Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO 2 dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO 2 dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO 2 dark fixation rates, ranging from 0.7 to 1.4 mol liter ؊1 day ؊1 , were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO 2 dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 mol liter ؊1 CO 2 day ؊1 . Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 mol liter ؊1 NO 3 ؊ day ؊1 . Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines.Chemolithoautotrophic bacteria play an important role in biogeochemical cycles of aquatic habitats. Molecular hydrogen and reduced nitrogen (NH 4 ϩ and NO 2 Ϫ ), sulfur (e.g., H 2 S and S 2 O 3 2Ϫ ), metals (e.g., Fe 2ϩ and Mn 2ϩ ) and carbon (e.g., CO and CH 4 ) compounds serve as electron donors for these bacteria (37), whereas oxygen and nitrate mostly serve as electron acceptors. CO 2 dark fixation has been determined in different pelagic redoxclines worldwide. For example, Taylor et al. (42) showed that bacterial chemoautotrophy, fueled by reduced sulfur species, supported an active secondary microbial food web in the redox transition zone of the Cariaco Basin. Depending on the season, dissolved inorganic carbon assimilation (27 to 159 mmol C m Ϫ2 day Ϫ1 ) in this zone was equivalent to 10% to 333% of phytoplankton primary production. Madrid et al. (24) hypothesized that sulfide-oxidizing epsilon symbiont relative clones were responsible for the dark CO 2 fixation. Nitrate, manganese, and iron as potential electron acceptors were available for the epsilon symbiont relatives, but their reduction was not investigated. Jannasch et al. (17) isolated nine chemolithoautotrophic bacterial strains from the anoxic interface of the Black Sea. These isolates were unable to utilize nitrate, manganese,...

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [ 13 C]acetate and H 2 -13 CO 2 , were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [ 13 C]methane was linked to acetoclastic methanogenesis in both the [ 13 C]acetate-and the H 2 -13 CO 2 -amended cultures of coal and timber. H 2 -13 CO 2 was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H 2 conditions found in coal mines.

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Despite the significance of biogenic methane generation in coal beds, there has never been a systematic long-term evaluation of the ecological response to biostimulation for enhanced methanogenesis in situ . Biostimulation tests in a gas-free coal seam were analysed over 1.5 years encompassing methane production, cell abundance, planktonic and surface associated community composition and chemical parameters of the coal formation water. Evidence is presented that sulfate reducing bacteria are energy limited whilst methanogenic archaea are nutrient limited. Methane production was highest in a nutrient amended well after an oxic preincubation phase to enhance coal biofragmentation (calcium peroxide amendment). Compound-specific isotope analyses indicated the predominance of acetoclastic methanogenesis. Acetoclastic methanogenic archaea of the Methanosaeta and Methanosarcina genera increased with methane concentration. Acetate was the main precursor for methanogenesis, however more acetate was consumed than methane produced in an acetate amended well. DNA stable isotope probing showed incorporation of 13 C-labelled acetate into methanogenic archaea, Geobacter species and sulfate reducing bacteria. Community characterisation of coal surfaces confirmed that methanogenic archaea make up a substantial proportion of coal associated biofilm communities. Ultimately, methane production from a gas-free subbituminous coal seam was stimulated despite high concentrations of sulfate and sulfate-reducing bacteria in the coal formation water. These findings provide a new conceptual framework for understanding the coal reservoir biosphere.