[MIS25-09] 海洋性アナモックス細菌"Ca. Scalindua sp." の酸素同位体分別
キーワード：アナモックス、酸素同位体分別、窒素循環
Natural abundance of stable nitrogen and oxygen isotopes are invaluable biogeochemical tracers for assessing the N transformations in the environment. To fully exploit these tracers, the N and O isotope effects (15ε and 18ε) associated with the respective nitrogen transformation processes must be known. Anaerobic ammonium oxidation (anammox) and denitrification are the two major sinks of fixed nitrogen (N). In addition, anammox bacteria contribute to re-oxidation of nitrite to nitrate, because they fix CO2 into biomass with reducing equivalents generated from oxidation of nitrite to nitrate. Nitrate production by anammox bacteria influences the nitrite and nitrate N and O isotope effects in freshwater and marine systems. Despite the significant importance of anammox bacteria in the global N cycle, the N and O isotope effects of anammox are not well known. Especially, the oxygen isotope effect of anammox metabolism has never been determined yet. This is probably because the O isotope effect of nitrite is affected by three simultaneously occurring reactions; (1) nitrite reduction to N2 gas, (2) nitrite oxidation to nitrate, and (3) incorporation of a water-derived O atom into nitrate during nitrite oxidation to nitrate, and thus is difficult to determine. Furthermore, the δ18ONO2 value is affected by abiotic O isotope exchange between nitrite and water. Here we analyzed the O isotope effects and oxygen atom exchange associated with anammox metabolism by a marine anammox species “Ca. Scalindua sp.”.
The rate of abiotic oxygen atom exchange rate was measured using the medium with different δ18O values (δ18OH2O= -12.3, 27.1, 60.0, and 114.3‰) at a temperature of 30°C and pH=7.5 which was same as the experimental condition of anammox batch incubation. The approach of δ18ONO2 to isotope equilibrium (δ18ONO2, eq) is modeled with the following formula: δ18ONO2 = δ18ONO2,eq + (δ18ONO2,initial - δ18ONO2,eq )*exp(-keq*Time). keq (in units of h-1) represents the rate constant for abiotic equilibration of oxygen atoms (Fig. 1). The model fitting approach of δ18ONO2 at different medium δ18O values yielded a rate constant (keq) of (1.13 ± 0.007) × 10-2 (h-1), and the equilibrium oxygen isotope effect between nitrite and water (18εeq) of 12.95 ± 0.16‰ (tentative result).
To determine oxygen isotope effects of (1) nitrite reduction to N2 gas (18εNO2→N2) and (2) nitrite oxidation to nitrate (18εNO2→NO3), anammox batch experiments were conducted using the medium with different δ18O values (δ18OH2O= -12.3, 27.1, 60.0, and 114.3‰) in triplicate. In these batch experiments, we used highly enriched (Percoll-separated) cultures (>99.9%) of “Ca. Scalindua sp.”. The stoichiometric ratios of consumed nitrite and consumed ammonium (ΔNO2-/ΔNH4+, average 1.35) and produced nitrate and consumed ammonium (ΔNO3-/ΔNH4+, average 0.29) agreed with previously observed stoichiometry of anammox process (Fig. 2). During the anammox reaction, δ18O of produced nitrate appeared to depend on the δ18OH2O of medium. This observation suggested that a water-derived O atom was incorporated into nitrate during nitrite oxidation to nitrate. Rapid increase in δ18O of nitrite overtime was observed in high δ18OH2O media as compared to abiotic exchange. A numerical model is currently being developed to estimate the oxygen isotope effect of each reaction (18εNO2→N2 and 18εNO2→NO3). The obtained O isotopic effects of a marine anammox species “Ca. Scalindua sp.” could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO2 - reoxidation (N recycling) in the ocean.
The rate of abiotic oxygen atom exchange rate was measured using the medium with different δ18O values (δ18OH2O= -12.3, 27.1, 60.0, and 114.3‰) at a temperature of 30°C and pH=7.5 which was same as the experimental condition of anammox batch incubation. The approach of δ18ONO2 to isotope equilibrium (δ18ONO2, eq) is modeled with the following formula: δ18ONO2 = δ18ONO2,eq + (δ18ONO2,initial - δ18ONO2,eq )*exp(-keq*Time). keq (in units of h-1) represents the rate constant for abiotic equilibration of oxygen atoms (Fig. 1). The model fitting approach of δ18ONO2 at different medium δ18O values yielded a rate constant (keq) of (1.13 ± 0.007) × 10-2 (h-1), and the equilibrium oxygen isotope effect between nitrite and water (18εeq) of 12.95 ± 0.16‰ (tentative result).
To determine oxygen isotope effects of (1) nitrite reduction to N2 gas (18εNO2→N2) and (2) nitrite oxidation to nitrate (18εNO2→NO3), anammox batch experiments were conducted using the medium with different δ18O values (δ18OH2O= -12.3, 27.1, 60.0, and 114.3‰) in triplicate. In these batch experiments, we used highly enriched (Percoll-separated) cultures (>99.9%) of “Ca. Scalindua sp.”. The stoichiometric ratios of consumed nitrite and consumed ammonium (ΔNO2-/ΔNH4+, average 1.35) and produced nitrate and consumed ammonium (ΔNO3-/ΔNH4+, average 0.29) agreed with previously observed stoichiometry of anammox process (Fig. 2). During the anammox reaction, δ18O of produced nitrate appeared to depend on the δ18OH2O of medium. This observation suggested that a water-derived O atom was incorporated into nitrate during nitrite oxidation to nitrate. Rapid increase in δ18O of nitrite overtime was observed in high δ18OH2O media as compared to abiotic exchange. A numerical model is currently being developed to estimate the oxygen isotope effect of each reaction (18εNO2→N2 and 18εNO2→NO3). The obtained O isotopic effects of a marine anammox species “Ca. Scalindua sp.” could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO2 - reoxidation (N recycling) in the ocean.