Identification and characterisation of luxury uptake proteins in Chlamydomonas for enhanced wastewater phosphorus removal

Abstract

Microalgal treatment processes form a promising alternative to traditional wastewater treatment processes for the removal of Phosphorus. Microalgae are able to treat wastewater to a threshold acceptable for future regulations without requiring exponential chemical input, while also leaving phosphorus locked in a recoverable form. Despite these benefits, current algal treatment processes suffer from a number of setbacks, notably low settlement capacity, growth rates, and the prohibitive space requirements required by raceway ponds due to a reliance on light for growth, limiting pond depth. This project aims to contribute towards the design of microalgal treatment processes centred around microalgal “luxury uptake” behaviours in which cells take in nutrients based on previous nutrient status. Working to understand the basic processes that control observed behaviour, this project took a genetics-based approach to identify and attempt to characterise proteins associated with phosphorus storage in Chlamydomonas reinhardtii. An investigation into the dynamics of polyphosphate (PolyP) during starvation through fractional extraction confirmed a potential link between the phosphorus starvation response gene Psr1, which oversees global starvation response to the absence of environmental phosphorus. Mutant Chlamydomonas reinhardtii with the Psr1-1 mutation, preventing activation of downstream response genes, were unable to mobilise a fraction of their internal polyphosphate reserves and preventing some extent of further growth. Following this finding, the C. reinhardtii genome was investigated for polyphosphate-related proteins. A RNAseq database was also examined using BLAST2GO with a shortlist of genes identified with characteristics indicative of putative polyphosphatase/ polyphosphate kinase activity. Identified proteins were targeted for preliminary expression study, with some progress made. Candidate Psr1-controlled genes were cloned to pJET but no further expression was possible due to reagent limitations. In parallel, candidate actin-related proteins with possible polyphosphate kinase activity were transformed to E. coli and limited evidence of expression was gained. With further work, these proteins could be purified, assayed, and characterised as a potential tool to actively manipulate microalgal nutrient status and improve future implementation of microalgal water treatment processes

Date of Award	16 Nov 2022
Original language	English
Awarding Institution	University of Bath
Supervisor	James Doughty (Supervisor) & Tom Arnot (Supervisor)