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A study of over production and enhanced secretion of enzymes. Quarterly report 1

Description: The current project is concerned with the over-production and enhanced secretion of PPO, cellulase and lignin peroxidase. The project is divided into two segments: over-production of lignocellulolytic enzymes by genetic engineering methodologies and hyper-production and enhanced secretion of these enzymes by biochemical/electron microscopical techniques. The former approach employs recombinant DNA procedures, ligation of appropriate nuclease generated DNA fragments into a vector and the subsequent transformation of Escherichia coli to yield E. coli harboring a C. versicolor DNA insert. The biochemistry/electron microscopical method involves substrate induction and the time-dependent addition of respiration and PPO inhibitors to elevate C.versicolor`s ability to synthesize and secrete lignocellulosic enzymes. In this connection, cell fractionation/kinetic analysis, TEM immunoelectron microscopic localization and TEM substrate localization of PPO are being employed to assess the route of secretion. Both approaches will culminate in the batch culture of either E. coli or C. versicolor, in a fermentor with the subsequent development of rapid isolation and purification procedures to yield elevated quantities of pure lignocellulosic enzymes. During the past year, research effort were directed toward determining the route of polyphenol oxidase (PPO) secretion by the wood-decay fungus, Coriolus versicolor. In addition, research activities were continued to over-produce and to purify PPO as well as define the time-dependent intra- and extra-cellular appearances of C. versicolor ligninases and cellulases.
Date: December 28, 1992
Creator: Dashek, W.V.
Partner: UNT Libraries Government Documents Department

A study of over-production and enhanced secretion of enzymes. Quarterly report 2

Description: This project is concerned with the over-production of ligno-cellulolytic enzymes which are relevant to the paper-pulp industry and agricultural community. Since ligno-cellulosics are components of wood, the project involves the forest, a renewable energy resource. Attention is focused on the following: over-production of polyphenol oxidase; establishment of the route of polyphenol oxidase secretion; regulation of polyphenol oxidase secretion; purification of extracellular oxidase.
Date: April 8, 1993
Creator: Dashek, W.V.
Partner: UNT Libraries Government Documents Department

Ethanol production from dry-mill corn starch in a fluidized-bed bioreactor

Description: The development of a high-rate process for the production of fuel ethanol from dry-mill corn starch using fluidized-bed bioreactor (FBR) technology is discussed. Experiments were conducted in a laboratory scale FBR using immobilized biocatalysts. Two ethanol production process designs were considered in this study. In the first design, simultaneous saccharification and fermentation was performed at 35 C using {kappa}-carageenan beads (1.5 mm to 1.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. For dextrin feed concentration of 100 g/L, the single-pass conversion ranged from 54% to 89%. Ethanol concentrations of 23 to 36 g/L were obtained at volumetric productivities of 9 to 15 g/L-h. No accumulation of glucose was observed, indicating that saccharification was the rate-limiting step. In the second design, saccharification and fermentation were carried out sequentially. In the first stage, solutions of 150 to 160 g/L dextrins were pumped through an immobilized glucoamylase packed column maintained at 55 C. Greater than 95% conversion was obtained at a residence time of 1 h, giving a product of 165 to 170 g glucose/L. In the second stage, these glucose solutions were fed to the FBR containing Z. mobilis immobilized in {kappa}-carageenan beads. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L was achieved, giving an overall productivity of 23 g/L-h.
Date: August 1, 1998
Creator: Krishnan, M. S.; Nghiem, N. P. & Davison, B. H.
Partner: UNT Libraries Government Documents Department

Regulation of coal polymer degradation by fungi. Eighth quarterly report, [April--June 1996]

Description: This project addresses the solubilization of low-rank coal (leonardite) by lignin degrading fungi. During this reporting period efforts were focused on determining the effect of pH on coal solubilization by oxalate ion and other biologically important compounds that might function as metal chelators, on the role of laccase in coal solubilization and metabolism, on decolorization of soluble coal macromolecule by Phanerochaete chrysosporium and T. versicolor in solid agar media, and on solubilization of coal in slurry cultures and solid phase reactors.
Date: July 28, 1996
Creator: Irvine, R.L. & Bumpus, J.A.
Partner: UNT Libraries Government Documents Department

Enzymatic degradation of plutonium-contaminated cellulose products

Description: Enzyme solutions produced for commercial purposes unrelated to waste management have the potential for reducing the volume of wastes in streams containing cellulose, lipid and protein materials. For example, the authors have shown that cellulases used in denim production and in detergent formulations are able to digest cellulose-containing sorbents and other cellulose-based wastes contaminated either with crude oil or with radionuclides. This presentation describes the use of one such enzyme preparation (Rapidase{trademark}) for the degradation of cotton sorbents intentionally contaminated with low levels of plutonium. This is part of a feasibility study to determine if such treatments have a role in reducing the volume of low level and transuranic wastes to minimize the amount of radionuclide-contaminated waste that must be disposed of in secured storage areas.
Date: March 1, 1999
Creator: Heintz, C.E.; Rainwater, K.A.; Swift, L.M.; Barnes, D.L.; Worl, L. & Avens, L.
Partner: UNT Libraries Government Documents Department

Preparation for commercial demonstration of biomass-to-ethanol conversion technology. Final report

Description: The objective of this program was to complete the development of a commercially viable process to produce fuel ethanol from renewable cellulosic biomass. The program focused on pretreatment, enzymatic hydrolysis, and fermentation technologies where Amoco has a unique proprietary position. Assured access to low-cost feedstock is a cornerstone of attractive economics for cellulose to ethanol conversion in the 1990s. Most of Amoco`s efforts in converting cellulosic feedstocks to ethanol before 1994 focused on using paper from municipal solid waste as the feed. However, while many municipalities and MSW haulers expressed interest in Amoco`s technology, none were willing to commit funding to process development. In May, 1994 several large agricultural products companies showed interest in Amoco`s technology, particularly for application to corn fiber. Amoco`s initial work with corn fiber was encouraging. The project work plan was designed to provide sufficient data on corn fiber conversion to convince a major agriculture products company to participate in the construction of a commercial demonstration facility.
Date: July 1, 1997
Partner: UNT Libraries Government Documents Department

Seventeenth symposium on biotechnology for fuels and chemicals. Program and abstracts

Description: This volume contains the abstracts of oral and poster presentations made at the Seventeenth Symposium on Biotechnology for Fuels and Chemicals. Session titles include Thermal, Chemical, and Biological Processing; Applied Biological Research; Bioprocessing Research; Special Topics Discussion Groups; Process Economics and Commercialization; and Environmental Biotechnology.
Date: May 1, 1995
Partner: UNT Libraries Government Documents Department

Over production of lignocellulosic enzymes of Coriolus versicolor by genetic engineering methodology. Final report

Description: The project seeks to understand the biological and chemical processes involved in the secretion of the enzyme polyphenol oxidase (PPO) by the hyphae, the basic unit of the filamentous fungus Coriolus versicolor. These studies are made to determine rational strategies for enhanced secretion of PPO, both with the use of recombinant DNA techniques and without. This effort focuses on recombinant DNA techniques to enhance enzyme production. The major thrust of this project was two-fold: to mass produce C. versicolor tyrosinase (polyphenol oxidase) by genetic engineering as well as cultural manipulations; and to utilize PPO as a biocatalyst in the processing of lignocellulose as a renewable energy resource. In this study, the assessment of genomic and cDNA recombinant clones with regards to the overproduction of PPO continued. Further, immunocytochemical techniques were employed to assess the mechanism(s) involved in the secretion of PPO by the hyphae. Also, factors influencing PPO secretion were examined.
Date: July 1, 1998
Creator: Williams, A. L.
Partner: UNT Libraries Government Documents Department

Cyclodextrin-based surface acoustic wave chemical microsensors

Description: Cyclodextrin thin films were fabricated using either self-assembled monolayer (SAM) or solgel techniques. The resulting host receptor thin films on the substrates of surface acoustic wave (SAW) resonators were studied as method of tracking organic toxins in vapor phase. The mass loading of surface-attached host monolayers on SAW resonators gave frequency shifts corresponding to typical monolayer surface coverages for SAM methods and ``multilayer`` coverages for sol-gel techniques. Subsequent exposure of the coated SAW resonators to organic vapors at various concentrations, typically 5,000 parts per millions (ppm) down to 100 parts per billions (ppb) by mole, gave responses indicating middle-ppb-sensitivity ({approximately}50 ppb) for those sensor-host-receptors and organic-toxin pairs with optimum mutual matching of polarity, size, and structural properties.
Date: July 1, 1996
Creator: Li, D.Q.; Shi, J.X.; Springer, K. & Swanson, B.I.
Partner: UNT Libraries Government Documents Department

Enzymes for Degradation of Energetic Materials and Demilitarization of Explosives Stockpiles - SERDP Annual (Interim) Report, 12/98

Description: The current stockpile of energetic materials requiring disposal contains about half a million tons. Through 2001, over 2.1 million tons are expected to pass through the stockpile for disposal. Safe and environmentally acceptable methods for disposing of these materials are needed. This project is developing safe, economical, and environmentally sound processes using biocatalyst (enzymes) to degrade energetic materials and to convert them into economically valuable products. Alternative methods for destroying these materials are hazardous, environmentally unacceptable, and expensive. These methods include burning, detonation, land and sea burial, treatment at high temperature and pressure, and treatment with harsh chemicals. Enzyme treatment operates at room temperature and atmospheric pressure in a water solution.
Date: January 18, 1999
Creator: Shah, M.M.
Partner: UNT Libraries Government Documents Department

Enzymatic degradation of plutonium-contaminated cellulose products

Description: Enzyme solutions produced for commercial purposes unrelated to waste management have the potential for reducing the volume of wastes in streams containing cellulose, lipid and protein materials. For example, the authors have shown previously that cellulases used in denim production and in detergent formulations are able to digest cellulose-containing sorbents and other cellulose-based wastes contaminated either with crude oil or with uranium. This presentation describes the use of one such enzyme preparation (Rapidase{trademark}, manufactured by Genencor, Rochester, NY) for the degradation of cotton sorbents intentionally contaminated with low levels of plutonium. This is part of a feasibility study to determine if such treatments have a role in reducing the volume of low level and transuranic wastes to minimize the amount of radionuclide-contaminated waste destined for costly disposal options.
Date: June 1, 1999
Creator: Heintz, C.E.; Rainwater, K.A.; Swift, L.M.; Barnes, D.L. & Worl, L.A.
Partner: UNT Libraries Government Documents Department

Identification of the primary mechanism for fungal lignin degradation. Progress report

Description: Many lignin-degrading fungi appear to lack lignin peroxidase (LiP), an enzyme generally thought important for fungal ligninolysis. The authors are working with one of these fungi, Ceriporiopsis subvermispora, an aggressive white-rotter that selectively removes lignin from wood. During this project period, they have obtained the following principal results: new polymeric lignin model compounds were developed to assist in the elucidation of fungal ligninolytic mechanisms; experiments with one of the polymeric lignin models showed that C. subvermispora cultures which express no detectable LiP activity are nevertheless able to degrade nonphenolic lignin structures, this result is significant because LiPs were previously considered essential for fungal attack on these recalcitrant structures, which constitute about 90% of lignin; manganese peroxidases (MnPs), which C. subvermispora does produce, catalyze the peroxidation of unsaturated fatty acids to give fatty acid hydroperoxides, fatty acid hydroperoxides are also used by MnP as oxidants (in place of H{sub 2}O{sub 2}) that support the MnP catalytic cycle, these results indicate that MnP turnover in the presence of unsaturated lipids generates reactive lipid oxyradicals that could act as oxidant of other molecules; MnP-mediated lipid peroxidation results in the co-oxidative cleavage of nonphenolic lignin structures, the MnP/lipid peroxidation system may therefore provide C. subvermispora and other LiP-negative fungi with a mechanism to degrade the principal structures of lignin.
Date: June 1, 1997
Partner: UNT Libraries Government Documents Department

Carbonic Acid Pretreatment of Biomass

Description: This project sought to address six objectives, outlined below. The objectives were met through the completion of ten tasks. 1) Solidify the theoretical understanding of the binary CO2/H2O system at reaction temperatures and pressures. The thermodynamics of pH prediction have been improved to include a more rigorous treatment of non-ideal gas phases. However it was found that experimental attempts to confirm theoretical pH predictions were still off by a factor of about 1.8 pH units. Arrhenius experiments were carried out and the activation energy for carbonic acid appears to be substantially similar to sulfuric acid. Titration experiments have not yet confirmed or quantified the buffering or acid suppression effects of carbonic acid on biomass. 2) Modify the carbonic acid pretreatment severity function to include the effect of endogenous acid formation and carbonate buffering, if necessary. It was found that the existing severity functions serve adequately to account for endogenous acid production and carbonate effects. 3) Quantify the production of soluble carbohydrates at different reaction conditions and severity. Results show that carbonic acid has little effect on increasing soluble carbohydrate concentrations for pretreated aspen wood, compared to pretreatment with water alone. This appears to be connected to the release of endogenous acids by the substrate. A less acidic substrate such as corn stover would derive benefit from the use of carbonic acid. 4) Quantify the production of microbial inhibitors at selected reaction conditions and severity. It was found that the release of inhibitors was correlated to reaction severity and that carbonic acid did not appear to increase or decrease inhibition compared to pretreatment with water alone. 5) Assess the reactivity to enzymatic hydrolysis of material pretreated at selected reaction conditions and severity. Enzymatic hydrolysis rates increased with severity, but no advantage was detected for the use of carbonic acid compared to ...
Date: May 31, 2003
Creator: Walsum, G. Peter van; Jayawardhana, Kemantha; Yourchisin, Damon; McWilliams, Robert & Castleberry, Vanessa
Partner: UNT Libraries Government Documents Department

Dynamic Impregnator Reactor System (Poster)

Description: IBRF poster developed for the IBRF showcase. Describes the multifarious system designed for complex feedstock impregnation and processing. IBRF feedstock system has several unit operations combined into one robust system that provides for flexible and staged process configurations, such as spraying, soaking, low-severity pretreatment, enzymatic hydrolysis, fermentation, concentration/evaporation, and distillation.
Date: September 1, 2012
Partner: UNT Libraries Government Documents Department

Integrated Biorefinery Project: Cooperative Research and Development Final Report, CRADA Number CRD-10-390

Description: The Amyris-NREL CRADA is a sub-project of Amyris?s DOE-funded pilot-scale Integrated Biorefinery (IBR). The primary product of the Amyris IBR is Amyris Renewable Diesel. Secondary products will include lubricants, polymers and other petro-chemical substitutes. Amyris and its project partners will execute on a rapid project to integrate and leverage their collective expertise to enable the conversion of high-impact biomass feedstocks to these advanced, infrastructure-compatible products. The scope of the Amyris-NREL CRADA includes the laboratory development and pilot scale-up of bagasse pretreatment and enzymatic saccharification conditions by NREL for subsequent conversion of lignocellulosic sugar streams to Amyris Diesel and chemical products by Amyris. The CRADA scope also includes a techno-economic analysis of the overall production process of Amyris products from high-impact biomass feedstocks.
Date: June 1, 2013
Creator: Chapeaux, A. & Schell, D.
Partner: UNT Libraries Government Documents Department

Applications of a single-molecule detection in early disease diagnosis and enzymatic reaction study

Description: Various single-molecule techniques were utilized for ultra-sensitive early diagnosis of viral DNA and antigen and basic mechanism study of enzymatic reactions. DNA of human papilloma virus (HPV) served as the screening target in a flow system. Alexa Fluor 532 (AF532) labeled single-stranded DNA probes were hybridized to the target HPV-16 DNA in solution. The individual hybridized molecules were imaged with an intensified charge-coupled device (ICCD) in two ways. In the single-color mode, target molecules were detected via fluorescence from hybridized probes only. This system could detect HPV-16 DNA in the presence of human genomic DNA down to 0.7 copy/cell and had a linear dynamic range of over 6 orders of magnitude. In the dual-color mode, fluorescence resonance energy transfer (FRET) was employed to achieve zero false-positive count. We also showed that DNA extracts from Pap test specimens did not interfere with the system. A surface-based method was used to improve the throughput of the flow system. HPV-16 DNA was hybridized to probes on a glass surface and detected with a total internal reflection fluorescence (TIRF) microscope. In the single-probe mode, the whole genome and target DNA were fluorescently labeled before hybridization, and the detection limit is similar to the flow system. In the dual-probe mode, a second probe was introduced. The linear dynamic range covers 1.44-7000 copies/cell, which is typical of early infection to near-cancer stages. The dual-probe method was tested with a crudely prepared sample. Even with reduced hybridization efficiency caused by the interference of cellular materials, we were still able to differentiate infected cells from healthy cells. Detection and quantification of viral antigen with a novel single-molecule immunosorbent assay (SMISA) was achieved. Antigen from human immunodeficiency virus type 1(HIV-1) was chosen to be the target in this study. The target was sandwiched between a monoclonal capture antibody and ...
Date: October 15, 2008
Creator: Li, Jiangwei
Partner: UNT Libraries Government Documents Department

Countercurrent Process for Lignin Separation from Biomass Matrix

Description: The overall goal of the project was to test the concept of using a twin-screw extruder to conduct autohydrolysis pretreatment of wheat straw in countercurrent fashion, demonstrate in situ solid/liquid separation, and produce a low-lignin cellulose product using ethanol as an extractant. The resultant solid product is suitable for sugar production through enzymatic hydrolysis and for pulp applications. Pilot-scale equipment was used to successfully demonstrate the process both for sugar and pulp applications.
Date: March 31, 2006
Creator: Kadam, Kiran & Lehrburger, Ed
Partner: UNT Libraries Government Documents Department

Regulation of coal polymer degradation by fungi. Eighth quarterly report, [January--March 1996]

Description: Progress is reported on solubilization of low-rank coal by enzyme activity derived from Trametes versicolor or P. chrysosporium. Specifically during the reporting period efforts were directed towards the determining the effect of pH on solubilization of leonardite, the role of laccase in low coal solubilization and metabolism, the decolorization of soluble coal macromolecule by P. chrysosprium and T. versicolor in solid agar gel, and the solubilization of low rank coal in slurry cultures and solid phase reactors.
Date: July 28, 1996
Creator: Irvine, R.L. & Bumpus, J.A.
Partner: UNT Libraries Government Documents Department

Optimizing cellulase mixtures for maximum rate and extent of hydrolysis. Final report

Description: Pure Thomomonospora fusca and Trichoderma reesei cellulases and their mixtures were studied to determine the optimal set of cellulases for biomass hydrolysis. The objective was to reduce the cost of cellulase in order to help lower the overall processing cost of the enzymatic conversion of biomass cellulose to sugars, which can then be fermented into fuels and other energy-intensive chemicals. No cellulase mixture was obtained that was much better than the best commercially available preparations. However, the study has greatly increased knowledge of T. fusca cellulases, synergism, and cellulose binding, and provide evidence that future work will produce cellulases with higher activity in degrading crystalline cellulose. T. fusca cellulases may have good industrial potential because: (1) they are compatible with industrial processes that operate at elevated temperatures; (2) they retain 90% of their activity under neutral or basic conditions, which provides a great deal of flexibility in reactor design and operation; and (3) tools are now available to change specific amino acid residues in their catalytic domains and to assess how these changes influence catalysis. 74 refs.
Date: March 1, 1997
Creator: Walker, L.P. & Wilson, D.B.
Partner: UNT Libraries Government Documents Department