Dominant sugar utilizers in sediment of Lake Constance depend on syntrophic cooperation with methanogenic partner organisms

Abstract

Six strains of novel bacteria were isolated from profundal sediment of Lake Constance, a deep freshwater lake in Germany, by direct dilution of the sediment in mineral agar medium containing a background lawn of the hydrogen-scavenging Methanospirillum hungatei as a syntrophic partner. The numbers of colony-forming units obtained after incubation for more than 2 months were in the same range as those of total bacterial counts determined by DAPI staining (up to 10(8) cells per millilitre) suggesting that these organisms were dominant members of the community. Identical dilution series in the absence of methanogenic partners yielded numbers that were lower by two to three orders of magnitude. The dominant bacteria were isolated in defined co-culture with M. hungatei, and were further characterized. Growth was slow, with doubling times of 22-28 h at 28 degrees C. Cells were small, 0.5 x 5 microm in size, Gram-positive, and formed terminal oval spores. At 20 degrees C, glucose was fermented by the co-culture strain BoGlc83 nearly stoichiometrically to 2 mol of acetate and 1 mol of methane plus CO(2). At higher temperatures, also lactate and traces of succinate were formed. Anaerobic growth depended strictly on the presence of a hydrogen-scavenging partner organism and was inhibited by bromoethane sulfonate, which together indicate the need for a syntrophic partnership for this process. Strain BoGlc83 grew also aerobically in the absence of a partner organism. All enzymes involved in ATP formation via glycolysis and acetyl CoA were found, most of them at activities equivalent to the physiological substrate turnover rate. This new type of sugar-fermenting bacterium appears be the predominant sugar utilizer in this environment. The results show that syntrophic relationships can play an important role also for the utilization of substrates which otherwise can be degraded in pure culture.

Bacillus stamsii sp. nov., a facultatively anaerobic sugar degrader that is numerically dominant in freshwater lake sediment

“…This bacterium depended on cooperation with a partner organism; inhibition of the methanogenic partner by bromoethane sulfonate prevented growth and substrate utilization completely. Phylogenetic analysis indicated that this new isolate is related to aerobic sporeformers of the genus Bacillus, most closely related to Bacillus jeotgali [18]. Growth tests indicated that our isolate strain BoGlc83 could also grow aerobically, however, aerobic growth was found only in complex growth media and was not easy to reproduce.…”

“…In a study on anaerobic saccharolytic bacteria in the profundal sediment of Lake Constance, Germany, we isolated a slow-growing, spore-forming bacterium which grew with glucose, fructose, and few other sugars only in syntrophic association with hydrogenor formate-oxidizing methanogenic partner organisms [18]. This bacterium depended on cooperation with a partner organism; inhibition of the methanogenic partner by bromoethane sulfonate prevented growth and substrate utilization completely.…”

“…These stock solutions were made anoxic by repeatedly stirring under vacuum and gassing with 100% nitrogen as reported before [18].…”

“…Growth under anoxic, reducing conditions was tested in oxygenfree freshwater medium buffered with 30 mM sodium bicarbonate plus 1 mM sodium sulfide as reducing agent as described before [18,51,52]. Cultivation was done in 15-ml or 25-ml glass tubes sealed with butyl rubber stoppers that were rendered anoxic by flushing the headspace with a N 2 /CO 2 mixture (80%/20%).…”

“…The basal medium was autoclaved at 121 • C and 1 bar overpressure for 20 min. After cooling to room temperature, the following supplements were added to the medium from concentrated sterile stock solutions: CaCl 2 ·2H 2 O (1 mM), K-Na-phosphate buffer, pH 7.0 (1 mM), 7-vitamin-solution (1×, after Widdel and Pfennig [53]), trace element solution SL13 (1×, after Widdel et al [51] and Müller et al [18]). Thiosulfate was added as sulfur source at concentrations between 2 and 10 mM.…”