Global food production management has become a challenge with an anticipated population of 10 billion people by 2050 and the ongoing COVID-19 epidemic. Seafood is a vital food source due to its widespread consumption, excellent nutrient profile, and low feed conversion ratio, rendering its sustainable production quintessential. Cellular agriculture or cultured meat can increase seafood production; however, the conventional use of Fetal Bovine Serum (FBS) in culture media restricts its utilization at an industrial level. FBS is effective but has many limitations: unethical animal extraction, high demand and low supply, poorly defined ingredients, variable performance, and high cost that impedes the feasibility and commercial viability of cellular agriculture. Thus, employing serum-free media becomes a quintessential need for cellular agriculture. This project aims to replace or reduce the typical 10% serum usage in Zebrafish embryonic stem cell (ESC) production media with protein hydrolysates derived from low-cost natural sources with high protein content. Enzymatic hydrolysis was performed on nine sources: insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine invertebrates (oyster, mussel, and lugworm). The resulting hydrolysates were evaluated for serum replacement in zebrafish ESCs. All hydrolysates were used at five different concentrations (10, 1 0.1, 0.01 and 0.001 mg/mL) in serum concentrations of 10%, 5%, and 0% with four biological replicates. The best hydrolysate sources and concentrations were selected for further testing at 2.5% and 1% serum concentrations. All hydrolysates, except for cricket, could restore or significantly increase cell growth with 50% less serum at a concentration of 0.1-0.001mg/mL. Protein hydrolysate concentration of 10 and 1mg/mL was toxic for cells. Additionally, the eight hydrolysates could reduce serum concentrations up to 75–90%. However, no protein hydrolysate could completely replace serum, as cells using only protein hydrolysates exhibited morphological aberrations and decreased growth. Replacing serum with protein hydrolysates lowers cellular agriculture's overall cost, thus enabling the commercialization of cultured meat and the development of a sustainable food system. In the future, blending various protein hydrolysate sources with or without the addition of conventional growth factors could be done to create the ideal serum-free media. / Doctor of Philosophy / With a predicted population of 10 billion by 2050 and the ongoing COVID-19 outbreak, the management of global food production has become a dilemma. However, due to its widespread consumption and good nutrient profile, seafood is an essential food supply, making its sustainable production indispensable. Both capture fisheries and aquaculture are conventional ways to produce seafood. However, they are under tremendous pressure and require alternatives that can alleviate this demand and contribute to the sustainable growth of seafood. In-vitro cultured meat, also known as lab-grown meat, is a novel technique with the potential to supplement the traditional fish sector. It appears a great option, as it completely imitates meat and offers numerous environmental, financial, and health advantages. A culture medium supports the existence, survival, growth, and multiplication of meat-producing cells and tissues in cell-based meat. However, the culture medium uses a Fetal Bovine Serum (FBS) supplement, which dramatically increases the cost and raises many ethical concerns as it is derived from a cow's fetus. In this thesis, we substitute FBS with protein hydrolysates derived from nine distinct sources. Hydrolysing proteins with enzymes produce protein hydrolysates, rich in nutrients and peptides that promote cell development. Enzymes were used to hydrolyse nine unique and protein-rich sources, including insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine creatures (oyster, mussel, and lugworm). The resultant hydrolysates were investigated for replacement of serum in cell culture. Eight protein hydrolysates successfully replaced 90% of serum without impairing cell growth and structure but could not replace serum entirely. In the future, serum-free media could be created by combining these various protein hydrolysates with or without adding other growth-promoting components.

<p dir="ltr">Feedstock variability represents a challenge in the adoption of lignocellulosic biomass for biofuels and biochemicals production, due to the differences in critical chemical and physical properties like lignin content, and water absorption respectively. Thus, difficult continuous manufacturing processes in biorefineries, hinder the transition from liquid feedstocks to renewable materials that consisting of solid particles. Modeling of flow properties based on rheological measurements of treated biomass is a quantitative metric for identifying if different feedstocks form pumpable slurries. Additionally, the correlation of yield stress to physical and chemical properties gives a measure that accounts for the variability in the processing design. This research models rheological properties and relates these to compositional data from different non-pretreated fractions of corn stover biomass slurries. Slurries were formed with solids concentrations of 300 g/L in a 6 hours fed-batch process using the commercial enzymes Celluclast 1.5L or Ctec-2 at 1FPU/g or 3 FPU/g of dry solids, basis to enable the liquefaction (i.e., slurry-forming) mechanism. We found that insoluble lignin content of the different fractions was related to water absorption in pellets and free water on slurries and that free water was a good indicator of the potential for a material to form slurry. Higher flowability (lower yield stress) was found at higher content of lignin, particularly for materials containing 26% lignin where yield stress was reduced to 254Pa when compared with mixtures of 14% lignin that presented yield stresses of around 4000 Pa. We show that rheology modeling linked to compositional characteristics for biomass slurries can be used to predict material flow behavior in a biorefinery to optimize and achieve high solids loadings that enhance the production of ethanol for biofuels. This insight and the ability to form high concentration slurries before pretreatment holds the potential to develop new processing strategies that could help to foster a more efficient and sustainable bio-based industry. </p>

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

This dissertation is composed of two projects dedicated to the development of techniques and technologies for improving the quality of life for patients in both clinical and resource-limited settings. The purpose of the first project was to design a rapid diagnostic device to screen whole blood samples for the presence of infectious agents. Point-of-care (PoC) technologies are becoming increasingly important for the detection of infectious agents in resource-limited settings (RSLs) where state-of-the-art blood screening practices are not feasible for implementation. For this project, a rapid diagnostic device was developed to directly detect pathogen content within freshly drawn whole blood samples using a ligand-binding assay format. The assay is completely self-contained within a hermetically sealed device to minimize operational complexity and ensure operator safety. The diagnostic device is capable of processing complex sample matrices by selectively capturing, concentrating, and labeling infectious agents upon functionalized surfaces. Following sample processing, the assay is optically interrogated with a fluorescence-based reader to provide rapid feedback regarding sample purity. Designs of the rapid diagnostic platform evolved over several prototype generations corresponding to project milestones emphasizing ergonomic performance, military specification testing for environmental resilience, and manufacture to yield production-grade devices for future diagnostic performance data collection. The goal of the regenerative therapy-based portion of this research was to develop a novel technique for the selective enrichment of cells demonstrating enhanced regenerative capacity in tissue-extracted cell samples. Adherent cell cultures of stromal vascular fractions (SVFs) extracted from adipose tissues were exposed to nutrient deficient conditions' eliciting a bimodal cellular response between two dissimilar cell culture subpopulations. The regenerative capacity of these two distinct subpopulations was evaluated by assessing their characteristic morphology, metabolic activity, and ability to undergo multilineage differentiation. The SVF subpopulation which demonstrated sensitivity to the nutrient deficient conditions expressed typical morphological expression of adherent cell cultures, elevated metabolic activity, and the ability to differentiate along adipogenic, chondrogenic, and osteogenic lineages. The SVF subpopulation which demonstrated resistance to the nutrient deficient conditions, however, expressed atypical morphologies, impaired metabolic activity, and did not survive culture with differentiation growth media. Based on the data, the 'treatment-sensitive' SVF subpopulation demonstrated a greater regenerative capacity than the‘treatment-resistant' subpopulation. Furthermore, the treatment-resistant subpopulation of the SVF may be representative of the damaged, senescent, and otherwise less-functional cells that comprise a significant portion of tissue-extracted cell samples and pose a significant risk to therapeutic efficacy and reproducibility. Ultimately, this expedient and inexpensive bioprocessing technique may serve to improve cell-based regenerative therapies by eliminating undesirable cells from culture.

Protein misfolding and aggregation are the cause of many problems within the biopharmaceutical industry and medical fields. Although many experimental studies have been implemented in vivo in order to understand this process, the mechanism occurs in time and length scales inaccessible to conventional experiments. On the other hand, computational studies have shown significant improvement in elucidating key aspects of the aggregation pathways and gain insights to the folding behavior of the proteins. Consequently, this makes computational modeling an ideal complement to experiment in understanding the generic behavior and mechanisms of aggregation. This study is concerned with DynamO, a coarse-grained, off-lattice, general event-driven discontinuous molecular-dynamics simulation package. This simulator offers a unique opportunity to gain insight into the process of protein aggregation by displaying the optimal O(N) asymptotic scaling of the computational cost with the number of particles N, rather than O(NlogN) scaling found in most standard algorithms. The study was split into two loosely related projects: in the first project, a computer model was developed in which the effect of model parameters on folding behavior and characteristics of isolated peptides is investigated. The model parameters include chain stiffness (an overlap parameter defined as the ratio of the hard-core diameter to bond length 'sigma/l'), range of interaction potential 'Gamma', sequence, and chains length 'N '. Based on the model chosen from systems of isolated chains, aggregation in multichain systems is studied. In another project, we simulate various square-well fluid systems with different ranges of interaction potential in order to understand the phase behavior of proteins due to its relevance to aggregation and many bioprocessing events. Changing the model parameters shows different folding behaviors. The model-chains with 64 residues, Gamma equal to 1.1 and sigma/l equal to 1.9 is the least computationally expensive model displaying all the characteristics found in real proteins. We introduce a new order parameter which divides the conformational space into folded and unfolded ensemble-structures, this order parameter corresponds to a transition in the folding behavior of the chains. We define a native state ensemble as an ensemble of structures with small deviation in contact maps for spheres inaccessible to the solvent defined as the core of the chain. This native ensemble corresponds to the structures exhibiting low-temperature fluctuations simulating the 'breathing motions' of real proteins which is considered responsible for their catalytic activities. On the other hand, the non-native ensemble unfolds at higher temperatures, which increases the propensity for aggregation by forming intermolecular contacts, and therefore reproduce the behavior of proteins under severe solution conditions which occurs in bio- processing (this includes high concentration, temperature, pressure, pH ...). The behavior of multichain systems shows that it is possible to correlate the aggregation propensity of chains at room temperature from the behavior of chains in isolated system at the collapse temperature, which in turn correlate with the stability of the low-T ensemble. In the second project, we developed a more efficient way of calculating the critical temperature in SW fluids even for strongly short-ranged systems which are especially difficult to simulate. In the supercritical region, every isotherm obeys the linear equation for the pressure with a high precision within the bounds of uncertainty. The linear equation pm = p0 + Rm with Rm being the constant isothermal rigidity (dp/d)T . The constant rigidity can be used to estimate directly a critical temperature (Tc) and critical pressure (pc), respectively, and also to obtain the pressures and densities of the percolation loci based on an empirical quadratic nature of change in pressure with densities outside the percolation loci. Identifying the critical temperature and how it depends on the pair potential is very important in formulations with a growing need to predict when the solution will go opalescent.

The maximum potential of biorefineries cannot be achieved without the valorisation of their residues. The two main residues from wheat biorefineries, bran and wet distiller's grains and solubles (WDGS), have a projected production of 7 and 3 million tonnes/year respectively by 2025 just in the European Union. These residues can be mixed to form moist animal feed. However, the product can undergo growth of contaminating fungi. This bio-deterioration causes significant losses and represents a hazard for the animals. Currently, commercial preservatives (from the petrochemical industry) are added to prevent bio-deterioration but these add to the production cost. The research reported in this thesis was focused on the utilisation of solid state bioprocessing (SSB) to prevent the bio-deterioration of the moist feed. The method is based on the inhibition of contaminating fungi by an edible fungus, R. oryzae, considered safe for animal consumption which is a lactic acid producer. This fungus is known to produce very dense mycelia causing significant oxygen transfer limitations and this, together with the reduction in pH and substrate available for contaminating fungi, was explored as a potential mechanism to bio-preserve the moist feed. SSB, has been targeted as a key technology for the treatment of agro-industrial solid residues. However, the measurement of parameters in SSB is very complicated, hampering the development of the technology. Consequently, methods for the measurement of pH and growth in SSB were also studied. The measurement of pH is normally performed in extract solutions but, although widely used, the method has not been standardised. There are many extraction variables, such as contact time, type of solids and solvent, solid:water ratio and agitation velocity, involved in the measurement. Experiments revealed that changes in the extraction conditions affect pH readings. The degree of impact depends upon the variable but results presented in this thesis clearly highlight the importance of providing a precise and comprehensive report of the extraction conditions. For the estimation of growth, a method based on digital imaging analysis (DIA) was developed in this work. DIA is a non-destructive, quick and simple method, which uses computational analysis of digital images to measure areas and colour changes on a surface. The technique provided data that allowed an objective comparison of fungal colonies on plates, making it of higher quality than simple visual evaluation. It was determined that C*Traffordgold, the model moist feed used in this research, has starch and moisture contents of 10% and 50% respectively. Bio-deterioration is caused by indigenous organisms in the bran and is observable after only 2 days of incubation at 20°C. R. oryzae was able to bio-preserve the material for at least a month. This is a very promising result since preservative treated C*Traffordgold shows considerable bio-deterioration after just 2 weeks. DIA was used for the estimation of R. oryzae growth at different temperatures, moisture contents and inoculum sizes. The fungus was able to grow at temperatures between 15° and 37°C, with optimum growth at 30°C. An increase in the moisture content resulted in faster growth of the fungus and it was determined that a minimum inoculum size of 103 spores/gC*Traffordgold is necessary to avoid bio-deterioration. The bio-preservation is a result of the reduction of substrate and oxygen available for contaminating microorganisms. Studies revealed that lactic acid does not inhibit growth of contaminating fungi even at pH 4. On the other hand, R. oryzae showed radial growth rates up to 6 times faster than those from contaminating cultures. The fungus is able to surround the competitor colony and 'enclose' it. The formation of a fungal barrier limits the oxygen available for the undesired colony, hampering its growth. It was estimated that by implementing the bio-preservation method a wheat biorefinery producing 180,000 tpa of moist feed could save circa 500,000 USD per annum. Integration of residue processing in biorefineries is necessary to make them sustainable. The application of SSB for bio-preservation could enhance the overall value of wheat biorefineries and simultaneously reduce dependency on the petrochemical industry.

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.

Synechocystis PCC 6803 is a photosynthetic bacterium that has the potential to make bioproducts from carbon dioxide and light. Biochemical production from photosynthetic organisms is attractive because it replaces the typical bioprocessing steps of crop growth, milling, and fermentation, with a one-step photosynthetic process. However, low yields and slow growth rates limit the economic potential of such endeavors. Rational metabolic engineering methods are hindered by limited cellular knowledge and inadequate models of Synechocystis. Instead, inverse metabolic engineering, a scheme based on combinatorial gene searches which does not require detailed cellular models, but can exploit sequence data and existing molecular biological techniques, was used to find genes that (1) improve the production of the biopolymer poly-3-hydroxybutyrate (PHB) and (2) increase the growth rate. A fluorescence activated cell sorting assay was developed to screen for high PHB producing clones. Separately, serial sub-culturing was used to select clones that improve growth rate. Novel gene knock-outs were identified that increase PHB production and others that increase the specific growth rate. These improvements make this system more attractive for industrial use and demonstrate the power of inverse metabolic engineering to identify novel phenotype-associated genes in poorly understood systems. / Singapore-MIT Alliance (SMA)