We have developed an analytical system to determine stable isotopic compositions (delta13C and delta18O) of sub-microgram quantities of CaCO3 for the purpose of analyzing individual foraminiferal shells, using continuous-flow isotope ratio mass spectrometry (CF-IRMS). The system consists of a micro-volume CaCO3 decomposition tube, stainless steel CO2 purification vacuum line with a quantity-regulating unit, helium-purged CO2 purification line, gas chromatograph, and a CF-IRMS system. By using this system, we can determine stable carbon and oxygen isotopic compositions as low as 0.2 microg of CaCO3, with standard deviations of +/-0.10 per thousand for delta13C and +/-0.18 per thousand for delta18O within a 4-h reaction time and 30-min analysis period.

Recent single-gene-based surveys of deep continental aquifers demonstrated the widespread occurrence of archaea related to Candidatus Methanoperedens nitroreducens (ANME-2d) known to mediate anaerobic oxidation of methane (AOM). However, it is unclear whether ANME-2d mediates AOM in the deep continental biosphere. In this study, we found the dominance of ANME-2d in groundwater enriched in sulfate and methane from a 300-m deep underground borehole in granitic rock. A near-complete genome of one representative species of the ANME-2d obtained from the underground borehole has most of functional genes required for AOM and assimilatory sulfate reduction. The genome of the subsurface ANME-2d is different from those of other members of ANME-2d by lacking functional genes encoding nitrate and nitrite reductases and multiheme cytochromes. In addition, the subsurface ANME-2d genome contains a membrane-bound NiFe hydrogenase gene putatively involved in respiratory H oxidation, which is different from those of other methanotrophic archaea. Short-term incubation of microbial cells collected from the granitic groundwater with C-labeled methane also demonstrates that AOM is linked to microbial sulfate reduction. Given the prominence of granitic continental crust and sulfate and methane in terrestrial subsurface fluids, we conclude that AOM may be widespread in the deep continental biosphere.

To detect the relationship between ambient temperature and otolith stable oxygen isotope (δ O, −0.17 ‰) were lower, but were not significantly different from those of larvae reared in + 0.31 ‰ water, which is the δ 18 O value of the water in which the spawners were raised. These results suggest that the sample size for the 32 psu experiment was too small and that the rearing durations were too short to affect the otoliths completely. Our results demonstrate that otolith δ

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