The link between Denitrifying Ecology, Environmental Conditions, and Nitrous Oxide Production in Wastewater Treatment

Abstract

Nitrous oxide (N2O) is a potent greenhouse gas and ozone-depleting compound known to be emitted from wastewater treatment. Emissions of N2O results from the activity of the microorganisms employed in the transformation of reactive nitrogen species (NOx). N2O emissions from wastewater treatment are currently underestimated by United Nations (UN) and the United States (US) Environmental Protection Agency (EPA) guidelines, and treatment plants show differing rates of N2O emissions. Improving our understanding of the mechanisms that drive N2O production and subsequent emissions are essential for its mitigation. N2O is produced by a variety of microbial metabolic pathways; this thesis primarily focuses on N2O production via the denitrification pathway. Denitrification, conducted by heterotrophic bacteria is the Dissimilatory reduction of nitrogen oxides such as nitrate (NO3-) or nitrite (NO2-) to di-nitrogen gas (N2), using carbon as electron donors. The process occurs in a cascade reducing NO3-, to NO2-, nitric oxide (NO), N2O and finally N2. This process is catalyzed by several enzymes including, nitrate reductase (Nar, and Nap), nitrite reductase (Nir) nitric oxide reductase (Nor) and nitrous oxide reductase (NosZ). Denitrification is a widespread trait, and the pathway is encoded by a variety of genes, which produce functionally equivalent enzymes but form differing denitrifying phylogenies. For example, nitrite reductase is encoded by nirS and nirK the genes for the cytochrome-cd1 and copper-based dissimilatory nitrite reductases, respectively, while nitrous oxide reductase is encoded by either the truncated or non-truncated gene nosZII or nosZI respectively. It has been proposed that differences in the microbial population may explain the difference in N2O emission observed between treatment plants. This hypothesis is supported by the recent discovery of non-denitrifying N2O reducing organisms encoded by the gene nosZII. These organisms lack the preceding enzymes of the denitrifying pathway but retain the ability to reduce N2O, they have a high abundance in soils, and nosZII abundance shows a strong correlation with reduced N2O emissions. The understanding of denitrifying microbial ecology and its role in the production of N2O from wastewater treatment is currently limited. The primary aim of this thesis is to address the hypothesis: Denitrifying ecology determines the magnitude and rate of N2O accumulation in Wastewater treatment plant (WWTP) sludge. To address this hypothesis, a stepwise approach has been taken. 1) A survey of the microbial ecology of three New Zealand (NZ) WWTPs was conducted to determine denitrifying ecology by the distribution of denitrifying functional markers (nirS, nirK, nosZI, and nosZII) within each population. The following hypotheses were tested, a) denitrifying populations will differ between WWTPs, and b) Wastewater is a disadvantageous environment for non-denitrifying organisms (nosZII), and they will be under-represented. 2) Using the sludges from the surveyed WWTPs, sludge metabolism was examined under ideal denitrifying conditions for differences in NOx profiles including N2O accumulation. Accumulation of NOx intermediaries was correlated with the relative abundance of denitrifying functional markers, testing the hypotheses c) differing denitrifying populations accumulate different amounts of N2O, and d) N2O accumulation will correlate with non-denitrifying organism abundance. 3) As wastewater treatment plants are not stable environments and can suffer from process perturbations, sludges with now known populations, and NOx profiles under control conditions, were exposed to environmental shocks (pH and nitrite (NO2-). Testing the hypothesis: e) Within a specific range of environmental shock, denitrifying population structure will determine N2O accumulation. Survey of NZ microbial populations supports the hypotheses, showing differing total microbial and denitrifying populations, with low abundance of non-denitrifying N2O reducing organisms(nosZII). Analysis of the relative abundance of denitrifying functional markers suggests NZ WWTPs have a genetic predisposition to N2O emissions. Additional findings include the identification of co-occurring nirK and nirS in the same organisms in each treatment plant and the observation that NZ treatment plants show the opposite trend to most of the literature reports with a markedly dominant nirK over nirS population. Examination of the NOx profiles of the three sludges under ideal denitrifying conditions supports the hypotheses showing differing N2O accumulation in each sludge and increasing abundance of nosZII correlating with a decrease in the maximum N2O concentration. All sludges acclimatized to laboratory conditions quickly accumulating minimal N2O after just four sequence batch cycles. Analysis of changes in the rate of NO3- reduction and the Max N2O accumulation suggests that under stable conditions N2O accumulation will be minimal. Examination of the population's response to environmental shock supports the hypothesis, with different populations accumulating N2O when exposed to different environmental shocks. No population was susceptible to N2O accumulation under all conditions. Populations that exhibited NO2- accumulation were resistant to NO2- shock but more susceptible to changes in pH, while populations exhibiting minimal NO2- accumulation were sensitive to NO2- shock but more resistant to changes in PH accumulation. This research concludes that denitrifying microbial populations determine the accumulation of N2O in response to changes in environmental conditions. Denitrifying populations differ between WWTPs. However, results suggest, irrespective of the denitrifying microbial population's structure, stable conditions will result in minimal N2O accumulation. When exposed to environmental shock differing populations showed susceptibility to accumulated N2O under differing environmental conditions. These results suggest that management of N2O accumulation and subsequent emissions will need to be on a case by case basis. Emissions may be addressed by the introduction of a microbial population less susceptible to the N2O accumulation under the environmental conditions of a given treatment plant. Further research is required to test the feasibility of this management strategy.