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    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, Alexander
    Many 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.
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    Elevating phosphorus accumulation in waste stabilisation pond algae : 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, 2019) Sells, Matthew
    Facultative waste stabilisation ponds (WSP) are used globally for wastewater treatment due to their low cost and simple operation. While WSPs can be effective at removing organic pollutants and pathogens, phosphorus removal is typically poor. Algae that are common in WSPs are known to accumulate phosphorus and increase their phosphorus content in the biomass from 1% up to 3.8% (gP/gSS), which is believed to be from the production of intracellular polyphosphate granules. This phenomenon, known as luxury uptake, may be possible to manipulate to improve phosphorus removal in WSPs; however, its occurrence is sporadic and poorly understood. This PhD thesis was undertaken to investigate the conditions that influence phosphorus accumulation in WSP algae. Phosphorus accumulation was quantified using two methods: (1) the traditional phosphorus content in the biomass (gP/gSS), and (2) a new image analysis method developed in this thesis that quantifies stained polyphosphate granules within individual algal cells (μm2 granule/μm2 cell). Following a literature review and screening experiments that sought to identify variables that could affect the phosphorus content in the biomass (gP/gSS), six variables: temperature, phosphorus concentration, light intensity, mixing intensity, organic load, and pH were comprehensively examined using 40 batch factorial experiments (26-1) and a mixed genus culture from a full-scale WSP. Nine variables and interactions had a significant effect on the phosphorus content in the biomass and were incorporated into a regression equation. This ‘mixed genus’ regression equation was tested against literature data, where seven out of the eight batch experiments from the literature were successfully predicted. In order to identify if the batch findings could be applied to a continuous process, which is more typical of full-scale WSPs, a bench-scale novel ‘luxury uptake’ process was designed, built, and operated under five different scenarios. The regression equation successfully predicted the experimental results for three of the five conditions examined. It was theorised that differences in behaviour at the genus level might explain why all five conditions were not successfully predicted. In an attempt to improve the prediction capability, the ‘black-box’ of mixed genus analysis was ‘opened’ to allow the effects of variables on phosphorus accumulation at the genus level to be directly examined. To achieve this, a new image analysis method was developed that quantified stained polyphosphate granules in individual algal cells. To ensure the granules being measured were indeed polyphosphate, algal cells were analysed using transmission electron microscopy coupled with energy dispersive X-ray spectroscopy, which confirmed the granules contained higher levels of phosphorus compared to the remaining cell. The image analysis method was then used to quantify stained polyphosphate granules in individual cells from the 40 batch factorial experiments mentioned previously. The results using the image analysis method showed that, for the five most abundant algal genera, Micractinium/Microcystis had the highest average accumulation of polyphosphate granules (17% μm2 granule/μm2 cell), followed by Scenedesmus (12%), Pediastrum (11%), Monoraphidium (8%), and Actinastrum (4%). Although none of the genera studied had the same combination of significant variables, all five genera preferred a high phosphorus concentration to elevate polyphosphate granule accumulation. Furthermore, a high light intensity, high organic load, or high temperature was preferred by the algae if the variable was significant for that genus. The culture used in the bench-scale continuous flow ‘luxury uptake’ process originated from a mixed genus WSP culture; however, it had become dominated by the Scenedesmus genus. Therefore, the regression equation was refined to use the batch data for this genus alone. This new Scenedesmus regression equation was compared against the experimental data from the ‘luxury uptake’ process previously mentioned. Polyphosphate granule accumulation was now successfully predicted in all five experimental conditions at the 95% confidence level. This improved prediction capability indicates that an understanding of the algal genus present in a WSP system is required for accurate predictions of the phosphorus accumulation to be obtained, and the batch data can indeed be applied to a continuous process. An unexpected result of the research was that, contrary to what was believed in the literature, an increase in the phosphorus content in the biomass did not necessarily increase the polyphosphate granule accumulation. Further examination identified that individual cells from the same algal species had varying polyphosphate granule contents from 0% to over 20% (μm2 granule/μm2 cell) when exposed to the same conditions. This variation was hypothesised to be from cellular functions influencing the granules differently depending on the individual alga’s cell cycle. In addition, when the phosphorus content in the biomass was increased above 2.1% (gP/gSS), no significant effect on the average quantity of polyphosphate granules was observed. This finding indicates that other forms of phosphorus storage must be responsible for attaining a highly elevated phosphorus content in the biomass.