Doctoral Degrees (Chemical Engineering)

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Browsing Doctoral Degrees (Chemical Engineering) by Subject "Anaerobic bacteria -- Growth"

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    Engineering strategies for enhancement of bio-hydrogen production by phototrophic bacterium Rhodopseudomonas palustris
    (Stellenbosch : Stellenbosch University, 2021-03) Du Toit, Jan-Pierre; Pott, Robert William M.; Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering.
    ENGLISH ABSTRACT: The photosynthetic bacterium Rhodopseudomonas palustris demonstrates an exceptional metabolic diversity and is capable of consuming a wide variety of organic compounds including those toxic to other organisms. Anaerobic photoheterotrophic growth results in a cellular redox imbalance due to accumulation of excess reducing equivalents arising from substrate breakdown. This favourable energy state drives energy-intensive pathways including nitrogenase-mediated hydrogen production, raising the potential for generation of a clean renewable energy source from a multitude of organic waste streams. Realising the promise of biohydrogen production via photofermentation as part of a nascent circular bioeconomy requires technological development to address two key issues: i. low volumetric production rates, and ii. means of sustaining long-term continuous production. The research herein explores strategies comprising process engineering and optimisation, materials science and metabolic engineering to overcome these barriers to process feasibility. The first objective was to definitively determine optimal temperatures for growth and hydrogen production by R. palustris; a fundamental process parameter with significant impact on enzyme and metabolic efficiency. By acclimatising two closely-related laboratory strains to higher temperatures, temperature optima 5 to 10°C higher than the widely-accepted 30°C were seen. Higher optima are advantageous for outdoor sunlit bioreactors which experience high temperatures, reducing considerations for temperature control. At 35°C, strain CGA009 showed 53% faster growth and 2.4-fold higher hydrogen production versus 30°C. Strain ATH 2.1.37 grew optimally at 40°C, with 86% faster growth and 4-fold higher productivity. In context of the strains’ high genome similarity, long-term laboratory cultivation seems to diminish temperature resistance over time, informing selection criteria for high-temperature, catalytically-efficient strains. Hydrogen production is not growth-associated in R. palustris, and non-growing biomass supports higher production rates due to reduced energetic competition from cell division. Immobilisation of cells in a suitable solid matrix is thus an attractive means of retaining biomass in a continuous reactor independent of hydraulic retention time. To this end, a novel transparent cryogel composed of poly vinyl-alcohol was characterised and optimised to yield properties suited to entrapment of photosynthetic bacteria, aided by newly-devised in situ imaging techniques. High transparency, mechanical resilience and biocompatibility, and low resistance to substrate diffusion was demonstrated. Immobilised R. palustris showed higher specific hydrogen production rates which continued longer than planktonic controls. Continuous cultures further maintained productivity for at least 67 days, verifying suitability of the PVA cryogel for long-term photofermentation and indeed wider applications where high biocompatibility and resilience is desirable. Metabolic engineering is a powerful tool for optimising the productivity of specific pathways including nitrogenase-mediated hydrogen production. The lack of efficient tools for genetic manipulation of R. palustris was thus addressed by development of a rapid, electroporation-based technique for chromosomal modification. Multiple refinements effectively halve the time required to generate markerless strains to 12 days versus previous methods. This system was used to over-express alternative nitrogenase genes with the potential to improve low enzyme efficiency; hypothesised to be rate-limiting for hydrogen production overall. By insertion of strong promoters upstream of native genes, up to 4000-fold overexpression was achieved. While hydrogen productivity was not ultimately improved, these tools facilitate further efforts and advance R. palustris as a biotechnological chassis for high value, energy-intensive bioproducts. These advancements in temperature optimisation, bacterial immobilisation and metabolic engineering as an integrated strategy have the potential to enable maturation of photosynthetic biohydrogen towards larger-scale viability.