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Item Tillage-induced soil nitrous oxide fluxes from tow soils in the Manawatu : a thesis presented in partial fulfilment of the requirements for the degree of Master of Applied Science in Agricultural Engineering, Institute of Technology and Engineering, Massey University, Palmerston North, New Zealand(Massey University, 2000) Akramkhanov, AkmalEnhanced greenhouse gas emissions of nitrous oxide (N₂O) induced by agricultural practices is believed to be the major anthropogenic source. Studies conducted in New Zealand generally from pasture suggest low N₂O emission, however, there is little information for arable farming systems. Therefore, there is a need for a site-specific assessment of the impact of tillage practices on N₂O fluxes. This paper evaluates tillage system and land use effects on N₂O emissions at two sites using a closed chamber technique. Sites included a Kairanga silt loam where maize/barley was grown continuously for either 17 (K17) or 34 (K34) years, with a conventional tillage system (Kairanga), and an Ohakea silt loam where winter oats and summer fodder maize was double-cropped for five years with conventional (CT) and no-tillage (NT) systems (Massey). At both sites permanent pasture (PP) soil was used as a control. Spatial measurements for all treatments at Massey site showed large inherent variations in N₂O fluxes (a mean CV=119%) which reflected natural soil heterogeneity, and perhaps the measurement technique used rather than the real differences due to the tillage and cropping systems evaluated. N₂O emissions measured from December 1998 to September 1999 from the PP were significantly lower (1.66 kg N₂O-N/ha/year) than the CT and NT plots at 9.20 and 12.00 kg N₂O-N/ha/year respectively. However, there were no differences in N₂O emission rates between the CT and NT treatments. Cumulative coefficient of variation (CV) of treatments ranged from 39 to 140%. Seedbed preparation using power-harrow which was done within few days of ploughing the CT plots reduced N₂O emissions by 65% within the first hour after power-harrowing. However. N₂O emission rates returned to the pre-power harrowing levels one month after power-harrowing. There was strong relationship between log-transformed values of soil moisture content (SMC) and N₂O emissions in all treatments. PP (r = 0.73), CT (r = 0.75) and NT (r = 0.86). Seasonal variation in N₂O emission from the PP was in the order of winter=autumn>summer. Although fluxes in the CT were higher in winter than in the autumn season, there were no differences between the summer and autumn data. Similar to the PP. the seasonal variations in N₂O emission in the NT treatment were in the order of winter>autumn=summer. The estimated annual N₂O emissions from the PP. K17 and K34 (calculated as the mean of all individual closed cover chamber measurements between November 1998 and September 1999) from Kairanga site were similar at 3.24, 3.42 and 2.37 kg N₂O-N/ha/year, respectively. There were large variations in N₂O emissions during the year with the mean flux rates ranging from 0.175 to 13.32, 0.175 to 16.91 and 0.088 to 30.05 kg N₂O-N/ha/year in the PP, K17 and K34 fields, respectively. Although overall comparison of treatment means did not show any discernible differences between management practices, there were signs that the K34 had lower emissions compared to the PP. N₂O fluxes from the K17 and PP field appeared to be influenced by SMC. There is clear indication that low or negligible emissions occur when gravimetric soil water content is less than 30% in the PP. Although N₂O fluxes did not follow the rainfall patterns in the K17 and PP, linear regression analyses indicated low but significant relationship r = 0.46 and 0.53 (0.72 when log-transformed), respectively. In the K34 field. SMC did not seem to govern fluxes which were especially apparent during wet months of April and May. The linear regression analysis using the measured data revealed no relationship (r = 0.12) between the SMC and N₂O fluxes in the K34 treatment. Seasonal grouping of monthly log-transformed N₂O emissions showed significant differences in all treatments. Summer season N₂O emissions in the PP were the lowest than other seasons whereas no discernible differences were observed among other seasons. Although N₂O fluxes during spring and summer were similar in the K17 field, they were significantly lower than the winter and higher than autumn fluxes. There were considerably higher emissions in summer than in autumn in the K34 but seasonal variation between winter and spring was less profound. Spatial variability in N₂O fluxes was large during the year with coefficients of variation (CV) ranging from 10 to 82%, 12 to 99% and 9 to 137% for the PP, K17 and K34 fields, respectively.Item 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, MaxenceOver 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.Item The influence of soil parameters and denitrifiers on N₂O emissions in New Zealand dairy-grazed pasture soils : a thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy in Soil Science, Massey University Palmerston North New Zealand(Massey University, 2015) Jha, NehaDenitrification is a primary source of nitrous oxide (N2O) production found globally in temperate grasslands and in New Zealand pasture soils. The various reductase enzymes coded by specific denitrifier genes, and influenced by soil and environmental factors, regulate the N2O production and reduction during the denitrification process. An understanding of the soil and environmental factors that have the potential to enhance the activity of denitrifiers reducing N2O to dinitrogen (N2) contributes to the development of novel and effective N2O mitigation technologies. This thesis attempts to address a critical gap in our understanding of the role of bacterial denitrifier communities and their abundance in denitrification under various field-moist and incubation conditions in New Zealand dairy-grazed pasture soils. This thesis consists of a literature review (Chapter 2) that describes the biochemical and molecular aspect of denitrification, and identifies key factors that affect the process and the denitrifier community structure and its abundance. It addresses the advantages and limitations of available techniques to measure denitrification, the denitrifier community structure and abundance, and also any available mitigation options to reduce denitrification losses. The review concludes by identifying gaps in our knowledge of the denitrifier communities, and their abundance and activities in New Zealand soils. The acetylene (C2H2) inhibition technique (AIT) was standardised (Chapter 3) for the measurements of denitrification enzyme activity (DEA) and denitrification rate (DR) used during the research. Similarly, terminal restriction fragment length polymorphism (T-RFLP) and quantitative polymerase chain reaction (qPCR) were standardised to assess denitrifier community structure (numbers of nirS+nirK and nosZ gene T-RFs) and abundance (nirS+nirK and nosZ gene copy numbers) in soils. Soil samples were obtained from 10 sites with known management histories, representing dairy farms across the North and South Islands of New Zealand, to determine the soil factors contributing to spatial variability in DEA, DR and denitrifier community structure and abundance within and across dairy-grazed pastures (Chapter 4). Despite the spatial variability at each site, the DEA results show large differences in potential denitrification in these soils. The outcome of this study painted an overall picture of the distribution of denitrification enzyme The bacterial denitrifier genes richness correlated significantly (P < 0.05) to Olsen P, microbial biomass C (MBC), and NH4+-N contents of soil, whereas these gene abundances correlated to MBC and NO3–-N contents in soils, thus confirming inherent soil characteristics that influence denitrifier populations in soils. NirS and nirK gene copy numbers correlated positively with N2O emissions (from nitrification and denitrification). There was no clear relationship between nosZ gene copy numbers and denitrification rate in the field-moist soils, which could be due to less anaerobic condition for denitrifiers to carry denitrification. To determine the effect of increasing soil water content (SWC) on changes in denitrification, 5 soils contrasting in DEA were incubated at field capacity (FC) and saturation SWC (Chapter 5). The measured DRs were higher in soils incubated at saturation, ranging from 21.5 to 73.9 µg N2O-N kg–1soil hr–1, than in soils incubated at FC, in which DR varied from 0.8 to 50.4 µg N2O-N kg–1soil hr–1. Although the direction of change in denitrification with the increase in SWC was similar among the soils, the magnitude of increase was variable among the five soil types. This variability was mainly driven by their inherent biochemical (NO3–-N, TC, TN, MBC, Olsen P, DEA,) and molecular characteristics (denitrifier richness and denitrifier abundance). A subsequent incubation experiment was conducted to investigate the effect of water only, cattle urine, and cattle urine with added nitrification inhibitor dicyandiamide (DCD) on denitrification and numbers of denitrifier gene T-RFs and their copy numbers in three pasture soils with contrasting DEA. The results of this incubation are described in Chapters 6 and 7. The results described in Chapter 6 explore the effect of saturation (only water addition) on DRs, denitrifier gene richness, and denitrifier gene copy numbers in three soils. At saturation soil water content in the incubated soils, the increases or decreases in DRs with incubation time were variable in three soils and depended on their TC, TN, Olsen P, MBC, and denitrifier gene richness. It is inferred from the results that, with increasing SWC, a denitrifier community of a constant size was maintained in the incubated soils. The results in Chapter 7 describe the effect of urine with and without DCD on denitrification, denitrifier richness, and denitrifier gene abundance. There were changes in soil pH, NO3–-N, NH4+-N, soluble C, and microbial biomass C in soils with urine application. The numbers of denitrifier gene T-RFs and copy numbers either remained unchanged or were lower under urine + DCD treatments than urine only and related to N2O productions (from both nitrification and denitrification) during the incubation. There was an overall increase in DR with application of urine and urine + DCD to soils. Comparatively higher N2O-NA (N2O productions from non- acetylene jars) were observed in urine-only treatments than in urine + DCD. The cumulative N2O-NA with the addition of urine and urine + DCD were variable among the three soils. During 4 weeks incubation the N2O-NA ranged from 20.9 to 26.7 mg N2O-N kg–1 soil in urine treatments and from 19.6 to 21.7 mg N2O-N kg–1 soil in urine + DCD treatments in three soils. The proportion of total N denitrified during the entire incubation ranged from 6.3 to 22.4 % in urine treatments and 3.6 to 5.6 % in urine + DCD treatments in three soils. Denitrifier gene richness and gene copy numbers during incubation helped identify overall changes in the denitrifier community with the application of treatments and link these changes to N2O-NA across various soils. Overall, in the urine-applied samples the N2O-NA (positively) was significantly correlated with their DEA, MBC, Olsen P, and numbers of denitrifier gene richness and denitrifier gene copy numbers. The information obtained in this research helped enhance the understanding of the variability in denitrification and the denitrifier community in the New Zealand dairy pasture soils. The molecular measurements of the soils helped identify differences in N2O productions in soils at similar incubation conditions. Soils with low denitrifier richness and, more importantly, lower abundance of complete denitrifiers had limited capacity to denitrify available inorganic N. Measurements of DEA, denitrifier richness, and denitrifier abundance in soils, along with their N2O productions (with and without acetylene), indicated the inherent potential of soils to carry denitrification. Under urine application, soil with the most abundant denitrifier population and the highest DEA responded to added treatment quickly and produced more N2O-NA and N2O-A than the other two soils did. On the other hand, soils with lower DEA and less abundant denitrifier community showed lower N2O production (with and without acetylene) even under anaerobic condition than did other soils.