Isotope Signatures of N2O in a Mixed Microbial Population System: Constraints on N2O Producing Pathways in Wastewater Treatment

We present measurements of site preference (SP) and bulk N-15/N-14 ratios (delta N-15(N2O)bulk) of nitrous oxide (N2O) by quantum cascade laser absorption spectroscopy (QCLAS) as a powerful tool to investigate N2O production pathways in biological wastewater treatment. QCLAS enables high-precision N2O isotopomer analysis in real time. This allowed us to trace short-term fluctuations in SP and delta N-15(N2O)bulk and, hence, microbial transformation pathways during individual batch experiments with activated sludge from a pilot-scale facility treating municipal wastewater. On the basis of previous work with microbial pure cultures, we demonstrate that N2O emitted during ammonia (NH4+) oxidation with a SP of -5.8 to 5.6 parts per thousand derives mostly from nitrite (NO2-) reduction (e.g., nitrifier denitrification), with a minor contribution from hydroxylamine (NH2OH) oxidation at the beginning of the experiments. SP of N2O produced under anoxic conditions was always positive (1.2 to 26.1 parts per thousand), and SP values at the high end of this spectrum (24.9 to 26.1 parts per thousand) are indicative of N2O reductase activity. The measured delta N-15(N2O)bulk at the initiation of the NH4+ oxidation experiments ranged between -42.3 and -57.6 parts per thousand (corresponding to a nitrogen isotope effect Delta delta N-15 = delta N-15(substrate) - delta N-15(bulk) (N2O) of 43.5 to 58.8 parts per thousand), which is considerably higher than under denitrifying conditions (delta N-15(N2O)bulk 2.4 to -17 parts per thousand; Delta delta N-15 = 0.1 to 19.5 parts per thousand). During the course of all NH4+ oxidation and nitrate (NO3-) reduction experiments, delta N-15(N2O)bulk increased significantly) indicating net N-15 enrichment in the dissolved inorganic nitrogen substrates (NH4+, NO3-) and transfer into the N2O pool. The decrease in delta 15N(N2O)(bulk) during NO2- and NH2OH oxidation experiments is best explained by inverse fractionation during the oxidation of NO2- to NO3-.