Being the most abundant carbon-containing terrestrial biopolymer, lignocellulose serves as one of the best candidate feedstocks for biofuel production. The current cost-ineffective method for lignocellulose pretreatment is one of the major barriers that hinder the development of biofuel production. This leads to an exploration in the potential application of lignocellulolytic enzymes in this biorefinery process. Taking advantage of the strong activity of ligninolytic enzymes in L. edodes, we aimed at cloning and heterologously expressing these enzymes. The present project applied a yeast expression system, Pichia pastoris, as a laboratory-scale platform for heterologous expression of one of our target ligninolytic enzymes, manganese peroxidase (MnP). We successfully cloned and expressed recombinant MnP. Its enzymatic activity was the highest when grown in the presence of hemoglobin. Our long-term goal is to establish a platform for the large-scale production of recombinant lignocellulotyic enzymes at low-cost, which would strengthen their application in biofuel production. / The shitake mushroom, Lentinula edodes, is one of the most commonly consumed edible mushrooms in Asian countries. It is a saprophyte that naturally colonizes dead wood. As a member of wood-decaying white rot basidiomycete, L. edodes is able to depolymerize lignin and hydrolyze wood polysaccharides. However, the enzymatic mechanism for its lignocellulolytic system is poorly understood. Examination on the L. edodes genome and transcriptome revealed a unique lignocellulolytic system. L. edodes has a diverse enzymatic arsenal for lignin degradation. The enzymes include laccase, manganese peroxidase, cellobiose dehydrogenase and various lignin degrading auxiliary enzymes. When compared to another white rot fungus Phanerochaete chrysosporium, L. edodes possesses more hemicellulase- and pectinase-coding genes, and fewer genes encoding cellulases, suggesting that it preferentially attacks non-cellulosic polysaccharides. The transcription analysis on genes related to antioxidative mechanisms also offers insights to the oxidative stress encountered by mycelium during the free radical-mediated lignin degradation. / Kwok, Sze Wai. / Adviser: Hoi-Shan Kwan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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The method was tested on a gram-positive (Micrococcus lutea) and one gram-negative (E-coliJM109) bacteria. The study was begun by cultivation of the two microorganisms. The cultivation was done to calculate the generation times of the bacteria and to obtain growth curves. Cells from these cultivations were also frozen (-70ºC) and later used for inoculation. At STFI-Packforsk AB the total charge on the mass was measured and later a conductivity titration on the mass was executed as well, all to find out more about the different properties of the mass. Properties that in a later part of this study could possibly be linked to the adhesion of cells to the pulp. The adhesion experiments that were executed gave poor results. The adhesion experiment with M. lutea was the only experiment that gave a reproducible result. In this experiment M. lutea was contacted with bleached leaf. A reduction of cells was observed in all of the dilutions where M. lutea had been in contact with the mass. The number of colony forming units of the culture was 1,2×107 before the adhesion and 2×106 subsequently.

The efficiency of cellulose hydrolysis under straight saccharification and simultaneous saccharification and fermentation (SSF) conditions was evaluated using three lignocellulosic materials (switchgrass, cornstover, and poplar), which had been pretreated with dilute sulfuric acid under conditions which optimized xylose concentrations in the prehydrolysate liquid. Yields of glucose, cellobiose and ethanol obtained from the pretreated feedstocks were measured over 168 hrs. The final theoretical conversions of cellulose from pretreated switchgrass, cornstover, and poplar in straight saccharification were 85-100% (average 94%), 84-100% (average 96%), and 75-100% (average 87%), respectively, while in SSF the conversions were 84-90% (average 87%), 91-96% (average 90%), 72%-82% (average 76%), respectively. The conversion rates of poplar in straight saccharification and SSF were significantly lower than those of switchgrass and cornstover. The effects of reaction parameters such as enzyme activity, cellulose availability, and yeast cell viability on the extent of hydrolysis in straight saccharification and SSF were also studied. Results indicate that the lower glucose or ethanol yields associated with some of the poplar were due to the recalcitrant nature of its cellulose. To compare accurately the efficiencies between straight saccharification and SSF, a direct method for determining the cellulose content of the feedstocks residues resulting from SSF experiments has been developed and evaluated. The method improves on classical cellulose assays by incorporating a yeast lysing enzyme to remove yeast glucans from the feedstocks residue prior to acid hydrolysis and subsequent quantification of cellulose derived glucose. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzyme lysis. The method was applied to the analysis of the cellulose and yeast-glucan content of SSF residues from the three pretreated feedstocks. Cellulose assays employing the lysing enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucan were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to the subsequent chemical and physical analysis of SSF residues. / Graduation date: 1996

Controlling the orientation and spatial distribution of discontinuous fibers in composite materials enables product properties to be tailored to anticipated use. Electric fields are already (albeit rarely) used to affect alignment in lignocellulosic (LC) fiber composites. The use of magnetic fields has not, however, been suggested or explored; this is apparently because LC fibers are essentially non-magnetic. The approach may offer, however, some considerable advantages, as long as ferromagnetism may be imparted to the fibers. In the present research several fiber modification processes were considered and two, electroless nickel plating and spray application of a coating containing nickel in suspension, were investigated in more depth. The latter was chosen to render highly engineered, elongated wood particles responsive to magnetic fields. Individual treated particles were suspended in viscous, newtonian silicone fluids and their rotation under the influence of a controlled magnetic field was video recorded. The magnetic torque on the particle was, under the above conditions, directly proportional to the fluid viscosity, to the particle's angular velocity and to a characteristic shape constant. The maximum of the specific magnetic torque (magnetic torque divided by the shape constant) was found to reflect the influence of field strength and particle Ni-treatment on rotation. Results were scaled to an arbitrarily chosen viscosity for comparison. The dependencies of the magnetic torque found in the present research compare with those theoretically predicted for ellipsoidal and cylindrical bodies. For field strengths ranging from 0.07T to 0.15T (below magnetic saturation) the magnetic torque increased almost linearly with increasing field strength. Magnetic torque was also found to increase nearly linearly with increasing bulk Ni-concentration (5g/kg - 50g/kg). Rotational motion was sometimes impeded at low field strengths and this was attributed to a permanent magnetic moment obtained by the particle. A coercive field strength of 7600A/m supported this hypothesis. Judiciously switched field polarity increased magnetic torque at small alignment angles. The present research indicates that orienting LC fibers with magnetic fields is possible and promising. To study dynamics of fiber motion in low viscosity fluids, such as air, a different experimental method is necessary; however, dependencies of the magnetic torque found in the present study still hold true. / Graduation date: 1993

Because of widespread availability, low cost, sustainability, and potential supply far greater than that of food crops, lignocellulosic biomass is one of the most promising feedstocks for producing biofuels through fermentation processes. Among lignocellulose choices, poplar wood is appealing because of high energy potential, above-average carbon mitigation potential, fast growth, and high yields. Lignocellulose structural features limit accessibility of enzymes or microorganisms. To overcome these limitations, pretreatment is required. Among several choices of pretreatment, lime pretreatment is preferred because lime is the cheapest alkali, safest to handle, easy to recover, and compatible with oxidants. The main effect of lime pretreatment is to degrade lignin, which occurs with good carbohydrate preservation and is enhanced with oxidants. Among several choices of oxidant, oxygen and air are preferred because of low cost and widespread availability. This study systematically assesses the effects of lime pretreatment on poplar wood using four different modes: long-term oxidative, long-term non-oxidative, short-term constant pressure, and short-term varying pressure. Long-term pretreatments use temperatures between 25 and 65° C, air if oxidant is used, and last several weeks. Short-term pretreatments use temperatures between 110 and 180° C, pressurized oxygen, and last several minutes to hours. Pretreatment was assessed on the basis of 3-day enzymatic digestibility using enzyme loadings of 15 FPU/g glucan in raw biomass. The results were used to recommend pretreatment conditions based on highest overall yield of glucan (after combined pretreatment and enzymatic hydrolysis) for each pretreatment mode. For each pretreatment mode, kinetic models for delignification and carbohydrates degradation were obtained and used to determine the conditions (temperature, pressure, and time) that maximize glucan preservation subjected to a target lignin yield. This study led to conclude that the most robust, and selective mode of lime pretreatment is varying pressure.

Lignocellulosic biomass (e.g., poplar wood) provides a unique and sustainable resource for environmentally safe organic fuels and chemicals. The core of this study is the pretreatment step involved in bioconversion processes. Pretreatment is required to realize high yields vital to commercial success. The focus of the pretreatment step is to methodically change key features of the biomass to favor enzymatic hydrolysis. This work assesses the compositional changes due to oxidative and non-oxidative longterm lime pretreatment of poplar wood (up to 4 weeks of pretreatment) at mild temperatures (25ºC to 65ºC), and their effect on the enzymatic yield of glucan and xylan. The most important pretreatment yield of lignin was 54 g lignin remaining/100 g lignin in raw biomass, and was accomplished for 4-week lime pretreatment at 65ºC in oxidative conditions. The corresponding pretreatment yields of glucan and xylan were 85.9 g glucan recovered/100 g glucan in raw biomass and 80.2 g xylan recovered/100 g xylan in raw biomass respectively. For poplar wood oxidatively pretreated with lime for 4 weeks at 65ºC and enzymatically hydrolyzed with an enzyme loading of 15 FPU/g glucan in raw biomass during a 3-day period, the best overall yields of glucan and xylan, were 80.7 g glucan hydrolyzed/100 g glucan in raw biomass and 66.9 g xylan hydrolyzed/100 g xylan in raw biomass respectively. The corresponding hydrolysis yields were 94.0 g glucan hydrolyzed/100 g glucan in treated biomass and 83.5 g xylan hydrolyzed/100 g xylan in treated biomass respectively. Because there is a previous study of long-term lime pretreatment of corn stover (Kim, 2004), the data obtained in this work show the effect of using woody lignocellulose as substrate. From the comparison, resulted that in the case of poplar wood oxidatively pretreated at 65ºC for 4 weeks, less lignin was removed and more carbohydrates were solubilized, however the hydrolysis yield of glucan was almost equal and the hydrolysis yield of xylan was higher than the reported by Kim for corn stover oxidatively pretreated at 55ºC for 4 weeks. The overall yield of glucan resulted lower in the case of poplar wood because of the lower pretreatment yield of glucan. Thus, it is important to complete the mass balances including an analysis on the pretreatment liquor to determine if the solubilized glucan was degraded.

Thesis--Auburn University, 2009. / Abstract. Vita. Includes bibliographic references (p.64-77).

Thesis (M.S.)--Mississippi State University. Dave C. Swalm School of Chemical Engineering. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.)--Kansas State University, 2008. / Advisers: Donghai Wang, Scott Bean. Includes bibliographical references.

Thesis (M.A.)--University of Oregon, 2008. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 109-111). Also available online.