Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The need to mitigate the twin crises of peak oil and climate change has driven a headlong rush to biofuels. This study was aimed at the development of a process to efficiently convert steam explosion pretreated (STEX) sugarcane bagasse into ethanol by using combinations of commercial enzyme cocktails and recombinant Saccharomyces cerevisiae strains. Though enzymatic saccharification is promising in obtaining sugars from lignocellulosics, the low enzymatic accessibility of the cellulose and hemicellulose is a key impediment thus necessitating development of an effective pretreatment scheme and optimized enzyme mixtures with essential accessory activities. In this context, the effect of uncatalysed and SO2 catalysed STEX pretreatment of sugarcane bagasse on the composition of pretreated material, digestibility of the water insoluble solids (WIS) fraction and overall sugar recovery was investigated. STEX pretreatment with water impregnation was found to result in a higher glucose recovery (28.1 g/ 100 bagasse) and produced WIS with a higher enzymatic digestibility, thus was used in the optimization of saccharification and fermentation. Response surface methodology (RSM) based on the 33 factorial design was used to optimize the composition of the saccharolytic enzyme mixture so as to maximize glucose and xylose production from steam exploded bagasse. It was established that a combination of 20 FPU cellulase/ g WIS and 30 IU -glucosidases/ g WIS produced the highest desirability for glucose yield. Subsequently the optimal enzyme mixture was used to supplement enzyme activities of recombinant yeast strains co-expressing several cellulases and xylanases in simultaneous saccharification and fermentations SSFs. In the SSFs, ethanol yield was found to be inversely proportional to substrate concentration with the lowest ethanol yield of 70% being achieved in the SSF at a WIS concentration of 10% (w/v). The ultimate process would however be a one-step “consolidated” bio-processing (CBP) of lignocellulose to ethanol, where hydrolysis and fermentation of polysaccharides would be mediated by a single microorganism or microbial consortium without added saccharolytic enzymes. The cellulolytic yeast strains were able to autonomously multiply on sugarcane bagasse and concomitantly produce ethanol, though at very low titres (0.4 g/L). This study therefore confirms that saccharolytic enzymes exhibit synergism and that bagasse is a potential substrate for bioethanol production. Furthermore the concept of CBP was proven to be feasible. / AFRIKAANSE OPSOMMING: Die behoefte om die twee krisisse van piek-olie en klimaatsverandering te versag, het veroorsaak dat mense na biobrandstof as alternatiewe energiebron begin kyk het. Hierdie studie is gemik op die ontwikkeling van 'n proses om stoomontplofde voorafbehandelde (STEX) suikerriet bagasse doeltreffend te omskep in etanol deur die gebruik van kombinasies van kommersiële ensiem mengsels en rekombinante Saccharomyces cerevisiae stamme. Alhoewel ensiematiese versuikering belowend is vir die verkryging van suikers vanaf lignosellulose, skep die lae ensiematiese toeganklikheid van die sellulose en hemisellulose 'n hindernis en dus is die ontwikkeling van' n effektiewe behandelingskema en optimiseerde ensiemmengsels met essensiële bykomstige aktiwiteite noodsaaklik. In hierdie konteks, was die effek van ongekataliseerde en SO2 gekataliseerde stoomontploffing voorafbehandeling van suikerriet bagasse op die samestelling van voorafbehandelde materiaal, die verteerbaarheid van die (WIS) breuk van onoplosbare vastestowwe in water (WIS), en die algehele suikerherstel ondersoek. Daar was bevind dat stoomontploffing behandeling (STEX) met water versadiging lei tot 'n hoër suikerherstel (21.8 g/ 100g bagasse) en dit het WIS met ‘n hoër ensimatiese verteerbaarheid vervaardig en was dus gebruik in die optimalisering van versuikering en fermentasie. Reaksie oppervlak metodologie (RSM), gebasseer op die 33 faktoriële ontwerp, was gebruik om die samestelling van die ‘saccharolytic’ ensiemmengsel te optimaliseer om sodoende die maksimering van glukose en ‘xylose’ produksie van stoomontplofde bagasse te optimaliseer. Daar was bevestig dat ‘n kombinasie van 20 FPU sellulase/ g WIS en 30 IU ‘ -glucosidases/ g’ WIS die hoogste wenslikheid vir glukose-opbrengs produseer het. Daarna was die optimale ensiemmengsel gebruik om ensiemaktiwiteit van rekombinante gisstamme aan te vul, wat gelei het tot die medeuitdrukking van verskillende ‘cellulases’ en ‘xylanases’ in gelyktydige versuikering en fermentasie SSFs. In die SSFs was daar bevind dat die etanol-produksie omgekeerd proporsioneel is tot substraat konsentrasie, met die laagste etanolopbrengs van 70% wat bereik was in die SSF by ‘n WIS konsentrasie van 10% (w/v). Die uiteindelike proses sal egter 'n eenmalige "gekonsolideerde" bioprosessering (CBP) van lignosellulose na etanol behels, waar die hidrolise en fermentasie van polisakkariede deur' n enkele mikroorganisme of mikrobiese konsortium sonder bygevoegde ‘saccharolytic’ ensieme bemiddel sal word. Die ‘cellulolytic’ gisstamme was in staat om vanself te vermeerder op suikerriet bagasse en gelyktydig alkohol te produseer, al was dit by baie lae titres (0.4 g/L). Hierdie studie bevestig dus dat ‘saccharolytic’ ensieme sinergisme vertoon en dat bagasse 'n potensiële substraat is vir bio-etanol produksie. Daar was ook onder meer bewys dat die konsep van CBP uitvoerbaar is. / The National Research Foundation (NRF) for financial support

Master of Science / Department of Biological and Agricultural Engineering / Mei He / Thermobifida fusca, an aerobic moderately thermophilic, filamentous soil bacterium is capable of producing butyric acid. Butyric acid is a 4-carbon short chain fatty acid that is widely used in the chemical, food, and pharmaceutical industries. Currently, butyric acid is primarily produced through petroleum-based chemical synthesis, but could be a candidate to be produced by fermentation. By producing through a fermentation platform, production of butyric acid can be shifted from a non-renewable to a renewable source. In an effort to make T. fusca produce a high yield of butyric acid, multiple fermentation parameters were explored and optimized. The effect of different carbon sources (mannose, xylose, lactose, cellobiose, glucose, sucrose, and acetates) on butyric acid production was studied, where cellobiose produced the highest yield of 0.67 g/g C (g-butyric acid/g-carbon input). The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. Fermentation was performed on ground corn stover as a substrate to evaluate the production of butyric acid on lignocellulosic biomass, and the optimized conditions resulted in a titer of 2.37 g/L butyric acid. The butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanyol-CoA in T. fusca. A study into the transcriptomics of T. fusca was begun by growing T. fusca under a variety of fermentation conditions, isolating the messenger RNA, and performing a sequence of the mRNA using whole transcriptome shotgun sequencing. The results of sequencing of various samples were plotted to determine correlation across numerous fermentation parameters. This correlation based analysis determined that the carbon to nitrogen ratio has the largest overall impact on gene transcription of T. fusca among all of the fermentation parameters studied. Overall, the work from this study proves that production of butyric acid is possible from a renewable cellulosic feedstock.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The search for a cost-effective, environmentally friendly replacement for fossil fuels resulted in bio-ethanol production receiving a lot of attention. Lignocellulose, is considered to be the most abundant renewable source on earth, and consists of cellulose, hemicellulose and lignin. Exploitation thereof as a substrate for ethanol production, can serve as solution in producing bio-ethanol as an adequate replacement for fossil fuels. Hemicelluloses, contributing up to a third of the lignocellulosic substrate, consists mainly of xylan and mannan and can be degraded by hemicellulolytic enzymes that are produced by plant cell wall degrading organisms. Galactoglucomannan is the most complex form of mannan and requires a consortium of enzymes for complete hydrolysis. These enzymes include β-mannanase, β-mannosidase, α-galactosidase, β-glucosidase and galactomannan acetylesterases. Saccharomyces cerevisiae is a well-known fermentative organism that has been used in various industrial processes and is able to produce ethanol from hexose sugars. Although this organism is unable to utilize complex lignocellulosic structures, DNA manipulation techniques and recombinant technology can be implemented to overcome this obstacle. Strains of S. cerevisiae pose other shortcomings like hyperglycosylation and therefore other non-conventional yeasts (such as Kluyveromyces lactis) are now also being considered for heterologous protein production. The mannanase gene (manI) of Aspergillus aculeatus was expressed in K. lactis GG799 and S. cerevisiae Y294. K. lactis transformants were stable for two weeks in consecutive subcultures and secreted a Man1 of 55 kDa. The recombinant Man1 displayed an optimum temperature of 70°C and a pH optimum of 5 when produced by K. lactis. Activity levels of about 160 – 180 nkat/ml was obtained after 86 hours of cultivation, which was similar to the activity observed with S. cerevisiae under the same conditions. Disruption of the ku80 gene did not contribute to the stability of the cultures and a heterogeneous culture developed for 10 days of consecutive subculturing. The mannosidase gene (man1) from A. niger and mannanase gene (manI) from A. aculeatus were constitutively expressed in S. cerevisiae Y294 and S. cerevisiae NI-C-D4. The MndA and Man1 proteins appeared as a 140 kDa and 58 kDa species on the SDS-PAGE analysis when expressed in S. cerevisiae Y294, respectively. MndA had an optimum temperature of 50°C and optimum pH 5. Man1 produced by S. cerevisiae Y294 indicated a pH optimum of 6 and temperature optimum of 70°C. The MndA displayed low levels of endomannanase activity and no β-mannosidase activity could be detected. Co-expression of man1 and mndA in either S. cerevisiae Y294 and S. cerevisiae NI-C-D4, resulted in less hydrolysis of galactoglucomannan. An increase in the size of the plasmid generally results in a decrease in the copy number, leading to a decrease in the amount of ManI protein being produced. The co-expression of ManI and MndA could also have resulted in a higher metabolic burden on the cell, hence the amount of ManI are produced. This study confirms that more research should be done on the evaluation of alternative hosts for expression of foreign proteins. Furthermore, producing enzymes cocktails for industrial application should be considered rather than co-expression of various enzymes in one host. / AFRIKAANSE OPSOMMING: ‘n Behoefte na ‘n koste-effektiewe en omgewingsvriendelike vervoer brandstof is besig om toe te neem. Lignosellulose word beskou as die volopste hernubare bron vir biobrandstof en lignosellulose bestaan uit sellulose, hemisellulose en lignien. Die gebruik daarvan vir die produksie van bio-etanol kan ’n voldoende alternatief vir fossielbrandstowwe bied. Verbruik van lignosellulose as bron vir die produksie van biobrandstof bied ’n oplossing vir die energie krises. Hemisellulose vorm ’n derde van lignosellulose substraat en bestaan uit xilaan en mannaan en word deur hemisellolitiese ensieme afgebreek wat algemeen by plantselwand-verterende organismes voorkom. Galaktoglukomannaan is die mees komplekse vorm van mannaan en benodig verskeie ensieme vir volkome hidroliese. Hierdie ensieme sluit in β-mannanase, β-mannosidase, α-galaktosidase, β-glukosidase en galaktomanaan asetielesterases. Saccharomyces cerevisiae is ‘n bekende fermenterende organisme wat gereeld in verskeie industriële prosesse gebruik word en kan etanol van heksose suikers produseer. Die organisme beskik nie oor die vermoë om komplekse polisakkarides wat in lignosellulose voorkom te hidroliseer nie maar. DNS-manipuleringstegnieke en rekombinante tegnologie maak dit egter moontlik die probellm te oorbrug. S. cerevisiae het nogtans tekortkominge soos hiperglikosilering en daarom word ander nie-konvensionele giste (soos Kluyveromyces lactis) tans ook vir die produksie van rekombinante proteine ondersoek. Die mannanase geen (manI) vanaf Aspergillus aculeatus is in K. lactis GG799 en S. cerevisiae Y294 uitgedruk. K. lactis transformante was stabiel vir twee weke in opeenvolgende subkluture en het ‘n Man1 van 55 kDa geproduseer. Die rekombinante Man1 ensiem het ‘n temperatuur optimum van 70°C en pH optimum van 5.0 getoon in K. Lactis. Aktiwiteitsvlakke van 160 – 180 nkat/ml was bereik na 86 uur klutivering, In vergelyking met S. cerevisiae was aktiwiteitsvlakke eenders oor ‘n periode Die disrupsie van die ku80 geen het geen effek op die stabiliteit van die transformante in 10 dae opeenvolgende sub-kulture getoon nie. Die mannosidase geen (mndA) vanaf Aspergillus niger en die mannanase geen (man1) van Aspergillus aculeatus is konstitutief in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk. Uitdrukking van die MndA en Man1 proteïen in S. cerevisiae Y294 het onderskeidelik ‘n 140 kDa en 58 kDa spesie getoon met SDS-PAGE analisering. Die MndA ensiem het ‘n temperatuur optimum van 50°C and pH optimum van 5.0 getoon. Man1 het ‘n pH optimum van 6.0 en ‘n temperatuur optimum van 70°C getoon. MndA het lae hidrolitiese aktiwiteit op galaktoglukomannaan, maar geen β-mannosidase aktiwiteit getoon nie. Wanneer man1 and mndA saam in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk is, het die hidroliese van galaktoglukomannan dramaties afgeneem. ‘n Toename in die grootte van ‘n plasmied veroorsaak dikwels ‘n afname in kopiegetal wat die produksie van ManI verlaag. Die ko-uitdrukking van ManI en MndA kan ook tot ’n hoër metaboliese las lei en dus die laer produksie van ManI. Resultate in hierdie studie wys daarop dat meer navorsing benodig word in die soeke na alternatiewe gashere vir uitdrukking van mannanases. Ensiem mengsels vir industriële toepassings behoort eerder gebruik te word as die ko-ekspressie van verskeie ensieme in ’n enkel gasheer.

Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be "readily" converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres. Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis. In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters. Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be „readily‟ converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres. Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis. In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters. / AFRIKAANSE OPSOMMING: Biobrandstof word beskou as die ideale plaasvervanger vir fossielbrandstof weens die beskikbaarheid en herwinbare aard daarvan. Bio-etanol wek tans groot mark-verwante belangstelling as alternatiewe brandstof weens die potensiaal om petroleum as vervoerbrandstof aan te vul. Lignosellulose biomassa, 'n hernubare energiebron, kan "maklik" tot bio-etanol omgeskakel word. Die groot struikelblok in die omskakelingsproses is die weerstandbiedendheid van die lignosellulose komponente (sellulose, hemisellulose en lignien) teen ensiematiese hidroliese asook die gebrek aan beskikbaarheid van lae koste tegnologie. Gekonsolideerde Bioprosessering (KBP) is 'n enkel stap proses wat 'n koste-effektiewe en ekonomiesvatbare strategie voorstel vir bio-etanolproduksie. Die proses benodig 'n mikroorganisme wat daartoe instaat is om etanol teen hoë vlakke en tempo te kan produseer. Saccharomyces cerevisiae het potensiaal as 'n KBP kandidaat weens sy hoë vlakke van etanolproduksie, gehardheid in industriële prosesse, goed-ontwikkelde geenuitdrukking sisteme en veiligheidstatus. Ongelukkig kan S. cerevisiae nie polisakkariede afbreek nie en benodig derhalwe heteroloë uitdrukking van sellulases. Die genetiese manipulering van S. cerevisiae vir sellulose hidroliese dien as 'n belangrike stap in gisrasontwikkeling vir KBP en dien as 'n “stepping stone” vir die kommersialisasie van lignosellulose bio-etanol. Alhoewel sellulose-benuttende S. cerevisiae rasse reeds gekonstrueer is, is sellulose omskakeling stadig en belemmer dit optimale etanolproduksie. 'n Hoogs aktiewe glukosidase word derhalwe benodig om die hidroliese van sellobiose te versnel. Die studie behels die konstruksie van verskeie sellobiose-fermenterende S. cerevisiae rasse. Ektrasellulêre, fungiese -glukosidase-koderende gene was suksesvol in S. cerevisiae uitgedruk onderhewig aan die transkripsionele beheer van die ENO1 (enulase) promoter en termineerder DNS-volgordes. Die geproduseerde, rekombinante ensieme is gekarakteriseer op grond van optimale pH en temperatuur, asook kinetiese eienskappe. Die intrasellulêre benutting van sellobiose is 'n ideale benadering tot sellobiose hidroliese siende dat dit die risiko van kontaminasie verminder wat veroorsaak word deur die glukose wat vrygestel word in die ekstrasellulêre omgewing. Tog beskik S. cerevisiae nie oor 'n vervoer meganisme om sellobiose in die sel in te bring nie. Derhalwe is die intrasellulêre Phanaerochaete chrysosporium -glukosidase-koderende geen suksesvol saam met die Kluyveromyces lactis laktose permease uitgedruk. Alle rekombinante rasse is vir groei op sellobiose geevalueer. Die mees belowendste esktrasellulêre -glukosidase-produserende S. cerevisiae Y294[Pccbgl1] ras toon 'n aktiwiteit van 3.85 nkat.g-1, 1.85 keer meer die aktiwiteit van die S. cerevisiae Y294[SFB] ras (2.07 nkat.g-1). S. cerevisiae Y294[Pccbgl1] het ook 'n maksimum groei tempo van 0.25 h-1 onder anearobiese kondisies in vergelyking met die 0.064 h-1 van S. cerevisiae Y294[iPcbglB+lac12] toon. Onder anaërobe kondisies het S. cerevisiae Y294[Pccbgl1] 7.95 g.l-1 sellobiose verbruik en 4.05 g. l-1 etanol geproduseer oor 'n tydperk van 116 uur, terwyl S. cerevisiae Y294[iPcbglB+lac12] 0.41 g.l-1 sellobiose verbruik het en 0.21 g.l-1 etanol oor dieselfde tydperk geproduseer het. Die rekombinante rasse wat in die studie gekonstrueer is, is 'n belangrike stap in die ontwikkeling van S. cerevisiae as KBP sellulolitiese gis. / The South African National Research Institute (SANERI) for financial support

Consolidated bioprocessing (CBP) provides a cost effective cellulose processing strategy, in which enzyme production, substrate hydrolysis, and fermentation of sugars to ethanol are all carried out in a single step by microorganisms. For industrial-scale bioethanol production, CBP-enabling microbes must be able to both efficiently degrade lignocellulosic material to fermentable sugars and synthesize bioethanol with high yields. Microbes with these properties have so far not been identified. Developing naturally occurring cellulolytic isolates with CBP-relevant properties requires a comprehensive understanding of their lignocellulosic hydrolysis mechanism and metabolism. In my quest to find a suitable organism for potential use in CBP, I took to investigate the under-characterized anaerobic bacterium, Clostridium termitidis strain CT1112. C. termitidis produces fermentative hydrogen and ethanol from a variety of lignocellulose derived substrates. I sought to investigate the metabolism of C. termitidis on different substrates and the mechanisms of substrate hydrolysis using a combination of microscopy, comparative bioinformatics, and ‘Omic (transcriptomic and proteomic) analyses. Comparative bioinformatics analyses revealed higher numbers of genes encoding carbohydrate active enzymes (CAZymes) with the potential to hydrolyze a wide-range of carbohydrates, and ‘Omic analyses were used to quantify the levels of expression of CAZymes, including endoglucanases, exoglucanases, hemicellulases and cellulosomal components. While cellulases and cellulosome components were highly expressed on cellulose, xylanases and glucosidases were predominantly expressed on pentoses, and chitinases (as well as cellobiose phosphorylases) were significantly up-regulated on cellobiose. In addition to growth on xylan, the simultaneous consumption of two important lignocellulose constituents, cellobiose and xylose was also observed. The ability to metabolize both hexose and pentose sugars is a highly desirable feature of CBP-relevant organisms. Metabolic profiles in association with ‘Omics analyses showed that hexoses and pentoses are consumed via the Embden-Meyerhof-Parnas and Pentose-Phosphate pathways, respectively, and that the genome content and expression profiles dictate end-product synthesis patterns. Genes and gene-products of enzymes in central metabolism and end-product synthesis were detected in high abundance under all substrate conditions, regardless of the amounts of end-products synthesized. The capabilities described thus far, identifies C. termitidis as a strain of interest for CBP. Further studies are, however, required for its development in to an industry-ready strain for biofuel production. / February 2016

The potential value of the chitin biomass (e.g. food waste) is recently considered being ignored by landfill. Chitin can be a potential cheap carbon source for converting into value-added chemicals by microorganisms. Serratia marcescens is a chitinolytic bacterium that harbors endogenous chitinase systems. With goals of characterzing S. marcescens chitinolytic capabilities and applying S. marcescens to chemical production from chitin, my dissertation main content includes five chapters: 1) Chapter 1 highlights background information of chitin source, S. marcescens and potential metabolic engineering targets using chitin as a substrate; 2) Chapter 2 demonstrates that ChiR is a key regulator in regulating 9 chitinase-related genes in S. marcescens Db11 and manipulation of chiR can be a useful and efficient genetic target to enhance chitin utilization; 3) Chapter 3 reports the production of N-acetylneuraminic acid (Neu5Ac) from chitin by a bottom-up approach of engineering the nonconventional chitinolytic bacterium, Serratia marcescens, including native constitutive promoter characterization and transcriptional and translational pathway balancing; 4) Chapter 4 describes improvement of S. marcescens chitinolytic capability by an adaptive evolution approach; 5) Chapter 5 elucidates S. marcescens intracellular metabolite profile using a constraint-based genome-scale metabolic model (iSR929) based on genomic annotation of S. marcescens Db11. Overall, the dissertation work is the first report of demonstrating the concept of chitin-based CBP using S. marcescens and the computational model and genetic molecular tools developed in this dissertation are valuable but not limited to design-build-test of S. marcescens for contributing to the field of biological science and metabolic engineering applications.

A major obstacle to achieving commercially viable lignocellulosic biofuels through consolidated bioprocessing (CBP) is the lack of “industry-ready” microorganisms. Ideally, a CBP-relevant organism would achieve efficient and complete hydrolysis of lignocellulose, simultaneous utilization of the diverse hydrolysis products and high yields of the desired biofuel. To date, no single microbe has been identified that can perform all of these processes at industrially significant levels. As such, thermophilic decaying woodchip compost was investigated as a source of novel lignocellulolytic, biofuel producing bacteria. From a single sample, a collection of physiologically diverse strains were isolated, which displayed differences in substrate utilization and biofuel production capabilities. Molecular characterization of these isolates, and development of a genome relatedness prediction model based on the chaperonin-60 universal target sequence, identified these isolates as strains of Thermoanaerobacter thermohydrosulfuricus. Application of this model to other Thermoanaerobacter spp. further identified that these isolates belong to a divergent and lesser characterized lineage within the genus. Based on this, the CBP-potential of a single isolate, T. thermohydrosulfuricus WC1, was selected for further investigation through metabolic, genomic and proteomic analyses. Its ability to grow on polymeric xylan, potentially catalyzed by an endoxylanase found in only a few Thermoanaerobacter strains, distinguishes T. thermohydrosulfuricus WC1 from many other strains within the genus. The simultaneous consumption of two important lignocellulose constituent saccharides, cellobiose and xylose was also observed and represents a desirable phenotype in CBP-relevant organisms. However, at elevated sugar concentrations, T. thermohydrosulfuricus WC1 produces principally lactate, rather than the desired biofuel ethanol, as the major end-product. Proteomic analysis identified that all likely end-product forming proteins were expressed at high levels suggesting that the end-product distribution patterns in T. thermohydrosulfuricus WC1 are likely controlled via metabolite-based regulation or are constrained by metabolic bottlenecks. The xylanolytic and simultaneous substrate utilization capabilities of T. thermohydrosulfuricus WC1 identify it as a strain of interest for CBP. However, for its development into an “industry-ready” strain as a co-culture with a cellulolytic microorganism, improved biofuel producing capabilities are needed. The practical implications of CBP-relevant phenotypes in T. thermohydrosulfuricus WC1 in relation to other Thermoanaerobacter spp. will be discussed.

Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes. Two of the biggest challenges for its commercialization are: effective release of fermentable sugars from pretreated biomass, and preparations of thermostable enzymes with low-cost. The hydrolysis performance of 21 reconstituted bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase, family 9 Clostridium phytofermentans processive endoglucanase, and family 48 Clostridium phytofermentans cellobiohydrolase was investigated on microcrystalline cellulose (Avicel) and regenerated amorphous cellulose (RAC). The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was most important for high cellulose digestibility regardless of substrate type. These results suggested that the hydrolysis performance of reconstituted cellulase cocktail strongly depended on experimental conditions. Thermobifida fusca YX was hypothesized to have a thermophilic polyphosphate glucokinase. T. fusca YX ORF Tfu_1811 encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was over expressed in Escherichia coli. By a simple one-step immobilization, the half-life time increased to 2 h, at 50 °C. These results suggest that this enzyme was the most thermostable PPGK reported. My studies would provide important information for the on-going project: high-yield hydrogen production from cellulose by cell-free synthetic enzymatic pathway. / Master of Science

Thesis (MBA (Business Management))--University of Stellenbosch, 2007. / ENGLISH SUMMARY: Fossil fuel has been the preferred source for the production of transportation fuel for many years. However, this is not a renewable resource. Many conflicting reports have been published as to how long this resource will last. One thing is certain: eventually the supply of cheap crude oil will run out. It is therefore crucial to start the search for renewable alternatives now. There are a number of possible candidates vying for replacing fossil fuel as primary transportation fuel. Hydrogen, methanol, biodiesel and bioethanol all have the characteristics required of a good transportation fuel. It is unlikely that only one of these will replace oil. A more likely scenario would be that they all play a role in transportation in the future. Apart from being renewable, these alternatives have the further advantage of being less damaging to the environment, something that will become essential in future. Among the renewable alternatives, bioethanol has the second highest energy density. Currently, ethanol production worldwide almost exclusively uses sugarcane and maize as raw material. However, both these are food crops and using them for ethanol could lead to an increase in food prices. Furthermore, there is not enough agricultural land available to produce sufficient quantities of sugarcane and maize for ethanol to replace fossil fuel. Producing ethanol from plant material has the potential to meet the capacity requirements without impacting directly on food production. Approximately 180 million tons of agricultural biomass are produced in the United States each year, sufficient to produce 75 to 110 billion litres of ethanol. Despite its abundance, the technical challenges in converting cellulose to ethanol are significant. One major obstacle to the production of ethanol out of plant material is that most of the sugar in plant material is unavailable for fermentation by micro-organisms. In order to render the sugars in the cellulose fraction accessible to conversion, it is necessary to treat the plant fibres with a combination of chemical and enzymatic processes. Only when a complex mixture of enzymes is used, does it become possible to break down cellulose to glucose for subsequent fermentation to ethanol. Biomass processing by means of enzymes currently involves four separate biological steps: (i) production of enzymes (cellullases and hemicellulases), (ii) hydrolysis of cellulose and hemicellulose to sugars, (iii) fermentation of hexose sugars and (iv) fermentation of pentose sugars. Consolidated BioProcessing (CBP) will combine all these steps into one. However, CBP is not yet possible and the magnitude of research and developmental advancement required to realize this goal is significant. Both sugar and starch ethanol technologies are well established and major process advances are therefore unlikely. Currently there are no commercial-sized plants for the production of ethanol from lignocellulosics, however this is likely to change in the near future considering the progress made in this field during recent years. This study will focus on the current status of the bioethanol industry, as well as on the potential for future development. / AFRIKAANSE OPSOMMING: Fossielbrandstof was vir baie jare die hoofbron vir die produksie van brandstof vir die vervoerbedryf. Fossielbrandstof is nie ’n hernubare energiebron nie en daar is al baie gespekuleer oor presies hoe lank daar nog goedkoop olie beskikbaar sal wees. Baie min van die gepubliseerde bronne stem ooreen, maar almal is dit eens dat olie op een of ander stadium sal opraak. Om hierdie rede is dit noodsaaklik om nou reeds te soek na alternatiewe. Daar is ’n hele aantal hernubare alternatiewe wat gebruik kan word in die plek van olie. Waterstof, metanol, biodiesel en bioetanol beskik almal oor die nodige eienskappe om ’n effektiewe vervoerbrandstof te wees. Die hoofvoordeel van hierdie brandstowwe is dat hulle minder skadelik is vir die omgewing as olie, ’n eienskap wat baie belangrik sal wees in die toekoms. Die kans is eger skraal dat een van bogenoemde bronne die mark totaal sal oorheers soos wat olie tot op hede oorheers het. ’n Meer waarskynlik uitkoms sou wees dat al hierdie bronne op een of ander manier ’n rol gaan speel in die vervoerbedryf in die toekoms. Etanol het die tweede hoogste energie digtheid van die vier genoemde hernubare brandstowwe. Etanol word tans uitsluitlik van suikerriet en mielies geproduseer. Beide suikerriet en mielies is voedselgewasse en die gebruik daarvan vir brandstof kan lei tot ’n toename in voedselpryse. Daar is ook nie genoeg landbougrond beskikbaar vir die verbouing van suikerriet en mieles sodat genoeg etanol geproduseer kan word om fosielbranstof te vergang nie. Die vervaardiging van etanol vanaf lignosellulose het die potensiaal om etanolkapasiteitprobleme te oorkom sonder om direk met voedselproduksie te kompeteer. Ongeveer 180 miljoen ton landbouafval word jaarliks in die Verenigde State geproduseer, genoeg vir die vervaardiging van tussen 75 en 110 biljoen liter etanol. Die tegniese kompleksiteit gekoppel aan die omskakeling van sellulose na etanol is beduidend. Die belangrikste hindernis vir die produksie van etanol vanaf plantmateriaal is die feit dat die meeste van die suiker nie beskibaar is vir fermentasie deur mikroörganismes nie. Plantvesels moet daarom met ’n kombinasie van chemikalieë en ensieme behandel word om sodoende die suiker beskikbaar te maak vir omskakeling. Sellulose kan slegs met ’n komplekse mengsel van ensieme afgebreek word tot glukose wat dan daarna gefermenteer kan word tot etanol. Die verwerking van biomassa met behulp van ensieme behels tans vier afsonderlike biologiese stappe: (i) ensiemproduksie (sellulases en hemisellulases), (ii) hidrolise van sellulose en hemisellulose tot fermenteerbare suikers, (iii) fermentasie van heksose suikers en (iv) fermentasie van pentose suikers. Consolidate BioProcessing (CBP) poog om al vier hierdie stappe te kombineer. Ongelukkig is die CBP proses nog nie moontlik nie en daar moet nog baie navorsing en ontwikkeling gedoen word om dit ’n realiteit te maak. Beide die metodes vir suiker- en styseletanolproduksie is goed gevestig, dus is die kans vir beduidende verbeteringe klein. Daar is tans geen aanlegte van kommersiële grootte vir die produksie van etanol vanaf lignocellulose nie, maar dit gaan waarskynlik binnekort verander as ’n mens die vordering in ag neem wat daar onlangs gemaak is in hierdie veld. Hierdie studie fokus op die huidige stand van sake in die etanolbedryf en die ontwikkelingsmoontlikhede vir die toekoms.

Lignocellulosic biomass is an abundant renewable resource targeted for biofuel production. Cellulose and hemicellulose from biomass both contain fermentable sugars and other moieties that can be converted to biofuels or other commodity chemicals. Enzymatic hydrolysis of these biopolymers is a critical step in the liberation of sugars for fermentation into desired products. In nature, anaerobic microbes produce protein nanostructures called cellulosomes that efficiently degrade cellulose substrates by combining multiple enzyme activities onto a scaffolding protein. However, current enzyme cocktails used in industry contain secretomes of aerobic microbes and are not efficient enough to be highly economical. Furthermore, most bio-processes focus on cellulose, rendering hemicellulose under-utilized. The three main objectives of this dissertation are to 1) develop multi-functional, self-assembling protein nanostructures for hemicellulose degradation using the architecture provided by cellulosomes, 2) understand the self-assembly mechanism at conditions for consolidated bioprocessing applications, and 3) compare the effectiveness of structured to non-structured hemicellulases in the hydrolysis of biomass. Xylan is a major type of hemicellulose in biomass feedstocks targeted for biofuel production. Six different xylanosomes were designed for hydrolysis of xylan within multiple biomass substrates using the cohesin-dockerin domain systems from Clostridium thermocellum, Clostridium cellulovorans, and Clostridium cellulolyticum. Each two-unit structure contained a xylanase for internal cleavage of the xylan backbone and one side-chain acting enzyme, either a ferulic acid esterase or bi-functional arabinofuranosidase/xylosidase. Expansion to three-unit xylanosomes included a family 10 or 11 xylanase, a bi-functional arabinofuranosidase/xylosidase, and bi-functional ferulic acid esterase/acetylxylan esterase. These multi-functional biocatalysts were used to degrade hemicellulose-rich wheat arabinoxylan and cellulose-containing destarched corn bran. Synergistic release of soluble sugars and ferulic acid was observed with select xylanosomes and in some cases required addition of an endoglucanase and cellobiohydrolase for enhanced hydrolysis. Furthermore, a putative ferulic acid esterase gene from the soil bacterium Cellvibrio japonicus was characterized and its role in xylan hydrolysis investigated. Information for the development of stable and functional cellulosome-like biocatalysts in metabolically-engineered microbes was collected using surface plasmon resonance. The protein-protein interaction of cohesin and dockerin domains for xylanosome self-assembly was examined at various temperatures and in the presence of ethanol to mimic different hydrolysis and fermentation processes and found to retain high affinities at the selected conditions. Moreover, the high-affinity interaction of cohesin and dockerin domains in the presence of non-specific proteins eliminated the need for protein purification for xylanosome construction. In addition to development of the first cellulosome-like biocatalysts targeted for hemicellulose degradation, this dissertation provides insight on possible improvements for the enzymatic hydrolysis of biomass, as well as the applicability of xylanosomes in consolidated bioprocessing.