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 (15)N/(14)N ratios (δ(15)N(bulk)(N2O)) of nitrous oxide (N(2)O) by quantum cascade laser absorption spectroscopy (QCLAS) as a powerful tool to investigate N(2)O production pathways in biological wastewater treatment. QCLAS enables high-precision N(2)O isotopomer analysis in real time. This allowed us to trace short-term fluctuations in SP and δ(15)N(bulk)(N2O) 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 N(2)O emitted during ammonia (NH(4)(+)) oxidation with a SP of -5.8 to 5.6 ‰ derives mostly from nitrite (NO(2)(-)) reduction (e.g., nitrifier denitrification), with a minor contribution from hydroxylamine (NH(2)OH) oxidation at the beginning of the experiments. SP of N(2)O produced under anoxic conditions was always positive (1.2 to 26.1 ‰), and SP values at the high end of this spectrum (24.9 to 26.1 ‰) are indicative of N(2)O reductase activity. The measured δ(15)N(bulk)(N2O) at the initiation of the NH(4)(+) oxidation experiments ranged between -42.3 and -57.6 ‰ (corresponding to a nitrogen isotope effect Δδ(15)N = δ(15)N(substrate) - δ(15)N(bulk)(N2O) of 43.5 to 58.8 ‰), which is considerably higher than under denitrifying conditions (δ(15)N(bulk)(N2O) 2.4 to -17 ‰; Δδ(15)N = 0.1 to 19.5 ‰). During the course of all NH(4)(+) oxidation and nitrate (NO(3)(-)) reduction experiments, δ(15)N(bulk)(N2O) increased significantly, indicating net (15)N enrichment in the dissolved inorganic nitrogen substrates (NH(4)(+), NO(3)(-)) and transfer into the N(2)O pool. The decrease in δ(15)N(bulk)(N2O) during NO(2)(-) and NH(2)OH oxidation experiments is best explained by inverse fractionation during the oxidation of NO(2)(-) to NO(3)(-).

N2O emissions: modeling the effect of process configuration and diurnal loading patterns.

The objective of this research was to develop a mechanistic model for quantifying N2O emissions from activated sludge plants and demonstrate how this may be used to evaluate the effects of process configuration and diurnal loading patterns. The model describes the mechanistic link between the factors recognized to correlate positively with N2O emissions. The primary factors are the presence of ammonia and nitrite accumulation. Low dissolved oxygen concentrations also may be implicated through differential impacts on ammonia-oxidizing bacteria (AOB) versus nitrite-oxidizing bacteria (NOB) activity. Factors promoting N2O emissions at treatment plants are discussed below. The model was applied to data from laboratory and pilot-scale systems. From a practical standpoint, plant configuration (e.g., plug-flow versus complete-mix), influent loading patterns (and peak load), and certain operating strategies (e.g., handling of return streams) are all important in determining N2O emissions.

N2O emission in full-scale wastewater treatment: Proposing a refined monitoring strategy.

Nitrous oxide (N2O) emissions from wastewater treatment contribute significantly to greenhouse gas emissions. They have been shown to exhibit a strong seasonal and daily profile in previously conducted monitoring campaigns. However, only two year-long online monitoring campaigns have been published to date. Based on three monitoring campaigns on three full-scale wastewater treatment plants (WWTPs) with different activated sludge configurations, each of which lasted at least one year, we propose a refined monitoring strategy for long-term emission monitoring with multiple flux chambers on open tanks. Our monitoring campaigns confirm that the N2O emissions exhibited a strong seasonal profile and were substantial on all three plants (1-2.4% of the total nitrogen load). These results confirm that N2O is the most important greenhouse gas emission from wastewater treatment. The temporal variation was more distinct than the spatial variation within aeration tanks. Nevertheless, multiple monitoring spots along a single lane are crucial to assess representative emission factors in flow-through systems. Sequencing batch reactor systems were shown to exhibit comparable emissions within one reactor but significant variation between parallel reactors. The results indicate that considerable emission differences between lanes are to be expected in cases of inhomogeneous loading and discontinuous feeding. For example, N2O emission could be shown to depend on the amount of treated reject water: lanes without emitted <1% of the influent load, while parallel lanes emitted around 3%. In case of inhomogeneous loading, monitoring of multiple lanes is required. Our study enables robust planning of monitoring campaigns on WWTPs with open tanks. Extensive full-scale emission monitoring campaigns are important as a basis for reliable decisions about reducing the climate impact of wastewater treatment. More specifically, such data sets help us to define general emission factors for wastewater treatment plants and to construct and critically evaluate N2O emission models.

Isotopic evidence for nitrous oxide production pathways in a partial nitritation-anammox reactor.

Nitrous oxide (N2O) production pathways in a single stage, continuously fed partial nitritation-anammox reactor were investigated using online isotopic analysis of offgas N2O with quantum cascade laser absorption spectroscopy (QCLAS). N2O emissions increased when reactor operating conditions were not optimal, for example, high dissolved oxygen concentration. SP measurements indicated that the increase in N2O was due to enhanced nitrifier denitrification, generally related to nitrite build-up in the reactor. The results of this study confirm that process control via online N2O monitoring is an ideal method to detect imbalances in reactor operation and regulate aeration, to ensure optimal reactor conditions and minimise N2O emissions. Under normal operating conditions, the N2O isotopic site preference (SP) was much higher than expected - up to 40‰ - which could not be explained within the current understanding of N2O production pathways. Various targeted experiments were conducted to investigate the characteristics of N2O formation in the reactor. The high SP measurements during both normal operating and experimental conditions could potentially be explained by a number of hypotheses: i) unexpectedly strong heterotrophic N2O reduction, ii) unknown inorganic or anammox-associated N2O production pathway, iii) previous underestimation of SP fractionation during N2O production from NH2OH, or strong variations in SP from this pathway depending on reactor conditions. The second hypothesis - an unknown or incompletely characterised production pathway - was most consistent with results, however the other possibilities cannot be discounted. Further experiments are needed to distinguish between these hypotheses and fully resolve N2O production pathways in PN-anammox systems.

Mechanisms of N2O production in biological wastewater treatment under nitrifying and denitrifying conditions.

Nitrous oxide (N2O) is an important greenhouse gas and a major sink for stratospheric ozone. In biological wastewater treatment, microbial processes such as autotrophic nitrification and heterotrophic denitrification have been identified as major sources; however, the underlying pathways remain unclear. In this study, the mechanisms of N2O production were investigated in a laboratory batch-scale system with activated sludge for treating municipal wastewater. This relatively complex mixed population system is well representative for full-scale activated sludge treatment under nitrifying and denitrifying conditions. Under aerobic conditions, the addition of nitrite resulted in strongly nitrite-dependent N2O production, mainly by nitrifier denitrification of ammonia-oxidizing bacteria (AOB). Furthermore, N2O is produced via hydroxylamine oxidation, as has been shown by the addition of hydroxylamine. In both sets of experiments, N2O production was highest at the beginning of the experiment, then decreased continuously and ceased when the substrate (nitrite, hydroxylamine) had been completely consumed. In ammonia oxidation experiments, N2O peaked at the beginning of the experiment when the nitrite concentration was lowest. This indicates that N2O production via hydroxylamine oxidation is favored at high ammonia and low nitrite concentrations, and in combination with a high metabolic activity of ammonia-oxidizing bacteria (at 2 to 3 mgO2/l); the contribution of nitrifier denitrification by AOB increased at higher nitrite and lower ammonia concentrations towards the end of the experiment. Under anoxic conditions, nitrate reducing experiments confirmed that N2O emission is low under optimal growth conditions for heterotrophic denitrifiers (e.g. no oxygen input and no limitation of readily biodegradable organic carbon). However, N2O and nitric oxide (NO) production rates increased significantly in the presence of nitrite or low dissolved oxygen concentrations.

Nitric oxide and nitrous oxide turnover in natural and engineered microbial communities: biological pathways, chemical reactions, and novel technologies.

Nitrous oxide (N(2)O) is an environmentally important atmospheric trace gas because it is an effective greenhouse gas and it leads to ozone depletion through photo-chemical nitric oxide (NO) production in the stratosphere. Mitigating its steady increase in atmospheric concentration requires an understanding of the mechanisms that lead to its formation in natural and engineered microbial communities. N(2)O is formed biologically from the oxidation of hydroxylamine (NH(2)OH) or the reduction of nitrite (NO(-) (2)) to NO and further to N(2)O. Our review of the biological pathways for N(2)O production shows that apparently all organisms and pathways known to be involved in the catabolic branch of microbial N-cycle have the potential to catalyze the reduction of NO(-) (2) to NO and the further reduction of NO to N(2)O, while N(2)O formation from NH(2)OH is only performed by ammonia oxidizing bacteria (AOB). In addition to biological pathways, we review important chemical reactions that can lead to NO and N(2)O formation due to the reactivity of NO(-) (2), NH(2)OH, and nitroxyl (HNO). Moreover, biological N(2)O formation is highly dynamic in response to N-imbalance imposed on a system. Thus, understanding NO formation and capturing the dynamics of NO and N(2)O build-up are key to understand mechanisms of N(2)O release. Here, we discuss novel technologies that allow experiments on NO and N(2)O formation at high temporal resolution, namely NO and N(2)O microelectrodes and the dynamic analysis of the isotopic signature of N(2)O with quantum cascade laser absorption spectroscopy (QCLAS). In addition, we introduce other techniques that use the isotopic composition of N(2)O to distinguish production pathways and findings that were made with emerging molecular techniques in complex environments. Finally, we discuss how a combination of the presented tools might help to address important open questions on pathways and controls of nitrogen flow through complex microbial communities that eventually lead to N(2)O build-up.

Nitrous oxide emissions from the oxidation tank of a pilot activated sludge plant.

This study discusses the results of the continuous monitoring of nitrous oxide emissions from the oxidation tank of a pilot conventional wastewater treatment plant. Nitrous oxide emissions from biological processes for nitrogen removal in wastewater treatment plants have drawn great attention over the last years, due to the high greenhouse effect. However, even if several studies have been carried out to quantify nitrous oxide emission rates from different types of treatment, quite wide ranges have been reported. Only grab samples or continuous measurements over limited periods were considered in previous studies, which can account for the wide variability of the obtained results. Through continuous monitoring over several months, our work tries to fill this gap of knowledge and get a deeper insight into nitrous oxide daily and weekly emission dynamics. Moreover, the influence of some operating conditions (sludge age, dissolved oxygen concentration in the oxidation tank, nitrogen load) was studied to determine good practices for wastewater treatment plant operation aiming at the reduction of nitrous oxide emissions. The dissolved oxygen set-point is shown to play a major role in nitrous oxide emissions. Low sludge ages and high nitrogen loads are responsible for higher emissions as well. An interesting pattern has been observed, with quite negligible emissions during most of the day and a peak with a bell-like shape in the morning in the hours of maximum nitrogen load in the plant, correlated to the ammonia and nitrite peaks in the tank.