Understanding microalgal self-aggregation for economic and sustainable harvesting : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering at Massey University, Manawatū, New Zealand

Abstract

The potential of microalgae biotechnology is often limited by the costs of biomass harvesting. Chemical flocculation is often used to increase particle size and improve harvesting efficiency; however, flocculants risk contamination and limit downstream applications. Self-aggregation offers a biological alternative to flocculation: many unicellular microalgae form multicellular structures as a defence response to stress. Predator-released infochemicals are especially potent triggers that act at extremely low concentrations and could become economic alternatives to traditional flocculants. Literature showed that predator-induced aggregation can involve changes to motility, the cell wall, the extracellular matrix, and extracellular polysaccharides, but the identity of active compounds and the specific mechanisms of infochemical perception and aggregation remain poorly understood. As a first step to this research, an assay was developed to induce a significant and reproducible self-aggregation response in microalgae. Thus, for the first time, the aggregation responses of Chlamydomonas reinhardtii, Tetradesmus obliquus, and Chlorella vulgaris were systematically studied under exposure to the live zooplankton Daphnia or concentrated Daphnia extracts. C. reinhardtii cultures treated with Daphnia extract formed palmelloid colonies of 4–32 cells within 24 hours, with the mean colony size exceeding four cells. This rapid and uniform response was ideal for use in experimental transcriptomic and metabolomic studies. Subsequent transcriptomic analyses of C. reinhardtii revealed significant downregulation of flagella-related genes (e.g., genes encoding flagella-associated proteins or intraflagellar transport components) in palmelloid-rich cultures relative to unicellular controls. Changes were also observed in genes encoding matrix metalloproteinases (MMPs), pherophorins (PHCs), calcium signalling, the ubiquitin-proteasome system, and TRP channels. Mutant phenotypes confirmed the likely role for MMP13 as a key MMP required for cell wall degradation, and established TRP13 as a new candidate for external signal sensing linked to palmelloid formation. These findings suggested, for the first time, that predator infochemicals induced palmelloid formation by influencing the kinases and protein turnover controlling flagella assembly, thus preventing flagella reassembly following cell division. Without flagella, cells were not able to degrade the mother cell wall and instead continued to divide within palmelloid colonies. Untargeted metabolomic profiling using GC-MS was also carried out on Daphnia-extract treated C. reinhardtii cultures, and unicellular controls, for the first time. Hundreds of peaks were detected, from which 22 consistent compounds were identified. However, most were also present in the Daphnia extract, underscoring the difficulty of distinguishing between predator- and algal-derived compounds. Despite these challenges, several amino acids, including tyramine and tyrosine, and urea were identified as possible candidate Daphnia infochemicals, while amino acids, polyamines, and fatty acids could represent C. reinhardtii alarm signals during aggregation. If validated through targeted bioassays, these compounds could be used for controlled induction of palmelloids prior to harvesting or for further mechanistic studies. The potential of using self-aggregation for harvesting was finally evaluated based on the observed aggregation dynamics in C. reinhardtii: the extremely low reported active concentrations of Daphnia infochemicals would make induction of self-aggregation more economically sustainable compared to chemical flocculation, and the natural origin may remove the barrier that exists for common flocculants to food and feed applications. However, using live zooplankton or extracts would likely require biomass at significant volumes that would be economically unviable. Instead, synthetic infochemicals could be a more affordable option. Light, temperature, cell density, the type of infochemical, and the timing of addition are key operational parameters to be controlled and their impacts on self-aggregation responses are species-specific. The aggregate phenotypes (i.e., colonies or adhered cell flocs) also determine suitability for certain harvesting processes, such as sedimentation, filtration, and centrifugation, as the size, shape, and density of aggregates, and presence of polymers can influence harvesting efficiency. Overall, this thesis developed an aggregation assay protocol and provided the first combined transcriptomic and metabolomic investigation of Daphnia-induced palmelloid formation in C. reinhardtii. This research showed that predator-derived extracts could reproducibly trigger aggregation, and identified new candidate molecular regulators and pathways, and crucial factors to be considered when designing an aggregation-based harvesting system. While the self-aggregation response was not scaled, the variability observed across species and strains presents both challenges and opportunities for designing effective bespoke systems. Together, these findings provide new insights into the mechanisms regulating palmelloid formation and establish a foundation to design protocols and evaluate the technical and economic feasibility of a self-aggregation-based harvesting method.