Enzymatic hydrolysis of steam pretreated bagasse : Enzyme preparations for efficient cellulose conversion and evaluation of physiochemical changes during hydrolysis

Wallace, Joshua (2013-03)

Thesis (MScEng)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Biomass recalcitrance represents the major technical obstacle in 2nd generation bio-ethanol production, as it impedes efficient enzyme action. Elucidating how the recalcitrant nature of lignocellulosic biomass inhibits the enzymatic degradation of cell wall polysaccharides could help to overcome these obstacles. This decrease in overall enzymatic hydrolysis performance in the industry, calls for the addition of different enzymes that work synergistically to alleviate slow cellulose to glucose conversion rates and low final glucose yields. Furthermore, high enzyme dosages (greater than 25FPU/g cellulose ~ approximately 75g enzyme/ kg cellulose) are required for rapid biomass conversion rates and complete hydrolysis, which have a negative effect on the viability of the process, as enzyme addition is a cost intensive part of biomass derived bio-ethanol production. In this regard, this study strived to improve the 2nd generation bio-ethanol industry by elucidating the slowdown phenomenon related to biomass recalcitrance during enzymatic hydrolysis. The substrate and enzyme properties that affect enzymatic hydrolysis rate and yield were investigated. Additionally, screening and optimization of a commercially available enzyme cocktail for bio-ethanol production from steam pretreated sugarcane bagasse (SB) was performed. Therefore, in the present study, sugarcane bagasse was steam pretreated and subjected to enzymatic hydrolysis (Spezyme + Novozym 188) whereafter the kinetic of the conversion was studied. Furthermore, new commercially available cocktails (Cellic CTec2, Optiflow) were compared, and the dosage of the preferred cocktail (Cellic CTec2) optimized for the production of bio-ethanol from steam pretreated sugarcane bagasse. Additionally, xylanase (Cellic HTec2 and Multifect Xylanase), pectinase (Pectinex Ultra) and surfactant (Tween 80) supplementation was investigated for positive effects on glucose and xylose release, and on ethanol production. Three distinctive phases of cellulose conversion rates were observed in succession of each other: (1) initial fast phase, (2) intermediate, slower phase, and (3) the slowest recalcitrant phase. The material was characterised by physicochemical analysis during each of the stages during enzymatic hydrolysis. The results indicated that the slowdown was caused by an increased lignin/cellulose ratio combined with changes in accessible surface area for enzymatic action (determined by Simons‘ staining). Xylanase supplementation was the only significant factor improving the cellulase cocktails. The positive effect of xylanase on glucose and xylose release (rate and yield) was probably due to synergism between xylanases and cellulases, as well as the increased accessible surface area due to hemicellulose removal. Decreased solid loadings % (w/v) were also favorable for maximal cellulose conversion yield and rate with all enzymes studied. The optimized cocktail (0.15ml/g WIS Cellic CTec2 + 0.213ml/g WIS HTec2) resulted in 79.2% ethanol yield compared with 55% for the control cocktail, at equivalent volumetric dosages. The increased ethanol production yield and rate with the optimized cocktail was due to the presence of greater amounts of cellulase, xylanase, β-glucosidase and oxidative enzymes (GH61s). These enzymes possibly increased the accessible surface area for cellulose degradation vs. the control cocktail. Altogether, the recalcitrance caused by increasing lignin/cellulose ratio with subsequent decreases in accessible surface area was the most important factors slowing down enzymatic hydrolysis rate and yield. However, it seems that newer generations of enzymes, such as CTec2, were less susceptible to the increasing biomass recalcitrance during enzymatic hydrolysis of lignocellulose resulting in higher product formation.

AFRIKAANSE OPSOMMING: Die inherente weerstand van biomassa verteenwoordig die grootste tegniese probleem in 2de generasie bio-etanol produksie. Deur te verstaan hoe die natuurlike weerstand van lignosellulosiese biomassa die ensiematiese hidroliese van die selwandsuikers vertraag kan help om hierdie weerstand te oorkom. In die lignosellulosiese bio-etanol indusitrie, lei hierdie vertraging in die ensiematiese reaksietempo en verlaagde suiker opbrengs, tot die gebruik van groot hoeveelhede ensieme. Bo en behalwe die vertraging van die reaksietempo, is die gebruik van hierdie hidrolitiese ensieme duur. Dus is die doel van hierdie projek, om die fisiese, asook chemiese verandering op die substraat wat tot die vertraging van die ensiematiese hidroliese lei, te ondersoek. Verder het hierdie studie daarna gestrewe om ‘n kommersiëel beskikbare optimale ensiem oplossing vir die produksie van bio-etanol, van stoom behandelde suikerriet bagasse, te identifiseer. Dus is die stoom behandelde suikerriet bagasse ensiematies gehidroliseer (Spezyme + Novozym 188) waarna die reaksie-kinetieka bestudeer was. Nuwe generasie ensieme (Cellic CTec2, Optiflow) was ook met mekaar vergelyk en 'n optimale ensiem dosis was vasgestel vir die verkose ensiem (Cellic CTec2). Ondermeer was die voordele van die toevoeging van addisionele ensieme (Cellic HTec2, Multifect Xylanase, Pectinex Ultra) en Tween 80, op glukose, xylose en etanol produksie, bestudeer. Drie onderskeidende fases van sellulose hidroliese tempo was opgemerk: (1) inisiële vinnige fase, (2) intermediêre fase en (3) verhardings fase. Die materie was onderworpe aan fisiese en chemiese analiese tydens al die stadiums gedurende ensiematiese hidroliese. Die resultate het aangedui dat die vertraging van ensiematiese hidroliese veroorsaak word deur verhoogde lignien/sellulose verhoudings, asook veranderings in die beskikbare oppervlakarea vir ensiem aksie. Xylanase toevoeging was die engiste addisionele ensiem wat 'n voordelige uitwerking op sellulose hidroliese deur ensieme, veroorsaak het. Hierdie positiewe interaksie was heelwaarskynlik die gevolg van sinergie tussen die xylanase en sellulase ensieme, sowel as 'n vergrote oppervlakarea vir ensiem aksie as gevolg van hemisellulose hidroliese. Die geoptimeerde ensiem mengsel (0.15ml/g WIS Cellic CTec2 + 0.213ml/g WIS HTec2) het tot 79.2% etanol opbrengs gelei, teenoor 55% vir die kontrole. Hierdie verhoogde opbrengs was moontlik as gevolg van die verhoogte ensiem aktiwiteit in die geoptimeerde ensiem. Die addisionele ensieme teenwoordig in die geoptimeerde ensiem mengsel het waarskynlik gelei tot groter oppervlakarea vir die sellulase ensieme, wat hoër etanol opbrengste tot gevolg gehad het. Ten slotte, biomassa verharding was meestal toegeskryf aan die verhoogte lignien/sellulose verhouding soos ensiem hidroliese geskied, wat 'n verlaagde oppervlakarea vir ensiem-aksie tot gevolg gehad het. Die verlaagde oppervlakarea is verantwoordlik vir vertraagde ensiem reaksie-tempos en lae opbrengste. Nie te min, dit lyk asof nuwer ensieme, soos CTec2, hierdie verharding kan bestry, wat lei tot verhoogde produk vormasie in vergelyking met die kontrole ensiem mengsels.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/79842
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