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

Abstract

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.