Akinari Hirota

Sensitive determinations of stable nitrogen isotopic composition of organic nitrogen through chemical conversion into N2O

We present a method for high-sensitivity nitrogen isotopic analysis of particulate organic nitrogen (PON) in seawater and freshwater, for the purpose of determining the aquatic nitrogen fixation rate through the 15N2 tracer technique for samples that contain a low abundance of organisms. The method is composed of the traditional oxidation/reduction methods, such as the oxidation of PON to nitrate (NO3*) using persulfate, the reduction of NO3* to nitrite (NO2*) using spongy cadmium, and further reduction of NO2* to nitrous oxide (N2O) using sodium azide. Then, N2O is purged from the water and trapped cryogenically with subsequent release into a gas chromatography column to analyze the stable nitrogen isotopic composition using continuous-flow isotope ratio mass spectrometry (CF-IRMS) by simultaneously monitoring the NO+ ion currents at masses 30, 31, and 32. The nitrogen isotopic fractionation was consistent within each batch of analysis. The standard deviation of sample measurements was less than 0.3 per thousand for samples containing PON of more than 50 nmolN, and 0.5 per thousand for those of more than 20 nmolN, by subtracting the contribution of blank nitrogen, 8 +/- 2 nmol at final N2O. By using this method, we can determine delta15N for lower quantities of PON better than by other methods, so we can reduce the quantities of water samples needed for incubation to determine the nitrogen fixation rate. In addition, we can expand the method to determine the nitrogen isotopic composition of organic nitrogen in general, such as that of total dissolved nitrogen (TDN; sum of NO3*, NO2*, ammonium, and DON), by applying the method to filtrates.

Simultaneous determination of δ15N and δ18O of N2O and δ13C of CH4 in nanomolar quantities from a single water sample

We have developed a rapid, sensitive, and automated analytical system to simultaneously determine the concentrations and stable isotopic compositions (delta(15)N, delta(18)O, and delta(13)C) of nanomolar quantities of nitrous oxide (N(2)O) and methane (CH(4)) in water, by combining continuous-flow isotope-ratio mass spectrometry and a helium-sparging system to extract and purify the dissolved gases. Our system, which is composed of cold traps and a capillary gas chromatograph that use ultra-pure helium as the carrier gas, achieves complete extraction of N(2)O and CH(4) in a water sample and separation among N(2)O, CH(4), and the other component gases. The flow path following exit from the gas chromatograph was periodically changed to pass the gases through the combustion furnace to convert CH(4) and the other hydrocarbons into CO(2), or to bypass the combustion furnace for the direct introduction of eluted N(2)O into the mass spectrometer, for determining the stable isotopic compositions through monitoring the ions of m/z 44, 45, and 46 of CO(2) (+) and N(2)O(+). The analytical system can be operated automatically with sequential software programmed on a personal computer. Analytical precisions better than 0.2 per thousand and 0.3 per thousand and better than 1.4 per thousand and 2.6 per thousand were obtained for the delta(15)N and delta(18)O of N(2)O, respectively, when more than 6.7 nmol and 0.2 nmol of N(2)O, respectively, were injected. Simultaneously, analytical precisions better than 0.07 per thousand and 2.1 per thousand were obtained for the delta(13)C of CH(4) when more than 5.5 nmol and 0.02 nmol of CH(4), respectively, were injected. In this manner, we can simultaneously determine stable isotopic compositions of a 120 mL water sample with concentrations as low as 1.7 nmol/kg for N(2)O and 0.2 nmol/kg for CH(4).

Biogeochemical Signals from Deep Microbial Life in Terrestrial Crust

In contrast to the deep subseafloor biosphere, a volumetrically vast and stable habitat for microbial life in the terrestrial crust remains poorly explored. For the long-term sustainability of a crustal biome, high-energy fluxes derived from hydrothermal circulation and water radiolysis in uranium-enriched rocks are seemingly essential. However, the crustal habitability depending on a low supply of energy is unknown. We present multi-isotopic evidence of microbially mediated sulfate reduction in a granitic aquifer, a representative of the terrestrial crust habitat. Deep meteoric groundwater was collected from underground boreholes drilled into Cretaceous Toki granite (central Japan). A large sulfur isotopic fractionation of 20–60‰ diagnostic to microbial sulfate reduction is associated with the investigated groundwater containing sulfate below 0.2 mM. In contrast, a small carbon isotopic fractionation (<30‰) is not indicative of methanogenesis. Except for 2011, the concentrations of H2 ranged mostly from 1 to 5 nM, which is also consistent with an aquifer where a terminal electron accepting process is dominantly controlled by ongoing sulfate reduction. High isotopic ratios of mantle-derived 3He relative to radiogenic 4He in groundwater and the flux of H2 along adjacent faults suggest that, in addition to low concentrations of organic matter (<70 µM), H2 from deeper sources might partly fuel metabolic activities. Our results demonstrate that the deep biosphere in the terrestrial crust is metabolically active and playing a crucial role in the formation of reducing groundwater even under low-energy fluxes.

Deep microbial life in high-quality granitic groundwater from geochemically and geographically distinct underground boreholes.

Deep granitic aquifer is one of the largest, but least understood, microbial habitats. To avoid contamination from the surface biosphere, underground drilling was conducted for 300 m deep granitic rocks at the Mizunami underground research laboratory (URL), Japan. Slightly alkaline groundwater was characterized by low concentrations of dissolved organic matter and sulfate and the presence of > 100 nM H2 . The initial biomass was the highest (∼10(5)  cells ml(-1) ) with the dominance of Hydrogenophaga spp., whereas the phylum Nitrospirae became predominant after 3 years with decreasing biomass (∼10(4)  cells ml(-1) ). One week incubation of groundwater microbes after 3 years with (13) C-labelled bicarbonate and 1% H2 and subsequent single-cell imaging with nanometer-scale secondary ion mass spectrometry demonstrated that microbial cells were metabolically active. Pyrosequencing of microbial communities in groundwater retrieved at 3-4 years after drilling at the Mizunami URL and at 14 and 25 years after the drilling at the Grimsel Test Site, Switzerland, revealed the occurrence of common Nitrospirae lineages at the geographically distinct sites. As the close relatives of the Nitrospirae lineages were exclusively detected from deep groundwaters and terrestrial hot springs, it suggests that these bacteria are indigenous and potentially adapted to the deep terrestrial subsurface.

Ecological and genomic profiling of anaerobic methane-oxidizing archaea in a deep granitic environment

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 H2 oxidation, which is different from those of other methanotrophic archaea. Short-term incubation of microbial cells collected from the granitic groundwater with 13C-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.