<p dir="ltr">Corn stover is a promising resource for biofuels, but its high viscosity makes it challenging to convert into a pumpable slurry. This thesis covers the scale-up, techno-economic assessment, and life cycle assessment to ensure profitable and sustainable biofuel production. In this study, a slurry was created using corn stover pellets, enzymes, and deionized water with a moisture content of 16%. The viscosity of the slurry was measured at different concentrations and shear rates using an Anton Parr rheometer. Important parameters such as the consistency index and flow behavior index were obtained through linear regression, which were then used to predict energy usage by correlating them with slurry concentration. Steady and unsteady state mass and energy balances were conducted to determine operation time, verify power consumption, and provide values for techno-economic and life cycle assessments.</p><p dir="ltr">Our study found that slurry can be pumped up to 300g/L concentrations with a viscosity of less than 300 Pa*s at shear rate close to 3Hz. We established a correlation between consistency and flow behavior index with solids content, predicting power consumption in the mixing tank. Our findings enabled us to calculate the production cost of $0.0155 per pound of the pumpable slurry, which is 38% less than the sale price to biorefineries.</p><p dir="ltr">This study confirms that it is economically viable to convert corn stover pellets into pumpable slurry for biofuel production without pretreatment. Although the process faces challenges with viscosity, it ensures a steady supply of feed for the biorefinery and results in profitability. The research takes a comprehensive approach that includes benchtop scale-up and assessments, establishing the practicality and cost-effectiveness of slurry production. Experimental findings and correlation analysis provide accurate predictions of power consumption, while steady-state energy balance results closely align with predictions. Techno-economic and life cycle assessments further support the competitiveness of slurry conversion. In essence, this research offers a sustainable solution to challenges in energy generation.</p>

No description available.

<p> Improved and efficient bioprocessing technology is a key requirement in the manufacture of biopharmaceuticals. The increasing need to reduce biopharmaceutical cost is driven by the business challenges such as the emerging biogenerics. The great advances in upstream technologies make bioseparation the major cost in bioprocessing. Developing efficient bioseparation technologies is therefore strongly desired. Membrane chromatography is a promising bioseparation technology which combines the advantages of membrane technology and chromatography, thus making high-throughput and high-resolution bioprocesses feasible. This thesis focuses on the novel applications and improvements of membrane chromatography: bioprocessing of transgenic tobacco derived monoclonal antibodies and PEGylated proteins, integrated bioprocessing for antibody fragmentation, study of antibody binding on membranes and the development of novel membranes.</p> <p> Membrane chromatography based bioprocesses were developed for purification of monoclonal antibody (mAb) from transgenic tobacco, primarily addressing the challenge of low mAb abundance in the feed material. PEGylated proteins were purified, demonstrating that high-throughput and high-resolution purification of low-binding-propensity proteins was feasible using membrane chromatography.</p> <p> Membrane chromatography based reactant adsorptive membrane bioreactor separator (RAMBS) systems were developed to integrate enzymatic fragmentation of human IgG with the purification of target fragment. This novel system facilitated the process intensification and led to higher IgG digestion than in liquid phase reaction.</p> <p> The mechanism of hydrophobic interaction based IgG binding on synthetic membranes was studied using the RAMBS system. The results showed that the binding took place primarily through a combination of the hinge and CH2 domain of Fc. This study provides a new approach for studying antibody interaction with membranes and surfaces and could help design membrane-based antibody purification, immunoassay and biomaterials.</p> <p> PEG grafted filter paper was developed as an inexpensive alternative to commercial synthetic membranes. These novel membranes possessed high permeability and low fouling tendency and demonstrated good selectivity and reusability in monoclonal antibody purification.</p> / Thesis / Doctor of Philosophy (PhD)

Cellulosic biomass is an incredibly abundant resource and a capable feedstock for production of energy, biofuels, and commodity chemicals. Current technologies for bioprocessing of cellulose utilize a three-step process in which enzymes capable of cellulose hydrolysis are expressed and purified, cellulose is hydrolyzed, and then product is formed in separate processes. This multi-step processing increase costs. As such, one approach to lowering these costs it to develop on consolidated system in which all three of these processes occur in a single step. Toward this aim, the three main goals of this dissertation are (1) characterization of a new hydrolytic enzyme and its application to fermentation of relevant sugars, (2) selection of proteins capable of intracellular cellobiose transport, and (3) development of a minimal set of cellulases capable of extensive hydrolysis under physiological conditions. A mixture of cellodextrins is produced by enzymatic hydrolysis of cellulose and Ced3A, a cellodextrinase, was shown to hydrolyze all of these completely to glucose and confer the ability to metabolize these sugars to E. coli when expressed. Activity on cellobiose, however, was lower than on other species. Co-expression of Cep94A, a cellobiose phosphorylase, and Ced3A was shown to improve the cellobiose metabolism of E. coli. In order to facilitate conversion of cellobiose to glucose by Cep94A, cellobiose must be transported into the cytoplasm. Three cellobiose permease enzymes, LacY, CP1, and CP2, were expressed in E. coli. It was shown that each protein has affinity for cellobiose transport and expression of each 126 allowed fermentation of cellobiose by E. coli strains expressing a cytoplasmic cellobiase. All three proteins are likely suitable for cellobiose transport during a consolidated bioprocess. Finally, a system of three cellulase enzymes Cel5H, Cel9R, and Cel48S were evaluated at E. coli physiological conditions and it was shown that extensive hydrolysis occurred at over half of the compositions tested. Additionally, when strains expressing cellulases were grown in binary culture with strains previously engineered for cellodextrin metabolism substantial product formation was observed, representing suitable performance of a consolidated cellulose bioprocess. This dissertation presents successful performance of all three components necessary for consolidated bioprocessing both individually and when working in tandem. Furthermore, the technologies developed in this dissertation demonstrate the capacity for consolidated bioprocessing of cellulose.

Processing of lignocellulosic biomass for transportation fuels and other biocommodities in integrated biorefineries has been proposed as the future for emerging sustainable economies. Currently bioprocessing strategies are all multi-step processes involving extensive physicochemical pretreatments and costly amounts of exogenous enzyme addition. Consolidated bioprocessing (CBP), or direct microbial conversion, is a strategy that combines all the stages of production into one step, thus avoiding the use of expensive pretreatments and exogenous enzymes that reduce the economic viability of the products produced. With a growing trend towards increased consolidation, most of the reported work on CBP has been conducted with soluble sugars or commercial reagent grade cellulose. For CBP to become practical fermentative guidelines with native feedstocks and purified cellulose need to be delineated through specific substrate characterization as it relates to possible industrial fermentation. By carefully reviewing the fundamentals of biomass pretreatments for CBP, a comparative assessment of the fermentability of non-food agricultural residue and processed biomass was conducted with Clostridium thermocellum DSMZ 1237. Cell growth, and both gaseous and liquid fermentation end-product profiles of C. thermocellum as a CBP processing candidate was characterised. Batch fermentation experiments to investigate the effect of cellulose content, pretreatment, and substrate concentration, revealed that higher yields were correlated with higher cellulose content. Pretreatment of native substrates that increased access of the bacterial cells and enzymes to cellulose chains in the biomass substrate were key parameters that determined the overall bioconversion of a given feedstock to end-products. The contribution of amorphous cellulose (CAC) in different biomass substrates subjected to the same pretreatment conditions was identified as a novel factor that contributed to differences in bioconversion and end-product synthesis patterns. Although the overall yield of end products was low following bioaugmentation with exogenous glycosyl hydrolases from free-enzyme systems and cellulosome extracts. Treatment of biomass substrates with glycosyl hydrolase enzymes was observed to increase the rate of bioconversion of native feedstocks in biphasic manner during fermentation with C. thermocellum. A “quotient of accessibility” was identified as a feedstock agnostic guideline for biomass digestibility. / October 2015

Protein A (ProA) chromatography is a bioseparations technique employed throughout the biopharmaceutical industry for the selective capture and purification of IgG-class monoclonal antibodies (mAbs) and Fc-fusion proteins. The rapid growth of mAbs as commercial therapeutics has motivated the need for improved, efficient, and high-throughput purification processes during manufacturing. In direct response, the work presented in thesis aims to 1) increase the scientific community’s understanding of IgG adsorption behavior on ProA chromatography resins and 2) improve the performance of ProA chromatography with ligands that are chemically modified using polyethylene glycol (PEGylated). The results of this thesis suggest that IgG molecules of varying binding strength, or varying elution pH, are capable of competing for binding sites on ProA chromatography resins in simultaneous or sequential adsorption. The competitive phenomenon derives from variance in IgG binding strength, or IgG elution pH, due to differences in sub-class behavior as well as secondary IgG binding interactions with the ProA ligand. Competition is readily apparent in the adsorption of human polyclonal IgG, which has a wide variety of IgG sub-classes and binding epitopes. Additionally, the results presented in this thesis suggest that ProA chromatography resins with PEGylated ligands are a viable path to increase resin robustness and real-world chromatographic selectivity. It is demonstrated that ligand PEGylation can increase resistance to proteolytic digestion, mitigate impurity interactions with mAbs that are bound to ProA, and increase process selectivity against Chinese Hamster Ovary host cell proteins by up to 37%. However, resins with large volumes of conjugated PEG significantly decrease IgG static binding capacity and decrease the available pore space for diffusion, resulting in losses in dynamic binding capacity and productivity. Lighter modifications appear to avoid losses in dynamic binding capacity, however, they do not appear to be effective at mitigating impurity interactions with mAbs that are bound to ProA, which is key to increasing process selectivity. PEGylation of ProA also universally increases the elution pH of IgG molecules by weakening the binding interaction. This last result opens another path of viability for PEGylated ProA ligands for purification of mAbs of Fc-fusion proteins that are sensitive to low pH environments.

As the worldwide demand for recombinant proteins and valuable metabolites continues to grow, and as the biological toolset at our disposal continues to expand, the development of novel, robust, and effective platforms for the production of these bioproducts represents an area of ever-increasing interest. Although many such bioprocesses are currently economically viable, many more, though holding considerable promise, remain uncompetitive. The development of novel, more productive systems increases the versatility and industrial applications of bioprocesses. The work described in this thesis explores several aspects of bioprocessing, both on the upstream side, concerned with the development of novel recombinant protein expression platforms or the isolation of novel genes with products possessing characteristics of interest, and on the downstream side, through the improvement of fermentation-based bioprocesses. Thirty-six homoplasmic recombinant strains of the microalga Chlamydomonas reinhardtii were developed having integrated genes for phytase or xylanase under the control of psbA and psbD promoters, codon optimized using novel algorithms, at two different genetic loci, in chloroplasts, to be used as novel animal feed additives. Enzyme production was characterized, and results, when compared to other published work in this field, may provide insight into the factors impacting recombinant protein production in microalgae. Using a “bio-prospecting approach”, the microflora of the digestive tract of a Canadian beaver was screened for cellulase-producing microorganisms. Although the screening approach did successfully identify a novel β-glucosidase gene from an isolated strain of Bacillus thuringiensis, the sequence was not significantly different from those already characterized. Two bioprocessing studies were performed to improve recombinant protein production in Pichia pastoris. In the first, the composition of standard Basal Salt Medium (BSM) was systematically optimized for the production of recombinant phytase, and the optimized media produced significantly more enzyme than the standard one, while also containing significantly reduced concentrations of KH2PO4 and MgSO4·7H2O (27.9 g/l and 4.8 g/l respectively), lowering the price of process inputs. The second was based on the screening of unconventional carbon sources for candidates that could sustain the growth and enzyme production using the same P. pastoris strain. Fructose and ethanol have shown to be viable alternatives to glucose or glycerol as sole carbon sources, and provide flexibility in terms of process design.

Compressed and supercritical fluids, such as pressurized CO2, ethane, orpropane, provide a versatile and environmentally acceptable alternative to conventionalliquid organic solvents in bioprocessing applications – specifically in the areas ofproduct extraction, protein purification, microbial sterilization, and enzymatic and wholecellbiocatalysis. While their advantages have been well demonstrated, the effects ofcompressed and supercritical fluids on whole cells are largely unknown.Metabolic and structural perturbations of whole cells by compressed andsupercritical fluid solvents were examined. These perturbations exist as cell metabolismand membrane structure are influenced by pressure and the presence of a solventphase. Continuous cultures of Clostridium thermocellum (a model ethanol-producingthermophilic bacterium) were conducted under elevated hydrostatic and hyperbaricpressure to elucidate pressure- and solvent-effects on metabolism and growth.Fluorescence anisotropy was employed to study liposome fluidization due to thepresence of compressed and supercritical fluids and their partitioning/accumulation inthe phospholipid bilayer.Under elevated hydrostatic pressure (7.0 and 13.9 MPa; 333 K), significantchanges in product selectivity (towards ethanol) and growth were observed in C.thermocellum in conjunction with reduced maximum theoretical growth yields andincreased maintenance requirements. Similarly, metabolism and growth were greatlyinfluenced under hyperbaric pressure (1.8 and 7.0 MPa N2, ethane, and propane; 333K); however, severe inhibition was observed in the presence of supercritical ethane andliquid propane. These changes were attributed to mass-action effects on metabolicpathways, alterations in membrane fluidity, and the dominant role of phase toxicityassociated with compressed and supercritical fluids.Fluorescence anisotropy revealed fluidization and melting point depression ofdipalmitoylphosphatidylcholine liposomes in the presence of CO2, ethane, and propane(1.8 to 20.7 MPa; 295 to 333 K). The accumulation of these fluids within the bilayerupon pressurization and the ordering effects of pressure influenced liposome fluidity, themelting temperature, and the gel-fluid phase transition region. These resultsdemonstrate the disordering effects of compressed and supercritical fluids on biologicalmembranes and the ability to manipulate liposomes.

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

Continuous counter current extraction (CCCE) or dual flow counter current chromatography (DFCCC) is a promising technique where components can be separated continuously by two liquid phases that flow in opposite directions through a continuous length of coiled tubing. Specially designed end connectors and a coil planet centrifuge allow each respective phase to be pumped into each end of the tubing and the other phase to elute at each opposite end. In this thesis the feasibility and the mechanics of CCCE are investigated using stroboscopic photography on an experimental rig and a specially built pilot-scale CCCE centrifuge. The mechanics of the hydrodynamics in the coil was investigated systematically by comparing the measured volumes with photographic images of the process. This investigation revealed that the phases are not distributed evenly throughout the coil, which was previously assumed, but that there is a transition area where the phases switch from mainly upper phase at the head end of the tubing to mainly lower phase at the tail end. This means that the sample encounter three different phase distribution zones in the coil. At the head the upper phase is the dominant phase with a small volume of lower phase running through. At the tail the reverse situation is found and lower phase is dominant. The third zone is a short segment of the coil where there is a transition between the dominant phase conditions that exist at each end. The position of the transition zone and the volume of the other two zones are profoundly affected by the relative flow rates of the two phases. This work indicates that the volume distribution in the coil is affected most by the upper phase flow rate. The pilot-scale CCCE centrifuge was used to successfully separate industrially supplied samples. Crude reaction liquor was processed in both batch and continuous modes achieving the separation of the multi-component mixture into two groups. Changing the flow rate combinations changed the location of elution of some of the components in the mixture. Separation efficiency was maintained even when sample loading was increased. The separations were shown to be predictable with the dual flow theoretical model.