INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1996, p. 1131-1 137 0020-7713/96/$04.00 + 0 Copyright 0 1996, International Union of Microbiological Societies Vol. 46, No. 10 Themosyntropha lipolytica gen. nov., sp. nov., a Lipolytic, Anaerobic, Alkalitolerant, Thermophilic Bacterium Utilizing Short- and Long-Chain Fatty Acids in Syntrophic Coculture with a Methanogenic Archaeum VITAL11 SVETLITSHNY1,l FRED RAINEY,2 AND JUERGEN WIEGEL1* Department of Microbiology and Center for Biological Resource Recoveiy, University of Georgia, and Cell Cultures, 3300 Bruunschweig, Germany2 Athens, Georgia 30602-2605, and German Collection of Microorganisms Three strains of an anaerobic thermophilic organoheterotrophic lipolytic alkalitolerant bacterium, Thermo- syntropha fipolytica gen. nov., sp. nov. (type strain JW/VS-265T; DSM 11003), were isolated from alkaline hot springs of Lake Bogoria (Kenya). The cells were nonmotile, noon-spore forming, straight or slightly curved rods. At 60°C the pH range for growth determined at 25°C [pHZ5 c] was 7.15 to 9.5, with an optimum between 8.1 and 8.9 (pH600C of 7.6 and 8.1). At a pHZ5OC of 8.5 the temperature range for growth was from 52 to 70°C, with an optimum between 60 and 66°C. The shortest doubling time was around 1 h. In pure culture the bacterium grew in a mineral base medium supplemented with yeast extract, tryptone, Casamino Acids, betaine, and crotonate as carbon sources, producing acetate as a major product and constitutively a lipase. During growth in the presence of olive oil, free long-chain fatty acids were accumulated in the medium but the pure culture could not utilize olive oil, triacylglycerols, short- and long-chain fatty acids, and glycerol for growth. In syntrophic coculture (Methanobacterium strain JW/VS-M29) the lipolytic bacteria grew on triacylglycerols and linear saturated and unsaturated fatty acids with 4 to 18 carbon atoms, but glycerol was not utilized. Fatty acids with even numbers of carbon atoms were degraded to acetate and methane, while from odd-numbered fatty acids 1 mol of propionate per mol of fatty acid was additionally formed. 16s rDNA sequence analysis identified Syntruphospora and Syntrophomonas spp. as closest phylogenetic neighbors. Until now, thermophilic anaerobic lipolytic bacteria able to utilize long-chain fatty acids in syntrophic cocultures were un- known. This is surprising since a large number of obligately anaerobic thermophilic and extremely thermophilic organotro- phic hydrolytic (eu)bacteria and archaea have been isolated from geothermally heated and anthropogenic thermobiotic en- vironments (41). Most of these microorganisms grow at pH values close to neutral except for a few recently isolated pro- teolytic anaerobic alkali(to1erant)-thermophiles (8, 20, 21). Most of the known lipolytic microorganisms are aerobes, which include some recent examples of thermophiles and alkaliphiles (7, 16, 17,36,38). All known anaerobic lipolytic bacteria which produce true lipases, such as Anaerovibrio lipolytica (29) and a Butyn'vibrio sp. (12) from rumina, are mesophilic neutrophiles (24). They are able to grow anaerobically on triacylglycerides, hydrolyzing the acylglycerol linkage and utilizing the liberated glycerol for growth, but do not utilize the long-chain fatty acids. The short- and long-chain fatty acids under anaerobic conditions are degraded by nonlipolytic syntrophic proton-re- ducing microorganisms (1, 2, 4, 14, 23-26, 34, 35, 37, 43, 44) and by some nonlipolytic sulfate-reducing bacteria (32, 40); however, they depend on other lipolytic organisms, such as A. lipolytica (29). Only two nonlipolytic, neutrophilic proton-re- ducing mesophiles have been described, the syntrophic Syntro- phomonas sapovoruns and Syntrophomonas wolfei subsp. saponavida, which are able to degrade long (lt?-carbon)-chain fatty acids via P-oxidation in syntrophic associations with H,- utilizing methanogens or sulfate reducers (23, 34). * Corresponding author. Phone: (706) 542-2651. Fax: (706) 542- 2674. Electronic mail address: jwiegel@uga.cc.uga.edu. To our knowledge, no lipolytic, obligately anaerobic neutro- philic or alkaliphilic thermophile from any natural volcanic or man-made thermobiotic environments nor an anaerobic ther- mophile which hydrolyzes triglycerides and utilizes the liber- ated short- and long-chain fatty acids for growth has been described. Recently, an anaerobic thermophilic (55°C) enrich- ment culture able to degrade a wide range of short- and long- chain fatty acids was described (1). Here, we report on the first thermophilic syntrophic anaerobic alkalitolerant Iipolytic eu- bacterium, Thermosyntropha lipolytica gen. nov., sp. nov., iso- lated from alkaline volcanic environments of Lake Bogoria, Kenya. It presents a novel physiological type among the ther- mophiles. When incubated in the presence of triglycerides, this bacterium hydrolyzed triglycerides and grew on the liberated fatty acids in a syntrophic association with an alkalitolerant H,-utilizing methanogen, isolated from the same environment. MATERIALS AND METHODS Isolation and culture conditions. The prereduced basal medium used for enrichment, isolation, and cultivation of lipolytic bacteria was prepared by the modified Hungate technique (22) under nitrogen gas phase. The basal medium contained (per liter) 0.3 g of K2HP04, 0.3 g of KCI, 0.5 g of NaCI, 1.0 g of NH,CI, 0.3 g of MgCI, - 6 H20; 0.05 g of CaCI2 - 2H20, 3.0 g of NaHCO,, 3.0 g of Na2C0,, 0.5 g of Na,S - 9H@, 0.15 g of cysteine, 2 rnl of vitamin solution (ll), and 2.5 ml of a trace element solution (11). Substrates were added in concen- trations indicated below. After addition of the substrates, the pH of the medium was adjusted to values indicated below at 25°C (pHZ5OC) or at cultivation tem- perature (e.g., pH600C). For the enrichment of lipolytic bacteria, 20 ml of commercial olive oil (Kroger brand) per liter and 5 g of yeast extract per liter were added to the basal medium and the pH250C was adjusted to 8.8 and 9.5. The enrichments were incubated at 60,70, and 80°C. Serial dilutions of positive incubations were made into the same medium ( PH~"~~ KO), and the mixtures were incubated at 60°C. Partially purified lipolytic cocultures obtained by serial dilutions in liquid medium were serially diluted and used to inoculate agar-shake-roll tubes containing basal medium (pH6OoC 7.8) with 5 ml of emulsified olive oil, 0.5 g of yeast extract, and 3 mg of rhodamine B (19) per liter and 2% agar. The agar-shake-roll tubes were incu- 1131