The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.

No description available.

A dissertation submitted to the Faculty of Engineering and the Built Environment, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Master of Science in Engineering. March 2019 / Depleting fossil fuels and the increasing energy demand has necessitated the move to alternative renewable forms of energy. Lignocellulosic biomass is a renewable and sustainable source for highly valuable bio-based chemicals and material production in a biorefinery system. The effective fractionation of the main components of lignocellulosic biomass (cellulose, hemicellulose and lignin) into usable forms is a crucial step in unlocking an economically viable, high-value product producing biorefinery. The main concern associated with the conversion of lignocellulose is overcoming biomass recalcitrance using pretreatment while still maintaining a green, cost-effective and energy efficient process. Over the last decade, molten hydrate salts have been used for isolated cellulose dissolution, however very few studies have been done to check their ability in lignocellulosic biomass pretreatment. The aim of the study was to compare seven molten hydrate salt solvent systems including unary, binary and ternary mixtures of ZnCl2.4H2O, LiClO4.3H2O and Urea for the effective pretreatment of corncob in terms of physicochemical properties and pretreatment efficiencies and to optimise these efficiencies. The molten salt hydrate pretreatment systems used in this study are aimed at fractionating the corn cobs biomass into a solid fraction which mostly contains cellulose and lignin as the major components, while the liquid fraction contains hemicellulose as the main component. The pretreatment experiments were carried out at 70 for 60 minutes at a biomass: solvent ratio of 1:10. Physicochemical change after pretreatment was checked by FTIR, XRD and SEM. The most efficient solvent mixture was identified by gravimetric analysis for its ability to fractionate the biomass into a cellulose and lignin rich solid fraction and a hemicelluloserich liquid fraction. The effect of solvent pretreatment operating variables (temperature, time and solvent concentration) was investigated to maximize cellulose recovery, hemicellulose recovery in the liquid fraction and lignin recovery from the biomass by response surface methodology (RSM) approach using a central composite design (CCD). Physicochemical analysis showed a decrease in crystallinity and an increase in surface area after the pretreatment in all the MHS solvents tested. This work has successfully shown the use of ZnCl2.4H2O/ Urea, to pre-treat and fractionate corn cob with high recovery of cellulose (100%), low recovery of hemicellulose (42%) and lignin (44%) when compared to the other proposed systems. Through the RSM approach, optimum pretreatment conditions obtained Abstract were: 90 min, 120 oC and concentration of 71.32%/28.68 (w/w) ZnCl2.4H2O/ Urea. At these conditions, the predicted recovery for cellulose, hemicellulose and lignin 99.03%, 27.18% and 72.43% respectively with a desirability of 0.902. The actual recovery was 91%, 29% and 68% for cellulose, hemicellulose and lignin respectively at the same conditions. For a better understanding of the dissolution kinetics and thermodynamics of cellulose, hemicellulose and lignin dissolution in ZnCl2.4H2O/ Urea solvent system, a kinetic study was carried out. The results reveal the dissolution to be a 1st order kinetics and the obtained activation energy for cellulose, hemicellulose and lignin dissolution were 14.10 kJ.mol-1, 11.29 kJ.mol-1 and 7.606 kJ.mol-1 ,respectively. that the dissolution process for all three components are endothermic and endergonic. The -0.190; -0.195 kJ.mol-1) showed that the process of dissolution of hemicellulose occurred more rapidly and produced more stable products. It was concluded that ZnCl2.4H2O/ Urea pretreatment provided a potential way to fractionate lignocellulosic biomass which can improve the effective utilization of all feedstock fractions. / E.K. 2020

Cellulose from energy crops or agriculture residues can be utilized as a sustainable energy resource to produce biofuels such as ethanol. The process of converting cellulose into solvents and biofuels requires the saccharification of cellulose into soluble, fermentable sugars. However, challenges to cellulosic biofuel production include increasing the activity of cellulose-degrading enzymes (cellulases) and increasing solvent (ethanol) yield while minimizing the co-production of organic acids. This work applies novel surface analysis techniques and fermentation reactor perturbations to quantify, manipulate, and model enzymatic and metabolic processes critical to the efficient production of cellulosic biofuels. Surface analysis techniques utilizing cellulose thin film as the model substrate are developed to quantify the kinetics of cellulose degradation by cellulase as well as the interactions with cellulase at the interfacial level. Quartz Crystal Microbalance with Dissipation (QCM-D) is utilized to monitor the change in mass of model cellulose thin films cast. The time-dependent frequency response of the QCM simultaneously measures both enzyme adsorption and hydrolysis of the cellulose thin film by fungal cellulases, in which a significant reduction in the extent of hydrolysis can be observed with increasing cellobiose concentrations. A mechanistic enzyme reaction scheme is successfully applied to the QCM frequency response for the first time, describing adsorption/desorption and hydrolysis events of the enzyme, inhibitor, and enzyme/inhibitor complexes. The effect of fungal cellulase concentration on hydrolysis is tested using the QCM frequency response of cellulose thin films. Atomic Force Microscopy (AFM) is also applied for the first time to the whole cell cellulases of the bacterium C. thermocellum, where the effect of temperature on hydrolysis activity is quantified. Fermentation of soluble sugars to desirable products requires the optimization of product yield and selectivity of the cellulolytic bacterium, Clostridium thermocellum. Metabolic tools to map the phenotype toward desirable solvent production are developed through environmental perturbation. A significant change in product selectivity toward ethanol production is achieved with exogenous hydrogen and the addition of hydrogenase inhibitors (e.g. methyl viologen). These results demonstrate compensatory product formation in which the shift in metabolic activity can be achieved through environmental perturbation without permanent change in the organism’s genome.

Doctor of Philosophy / Department of Chemical Engineering / Keith L. Hohn / Extensive research has gone into developing and modeling the three-way catalyst (TWC) to reduce the emissions of hydrocarbons, NOx and CO from gasoline-fueled engines level. However, much less has been done to model the use of the three-way catalyst to treat exhaust from natural gas-fueled engines. Our research address this gap in the literature by developing a detailed surface reaction mechanism for platinum based on elementary-step reactions. A reaction mechanism consisting of 24 species and 115 elementary reactions was constructed from literature values. All reaction parameters were used as found in the literature sources except for steps modified to improve the model fit to the experimental data. The TWC was simulated as a one-dimension, isothermal plug flow reactor (PFR) for the steady state condition and a continuous stirred-tank reactor (CSTR) for the dithering condition. This work describes a method to quantitatively simulate the natural gas engine TWC converter performance, providing a deep understanding of the surface chemistry in the converter. Due to the depletion of petroleum oil and recent volatility in price, synthesizing value-added chemicals from biomass-derived materials has attracted extensive attention. 1, 3-butadiene (BD), an important intermediate to produce rubber, is conventionally produced from petroleum. Recently, one potential route is to produce BD by dehydration of 2, 3-butanediol (BDO), which is produced at high yield from biomass. This reaction was studied over two commercial forms of alumina. Our results indicate acid/base properties greatly impact the BD selectivity. Trimethylamine can also modify the acid/base properties on alumina surface and affect the BD selectivity. Scandium oxide, acidic oxide or zirconia dual bed systems are also studied and our results show that acidic oxide used as the second bed catalyst can promote the formation of BD, while 2,5-dimethylphenol is found when the zirconia is used as the second bed catalyst which is due to the strong basic sites.

The biorefinery concept is attractive. Increasing international concerns over issuessuch as climate change have led to political as well as social pressures for a shift fromfossil fuels to renewable resources and biomass is one abundant renewable resource.Biomass has the potential of supplying many of the fuels and chemicals which arecurrently dependent on petroleum. Much development is still needed in the field ofbiorefineries and a systematic approach to evaluate and compare process technologiesand to suggest optimizations seems necessary.The objective of this thesis is to develop entropy analysis as a possible evaluation toolfor optimization of biorefinery processes. This is a new application of entropyanalysis which is rarely discussed in the literature. The scientific basis of the entropyanalysis is described and the proposed methodology is explained. The position ofentropy analysis among other system analysis tools such as exergy analysis and lifecycle assessment is discussed along with entropy analysis earlier applications.A case study is introduced which is the IBUS (Integrated Biomass Utilization System)project in Denmark. The idea in IBUS is to integrate the biomass plant with a powerplant to utilize the surplus steam from the power plant for the internal use of thebiorefinery. The suggested method of entropy analysis is applied to this case study tocompare different processes for production of ethanol along with solid biofuel andanimal feed from Danish wheat straw. The evaluation is a gate to gate analysis inwhich production of energy carriers are also included in addition to biorefining ofwheat straw. A parallel life cycle assessment study with equivalent system boundariesis also carried out to compare the results with a conventional environmental systemsanalysis method.The results from the entropy analysis of the IBUS case study show that fermentationof C5 and C6 sugars by yeast is the most efficient process thermodynamically whilefermentation of only C6 sugars by yeast is the least efficient among the three casesstudied. Integration of the biorefinery with a coal fired CHP plant is identified as awise choice by the results of the entropy analysis method.For the IBUS process alternatives investigated in this study, the entropy results andthe LCA results (aggregated environmental load) are in correlation; entropy results areconsistent with weighting results based on two different weighting methods namelyEco indicator 99 and EPS 2000. Entropy generation is also in correlation withproduction cost for the processes analyzed in this evaluation. Another observation isthat cooling in the biorefining process contributes highly in the generation of entropy.This potential improvement option is not surfaced by the LCA conducted.The potential for further investigation and development of the tool is recognizedreflecting on some interesting observations in the results. Improvement of the tool ishighly possible for example by supplementing other implications of entropy inprocess design such as "waste potential entropy" concept which is developed as aneco-toxicity measure. / Uppsatsnivå: D

Lignin is the second most abundant biopolymer on Earth, right after cellulose, with a highly complex chemical structure that hinders its possible utilizations. Applications that utilize lignin in different manners are of great interest, due to its inexpensive nature. Present work is based on the notion of converting lignin into different biofuels that have only a few, however important, advantages over lignin as a direct energy source. The first part of current work (pyrolysis) details the analysis of lignin from a relatively new lignin isolation process called LignoBoost. It is obtained from the pulp and paper industry via CO₂ precipitation of lignin from black liquor (BL). This method is environment friendly, results lignin with minimal oxidation, eliminates the main bottleneck of the Kraft cycle (recovery boiler capacity), and yet leaves enough lignin in the process stream to recover pulping chemicals and generate energy for the pulp mill. Pyrolysis had converted this lignin into bio-oil with high aliphatic content and low oxidation level, all advantageous for application as liquid fuel. The second part of this dissertation proved the theory that lignin degradation and lipid accumulation metabolic pathways can be interconnected. Gram-positive Rhodococcus opacus species, DSM 1069 and PD630 were used to evaluate lignin to lipid bioconversion, starting with ethanol organosolv and Kraft lignin. This conversion is a first step in a multistep process towards biodiesel production, which includes transesterification, after lipids are extracted from the cells. Results clearly indicated that the lignin to lipid bioconversion pathway is viable, by cells gaining up to 4 % of their weight in lipids, while growing solely on lignin as a carbon and energy source.

The objective of this study was to investigate the possibility of recycling enzymes during saccharification of cellulose for the production of ethanol from woodchips. To make enzyme recycling feasible and economical when woodchips are processed for ethanol, the lignin in the wood is to be removed before the enzymes are added. Since enzymes constitute a major part of the input costs, second only to the feedstock, the ability to reuse the enzymes could lead to a considerable decrease in the production cost of ethanol. Tulip poplar woodchips were selected as the feedstock. Different delignification methods with recovery of byproducts were investigated. Alkali extraction, using dilute NaOH for the removal of lignin after steam pretreatment, was used as the base case against which all other processes were compared. Recovery of furfural and methanol, produced during the pretreatment of the woodchips, for sale as byproducts was one modification to the alkali extraction process that was investigated. The conversion of xylose to furfural and the recovery of the furfural as a byproduct was the third case explored. Solvent extraction using a 50:50 ethanol-water mixture instead of extraction with NaOH was the fourth case examined. Process flow sheets were then developed to recycle the enzymes during the hydrolysis and fermentation of this prehyrolyzed and delignified wood. Two reactor setup schemes were examined for enzyme recycling. One scheme involved a single train of reactors, with the whole pretreated slurry flowing from one reactor to the next, whereas, in the other scheme, the slurry was split among parallel trains of reactors. The activity loss of the enzymes was modeled such that a part of the enzymes entering the reactor lost all their activity. The loss of activity in multiple steps, with enzymes losing only some of their activity, was also modeled. Here the enzymes entering the reactor constituted a mixture with different activities instead of all the enzymes having the same activity like in the previous single step model. Recovering methanol and furfural reduced the minimum ethanol selling price. High temperature ethanol water pretreatment and lignin extraction reduced the minimum ethanol selling price compared to the base case of steam pretreatment followed by alkali extraction. Enzyme recycling also reduces the minimum ethanol selling price. The magnitude of the price reduction depends on the recycling scheme selected and the rate of enzyme deactivation, which has not been measured.

Thesis (M.S.)--Brigham Young University. Dept. of Chemical Engineering, 2006. / Includes bibliographical references (p. 89-95).

Cette thèse porte sur l'hydrogénation en phase gazeuse du furfural sur des oxydes de fer et de magnésium. De nombreux catalyseurs avec différents ratio molaires en fer et magnésium ont été préparés par des méthodes de co-précipitation ou d'imprégnation. Ils ont été ensuite testés lors de la réduction du furfural (FU) en utilisant du méthanol comme donneur d'hydrogène. L'alcool furfurylique (FAL) et le 2-methyl furfural (MFU) étaient les principaux produits obtenus démontrant alors que les systèmes Mg/Fe/O peuvent favoriser les réactions d'hydrogénation séquentielles et d'hydrogénolyse. Les catalyseurs imprégnés se sont révélés plus actif et sélectif vis-à-vis des MFU que ceux préparés par co-précipitation. Les données rapportées ont montré que la distribution du produit était fortement influencée par la teneur en fer et par l'acide résultant, ainsi que les propriétés d'oxydoréduction du matériau. En effet, l'introduction de fer à la surface d'oxyde basique a conduit à l'addition d'acidité de Lewis et de potentiel d'oxydoréduction dans le système, améliorant significativement la conversion de FU et la production de MFU. L'activation des différentes espèces à la surface du catalyseur a été étudié in-situ par DRIFTS et FTIR. Les résultats révèlent que la basicité du MgO favorise l'activation du méthanol et que le potentiel d'oxydoréduction du FeOx pourrait être responsable de l'hydrogénolyse de l'alcool furfurylique / This PhD project is focused on the gas phase hydrogenation of furfural over iron and magnesium oxides. Numerous catalysts with different iron and magnesium molar ratios, were prepared by co-precipitation or impregnation methods and were tested for the reduction of furfural (FU) using methanol as hydrogen donor. Furfuryl alcohol (FAL) and 2-methyl furfural (MFU) were the main products obtained, demonstrating that Mg/Fe/O systems can promote sequential hydrogenation and hydrogenolysis reactions. Impregnated catalysts demonstrated to be more active and selective towards MFU than co-precipitated ones. Reported data demonstrated that product distribution was strongly influenced by the iron content and from the resulting acid and redox properties of the material. As a matter of fact, the introduction of iron on the surface of the basic oxide led to the addition of Lewis acidity and redox capacity in the system, significantly enhancing FU conversion and MFU production. The activation of different species on the catalyst surface has been studied by in situ DRIFTS and FTIR. The results reveal that the MgO basicity favors methanol activation and FeOx redox capacity might be the responsible of furfuryl alcohol hydrogenolysis