An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers

Abstract

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H₂ Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH₄ to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic β-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H₂ oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.

Keywords: active subsurface environment; inverted biomass pyramid; metabolic interactions; sulfur-driven autotrophic denitrifiers; syntrophy.

Fig. 1.

Metabolic pathways expressed in the deep subsurface. Pathways of N, S, and C metabolisms are enclosed in blue, yellow, and brown envelopes, respectively. Black and gray solid lines indicate reactions mediated by enzymes, whereas black dotted lines in the N cycle indicate abiotic reactions. PEG transcripts and the encoded enzymes detected in this study are denoted by ellipses, appended with coverage (mapped read counted normalized to PEG transcript length) and, if detected, the number of peptide spectral matches in metaproteome. Green-outlined enzymes are encoded by PEG transcripts related to Sulfuritalea and Thauera. Enzymes in gray operate in both autotrophic and heterotrophic C metabolisms, and the PEG transcripts detected in this study were closely related to those of autotrophs and heterotrophs. NifH gene, encoding for dinitrogenase reductase subunit H, was detected by RT-PCR cloning (SI Appendix, SI Materials and Methods, Table S4). A complete list of proteins detected in the PEG transcriptome and metaproteome is provided in SI Appendix, Table S6. ANAMMOX, anaerobic ammonium oxidation; ANME, anaerobic methane oxidation (upward direction); CBB, Calvin–Benson–Bassham cycle or reductive pentose phosphate cycle; DNRA, dissimilatory nitrate reduction to ammonia; EMP, Embden–Meyerhof–Parnas glycolysis (downward direction); GLUCO, gluconeogenesis (upward direction); 3-HP/4-HB, 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle; MEGEN, methanogenesis (downward direction); nd, not detected; 3PG, 3-phosphoglycerate; rTCA, reverse tricarboxylic acid cycle; SERINE, formaldehyde assimilation via serine pathway; WL, Wood–Ljundahl or reductive acetyl-CoA pathway.

Fig. 2.

Metabolic landscape in the deep subsurface. Bubbles represent the gas in the fracture head space, with font size representing the relative abundance of the species. Radiolytic species are originated from the rock. Blue, yellow, and brown arrows, respectively, represent the N, S, and C metabolic pathways detected, with the large arrows representing the principal pathways and the smaller arrows representing the less expressed. CBB stands for Calvin–Benson–Bassham cycle, the dominant CO₂ fixation pathway. Circles are numbered to show various microbial taxa and are colored based on their phylogenetic origins: 1 for Sulfuritalea- and Sulfuricella-like taxa, 2 for Thiobacillus- and Thauera-like taxa (class β-proteobacteria); 3 for Rhodospirillaceae (class α-proteobacteria); 4 for Ectothiorhodospiraceae, 5 for Methylococcaceae (class γ-proteobacteria); 6 for Brocadiaceae (phylum Planctomycetes); 7 for Desulfobulbaceae and Desulfovibrionaceae (class δ-proteobacteria); 8 for Bacillaceae, 9 for Peptococcaceae including Candidatus Desulforudis (phylum Firmicutes); 10 for ANME, 11 for methanogenic Methanosarcinaceae (phylum Euryarchaeota). Orange bars indicate S–N coupling due to the abundant autotrophic, sulfur-oxidizing, denitrifiers Sulfuritalea, Thauera, Thiobacillus, and Sulfuricella. Isotopic data (SI Appendix, Table S1) are given in rectangles.

Fig. 3.

C1 carbon flow map in the oligotrophic deep subsurface. Microbial groups are represented by rectangles. Rectangle sizes are not scaled to their relative abundances derived from ribosomal proteins in the PEG transcriptome, which are indicated to the top right corners. The color scheme for sulfur oxidizers (SOB), sulfate reducers (SRB), anaerobic methane oxidizers (ANME), and methanogens (Met) is the same as in Fig. 2. Arrows indicate the direction of metabolic transfers between microbial groups and do not imply that direct physical contact is involved. Three electron-shuttling systems occur between the microbial groups, which resulted in three pairs of syntrophic partners. The C in CO₂ is cycled through the syntrophic pair of methanogens and ANME. The CO₂ from anaerobic methane oxidation is being incorporated into the more abundant SRB and SOB. The metabolic versatility of denitrifying SOB to use various electron donors enables them to outnumber their syntrophic partner SRB and become the most dominant chemolithoautotroph in the studied SLiME.