ISOLATION AND CHARACTERIZATION OF ZYMOMONAS MOBILIS FROM SUGARCANE JUICE AND NIPA SAP ASUNCION K. RAYMUNDO, E.D. GUEVARRA and N.F. PAE Microbiology Laboratory, Institute of Biological Science; BIOTECH, University of the Philippines at Los Bofios, Laguna ABSTRACT Samples of fermenting sugarcane juice and nipa sap were used as sources of alcohol-producing, bacteria, From hundreds of colonies on isolation plates, 13 isolates produced alcohol higher than 3% by volume, Nine were obtained from fermenting nipa sap and fous Jom fermenting sugarcane juice, Through morphological and physiological characterization, all isolates were identified to be Zymoma- nas mobilis subsp, mobili. INTRODUCTION Alcohol is now being tapped as an alternative source of energy to reduce dependence on oil. Interest in alcohol production by fermentation has been renewed with the discovery that besides yeast, bacterial species like Zymomonas mobilis can also be used for large scale production of ethanol (Rogers ef al, 1979). Some of the reported advantages of Z. mobills over Yeast are high sugar uptake and ethanol productivities on cell recycle systems, no oxygen requirement and genetic manipulation potential (Lavers et al, 1980; Lee et al, 1979; Rogers et al, 1979). In the tropics, Z. mobilis has been isolated from various fermenting juices and is being used to make palm wines (Swings and de Ley, 1977). However, the potential of this bacterium for commercial scale production has been sealized only recently (Rogers et al, 1979). The use of Z, mobilis in the Philippines hinges on the,selection of the right strain and the development of technology appropriate to local conditions. There is a need, therefore, to isolate strains from local sources which might be more suited to local conditions. So far work of this kind has not been done here. MATERIALS AND METHODS Isolation Fresh sugarcane and nipa juice were obtained from Canlubang, Laguna and Paom- bong, Bulacan. Twenty-five mL samples were inoculated into RM broth (Rogers et al, 1979) using 4% sucrose instead of 1% glucose and with 100 mg/L of an antifungal agent, Kabicidin. After incubation at room temperature for 24h, 5 mL Schiff's reagent (Dennis and Young, 1982) was added to 10 mL aliquots. Samples which produced purple color characteristic of Zymomonas species were plated out on RM agar and incubated anaerobically in Bray dishes at 30°C. After 24-48 h, colonies typical of Zymomonas 123, 124 The Philippine Journal of Science 1986 species were transferred to RM broth tubes and incubated at 30°C for 24 h. Isolates which released gas upon shaking were inoculated on RM broth with 15% glucose, incubated for 24 h at room temperature and alcoho! production determined with an ebulliometer. Isolates which produced alcohol higher than 3% by volume were subjected to further alcohiol production, characterization and identification tests. Alcohol Production Alcohol yields of the different isolates using RM as the basal medium and two substrates, 15% glucose and 15% total sugars in molasses were determined. Fifteen mL of a 24-h-old RM broth culture of each isolate was inoculated on 85 mL of the fermen- tation medium in 125 mL Erlenmeyer flasks covered with “bungs” (rubber stoppers with bended glass tubings). After inocalation, the tips of the glass tubings were immersed in test tubes filled with water and incubated at 30°C for 24 h. Alcohol yield was deter- mined using an ebulliometer. Characterization and Identification ‘The isolates were characterized morphologically, culturally and physiologically using standard methods (Collins and Lyne, 1975). Z mobilis ATCC 10988 was used as the control strain, The following tests were conducted: Gram reaction, motility, catalase, gelatin liquefaction, hydrogen sulfide production, methyl red-Voges Proskauer, levan production, growth factor requirement and utilization of ten sugars, namely: glucose, fructose, sucrose, galactose, lactose, maltose, mannitol, raifinose, rhamnose and xylose. These isolates were identified according to the established taxonomic key of classification contained in Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974). RESULTS AND DISCUSSION Jsolation ‘Streak plates from fermenting sugarcane juice and nipa sap which produced deep purple color upon the addition of Schiil’s reagent exhibited colonies which resembled the control, Z. mobilis ATCC 10988. These colonies were circular, entire-edged and cream-colored measuring about 0.1 to 1.5 mm in diameter alter 24h incubation. About 25 such colonies were picked from the isolation plates per sample using three samples from sugarcane juice and three from nipa sap. These isolates were inoculated on RM broth and a total of 26 isolates showed signs of gas production. When these isolates were restreaked on RM agar, further purified and re-inoculated on RM broth, only 13 pro- duced vigorous bubbling in RM broth. Nine of these, BZ18, BZ19, BZ21, BZ22, BZ23, BZ24, BZ25 and BZ26 were obtained from fermenting nipa sap and four, BZ22A, BZ13, BZ14, BZ16 from fermenting sugarcane juice Alcohol Production The alcohol yield (% by volume) of the 13 presumptive Zymomonas isolates on the two fermentation media used after 24 h incubation at room temperature are shown. in Table 1. In RM containing glucose, the alcohol yield ranged from 5.58-7,83% with118:2, Raymando et al: Isolation and Characterization of Z. mobilis 125 the reference culture yielding 7.82%, The isolates were ranked based on their yields in descending order as follows, BZ16, BZ2A, BZ14, BZ13, BZ220, BZ23, BZ26, BZ21, BZ24, BZ25, BZ22, BZi8, BZ19. Four isolates from sugarcane juice and one from nipa sap gave yields comparable to that of Z. mobilis ATCC 10988. In molasses with 15% total sugar, the alcohol yield of the local isolates ranged from 2.69-4.73% with the reference culture producing 4.80% by volume. BZ22 produced a yield (4.73%) comparable to ATCC 10988 (4.84%). The yields obtained from glucose were greater than those from molasses for all the isolates tested including the reference culture ATCC 10998. The same results were obtained by Van Vuuren and Meyer (1982). The lower alcohol yield may be ascribed to the presence of certain compounds in molasses which are inhibitory to growth and ethanol production of Zymomonas. Molasses contains 50-60% sugars mainly in the form of sucrose; other components are vitamins, non-nitrogenous acid, pigmented materials, waxes, sterols, lipids, odorants and inorganic components (Binkley and Wolfrom, 1953). The components and quality of molasses vary according to the treatments on sugarcane juice used to remove interfering com- pounds and impurities. Some of these substances in molasses like salts have been estab- lished to be inhibitory to the fermentative capability and growth of the cells (Rogers et al, 1982; Van Vuuren and Meyer, 1981). The fermentation performance of the local isolates differed with the media used. BZ20 produced higher yield, (7.27%) than BZ23 (6.53%) in glucose. However, in molas- ses, BZ23 produced higher yield, 4.73%, than BZ20 with only 3.44%. Two isolates, BZ23 and BZ26 produced the same yield of 7.2% in glucose but differed in their yields in molasses. The opposite was true with BZ25 and BZ26 which differed in their yields in glucose but produced the same yield in molasses. Isolates BZ[3 and BZ14 produced very high yields in 15% glucose but very low yields in molasses. These differences in yields may be attributed to the varying abilities of these isolates to tolerate the inhibitory substances present in molasses. Characterization and Identifiostion Morphological examination of the isolates revealed Gram-negative, plump rod cells with distinct rounded ends. No endospores were observed. Most of the cells occur singly or in pairs. All of them are motile. The same characteristics were observed in the reference organism, Z, mobilis. ATCC 10988. Such characteristics were also reported by earlier workers (Buchanan and Gibbons, 1974. Swing and de Lay, 1977). When plates were incubated aerobically, the resulting colonies were smalfer than those incubated anaerobically, The average colony diameter for isolates grown aero- bically after 48 h of incubation was 1-1.2 u while those isolates incubated anaerobically ranged from 1,8-3.0 u. The same results were observed with Z mobilis strains CP3 and CP, obtained from fermenting sugarcane juice in Brazil and Ag 1! isolated from ferment- ing agave juice in Mexico (Swings and de Lay, 1977). Physiological and biochemical tests showed uniform reaction of all the isolates. All of them were catalase positive, produced levan, required biotin and panthothenate for growth, failed to hydrolize gelatin, did not produce HS, negative in methyl red test but,126 The Philippine Journal of Science 1986 positive for Voges-Proskauer test and fermented only sucrose, fructose and glucose. Based ‘on the results mentioned, the 13 local isolates were identified as Z. mobilis (Lindner) Kluyver and Van Neil. Kluyver (1956) (as cited in Swings and de Lay, 1977) proposed two species of Zymomonas based on sucrose fermentation. Strains which could ferment glucose, fruc- tose and sucrose were assigned to the species of Z. mobilis and isolates which cannot ferment sucrose to Z, anaerobia. The proposal was adopted in the eighth edition of Bergey’s Manual of Determinative Bacteriology (Buchanan and Gibbons, 1974). However, the ability to ferment sucrose was established to be an inducible phenotype and that the G-C ratios of the two Zymomonas species are very close (Dadds and Martins, 1983). These results prompted Swings and de Lay (1977) to propose only one species: Z. mo- bilis. The strains which can ferment glucose, fructose and sucrose wili be called Z. mo- bilis subsp. mobilis, In view of this, the strains isolated were identified as Z, mobilis subsp. mobilis, This type of classification has been adapted and is included in the “Ap- proved Lists of Bacterial Names” (Skerman et al, 1980). ACKNOWLEDGMENT This work was supported by the National Institutes of Biotechnology and Applied Microbiology (BIOTECH), a research and development facility established jointly by the Ferdinand E. Marcos Foundation, Ministry of Energy and UPLB. REFERENCES 1. Binkley, W.W., and ML. Wolfrom, 1953. Composition of cane juice and cane final molasses. Adv. Carbohydrate Chem, 8:291-303. 2, Buchanan, R., and N, Gibbons (eds.). 1974. Bergey’s Manual of Determinative Bacteriology. 8th ed. Baltimore. The Williams and Wilkins Company. 1268 p. 3. Collins, CH. and PM. Lyne. 1970. Microbiological Methods. University Park Press. Baltimore. 454 p. 4. Dadds, M., and P. Martin. 1973, The doubtful status of the species of Z. anaerobia and Z, mobilis. J. Appl. Bacteriol, 36:531-539. Dennis, R., and T. Young. 1982. A simple rapid method for the detection of sub- species of Z. mobilis. J, Inst. Brew. London, 88. 25-29. 6. Lavers, BH, P. Pang, C:R. Mackenzie, G.R. Lawford, J, Pik and H.G. Lawford. 1980, Industrial alcohol production by high performance bacterial fermenta- tion, Abstract. 6th Int’l, Fermentation Symp. p. 81. 7, Lee, K., M. Lefervre, D. Tribe and P. Rogers. 1980. Migh productivity ethanol fermentations with Z. mobilis using continuous cell recycle: Biotechnol. Letters. 2. 487 492 8 Lee, K., M_Skotnicki, D. Tribe and P, Rogers. 1980. Kinetic studies on a highly productive strain of Z. mobilis. Biotechnol. Letters. 2, 339-344. 9. Lee, K.J., DE. Tribe and P.L. Rogers. 1979. Ethanol production by Zymomonas mobilis in continuous culture at high glucose concentrations Biotechnol. Letters. 1, 421-426.116:2 Raymundo et al: Isolation and Characterization of Z. mobilis 127 10. Rogers, P.L., KJ. Lee, JLH. Lee, ML. Skotnicki, R.J. Pagan and D.E. Tribe. 1982. The Zymomonas process for ethanol production. Paper presented at the Regional Workshop on the Technology of Fuel Alcohol Production, Oct. 25-30, 1982, University of the Phil, at Los Banos, College, Laguna. 11, Rogers, P., K. Lee, M. Skotnicki and D. Tribe. 1979, Ethanol fermentation by highly productive strains of Z, mobilis. Biotechnol. Letters 1(4), 165-170. 12. Skerman, V.B.D., V. McGowan and P.H.A. Salath, 1980. Approved lists of bacterial ‘names. Int. J. Sys. Bacteriol. 30, 225-420. 13. Swings, J., and De Ley. 1977. The biology of Zymomonas. Bacteriol. Rev. 41: 1 “46. 14. Van Vuuren, J.J.J., and L. Meyer. 1982. Production of ethanol from sugarcane molasses by Zymomonas mobilis. Biotechnol, Letter. 4(4) , 253-256.128 The Philippine Journal of Science 1986 Table 1. Alcohol yield of Zyntomonas isolates in 15% glucose and molasses (15% total sugar) substrates incubated for 24 h at 30°C. Percent alcohol yield by volume Isolate Number 15% Giucose Total Sugar BZ2A 118 4.02 BZI3 75) 2.69 BZIg 767 2.28 BZ16 7.83 356 BZ18 5372 4.00 BZI9 558 3.48 BZ20 721 3.44 BZ21 692 357 BZ22 653 4.73 BZ23 720 424 Bz24 6.90 4.66 BZ25 6.60 3.44 BZ26 7.20 344 ATCC 10988 7.82 484