Engineering microorganisms at the systems level is recognized to be the future of metabolic engineering. Thanks to the development of genome annotation, mcroorganisms can be understood, as never before, and be reconstructed in the form of computational models. Flux balance analysis provides a deep insight intocellular metabolism and can guide metabolic engineering strategies. In particular, algorithms can assess the cellular complexity of the metabolism and hint at genetic interventions to improve product productivity. In this work, Synechosystis PCC6803 metabolism was invesetigated in silico. Genetic interventions could besuggested to couple butanol synthesis to growth as a way to improve currentproductivities. Cofactor recycling and, in particular, buffering mechanisms were shown to be important targets. Creating a cofacor imbalance and removing thesebuffering mechanisms is an important driving force. This forces a carbon flux through butanol synthesis to maintain cofactor balance and sustain growth.

Thesis (Ph.D. (Microbiology)) -- University of Limpopo, 2021 / Biofuel production from lignocellulose material is an attractive alternative to fossil fuel. The use of lignocellulose material for biofuel production is imperative because of the numerous advantages that it offers. Biofuel is environmentally friendly and in developing countries such as South Africa, it has the potential to reduce the use of imported fuel and create jobs. Currently, several constraints are affecting the implementation of biofuel. One of the constraints is the cost-effectiveness and the efficiency of the enzymes involved in the enzymatic degradation of lignocellulose polymers to monomers, which can further be fermented to bioethanol. The potential way to reduce enzymatic degradation cost could be by supplementing the fungal enzymes with accessory enzymes such as endo-xylanase. The enzyme production cost is also dependent on the carbon source used. Lignocellulose materials that are regarded as waste must be assed for their use as enzyme inducer carbon sources and as biomass for biofuel production. This is a potential route that will reduce enzyme and biofuel production costs. Biofuel production cost can further be reduced by finding a yeast that can ferment xylose and ferment in the presence of inhibitors released during lignocellulose pretreatment. This study sought to tackle the enzymatic hydrolysis constraints and also search for xylose-fermenting yeast by exploring the gut microbiota of dung beetle. The gut of the dung beetle has recently received great attention since it is proposed to be a bioreactor for lignocellulolytic microorganisms that can be used in biofuel applications. This is because dung beetles feed on the dung of herbivorous animals and the dung is composed of 80% undigested plant material. In this study the guts of four Scarabaeidae dung beetles Kheper nigroanaeus Boheman, Heteronitis castelnaui, Pachylomerus femoralis, Anachalcos convexus and dung beetle larvae, Euoniticellus intermedius were screened for hemicellulolytic fungi and xylose-fermenting yeast. Hundred and thirty-two yeast isolates and two-hundred and twenty-two filamentous fungi were isolated and identified using ITS and D1/D2 regions. The yeast isolates were assigned to 8 genera and 18 species, Trichosporon was the most dominant genus while Candida tropicalis was the most dominant specie. Some of the yeast isolates were identified as uncultured fungi. This yeast must be characterised to be certain if they are novel species. The fungal isolates were assigned to 12 genera and 25 species, Aspergillus was the most dominant genus while Hypocrea lixii was the most dominant specie. The yeast isolated could assimilate xylose and could grow at a maximum temperature of 40 °C. Furthermore, these yeast isolates could also grow in the presence of 3 g/L acetic acid. Most of the fungal isolates had xylanolytic activity. The phylogenetic analysis revealed close genetic relatedness between isolates from the different dung beetle species and dung beetle larvae. The profile of the fungal genera was similar in the different dung beetles. Both guts and the larvae had Aspergillus, Hypocrea, Trichoderma, Talaromyces and Penicillium. The filamentous fungi that showed good xylanolytic activity were further screened for their ability to produce xylanase enzyme using thatch grass as an inductive carbon source. Thatch grass was selected in this study since it is in-house plant-based biomass. Thatch grass is abundantly available in South Africa; it is used for animal grazing but the more it grows it loses its nutritional content. Once it reaches this stage, it is no longer used and most of it is burnt. The fire from burning grass contains higher levels of nitrogen-containing chemicals that pollute the environment. Its compositional analysis (cellulose 46%, hemicellulose 27% and lignin 10%) also attributed to its selection as potential inductive carbon and attractive lignocellulose biomass for biofuel production. The higher xylanase activity of 283.43, 270 and 287.03 nkat/ml were observed from Aspergillus fumigatus L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus), respectively. This was achieved when acid pretreated thatch grass was used as an inductive carbon source. Aspergillus fumigatus L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus) showed xylanase activity of 393,22, 313,06 and 200 nkat/ml when grown on synthetic xylan. Neosartotya sp AB2XYL20 showed higher xylanase activity on thatch grass. The suitable production process for xylanase enzyme on acid pretreated thatch grass was assessed by conducting a comparative study on solid-state and submerged fermentation using L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus) as the best xylanase producer on acid pretreated thatch grass. The strain showed better xylanase activity when submerged fermentation was used. In this study, Hypocrea lixii AB2A3 was selected for further studies since it was the most dominant species and also showed good xylanase activity. Thatch grass was pretreated differently to evaluate the suitable chemical for pretreating thatch grass. Thatch grass was pretreated with dilute sulphuric acid 1.2% and maintained the pH of 5.5 by using sodium hydroxide while another batch was pretreated the same way and was washed with distilled water till pH of 5.5. The other batch was then pretreated with ammonium solution and was also washed with distilled water to maintain a pH of 5.5. The above-mentioned pretreated thatch grass was tested as an inductive carbon source as well as untreated thatch grass. The xylanase activity was determined to assess a good inductive carbon. All the thatch grass pretreated and washed with distilled water showed very low xylanase activity. The untreated thatch grass resulted in lower xylanase activity as compared to xylanase activity achieved when pretreated thatch grass was used. Parameters such as agitation speed and initial inoculum size were also assessed during xylanase production by Hypocrea lixii AB2A3 on acid pretreated thatch grass. Xylanase activity increased from 525 nkat/ml (Inoculum size 2×106 spore/ml and agitation speed 150 rpm) to 584.8 nkat/ml (Inoculum size 2×106 spore/ml and agitation speed 200 rpm). The crude xylanase from Hypocrea lixii AB2A3 was used to hydrolyse acid pretreated thatch grass. This resultant in xylose yield of 138 mg/g of substrate and glucose yield of 49 mg/g of substrate. Crude xylanase was mixed with commercial celluclast™. This enzyme mixture resulted in a xylose yield of 128 mg/g substrate and a glucose yield of 549 mg/g of substrate. The results obtained in this study show that indeed gut of the dung beetles and dung beetle larvae are a rich source of microorganisms that can play an important role in biofuel application and remediating the environment by degrading plant-based biomass regarded as waste into valuable products. It is imperative to evaluate the gut microbiota of dung beetles from different regions in South Africa for their application in the biofuel industry to reinforce its implementation. Thatch grass is a potential inductive carbon and lignocellulose biomass for biofuel production. / NRF (National Research Foundation)

abstract: Redox enzymes represent a big group of proteins and they serve as catalysts for biological processes that involve electron transfer. These proteins contain a redox center that determines their functional properties, and hence, altering this center or incorporating non-biological redox cofactor to proteins has been used as a means to generate redox proteins with desirable activities for biological and chemical applications. Porphyrins and Fe-S clusters are among the most common cofactors that biology employs for electron transfer processes and there have been many studies on potential activities that they offer in redox reactions. In this dissertation, redox activity of Fe-S clusters and catalytic activity of porphyrins have been explored with regard to protein scaffolds. In the first part, modular property of repeat proteins along with previously established protein design principles have been used to incorporate multiple Fe-S clusters within the repeat protein scaffold. This study is the first example of exploiting a single scaffold to assemble a determined number of clusters. In exploring the catalytic activity of transmetallated porphyrins, a cobalt-porphyrin binding protein known as cytochrome c was employed in a water oxidation photoelectrochemical cell. This system can be further coupled to a hydrogen production electrode to achieve a full water splitting tandem cell. Finally, a cobalt-porphyrin binding protein known as cytochrome b562 was employed to design a whole cell catalysis system, and the activity of the surface-displayed protein for hydrogen production was explored photochemically. This system can further be expanded for directed evolution studies and high-throughput screening. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18332号 / 農博第2057号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4839(農学部図書室) / 31190 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 渡邊 隆司, 教授 梅澤 俊明 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20425号 / 農博第2210号 / 新制||農||1047(附属図書館) / 学位論文||H29||N5046(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 加納 健司, 教授 宮川 恒, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

This dissertation studies two important problems in the field of biomass supply chain network. In the first part of the dissertation, we study the impact of different carbon regulatory policies such as carbon cap, carbon tax, carbon cap-and-trade and carbon offsetmechanism on the design and management of a biofuel supply chain network under both deterministic and stochastic settings. These mathematical models identify locations and production capacities for biocrude production plants by exploring the trade-offs that exist between transportations costs, facility investment costs and emissions. The model is solved using a modified L-shaped algorithm. We used the state of Mississippi as a testing ground for our model. A number of observations are made about the impact of each policy on the biofuel supply chain network. In the second part of the dissertation, we study the impact of intermodal hub disruption on a biofuel supply chain network. We present mathematical model that designs multimodal transportation network for a biofuel supply chain system, where intermodal hubs are subject to site-dependent probabilistic disruptions. The disruption probabilities of intermodal hubs are estimated by using a probabilistic model which is developed using real world data. We further extend this model to develop a mixed integer nonlinear program that allocates intermodal hub dynamically to cope with biomass supply fluctuations and to hedge against natural disasters. We developed a rolling horizon based Benders decomposition algorithm to solve this challenging NP-hard problem. Numerical experiments show that this proposed algorithm can solve large scale problem instances to a near optimal solution in a reasonable time. We applied the models to a case study using data from the southeast region of U.S. Finally, a number of managerial insights are drawn into the impact of intermodal-related risk on the supply chain performance.

Marine ecosystems can be a promising reservoir of various kinds of chemical components, applicable as pharmaceutical materials, food, cosmetics, nutraceuticals, and others for different industry. As an example, Tunicates, a group of marine animals, have been attracted a lot of attention in medical application, food market, water pollution issues, and Cellulose nanomaterial production due to their consisting of chemical compounds such as cellulose, amino-sugars, and proteins or protein-polysaccharide complexes e.g. collagen, glycosaminoglycan, chitin, scleroprotein, iodine-binding proteins, and elastin. In this project,  two dominant species of Scandinavian Tunicates, i.e. Ciona intestinalis and Clavelina lepadiformis, harvested from Norwegian ocean have been classified according to body sizes, depths from the ocean surface, ages and species, and separated physically into outer layer and internal organs, followed by measurements of sugar composition, oil content, and  protein content. Application potentials have been investigated by trials for production of pure crystalline cellulose, bioethanol, and biodiesel, and by analysis of amino acid composition of the samples. The cellulose percentage and cellulose yield for the chemically pure cellulose obtained, is around 96% and 54% respectively, and the protein content is decreased step by step by the acid, alkali, and bleaching process applied. Bioethanol can be obtained by fermentation of tunicate hydrolysate with strains A and C which are derived from Saccharomyces cerevisiae. The biodiesel yield of tunicate samples is around 4-6% as an average. The amino acid compositions in our tunicate samples are similar to egg albumin, implying tunicate being an alternative material for animal feed production. Several processing treatments have been conducted with the aims to fractionate tunicate biomass components or enhance the cellulose accessibility and reactivity. After a single processing step, Ba(OH)2 treated samples seemed to be the best in terms of both cellulose preservation (66.5% cellulose) and protein removal (6% protein in the treated residue). Results from the physical separation plus washing reveal that the highest amount of cellulose and protein presents is found in the outer (Tunic) part and internal organs of Tunicate samples respectively. Data obtained from FTIR(Fourier Transform Infrared Spectroscopy) and SEM(Scanning Electron Microscope) indicate that among all processing trials, H3PO4 is the most effective in decreasing the cellulose crystallinity, which renders a higher accessibility for acidic or enzymatic reaction during bioethanol production due to a higher amount of amorphous structure of cellulose. From the analysis results of component contents and structures, it could be concluded that increase of deepness results in a decrease of sugar content of the Tunicate samples while there are no differences in protein and carbohydrate content in different tunicate species. The body size has a positive influence on the protein content and the sample age alters the contents of both sugar and protein. In addition, Tunicate oil has high phospholipid content instead of glycerol ester, the latter being the common oil from vegetable origins. Moreover, lots of free fatty acid is present, and the composition profile of Tunicate fatty acids seems to be similar to fish oil, as revealed by NMR (Nuclear Magnetic Resonance Spectroscopy), FTIR, and GC-MS (Gas Chromatography-Mass Spectrometry).

Biofuel additives have been designed to reduce vehicular emissions to the atmosphere to limit the effects of greenhouse gases on global climate change. The chemical properties of common biofuel additives exhibit ideal characteristics for use in gasoline and diesel, while limiting emissions from exhaust. As biofuel additives begin to be administered regularly to gasoline and fuel sources, the compounds will appear in spill sites, posing a risk to groundwater sources. The interactions that occur between common biofuel additives and trapped gases below the water table were analyzed in this work to further understand the potential consequences on quasi-saturated groundwater zones. The behaviour of trapped gases contaminated with different biofuel additives were analyzed in laboratory experiments conducted in a two-dimensional flow cell to demonstrate the mechanisms of gas flow through a capillary barrier resulting from modified interfacial properties in the presence of a chemical surfactant. Contamination of gas-fluid interfaces by applied biofuel additives at the pore scale resulted in the breakthrough of gas through the capillary barrier. Gas migration terminated at a critical pool height proportional to the reduction in interfacial tension induced by the administered biofuel additives. To further demonstrate the relationship between interfacial tension and critical gas pool height, an interfacial tension-macroscopic invasion percolation model was developed to simulate the transport mechanisms and behaviours of gas flow when an immobile pool is contaminated with 1-Butanol. The findings in this study provide a fundamental understanding of the mechanisms and behaviours of gas mobilization in the presence of common biofuel additives. / Thesis / Master of Science (MSc) / The use of biofuel additives in gasoline and diesel fuels has become an attractive alternative to fully petroleum-based fuels to reduce the release of vehicular greenhouse gases to the atmosphere. As fuel spills and storage tank leaks continue to be a primary source of groundwater contamination, the appearance of biofuel additives in contaminated systems will appear below the subsurface as they continue to be administered to modern gasoline and diesel fuels. This work investigated the consequences of biofuel contamination of groundwater systems containing gas trapped within pore spaces through the use of laboratory experiments and numerical modelling. Contamination of these systems with different biofuel additives displayed a similar response, in which gas had mobilized from within pore spaces and released to the atmosphere. Mobilization of trapped gas in groundwater can alter the primary hydraulic properties that characterize a particular hydrogeologic system.

Marine ecosystems can be a promising reservoir of various kinds of chemical components, applicable as pharmaceutical materials, food, cosmetics, nutraceuticals, and others for different industry. As an example, Tunicates, a group of marine animals, have been attracted a lot of attention in medical application, food market, water pollution issues, and Cellulose nanomaterial production due to their consisting of chemical compounds such as cellulose, amino-sugars, and proteins or protein-polysaccharide complexes e.g. collagen, glycosaminoglycan, chitin, scleroprotein, iodine-binding proteins, and elastin. In this project,  two dominant species of Scandinavian Tunicates, i.e. Ciona intestinalis and Clavelina lepadiformis, harvested from Norwegian ocean have been classified according to body sizes, depths from the ocean surface, ages and species, and separated physically into outer layer and internal organs, followed by measurements of sugar composition, oil content, and  protein content. Application potentials have been investigated by trials for production of pure crystalline cellulose, bioethanol, and biodiesel, and by analysis of amino acid composition of the samples. The cellulose percentage and cellulose yield for the chemically pure cellulose obtained, is around 96% and 54% respectively, and the protein content is decreased step by step by the acid, alkali, and bleaching process applied. Bioethanol can be obtained by fermentation of tunicate hydrolysate with strains A and C which are derived from Saccharomyces cerevisiae. The biodiesel yield of tunicate samples is around 4-6% as an average. The amino acid compositions in our tunicate samples are similar to egg albumin, implying tunicate being an alternative material for animal feed production. Several processing treatments have been conducted with the aims to fractionate tunicate biomass components or enhance the cellulose accessibility and reactivity. After a single processing step, Ba(OH)2 treated samples seemed to be the best in terms of both cellulose preservation (66.5% cellulose) and protein removal (6% protein in the treated residue). Results from the physical separation plus washing reveal that the highest amount of cellulose and protein presents is found in the outer (Tunic) part and internal organs of Tunicate samples respectively. Data obtained from FTIR(Fourier Transform Infrared Spectroscopy) and SEM(Scanning Electron Microscope) indicate that among all processing trials, H3PO4 is the most effective in decreasing the cellulose crystallinity, which renders a higher accessibility for acidic or enzymatic reaction during bioethanol production due to a higher amount of amorphous structure of cellulose. From the analysis results of component contents and structures, it could be concluded that increase of deepness results in a decrease of sugar content of the Tunicate samples while there are no differences in protein and carbohydrate content in different tunicate species. The body size has a positive influence on the protein content and the sample age alters the contents of both sugar and protein. In addition, Tunicate oil has high phospholipid content instead of glycerol ester, the latter being the common oil from vegetable origins. Moreover, lots of free fatty acid is present, and the composition profile of Tunicate fatty acids seems to be similar to fish oil, as revealed by NMR (Nuclear Magnetic Resonance Spectroscopy), FTIR, and GC-MS (Gas Chromatography-Mass Spectrometry).

Abiotic stresses such as drought, salt and exposure to toxic metals adversely affect the growth and productivity of crop plants and are serious threats to agriculture. We are currently working with an Arabidopsis family of proteins known as Stress Associated Proteins (SAPs). There are a total of 14 proteins in the Arabidopsis SAP family whose members have been suggested to provide tolerance to abiotic stresses in plants. For this project, we aim to characterize AtSAP13, which codes for a protein of 249 amino acid residues. Through overexpression, we investigated the sensitivity or tolerances provided by the overexpressed protein in comparison to wild-type plants. Our preliminary results showed that Arabidopsis plants overexpressing SAP13 showed strong tolerance to arsenite, cadmium, and zinc. A semi-quantitative RT-PCR was performed to analyze SAP13’s mRNA levels in wild type plants exposed to the same set of stresses. Tissue specific expression was analyzed using a GUS histochemical assay. Sub-cellular localization of AtSAP13 was analyzed by creating an in-frame fusion of SAP13 and enhanced GFP (eGFP). We set out to optimize growth conditions for Crambe abyssinica in order to determine if Crambe could be grown as a biofuel crop in New England. We have determined that Crambe can be grown successfully in the New England climate. We tested three fertilizer application rates, two different cultivars, and two different soil types for changes in yield. In the end, we observed the greatest increase in yield when planted on well drained soil.