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
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2017
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Massey University
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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 N2O 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 (0.0 – 38 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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Keywords
Microalgae, Biotechnology, Environmental aspects, Atmospheric nitrous oxide, Nitrous oxide, Research Subject Categories::TECHNOLOGY::Bioengineering::Biochemical process engineering