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Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways was observed but with a predominance of aerobic dioxygenases. The putative low-molecular-weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study—subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes—offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon.

 

Abstract. The kinetics of urea hydrolysis (ureolysis) and induced calcium carbonate(CaCO3) precipitation for engineering use in the subsurface wasinvestigated under aerobic conditions using Sporosarcina pasteurii(ATCC strain 11859) as well as Bacillus sphaericus strains 21776and 21787. All bacterial strains showed ureolytic activity inducingCaCO3 precipitation aerobically. Rate constants not normalized tobiomass demonstrated slightly higher-rate coefficients for both ureolysis(kurea) and CaCO3 precipitation (kprecip)for B. sphaericus 21776 (kurea=0.10±0.03 h−1, kprecip=0.60±0.34 h−1) compared toS. pasteurii (kurea=0.07±0.02 h−1,kprecip=0.25±0.02 h−1), though these differences werenot statistically significantly different. B. sphaericus 21787showed little ureolytic activity but was still capable of inducing someCaCO3 precipitation. Cell growth appeared to be inhibited duringthe period of CaCO3 precipitation. Transmission electron microscopy (TEM) images suggest this is dueto the encasement of cells and was reflected in lower kureavalues observed in the presence of dissolved Ca. However, biomass regrowthcould be observed after CaCO3 precipitation ceased, which suggeststhat ureolysis-induced CaCO3 precipitation is not necessarilylethal for the entire population. The kinetics of ureolysis andCaCO3 precipitation with S. pasteurii was furtheranalyzed under anaerobic conditions. Rate coefficients obtained in anaerobicenvironments were comparable to those under aerobic conditions; however, nocell growth was observed under anaerobic conditions with NO3-,SO42- or Fe3+ as potential terminal electronacceptors. These data suggest that the initial rates of ureolysis andureolysis-induced CaCO3 precipitation are not significantlyaffected by the absence of oxygen but that long-term ureolytic activity mightrequire the addition of suitable electron acceptors. Variations in theureolytic capabilities and associated rates of CaCO3 precipitationbetween strains must be fully considered in subsurface engineering strategiesthat utilize microbial amendments.