Isotopomeric characterization of nitrous oxide produced by reaction of enzymes extracted from nitrifying and denitrifying bacteria
Nitrous oxide (N 2O) is a potent greenhouse gas and produced in denitrification and nitrification by various microorganisms. Site preference (SP) of 15N in N 2O, which is defined as the difference in the natural abundance of isotopomers 14N 15NO and 15N 14NO relative to 14N 14NO, has been reported to be a useful tool to quantitatively distinguish N 2O production pathways. To determine representative SP values for each microbial process, we firstly measured SP of N 2O produced in the enzyme reaction of hydroxylamine oxidoreductase (HAO) purified from two species of ammonia oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosococcus oceani, and that of nitric oxide reductase (NOR) from Paracoccus denitrificans. The SP value for NOR reaction (−5.9 ± 2.1‰) showed nearly the same value as that reported for N 2O produced by P. denitrificans in pure culture. In contrast, SP value for HAO reaction (36.3 ± 2.3‰) was a little higher than the values reported for N 2O produced by AOB in aerobic pure culture. Using the SP values obtained by HAO and NOR reactions, we calculated relative contribution of the nitrite (NO 2–) reduction (which is followed by NO reduction) to N 2O production by N. oceani incubated under different O 2 availability. Our calculations revealed that previous in vivo studies might have underestimated the SP value for the NH 2OH oxidation pathway possibly due to a small contribution of NO 2– reduction pathway. Further evaluation of isotopomer signatures of N 2O using common enzymes of other processes related to N 2O would improve the isotopomer analysis of N 2O in various environments.