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    N₂O synthesis by microalgae : pathways, significance and mitigations : a thesis presented in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Environmental Engineering at Massey University, Palmerston North, New Zealand
    (Massey University, 2017) Plouviez, Maxence
    Over the last decades, various studies have reported the occurrence of emissions of nitrous oxide (N₂O) from aquatic ecosystems characterised by a high level of algal activity (e.g. eutrophic lakes) as well as from algal cultures representative of the processes used by the algae biotechnology industry. As N₂O is a potent greenhouse gas (GHG) and ozone depleting pollutant, these findings suggest that large scale microalgae cultivation (and possibly, eutrophic ecosystems) could contribute to the global N₂O budget. Considering the current rapid development of microalgal biotechnologies and the ubiquity of microalgae in the environment, this PhD research was undertaken to determine the biochemical pathway of microalgal N₂O synthesis and evaluate the potential significance of microalgal N₂O emissions with regard to climate change. To determine the pathway of N₂O synthesis in microalgae, Chlamydomonas reinhardtii and its associated mutants were incubated in short-term (24 h) laboratory in vitro batch assays. For the first time, axenic C. reinhardtii cultures (i.e. culture free of other microorganisms such as bacteria) fed nitrite (NO₂⁻) were shown to synthesise N₂O under aerobic conditions. The results evidenced that N₂O synthesis involves 1) NO₂⁻ reduction into nitric oxide (NO), followed by 2) NO reduction into N₂O by nitric oxide reductase (NOR). With regard to the first step, the results show that NO₂⁻ reduction into NO could be catalysed by the dual system nitrate reductase-amidoxime reducing component (NR-ARC) and the mitochondrial cytochrome c oxidase (COX). Based on our experimental evidence and published literature, we hypothesise that N₂O is synthesised via NR-ARC-mediated NO₂⁻ reduction under physiological conditions (i.e. low/moderate intracellular NO₂⁻) but that under NO₂⁻ stress (i.e. induced by high intracellular NO₂⁻), N₂O synthesis involves both NR-ARC-mediated and COXmediated NO₂⁻ reductions. RNA sequencing analysis on C. reinhardtii samples confirmed that the genes encoding ARC, COX and NOR were expressed in NO₂⁻-laden culture, although NO₂⁻ addition did not trigger significant transcriptomic regulation of these genes. We therefore hypothesise that the microalgal N₂O pathway may be involved in NO regulation in microalgae where NOR acts as a security valve to get rid of excess NO (or NO₂⁻). To evaluate emissions during microalgal cultivation, N₂O emissions were quantified during the long term outdoor cultivation of commercially relevant microalgae species (Chlorella vulgaris, Neochloris sp. and Arthrospira platensis) in 50 L pilot scale tubular photobioreactors (92 days) and during secondary wastewater treatment in a 1000 L high rate algal pond (365 days). Highly variable N₂O emissions were recorded from both systems (μmol N₂O·m⁻²·h⁻¹, n = 510 from the 50 L photobioreactors; 0.008–28 μmol N₂O·m⁻²·h⁻¹, n = 50 from the high rate algal pond). Based on these data, we estimated that the large scale cultivation of microalgae for biofuel production in order to, for example, replace 30% of USA transport fuel with algal-derived biofuel (i.e. a commonly used sustainability target), could generate N₂O emissions representing up to 10% of the currently budgeted global anthropogenic N₂O emissions. In contrast, N₂O emissions from the microalgae-based pond systems commonly used for wastewater treatment would represent less than 2% of the currently budgeted global N₂O emissions from wastewater treatment. As emission factors to predict N₂O emissions during microalgae cultivation and microalgae-based wastewater treatment are currently lacking in Intergovernmental Panel for Climate Change methodologies, we estimated these values to 0.1 – 0.4% (0.02–0.11 g N–N₂O·m⁻³·d⁻¹) of the N load on synthetic media (NO₃⁻) during commercial cultivation and 0.04 – 0.45% (0.002–0.02 g N–N₂O·m⁻³·d⁻¹) of the N load during wastewater treatment. The accuracy of the emission factors estimated is still uncertain due to the variability in the N₂O emissions recorded and by consequence further research is needed. Nevertheless, further monitoring showed that the use of ammonium as N source and/or the cultivation of microalgae species lacking the ability to generate N₂O (e.g. A. platensis) could provide simple mitigation solutions.
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    An investigation of the spatial distribution of N2O emissions from sheep grazed hill country pastures in New Zealand :|ba thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Environmental Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Letica, Selai Ahovelo
    New Zealand’s (NZ) greenhouse gas (GHG) profile is unique amongst developed countries as almost 50% of GHG emissions are derived from agriculture. In contrast, agricultural sectors of other developed countries typically contribute <10% to the national total GHG profile. In NZ, agricultural GHG emissions are dominated by methane (CH4) from enteric fermentation and nitrous oxide (N2O) from excreta deposition and nitrogen (N) fertiliser application. Nitrous oxide emissions from agricultural soils are largely affected by N inputs and soil moisture conditions, and contribute 33% of agricultural GHG emissions. In pastoral hill country these factors are inherently more variable than in flat land pastures due to topographydriven differences in excretal N returns and in soil moisture. This limits the application of N2O emission data collected from trials conducted on flat land to hill country situations. The objective of this thesis was to determine the influence of topography and fertiliser N inputs to soil on N2O emissions in hill country. Small scale trials were conducted to measure these aspects of N cycling. Three trials were conducted to measure the effect of slope and fertiliser N input on nitrification potential (NP) and N2O emissions. The results of these short term trials suggested that slope class and fertiliser N rates significantly affected nitrification rates and N2O emissions in hill country due to differences in N inputs and moisture status, as affected by slope. Both NP and N2O emissions were highly spatially variable during the measurement periods and the results presented in this thesis suggest that the majority of N2O emissions in sheep grazed hill country are produced from low slope/stock camping areas. Based on our findings it is recommended that mitigation options to reduce the risk of N loss from sheep grazed hill country should be targeted at low slope/stock campsite areas. Due to the significant relationship between slope class and N2O emissions, slope class may be a suitable parameter for up-scaling estimates of N2O emissions from sheep grazed hill country.
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    Estimating direct N2O emissions from sheep, beef, and deer grazed pastures in New Zealand hill country: accounting for the effect of land slope on N2O emission factors from urine and dung
    (Elsevier, 2015-03) Saggar SK; Giltrap DL; Davison R; Gibson R; de Klein CAM; Rollo M; Ettema P; Rys G
    Nearly one-half of New Zealand's ruminant livestock graze on hill country pastures where spatial differences in soil conditions are highly variable and excretal deposition is influenced by pasture production, animal grazing and resting behaviour that impact the nitrous oxide (N2O) emission factor from excreta (EF3). New Zealand currently uses country-specific EF3 values for urine and dung of 0.01 and 0.0025, respectively, to estimate direct N2O emissions from excreta. These values have largely been developed from trials on flat pastoral land. The use of the same EF3 for hill pasture with medium and steep slopes has been recognised as a possible source of overestimation of N2O emissions in New Zealand. The objectives of this study were to develop and describe an approach that takes into account the effects of slope in estimating hill country N2O emissions from the dung and urine of ruminant animals (sheep, beef cattle, and deer) across different slope classes, and then compare these estimates with current New Zealand inventory estimates. We use New Zealand as a case study to determine the direct N2O emissions between 1990 and 2012 from sheep, beef cattle and deer excreta using updated estimates of EF3 for sloping land, the area of land in different slope classes by region and farm type, and a nutrient transfer model to allocate excretal-N to the different slope classes, and compare the changes between these hill pastures-specific and current inventory estimates. Our findings are significant - the proposed new methodology using New Zealand specific EFs calculated from a national series of hill country experiments resulted in 52% lower N2O estimates relative to using current inventory emission factors, for the period between 1990 and 2012 and reduces New Zealand's total national agricultural N2O greenhouse inventory estimates by 16%. The improved methodology is transparent, and complete, and has improved accuracy of emission estimates. On this basis, the improved methodology of estimating N2O emission is recommended for adoption where hill land grasslands are grazed by sheep, beef cattle and deer.