Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane

Abstract

Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.

Fig. 1. Conceptual representation of BONCAT-FACS experimental setup and metagenomic sequencing workflow.

We performed down-well incubation of sterile, crushed coal in the SES allowing for microbial colonization and retrieval under in situ pressure and anaerobic conditions. Samples were allocated into sterile gassed out serum bottles for addition of the bioorthogonal amino acid (HPG) in triplicate 24 hr incubations. We then sorted click-labeled BONCAT active cells (FAM Picolyl dye; Ex: 488 nm/Em: 530 nm) and total cells (SYTO59; Ex: 640 nm/Em: 655–685 nm) from each biological replicate. This was followed by DNA extraction, MDA amplification, sequencing, and analysis. The upper left coal seam stratigraphy panel was modified from Barnhart et al., 2016 [6].

Fig. 2. Phylogenetic positions of M. paradoxum PRB (A) and Chlorobi PRB (B).

Bayesian trees were constructed from concatenated alignments of 16 ribosomal proteins. Posterior probabilities (between 0.00 and 1.00) are displayed at branch nodes. The tree scale represents the average number of substitutions per site.

Fig. 3. Metabolic reconstruction of Chlorobi PRB compared to Chlorobi NICIL-2.

This figure is modified from a previous version published by Hiras and colleagues [41] for comparison with Chlorobi PRB. Colored enzyme numbers indicate which genome contained the respective gene (teal = Chlorobi PRB and NICIL-2; purple = Chlorobi PRB only; orange = NICIL-2 only; red = neither). Enzymes are numbered and are defined in Supplementary Table 3. [xyl xylose, xylu xyluose, ribu ribulose, rib ribose, glu glucose, pyru pyruvate; standard three-letter abbreviations are used for amino acids].

Fig. 4. Biogenic CBM production in the PRB, from aromatic hydrocarbon degradation to acetoclastic methanogenesis.

Translationally active MAGs in the PRB harbor the putative ability to degrade a variety of aromatics, including ethylbenzene, phenylethanol, phenol, phenylphosphate, toluene, xylene, and phenylacetate. Arrows representing genes or deduced enzymes are colored by the host microbial population (orange = Chlorobi PRB, purple = Geobacter PRB, blue = Bacteroidetes PRB, teal = M. paradoxum PRB) and indicate that either carbon or energy may be derived from the putative reaction. Dashed lines indicate putative oxygen-consuming reactions. Detailed metabolic potential of the Chlorobi PRB MAG is presented in Fig. 3. [ebd = ethylbenzene dehydrogenase, ped = phenylethanol dehydrogenase, pps = phenylphosphate synthase, ppc = phenylphosphate carboxylase, bss = putative benzylsuccinate synthase, ack = acetate kinase, pta = phosphotransacetylase, acs = acetyl CoA synthase, cdh = carbon monoxide dehydrogenase, mtr = methyltransferase, mcr = methyl CoM reductase, elh = extradiol dioxygenase: LigB superfamily: homoprotocatechuate].