ISOLATION AND IDENTIFICATION OF ACETIC ACID BACTERIA FROM DIFFERENT ECOSYSTEMS* BY R. S. KAHLON AND S. R. VYAS

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1 SOLATON AND DENTFCATON OF ACETC ACD BACTERA FROM DFFERENT ECOSYSTEMS* BY R. S. KAHLON AND S. R. VYAS (Department of Microbiology, Haryana Agricultural University, tissar) Received January ll, 97 (Communicated by Prof. J.V. Bhat, F.A.SC.) ABSTRACT Forty-five cultures of acetic acid bacteria were isolated from different ecosystems including fruits, vegetables, flowers, vinegars, etc. On the basis of morphological, physiological and biochemical characteristics they were identified as A. aceti, A. xylinum, A. suboxydans, A. melanogenum and Bacterium xylinoides. t was obvious that A. aceti and A. xylinum were prevalent in fruits, vinegars and cane juice while A. rancens was to be found in flowers and vegetables. Grapes seem to be a fine source for isolation of common species. Alcohol tolerance and ability to oxidize alcohol to acetic acid showed that A. aceti and A. xylinum possess higher alcohol tolerance and produce more acetic acid than the other species. NTRODUCTON THE acetic acid bacteria are known to exist in a wide variety of natural sources and a number of brewery products. Their morphology and physiology have been the subject of extensive study by various workers (Frateur, 950; Asai and Shoda, 958; Brown and Rainbow, 956; DeLey, 96 ; Cooksey and Rainbow, 96; Ameyama and Knodv, 967) and have been ably reviewed by Rao (957), Carr and Shimwell (96), Loitsyanksaya (962) and Janke (962). Yet their characterization and grouping pose serious problems probably because of their high morphological and biochemical mutation frequency as shown by Shimwell and Carr (96 and 964), Shimwell (959) and Schram and Hestrin (954), though Schell and DeLey (962) have reported different results. * Part of the thesis submitted by R. S. Kahlon in partial fulfilment of the degree of Master of Science in Microbiology to Punjab A~ieu!tuml University, Hissar (Now known as Haryana Agricultural University,), 29

2 294 R.S. KAHLON AND S. R. VYAS The present work was undertaken with a view to elucidate some of the biological and biochemical aspects of acetic acid bacteria and to understand the basic differences observed in the organisms isolated from different ecological environments. n this paper are described the properties of the forty-five cultures isolated from different natural ecosystems. MATERALS AND METHODS Enrichment and pure culture isolation.--forty-five isolates of acetic acid bacteria were isolated from various sources as fruits, vegetables, flowers and vinegars, by setting up the enrichments in yeast extract (YE) alcohol medium. The cultures were purified by repeated streaking on alcohol medium containing CaCO. The pure cultures so obtained were maintained on YE-glucose-CaCO agar slants (DeLey, 96). Characterization of isolates.--the colony characteristics, gram staining, capsule staining, motility and pigment production on glucose-peptone medium were studied. Several other properties of the organisms were examined along with their biochemical characteristics as catalase production, oxidation of ethyl alcohol and lactate, ketogenesis on mannitol, glycerol, sorbitol and i-erythritol (Frateur, 950; DeLey, 96), 5-ketogluconate formation, growth in Hoyer's solution, fermentation of sugars and sugar alcohols, gelatin liquifaction and cellulose production. The extent of their acid production and alcohol tolerance were tested in suitable media by titrating the acid produced and ascertaining the percentage of the alcohol tolerated. RESULTS AND DSCUSSON A glance at Tables and will show that all the cultures were gram negative, catalase positive and gelatine negative. On the basis of all the characteristics (Bhat and Rajsinghani, 955 ; Bergey's Manual, 957; Shimwell, 959; D eley, 96, 96) they could be grouped into three groups: Group, comprising 4 cultures, tagged: A-l, A-, A-4, G-, G-4, G-5, G-7, St-l, M-2, M-, Or-l, V-l, V-2, and Cj-, included those isolates which oxidized ethanol to CO2 and utilized (NH4)2SO4 as the sole source of nitrogen. All of them gave positive results for lactate oxidation, dihydroxyacetone formation from glycerol, fructose from mannitol and grew well in Hoyer's solution. None produced erythrulose from i-erythritol. They could oxidize clearly only ethanol and glucose to acid and were unable to do so in ~ase of arabinose, glycerol, inulin, rhamnose~ xylo s,

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4 296 R.S. KAHLON AND S. R. VYAS butanol and isopropanol. Their reactions to other substrates were variable in the sense whereas some produced acid, others did not. TABLE Physiological and biochemical characteristics-- (acid production) Ethanol Glucose Arabi- Dextrin Duleitol Fructose Galac- Glycerol nulin Lactose Maltose nose rose Group,4. a "eli.. - 5: Group.4. xylinum.. _ S B. a yli~toides..,4. "a~cgpt$. 5: ± G, oup [ A. melanogennm A. suboxydan; 5: S - Mannitol Ethylene Methanol so- so- Mannose Rhamnose Sorbitol Sucrose Xylose g ycol Butanol butanol propanol Group A. aceti.. ± Groap A. xylinum.. B. xelinoides,d. ratt c~t$.. _s s 5= 5: Group A. melano. genu~.4. suboxtdans = Positive reaction. - = Negative reaction. = Some give positive, some negative reaction. S = Some give slight and some negative reaction. They tolerated alcohol to the extent of 0 per cent. Morphologically all were similar, gram negative and cylindrical to ellipsoidal and non-capsulating; motility was restricted to only a few. All the strains were clearly of the species Acetobactcr ace~i,

5 Acetic Acid Bacteria from Different Ecosystems 297 n Group were placed twenty-six cultures which oxidized ethyl alcohol but failed to utilize (NH4)2SO4. All of them produced acid from glucose and ethanol and oxidized lactate but only thirteen clearly produced acid from glycol, dihydroxyacetone from glycerol, fructose from mannitol and 5-ketogluconate from glucose. Only a few formed sorbose from sorbitol. They failed to grow in Hoyer's solution and to produce erythrulose from i-erythritol. Fructose, galactose, glycerol, maltose, mannose and butanol were acidified by few but all gave no reaction in other substrates, xylose and lactose being exception in that a few showed slight reaction. These thirteen strains were recognised as A. xylinum species, occurring singly or in chains, microscopically non-capsulated a few being motile. All produced cellulosic fibrils. The isolates comprising this group were : A-2, A-5, G-l, G-2, (-9, P-l, Gh-, J-l, V-, Cj-2, Cj-, Cj-4 and Asv-. Two other strains (G-8, and M-l) differed from the above in that they oxidized ethanol to CO2 but did not form fructose or sorbose from mannitol and sorbitol and were of doubtful character in so far as formation of 5-ketogluconic acid from glucose and dihydroxyacetone from glycerol was concerned. These, therefore, have been designated as Bacterium xylinoides species. One of them gave the cellulose positive test. Other eleven strains showed no ketogenesis on glycerol, mannitol, sorbitol, i-erythritol and glucose. Moreover, they did not grow in Hoyer's solution and did not produce cellulose. They oxidized lactate and acidified butanol while had variable reaction on all the substrates inulin, rhamnose, methanol and isopropanol being exceptions in that their reaction in them agrees with that of A. xylinum. Presumably, these strains were A. rancens. All these strains were motile and produced a slight pellicle in glucose-ye broth. The isolates which fell in this category were: Pm-, H-l, C-l, Mrg-, K-, Bg-, and Asv-2. G-6, G-0, D-l, B-l, Other five isolates were placed in Group!, their characteristics being that they oxidized ethanol only upto acetic acid and never to COs and consequently showed no redeposition of CaCO around the colonies on prolonged incubation. They did not oxidize lactate but showed marked ketogenic property for glycerol, mannitol, sorbitol, i-erythritol and glucose. They clearly displayed fermentation reactions with glucose, ethanol, maltose, arabinose, and only variable reactions with galactose, lactose, mannitol, mannose, sorbitol and sucrose and no reaction with other substrates.

6 298 R.S. KAHLON AND S. R. VYAS Two isolates (Gu-2 and T-l) produced dark brown pigment on glucosepeptone medium. The cells were coccoidal rods and motile They were recognized as A. melanogenum. Other three isolates (G-l, Lf-2, KA-) which did not produce any pigment were non-motile small rods, occurring singly, were recognized as A. subovydans as they characteristically oxidized sorbitol to sorbose although DeLey (96) recommended the name Gluconobacter for acetic acid bacteria showing this type of behaviour. From the above it is clear that acetic acid bacteria display considerable variation in properties and it is of interest to consider in this context their ecological distribution From Table, it would seem that some species TABLE Ecological distribution of various species Source i A. aceti A, x)liuum A. rancem A. suboxy. A. mdana- A. xvli Total number of darts ge um moides isolates Apple.. 2 o,o, a 5 Grapes. 2 0 Malta.. * Guava.. * 2 Orat~ge...,g Custard Apple.. * Pear,,~ o* Pomegranate o* ol i, Banana ** Date.. *. o* Vinegar 2 ~* J, Cane luice.. 4 Vegetable*.. ai Flowerst.. *o oo QO 5 Ay urvedic Medicines i* OO *Ghia, Lady's finger, Tomato. t Jenia, Marigold, Cosmos, Kaner, Bougainvillea.

7 Acetic Acid Bacteria from Different Ecosystems 299 are common in type of environment they are encountered while others exist in other environments. A. aceti and A. xylinum are far more common in fruits, vinegars and cane juice than the other species A. rancens which seem to find flowers as a preferential habitat. Apparently, grapes is a fine source for isolation of common species. Alcohol tolerance and acid production.--the amount of acid produced from 2, 6 and 0 per cent alcohol in broth was determined. solates belonging to Groups and showed high tolerance for alcohol and produced higher amount of acid than did those of Group t. A. aceti produced -6 to 2- per cent acid from 2 per cent alcohol, thereby converting per cent alcohol into acetic acid. Eleven of these tolerated upto l0 per cent alcohol and produced 2.7 to 4.28 per cent acid only. Other three grew fairly well in 6 per cent alcohol and converted 49. per cent of this to give.84 per cent acetic acid. May be they could not tolerate higher acid concentration and as such they did not oxidize remaining alcohol. A. xylinum from Group showed similar results in 2.0 per cent alcohol while only half the number could grow in 0.0 per cent alcohol, others could grow in 6-0 per cent alcohol producing acid concentration between per cent. Both the B. xylinoides strains grew in 6 per cent alcohol but not in 0-0 per cent. Except four isolates (G-0, D-l, K-l, Asv-2),4. rancens failed to grow in 0.0 per cent and produced acid to a final concentration of 2.7 per cent. solates to Group could attain acid concentration of only 0.84 to.2 per cent utilizing for the purpose about per cent of the 2"0 per cent available alcohol. Except one (Lf-) they even failed to tolerate 6.0 per cent alcohol. This gave a final acid concentration of.68 per cent utilizing threrefor only 2.5 per cent of the 6.0 per cent available concentration of alcohol. The results showed that A. aceti and A. xylinum tolerated higher alcohol concentration and produced more acetic acid as compared to A. rancens; A. melanogenum and A. suboxydans, the last two species displaying rather poor tolerance to alcohol. REFERENCES Ameyama, M. and Kondo, K. Asai, T. and Shoda, K. "Carbohydrate metabolism by acetic acid bacteria. V. Characteristics of intermodiate type strains," Agri. BioL Chem., 967,, 727. "The taxonomy of Acetobacter and allied oxidative bacteria,', J. Gen. Appl. Microbiol., 958, 4, 289.

8 00 R. S. KAHLON AND S. R. VYAS Bhat, J. V. and Rajsinghani, K. Brown, G. D. and Rainbow, C. Carr, J. G. and Shimwell, J. L. Cooksey, K. E. and Rainbow, C. DeLey, J. Frateur, J. Jank, J. Loitsyanskaya, M.S... Ran, M. R. R. and Stokes, J. L. Shell, J. and DeLey, J... "Variability in acetic acid bacteria," Antonie van Zeeuwenhoek J. Microbiol and SeroL, 962, 28, Sehram, N. and Hestrin, S... "Factors affecting production of cellalose at the air liquid inter face of a culture of Acetobacter xylinum," J. Gen. MicrobioL, 954,, Shimwel, J. L. and Carr, J. G. Bergey's Mannual of Determinative Bacteriology, 957, R. S. Breed E. G. D., Murray and N. Smith. Baltimore: William', and Willams, Co. "Studies on Acetobacter.. solation and characterization, of species," Proc. nd. Acad. Sci., 955, 4 B, "Nutritional patterns in acetic acid baclcria," J. Gen. Microbiol. 956, 5, "The acetic acid bacteria: A critical review,'" Autonie van Leeuwehoeles J. Microbiol. and Serol., 96, 27, "Metabolic patterns of acetic acid bacteria," J Gen. MicrobioL 96, 27, "Co.nparative carbohydrate metabolism and a proposal for a phylogenedc relationship of acetic acid bacteria," bid., 96, 24, "The use of i-erythritol for the classification of acetic acid bacteria," Antonie van Leeuwenhoek J. MicrobioL Serol., 96, 29, "Essai sur la systematique des Acetobacter," La Cellule, 950, 5, "Recent studies on the metabolism, th: systematics and evolution of the acetic acid bacterm," Arch. Microbiol., 962., 4, "Systematics of acetic acid bacteria," Mikrobiologiya (Trans.), 962,, from, "Acetic acid bacteria," Ann, Rev. MicrobioL, 957,, 7-8. "Util!zation of ethanol by acetic acid bacteria," J. BacterioL, 95, 65, "Rcassessment of the genus Aectobacter," Autonie van Leeuwenhoek J. MicrobioL and SeroL, 959, 25, "Derivation of non-acetifying quasi Acetobacters from a true- Acetobacter strain and vice versa," bid., 960, 26, "Mutation frequency in Acetabacter," Nature, 964, 20,