Crossbreeding of natural Saccharomyces cerevisiae strains for enhanced bio-ethanol production

Blaauw, Domecia (2015-12)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: The fluctuating fuel prices, possible future shortage of fossil fuels, the increasing demand and the negative impacts on the ecosystem have all contributed to the search and development of alternatives during the last two decades. Biofuels, bio-ethanol in particular, is a reliable substitute for fossil fuel (petroleum) and can be produced from inexpensive, non-edible feedstock such as lignocellulosic biomass. Lignocellulose is, however, a problematic substrate as the hydrolysis results in inhibitor formation that hinders the fermentation ability of the fermenting microorganism (Saccharomyces cerevisiae yeast strain). The problem can be circumvented by the construction of robust S. cerevisiae strains that can withstand the effect of inhibitors in addition to exhibiting fermentation vigour, ethanol tolerance, inhibitor tolerance, osmotolerance and thermotolerance. In this study, four natural strains of S. cerevisiae (HR14, YI64, YI2 and MF15) with different superior characteristics (fermentation vigour, inhibitor-, osmo-, thermo- and ethanol tolerance) were selected for mating experiments to generate hybrid progeny with superior traits. The HO gene of the diploid homothallic yeast strains was disrupted to produce haploid heterothallic strains. Haploid strains with the opposite mating-types and displaying different characteristics were mated to produce hybrid strains with combined / superior characteristics. Six hybrid strains (YH1, YH2, YH3, MY3, MY5 and MY7) were selected for the screening process. The parent and hybrid strains were screened for fermentation vigour, ethanol tolerance, inhibitor tolerance, growth at temperatures above 30°C and osmotolerance. The YH3 and MY5 hybrid strains displayed the highest fermentation vigour (productivity) of the hybrid strains and were able to consume all available glucose (200 g/L) and produce approximately 100 g/L and 81 g/L ethanol, respectively. These hybrid strains did however, not display superior fermentation abilities when compared to the parent YI64 and YI2 strains as these strains produced the same amount of ethanol during the fermentation trials. The MY5 hybrid exhibited an inhibitor tolerance, similar to the MF15 parental strain in the presence of 25% inhibitor cocktail. The HR14 and YI64 parental strains and their YH1, YH2 and YH3 hybrid strains were unable to grow and ferment in the presence of 25% inhibitor cocktail. None of the strains was able to grow and ferment in the presence of 10% ethanol. Some inherited characteristics (fermentation vigour and inhibitor tolerance) of the hybrid strains were not superior to that displayed by the parental strains. The inherited osmotolerance and thermotolerance were, however, superior to that displayed by the parent strains as the best performing hybrids managed to grow at 43°C and grew slightly faster than the parent strains in the presence of 65% glucose. The mating experiments yielded hybrid strains with combined characteristics such as fermentation vigour, inhibitor tolerance, osmotolerance and thermotolerance. Mating of yeast strains to combine and generate superior traits in the progeny is thought to be the best method to use. Hybrid strains generated during this method are produced through minimum gene manipulation. The use of these strains in the production of bio-ethanol should not cause public concerns nor should it infringe on legislation. The mating experiments can be followed by an adaptation to inhibitory compounds, as the hybrid strains in this study were slightly more tolerant to ethanol during the fermentation trials after adaptation.

AFRIKAANSE OPSOMMING: Die wisselende brandstofpryse, moontlike toekomstige tekort van fossielbrandstowwe, die toenemende aanvraag en die negatiewe impak op die ekosisteem het alles bygedra tot die soek en die ontwikkeling van alternatiewe gedurende die laaste twee dekades. Biobrandstof, bio-etanol in besonder, is 'n betroubare plaasvervanger vir fossielbrandstof (petroleum) en kan geproduseer word van goedkoop, nie-eetbare roumateriaal soos lignosellulose biomassa. Lignosellulose is egter 'n problematiese substraat, want die hidrolise daarvan lei tot die vorming van inhibitore wat die fermentasievermoë van die vergistende mikroörganisme (Saccharomyces cerevisiae gisras) verhinder. Die probleem kan egter omseil word deur die konstruksie van robuuste S. cerevisiae gisrasse wat die effek van inhibitore kan weerstaan bykomend tot die toon van fermentasiekrag, etanolverdraagsaamheid, inhibitorverdraagsaamheid, osmotoleransie en termotoleransie. In hierdie studie is vier natuurlike stamme van S. cerevisiae (HR14, YI64, YI2 en MF15) met verskillende voortreflike eienskappe (fermentasiekrag, inhibitor-, osmo-, termo- en etanol verdraagsaamheid) gekies vir parings eksperimente om ʼn hibried nageslag te genereer met verbeterde eienskappe. Die HO-geen van die diploïede homotalliese gisstamme was ontwrig om haploïede heterotalliese stamme te produseer. Haploïede stamme met die teenoorgestelde paring-tipes wat verskillende eienskappe getoon het, was gekruis om hibried stamme met gekombineerde / verbeterde eienskappe te produseer. Ses hibried stamme (YH1, YH2, YH3, MY3, MY5 en MY7) was gekies vir die keuringsproses. Die ouers en hibried stamme was gekeur vir hul fermentasiekrag, etanolverdraagsaamheid, inhibitorverdraagsaamheid, groei by temperature bo 30°C en osmotoleransie. Die YH3 en MY5 hibried stamme het die hoogste fermentasiekrag (produktiwiteit) van al die hibried stamme vertoon en was in staat om alle beskikbare glucose (200 g/L) te verbruik en het ongeveer 100 g/L en 81 g/L etanol, onderskeidelik geproduseer. Hierdie hibried stamme het egter nie beter fermentasie vermoëns in vergelyking met die ouers YI64 en YI2 vertoon nie, want die ouers het dieselfde hoeveelheid etanol tydens die fermentasie proewe geproduseer. Die hibried MY5 het 'n inhibitor verdraagsaamheid, soortgelyk aan die MF15 ouerstam in die teenwoordigheid van 25% inhibitor mengsel getoon. Die HR14 en YI64 ouer stamme en hul YH1, YH2 en YH3 hibried stamme was nie in staat om te groei en te fermenteer in die teenwoordigheid van 25% inhibitor mengsel nie. Nie een van die stamme was in staat om te groei en te fermenteer in die teenwoordigheid van 10% etanol nie. Sommige oorgeërfde eienskappe (fermentasiekrag en inhibitorverdraagsaamheid) van die hibried stamme was nie beter as wat vertoon was deur die ouer stamme nie. Die oorgeërfde osmotoleransie en termotoleransie was egter beter as wat vertoon was deur die ouer stamme, want die bes presterende hibriede het daarin geslaag om te groei by 43°C en het effens vinniger as die ouer stamme in die teenwoordigheid van 65% glukose gegroei. Die parings eksperimente het dus hibried stamme opgelewer met gekombineerde eienskappe soos fermentasiekrag, inhibitorverdraagsaamheid, osmotoleransie en termotoleransie. Paring van gisstamme om verbeterde eienskappe in die nageslag te kombineer en te genereer is van mening dat die beste metode om te gebruik. Hibried stamme wat tydens hierdie metode gegenereer word bevat minimum geen manipulasie. Die gebruik van hierdie stamme in die produksie van bio-etanol hoort nie openbare kommer veroorsaak of teen wetgewing gaan nie. Die paringeksperimente kan gevolg word deur 'n aanpassing teen inhiberende verbindings, want die hibried stamme in hierdie studie was effens meer verdraagsaam teen etanol tydens die fermentasie proewe na die aanpassing.

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