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Item Environmental influences on polyphosphate accumulation in microalgae : an investigation into species differences and transcriptional responses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Manawatū, New Zealand(Massey University, 2022) Cliff, AlexanderMany species of microalgae can store phosphorus (P) as polyphosphate (polyP) granules during the process of ‘luxury uptake’, a term describing intracellular P accumulation above levels required for normal metabolism (0.2 – 1% P by dry weight). Environmental conditions can influence P luxury uptake but it is not known whether the effects of environmental conditions on luxury P uptake are the same for all microalgae or whether all microalgae can in fact store P as polyP. The broad aim of the work described in this thesis was to extend the current knowledge of polyphosphate (polyP) synthesis in microalgae, enabling improved exploitation of their ability to sequester P from water sources and enhance recovery of a vital nutrient. Specifically, the mechanisms by which environmental conditions influence luxury uptake and potential species differences need to be studied to better understand observations at the population level and make informed decisions in the design of treatment processes. The experimental work was therefore divided into two main objectives: Objective 1 sought to determine whether there were in fact differences in luxury uptake ‘abilities’ between species. In Chapter 3, this is explored through the use of genetic database searches, biochemical assays, and protein modelling. Objective 2 examined the effects of environmental factors known to influence luxury uptake. In Chapter 4, the responses of the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris to P repletion were studied in a range of conditions to identify similarities and differences in environmental influences. Chapter 5 sought to determine whether the observed differences could be due to responses at the genetic level, by comparing the gene expression levels of P-related genes in C. reinhardtii under selected sets of conditions from the previous chapter. An additional experiment was conducted, to examine gene expression without inducing ‘noise’ through changes in growth conditions, and this is discussed in Chapter 6. Using protein sequence homology searches, phylogenetic tree generation, protein structure modelling, and biochemical assays (using the chlorophytes C. reinhardtii, C. vulgaris, Desmodesmus cf. armatus, Gonium pectorale, Pediastrum boryanum, and the cyanobacterium Microcystis aeruginosa), it was shown that the ability to store P as polyP is common among microalgae, as implied by the broad conservation of the polyP polymerase VTC4, but luxury uptake abilities vary between species. All six tested microalgae responded to P addition following a period of P depletion by accumulating P as granular polyP. Under the conditions tested, the total P assimilated over 24 hours was similar for five of the microalgae tested (2.6 – 3.6% P by dry weight) but C. vulgaris assimilated considerably less P (~1.2% P) than the others. The effects of environmental conditions on P uptake and polyP accumulation were assessed by triggering luxury uptake in C. vulgaris and C. reinhardtii in different conditions of light supply, temperature, and pH, with different P repletion doses following different P depletion times. P uptake and polyP accumulation were influenced by light supply, P depletion time, and P repletion dose in both microalgae but P dose had the strongest influence in C. reinhardtii versus light supply in C. vulgaris. PolyP was still accumulated by these two species in conditions suppressing growth and severely repressing metabolism (10 °C and darkness), evidencing that P uptake and polyP synthesis do not require light energy. The model alga C. reinhardtii was then used to evaluate, for the first time, whether the differences in P uptake and polyP accumulation observed with respect to differences in environmental conditions were associated with differences in gene expression. Although the genes assessed were downregulated (relative to controls) 24 hours after P repletion, as expected, in all experimental conditions, changing conditions at the start of the experiment also caused changes in gene expression in controls, making it hard to distinguish responses to P repletion from ‘global’ responses to changing conditions. Another experiment was therefore performed where temperature and light intensity were maintained constant before and after P repletion. The results confirmed that increased P repletion dose and P depletion time were associated with increased P uptake and polyP accumulation over 24 hours. The results evidenced the expected downregulation of PSR1, VTC4, VTCX, and PTB5 after 1 hour of P repletion, but this response was much more salient after 7 days of P depletion (compared to 3 days). Changes in gene expression were also associated with P repletion dose, but only after 7 days of P depletion. This showed that the response to P repletion is stronger after a longer P depletion time but the observed expression changes did not support the hypothesis that these changes were the reason for the higher observed P uptake and polyP accumulation. For the first time, it has been systematically shown that the ability to accumulate P as polyP is widespread among microalgae but that the kinetics of P accumulation vary between species and this species-dependence is influenced by environmental factors. These factors engender differences at the level of gene expression, involving both components of the VTC complex and phosphate transporters. However, the differences in P uptake and polyP accumulation may be better understood by investigating the structural differences and changes in activity of relevant proteins.Item UV radiation as a new tool to control microalgal bio-product yield and quality : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Industrial Biotechnology at Massey University, Palmerston North, New Zealand(Massey University, 2018) Schaap, RolandWhile ultraviolet (UV) radiation is most commonly known as an abiotic stress, various studies have shown targeted UV exposure increases bioproduct and biomass yields in microalgae. Microalgal cultivation processes face significant limitations in achievable bioproduct and biomass yields and thus improvements offered by targeted UV treatments during large-scale microalgae cultivation provide an opportunity for development of a novel UV treatment tool. Growing demand in microalgae (bio)products indicate there may be a substantial market for such UV treatment tools. No initiatives that explore the development of targeted UV treatments during large-scale microalgae cultivation have been found in the literature or in the industry. In collaboration with industrial partner BioLumic, a company specializing in applying targeted UV treatments in plants as a tool in agriculture, this PhD research examined if specific treatments of UV radiation (i.e. specific in UV waveband, irradiance and exposure duration) can reliably increase carotenoid accumulation in the microalga Dunaliella salina and if this new understanding can be feasibly used to develop an industrial system for UV treatment of microalgae. The PhD research was conducted utilizing D.salina after evaluation in four commercially relevant microalgae species: Arthrospira platensis, Chlorella vulgaris, Haematococcus pluvialis and D.salina. A UV-A induced carotenoid accumulated response was identified in D.salina (strain UTEX 1644). Targeted UV-A treatments reliably induced carotenoid accumulation in this species, and the magnitude of the response depended on the UV-A wavelength, UV irradiance, UV exposure duration, and UV dose. The UV-A carotenoid accumulation response was induced within 6 hours and was largely complete in 96 hours (24 h·d⁻¹ UV exposure). The highest UV-A dose tested induced the highest carotenoid accumulation rates and the highest total carotenoid concentrations after continuous UV exposure (24 h·d⁻¹) at the highest UV-A irradiance tested (30 W·m⁻²). Total carotenoid concentration increases of up to 162% were thus observed after 72 hr of UV-A exposure. UV-A exposure was associated with slowed or stopped cell proliferation as well as increased D.salina cell size (up to 15%) and altered intracellular structural organization. Carotenoid accumulation ceased and cell proliferation increased when UV-A exposure was stopped, leading to a subsequent resumption of cell proliferation. UV-A induced carotenoid accumulation was improved 51% during UV-A exposure concomitant with non-UV carotegenic stimuli (high PAR intensity and salinity) compared to UV-A exposure alone. The observations from experiments carried out in the thesis served as inputs in a techno-economic analysis (TEA) model developed to assess feasibility of large-scale UV treatment. The TEA model was developed to allow assessment of the most critical areas for improving profitability of large-scale UV treatment technology, rather than provide absolute economical outputs for revenue and profit. The TEA was based on two reference cultivation systems currently used for commercial D.salina cultivation. The TEA analysis considered four locations for the UV treatment system applied along the cultivation process: pre-cultivation stage (i.e. inoculum), main cultivation stage, post-cultivation stage (i.e. immediately prior to harvest) and during fluid transfer between stages. A dedicated post-cultivation UV treatment stage was shown to have a number of advantages over other treatment options. A model cultivation system for the case-study of D.salina was developed assuming an annual β-carotene production of 1,000 kg. The developed TEA model cultivation system and TEA UV treatment system were able to identify a potential increase in profitability generated from the application targeted UV treatment during large-scale D.salina cultivation. The maximum increase in profitability was achieved using a broad wavelength UV treatment system (irradiance = 30 W·m⁻², exposure duration = 24 h·d⁻¹, surface area coverage = 100%) applied during an intensive cultivation post-cultivation system. A relatively small contribution of the UV treatment system to CAPEX and OPEX to overall β-carotene production cost (i.e. < 10%) combined with the large increase in β-carotene production (711 kg·y⁻¹ and 895 kg·y⁻¹ for fluorescent UV tube and UV LED systems, respectively) leads to potentially large increases in profitability. The TEA analysis identified the magnitude of the UV-A induced carotenoid accumulation response to be the most important factor to influence the potential profitability. Moreover, the TEA indicated the increases in profitability are strongly influenced by optical efficiency, electrical efficiency and maximum optical power. The profitability estimates from the current TEA indicate that UV treatment during commercial microalgae cultivation has potential and justifies further research. To our knowledge the exploration of the fundamental UV photobiology in microalgae required to develop UV treatment regimes from discrete UV wavebands, complemented with a commercial microalgal-engineering insight, to produce UV treatment regimes and UV treatment technology for application during large-scale microalgae cultivation, has never been attempted. The multidisciplinary approach employed during this PhD research explored for the first time the development of a UV treatment system from laboratory observations to commercial cultivation. The current research described for the first time the UV exposure behaviour of D.salina (strain UTEX 1644) to varying UV waveband, UV irradiance, UV exposure durations as well as UV response interaction with PAR and salinity. The case-study of UV treatment during large-scale D.salina cultivation in this PhD research allowed recommendations to be made to the industrial partner BioLumic on potential areas of focus for continued research and development.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 An assessment of inexpensive methods for recovery of microalgal biomass and oils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand(Massey University, 2015) Chatsungnoen, TawanInexpensive processes for harvesting the microalgal biomass from the culture media and recovering oils from the harvested biomass are necessary for economically viable production of low-value products such as fuels. This study focused on harvesting of microalgae biomass from the culture broth by flocculation?sedimentation and recovery of oils from the harvested biomass using solvent-based extraction. Flocculation?sedimentation was explored for several marine and freshwater microalgae including Choricystis minor (freshwater), Neochloris sp. (freshwater), Chlorella vulgaris grown in freshwater; C. vulgaris grown in seawater; Nannochloropsis salina (seawater) and Cylindrotheca fusiformis (seawater), as a means for substantially concentrating the biomass prior to further dewatering by other methods. Aluminum sulfate and ferric chloride were investigated as cheap, highly effective, readily available in large quantities and innocuous flocculants. Flocculation?sedimentation behavior of the microalgae was evaluated with several flocculation conditions. The optimal microalgal biomass harvesting conditions identified in batch flocculation studies were applied to design and characterize a continuous flocculation?sedimentation system. The effect of the flocculant used and the water in the biomass paste on the extraction of oils were assessed in comparison with controls. The optimal solvent composition for extraction of the biomass paste was established. Using this solvent composition, the optimal extraction conditions (i.e. the volume of the solvent mixture relative to biomass, the extraction temperature and time) were identified using a 23 factorial experimental design. Removal of more than 95% of the biomass from the broth by flocculation? sedimentation was shown to be possible for all the microalgae, but the required dosage of the flocculant depended on the following factors: the microalgal species; the ionic strength of the suspending fluid; the initial concentration of the biomass in the suspension; and the nature of the flocculant. Irrespective of the algal species, the flocculant dosage was found to increase linearly with increasing concentration of the biomass in the culture broth. The flocculant dosage for a given level of biomass recovery under standardized processing conditions increased with an increase in the cell specific surface area in the range of 26–450 ?m2 cell?1. Al3+ was a better flocculant than Fe3+ for some algae, but the situation was reversed for some others. The continuous flow biomass recovery was performed with N. salina, as this alga had the highest oil productivity among the species studied. With an aluminum sulfate dosage of 229 mg L?1 and a total flow rate of 22.6 mL min?1, almost 86% of the N. salina biomass could be recovered from the broth within 148 min in the sedimentation tank. A prior flocculation–sedimentation treatment could greatly reduce the energy demand of subsequent dewatering by other methods. The flocculants adsorbed to the biomass were not removed by washing, but this did not hinder oil recovery from the biomass paste by solvent extraction. A modification of well-known Bligh and Dyer method could be used to recover more than 96% of the oils from N. salina biomass paste. The single-step modified extraction procedure was much superior to the Bligh and Dyer original. The optimal extraction conditions for N. salina biomass paste included a solvent mixture (chloroform, methanol and water in the volume ratio of 5.7:3:1) volume of 33 mL per g (dry basis) of the algae biomass; an extraction temperature of 25?C; and an extraction time of 2 h. This work represents the first detailed study of the continuous flocculation? sedimentation process for harvesting N. salina biomass from the culture broth and the specific suitable solvent combination of chloroform, methanol and water for extracting algal crude oils from the N. salina biomass paste without a prior drying step.Item Modelling the impact of temperature on microalgae productivity during outdoor cultivation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Environmental Engineering at Massey University, Palmerston North, New Zealand(Massey University, 2014) Béchet, QuentinAccurate predictions of algal productivity during outdoor cultivation are critically needed to assess the economic feasibility and the environmental impacts of full-scale algal cultivation. The literature shows that current estimations of full-scale productivities are mainly based on experimental data obtained during lab-scale experiments conducted under conditions poorly representative of outdoor conditions. In particular, the effect of temperature variations on algal productivity is often neglected. The main objective of this thesis was to develop a model able to predict algal productivity under the dynamic conditions of temperature and light representative of full-scale cultivation. In a first step, models were developed to predict broth temperature as a function of climatic, operational, and design parameters. The model developed for open ponds could predict temperature at an accuracy of ±2.6oC when assessed against experimental data collected in New Zealand over one year. The temperature model developed for closed photobioreactors was accurate at ±4.3oC when compared to experimental data collected in Singapore and New Zealand over a total of 6 months of cultivation. This second temperature model was then applied at different climatic locations to demonstrate that actively controlling temperature would seriously threaten the economics and sustainability of full-scale cultivation in photobioreactors. To quantify the impact of temperature variations on biomass productivity, a productivity model was developed using Chlorella vulgaris as a representative commercial species. To determine the best methodology, a review of more than 40 models described in the literature revealed that an approach accounting for light gradients combined with an empirical function of temperature for photosynthesis and first-order kinetics for respiration would offer the most pragmatic compromise between accuracy and complexity. The model was parameterized using short-term indoor experiments and subsequently validated using independent benchscale indoor (> 160 days) and pilot-scale outdoor (> 140 days) experiments, showing prediction accuracies of ± 13%. The outdoor data set was obtained from 13 different experiments performed in 4 different reactors operated under various regimes and climatic conditions. The productivity model was found to be accurate enough to significantly refine previous assessments of the economics and the environmental impacts of full-scale algal cultivation. The productivity model was then used in different case studies in order to investigate the impact of location/climate, design (pond depth or reactor diameter), and operation (hydraulic retention time or HRT) on productivity and water demand. Although the qualitative impact of the HRT on process was already known, this application enabled the first quantification of the HRT value on the productivity. Low HRT values around 3 days were found to maximize productivity at most locations investigated but these operating conditions were associated with a large water demand, illustrating a poorly acknowledged trade-off between sustainability and revenues. The model was also used to demonstrate that actively controlling the pond depth can increase the productivity by up to 23% while minimizing the water demand by up to 46%. This thesis therefore revealed that the choice of a location for algal full-scale production must be based on the comparison of optimized systems, contrarily to current assessments assuming the same design and operation at different locations.Item Raceway-based production of microalgae for possible use in making biodiesel : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University, Palmerston North, New Zealand(Massey University, 2014) Tahir, SadiaOils from microalgae are of interest as a potential feedstock for producing renewable transport fuels including gasoline, diesel, biodiesel and jet fuel. For producing feedstock oils, an alga must be capable of being grown easily in readily available seawater and have a high productivity of biomass and oil. This study explored the biomass and lipid production potential of the microalga Chlorella vulgaris in seawater media, as a potential producer of feedstock oils. The alga was grown photoautotrophically under various conditions in ~2 L Duran bottles and a pilot scale (~138 L) raceway system. Initially, eight species of microalgae of different classes were assessed under nutrient sufficient growth conditions for the production of biomass and lipids in ~2 L Duran bottles. Two of the promising species (C. vulgaris and Nannochloropsis salina) were then further evaluated extensively under various conditions (i.e. salinity stress, different levels of nitrogen in growth media, continuous light and light-dark cycling). Based on these assessments C. vulgaris stood out as the best alga for further detailed study. C. vulgaris was evaluated for biomass production and lipid production. The consumption rates of major nutrients (N and P) were quantified. Biomass was characterized for elemental composition and energy content at the end of the growth cycle. A maximum lipid productivity of ~31 mg L-1 d-1 was attained in Duran bottle batch culture under nitrogen starvation in continuous light with a lipid content in the biomass of 66% (dry weight). This appears to be the highest lipid content reported for C. vulgaris grown in seawater and demonstrates an excellent ability of this alga to accumulate high levels of oil. Under a 12:12 h light-dark cycle, the lipid content and productivity in Duran bottle batch culture were decreased by 13% and 41%, respectively, relative to the case for continuous illumination. Energy content of the biomass produced in Duran bottle batch culture exceeded 30 kJ g-1 both in continuous light and the 12: 12 h light-dark cycle. Batch and continuous culture kinetics of C. vulgaris in the raceway system were assessed. The alga was subjected to various light regimes and nitrogen starvation conditions. Although the N starvation enhanced the lipid accumulation by 42% relative to nutrient sufficient growth in batch culture, the highest biomass and oil productivities were attained under nutrient sufficient conditions in continuous mode of cultivation. Under nutrient sufficiency in continuous culture with a constant illumination of 91 μmol.m-2s-1, the productivities of biomass and lipid in the raceway were >61 mg L-1 d-1 and >8 mg L-1 d-1, respectively. This work represents the first detailed study of C. vulgaris in a raceway pond in full strength seawater media. Previous studies of this alga were almost always carried out in freshwater media.