Engineered yeast and microalgae mutualisms: Synthetic ecology applied to species isolated from winery wastewater

Simpson, Zoe Faith (2018-03)

Thesis (MSc)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Large volumes of winery wastewater, classified as biodegradable industrial effluent, are generated annually. The development of a cost-effective treatment system is difficult due to the variable and batch nature of winery wastewater. Research has focused on the development of dynamic biological treatment systems using microorganisms including yeast and microalgae, as it has become apparent that these microbes have bioremediation capabilities in various wastewaters. However, no biological winery wastewater treatment system, employing multi-species ecosystems with known species of yeast and microalgae, currently exists. In this study, yeast and microalgae were previously isolated from natural winery wastewater. The first aim of this study was to characterise the bioremediation potential of the yeast, Saccharomyces cerevisiae, and microalga, Parachlorella beijerinckii, in synthetic and raw winery wastewater. P. beijerinckii was physiologically characterised and was able to tolerate salinity and ethanol levels commonly associated with winery wastewater, making it a suitable candidate for further bioremediation studies. Both S. cerevisiae and P. beijerinckii were able to decrease the chemical oxygen demand of winery wastewater and P. beijerinckii monoculture was able to increase the pH of the acidic wastewater. S. cerevisiae out-competed P. beijerinckii in co-culture growth experiments. Interestingly, yeast growth was improved in the presence of the microalgae in this system, suggesting a potential for symbiotic association. The increased yeast growth however had no impact on the bioremediation potential of the co-culture system. To overcome this drawback, a synthetic ecology approach was used to engineer stable symbiotic associations between these evolutionarily unrelated strains of yeast and microalgae. Engineered mutualisms between S. cerevisiae and P. beijerinckii were established under strongly selective conditions based on the nutrient exchange of carbon and nitrogen. These mutualistic associations were relatively easy to establish as the complementary metabolic abilities of each organism were key elements in the mutualism design. The impact of temperature and pH were assessed in these obligatory mutualistic conditions to determine whether the co-culture functions optimally in specific environmental conditions and whether such conditions are similar or different from the optimal conditions required for single species growth. Experiments were first conducted in small scale and continued in larger scale bioreactor studies. The bioreactor conditions were evaluated to generate a more constant continuous culture system. Such continuous culture system would provide an ideal tool to conduct studies on the evolutionary development of mutualistic associations, and may be the first step in developing a multi-species approach to winery wastewater treatment with enhanced bioremediation capabilities. We propose that in the long run such co-culture systems might serve to overcome the limitations associated with single culture system and might improve biotechnological processes.

AFRIKAANSE OPSOMMING: Groot volumes kelderafvalwater, geklassifiseer as bioafbreekbare nywerheidsafvloeisel, word jaarliks gegenereer. Die ontwikkeling van 'n koste-effektiewe behandelingstelsel is moeilik as gevolg van die konstante variasie en groep-aard van kelderafvalwater. In die verlede het navorsing gefokus op die ontwikkeling van dinamiese biologiese behandelingstelsels deur mikroörganismes te gebruik, insluitend gis en mikroalge. Dit het duidelik geword dat hierdie mikrobes bioremediëringsvermoëns in verskeie afvalwater het. Daar bestaan egter geen biologiese kelderafvalwaterstelsel wat bekende spesies gis en mikroalge gebruik as multi-spesiesekosisteme nie. In hierdie studie is gis en mikroalge, voorheen geïsoleer uit natuurlike kelderafvalwater, gebruik. Die eerste doelwit van die studie was om die bioremediëringspotensiaal van die gis, Saccharomyces cerevisiae, en die mikroalge, Parachlorella beijerinckii, in sintetiese en rou kelderafvalwater te karakteriseer. P. beijerinckii is fisiologies gekarakteriseer en was in staat om die sout- en etanolvlakke in kelderafvalwater te weerstaan en dus is P. beijerinckii ‘n geskikte kandidaat vir verdere bioremediëringsstudies. Beide S. cerevisiae en P. beijerinckii was in staat om die chemiese suurstofaanvraag te verminder in kelderafvalwater en P. beijerinckii monokultuur was in staat om die suur pH van die water te verhoog. S. cerevisiae het in multikultuur groei-eksperimente beter as P. beijerinckii gegroei. Interessant genoeg is gisgroei verbeter in die teenwoordigheid van die mikroalge, wat 'n potensiaal vir simbiotiese assosiasie voorstel. Die verhoogde gisgroei het egter geen impak gehad op die bioremediëringspotensiaal van die ko-kultuurstelsel nie. Om hierdie nadeel te oorkom, is 'n sintetiese ekologiebenadering gebruik om stabiele simbiotiese assosiasies tussen hierdie evolusionêr onverwante stamme van gis en mikroalge te bou. S. cerevisiae en P. beijerinckii is onder sterk selektiewe toestande toegelaat om ‘n mutualistiese verhouding te vorm, gebaseer op die uitruiling van koolstof en stikstof. Hierdie mutualistiese assosiasies was relatief maklik om te vestig, aangesien die komplementêre metaboliese vermoëns van elke organisme sleutelelemente in die mutualisme-ontwerp was. Die impak van temperatuur en pH op hierdie obligate mutualistiese kondisies is geëvalueer om vas te stel of die ko-kultuur optimaal funksioneer onder spesifieke omgewingskondisies en of sulke kondisies ooreenstem of verskil van die optimale toestande wat benodig word vir enkelspesiegroei. Die eksperimente is eers op klein skaal uitgevoer, gevolg deur bioreaktorstudies op groot skaal. Die bioreaktor kondisies is uitgevoer om 'n meer konstante aaneenlopende kultuurstelsel te genereer. So 'n deurlopende kultuurstelsel sal 'n ideale hulpmiddel wees om studies uit te voer oor die evolusionêre ontwikkeling van mutualistiese assosiasies. Hierdie kan die eerste stap wees in die ontwikkeling van 'n multi-spesie benadering tot kelderafvalwaterbehandeling met verhoogde bioremediëringsvermoëns. Ons stel voor dat hierdie ko-kultuurstelsels op die langtermyn die beperkinge geässosieer met enkelkultuurstelsel kan oorkom en sodoende biotegnologiese prosesse verbeter.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/103669
This item appears in the following collections: