Microbial Life in the Deep Subsurface Aquifer Illuminated by Metagenomics

Abstract

To get insights into microbial diversity and biogeochemical processes in the terrestrial deep subsurface aquifer, we sequenced the metagenome of artesian water collected at a 2.8 km deep oil exploration borehole 5P in Western Siberia, Russia. We obtained 71 metagenome-assembled genomes (MAGs), altogether comprising 93% of the metagenome. Methanogenic archaea accounted for about 20% of the community and mostly belonged to hydrogenotrophic Methanobacteriaceae; acetoclastic and methylotrophic lineages were less abundant. ANME archaea were not found. The most numerous bacteria were the Firmicutes, Ignavibacteriae, Deltaproteobacteria, Chloroflexi, and Armatimonadetes. Most of the community was composed of anaerobic heterotrophs. Only six MAGs belonged to sulfate reducers. These MAGs accounted for 5% of the metagenome and were assigned to the Firmicutes, Deltaproteobacteria, Candidatus Kapabacteria, and Nitrospirae. Organotrophic bacteria carrying cytochrome c oxidase genes and presumably capable of aerobic respiration mostly belonged to the Chloroflexi, Ignavibacteriae, and Armatimonadetes. They accounted for 13% of the community. The first complete closed genomes were obtained for members of the Ignavibacteriae SJA-28 lineage and the candidate phylum Kapabacteria. Metabolic reconstruction of the SJA-28 bacterium, designated Candidatus Tepidiaquacella proteinivora, predicted that it is an anaerobe growing on proteinaceous substrates by fermentation or anaerobic respiration. The Ca. Kapabacteria genome contained both the sulfate reduction pathway and cytochrome c oxidase. Presumably, the availability of buried organic matter of Mesozoic marine sediments, long-term recharge of the aquifer with meteoric waters and its spatial heterogeneity provided the conditions for the development of microbial communities, taxonomically and functionally more diverse than those found in oligotrophic underground ecosystems.

Keywords: candidate phylum; deep subsurface; metagenome; microbial diversity; uncultivable bacteria.

FIGURE 1

The relative abundance of taxonomic groups of microorganisms in the metagenome.

FIGURE 2

Phylogenomic placement of the Ch6, Ch128a, and Ch128b genomes in the maximum likelihood concatenated protein phylogeny of the Bacteroidetes/Chlorobi group. The level of support for internal branches was assessed using the Bayesian test in PhyML. The numbers of genomes used for the analysis in the collapsed clusters is shown after the name of the taxon. Taxonomy is shown according to the GTDB.

FIGURE 3

An overview of the metabolism of Ignavibacteriae bacterium Ch128a (Candidatus Tepidiaquacella proteinivora) reconstructed from its genome. Enzyme abbreviations: GK, glukokinase; GPI, glucose-6-phosphate isomerase; PFK, 6-phosphofructokinase; FBA, fructose-bisphosphate aldolase; TIM, triosephosphate isomerase; GPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PYK, pyruvate kinase; PEPS, phosphoenolpyruvate synthase; FBP, fructose-1,6-bisphosphatase; PEPCK, phosphoenolpyruvate carboxykinase; RPE, ribulose-phosphate 3-epimerase; RPI, ribose 5-phosphate isomerase; TKT, transketolase, TAL, transaldolase, PFL, pyruvate formate lyase; POR, pyruvate ferredoxin oxidoreductase; ACS, acetyl-CoA synthetase, Mdh, malate dehydrogenase; Mae, malic enzyme; Fum, fumarate hydratase; SDH, succinate dehydrogenase; SCS, succinyl-CoA ligase; OOR, 2-oxoglutarate ferredoxin oxidoreductase; Icd, isocitrate dehydrogenase; ATR, aminotransferase; IOR, indolepyruvate ferredoxin oxidoreductase; FNOR, ferredoxin-NAD(P) + reductase; PPase, pyrophosphatase; NDH, NADH dehydrogenase (subunits NuoABCDHIJKLMN); Otr, octaheme tetrathionate reductase. Other abbreviations: F-6-P, fructose 6-phosphate; F-1,6-bisP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3 phosphate; 1,3–bis–PG, 1,3–biphosphoglycerate; 3–PG, 3–phosphoglycerate; 2–PG, 2–phosphoglycerate; PEP, phosphoenolpyruvate; Xyl-5P, xylulose 5-phosphate; Er-4-P, erythrose 4-phosphate; Sed–7-P, sedoheptulose–7–phosphate; Pi, phosphate; PPi, pyrophosphate.

FIGURE 4

An ecological model of the deep subsurface aquifer. OR, aerobic respiration; F, fermentation; SR, sulfate reduction; AR, anaerobic respiration (other than SR); M, methanogenesis. The sizes of the circles roughly reflect the relative abundance of the respective metabolic groups in the metagenome.