Engineering strategies for enhancement of bio-hydrogen production by phototrophic bacterium Rhodopseudomonas palustris
| dc.contributor.advisor | Pott, Robert William M. | en_ZA |
| dc.contributor.author | Du Toit, Jan-Pierre | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering. | en_ZA |
| dc.date.accessioned | 2021-03-08T11:42:45Z | |
| dc.date.accessioned | 2021-04-21T14:42:28Z | |
| dc.date.available | 2021-03-08T11:42:45Z | |
| dc.date.available | 2021-04-21T14:42:28Z | |
| dc.date.issued | 2021-03 | |
| dc.description | Thesis (PhD)--Stellenbosch University, 2021. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Die fotosintetiese bakterie Rhodopseudomonas palustris demonstreer ’n uitsonderlike metaboliese diversiteit en kan ’n wye verskeidenheid organiese samestellings verteer, insluitend die wat toksies is vir ander organismes. Anaerobiese fotoheterotrofiese groei lei tot ’n sellulêre redokswanbalans as gevolg van die akkumulasie van ’n oormaat reduseerekwivalente wat ontstaan vanuit substraatafbreking. Hierdie gunstige energietoestand dryf energie-intensiewe paaie insluitend nitrogenase-bemiddelde waterstofproduksie, wat die potensiaal wek vir ontwikkeling van ’n skoon, herwinbare energiebron vanuit ’n menigte organiese afvalstrome. Om die belofte van biowaterstofproduksie via fotofermentasie as deel van ’n nassente sirkulêre bio-ekonomie te realiseer, vereis tegnologiese ontwikkeling om twee sleutelkwessies aan te spreek: eerstens, lae volumetriese produksietempo’s, en tweedens die vermoë om langtermyn aaneenlopende produksie te handhaaf. Die navorsing hierin ondersoek strategieë wat uit prosesingenieurswese en optimering, materiaalkunde en metaboliese ingenieurswese bestaan, om hierdie hindernisse tot die proses se uitvoerbaarheid, te oorkom. Die eerste doelwit was om optimale temperature vir groei en waterstofproduksie deur R. palustris definitief te bepaal; ’n fundamentele prosesparameter met beduidende impak op ensiem- en metaboliese doeltreffendheid. Deur twee naby-verwante laboratoriumlyne na hoër temperature te akklimatiseer, is temperatuur optima 5 tot 10°C hoër as die wyd-aanvaarde 30 °C, waargeneem. Hoër optima is voordelig vir buitelug, sonverligte bioreaktors wat hoë temperature ondervind, en sodoende oorwegings vir temperatuurbeheer verlaag. By 35°C, het lyn CGA009 53% vinniger groei en 2.4-keer hoër waterstofproduksie getoon, teenoor by 30°C. Lyn ATH 2.1.37 het optimaal gegroei by 40°C, met 86% vinniger groei en 4-keer hoër produktiwiteit. In konteks van die lyne se hoë genoomooreenkoms, lyk dit asof langtermyn laboratorium kultivering temperatuurweerstand oor tyd verminder, wat seleksiekriteria vir hoë-temperatuur katalitiese-doeltreffende lyne inlig. Waterstofproduksie is nie groei-geassosieer in R. palustris nie, en nie-groeiende biomassa ondersteun hoër produksietempo’s as gevolg van verlaagde energieke kompetisie vanuit selverdeling. Immobilisasie van selle in ’n gepaste vaste matriks is dus ’n aantreklike manier om biomassa in ’n kontinue reaktor, onafhanklik van hidrouliese retensietyd, te behou. Hiertoe is ’n nuwe deursigtige kriojel, saamgestel uit polivinielalkohol (PVA), gekarakteriseer en optimeer om eienskappe gepas tot verstrikking van fotosintetiese bakterieë te lewer, met behulp van nuut-ontwerpte in situ beeldvormingstegnieke. Hoë deursigtigheid, meganiese veerkragtigheid en bioverenigbaarheid, en lae weerstand tot substraat diffusie is gedemonstreer. Geïmmobiliseerde R. palustris het hoër spesifieke waterstofproduksietempo’s getoon wat langer aangehou het as planktoniese kontroles. Verder het kontinue kulture produktiwiteit vir ten minste 67 dae gehandhaaf, wat geskiktheid van die PVA-kriojel vir langtermyn fotofermentasie verifieer en so ook wyer toepassings waar hoë bioverenigbaarheid en veerkragtigheid na wense is. Metaboliese ingenieurswese is ’n kragtige instrument om die produktiwiteit van spesifieke paaie te optimeer, insluitend nitrogenase-bemiddelde waterstofproduksie. Die gebrek aan doeltreffende instrumente vir genetiese manipulasie van R. palustris was dus geadresseer deur ontwikkeling van ’n vinnige elektroporasie-gebaseerde tegniek vir chromosoommodifikasie. Verskeie verfynings het die tyd wat dit neem om merkervrye lyne te genereer, na 12 dae gehalveer teenoor vorige metodes. Hierdie sisteem is gebruik vir die ooruitdrukking van alternatiewe nitrogenasegene met die potensiaal om lae ensiemdoeltreffendheid te verbeter; wat gehipoteseer is om tempo-beperkend te wees vir waterstofproduksie oor die algeheel. Deur sterk promotors stroomop van inheemse gene by te voeg, is tot en met 4000-maal ooruitdrukking bereik. Terwyl waterstofproduktiwiteit nie op die lange duur verbeter het nie, het hierdie instrumente verdere pogings gefasiliteer en R. palustris as ’n biotegniese raamwerk vir hoë waarde, energie-intensiewe bioprodukte bevorder. Hierdie vordering in temperatuuroptimering, bakteriese immobilisasie en metaboliese ingenieurswese as ’n geïntegreerde strategie het die potensiaal om rypwording van fotosintetiese biowaterstof tot groter skaal uitvoerbaarheid in staat gestel. | af_ZA |
| dc.description.sponsorship | The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at are those of the author and are not necessarily to be attributed to the NRF. | en_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.format.extent | xi, 161 pages : illustrations | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/110141 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Rhodopseudomonas palustris -- Growth | en_ZA |
| dc.subject | Hydrogen -- Biotechnology | en_ZA |
| dc.subject | Phototropism (Chemistry) | en_ZA |
| dc.subject | Anaerobic bacteria -- Growth | en_ZA |
| dc.subject | Renewable energy sources | en_ZA |
| dc.subject | Nitrogenase | en_ZA |
| dc.subject | UCTD | |
| dc.title | Engineering strategies for enhancement of bio-hydrogen production by phototrophic bacterium Rhodopseudomonas palustris | en_ZA |
| dc.type | Thesis | en_ZA |