Progress 07/01/04 to 06/30/07
Outputs
Protein Extraction:Solvents were screened to extract proteins from distiller's grains.Ethanol and weak bases like NH3 were ineffective,while a strong base like NaOH had a larger effect.Using 0.5M NaOH,2mM b-mercaptoethanol,and 0.3% SDS at 70C for 90 minutes,32% of the total protein was solubilized,while 22% of the remaining protein was solubilized during the subsequent fiber hydrolysis.Attempts to solubilize protein after hydrolysis saw low yields,with no more than 30% of the remaining protein being extracted in concentrated alkaline solutions.AFEX:The best AFEX treatment conditions for bagasse pith and CLM were found to be 100C for 30 min.,40% moisture,and NH3 loading at 2g NH3 per gram dry biomass.A process was developed for selective hydrolysis of hemicellulose using cellulase-free xylanases.For AFEX-treated CLM,about 52% of the xylan was converted to xylose while about 96% of the cellulose was left in the biomass.For AFEX-treated bagasse pith,about 56% of the xylan
and only 5% of the cellulose was hydrolyzed.Fermentability with Actinobacillus succinogenes FZ45 was assessed via separate hydrolysis and fermentation (SHF) processes.Nanowhiskers:Cellulose nanowhiskers (CNW) and microfibrillated cellulose (MFC) extracted from wheat straw and added as reinforcing fillers in polymers at 3% increased the composite modulus over 10% at 25C and 33% at 80C.The results indicate that CNW and MFC are about twice the modulus of glass fibers,can be obtained from a variety of agricultural waste products and have the potential to be a renewable,bio-based nanomaterial.Succinic Acid:This project focused on fermentative production of succinic acid utilizing AFEX-treated bagasse and CLM material.Succinic acid production with good yields was accomplished. Sugarcane biorefinery byproducts such as molasses, stillage and glycerol (a component in stillage) were evaluated as feedstocks in succinic acid fermentations.The sugars in molasses were efficiently converted to
succinic acid.Stillage was shown to be usable as a medium component in combination with additional supplements.Glycerol consumption was increased by 50% in a recombinant A. succinogenes strain. Biobased Technologies:BIO PacRim Meeting,The Biotechnology Industry Organization (BIO),in coordination with the State of Hawaii,the University of Hawaii,the Hawaii Life Sciences Council (HLSC),Enterprise Honolulu and the Oceanic Institute,Technology identified included Ethanol production using crop residue feedstocks;new biobased markets for sugars;marine biotech for industrial applications; Chemgenuity,emerging green chemistry company focused on the discovery and initial development of biobased chemicals and materials.Chemgenuity has no capacity for full-scale development, validation and commercialization.MBI evaluated Chemgenuity technology and their links to the Los Alamos Laboratory, University of New Mexico and other related entities in the Albuquerque/Santa Fe region.International
Biorefinery Workshop (DOE),The biorefinery has the potential to revolutionize rural economies and broaden the use of biobased energy and products in world markets.
Impacts
Distiller's grains are a major and growing byproduct of corn dry mills. But due to the extreme conditions required to remove even a fraction of the protein from DDGS, coupled with the poor amino acid ratios obtained and low sugar yields, this extraction/hydrolysis process does not currently appear to provide a potential industrial use. The production of fuels from sustainable, agricultural resources enhances the agricultural sector of our economy and lowers our dependence on petroleum. However, utilization of readily available sugars in agricultural products will not suffice to fulfill the demands of the food and fuel industry. The development of treatment processes to release sugars from lignocellulosic portion of biomass is imperative. Agricultural waste such as wheat straw can be utilized to produce a value-added, sustainable nanoparticle that can be a reinforcing additive to plastics. Both high strength and modulus and low density microfibrillated cellulose (MFC)
and cellulose nanowhiskers (CNWs) have been successfully extracted from wheat straw. The production of chemicals from agricultural by-products has the potential to reduce waste-disposal of dry-mill operations and enhance their profitability. Succinic acid could replace chemicals such as pyrrolidinones and butanediol (BDO) that are currently produced from petrochemicals. Fermentative production of succinic acid utilizes low-value dry-mill products and serves as a sink for CO2.
Publications
- Lu, J., Askeland, P., and Drzal, L. T.; "Surface Modification of Microfibrillated Nano-Web Cellulose for Epoxy Composite Applications", submitted to Polymer, July 13, 2007.
- Lu, J., and Drzal, L. T.; "Microfibrillated Cellulose/Cellulose Acetate Composites: Effect of Surface Treatment" submitted to Biomacromolecules, July 16, 2007
- Lu, J., and Drzal, L. T.; "Preparation And Properties of Polyvinyl Alcohol Composite Films Based on Microfibrillated Cellulose With Nano-Web Structure" submitted to Composites Part A, April, 2007.
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Progress 01/01/06 to 12/31/06
Outputs
Protein Extraction: Several solvents were screened to determine the best approach to removing proteins from distiller's grains. Alcohols like ethanol and minor bases like ammonia were ineffective, while a strong base like sodium hydroxide had a larger effect, with a protein yield of 22%. Using 0.5M NaOH at 70 degrees C, improved the extraction efficiency. A solution of 0.5M NaOH, 2mM b-mercaptoethanol, and 0.3% SDS was used to extract proteins at 70C for 90 minutes. Protein yields of 48% were obtained from DDGS when the extraction was followed by an enzymatic hydrolysis. AFEX Pretreatment of Bagasse and Cane Leaf Matter: The process parameters for the Ammonia Fiber Explosion (AFEX) process were investigated. A set of conditions that resulted in the highest glucose and xylose yields from several sources of lignocellulosic biomass were identified. The efficiency of AFEX pretreatment was evaluated via enzyme hydrolysis. The fermentability of the hydrolysates was
demonstrated in fermentations to ethanol using S. cerevisiae and Z. molbilis, and to the chemical succinic acid using Actinobacillus succinogenes. Design and Engineering of High Strength Compatibilized Green Nanocomposite Materials: Cellulose nanowhiskers were successfully extracted from wheat straw with a yield of about 13.6% by weight. Transmission Electron Microscopy images have shown the width of the whiskers to be about 5-10 nm and the length in the range of 100 to 200 nm. The crystallinity of the whiskers was studied by X-ray Diffraction. In order to evaluate the benefit of using the extracted nanowhiskers as reinforcement in polymer nanocomposites, polyvinyl alcohol was used as a matrix material to produce cellulose nanowhisker based composite films by a film casting technique. Excellent dispersion of the cellulose whiskers in the matrix was achieved. Characterization of the thermal and mechanical properties of the films is being performed. Preliminary results have shown
improved tensile properties of the composites. Succinic Acid Production from Biomass: The development of an economic process for the fermentative production of succinic acid that can be integrated into corn dry mill processing would be facilitated by the use of available dry mill products. Research efforts focused on the utilization of thin stillage (TS) as a complex medium component for succinic production using Actinobacillus succinogenes and corn endosperm as a potentially inexpensive sugar source. Both materials are readily available in corn dry mill ethanol operations. We demonstrated that TS was able to support succinic acid production at a productivity and titer needed for an industrial process. The conversion of endosperm to glucose was demonstrated in a 2-stage liquefaction/saccharification process under pasteurizing conditions. Identification of Commercially Attractive Biobased Technologies: MBI has entered into a three year agreement with a major ethanol producer to
demonstrate a continuous pilot scale AFEX pretreatment reactor in a cellulose-to-ethanol biorefinery. This agreement also calls for the validation of succinic acid at pilot scale as a byproduct in the cellulose-to-ethanol biorefinery.
Impacts
Distiller's grains are a major byproduct of corn dry mills. Extreme conditions are required to remove very little of the protein from DDGS. Due to poor amino acid ratios and low sugar yields, this extraction/hydrolysis process does not provide a potential industrial use. The production of fuels from sustainable, agricultural resources enhances the agricultural sector of our economy and lowers our dependence on petroleum. However, utilization of readily available sugars in agricultural products will not suffice to fulfill the demands of the food and fuel industry. The development of treatment processes to release sugars from lignocellulosic portion of biomass is imperative. Wheat straw, one of the major agricultural residuals in the United States, can serve as an inexpensive feedstock for the extraction of high strength and modulus and low-density cellulose nanowhiskers. The extracted nanowhiskers have promise to partially replace conventional inorganic fillers in
polymer composites. Nanocomposites based on cellulose nanowhiskers and biobased polymers will have good mechanical properties and will be degradable after their end of life, thus contributing to environmental sustainability. The production of chemicals from agricultural by-products has the potential to reduce waste-disposal of dry-mill operations and enhance their profitability. Succinic acid could replace chemicals such as pyrrolidinones and butanediol (BDO) that are currently produced from petrochemicals. Fermentative production of succinic acid utilizes low-value dry-mill products and serves as a sink for CO2.
Publications
- Bals, B., Dale, B.E., Balan, V. 2006. Enzymatic hydrolysis of distillers dry grain and solubles (DDGS) using ammonia fiber expansion. Energy & Fuels. 20: 2732-2736.
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Progress 01/01/05 to 12/31/05
Outputs
Protein Extraction: Our focus has been on extracting protein from distillers grains and switchgrass using aqueous ammonia. We acquired the necessary equipment (ASE-Accelerated Solvent Extractor) and are acquiring the necessary analytical techniques (chromatography, Bradford protein analysis) to extract protein from plant materials and do high quality mass balances on the resulting solid and liquid streams. So far we are unable to extract more than 25% of DDG protein with conventional approaches. Results with switchgrass are similar. We must develop other approaches to improve protein extraction. Cellulose Nanofibers: Cellulose nanofibers were successfully prepared from corn stover by chemical and mechanical treatments. They were then characterized by transmission electron microscopy (TEM). Cellulose nanofibers/polyvinyl alcohol (PVA) composite films were prepared by film casting. The crystallinity of the nanofibers and the composite films was measured by X-ray
diffraction (XRD). Corn stover has very low cellulose content and hence extracting cellulose nanowhiskers does not appear to be a viable process. AFEX Treatment of Biomass: A factorial design of experiments was performed for bagasse, CLM, and bagasse pith to optimize the AFEX process conditions which included reaction time, reaction temperature, ammonia loading, and moisture content of the biomass. The efficiency of the AFEX pretreatment was evaluated via enzymatic hydrolysis. AFEX treatment significantly increased the digestibility of all the tested biomass. For all three biomass, bagasse, CLM and bagasse pith, the maximum glucose and xylose yields were obtained from samples treated at 100-110 C, 40- 60 percent moisture content, 2:1 ratio of ammonia loading to dry biomass for 30 min. Fermentation of AFEX Treated Materials: AFEX treated Cane Leaf Matter (CLM) was loaded at 8 percent and 12 percent solid loading into the fermentation vessels and enzymatically hydrolyzed in the
fermentation medium routinely used for succinic acid fermentations. Typical yields for glucan and xylan conversion were reached. Higher biomass loading showed slightly diminished conversion yields. Subsequent fermentations of the hydrolysates to succinic acid consumed all available C6 and C5 sugars at near theoretical yields and produced 45.9 g/L succinic acid for the best case. Recombinant Organism Development: Previously we had identified fermentation conditions for a yeast strain that might allow succinic acid production without the need for neutralization and under anaerobic conditions. The strain was transformed with three succinic acid pathway genes, but the transformants continued to produce ethanol and no enhanced carbon routing towards succinic acid was detectable. Commercialization: MBI continuous to manage its projects through the Stage Gate Process towards commercialization. The succinic acid project and the AFEX project are at Stage 4, Validation. We are in the process of
developing a marketing plan with outside partners as we develop plans for a large scale pilot plant to demonstrate the commercial violability of these processes.
Impacts
Distillers grains are a major and growing byproduct of corn dry mills. New markets need to be found for this product and/or the protein content upgraded in some way. We have learned that protein extraction after DDG processing is not promising, thereby eliminating one processing approach. Cellulose nanowhiskers can become a very valuable product of biomass processing. We have also eliminated corn stover as a potential source of cellulose nanowhiskers. AFEX processing has been extended to include a much larger potential universe of raw materials, particularly bagasse, and some initially encouraging results obtained. Bagasse represents the most attractive single initial target for foreign application of AFEX technology and there are also some promising locations here in the U. S. to make ethanol and succinic acid from bagasse. Commercial development of AFEX and MBIs succinic acid organism are now both nearly ready for testing in industry.
Publications
- Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M.T., Ladisch, M.R. and Lee, Y.Y. 2005. Coordinated development of leading biomass pretreatment technologies. Bioresource Technology. 96: 1959-1966.
- Teymouri, F.A., Laureano-Perez, L.E., Alizadeh, H.Y. and Dale, B. E. 2005. Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover. Applied Biochemistry and Biotechnology. 96: 2014-2018.
- Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M.T., Ladisch, M.R. and Lee, Y.Y. 2005. Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover. Bioresource Technology 96:2026-2032.
- Alizadeh, H.Y, Teymouri, F.A., Gilbert, T.I. and Dale, B.E. 2005. Pretreatment of switchgrass by ammonia fiber explosion (AFEX). Applied Biochemistry and Biotechnology. 121:1133-1142.
- Laureano-Perez, L.E., Teymouri, F.A., Alizadeh, H.Y. and Dale, B.E. 2005. Understanding factors that limit enzymatic hydrolysis of biomass. Applied Biochemistry and Biotechnology. 121: 1081-1100.
- Mosier, N.A., Wyman, C.E, Dale, B.E., Elander, R.T., Lee, Y.Y., Holtzapple, M.T. and Ladisch, M.R. 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology. 96: 673-686.
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Progress 01/01/04 to 12/31/04
Outputs
Enzymes represent a major cost for cellulosic biomass conversion processes. Conventional cellulase mixtures were developed for acid treated biomass and lack adequate xylanase activity for neutral or basic pretreatments. We have used commercially available xylanases and cellulases to hydrolyze ammonia fiber explosion (AFEX) treated corn stover and have determined that the cellulase loading may be reduced by a factor of three with minimal amounts of xylanase while simultaneously increasing xylan conversion from under 70% to over 85% and maintaining glucan conversion at over 95% of theoretical. A.succinogenes fermentations using C6 or C5 sugars differ in their succinic acid yields, with glucose fermentations producing more succinic acid than comparable xylose or arabinose fermentations. Experiments showed that both types of sugars are consumed simultaneously and at the same rate, but 5-carbon sugars decreased succinic acid yields. When AFEX treated corn fiber hydrolyzed
by enzymes and dilute acid was used as the sole carbon source in A. succinogenes batch fermentations, growth and succinic acid production could only be achieved through medium supplementation. Further work will enhance succinic acid yields in fermentations using biomass derived sugars through strain adaptation and/or elimination of inhibitors. We obtained a broad host range plasmid and transformed this plasmid into A. succinogenes. The A. succinogenes phosphoenolpyruvate carboxykinase (PEPCK) gene and the malatedehydrogenase (MDH) genes were cloned into this vector and expression of each gene was verified by enzyme assays. A plasmid derivative was constructed containing the PEPCK promoter and expression of heterologous genes has been demonstrated. Thus a system has been established that allows metabolic alterations through over-expression of desirable genes to enhance succinic acid production. Ethanol fermentations will be used to establish the best conditions for AFEX-treatment of
corn fiber. Fermentations will be performed at the University of Florida. Corn fiber materials have been AFEX-treated and analyzed at MBI. Analyses results will be forwarded soon to Florida as will untreated corn fiber, AFEX treated corn fiber, and cellulase enzymes. The Millipore Pellicon Diafiltration system was set up to remove the residual acid molecules from the whisker suspension. Experiments were conducted to extract cellulose whiskers from corn stover and cellulose pulp. Yield was very low; work is continuing. Electron diffraction pattern of the whiskers was obtained by transmission electron microscope. Environmentally friendly fiber-reinforced poly(lactic acid) (PLA) laminated composites were successfully fabricated and developed. An invention disclosure details the process of preparing high performance laminated composites. Green composites were successfully fabricated through extrusion followed by injection molding from cellulose fiber (recycled newspaper)/talc and
polylactic acid (PLA). The tensile, flexural and impact strength of these hybrid composites are found to be significantly higher with the addition of 10 percent talc.
Impacts
Enzymes represent a major cost for cellulosic biomass conversion. Proper hemicellulase activities are particularly important for hydrolysis of ammonia treated biomass. We used commercially available xylanases and cellulases to hydrolyze ammonia fiber explosion (AFEX) treated corn stover. Cellulase loading is reduced by three fold with minimal xylanase while simultaneously increasing xylan conversion from under 70% to over 85% and maintaining glucan conversion at over 95%. These improvements reduce the estimated cost of biomass ethanol by approximately $0.20 per gallon. Metabolic flux analyses in A. succinogenes fermentations using C6 and C5 carbon sugars established that reducing power is the primary limiting factor in succinic acid fermentations. We demonstrated succinic acid production in A.succinogenes using only corn fiber-derived sugars as sole carbon source, and have established that carbon-recycling is feasible with this system. Our work has established
molecular biological tools for A. succinogenes. These tools will open the door to future targeted manipulations. The biopolymer polylactic acid (PLA) is considered too brittle for many commercial applications. Combining materials can overcome brittleness and poor processability of stiff and hard PLA, therefore PLA/cellulose( both virgin and recycled) fiber/talc hybrid composites were made by micro-compounding and injection molding system and evaluated. Overall, adding agro-based fibers to PLA composites provides a strategy to produce composites that are environmentally friendly, lower cost and biodegradable.
Publications
- Teymouri, F., Laureano-Perez, E. Alizadeh, H. and Dale, B.E. 2004. Ammonia Fiber Explosion Treatment of Corn Stover. Appl. Biochem. Biotech. 113-116:951-963.
- Mohanty, A. K., Drzal L. T. Misra, M. and Huda, M. S. Poly(lactic acid) (PLA)-based biocomposites and methods of making. (Note: submitted and assigned an Invention Disclosure Number (No. 02-042) by the Office of Intellectual Property at the Michigan State University in 2002)
- Huda, M. S. Mohanty, A. K. Misra, M. Drzal, L. T. Williams, K. and Mielewski, D. F. 2004. Mechanical, thermal and morphological studies of Poly (lactic acid) PLA/talc/recycled newspaper fiber hybrid Green Composites. BioEnvironmental Polymer Society 12th Annual Meeting, Monterrey, Mexico, 5 -10 December.
- Huda, M. S. Mohanty, A. K. Drzal, L. T. Williams, K. and Mielewski, D. F. Misra M. 2004. Thermo-mechanical & Morphological Properties of Composites from Poly(lactic acid) - Recycled Newspaper. AIChE 2004 Annual Meeting, Austin, TX, 7-12 November.
- Huda, M. S., Mohanty, A. K. Drzal L. T. and Misra, M. 2004. Effect of processing conditions on the physico-mechanical properties of Green composites from poly(lactic acid) and cellulose fibers, Proceedings of the Society of Plastics Engineers (SPE) ANTEC (Annual Technical Conference) 2004, Chicago, IL, 16-20 May.
- Huda, M. S., Mohanty, A. K. Drzal, L. T. Misra, M. and Schut, E. 2004. Physico-mechanical properties of Green Composites from poly(lactic acid) and cellulose fibers. Global Plastics Environmental Conference (GPEC 2004), Detroit, MI, 18-19 February.
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