The development of recombinant fungal enzyme cocktails for the hydrolysis of cellulosic waste products

Dobson, Rosemary Anne (2014-04)

Thesis (MSc)--Stellenbosch University,2014.

Thesis

ENGLISH ABSTRACT: Biofuels, such as bioethanol, provide an alternative, environmentally friendly transportation fuel. Renewable energy sources, such as lignocellulosic material, are therefore being explored for the production of biofuels, since they offer an attractive and sustainable source for bioconversion processes. However, the major obstacle in the use of lignocellulosic biomass is its recalcitrant nature, which decreases the enzyme accessibility to cellulose and thus affects the overall hydrolysis process. Current commercial enzyme cocktails are not yet sufficient to promote hydrolysis on an industrial scale, thus hampering biofuel production. A number of cellulase enzymes are needed to act in synergy to obtain complete hydrolysis of lignocellulosic material. The enzymatic hydrolysis of cellulose requires the synergistic action of three cellulase enzymes namely endoglucanases, exoglucanases and β-glucosidases. However, cellulolytic organisms do not produce significant amounts of ethanol, whereas strong fermentative organisms don’t produce enzymes for cellulose hydrolysis. A need has therefore arisen to develop recombinant technologies to obtain maximum production of cellulolytic enzymes that can be used (exogenously) in combination with a fermentative organism. Paper sludge is a lignocellulosic waste material that is generated in large quantities by the pulp and paper industry. Non-hazardous paper sludge can be converted to fermentable sugars, which can then be fermented to bioethanol. Biological conversion of paper sludge requires no pre-treatment, making it an ideal substrate for industrial use. The development of enzyme cocktails for efficient hydrolysis of paper sludge is therefore important in the pursuit of second-generation bioethanol production. A recombinant cellulase enzyme cocktail tailored for the degradation of paper sludge was developed using cellulases from recombinant Aspergillus niger and Saccharomyces cerevisiae strains. The recombinant strains were cultured and their supernatants used to develop an enzyme cocktail based on activity ratios. The core cellulases in the optimal cocktail included a cellobiohydrolase I, cellobiohydrolase II, endoglucanase and β-glucosidase. The enzyme cocktails were subsequently evaluated on triticale, Avicel and wheat bran. The activities (in Filter Paper Units) for the final cocktails were 0.7 and 0.45 for the CbhI:CbhII:EgA:Bgl1 and CbhI:CbhII:EgA:Bgl2 cocktails, respectively. The optimum enzyme ratio (based on protein concentrations) for the CbhI:CbhII:EgA:Bgl1 cocktail was 7.4:6.6:1:208 and 7.4:6.6:1:41 for the CbhI:CbhII:EgA:Bgl2 cocktail. Overall, hydrolysis with the Bgl2 cocktail allowed for longer incubation times and an improved degree of saccharification when the enzyme concentration was doubled. Comparison of paper sludge hydrolysis results with those from Avicel hydrolysis highlight the need to tailor enzyme cocktails based on natural substrates. Two industrial amylolytic S. cerevisiae yeast strains were compared in an SSF (10% wheat bran) process, using the Bgl2-cocktail. The maximum ethanol yield produced by S. cerevisiae S2[TLG, SFA] and S. cerevisiae MH1000[TLG, SFA], in the presence of the 1x enzyme cocktail, was 5.72 g.l-1 and 5.45 g.l-1, respectively. This study demonstrated that the addition of the recombinant cellulase cocktail improved the ethanol yields by 8.69% in the SSF process and that the S. cerevisiae S2[TLG, SFA] and MH1000[TLG, SFA] strains efficiently converted starch to ethanol. To our knowledge, this is the first report of the use of individual enzymes from recombinant strains, for the hydrolysis of paper sludge and wheat bran. This study has provided insight into the hydrolysis of cellulosic materials, using recombinant cellulase cocktails. The knowledge obtained could be applied in optimising lignocellulose hydrolysis, for efficient sugar release and ultimately improving ethanol production by recombinant yeast strains. This study also demonstrates the potential of using agricultural and industrial wastes as lignocellulosic feedstocks for biofuels production.

AFRIKAANSE OPSOMMING: Biobrandstof, bv.bioetanol, bied 'n alternatief vir 'n omgewingsvriendelike vervoerbrandstof. Hernubare energiebronne, soos lignosellulose-ryke materiaal, word dus vir die produksie van biobrandstof ondersoek, aangesien hulle 'n aantreklike en volhoubare bron vir bio-omskakelingsprosesse bied. Die grootste struikelblok in die gebruik van lignosellulose-ryke biomassa is hul weerstandige natuur, wat die hidrolitiese proses beïnvloed. Huidige kommersiële ensiemmengels is onvoldoende vir substraathidrolise op 'n industriële skaal wat dus biobrandstofproduksie belemmer. 'n Aantal sellulase ensieme, in sinergistiese samewerking is nodig vir volledige hidrolise van lignosellulose-ryke materiaal. Die ensiematiese hidroliese van sellulose vereis die sinergistiese aksie van drie sellulase ensieme naamlik endoglukanases, eksoglukanases en β-glukosidases. Sellulolitiese organismes produseer egter nie beduidende hoeveelhede etanol nie, en fermenterende organismes produseer nie sellulolitiese ensieme nie. Hiervolgens het 'n behoefte ontstaan om rekombinante tegnologie te ontwikkel waardeur groot hoeveelhede ensieme geproduseer kan word, wat dan eksogenies in aanvulling tot 'n fermenterende organisme gebruik kan word. Papierslyk is 'n lignosellulose-ryke afvalmateriaal wat in groot hoeveelhede deur die pulp-en-papierbedryf gegenereer word. Onskadelike papierslyk kan na fermenteerbare suikers omgeskakel word, wat dan na bioetanol gefermenteer kan word. Biologiese omskakeling van papierslyk vereis geen vooraf-behandeling nie en maak dit 'n ideale substraat vir industriële gebruik. Die ontwikkeling van 'n ensiemmengsel vir doeltreffende hidrolise van papierslyk is dus belanrik vir die nastrewing van tweede-generasie etanol produksie. 'n Rekombinante sellulase ensiemmengsel, aangepas vir die afbraak van papierslyk, is ontwikkel deur gebruik te maak van sellulases van rekombinante Aspergillus niger en Saccharomyces cerevisiae rasse. Die rekombinante stamme is gekweek en hul bostande gebruik om 'n ensiemmengsel, gebaseer op aktiwiteitsverhoudings, te ontwikkel. Die kern sellulases in die optimale mengsel sluit 'n sellobiohidrolase I, sellobiohidrolase II, endoglukanase en β-glukosidase in. Die ensienmengsels is geëvalueer op korog, Avicel en koringsemels. Die aktiwiteite (in Filter Paper eenhede) vir die finale ensiemmengsels was 0,7 en 0,45 vir die CbhI:CbhII:EgA:Bgl1 en CbhI:CbhII:EgA:Bgl2 ensiemmengsel, onderskeidelik. Die optimum ensiem verhouding (gebaseer op proteïen konsentrasies) vir die CbhI:CbhII:EgA:Bgl1 ensiemmengsel was 7.4:6.6:1:208 en 7.4:6.6:1:41 vir die CbhI:CbhII:EgA:Bgl2 ensiemmengsel. Algehele, hidrolise met die Bgl2 ensiemmengsel het 'n beter graad van versuikering te weeggebring met “n toename in inkubasie tyd en ‘n verdubbelling in ensiem konsentrasiel. Vergelyking van papierslyk hidrolise resultate met dié van Avicel hidrolise beklemtoon die noodsaaklikheid daarvan om ensiemmengsels aan te pas gebaseer op natuurlike materiale. Twee industriële amilolitieseS. cerevisiae-gisrasse is met mekaar vergelyking in 'n GVF (Gelyktydige Versuikering en Fermentasie) met 10 % koringsemels in die teenwooedigheid van die Bgl2-ensiemmengsel. Die maksimum etanolopbrengs deur S. cerevisiaeS2 [TLG, SFA] en S. cerevisiae MH1000[TLG, SFA], in die teenwoordigheid van die sellulase ensiemmengsel, was 5,72 g.l-1 en 5,45 g.l-1, onderskeidelik. Hierdie studie het getoon dat die toevoeging van die rekombinante sellulase ensiemmengsel die etanol opbrengs verbeter met 8,69% in die GVF proses en dat die S. cerevisiae S2 [TLG, SFA] en MH1000 [TLG, SFA] rasse doeltreffend stysel na etanol omskakel. Volgens ons kennis is dit die eerste verslag oor die gebruik van individuele ensieme vanaf rekombinante rasse vir die hidrolise van papierslyk en koringsemels. Hierdie studie lewer insig tot die hidroliese van sellulose-ryke materiaal deur rekombinante sellulase ensiemmengsels. Die kennis kan in die optimisering van lignosellulose hidrolise vir doeltreffende suikervrystelling en uiteindelik die verbetering van etanolproduksie deur rekombinante gisrasse toegepas word. Hierdie studie toon ook die potensiaal van landbou-en industriële afval as lignosellulose substrate vir biobrandstofproduksie.

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