Microbial carbon cycling in oligotrophic regional aquifers near the Tono Uranium Mine, Japan as inferred from δ13C and Δ14C values of in situ phospholipid fatty acids and carbon sources

Microorganisms are ubiquitous in deep subsurface environments, but their role in the global carbon cycle is not well-understood. The natural abundance delta C-13 and Delta C-14 values of microbial membrane phospholipid fatty acids (PLFAs) were measured and used to assess the carbon sources of bacteria in sedimentary and granitic groundwaters sampled from three boreholes in the vicinity of the Tono Uranium Mine, Gifu, Japan. Sample storage experiments were performed and drill waters analyzed to characterize potential sources of microbial contamination. The most abundant PLFA structures in all waters sampled were 16:0,16:1 omega 7c, cy17:0, and 18:1 omega 7c. A PLFA biomarker for type 11 methanotrophs, 18:1 omega 8c, comprised 3% and 18% of total PLFAs in anoxic sedimentary and granitic waters, respectively, sampled from the KNA-6 borehole. The presence of this biomarker was unexpected given that type II methanotrophs are considered obligate aerobes. However, a bacterium that grows aerobically with CH4 as the sole energy source and which also produces 56% of its total PLFAs as 18:1 omega 8c was isolated from both waters, providing additional evidence for the presence of type II methanotrophs. The Delta C-14 values determined for type II methanotroph PLFAs in the sedimentary (-86 parts per thousand) and granite (-867 parts per thousand) waters were very similar to the Delta C-14 values of dissolved inorganic carbon (DIC) in each water (similar to-850 parts per thousand). This suggests that type II methanotrophs ultimately derive all their carbon from inorganic sources, whether directly from DIC and/or from CH4 produced by the reduction of DIC. In contrast, delta C-13 values of type II PLFAs in the sedimentary (-93 parts per thousand) and granite (-60 parts per thousand) waters indicate that these organisms use different carbon assimilation schemes in each environment despite very similar delta C-13(CH4) values (-similar to 95 parts per thousand) for each water. The delta C-13(PLFA) values (-28 parts per thousand to -45 parts per thousand) of non-methanotrophic bacteria in the KNA-6 LTL water do not clearly distinguish between heterotrophic and autotrophic metabolisms, but Delta C-14(PLFA) values indicate that >65% of total bacteria filtered from the KNA-6 LTL water are heterotrophs. Ancient Delta C-14 values (similar to-1000 parts per thousand) of some PLFAs suggest that many heterotrophs utilize ancient organic matter, perhaps from lignite seams within the sedimentary rocks. The more negative range of delta C-13(PLFA) values determined for the KNA-6 granitic water (-42 parts per thousand to -66 parts per thousand) are likely the result of a microbial ecosystem dominated by chemolithoautotrophy, perhaps fuelled by abiogenic H-2. Results of sample storage experiments showed substantial shifts in microbial community composition and delta C-13(PLFA) values (as much as 5 parts per thousand) during 2-4 days of dark, refrigerated, aseptic storage. However, water samples collected and immediately filtered back in the lab from freshly drilled MSB-2 borehole appeared to maintain the same relative relationships between delta C-13(PLFA) values for sedimentary and granitic host rocks as observed for samples directly filtered under artesian flow from the KNA-6 borehole of the Tono Uranium Mine. (C) 2010 Elsevier Ltd. All rights reserved.