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Thesis (M. Sc. (Microbiology)) -- University of Limpopo, 2016 / Smart hydrogels could facilitate immobilisation of cellulases to allow recovery and decrease enzyme cost in the biofuel industry, as they have a soluble-gel transition. The aim of the study was to design and evaluate the use of smart hydrogels for immobilisation of cellulase system that can be recovered after hydrolysis of cellulosic biomass. Cellulases from Aspergillus niger FGSC A733 produced under solid state fermentation and commercial cellulases were used in immobilisation. Various support matrices prepared were poly-N-isopropylacrylamide (p-NIPAAm), poly-N isopropylacrylamide-co-Methacrylic acid (p-NIPAAm-co-MAA) and supermacroporous poly-crosslinked-Acrylamide-co-N,N’-Methylenebisacrylamide (p-crosslinked-AA-co MBA). Cellulases were coupled onto the support matrices by covalent attachment method through reactive groups of N-acryloxysuccinimide (NAS) or Methacrylic acid N-hydroxysuccinimide (NMS). The low critical solution temperature (LCST) of formed p-NIPAAm-co-MAA copolymer was determined by the inflection point method. The shrinking and swelling kinetics and pH sensitivity of p-NIPAAm-co-MAA copolymer and conjugates were characterised using a cloud point method. Hydrolysis of CMC using cellulase-microbeads-p-NIPAAm and cellulase-crosslinked-p-NIPAAm with different percentage gel showed activity trend of 0.05>1>10>5>0.1% and 5>2>10% respectively. HPLC analysis showed that supplementation of β-glucosidase in cellulase-crosslinked-p-NIPAAm conjugates increased glucose by 12 and 14-fold at 30 and 50 °C respectively in the avicel hydrolysate in comparison with no β glucosidase supplementation. In the hydrolysis of avicel using cellulase-crosslinked p-NIPAAm-co-MAA conjugate a total of 13.6 g/L of reducing sugar was liberated after three cycles. In comparison a total of 21.4 g/L of reducing sugars were released from avicel hydrolysis using cellulase-crosslinked-p-AA-co-MBA conjugate after 3 cycles. In contrast, reducing sugars released in thatch grass hydrolysis using free enzyme were 8 times greater than in cellulase-crosslinked-p-AA-co-MBA conjugate. Cellulase crosslinked-p-NIPAAm-co-MAA conjugates were more stable than free enzyme at 50 and 60 °C after 24 hour and 120 minutes of incubation respectively, but lost activities at 65 °C after 120 minute. Therefore the activity loss in the immobilised enzymes was more due to thermal inactivation during precipitation and recovery than incomplete recovery during precipitation cycles. The results show that cellulases immobilised on smart polymers with sol-gel transition could be used in hydrolysis of cellulose due to ease of recovery. Hydrolysis kinetics was efficient for both immobilised enzyme system (cellulase-crosslinked-p-AA-co-MBA and cellulase-crosslinked-p-NIPAAm-co MAA conjugate) since were re-used in hydrolysis of avicel. Therefore the use of these smart polymers for cellulase immobilisation can contribute in cost reduction of the enzymatic hydrolysis process in the biofuel industry. / National Research Foundation (NRF) , University of Limpopo financial aid office and Flemish Interuniversity Council (VLIR-UOS) fo

Since being discovered, combustion of fuels, especially fossil fuels in the last centuries, has been the dominant source of energy for human life. However, over the years, the adverse effects and shortcomings caused by the vast utilization of these energy sources have been observed; the three most important of which are unreliable resources, unfavorable natural outcomes, and limited performance. Using biofuels is one of the well-established proposed solutions to the scarcity and environmental issues of fossils as they are sustainable sources of energy with acceptable and even superior combustion characteristics. As a second-generation biofuel, anisole has shown promising results with high flame speed and high knock resistance. Therefore, the first chapter of this thesis is focused on experimental investigation of anisole laminar burning speed and stability properties so that it can be used as a benchmark for future kinetic mechanism validations. Stability is another important parameter in combustion systems, especially in diffusion jet flame combustion as used in many applications like thrusters or burners. Different methods are applied to improve the stability of such diffusion flames in propulsion systems, e.g., changing geometrical or flow characteristics of the burner. Most of these efforts have not been practically successful, due to the cost and compatibility issues. Another technique which minimizes such problems is to use electron impact excitation, dissociation and ionization and generate highly concentrated charged/excited species and active radicals. These methods include microwave, dielectric barrier, and repetitive nanosecond pulsed (RNP) discharge and the latter has shown promising results as one of the most effective low-temperature plasma (LTP) methods. In chapters 3 to 5, the benefits and issues associated with using RNP discharge in a single-element concentric methane-air inverse diffusion jet flame are discussed. It has been shown that RNP discharge with adequate discharge properties (voltage and repetition) can increase the stability of the flame and expand the flammability of the jet toward leaner compositions. However, the effectiveness is significant in a certain voltage-frequency ranges which results a non-thermal spark discharge mode. Hence, different modes of discharge were investigated and a parametric study on the transition between these modes were done.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24664号 / 農博第2547号 / 新制||農||1098(附属図書館) / 学位論文||R5||N5445(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 白井 理, 教授 菅瀬 謙治, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24671号 / 農博第2554号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5452(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 小川 順, 教授 阪井 康能, 教授 白井 理 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

In order to meet the energy needs of a growing world in a sustainable manner, new high efficiency, carbon-neutral fuels and chemical feedstocks are required. An emerging approach that shows promise for high efficiency production of renewable fuels and chemicals is the use of purified enzymes combined in one pot to catalyze complex conversions: synthetic pathway biotransformations (SyPaB). An exemplary technology in this burgeoning field is the production of hydrogen from biomass sugars. Lignocellulosic biomass, which includes agricultural residues, energy crops, and industrial waste streams, is an ideal substrate for SyPaB conversion, as it is abundant and cheap, second only to untaxed coal on a $/energy content basis. But the structure of biomass is highly recalcitrant, making high-yield biological conversion difficult to achieve. In order to increase susceptibility to enzymatic digestion, thermochemical pretreatments are applied, with the goals of removing of lignin, the simplest example being soaking in aqueous ammonia (SAA); hemicellulose removal, most often using dilute acid (DA); and increasing cellulose accessibility by cellulose solvent-based pretreatments, such as cellulose solvent- and organic solvent-based lignocellulose fractionation (COSLIF). In a comparison of the lignin removal (SAA) and accessibility increase (COSLIF) approaches, we found that certain levels of lignin removal (~15%) were important, but further lignin removal was less effective at achieving digestibility gains than increasing cellulose accessibility. Pretreated biomass forms an excellent substrate for SyPaB hydrogen generation, due to low cost and high sugar content. Following experiments demonstrating the high yield conversion of sucrose to hydrogen (97%) and SyPaB generation of hydrogen at a rate commensurate with the best biological rates achieved, 157 mmol/L/h. SyPaB was combined with enzymatic hydrolysis to enable the direct conversion of cellulosic biomass, including untreated, DA, and COSLIF corn stover. In addition, an updated kinetic model of the system was used to rationally increase the maximum hydrogen production rate by 70% while minimizing total enzyme loading and without increasing substrate concentration. Together, these results demonstrate the high level of engineering control in cell-free systems, which can enable conversion of a variety of substrates to hydrogen at the highest possible yield and rates as high as any biohydrogen production method. / Ph. D.

The Midwest US has experienced significant changes in agricultural land use and management practices in recent decades. Cropland expansion, crop rotation change, and crop phenology changes could lead to divergent environmental impacts on linked ecosystems. The overall objective is to examine agricultural land use and management changes and their impacts on water quality in the Midwest US, which is addressed in three separate studies. The first study examined spatial and temporal dimensions of agricultural land use dynamics in east-central Iowa, 2001-2012. Results of this study indicated that increases in corn production in response to US biofuel policies had been achieved mainly by altering crop rotation. This study also examined spatial relationships between cultivated fields and crop rotation practices with respect to underlying soils and terrain. The most intensively cultivated land had shallower slopes and fewer pedologic limitations than others, and the corn was planted on the most suitable soils. The second study characterized key crop phenological parameters (SOS and EOS) for corn and soybean and analyzed their spatial patterns to evaluate their change trends in the Midwest US, 2001-2015. Results showed that MODIS-derived SOS and EOS values are sensitive to input time-series data and threshold values chosen for crop phenology detection. The non-winter MODIS NDVI time-series input data, and a lower threshold value (i.e., 40%) both generated better results for SOS and EOS estimates. Spatial analyses of SOS and EOS values displayed clear south-north gradient for corn and trend analyses of SOS revealed only a small percentage of counties showed statistically significant earlier trends within a user-defined temporal window (2001-2012). The third study integrated remote sensing-derived products from the first two studies with the SWAT model to assess impacts of agricultural management changes on sediment and nutrient yields for three selected watersheds in the Midwest US. With satisfied calibration and validation results for stream flows, sediment and nutrient yields, considered under differing management scenarios, were compared at different spatial scales. Results showed that intensive crop rotation, advancing the planting date with the same length of growing season, and longer growing seasons, dramatically increased, maintained, and slightly reduced sediment, total nitrogen, and total phosphorous yields, respectively. Overall, these studies together illuminate relationships between broad-scale agricultural policies, management decisions, and environmental impacts, and the value of multi-temporal, broad-scale, geospatial analysis of agricultural landscapes. / Ph. D.

The goals of this research were 1) to produce hydrogen in high yields from cellulosic materials and water by synthetic pathway biotranformation (SyPaB), and 2) to increase the hydrogen production rate to a level comparable to microbe-based methods (~ 5 mmol H2/L/h). Cell-free SyPaB is a new biocatalysis technology that integrates a number of enzymatic reactions from four different metabolic pathways, e.g. glucan phosphorylation, pentose phosphate pathway, gluconeogenesis, and hydrogenase-catalyzed hydrogen production, so as to release 12 mol hydrogen per mol glucose equivalent. To ensure the artificial enzymatic pathway would work for hydrogen production, thermodynamic analysis was firstly conducted, suggesting that the artificial enzymatic pathway would spontaneously release hydrogen from cellulosic materials. A kinetic model was constructed to assess the rate-limited step(s) through metabolic control analysis. Three phosphorylases, i.e. α-glucan phosphorylase, cellobiose phosphorylase, and cellodextrin phosphorylase, were cloned from a thermophile Clostridium thermocellum, and heterologously expressed in Escherichia coli, purified and characterized in detail. Finally, up to 93% of hydrogen was produced from cellulosic materials (11.2 mol H2/mol glucose equivalent). A nearly 20-fold enhancement in hydrogen production rates has been achieved by increasing the rate-limiting hydrogenase concentration, increasing the substrate loading, and elevating the reaction temperature slightly from 30 to 32°C. The hydrogen production rates were higher than those of photobiological systems and comparable to the rates reported in dark fermentations. Now the hydrogen production is limited by the low stabilities and low activities of various phosphorylases. Therefore, non-biologically based methods have been applied to prolong the stability of α-glucan phosphorylases. The catalytic potential of cellodextrin phosphorylase has been improved to degrade insoluble cellulose by fusion of a carbohydrate-binding module (CBM) family 9 from Thermotoga maritima Xyn10A. The inactivation halftime of C. thermocellum cellobiose phosphorylase has been enhanced by three-fold at 70°C via a combination of rational design and directed evolution. The phosphorylases with improved properties would work as building blocks for SyPaB and enabled large-scale enzymatic production of cellulosic hydrogen. / Ph. D.

Although the 2005 EPAct was enacted to help bolster the emerging biofuel industry, 52% of advanced biofuel projects were closed or shut down by 2015. However, there are no complete lists of barriers that impeded these projects. The goal of this study was to develop a framework of barriers impeding success of advanced biofuel projects by conducting a literature review of barriers, spatial analysis of status, survey of barriers, and determination of coproducts and byproducts and their marketing and distribution barriers from the industry stakeholders. The spatial analysis indicated 59 biofuel projects were attempted, and their Eastern and Western location by status was not a barrier. Using Grounded Theory, nine barriers were derived and aggregated in major categories, including product development, strategy, technology, competition, energy costs, funding, government, suppliers, and third-party relations. A contingency analysis was conducted relating their status to internal and external barriers, indicating no relationship between type of closing and type of barrier. Next, the number of barriers was expanded to 23, and a survey was conducted to gain knowledge on these barriers from industry stakeholders. When comparing the barriers by stakeholders, there were differences based on status, type, and technology of the projects. In addition, the survey and discussion identified 79 barriers different across years, type of industry (pilot, demonstration, or commercial), status (open, closed, or planning), and technology (thermochemical, biochemical, or hybrid). Forty-seven coproducts and byproducts and many unknown barriers to their marketability and distribution were determined and ranked by primary and secondary barriers. These extensive lists of barriers and coproducts will aid future biofuels projects in their planning, research, and development stages. / Master of Science

Tyrimo tikslas – nustatyti įsitraukimo į biodegalų gamybą veiksnius ir įvertinti šalies įsitraukimą į biodegalų gamybą integralumo nacionalinėje ekonomikoje požiūriu. Darbo praktinė vertė. Biodegalų gamyba yra viena iš šiuolaikinės bioekonomikos sričių, kuria susidomi vis daugiau pasaulio šalių. Pastarasis dešimtmetis gali būti apibūdinamas kaip intensyvios biodegalų gamybos plėtros laikotarpis. Šalys, įsitraukdamos į biodegalų gamybą, siekia prisidėti prie darnios energetikos vystymosi. Darbe analizuojama, kaip šalies įsitraukimo į biodegalų gamybą veiksniai varijuoja priklausomai nuo šalies turtingumo naftos ištekliais. Nustatyta, kokie yra dominuojantys įsitraukimo į biodegalų gamybą veiksniai specifinėse šalių grupėse ir kokiu integralumu nacionalinėje ekonomikoje biodegalų gamyba pasižymi šiose šalyse. Tyrimas patvirtina, kad net ir paskatintas politinių iniciatyvų, biodegalų pramonės steigimasis šalyje paprastai grindžiamas išskirtinai didesniu apsirūpinimu biodegalų gamybos ištekliais arba didesne iškastinių energetinių išteklių stoka. Gautos tyrimo išvados gali pasitarnauti formuojant valstybės energetinės politikos gaires alternatyvios energetikos plėtotei. Mokslinio darbo naujumas. Šalies įsitraukimo į biodegalų gamybą vertinimo rezultatai papildo ekonomikos mokslo žinias apie egzistuojančius ryšius tarp ekonominių veiklų plėtojimo ir joms reikalingų gamybos išteklių šalyje gausos. Šių ryšių egzistavimas darbe atskleidžiamas analizuojant bioetanolio gamybą. Tyrimas... [toliau žr. visą tekstą] / Research aim – to identify factors behind engagement in biofuel production and evaluate engagement by a country in terms of production integration in the national economy. Practical value of the research. The production of biofuels is one of the most modern areas of the bioeconomy, which involves more and more countries around the world. The last decade can be described as an intense period of the development of biofuel production. Engaging in the production of biofuels countries seek to contribute to the sustainable energy development. In this research the analysis of factors of country’s engagement in biofuel production, depending on the oil richness of the country, is performed. It was analysed which factors determine the engagement in biofuel production in different groups of countries and which parameters of biofuel production integration in national economy are satisfied in these counties. The study confirms that biofuel industry establishment in the country, even promoted by political initiatives, is usually based on purely larger self-sufficiency in production resources or higher scarcity of fossil energy resource. The resulting findings can serve in shaping the state's energy policy guidelines for the development of alternative energy sources. Originality of the research work. The obtained results supplement the scientific knowledge in economics about the existence of the relationships between development economic activities and the abundance of necessary production... [to full text]