Study of enzymatic hydrolysis to improve sugar production from steam-pretreated sweet sorghum bagasse and triticale straw

Thesis (MScEng)--Stellenbosch University, 2013.ENGLISH ABSTRACT: The production of ethanol from lignocellulosic biomass (second generation biofuels) is currently being investigated worldwide for the development of biofuels that can be used as an alternative to fossil fuels. This follows the growing concerns over potential land protection, conflicts with food suppliers as well as arguments involving the reduction of greenhouse gas emission. Sweet sorghum bagasse and triticale straw are especially promising as these crops can produce high yields under a wide variety of conditions. They also have no food value and lower greenhouse gas emission compared to other feedstocks, making them attractive for second generation bio-ethanol production. Among the numerous different possibilities, enzyme-based processes are one of the most promising for the production of biofuel. This is mainly due to its specificity, higher yields, generation of lower amounts of inhibitory compounds formed during hydrolysis and fermentation as well as the high potential of improvement through biotechnology. At present, the enzymatic hydrolysis of lignocellulosic materials is a bottleneck in the process of bio-ethanol production and the associated production costs are still too high, thereby preventing the commercialisation of this process. The selection of feedstock varieties (selected based on their response to pretreatment and agronomic data) and optimisation of enzymatic hydrolysis for specific feedstock and pretreatment conditions is thus of key importance for obtaining an efficient sugar yield and therefore, ethanol yield. A need also exists for developing realistic and quick methods to evaluate feedstock digestibility. Increasing research is being directed towards the development of high-throughput systems based on distributing milled pretreated material into micro-well plates to evaluate multiple variables during enzymatic hydrolysis. Since the milling can be considered as an additional treatment, the application of other methods with minimum impact on lignocellulose structure is preferred. Such a method is based on handsheets as described in the TAPPI standard methodology. In this context, the main aim of this thesis was to improve enzymatic hydrolysis by identifying optimum enzyme combinations that are specific for feedstock varieties and steam-explosion pretreatment conditions of sweet sorghum bagasse and triticale straw. The specific goal was to obtain an optimum combination of enzyme preparations at minimum dosage that provide 80% of cellulose conversion. Based on preliminary results, two enzyme preparations characterised as cellulase and xylanase were selected for optimisation by central composite design and subsequent response surface plots with the use of Design Expert® software. A second aim of the thesis was to develop a micro-assay method that incorporates an additional homogenisation step prior to making the handsheets. The effects of different homogenisation treatments on fiber length and digestibility were evaluated and compared with lab-scale results. These enzymatic hydrolysis studies were conducted using pretreated bagasse and straw and the enzyme combinations selected in the optimisation study. Results showed that the optimum combination of cellulase and xylanase proved to be more effective during enzymatic hydrolysis compared to a conventional enzyme mixture. These optimised cocktails consisted of 0.15 mL.g-1 WIS Cellic® CTec2 + 0.32 mL.g-1 WIS Cellic® HTec2 for sorghum and 0.10 mL.g-1 WIS Cellic® CTec2 + 0.20 mL.g-1 WIS Cellic® HTec2 for triticale. This improvement is, however, dependent on the feedstock. Sorghum required double the enzyme dosage used for triticale to reach a cellulose conversion of 80%. This was possibly due to the higher cellulose content thereof and because the material was less digestible. With regards to the micro-assay, a homogenisation step showed to slightly improve the digestibility of the pretreated materials depending on the feedstock and enzyme combination applied. However, this method revealed the ability of the micro-assay to distinguish between the two different feedstocks as well as the two enzyme preparations. In conclusion, optimisation of enzymatic hydrolysis integrated with pretreatment can significantly improve the overall sugar yield. For sorghum, the optimised cocktail yielded 401.0 kg sugar.ton-1 bagasse compared to the 328.2 kg sugar.ton-1 bagasse obtained with the control enzyme combination. The optimised cocktail for triticale yielded 320.7 kg sugar.ton-1 straw and the control cocktail 275.5 kg sugar.ton-1 straw. Data confirmed that although feedstocks were similar in terms of biomass type and chemical composition, their different raw material properties and pretreatment conditions required them to have different optimum enzyme loadings for hydrolysis. The higher yields obtained with the optimised cocktails were also confirmed when enzymatic hydrolysis was performed at micro-cale. This is a useful screening method, but the differences observed should be kept in mind when high-throughput systems are applied.AFRIKAANSE OPSOMMING: Die vervaardiging van etanol vanaf lignosellulosiese biomassa (tweede generasie bio-brandstowwe) word tans wêreldwyd ondersoek vir die ontwikkeling van bio-brandstowwe wat as alternatief teenoor fossiel-brandstowwe gebruik kan word. Dit is die gevolg van toenemende besorgdheid oor potensiële landbewaring, konflik met voedselverskaffers asook argumente rondom die afname in die vrystelling van kweekhuisgasse. Soet sorghum bagasse en triticale strooi is veral twee belowende plantaardige grondstowwe, aangesien hul hoë opbrengste verskaf onder ‘n verskeidenheid kondisies. Hul het ook geen waarde as voedselbron nie en lei tot ‘n netto laer vrystelling van kweekhuisgasse, wat hul baie gewild maak vir die vervaardiging van tweede generasie bio-etanol. Onder die verskeie moontlikhede wat bestaan, is ensiem-gebaseerde prosesse die mees belowende vir die vervaardiging van bio-brandstowwe. Dit is grotendeels te danke aan die spesifisiteit daarvan, die hoër opbrengste, die generering van laer hoeveelhede inhiberende komponente tydens hidrolise en fermentasie sowel as die hoë potensiaal daarvan om te verbeter deur tegnologie. Tot op hede vorm die ensiematiese hidrolise van lignosellulosiese materiaal ‘n bottelnek in die proses van bio-etanol vervaardiging. Die produksiekostes wat daarmee geassosieer word, is ook steeds baie hoog en verhoed dus die kommersialisering van hierdie proses. Die seleksie van grondstof variëteite (geselekteer op grond van hul reaksie op vooraf-behandelings- en agronomiese data) en optimisering van die ensiematiese hidrolise stap vir spesifieke grondstof en vooraf-behandelingstoestande is dus van uiterste belang om ‘n voldoende suiker opbrengs, en gevolglik etanol opbrengs, te verkry. Daar is ook ‘n aanvraag om realistiese en vinnige metodes te ontwikkel vir die evaluering van grondstof verteerbaarheid. Toenemende navorsing word dus nou gerig op die ontwikkeling van hoë deurvoer sisteme wat gebaseer is op die verspreiding van gemaalde vooraf-behandelde materiaal in mikro-titer plate om verskeie veranderlike faktore tydens ensiematiese hidrolise te evalueer. Aangesien die maling van materiaal as ‘n addisionele behandeling beskou kan word, word die toepassing van ander metodes wat minimum impak op die lignosellulosiese struktuur het, verkies. So ‘n metode word gebaseer op handsheets soos beskryf in die TAPPI-standaard metode. Met hierdie as agtergrond, was die hoof doel van die tesis om ensiematiese hidrolise te verbeter deur optimum ensiem kombinasies saam te stel wat spesifiek is vir variëteite en stoom-voorafbehandelde toestande van sorghum bagasse en triticale strooi. Die spesifieke doel was om ‘n optimum kombinasie van ensiem-bereidings teen ‘n minimum dosis te verkry wat ‘n omskakeling van 80% van die sellulose na suikers, verskaf. Gebaseer op voorafgaande resultate is twee ensiem-bereidings wat as sellulase en xylanase gekarakteriseerd is, geselekteer vir optimisering deur ‘n sentrale saamgestelde ontwerp en die daaropvolgende respons-vlak grafieke met die hulp van Design® Expert sagteware. Die tweede doel van die tesis was om ‘n mikro-toets metode te ontwikkel wat ‘n addisionele homogeniserings-stap insluit voor die maak van die handsheets. Die effek van die verskillende homogeniserende behandelings op die vesellengtes en vertering was geëvalueer en vergelyk met laboratorium-skaal resultate. Hierdie ensiematiese hidrolise studies was uitgevoer met behulp van voorafbehandelde bagasse en strooi en deur gebruik te maak van die ensiem kombinasies wat geselekteer is in die optimiserings-studies. Resultate het getoon dat die optimum kombinasies van sellulase en xylanase meer effektief was gedurende ensiematiese hidrolise in vergelyking met ‘n konvensionele ensiem-mengsel. Die geoptimiseerde kombinasies het bestaan uit 0.15 mL.g-1 WIS Cellic® CTec2 + 0.32 mL.g-1 WIS Cellic® HTec2 vir sorghum en 0.10 mL.g-1 WIS Cellic® CTec2 + 0.20 mL.g-1 WIS Cellic® HTec2. Hierdie verbetering is egter afhanklik van die plantaardige grondstof. Sorghum het dubbel die hoeveelheid ensiem benodig as wat deur triticale gebruik is om ‘n 80% sellulose omskakeling te behaal. Dit was moontlik as gevolg van die hoër sellulose fraksie daarvan en omdat die materiaal minder verteerbaar is in vergelyking met triticale. Met betrekking tot die mikro-toets het ‘n homogeniserings-stap aangedui dat dit die verteerbaarheid van die vooraf-behandelde materiale tot ‘n mate kon verbeter, afhangend van die grondstof en ensiem kombinasies wat gebruik is. Hierdie metode het egter die vermoë van die mikro-toets om tussen twee verskillende plantaardige grondstowwe te onderskei, sowel as tussen die twee ensiem bereidings, onthul. Ten slotte, die optimisering van ensiematiese hidrolise wat geïntegreer is met voorafbehandeling kan die algehele suiker opbrengs merkwaardig verbeter. Vir sorghum het die geoptimiseerde ensiem kombinasie 401.0 kg suiker.ton-1 bagasse gelewer in vergelyking met die 328.2 kg suiker.ton-1 bagasse wat verkry is met die kontrole ensiem kombinasie. Die geoptimiseerde ensiem kombinasie vir triticale het 320.7 kg suiker.ton-1 strooi gelewer en die kontrole kombinasie 275.5 kg suiker.ton-1 strooi. Data het bevestig dat, alhoewel die grondstowwe eenders was in terme van die tipe biomassa en chemiese komposisie, hul verskillende rou materiaal eienskappe en vooraf-behandelingstoestande daartoe gelei het dat hulle verskillende optimum ensiem kombinasies tydens hidrolise vereis het. Die hoër opbrengste verkry met die geoptimiseerde kombinasies was ook bevestig met die uitvoer van ensiematiese hidrolise op mikro-skaal. Dit is ‘n bruikbare siftingsmetode, maar die verskille wat opgemerk is, moet in gedagte gehou word wanneer hoë deurvoer sisteme toegepas word