulatum, Acetobacter gludonioum, Acetobacter turbidans, and gels:- 39.29.13. viscosum required nicotinate, pantothenate, and p—uinobenseate Acid hydrolysed casein is a good source of nitrogen. honia could not be utilised as the sole nitrogen source. 01110383 WISH or TEE mansions um SPECIAL WW! 1'9. M It has been known for some time that ltcetobaoter and other pseudo- monads could oxidise glucose with the accumulation of various products 6 of partial oxidation of glucose. Butlin (1936) reported the oxidation of glucose by _A_. subogdans with the accumulation of gluconate. Kluy- ver and Boesaardt (1938) reported that g. suboxydans produced 2-keto- gluconate. Bernhauer and Knobloch (.1938), and Bernhauer and Riedl- lLl‘uinovu (1950) reported that strains of Estebacter would oxidize glu- cose to 2.. and 5-ketogluconate. Kulka and Walker (1951+) found that strains of égetobacter could accumulate ketogluconate or 2,5-diketo- gluconate in the medium. Working with cell free extracts of Pseudomonas aeginosa, Claridge and Workman (1953) noted the oxidation of glucose to gluconate and 2-ketogluconate. A great deal of interest arose in respect to the mechanism of these oxidations, and in the metabolic pathway involved in the utili- zation of glucose by members of this group of bacteria. . Wood and Schwerdt (1953) found that Pseudomonas fluorescens would oxidise glu- cose-l-phosphate, g1ucose-6-phosphate, fructose-B-phosphate, riboso- 5-phosphate, and 6-phosphogluconate. Stokes and Campbell (1951) found that dried cells of £1. aeruginosa would oxidize glucose or gluconate to Z-ketogluconate without involving phosphorylation. Wood and Schwerdt (1915) reported the oxidation of'glucose—l—phosphate, glucose-b-phos- phate, fructose-G-phosphate, ribose-E-phosphate, and 6-phosphog1ucon- ate with the evolution of 002 by cell-free extracts of as. fluorescens. In addition, they separated the two enzyme systems, and found that the oxidation of glucose proceeded to 2-ketogluconate without phosphoryla- tion, confirming Stokes and Campbell's (1951) finding. Koepsell (1950) found that 23. fluorescens oxidized 2-ketogluconate to pyruvate, and 7 Warburton, mgles and Campbell (1951) found the same to be true for 2.9.- aeruginosa. Working with resting cells of Pseudomonas saccharophila, Entner and Duodoroff (1952) observed the oxidation of 6-phosphoglucon_ ate to pyruvate and triose phosphate under anaerobic conditions. They postulated the formation of an intermediate, 2-keto-3-deozqr-6-phospho- gluconate. MacGee and. Doudoroff (195%) confirmed the oxidation of 6-phosphogluconate to pyruvate and triose phosphate and isolated an intermediate which was identified as 2-keto-3-deoxy-6—phosphogluconate dehydrase from extracts of as: ‘fluorescens. Kovachevich and Wood (1955a) found that this enzyme requires glutathione and ferrous ions for activity. The same authors (1955b), in a subsequent study, found the above enzyme in Agetobacter. Also, they isolated another enzyme, an aldolase, which splits 2.keto—3-deoxy-6-phosphogluconate to pyruvate and D-glyceraldehyde-3-phosphate. This enzyme was found in getobacter among other species. Recently, Rao (1955) did a considerable amount of work on the pyruvate and acetate metabolism of A. a_c_:_e_§_i_ and A. suboquans. He found that the dominant route of pyruvate oxidation in A. subogdans was through the aldehyde to acetate, whereas A. a_.c_§_t_A appeared to form acetate from pyruvate by means of an oxidative decarboxylation. He also found that no tricarboxylic acid cycle (TCAC) intermediates would be oxidized by resting cell suspensions of A. 3993;. However, cell free extracts would rapidly oxidize TCAC intermediates. This he attributed to a cell wall permeability barrier. In addition he found that A. aceti had all of the T0110 enzymes, whereas A. subogdans 8 possessed only detectable amounts of aconitase and fumarase. He noted that both species had.TPN linked acetaldehyde dehydrogenases. In addi- tion,.A, agg£1_had a DEN-specific dehydrogenase. .Aldehyde dehydrogen. ases were not CoA.dependent or acyl generating in either species. King and Cheldelin (1959) working with A, 9322;, and Lutwakhuann (1938) working with A, suboxydans found that the ethanol dehydrogen- ases of the two species were DPN linked. OPERATION OF VINEGIR GENERATORS Unfortunately, the literature is lacking in information perti- nent to the operation of vinegar generators. Much of the information available is outdated, and.no information is available relative to the physiology of the Acetobacter in generators. Both Brannt (191h) and Vaughn (195M) pointed out that air flow and pumping rate were important in the operation of a generator. Ap- parently, both the rate of acetification and the temperature of the generators could be controlled by regulating these two factors. Brannt (191”) noted four degrees of activity to be found in gen- erators: (a) normal oxidation which occurred when ethanol was converted to acetate, (b) underoxidation whiCh occurred when there was an insufk ficient air supply or when ethanol concentration was too high, (c) overoxidation which occurred when acetate which had been produced.was oxidized and which could be controlled by the addition of ethanol, and (d) superoxidation which occurred when there was an increase in the ethanol concentration in the circulating liquor along with a rise 9 in temperature of the generator. A.musty odor was evident when the latter condition existed. The author claimed that the superoxidation was caused by the incorporation of "too rich nutrients" in the medium; and concluded that this condition often resulted from.the use of fresh cider in generators. Brannt (19lh) also indicated that in most cases of under- and superoxidation, the only thing that could be done was to restart the generator. Brannt (1911;) noted that Acetobacter cultures must be adapted to ethanol stepwise either in the preparation of a starting culture or ”in the use of higher concentrations of ethanol in the generators. He stated that ethanol should not be increased more than 2 percent by volume at a time. vaughn (1959) stated that cider and wine vinegar were more easily produced in the recirculating generator than distilled.vinegar; also, it was easier to seed a cider or wine generator than a distilled vine- gar generator. He attributed this to distilled vinegar stock being a mixture of synthetic materials containing no natural nutritive sub- stances as does cider or wine. Thus, it was necessary to add organic and inorganic sources of nitrogen, phosphorous, etc. Most producers rely upon commercially available "bacteria food!f_which is added in given amounts to diluted ethanol, rather than develop their own foam- ulations. EEFECT OF THE VINEGAR EEL ON ACETIC FERMENTATIONS Due to the everpresence of the vinegar eel, Turbatrix aceti gar, aceti, in vinegar manufacture, it has been the subject of many studies. 10 Brannt (191“) felt that its presence was not desired under any condi- tions. Peters (1928), Potts (1910), Iyant (1919), and Zimerman (1921) all indicated that the eel impared vinegar production by destroying the film of acetic acid bacteria. However, in none of the cases has it been proven that the eel actually larmed the production capacity of acetic acid bacteria. Instenfeld (1930) took the opposite view by pointing out that the eel feeds mostly on available organic material which, in vinegar mem- facture, is predominantly acetic acid bacteria. He stated that in many cases it wasimpossible to operate generators unless they are present. He felt that the eel's metabolites might function as nutri- ents for the bacteria. mm and labian (1953), werking with A. MA, demonstrated that there was a higier acid production and greater efficiencies of conversion of ethanol to acetic acid in generators containing eels than in those without eels. EXPERD-IENTAL LEETI‘DDS Orflism: Tito strains of Acetobacter aceti designated as Nos. 578 and 581 were used. no cultures were supplied by Dr. W. C. Haynes of the Northern Utilisation Research Branch of the United States Depart- ment of Agriculture, Peoria, Illinois. @1133: Two mineral media were used: a) the mineral salts mix- ture described by Hennenberg (mm) (1926) -— 3.0 g (NHuh‘SOu, 3.0 g H2130”, 2.0 g MgSO,4 per liter of water; and b) the mineral salts mix- ture described by Frateur (FEM) (1950) - 1.0 g (NHQESOn, 0.1 g KZHPOI‘, 0.9 g KHZPOu, 0.25 g MgSOu per liter of water. To each of the above was added an appropriate carbon source( s). No appreciable difference should be noted in the growth of either strain of 5. 949311; in the two media. Ethanol, denatured by the addition of ethyl acetate (United States Industrial Chemicals Compamr 35-4) was used unless otherwise specified. THE EFFECT OF THE CARBON SOURCE ON THE CEOWTH OF ACETOBACTER 4931;; m MINERAL mam: The effect of various carbon sources on growth of A. 2521?. was tested in the following manner. Thirty m1 of medium in a 125 m1 Erlen- meyer flask was used for this and subsequent studies. mm was used as the basal medium and the following carbon sources tested: a) 2 percent dextrose b) 0.5 percent acetic acid adjusted to pH 6.0 (acetate). 12 c) 2 percent ethanol d) 2 percent dextrose and 0.5 percent acetate e) 2 percent dextrose and 2 percent ethanol. To test whether ;. algal; required vitamins or amino acids in a mineral medium with acetate as the carbon source, basal medium plus 0.5 percent acetate was supplemented with 0.5 percent Difco casamino acids, 0.5 percent Difco yeast extract, or a mixture of 9 B—complex vitamins. The vitamins and their concentrations (ug/ml are given as follows: DL 63 pantothenate, NO; nicotinamide, 20; praminobenzoic acid, 20; pyridoxine H01,'1&0: riboflavin, ho; thiamine H01, 20; biotin, 0.5; and irinositol, 20. . g The two strains of A. gg_e_t_i_ were maintained in mm containing 2 percent dextrose, 2 percent acetic acid, and 5 percent ethanol for a number of sub-cultures. An inoculum was prepared from.a 2kghr culture which had been grown at 30 c in 20 ml of medium contained in.a 125 ml Erlenmeyer flasks The culture was washed three times with sterile dis- tilled water and shaken with glass beads in 25 m1 of sterile distilled water. The latter procedure was necessary to minimize the amount of ”carry-over” of nutrients occluded in the slime produced by these or- ganisms. A volume of 0.25 ml was used to inoculate each test flask. A series of flasks containing various carbon sources was inoculated, incubated at 30 C, and observed for growth response. 13 THE VITAMIN AND AMINO ACID NUTRITION OF ACETOBACTER ACET]; IN MINERAL MEDIUM It is known that many nous-exacting organisms are stimulatedby the presence of various vitamins and amino acids included in the growth medium. Despite the fact that A. aceti will grow in dextrose plus mineral salts medium, it was necessary to determine whether its growth could be stimulated by vitamins or amino acids. mm containing 2 per- cent dextrose was used as the basal medium. . The vitamin stimulation of both strains of 5. goat; was tudied on one hand by excluding arw one of the nine B vitamins in various, sets of media and, on the other, by supplementing the basal medium with individual vitamins. Vitamins and concentrations employed were ‘those described previously. One—half percent casamino acids fortified by the addition of 20ug per 100 ml of L-tryptophane and Lcysteine was used as the amino acid source. Since _A_. §_c_¢_e_§_i_ is a film former and the formation of this film appears to follow a definite pattern in the life cycle of standing cultures, the time required for the first formation of a film on the surface of the medium was considered sufficient for determining growth response. In addition, titratable acidity produced after a given per- iod of time was determined in the experiment with single vitamins. MANOI-ETRIC STUDIES OF THE OXIDATION OF VARIOUS CARBON SOURCES BY ACETOBACTER ACETI The manometric studies of the oxidation of various carbon sources by A. aceti grown in mineral medium was carried out at 30 0 using the 1“ standard warburg technique (Umbreit, Burris and Stauffer, 1951). Cell suspensions of both strains of A._ac_:_e_t_:_i_ were prepared in the following manner. An inoculum was named by growing cells in 100 ml of PM with 0.1 percent dextrose, 0.1 percent yeast extract, and 5 percent ethanol contained in a 250 ml Erlenmeyer flasks. After #8 - 72 hours incubation, the contents of the inoculum flask were transferred to a Waring blender and mixed. The homogenized culture was transferred to- one liter of fresh medium, and approximately 10 ml portions were poured into sterile Petri dishes. These were incubated at 30 C for 36 hours. - The contents of the Petri dishes were then transferred to a Waring blendor, mixed for one-half minute, and strained through several layers of cheese-cloth. The suspension formed was concentrated by centrifuga- tion and washed four times in distilled water. The washed cells were. suspended in 0.075 M phthalate buffer at pH 5.0 containing 100 ppn MgSOu and stored in the frozen state until used. STUDIES OF PILOT-PLANT VINEGAR GENERATORS Four laboratory pilot—plant vinegar generators described by Zalkan and Fabian (1953) were used throughout this study. They consisted of glass towers approximately 15 cm 0.1). by 100 cm in height, with provis- ion for recirculation and forced aeration. Each generator was filled. with approximately one bushel of beechwood shavings, washed 30 times with hot distilled water and five. times with 5 percent acetic acid. The final acid wash was circulated until the time of inoculation. One liter of a “8-hour culture of strain 581 in MM with 1.0 per- cent dextrose, 2 percent acetic acid and 5 percent ethanol, and one 15 liter of fresh medium, wan added to each generator. The air flow was adjusted to approximately #00 ml per minute and the circulation rate was set at 50 ml per minute. The air flow rate was regulated with a needle valve and measured with either a rotameter or a permanent head loss flowmeter which had been calibrated in.mln of air per minute. The operation of the generator was studies as a function of dextrose and ethanol concentrations, and the rate of air flow. When recharging the generators, they were alldwed to drain for 15 to 30 minutes and were then filled with 2 liters of fresh medium. In. itial observations were made 10 - 12 hours after recharging. Twenty- five ml samples were drained from the generators at various time inter- vals and studies were made of the dextrose, ethanol, and acetic acid concentrations. Dextrose was determined colorimetrically as reducing sugar and ethanol was determined by dichromate oxidation (Neish, 1952). Total acid was titrated with 0.1 N'NaOH and calculated as percent acetic acid. The effect gf_the vinegar eel gg_the operation gf_vineg§£ gener- gfiggg; In commercial operation, it is practically impossible to keep vinegar eels out of vinegar generators. The eels appear to act as scavengers on acetic acid bacteria and much debate has transpired con- cerning the beneficial or detrimental effects of the eel on the aceti- fication process. Consequently, experimental generators were deliber- ately infected with these nematodes in the following manner. The sole in one—half gallon of commercial vinegar supplied by Libby, MacNeil and.Libby 00., Blue Island, Illinois, were concentrated by filtering 16 through a filter paper in a Buchner funnel. They were then washed five times with sterile saline and suspended for 15 minutes in a HgCl2 solution described by White (1931); waShed five more times and sus- pended in 100 ml of sterile saline. Fifty ml. of the suspension of eels were inoculated into each of two generators. Previous studies indicated that the above contact time in White's solution was suffic- ient to inhibit all organisms that would grow in nutrient agar acidi- fied to 5 percent acetic acid, without causing any apparent harm to the'eels. RESULTS m macs or mason SOURCE on THE GROWTH or ACETOBACI‘ER ACETI IN MINERAL MEDIW A great deal of difficulty was encountered with initial attempts to grow both strains of Acetobacter agg}i_in mineral medium. Eventual- ly, the organism grew in mineral medium containing dextrose as a car- bon scurce,’ however, it could not be grown if the dextrose were replaced with ethanol and/or acetate. Attempts were made to remedy this situ— ation by varying concentrations of carbon source(s), pH, 932,, but the organism would not grow with ethanol and/or acetate alone. It was observed that 5. 9.2.9.9.; grown in mineral medium with dextrose would continue growing in ethanol or acetate under mineral conditions if the entire flask contents were transferred. Therefore, it was suspected that the organism's inability to grow in the presence of ethanol and/or acetate was due to carbon source. Thusa study of the effect of car- bon source on the growth of A: aceti in mineral-salts-medium was made. The results of the study of the growth of two strains of £2222? bagggz.§gggi_in mineral medium with various carbon sources is presented in Table l. The organisms grew with dextrose but not with ethanol or acetate. Growth in a medium containing ethanol or acetate occurred upon the addition of dextrose. Subsequent experience with A. a__c_e_t_i_ pointed out that dextrose must always be included in.a.mineral-salts-ethanol medium for growth and acetic acid production. Apparently, only traces of dextrose are necessary, since 0.01 percent has been observed to satisfy this re- qui rement. 18 TABLE 1 Growth of _l_. aceti in mm with various carbon sources 7“ Carbon Source Strain No. 578 Strain No. 581 Dextrose (2%) w ‘4'. + Ethanol (2% by vol.) ‘ - .. Acetate (0.5%) - - Dextrose (2%) and ethanol (2% by vol.) 4- + Dextrose (2%) and acetate (0.5%) + + I " + = growth, - = no growth. TABLE 2 Growth of A. aceti in mm plus 0.5 percent acetic acid (pH 6.0) with vitamin and amino acid supplements. ‘ Supplement Strain No. 578 Strain No. 581 Yeast extract (0.5%) 4-. + 9 B-complex vitamins” as .. Cassmino acids (0.5%) - ,+ :1. , + 31. * + = growth, - = no growth, + s1. = slight growth. “ See text (p. 12) for vitamins and concentrations used. Pasteur (1868), Boyer (1898), and Beijerink (1898) reported that A. 22.9.5.2. could grow in a mineral-salts-e'thanol medium if acetate or acetic acid were added. Since our results did not confirm this find- ing it was necessary to determine if the organism would grow if acetate were. supplemented with various nutritional factors. Table 2 presents the results of the study of the growth of two strains of A. aceti in 19 mineral-salts-acetate medium supplaaented with yeast extract, vitmnins, and amino acids. Growth occurred in the presence of yeast extract, but there was no growth with the 9 D-complex vitamins. Slight growth was evident in the presence of Oasamino acids. It is possible that the factor in yeast extract may be some source of carbon which is available to the organism. This would parallel the finding of Rae and Stokes (1953b) with Acfigbacter subogdans. It is possible that the slight growth observed in the presence of amino acids and acetate could be attributed to the deamination of amino acid( s) such as alanine. The pseudomonads are generally quite capable of de- and transaminations, and it is assumed that masbers of the getobacter genus are no exceptions. THE EFFECT 01' VITAMINS AND AMINO ACIDS ON TH! BAT! 0? GROWTH AND ACID PROWCTION BI AQQBACI‘IB AGETI IN HINEBAI. MEDIUH PLUS WHOSE Although A. gg__et_i_ grew in mineral-salts-dextrose medium, the pos- sibility existed that it would be etimlated by the addition of various vitamins and/or amino acids. The following experiments were under- taken to deteimine if this did occur. Table 3 shows the results of the study of growth response of two strains of A. w; in mineral medium containing dextrose fortified with vitamins and/or nine acids. Vitamins or vitamins and amino acids were found to stimlate the growth of the organisms, but amino acids alone were not effective. It was of interest to know whether combinations of particular vitamins caused the observed stimulation, or whether it was the result of individual vitamins. 1n. results of thee-study of the growth TABLE 3 Time required for film formation of _A_. aceti in mm plus 2 percent dextrose with vitamin and amino acid supplements. Supplement Days required for film formation Strain no. 578 Strain no. 581 None I 2 h 9 B—complex vitamins" l 2 Casamino acids (0.55%) 2 9 Loomplex vitamins"I plus Casamino acids (0. 5%) l 2 I See text (p.127 for vitamins and concentrations used. want Effect of the omission of various vitamins from the vitamin supplement on the time required for film formation by A. aceti in mm plus 2 percent dextrose. Vitamin omitted Days required for film formation Strain No. 578 Strain no. 581 None Pantothenate folic acid nicotinamide p—aminobensoate pyridoxine riboflavin thiamine biotin HHHHHHHHH NNNNNNNNNN H inositol ‘ Bl response of g. a_.<_:_e_t_i_ in the presence of various mixtures of B—vitamins is presented in Table H. The growth response was the same in all cases for each strain. However, the stimulatory effect of the addition of single vitamins on the acid production and growth of A. M pre- sented in Table 5, ihowed that growth was more rapid in the presence of Ga pantothenate, folic acid, thiamine and biotin. There was no ap- preciable effect of these four vitamins on the amount of acid produced by strain 578; however, acid production by strain 581 was stimulated. These data indicate that the growth of A, 9222i.is stimulated by single vitamins,and not by'a combination of particular cofactors; in addition no vitamin requirement exists. TABLE 5 Effect of vitamins on the rate of film.formation and.acid production by A. aceti in mm plus 2 percent dextrose Hours required for Li :7 22 m Mls of 0.1 N'NaOH Supplement _g;pa formatiqufi’ Strain no. Strain no. Strain no. Strain no. 573 531 573‘ 531" None 21; he 2.17 1.67 pantothenate 12 36 1.92 3.62 folic acid 12 36 2.17 4.10 nicotinamide 2h #8 1.80 1.67 praminobenzoate 2h N8 1.90 1.M7. pyridoxine 2M 48 1.87 1.55 riboflavin 2h #8 1.82 1.50 thiamine 12 36 1 . 97 3 . 9O biotin 12 36 1.85 3.100 inositol 2h #8 1.85 1.52 all vitamins 12 36 1.97 I41.10 ‘ Average of duplicate 10 ml samples titrated to pH 7.0 after 36 hours incubation. ‘* Average of duplicate 10 m1 samples titrated to pl 7.0 after 72 hours incubation. 23 THE OXIDATION or VARIOUS CARBON SOURCES BY lcrromcm ACETI The information available on the oxidative characteristics of the Acetobacter has been obtained using suspensions or extracts of cells grown in enriched medium with either dextrose or glycerol as a carbon source. Since the primary purpose of this study was to obtain infom- ation concerning Acetobacter 849315; grown in mineral medium with the utilisation of ethanol as an energy source, it was necessary to study the oxidative characteristics of this organism grown under such con- ditions. In Table 6 and Figure 1 are presented the results of the study of the oxidation of various carbon sources by A. a_c_e_t_i_ grown in a mineral medium containing 0.1 percent yeast extract, 0.1 percent dex- trose, and 5 percent ethanol. No oxidation of dextrose, gluconate, pyruvate, or acetate was evident with either strain. Both strains oxidized ethanol, using one mole of oxygen per mole of ethanol. This would indicate the conversion of ethanol to acetate. Further studies were made combining ethanol and dextrose; but in each case the ongen uptake corresponded to the oxidation of ethanol to acetate. Since the growth of 5. age}; with dextrose was stimulated by the addition of certain vitamins, dextrose and ethanol were combined with 1 ug of pantothenate, folic acid, thiamine and llmug of biotin. Table 7 and Figure 2 present the results of this study. No oxygen uptake was evident with dextrose and vitamins; ‘The oxygen uptake with ethanol and vitamins corresponds to the oxidation to acetate. It had been demonstrated earlier in this work that both strains grew in a mineral medium containing either dextrose and ethanol, or TABLE 5 Oxygen uptake by A. aceti with various carbon sources. 3, \H Strain Oxygen uptake (ul) at various times (mim): substrate N0. 0 5 10 15 20 25 30 'ho Glucose (him) 578 0 -- 0 - 0 -- -- O 581 O 0 O 0 0 O -- .. Gluconate (Inn) 578 0 -- O -- 0 ' 0 O 0 581 O 0 0 0 0 0 -— -- Pyruvate (lull) 578 0 —. 0 -- 0 -- 0 O 581 0 0 0 0 O O -- —.. Acetate (511M) 578 0 -- 0 -- 0 -- 0 O 581 O 0 O O 0 0 -- .- Ethanol (5uM) 57s 0 -- 8O —. 106 -- 106 106 581 o 58 91 105 113 113 -- .. TABLE 7 Oxygen uptake by A. aceti strain 581 with dextrose and ethanol in the presence of vitamins.* m t various times (mind Dyson uptake (gg a ‘ 1 . 30 35 Substrate , 1‘0 Gluco so ( lull) O 0 O 0 0 O Ethanol (lOuM) 0 103 186 210 213 213 " For vitamins and concentrations used see text (p. 23). 25 600.38 confine c.9383 m3: Soon .4 an. 3335 nowhno 5m Edbm - m on ON 0. O n‘jllfillfiv ESPSE 2,: H I I 395an 21 H q a omobxom 2,: H «I4 339:“ 2,: m OIIIO 3:an 21 m ele say mum 5.93m - < Among: E: 259 0»... On ON .H 0.3%.; lfillmrllmr e fil l ON ov 0m cm 00. 9111mm uofifixo }0 smumrnw 26 .3233: Handgun can eaoApHoH. my? me 53pm goon . $3355 .258 no eonoaoam on» ca fl .3. 3399.9 comic. .N can”; oe on ON 0. 0 IT in + a Low omcbxon S? H «Illa W Bags 3? S e III. m. low m. w a m 10m. M an m .m low. m. a ivom i 27 dextrose and acetate. Therefore, it was evident that they could utilize dextrose for energy if necessary. Consequently, it was necessary to determine if A? aggti.grown in a dextrose containing medium would oxi- dize dextrose and acetate. A suspension of one strain was prepared as described previously with.the exception that cells which had been trans- ferred in mineral medium plus dextrose were grown in shake flasks for 12 hours at 30 C. The medium used was FMM containing 1 percent yeast extract and 1 percent dextrose. The oxidation of dextrose, ethanol and acetate by strain 578 is presented in Table 8 and Figure 3. The uptake was 1.5, 3.0 and 2.0 moles of oxygen per mole of dextrose, ethanol, and acetate respectively. This corresponds to a partial oxidation of dextrose, and complete oxi- dation of both ethanol and acetate. TABLE 8 Oxygen uptake with various carbon sources by A. aceti strain 578 grown in enriched medium 0 en take at various times min ASubstrate o 10 20 30 50 70 Glucose (luM) Trial 1 0 13 l2 19 25 29 36 37 Trial 2 0 1 12 17 2M 28 x 3h 36 Ethanol (luM) 0 7 16 25 37 146 5h 56 Acetate (luM) 0 9 18 28 to M5 h6 up £3302 concise nu macaw wpm sashes «noon .d hp accuses menace 3.839 A3: Samoan samba Hmwnsfifiv s85. ow 0v ON H fl 4 omobxom 25 H a a 4 change. 2.: H o 0 85:8 2: a . e o e 4 O .n 0.3m; 0_ ON on 0v Om Ow axsidn uofiixo 10 519111011117“ STUDIES OF PILOT - PLANT VINEGAR GENERATORS The effect g£_dextrose concentration gg_the operation g£_vineg§r generators - The generators were inoculated with a mineral medium con- taining 1 percent dextrose as previously described. According to Vaughn (19511), acid production subsequent to inoculation in generators will generally initiate in 7 - 10 days. In the present experhent, acid production began after 72 hours, continued for approximately 5 days, and then stopped. It was thought that the dextrose concentration was possibly too high, therefore, the generators were recharged with medium containing no dextrose. Table 9 and Figure 1} present changes in total acid, dextrose, and ethanol concentrations in generators after this modification. The acid concentration in the four generators did not change. There appeared to be a tendency for a slight decrease in the ethanol concentration followed by a slight rise. This my be due to a sampling error. The dextrose concentration increased, eventually reaching a constant level. The reason for the increase in dextrose is unknown at the present time. It may have been due to the slougiing off and/or hydrolysis of the slime present in the generators, or it my have been due to some as yet unexplained factor in the organism's metabolism. It has been reported (anonymous, 1955) that optimum acid production in experimental generators inoculated with Acetobacter glimm was achieved if the dextrose concentration was maintained be— tween 0.05 - 0.07 percent. The activity of the generators was re- stored on reducing the dextrose concentration to this level by dilution. In subsequent experiments, fresh medium contained 0.05 percent dextrose. Changes occurring in generators with high initial dextrose concentrations TABLE 9 —' r Generator Concentrations at various intervals - Days Nb. 0 1 2 3 *u 7 1 8 acid* 3.70 3.25 3.06 3.30 3.06 2.97 % dextrose-s 0.91 0.75 0.95 1.00 1.00 1.00 % ethan01*" “.68 “.30 h.3h ".08 9.NH 5.22 8 acid 3.90 u.16 n.17 n.26 n.02 3.90 2 8 dextrose 0.13 0.21 0.21 0.g& 0.h6 0.36 1 ethanol 6.20 5.8M 5.38 n. n.58 5.80 8 Acid n.38 n.60 n.71 n.59 n.56 u. 0 3 8 dextrose 0.12 0.11 0.17 0.32 0.37 0. s ethanol 5.76 5.16 n.80 n.6u n.88 n.52 % acid n.20 u.u2 n.50 n.32 n.50 u.uu h 8 dextrose 0.11 0.15 0.16 0.36 0.36 - 0.u1 % ethanol 5.39 n.66 n.52 u.h0 n.16 n.71 O % acid (gms/lOO ml) as acetic acid. " Reducing sugar calculated as percent dexteose. sss Percent ethanol. '/'° -1 '«Ant arctic l"§ P11 . ethanol wid. d1'xt1‘081' x 10 31 12 2 IC) ———11 ~43 -2 ’ 8- _ 6_— ,At/0 0/ 4 >-————L—%————!—--T 4 2~ L a) t 1 , l )( l J )S O L N 1 X l 2" )( IO 3 ”A“ 4 >< 835' _ 6’- 1—- is We 1 < , 1 1 ii 4_J 1 1 <1 1 O 2 4 “ 7 O 2 4 W 7 Time (days) . . ‘70 '«U id 0 0 % d(‘X11()SP A A (70 0111111101 Figure 11. Changes occurring in generators with high initial dextrose concentrations. 32 Changes occurring in generators with an 21.2. £191 9}; lg £1. no; 1313932 - An impericism guiding the vinegar industry states that the yield of cider vinegar from generators should be 0.25 gal. of 6 per- cent vinegar per bushel of shavings per 21‘ hours (Vinegar Handbook 1919). Since 1 mole of ethanol requires one mole of oxygen for oxi- dation to the acetate level, it is possible to calculate the air flow rate required. Based on Vaughn's (1954) recommendations, it was cal- culated that the generators used in this study required 125 ml of air per minute. This figure is based on an oxygen adéorption efficiency of 50 percent. This supply of air proved to be inadequate. Table 10 and Figure 5 show the effect of air supply on the changes in acetic acid, dextrose, and ethanol concentrations in generators. Acid concen- tration in the four generators did not change. Both dextrose and ethanol concentrations appeared to fluctuate. Here it appears that the organism could not obtain enough oxygen to oxidize the intermediates formed and would alternately take up and return carbon to the circulat— ing liquor. Initially, it was thought that too much ethanol was present but dilution to 1 percent did not remedy the situation. When air flow was increased, the generators began to produce acid within 1+8 hours. Changes occurring in ggnerators with a__n_ 23.; Q9! 9;: 593 Q pe_r 21223.2 - Table 11 and Figure 6 show changes occurring in acetic acid, dextrose, and ethanol concentrations in generators with ethanol concen- trations ranging from about It to 6 percent and an air flow of 1400 ml per minute. In general, the acid concentration increased at a slow rate and leveled off, despite the appreciable quantities of ethanol 33 TABLE 10 Changes occurring in generators with an air flow of 125 ml per minute r—‘r “ a—J .— Generator Concentrations at various intervals - Days Nb’ 0 1 2 3 u 5 6 8 acid‘ 1.68 1.65 1.65 1.68 1.68 1.71 1.68 1 8 dextrose** .029 .029 .092 .036 .036 .029 .032 8 ethan01*" 5.00 5.10 n.97 5.07 1.77 1.95 1.37 8 acid 1.71 1.68 1.68 1.7h 1.70 1.71 1.71 2 8 dextrose .032 .031 .ouo .035 .038 .030 .036 8 ethanol 5.05 5.10 5.15 5.15 5.72 5.20 n.70 8 acid 2.25 2.25 2.2 2.30 2. 30 2.33 2.33 3 8 dextrose .028 .028 .o .036 .039 .030 .035 8 ethanol 8.52 n.75 u.u2 3.90 n. 77 n.20 u.n7 8 acid 1.80 1.80 1.77 1.77 1.89 1.8 1.8 u 8 dextrose .095 .055 .050 .0h9 .059 .0 .0 8 ethanol 4.82 4.32 n.57 1.50 1.77 1.35 5.57 ' 8 acid (gms. /100 ml) as acetic acid. *9 Reducing sugar calculated as percent dextrose. *** Percent ethanol, w/v8 . ethanol —2 Percent acetic acid. dextrose x 10 6 1 2 t \\2 AHA—W/o\ 1M O\‘\fl \1 4 H /\ m o of w C 2H— + Le—e—+—— ——t———e - ——e-———-e———e~——a 0- O J l l l 6 as 2? E 1 0 2 4 Time (days) e % acid 0 Figure 5. 125 ml per minute. 0 0 % dextrose A % ethanol Changes occurring in generators with an air flow of L_ 6 3h TABLE 11 35 Changes occurring in generators with an air flow of 900 ml per minute -:_ r Concentrations at various intervals - Days Generator NO- 0 1 2 3 8 acid? 2.3 2.76 2.82 2.9“ 1 Edi, dextroset . .Onl e03“ e030 e030 8 ethan01*** 3.85 3.85 1.98 1.22 8 acid 1.95 2.95 3.u8 3.28 2 8 dextrose .039 .02h .022 .025 8 cthanol 5.50 n.27 2.15 1.29 8 acid 1.95 2.69 3.00 3.12 3 8 dextrose .038 .019 .015 .015 8 ethanol 6.10 n.87 2.u5 1.52 g 3616. 2e16 3e00 3e30 3e2ll' h 8 dextrose .035 .026 .019 .022 8 ethanol 5.35 3.99 2.u5 1.29 ‘ 8 acid (gms./.00 m1) as acetic acid. ‘* Reducing sugar calculated as percent dextrose. ’** Percent ethanol, w/v. -2 :1; id. dvxtz'ose x 10 1“‘(“I‘.t 11-. Mid Pt . ethanol 36 6 - ' - 2 l; 4 .- < 2 _ L L l O 3 6+ .- 4 4 r C 2 . L 1 1 O I 2 3 0 Time (days) 0 —————— o % .1: id 0 o % d< xtmso A A % (“11131101 Figure 6. Changes occurring in generators with an air flow of ’400 ml per minute. 37 still remaining in the generators. The dextrose concentration decreased in all cases, and there appeared to be a lag in the uptake of ethanol. There was considerably more ethanol taken up than can be accounted for by the acid produced. Stochiometrically, 1 gram of ethanol yields 1.26 grams of acetic acid. The ethanol was either lost by evaporation, or was converted to end products other than acetate. Upon dilution of the ethanol to 2 to 3 percent, normal fermentation began. Therefore, the above phenomena appear to have been caused by ethanol concentration. The effect of vinegar eels on the operation of vinegar generators - Table 12 and.Figure 7 show the effect of vinegar eels on the changes in acetic acid, dextrose, and ethanol concentrations in normally func- tioning generators with.an air flow rate of “00 ml per minute. The acid increased at a rapid rate in all cases. Dextrose and ethanol de- creased rapidly in.all instances, and no significant amounts of ethanol remained in the circulating liquor after 5 days. No measurable effect of the vinegar eel on the acetic acid fermentation could be seen in this experiment. However, there was less slime on the beechwood shav- ings in the generators infected with eels and they had a "brighter" appearance than in the shavings in the generators free of eels. In this and in similar experiments it was noted consistently that the decrease in ethanol during a given period of time was less than the corresponding acid increase. Calculated efficiencies of ethanol conversion ranged from 100 - 200 percent. The determinations for acid and alcohol proved to be accurate to the best of the author's know- ledge. Therefore, it was thought that the design or use of the generators TABLE 12 The effect of vinegar eels on the operation of normally functioning vinegar generators. Concentration at various intervals - Days_ 0 2 u 5 Generators Trial Trial Trial Trial Trial Trial Trial Trial 1 2 1 2 1 2 1 2 8 acidt 2.82 2.85 n.67 n.37 6.0 5.3h - 5.82 (controD% dextrose** .Oul .036 .026 .025 .017 .015 - .012 8 ethanol*** 2.13 2.0u 1.15 1.02 0.25 0.33 - 0.15 8 acid 2.82 3.72 n.61 n.80 5.70 5.98 - 6.00 (canuo1)8 dextrose .0u1 .030 .022 .021 .017 .012 - .007 8 ethanol 1.89 1.57 1.15 .076 0.u1 0.25 - 0.15 8 acid 1.98 3.66 n.15 5.05 5.57 6.00 - 5.88 (with 8 dextrose .0 7 .035 .020 .015 0.0 .010 - .007 eo1s) 8 ethanol 2.5 1.17 1.23 o.uu 0.82 0 - 0 8 acid 3.72 n.62 n.92 5.u6 5.85 5.9M — 6.00 (with 8 dextrose .037 .035 .026 .020 0.0 .012 - .007 eels) 8 ethanol 2.05 1.06 1.06 0.55 0.57 0 - e t 8 acid (gm./100 ml) as acetic acid. ** Reducing sugar calculated as percent dextrose. **‘ Percent ethanol w/v. -2 . ethanol Percent arctit‘ zu‘id. dextrose x 10 39 Without vols 24 6 \A: O 2 2i 60 2 4U6 Time (days) 0 % acid 0 0 % dcxtrom a A % (“thnnol Figure 7. The effect of vinegar eels on the operation of normally functioning vinegar generators. 140 may have been faulty. Figure 8 shows a drawing of a typical generator used in this study. It was noted that there was a hold-up volume of approximately 1 liter of medium above the air inlet, and it was pos- sible that the air bubbling through this medium might have a stripping effect, resulting in a higher ethanol concentration in the upper por— tion of the medium than in the area of sampling. 1 sample was drained from a generator and another was taken from the area above the air inlet by a hypodermic syringe and needle. The ethanol concentration in the upper area was 3.52 percent and that in the lower area was 2.38 percent. Consequently, the generators should to be modified in future studies by using a larger collection reservoir so that the surface of the alcoholic aolution will be below the air inlet. ’41 K- ('ondonsor 1 food distributor Lt— {also head Lb); lower area filled with shzu ings two liter mark ”TEL peristaltir pump air inlet —-|-’ {also bottom 1' 0801‘ vol 1‘ sum: ling: tuho rigors 8. Pile t-plant laboratory vinegar generator. DISMIOH The two strains of Acetobacter aceti studied could not be grown in a mineral medium containing ethanol or acetate unless dextrose was present. Growth would not occur in mineral salts acetate medium to any significant degree even when supplemented with vitamins and amino acids. This finding conflicted with previously reported data (Pasteur, 1868; Beijerink, 1898; Hoyer, 1898) which indicated that A. 9533; would grow in a mineral salts medium containing dextrose plus ethanol; and that acetate would replace the dextrose. Neither was it possible to grow the organism in Frateur's medium (1950) in which A. 93313; has been reported to grow utilising ethanol as the sole carbon source. In data which were not reported in this study it was impossible to grow the organism in this medium using cell suspensions which had been maintained in ethanol containing mineral medium for a number of sub- cultures as the inoculum. The observation of the need for dextrose along with ethanol in mineral medium agrees with the finding of Rae and Stokes (1953b) who found that Acetobacter subogdans would not grow in a basal medium supplemented with required vitamins and amino acids unless small quantities of dextrose or other reducing sugars were present. The physiology of the acetic acid bacteria pertinent to their growth in and utilization of ethanol is not well understood, and additional work should be done to clarify this issue. .Cell suspensions of A. 9333i; grown in a mineral medium plus 0.1 percent dextrose, 0.1 percent yeast extract, and 5 percent ethanol would not oxidize dextrose, gluconate, pyruvate, or acetate, and would “3 only oxidize ethanol to acetate. This is of notable significance since A, agg£i_has always been considered to be able to convert any available carbon source to carbont dioxide and water. The ability of the cells to oxidize dextrose or acetate was not restored upon the addition of four B—complex vitamins which stimulate A, aggti_in dextrose contain. ing mineral medium. However, evidence was obtained which demonstrated that cells of A. M grown in mineral medium containing 1 percent dextrose and 1 percent yeast extract would completely oxidize ethanol and acetate, and would take up 1.5 moles of oxygen per mole of glucose. This partial oxidation of glucose would correspond to the formation -of 2., 5—diketogluconate. This may be a step in the formation of slhme. Kulka and Walker (195a) have shown that Acetoba‘cter will accumulate this product in the growth medium. In addition Kondo and Takeda (1952), and Koepsell (1950) have shown that the degree of dextrose oxidation is dependent on the nitrogen content of the medium. Koepsell (1950) has also shown that the extent of dextrose oxidation depends upon the iron content of the medium. 4 These data indicate that A, aggti_grown under generator conditions is not a complete oxidizer, and, therefore, may be suitable for the production of grain or distilled vinegar commercially. It is possible that égetobacter glimm will behave as does A. $03.12. in this regard. It was possible to start and maintain pilot-plant vinegar genera- tors inoculated with A. 9311;; using a mineral medium as the substrate. The yield of acid from these generators was satisfactory -- approximately 1 percent per day during early stages of the fermentation. The maJor 1m factors to be controlled in generators as indicated in this study ap- peared to be concentrations of dextrose and ethanol, and air supply. The role played by dextrose in the generators is not understood. Ap- ‘ parently, it is not used for energy as previously claimed, however, it is necessary for growth. It has been pointed out by Alexander and lilson (195% that air supply is critical for sufficient production of cells of hotebacter. . Hromattea, lbner, and Ceolich (1951) found that Acetobacter in submerged fermentations would die rapidly in the ab. sence of air. 1 It is the opinion of .this author that insufficient air supply, and amounts of dextrose or ethanol in excess of optimum concentrations are all important factors in the physiological activity of the acetic acid bacteria. It appears that an optimum air-flow can be achieved: under which conditions, dextreee and ethanol concentrations can be varied without causing such drastic impairment of the fermentation as observed in this study. However, the maxilla air-flew allowed . under the present desigi of the generators used in this study is ’400 ll per minute. No effect of the vinegar eel upon the fermentation rate could be observed in this study. Since the phenomena observed by Zalhsn and rebien (1953) occurred in generators free of dextrose, the eel my supply some essential factor( e) which the acetic acid bacteria derive ' I from dextrose. SUMMARY AND CONCLUSIONS Little information is available regarding the acetic fermentation in mineral medium. This study was undertaken to determine factors af- fecting the growth in and oxidation of ethanob by Agatobacter aceti grown in mineral medium. Two strainsof;.e_.9_gt_i_ could be grown in mineral medium containing ethanol only upon the addition of dextrose. Growth did not occur in mineral medium plus acetate on the addition of vitamins, but slight growth occurred on the addition of amino acids. Growth occurred in the presence of yeast extract. The additions of pantothenic acid, folic acid, thiamine, and biotin stimulated the growth of both strains in dextrose mineral salts medium but stimulated acid production by one strain only. The growth of A. aceti was not stimulated by the addition of amino acids, and the organism did not demonstrate any vitae- min requirements. Cells of g. aceti grown in a mineral medium containing ethanol and dextrose oxidized ethanol to acetate, but would not oxidise dex- trose, gluconate, pyruvate, or acetate. However, cells of A. 949% grown in medium containing 1 percent dextrose and 1 percent yeast extract completely oxidized ethanol and acetate, and partially oxidized glucose. It is believed that glucose was oxidized to 2,5-diketogluconate, or similar end products. Further, it was postulated that the formation of slime by these bacteria may be a result of this partial oxidation. Experimental pilot-plant type vinegar generators inoculated with A. aceti were started and mintained on a mineral medium. This study 1&6 indicated that the major factors to be controlled in'the Operation of generators were the concentrations of dextrose and ethanol, and the air supply. With the above factors controlled, acid was produced at the rate of one percent per day initially. The vinegar eel appeared to have no effect on the fermentation in normally functioning generan tors. BIBLIOGRAPHY Alexander, M, and Wilson, P. W, 1951+. Large-scale production of the Azotobacter for enzymes. J. App. Microbiol., _2_, 135-1140. Anonymous, 1955. Glucose increases yield of vinegar generators, Fer- mentatio, 5_, 190-202, as quoted in Food Eng., .31, 175-176. Beijerinck, M. H., 1898. Uber die Arten der Essigbacterien. Zentr. Bakt. Parasitenk Abt., II, 5, 209-216. Bernhauer, H., and Knobloch, H., 1938. Der Abbau der Glucose durch Acetobacter suboqdans, Naturwissenschaften, 2_6_, 819. Bernhauer, K., and Riedl-Tumova, E., 1950. Oxidationen mittels Essig— bakterien. Zur Methodik der bakteriellen Oxidationen in der Sub— merslmltur, Biochem. z. 320, ”60-1471. Brannt, w. T., 1911i. 4. Practical Treatise 95 the manufacture gt; Vinegar, Henry Carey Baird and 00., Philadelphia. Butlin, K. H., 1936. Aerobic breakdown of glucose by Bact. subomdans. Biochem. J., 10, 1870-1877. 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