Variations in microbial carbon sources and cycling in the deep continental subsurface

Deep continental subsurface fracture water systems, ranging from 1.1 to 3.3km below land surface (kmbls), were investigated to characterize the indigenous microorganisms and elucidate microbial carbon sources and their cycling. Analysis of phospholipid fatty acid (PLFA) abundances and direct cell counts detected varying biomass that was not correlated with depth. Compound-specific carbon isotope analyses (δ¹³C and δ¹⁴C) of the phospholipid fatty acids (PLFAs) and carbon substrates combined with genomic analyses did identify, however, distinct carbon sources and cycles between the two depth ranges studied. In the shallower boreholes at circa 1kmbls, isotopic evidence indicated microbial incorporation of biogenic CH₄ by the in situ microbial community. At the shallowest site, 1.05kmbls in Driefontein mine, this process clearly dominated the isotopic signal. At slightly deeper depths, 1.34kmbls in Beatrix mine, the isotopic data indicated the incorporation of both biogenic CH₄ and dissolved inorganic carbon (DIC) derived from CH₄ oxidation. In both of these cases, molecular genetic analysis indicated that methanogenic and methanotrophic organisms together comprised a small component (<5%) of the microbial community. Thus, it appears that a relatively minor component of the prokaryotic community is supporting a much larger overall bacterial community in these samples. In the samples collected from >3kmbls in Tau Tona mine (TT107, TT109 Bh2), the CH₄ had an isotopic signature suggesting a predominantly abiogenic origin with minor inputs from microbial methanogenesis. In these samples, the isotopic enrichments (δ¹³C and δ¹⁴C) of the PLFAs relative to CH₄ were consistent with little incorporation of CH₄ into the biomass. The most ¹³C-enriched PLFAs were observed in TT107 where the dominant CO₂-fixation pathway was the acetyl-CoA pathway by non-acetogenic bacteria. The differences in the δ¹³C of the PLFAs and the DIC and DOC for TT109 Bh2 were ~-24‰ and 0‰, respectively. The dominant CO₂-fixation pathways were 3-HP/4-HB cycle>acetyl-CoA pathway>reductive pentose phosphate cycle.