Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

Abstract

Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H2-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities.

Figure 1

Relative abundance of microbial classes detected in metagenomes from NAPDC, TOLDC, SCADC and eight relevant environments (Supplementary Table S1), based on MG-RAST assignment of unassembled 454 metagenomic reads and expressed as the proportion of total reads in each metagenome.

Figure 2

Enzymes involved in anaerobic hydrocarbon degradation and the corresponding genes detected in the metagenomes of NAPDC, SCADC and TOLDC. (a) Known pathways (substrates) for anaerobic hydrocarbon metabolism and associated enzymes. (b) Heatmap showing the presence of various genes and putative genes involved in anaerobic hydrocarbon metabolism in the metagenomes. The color scale indicates the inferred enzyme completeness, in the case of enzymes with multiple subunits. Enzyme abbreviations: EbdABC, ethylbenzene dehydrogenase; Ped, (S)-1-phenylethanol dehydrogenase; Apc1 to Apc5, acetophenone carboxylase; Bal, benzoylacetate-CoA ligase; BssABC, benzylsuccinate synthase trimer; BbsEF, succinyl-CoA:(R)-benzylsuccinate CoA transferase; BbsG, (R)-benzylsuccinyl-CoA dehydrogenase; BbsH, phenylitaconyl-CoA hydratase; BbsCD, 2-[hydroxyl(phenyl)methyl]- succinyl-CoA dehydrogenase; BbsAB, benzoylsuccinyl-CoA thiolase; AbcDA, anaerobic benzene carboxylase; BzlA/BamY, benzoate-CoA ligase; BcrABCD, benzoyl-CoA reductase (ATP-dependent); BamB to BamI, benzoyl-CoA reductase (ATP-independent); NmsABC, naphthyl-2-methyl-succinate synthase; BnsEF, naphthyl-2-methyl-succinate CoA transferase; BnsG, naphthyl-2-methyl-succinate dehydrogenase; BnsH, naphthyl-2-methylene-succinyl CoA hydratase; BnsCD, naphthyl-2-hydroxymethyl-succinyl CoA dehydrogenase; BnsAB, naphthyl-2-oxomethyl-succinyl CoA thiolase; AncA, anaerobic naphthalene carboxylase; NcrABC, 2-naphthoyl CoA reductase; AssABC, alkylsuccinate synthase; AssK, AMP-dependent synthetase and ligase; Mcm, methylmalonyl CoA mutase; Mcd, methylmalonyl-CoA decarboxylase. Based on the models used, Mcd and benzoyl-CoA thiolase were abundant in all metagenomes. It was not known whether any of the genes identified were associated with hydrocarbon-degrading pathways or more general decarboxylase and thiolase functions, thus they have been omitted from the heatmap to curtail over-interpretation of the data.

Figure 3

Maximum likelihood tree of translated full-length and partial assA, bssA, and nmsA homologs recovered from TOLDC, SCADC and NAPDC metagenomes (bold font). Bootstrap support ⩾60% is indicated. Full-length translated pyruvate formate lyase (PFL) sequences were used as an outgroup (collapsed in figure). Sequence length of genes recovered from TOLDC, SCADC and NAPDC, and their corresponding GenBank accession numbers are indicated in parentheses. A tree with the same overall topology was obtained when including only full-length sequences with gaps (not shown).

Figure 4

Ternary plots showing three-way comparisons of functional categories in SEED subsystems level 3 shared among individual metagenomes or groups of metagenomes. (a) Comparison of individual NAPDC, SCADC and TOLDC metagenomes. (b) Comparison of metagenome groups from hydrocarbon-impacted communities: HC (TOLDC, SCADC and NAPDC); GM (three metagenomes from Gulf of Mexico deep marine sediments); and TP (two metagenomes from oil sands tailings ponds). Each point on the ternary plot represents a subsystem category in the three metagenomes or groups of metagenomes, with the proportion of each SEED being normalized to a value of 1. Data points are colored according to the source of each metagenome or group; gray dots represent functional categories present at low, statistically non-significant (P<0.01) abundance. Points located near the vertices are enriched within the metagenome or group associated with that vertex, points along each axis are shared only between the two vertices associated with that axis (labelled as Functional Groups I, II and III in b), and points located near the center of the plot have similar proportions in all three metagenomes or groups (that is, show no specific enrichment; Hug et al., 2012).

Figure 5

Principal component analysis of NAPDC, SCADC and TOLDC plus 41 additional relevant metagenomes available in MG-RAST (Supplementary Table S1); symbols are colored according to their environment of origin. All counts were normalized against total annotated sequences of each metagenome. Metagenomes from related environments are enclosed with broken lines.