Long-term succession in a coal seam microbiome during in situ biostimulation of coalbed-methane generation.

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 13C-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.

High-rate, High Temperature Acetotrophic Methanogenesis Governed by a Three Population Consortium in Anaerobic Bioreactors.

A combination of acetate oxidation and acetoclastic methanogenesis has been previously identified to enable high-rate methanogenesis at high temperatures (55 to 65°C), but this capability had not been linked to any key organisms. This study combined RNA-stable isotope probing on 13C-labelled acetate and 16S amplicon sequencing to identify the active micro-organisms involved in high-rate methanogenesis. Active biomass was harvested from three bench-scale thermophilic bioreactors treating waste activated sludge at 55, 60 and 65°C, and fed with 13-C labelled and 12C-unlabelled acetate. Acetate uptake and cumulative methane production were determined and kinetic parameters were estimated using model-based analysis. Pyrosequencing performed on 13C- enriched samples indicated that organisms accumulating labelled carbon were Coprothermobacter (all temperatures between 55 and 65°C), acetoclastic Methanosarcina (55 to 60°C) and hydrogenotrophic Methanothermobacter (60 to 65°C). The increased relative abundance of Coprothermobacter with increased temperature corresponding with a shift to syntrophic acetate oxidation identified this as a potentially key oxidiser. Methanosarcina likely acts as both a hydrogen utilising and acetoclastic methanogen at 55°C, and is replaced by Methanothermobacter as a hydrogen utiliser at higher temperatures.

Identification of toluene degraders in a methanogenic enrichment culture.

Methanogenic biodegradation involves the cooperative metabolism of syntrophic bacteria that catalyse the initial attack and subsequent degradation of hydrocarbons, and methanogens that convert intermediates such as hydrogen and carbon dioxide, formate, and/or acetate to methane. The identity of syntrophic microbes and the nature of their interactions with other syntrophs and methanogens are not well understood. Furthermore, it is difficult to isolate the organisms responsible for the initial activation and subsequent degradation of hydrocarbon substrates under methanogenic conditions due to the thermodynamic relationships that exist among microbes in methanogenic communities. We used time-resolved RNA stable isotope probing and RT-qPCR to identify the organisms involved in the initial attack on toluene and subsequent degradation reactions in a highly enriched toluene-degrading methanogenic culture. Our results reveal the importance of a Desulfosporosinus sp. in anaerobic toluene activation in the culture. Other organisms that appear to play roles in toluene degradation include Syntrophaceae, Desulfovibrionales and Chloroflexi. The high bacterial diversity observed in this culture and the extensive labelling of different phylogenetic groups over the course of the stable isotope probing experiment highlight the complexity of the relationships that exist in methanogenic ecosystems.

Investigation of the microbial metabolism of carbon dioxide and hydrogen in the kangaroo foregut by stable isotope probing.

Kangaroos ferment forage material in an enlarged forestomach analogous to the rumen, but in contrast to ruminants, they produce little or no methane. The objective of this study was to identify the dominant organisms and pathways involved in hydrogenotrophy in the kangaroo forestomach, with the broader aim of understanding how these processes are able to predominate over methanogenesis. Stable isotope analysis of fermentation end products and RNA stable isotope probing (RNA-SIP) were used to investigate the organisms and biochemical pathways involved in the metabolism of hydrogen and carbon dioxide in the kangaroo forestomach. Our results clearly demonstrate that the activity of bacterial reductive acetogens is a key factor in the reduced methane output of kangaroos. In in vitro fermentations, the microbial community of the kangaroo foregut produced very little methane, but produced a significantly greater proportion of acetate derived from carbon dioxide than the microbial community of the bovine rumen. A bacterial operational taxonomic unit closely related to the known reductive acetogen Blautia coccoides was found to be associated with carbon dioxide and hydrogen metabolism in the kangaroo foregut. Other bacterial taxa including members of the genera Prevotella, Oscillibacter and Streptococcus that have not previously been reported as containing hydrogenotrophic organisms were also significantly associated with metabolism of hydrogen and carbon dioxide in the kangaroo forestomach.

Capillary electrophoresis ribosomal RNA single-stranded conformation polymorphism: a new approach for characterization of low-diversity microbial communities.

Capillary electrophoresis (CE) has been the principle system for nucleic acid analysis since the early 1990s due to its inherent advantages such as fast analysis time, high resolution and efficiency, minimal sample requirement, high detection sensitivity, and automation. In the past few decades, microbial community fingerprinting methods such as terminal restriction fragment length polymorphism and single-stranded conformation polymorphism (SSCP) have migrated to CE to utilize its advantages over conventional slab gel electrophoresis. Recently, a gel-based direct rRNA fingerprint method was demonstrated. Different from other existing microbial community characterization approaches, this novel approach is polymerase chain reaction free and capable of providing information on the relative abundance of rRNA from individual phylotypes in low-diversity samples. As a gel-based method, it has a long analysis time and relatively large reagent and sample requirements. Here, we addressed these limitations by transferring the RNA fingerprint approach to the CE platform. Analysis time significantly improved from 24 h to 60 min, and the use of a fluorescently labeled hybridization probe as the detection strategy decreased the sample requirement by ten-fold. The combination of fast analysis time, low sample requirement, and sensitive fluorescence detection makes CE-RNA-SSCP an appealing new approach for characterizing low-diversity microbial communities.

Direct rRNA fingerprinting, a novel method to profile low diversity microbial communities.

In the past decade, an increasing number of methods in microbial ecology have been developed that address the questions of which microbes exist in the environment, what their roles are and, to some extent, what their abundance is. In the present paper, we propose and describe the proof of principle of a novel method for analysing shifts in microbial community composition that uses small RNA fragments directly derived from 16S rRNA. Community fingerprints are generated on the basis of sequence-dependent conformational differences of rRNA fragments. We applied this method to profile artificial and natural communities and to detect changes in community structure in enrichment cultures. This method constitutes a PCR-free alternative to microbial community characterisation and can provide information on the relative abundance of rRNA from individual phylotypes in low diversity samples.