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A microbial community analysis using 16S rRNA gene sequencing is performed on borehole water and granite rock core from Henderson Mine near Empire, Colorado. The studies have shown that geochemical variance has a key impact on microbial community structure in deep, subsurface systems.

Abstract: In order to determine if the δ 15N and δ 18O values of N2O produced during co-oxidation of NH4 + by methanotrophic (methane oxidizing) bacteria can be isotopically distinguished from N2O produced either by autotrophic nitrifying or denitrifying bacteria, we conducted laboratory incubation experiments with pure cultures of methanotrophic bacteria that were provided NH4Cl as an oxidation substrate. The N2O produced during NH4 + oxidation by methanotrophic bacteria showed nitrogen isotope fractionation between NH4 + and N2O (ε N2O–NH4 + ) of −48 and −55‰ for Methylomonas methanica and Methylosinus trichosporium, OB3b respectively. These large fractionations are similar to those previously measured for autotrophic nitrifying bacteria and consistent with N2O formation by multiple rate limiting steps that include NH4 +oxidation by the methane monooxygenase enzyme and reduction of NO2 − to N2O. Consequently, N2O formed by NH4 + oxidation via methanotrophic or autotrophic nitrifying bacteria might generally be characterized by lower δ 15NN2O values than that formed by denitrificaiton, although this also depends on the variability of δ 15N of available nitrogen sources (e.g., NH4 +, NO3 −, NO2 −). Additional incubations with M. trichosporium OB3b at high and low CH4 conditions in waters of different δ 18O values revealed that 19–27% of the oxygen in N2O was derived from O2 with the remainder from water. The biochemical mechanisms that could explain this amount of O2 incorporation are discussed. The δ 18O of N2O formed under high CH4 conditions was ~+15‰ more positive than that formed under lower CH4 conditions. This enrichment resulted in part from the incorporation of O2 into N2O that was enriched in 18O due to an isotope fractionation effect of −16.1±2.0‰ and −17.5±5.4‰ associated with O2 consumption during the high and low methane concentration incubations, respectively. Therefore, N2O formed by NH4 + oxidation via methanotrophic or autotrophic nitrifying bacteria can have very positive δ 18ON2O values if the O2 incorporated is previously enriched in 18O from high rates of respiration. Nitrous oxide was collected from various depths in soils overlying a coal-bed methane seep where methanotrophic bacteria are naturally enriched. In one sampling when soil methane concentrations were very high, the δ 18OVSMOW values of the N2O were highly enriched (+50‰), consistent with our laboratory experiments. Thus, soils overlying methane seeps could provide an 18O-enriched source of atmospheric N2O. [Copyright &y& Elsevier]

Emissions of N2O from cover soils of both abandoned (> 30 years) and active landfills greatly exceed the maximum fluxes previously reported for tropical soils, suggesting high microbial activities for N2O production. Low soil matrix potentials (< -0.7 MPa) indicate that nitrification was the most likely mechanism of N2O formation during most of the time of sampling. Soil moisture had a strong influence on N2O emissions. The production of N2O was stimulated by as much as 20 times during laboratory incubations, when moisture was increased from -2.0 MPa to -0.6 MPa. Additional evidence from incubation experiments and δ13C analyses of fatty acids (18:1) diagnostic of methanotrophs suggests that N2O is formed in these soils by nitrification via methanotrophic bacteria. In a NH3(g)-amended landfill soil, the rate of N2O production was significantly increased when incubated with 100 ppmv methane compared with 1.8 ppmv (atmospheric) methane. Preincubation of a landfill soil with 1% CH4 for 2 weeks resulted in higher rates of N2O production when subsequently amended with NH3(g) relative to a control soil preincubated without CH4. At one location, at the soil depth (9–16 cm) of maximum methane consumption and N2O production, we observe elevated concentrations of organic carbon and nitrogen and distinct minima in δ15N (+1.0‰) and δ13C (-33.8‰) values for organic nitrogen and organic carbon respectively. A δ13C value of -39.3‰ was measured for 18:1 carbon fatty acids in this soil, diagnostic of type II methanotrophs. The low δ15N value for organic nitrogen is consistent with N2 fixation by type II methanotrophs. These observations all point to a methanotrophic origin for the organic matter at this depth. The... [ABSTRACT FROM AUTHOR]

Abstract: Microorganisms are ubiquitous in deep subsurface environments, but their role in the global carbon cycle is not well-understood. The natural abundance δ13C and Δ14C 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, 16:1ω7c, cy17:0, and 18:1ω7c. A PLFA biomarker for type II methanotrophs, 18:1ω8c, 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 18:1ω8c was isolated from both waters, providing additional evidence for the presence of type II methanotrophs. The Δ14C values determined for type II methanotroph PLFAs in the sedimentary (−861‰) and granite (−867‰) waters were very similar to the Δ14C values of dissolved inorganic carbon (DIC) in each water (∼−850‰). 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, δ13C values of type II PLFAs in the sedimentary (−93‰) and granite (−60‰) waters indicate that these organisms use different carbon assimilation schemes in each environment despite very similar values (∼−95‰) for each water. The δ13CPLFA values (−28‰ to −45‰) of non-methanotrophic bacteria in the KNA-6 LTL water do not clearly distinguish between heterotrophic and autotrophic metabolisms, but Δ14CPLFA values indicate that >65% of total bacteria filtered from the KNA-6 LTL water are heterotrophs. Ancient Δ14C values (∼−1000‰) of some PLFAs suggest that many heterotrophs utilize ancient organic matter, perhaps from lignite seams within the sedimentary rocks. The more negative range of δ13CPLFA values determined for the KNA-6 granitic water (−42‰ to −66‰) are likely the result of a microbial ecosystem dominated by chemolithoautotrophy, perhaps fuelled by abiogenic H2. Results of sample storage experiments showed substantial shifts in microbial community composition and δ13CPLFA values (as much as 5‰) 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 δ13CPLFA 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. [Copyright &y& Elsevier]