Progress 07/01/11 to 06/30/16

Outputs
Target Audience:Our target audiences included: reasearchers and scientists, post-doctoral scholars, graduate students, undergraduate students, and high school students. Changes/Problems:Moisture control during the single pass harvesting of material other than grain during grain harvest was difficult. In all years, bales of stover and straw were harvested at a moisture content greater than 25%. This is considerably higher than 18% typically recommended. Both these factors made single pass harvest of corn stover and wheat straw difficult. Due to these issues bales were harvested at a high moisture and placed into storage. There were also challenges reported with taking representative samples from the bales to estimate realistic dry matter samples. A co-PI on the project (subaward to NC State) accepted a new job in industry and that move resulted in the integration of two research groups under another investigator at NC State. While the scope of work did not significantly change, there were delays in the project tasks. As a result, some of the data is still being collected and analyzed. In particular, information on gasification component was not available to be integrated into the on-farm processing system. Some harvesting equipment delays and sizing issues also delayed/changed some of the baling work at NC State. Delays in equipment deliveries (e.g. 1 year for the Christy Mill), equipment down time (field, analytical), waiting periods with contracted services, long measurement analysis times and slowed throughput for multiple lab users (5+ personnel) resulted in delayed outputs. From a producer outreach standpoint, a declining interest in biomass crops made it difficult to conduct on-farm research and hosting field-days to disseminate information on management practices for biomass crops. In the genetics, work, the original plan was to transform Setaria viridis.However, due to the recalcitrance of setaria to agrobacterium mediated transformation and even with various varieties of setaria as well as multiple protocols tested, the transformations were not successful. Therefore, the investigators refocused on heterologous expression studies in Arabidopsis to further characterize and better understand the functions and role of the identified setaria CesA genes. In addition, the goal of the project was widened to encompass a diverse range of cell wall regulatory mechanisms and not just the CesA genes that were originally targeted. Groups such as the laccases and microRNAs as well an in depth look into the phenylpropanoid pathway (RG studies) were investigated. Also, mutants that were created along the way have been studied and continue to be studied from a cell wall biosynthetic point of view. For the design of sugar-imprinted adsorbents, two challenges were encountered. First, the sugar groups in mixtures of sugar-based and cationic surfactants were found to be much more easily "buried" inside of micelles than expected. This made it difficult to use those micellar aggregates for imprinting of materials. This was overcome by modifying the surfactants to make the sugars more accessible, and using alternative approaches to materials synthesis that allowed the surface of particles to interact with the sugars. The second was the high degree of nonselective binding of sugars with silica. While enhanced adsorption was found due to imprinting, the "background" level of sugar binding was high - sometimes greater than 1 g of sugar / g of adsorbent. However, this has advantages for sugar concentration processes and is being pursued in a follow-on project as an approach to concentrating sugars from saccharification for subsequent fermentation. In the analysis of the saccharification using the QCM, developing a model able to match data from quartz crystal microbalance studies was challenging. This led to the novel development of a model in which accessible "surface" sites are distinguished from the sites located at the interior of cellulose fibers. This experience suggests that further progress in modeling this process can be made by extending this concept to more detailed models that account for accessibility of the cellulose surface in bulk lignocellulosic biomass. In the simulation of fermentation processes, the initial plan had been to use existing steady-state approaches (stoichiometric reactors) to describe the process. However, the stoichiometric reactions are completely insensitive to the variables most relevant to on-farm bioprocessing: time-dependent sugar concentrations, autocatalytic cell production, and product concentrations in recycle streams. Thus, an appropriately sensitive Monod-type model of ABE fermentation was identified and a new method to link these equations to an ASPEN batch reactor was developed. The NREL acid soluble and acid insoluble lignin assays indicated little or no lignin loss from Fenton treated biomass. The acetyl bromide soluble lignin confirmed this result. Results from the tests prompted the generation of a new hypothesis, Fenton chemistry does not degrade lignin but causes strand breaks in the polymer chain resulting in better enzymatic and bacterial access to cellulose. Experimental work conducted in the project support the hypothesis that Fenton chemistry results in cleavage of individual β-O-4 linkages in the lignin polymer generating defect sites that increase enzymatic and bacterial access to the underlying cellulose. What opportunities for training and professional development has the project provided?Number of extension training events/field days: 14 Smith, S. R. (2012). Bracken County Field Day Switchgrass Processing and AlternativeUses.Field Day.Bracken County, KY. Smith, S. R. (2012).Quicksand Field Day Switchgrass Research.Field Day.Eastern KY. Smith, S. R. (2013). Bracken County Fiscal Court - Switchgrass Research presentation.FieldDay.Lexington, KY. Smith, S. R. (2013).Campbell County Farmer's Tour - Switchgrass Energy Plots.Field Day.Quicksand Research Station, KY. Smith, S. R. (2013).Menifee County Farmer's Meeting - Renewable Energy Crop OptionsField Day.Northeast KY. Smith, S. R. (2013).UK Forage Workers Field Tour - Switchgrass Research presentation.Field Day.Lexington, KY. Smith, S. R. (2014).Boyd County Meeting - Switchgrass on Marginal LandField Day.Boyd County, KY. Smith, S. R. (2014).Bullitt County Farmer's Meeting - Renewable Energy Crop OptionsField Day.Bullitt County, KY. Smith, S. R. (2014). Growing Warm Season Grasses - UK Ext. Agent Update.Field Day.Winchester, KY. Smith, S. R. (2014).Renewable Energy Crops - Murray St. Biomass Day.Joint Field Day.M. S. University.Murray, KY. Smith, S. R. (2014).Woodford County Farmer's Meeting - Renewable Energy Crop Options.Field Day.Woodford County, KY. Smith, S. R. (2015).Alternative Warm Season Crops - UK Forage Workers TourField Day.Clark County, KY. Smith, S. R. (2015).Native Warm Season Grass adaptation on Reclaimed Surface Mines.American Society of Mining and Reclamation.Breathitt County, KY. Smith, S. R. (2015).Options for Summer Grass Production - Farm and Family Workshop,Maysville Comm. College.Field Day.Maysville, KY. Smith, S. R. (2015).Roundtable Discussion NRCS, KY Fish & Wildlife, & UK Cooperative Ext - Establishing Warm Season GrassesField Day.Harrodsburg, KY. Number of postdoctoral researchers: 2 Number of doctoral students trained: 17 Number of masters students trained: 16 Number of undergraduate students trained: 70 years equivalent How have the results been disseminated to communities of interest?Total number of peer reviewed publications: 40 Total number of extension publications: 5 Total number of presentations/publications: 79 Total number of completed dissertations/thesis: 27 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The discovery of a sorghum mutant is one of the unintended major accomplishments of this project. Sorghum was not part of the original bioenergy feedstock in the proposal but research that was being carried out into how to genetically manipulate switchgrass for low-lignin led to the discovery of this mutant which has generated considerable industry interest and to patent application. The grant enabled researchers to update the extension publication "Switchgrass for Biomass Production in Kentucky" by analyzing historic KY switchgrass agronomic data from on-farm demonstration fields (2007-2012) and from research results obtained in this project on 1) urea nitrogen and poultry litter fertilization rates, 2) cutting height in a dual use forage and biomass system, and 3) variable rate and timing nitrogen application. Studies have been conducted since 2012 on seeding rates, especially, but also different hybrids, nitrogen rates and row widths. In general, some of these practices increased grain yields when water was adequate. These practices had little influence on cob or fodder yields. Only for irrigated corn did extremely high populations (135,000 plants/ha or 60,000 plants/acre) increase fodder yields. These data suggest that farmers should focus on management practices that increase grain yield. Two levels of solids concentrations (5.7% and 12%) were compared in both white-rot (WRT) pretreatment and brown-rot (BRT) pretreatment. Corn stover of 5mm was used as the main substrate. The experiment verified the hypothesis that metabolic inhibition occurred under solid substrate concentration higher than 12%. There was no significant difference in the reducing sugars generated in the cellulolytic stage between these two levels of solids. In the cellulolytic stage by C.t, about 12.4-13 mg glucose/g biomass was accumulated using both 5.7% and 12% of corn stover pretreated by WRT. Around 4.2-4.8 mg glucose/g biomass was obtained using both levels of corn stover pretreated by BRT. Toward developing mass spectrometric strategies for the analysis of intact and pretreated lignin, a series of model lignin trimers were synthesized, isolated and characterized and studied fto understandthe ionization behavior of the model compounds. Compound dependent ionization indicates that oligomer specific approaches will be required for mass spectral elucidation of lignins. We developed the novel idea of pretreating biomass by recirculating a filtered, saturated lime solution in an up-flow, high solids (14-16% w/w) configuration at ambient conditions. In this system, lime solids were efficiently consumed, post-pretreatment washing of substrate did not significantly improve glucose yields, and energy and resources were conserved. Pretreatment effectiveness was assessed by glucose yield comparisons for both switchgrass and corn stover. Using mean glucose yields from 5mm corn stover, lime pretreatment required 350kgs of dry stover to produce 100kgs glucose at a chemical cost of $8.67 while NaOH required 300kgs at a cost of $22.38. Flushing of the hydrolysate and reusing the substrate was studied as a method for reducing inhibitory compounds affecting hydrolysis in order to increase overall glucose yields. Glucose conversions increased from 37-49% for conventional batch reactions and up to 73-99% for flushed reactions. While conversion of unwashed pretreated corn stover (PCS) was not as high as that of the washed PCS, the unwashed PCS with flushed hydrolysis still achieved significantly higher glucose concentrations than that of the washed PCS in conventional batch hydrolysis (73 g/L vs 48 g/L) with an enzyme loading of 15 FPU/g solids. The goal of this set of experiments was to develop a bench-scale high solids fermentation with intermittent flushing.Repeated flushing process was carried out in 2.5 cm I.D. and 10 cm length glass columns fitted with porous polyethylene discs at the bottom which allowed media to flow through yet retained the solid substrate. The columns were loaded with 5 g of miscanthus (particle size of 5mm) and inoculated with 2 ml of P. chrysosoporium to remove the lignin. After a 5-day fungal pretreatment at 35 oC, the fermentation of Clostridium thermocellum (C.t) ATCC 27405 was held at 65 oC for 2 days to hydrolyze cellulose to sugars. Then the sequential co culture was initiated by inoculating C. beirinjerckii (C.b) ATCC 51743 and held for 2 days for acids and solvents production. Glucose is the limiting factor that determines the metabolites production of co-culture system.The observation supports the necessity of re-incubation of C.t at 65 oC to hydrolyze cellulose into glucose in the flushing system.?In CFSM 5, re-inoculation of C.b produced 193.9 mM of total solvents and 77.7 mM of total acids which were 1.79 fold and 1.75 fold higher than those from CFSM 4 with no re-inoculation of C.b throughout all the flushing cycles.Re-inoculation of C.b cells increased the cell mass and accumulation of target acids and solvents by up to 1.7 fold and 2.85 fold respectively in the flushing co-culture system.The limited access to glucose negatively affected the cell metabolism and activity. Commercial cellulase was added (60 FPU/g biomass) and incubated for 3 days at 50C. Post-hydrolysisglucose levels were43 g/L, 20 g/L of xylose. One hundred grams per liter of initial corn stover concentration resulted in approximately 8 g/L of butanol. Low glucose concentration was identified as a primary limitation of the co-culturing approach. Therefore, low-energy technologies to increase sugar concentration in dilute aqueous systems were developed, including novel membrane separations and sugar-imprinted adsorption. Knowledge of the saccharification process, developed from interfacial measurements using model cellulose and lignin thin films, was used to understand the role of nonspecific enzyme binding and fermentation parameters on cellulose hydrolysis, with a goal of increasing sugar yields. An ASPEN simulation of the batch ABE fermentation with separation and recycle was developed to capture the key elements for the design of on farm biomass processing. ASPEN simulations incorporating measurements from commercial adsorbents were used to study the feasibility of multiple adsorbent beds in series to recover individual ABE products. A life-cycle analysis of the final system configuration predicted butanol production at a cost of $3.16 per gallon of gasoline equivalent (GGEE). If processed at a central facility using extraction-distillation, the butanol produced would result in a positive net energy value of 0.33 MJ/MJ with is superior to that of corn grain ethanol produced through the dry-grind process (0.18 MJ/MJ). The cost of production is estimated to be approximately $4.77 per GGEE. Required on-farm capital investment for a 2000 acre corn farm would be approximately $3.0 million.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: S. Das, S. Nagpure, R.K. Garlapalli, Q. Wu, S.Z. Islam, J. Strzalka and S.E. Rankin (2015) Pore Orientation Effects on the Kinetics of Mesostructure Loss in Surfactant Templated Titania Thin Films. Phyical Chemistry Chemical Physics, doi: 10.1039/C5CP05178A.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Daniel M. Schlipf, Stephen E. Rankin, Barbara L. Knutson, "Effects of Pore Size and Tethering on the Diffusivity of Lipids in Pore-Filled Mesoporous Silica," Submitted to Langmuir 2016
• Type: Journal Articles Status: Published Year Published: 2016 Citation: M. Kaitlyn Clark Wooten, Venkat R. Koganti, Shanshan Zhou, Stephen E. Rankin, and Barbara L. Knutson, "Nanofiltration Membranes Synthesized from Oriented Mesoporous Silica Thin Films on Macroporous Supports," Submitted to Journal of Membrane Science
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Shanshan Zhou, Hsin-Fen Li, Ravinder Garlapalli, Sue E. Nokes, Michael D. Flythe, Stephen E. Rankin, Barbara L. Knutson, Hydrolysis of Model Cellulose Films by Clostridium thermocellum: Extension of Quartz Crystal Microbalance Techniques to Cellulosomes
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kwabena Darkwah, Jeffrey R. Seay, Barbara L. Knutson, In silico analyses of cell-based unsteady-state batch Acetone-Butanol-Ethanol (ABE) fermentation and in situ gas stripping. Submitted to Computers & Chemical Engineering.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Kato DM, Elia N, Flythe M, Lynn BC. Pretreatment of lignocellulosic biomass using Fenton chemistry. Bioresource Technology 2014;162:273-8.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Joshua Jackson, Aaron Turner, Tyler Mark, Michael Montross, Densification of biomass using a pilot scale flat ring roller pellet mill, Fuel Processing Technology, Volume 148, July 2016, Pages 43-49, ISSN 0378-3820, http://dx.doi.org/10.1016/j.fuproc.2016.02.024. http://www.sciencedirect.com/science/article/pii/S0378382016300789
• Type: Other Status: Published Year Published: 2012 Citation: Stombaugh, T., Montross, M., Nokes, S., W. Adams. 2013. A new concept in on-farm biofuel production. AEN-112. Cooperative Extension Service, College of Agriculture, Food, and Environment, University of Kentucky.
• Type: Other Status: Published Year Published: 2012 Citation: Stombaugh, T., Nokes, S., and K. Gray. 2012. Butanol: The New Biofuel. AEN-111. Cooperative Extension Service, College of Agriculture, Food, and Environment, University of Kentucky.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yao, W. and S.E. Nokes. 2013. The use of co-culturing in solid substrate cultivation and possible solution to scientific challenges. Biofuels, Bioproducts, and Biorefining. Vol7(4):361-372
• Type: Journal Articles Status: Published Year Published: 2016 Citation: ��������� Hickman, A.N., Nokes, S.E., Sympson, W.S., Ruwaya, M.J., Montross, M.D., and B.L. Knutson. 2016. The confounding effects of particle size and substrate bulk density on Phanerochaete chrososporium pretreatment of Panicum virgatum. BioResources 11(3): 7500-7511. DOI: 10.15376/biores.11.3.7500-7511.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: ��������� Noelia M. El�a, Sue E. Nokes, Michael D. Flythe. 2016. Switchgrass (Panicum virgatum) fermentation by Clostridium thermocellum and Clostridium saccharoperbutylacetonicum sequential culture in a continuous flow reactor. AIMS Energy. 4(1):95-103. DOI: 10.3934/energy.2016.1.95
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Keene, T., K.L. Lea, K.J. Prince, T. Phillips, B.M. Goff, and S.R. Smith (2016) Switchgrass yield and quality with multiple fertilizer applications and harvest dates. Agronomy Journal, in preparation.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Sena, K, S.R. Smith, and B. Goff. (2016) Growth curve of switchgrass varieties for yield and fiber quality. Agronomy Journal, in preparation.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Switchgrass for Biomass Production in Kentucky. University of Kentucky Extension Bullentin AGR 201.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Sukumara, S. and�J. Seay�(2013): Multidisciplinary Optimization Model to Analyze Regional Supply Chains for Sustainable Biorefining,�Proceedings of the 3rd�International Conference on Sustainable Chemical Product and Process Engineering, CD-Volume, Dalian, China.
• Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: R.K. Garlapalli, B.L. Knutson, S.E. Nokes and S.E. Rankin, Investigation of cellulase binding to model lignin surfaces in presence of Tween 80 using quartz crystal microbalance, Kentucky Innovation and Entrepreneurship Conference, Louisville, KY, May 2012.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: S. Das, W. Xu, H.-J. Lehmler, B.L. Knutson and S.E. Rankin, Effects of Surfactant Structural Parameters on the Relative Position of Surfactant Head Groups in Mixed Cationic-Carbohydrate Surfactants Micelles, American Institute of Chemical Engineers Annual ?Meeting, Atlanta, GA, November 2014.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Development of Capillary Electrophoresis-Mass Spectrometry for Analysis of Fenton Chemistry Biomass Pretreatment for Biofuels Production, D. Kato, B.C Lynn, June 9-13, 2013 61st ASMS Conference on Mass Spectrometry, Minneapolis, MN
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: B. Lynn, "Progress in the nearly intractable problem of lignin analysis" FDA presentation and UK Seminars
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Sandoval, W., M.S. Chinn and J.M. Bruno-Barcena. Clostridium beijerinckii SA-1 is a butanol hyper-producing strain. Presented at the 2013 American Society of Microbiology General Meeting. Denver, CO. May 20, 2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Sandoval, W., M.S. Chinn and J.M. Bruno-Barcena. Inoculum Optimization of Clostridium beijerinckii for Bench-scale fermentation. Presented at the 2015 American Society of Microbiology, General Meeting. New Orleans, LA. May 30-June 2, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Mbaneme, V., M. Chinn, J. Bruno-Barcena and M. Veal. 2015. Influence of Synthesis Gas on Conversion of Annual and Perennial Bioenergy Crops using Clostridium Thermocellum. Presented at the 2015 ASABE Annual International Meeting. New Orleans, LA. July 26-29, 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Flythe. Metabolic control of Clostridium thermocellum via selective inhibition and compensatory product formation. Global Biofuels & Bioproducts Summit 2012, San Antonio, TX. Nov. 19-21, 2012
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Biofuel Production Potential, Midwest Outlook Conference. Louisville, Kentucky. August 14, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: H.S. Li, R. Garlapalli, M. D. Flythe, S.E. Nokes, S.E. Rankin and B.L. Knutson,  Inhibition of Enzymatic Hydrolysis by Soluble Sugars on Model Cellulose Thin Films using Quartz Crystal Microbalance, Oral presentation at the 2011 AIChE Annual Meeting, Minneapolis, MN, Oct. 16  21, 2011.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: H.F. Li, B.L. Knutson, S. E. Rankin, and S. E. Nokes, Modeling the Effect of Enzyme Concentration Dependence on Hydrolysis of Cellulose Thin Film as Measured by the Quartz Crystal Microbalance, Oral presentation at the 2012 AIChE Annual Meeting, Pittsburgh, PA., Oct. 28  Nov. 2. 2012.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Sukumara, S., J. Seay, F. Badurdeen, J. Amundson and W.�Faulkner (2012): Feed Flexible Process Optimization Model for Region Specific Sustainable Biorefining, AIChE Annual Meeting, Pittsburgh, Pennsylvania.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: S. Joshi, B.L. Knutson and S.E. Rankin, Surfactant imprinting of the surface of silica nanoparticles for selective adsorption of sugars with subtle structural differences, 87th ACS Colloid and Surfac Science Symposium, Riverside, CA, June 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: J. Seay and S. Sukumara (2013): Integrated Modeling for Region-Specific Biorefining, Invited presentation at the 3rd International Forum on Sustainable Manufacturing, Lexington, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Sukumara, S. and J. Seay (2013): Novel Framework To Examine Sustainability Of Biorefining Processes, AIChE Annual Meeting, San Francisco, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Sukumara, S., J. Seay, F. Badurdeen, W. Faulkner and J. Amundson (2013): A Framework to Examine Sustainability of Various Biorefining Processes, 3rd International Congress on Sustainable Science and Engineering, Cincinnati, OH.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: S.E. Rankin, R.K. Garlapalli, H.-F. Li, B.L. Knutson, S.E. Rankin and S.E. Nokes, Interfacial Engineering of Biomass Saccharification by Trichoderma reesei Enzymes, Kentucky Innovation and Entrepreneurship Conference, Lexington, KY, August 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: S.E. Rankin, Directing the Surfactant Templating of Oxide Materials for Adsorption, Catalysis and Energy,University of Tennessee, Knoxville, TN, 10/2013
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: S. Das, S. Joshi and S.E. Rankin, Interaction of Carbohydrates With Molecularly Imprinted Silica Particles, AIChE Annual Meeting, San Francisco, CA, November 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: K. Wooten, V.R. Koganti, S.E. Rankin and B.L. Knutson, Nanofiltration Membranes From Oriented Mesoporous Silica Thin Films, AIChE Annual Meeting, San Francisco, CA, November 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: S.E. Rankin, R.K. Garlapalli, H.-F. Li, B.L. Knutson and S.E. Rankin, Quartz Crystal Microbalance Study of Trichoderma Reesei Cellulase Interactions with Model Cellulosic Biomass Component Films, Kentucky Innovation and Entrepreneurship Conference, Louisville, KY, May 2012.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: D.M. Schlipf, S.E. Rankin and B.L. Knutson, Controlling the Pore Structure of Silica Platforms for the Isolation of Bioactive Molecules, Kentucky Innovation and Entrepreneurship Conference (KIEC), Louisville, KY, August 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: B.L. Knutson, "At the Interface of Advanced Materials and Plant Biotechnology," Invited Talk at Kentucky Innovation and Entrepreneurship Conference, Lexington, KY, August 2013.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: R. Garlapalli, B. L. Knutson, S.E. Nokes, and S.E. Rankin, Role of Tween-80 in Reduction of Nonproductive Cellulase Binding to Lignin, Oral presentation at the 2012 AIChE Annual Meeting, Pittsburgh, PA., Oct. 28  Nov. 2. 2012.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: E.A. McClean, S. E. Rankin, and B. L. Knutson Protein Diffusion Through Oriented Thin Film Silica Membranes, Poster presentation at undergraduate poster competition at the 2012 AIChE Annual Meeting, Pittsburgh, PA., Oct. 28  Nov. 2. 2012. 2nd Place winner in Separations division of posters
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: D.M. Schlipf, S.E. Rankin and B.L. Knutson, Effects of Nanopore Size on the Interactions of Lipid Bilayers with Mesoporous Silica Particles, Oral presentation at the American Institute of Chemical Engineers Annual Meeting, Atlanta, GA, November 2014
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: S. Zhou, R. Garlapalli, S.E. Nokes, S.E. Rankin and B.L. Knutson, Quartz Crystal Microbalance Investigation of Cellulose Hydrolysis By Clostridium Thermocellum on Model Cellulose Films, Poster presentation at the American Institute of Chemical Engineers Annual Meeting, Atlanta, GA, November 2014
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S. Zhou, D. Schlipf, S.E. Rankin, and B.L. Knutson, "Functionalization of composite thin film silica membranes with lipid bilayers," Poster presented at North American Membrane Society (NAMS), Boston, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S. Zhou, D. Schlipf, S.E. Rankin, and B.L. Knutson, "Recovery of dilute aqueous solutes with lipid-functionalized silica thin film membranes," Poster at North American Membrane Society (NAMS), Boston, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Sumesh Sukumara, Kwabena Darkwah, Jeffrey R. Seay Process Simulations Supporting a Techno-Economic Framework to Optimize Biorefinery Supply Chains, 25th European Symposium on Computer Aided Process Engineering (ESCAPE 2015), Copenhagen, Denmark
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S. Zhou, D. Schlipf, S.E. Rankin, and B.L. Knutson, "Functionalization of silica membranes with lipid bilayers for sugar recovery," Oral presentation presented at Salt Lake City, Utah, November 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: J. Seay, K. Darkwah, and B.L. Knutson, " Dynamic modeling and simulation of an on-farm bioconversion of lignocellulosic biomass in acetone-butanol-ethanal fermenation" Plenary talk at AIChE Annual Meeting, Salt Lake City, Utah, November 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: K. Darkwah, J. Seay, and B.L. Knutson, " Dynamic modeling and simulation of an on-farm bioconversion of lignocellulosic biomass in acetone-butanol-ethanal fermenation" Oral presentation at AIChE Annual Meeting, Salt Lake City, Utah, November 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: K. Darkwah, J. Seay, and B.L. Knutson, " A multidiscilinary framework decision support tool for assessing the economic viability of on farm biomass conversion," Poster presented at Salt Lake City, Utah, November 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S. Zhou, R. Garlapalli, S.E. Rankin, and B.L. Knutson, "Modeling of cellulose hydrolysis by Clostridium thermocellum monitored by quartz crystal microbalance," Paper presented at Salt Lake City, Utah, November 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S. Zhou, R. Garlapalli, S.E. Rankin and B.L. Knutson, Modeling of Cellulose Hydrolysis By Clostridium Thermocellum Monitored By Quartz Crystal Microbalance,American Institute of Chemical Engineers Annual Meeting, Salt Lake City, UT, November 2015.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: "B. Knutson, ""Engineering tools for advancing plant-derived natural products,"" Invited speaker, Monie A. Ferst Award Symposium In Honor of Charles Eckert, Georgia Institute of Technology, Feb. 10, 2016. "
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shanshan Zhou, Stephen Rankin, Barbara Knutson, Functionalization of silica membranes with lipid bilayers for sugar recovery, NAMS (North American Membrane Society) 2016
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Kwabena Darkwah, Jeffrey R. Seay, Barbara L. Knutson, In silico analyses of cell-based unsteady-state batch Acetone-Butanol-Ethanol (ABE) fermentation and in situ gas stripping,, 4th International Conference on Sustainable Chemical Product and Process Engineering (SCPPE 2016), Najing China
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: 9.����� Turner, A., M. Montross, W. Adams, C. Foster, J. Posselius, D. Lewis 2012. Single Pass Grain and Biomass Harvest for Wheat and Corn in Western Kentucky. ASABE International Meeting, Dallas, TX. ASABE International Meeting. July 28-August 1. Paper No. 121338326
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: "Structural effects on the ionization response of lignin model compounds during electrospray ionization". Fan Huang, B.C. Lynn, May 31- June 4, 2015, 63rd ASMS Conference on Mass Spectrometry, St. Louis, MO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Characterization of a B-O-4 trimeric lignin model compound and post Fenton chemistry products using HRAM mass spectrometry. Fan Huang, B.C. Lynn, June 5- June 9, 2016, 64th ASMS Conference on Mass Spectrometry, San Antonio, TX
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: 8.����� Adams, W., M. Montross, A. Turner, J. Jackson, L. Mathis, W. Hammond, N. Bush, M. Fogle. 2012. Evaluation of Current Technology for the Determination of Representative Bale Moisture Content. ASABE International Meeting, Dallas, TX. ASABE International Meeting. July 28-August 1. Paper No. 121338345
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 3.����� Koeninger, N.K., M.D. Montross, C.T. Agouridis, A.P. Turner. 2014. Impact of Varying Cover Factors on Soil Erosion due to Biomass Removal. ASABE International Meeting, Montreal, Canada. July 14-16, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 4.����� Schiavone, M.D. Montross, A.P. Turner, J.Jackson. 2014. Bale Moisture Measurement via Time-Domain Reflectometry. ASABE International Meeting, Montreal, Canada. July 14-16, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: 6.����� Schiavone, D., M. Montross, W. Adams. 2013. Hydraulic Conductivity of Baled Biomass. ASABE International Meeting, Kansas City, MO. ASABE International Meeting, Louisville, KY July 21-24. Paper no. 131619433
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: 7.����� Jackson, J. M. Montross. Pretreatment of Biomass Using an Alkaline Hydrogen Peroxide Spray. ASABE International Meeting, Kansas City, MO. ASABE International Meeting. Paper No. 131620350
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 5.����� Turner, A.P., M.D. Montross, N.K. Koeninger. 2014. Mechanical Properties of Radially Compressed Miscanthus and Switchgrass. ASABE International Meeting, Montreal, Canada. July 14-16, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: 1.����� Koeninger, N.K., M.D. Montross, C.T. Agouridis, A.P. Turner, J.J. Jackson. 2015. Determining soil erosion with varying corn stover cover factors. ASABE International Meeting, New Orleans, LA. July 26-29, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: 2.����� Jackson, J.J., A.P. Turner, N.K. Koeninger, M.D. Montross. 2015. Composition and enzymatic hydrolysis of biomass pretreated with alkaline hydrogen peroxide spray. ASABE International Meeting, New Orleans, LA. July 26-29, 2015
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: ��������� Modenbach, A., Nokes, S., Knutson, B., Rankin, S. 2011. Recovery of a Purified Stream of C5 Sugars from Lignocellulosic Hydrolyzate Using Microphase-Directed Imprinted Materials. Presented at the S-1041 Annual Symposium, Stillwater, OK, 2011.
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: ��������� Modenbach, A., Nokes, S., Knutson, B., Rankin, S. 2011. Recovery of a Purified Stream of C5 Sugars from Lignocellulosic Hydrolyzate Using Microphase-Directed Imprinted Materials. Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, Louisville, KY, 2011.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: ��������� Ruwaya, Mathew, Nokes, Sue, Flythe, Michael. 2012. Automated solid-substrate cultivation of the anaerobic, thermophilic bacterium Clostridium thermocellum. Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, Dallas, TX. July 29-August 1, 2012. Paper no. 121337913.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: ��������� Yao, Wanying, Nokes, Sue, Flythe, Michael. 2012. Characteristics of alcohol production from corn stover using a three-stage solid substrate cultivation process. Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, Dallas, TX. July 29-August 1, 2012. Paper no. 121337532.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: ��������� Modenbach, Alicia, Nokes, Sue, Montross, Michael, Knutson, Barbara. 2012. Characterization of the soluble and insoluble inhibitor effects on enzymatic hydrolysis at high solids using pretreated corn stover. Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, Dallas, TX. July 29-August 1, 2012. Paper no. 121336792.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: ��������� Yao, Wanying, Nokes, Sue, Flythe, Michael, Lynn, Bert, Kato, Dawn, and Montross, Michael. 2012. Phanerochaete chrysosporium pretreatment to enhance solvent production using solid-substrate fermentation. Poster presented at the S-1041 The Science and Engineering of the Biobased Economy. Washington, DC. August 6-7, 2012.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Hickman, A., Nokes, S. Scale up of P. Chrysosporium during solid state pretreatment Presented at the IBE Annual Meeting, Lexington, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Carey, B., Nokes, S. Rapid Genetic Identification of Microorganisms for Bioprocesses Presented at the IBE Annual Meeting, Lexington, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: ��������� Amanda Hickman and Sue Nokes. Is biological pretreatment effectiveness dependent on biomass particle size or biomass bulk density? Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, New Orleans, LA. July 26-29, 2015. Paper no. 152190146.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Amanda Hickman, Sue Nokes, William S. Sympson, Mathew J. Ruwaya, Michael Montross, and Barbara L. Knutson. The confounding effects of particle size and substrate bulk density on Phanerochaete chrososporium pretreatment of Panicum virgatum as a function of incubation time and percent of unsterilized substrate. Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, Orlando, FL. July 17-July 20, 2016. Paper no. 162461610.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: ��������� Amanda Hickman, Bobby Carey, Smruti Patel, William S. Sympson, and Sue Nokes. Pretreatment effectiveness of Phanerochaete chrososporium on Panicum virgatum. Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, Orlando, FL. July 17-July 20, 2016. Paper no. 162461639.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: ��������� Megan Walz, Danielle Empson, Sue Nokes, and Barbara Knutson. Use of mesoporous silicon nanoparticles to concentrate glucose from lignocellulosic hydrolysate for downstream fermentation. Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, Orlando, FL. July 17-July 20, 2016. Paper no. 162461640.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Jordyn Tucker, Amanda Hickman, and Sue Nokes. The effect of ?-glucosidase supplementation on the saccharification of pretreated switchgrass (NaOH and Phanerochaete chrososporium) using commercial cellulase enzymes. Presented at the American Society of Agricultural and Biological Engineering Annual Meeting, Orlando, FL. July 17-July 20, 2016. Undergraduate student poster competition.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Sept 22, 2012 - Presented the research being conducted at the Quicksand Field Day in eastern KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Oct 18, 2012  Presented to group of farmers the processing options and alternative uses for switchgrass at a field day in Braken County.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: May 8, 2013 - Presented the switchgrass research to the UK Forage Workers tour (tour for faculty and graduate students involved in forage research from the College of Agriculture).
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: July 10, 2013  Presented at discussion session on energy crops and was recognized by Bracken County Fiscal Court for cooperation with switchgrass research.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: July 23, 2013  Conducted tour with Campbell County farmers of energy plots (Switchgrass and other species) at Quicksand Research Station.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: July 25, 2013  Presentation to Breathitt County Farmers on renewable energy possibilities utilizing switchgrass.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: October 18, 2013 Presentation at Menifee county farmer meeting discussing renewable energy possibilities in northeastern Kentucky.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: March 11, 2014 Presentation to Woodford County farmers group on possible renewable energy crops for the future.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: October 14, 2014  Presented Growing Warm Season Grasses, UK Agent Update, Winchester, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: March 11, 2014 Presentation for Bullitt County farmers on possible renewable energy crops of the future.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: June 10, 2014 Switchgrass Field Day conducted on a Boyd County farm showcasing to farmers the possible uses of switchgrass on marginal land in eastern Kentucky.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: October 22, 2014  Participated with Murray State University on joint meeting on Renewal Energy Crops, Murray State Biomass Day, Murray, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Led discussion on Alternative Warm Season Crops, Univ. of Kentucky Forage Workers Tour, Clark County, KY
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Ray Smith, Tom Keene, Peter Robuck, Kelly Prince and Krista Lea. 2016. Switchgrass Quality and Biomass Suitability over Fertilizer Type and Rate and Maturity. Proceedings of the International Rangeland Congress. 17-22 July 2016. Saskatoon, Saskatchewan, Canada.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Caffrey, K.R., Veal, M.W. 2013. Conducting an Agricultural Life Cycle Assessment (LCA): Challenges and Perspectives. http://www.hindawi.com/journals/tswj/aip/472431/
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Flythe, M. , El�a, N. , Schmal, M. and Nokes, S. (2015) Switchgrass (Panicum virgatum) Fermentation by Clostridium thermocellum and Clostridium beijerinckii Sequential Culture: Effect of Feedstock Particle Size on Gas Production. Advances in Microbiology, 5, 311-316. doi: 10.4236/aim.2015.55031.
• Type: Other Status: Published Year Published: 2016 Citation: Halich, G., Kindred, S. 2016.��Switchgrass Budgets - Cost and Returns.� Cooperative Extension Service, College of Agriculture, Food, and Environment, Department of Agricultural Economics, University of Kentucky.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Nov 14, 2014 - Poster Presentation at the KY Academy of Science. Crisologo, A. and S.R. Smith. 2014. Analysis of forage quality in switchgrass at varied maturities using NIRS. In Proceedings of the Kentucky Academy of Science Annual Meeting. Lexington, KY. 14-16 November, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: January 12, 2015 Presented at the American Forage and Grassland Council annual meeting. Keene, T. and S.R. Smith. 2015. Harvest dates and fertility rates for switchgrass production. In Proc. AFGC Annual Meeting. Jan 11-13, 2015. St. Louis, MO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: February 5, 2015  Roundtable discussion with NRCS, KY Fish and Wildlife, and Cooperative Extension on establishing native warm season grasses on Mercer County farms. Harrodsburg, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: March 10, 2015  Presented Options for Summer Grass Production, Farm and Family Workshop, Maysville Community College, Maysville, KY.
• Type: Other Status: Published Year Published: 2016 Citation: Halich, G., Kindred, S. 2016.��Corn Stover�Budgets - Cost and Returns.� Cooperative Extension Service, College of Agriculture, Food, and Environment, Department of Agricultural Economics, University of Kentucky.
• Type: Theses/Dissertations Status: Published Year Published: 2012 Citation: Li, H.-F. Application of Thin Film Analysis Techniques and Controlled Reaction Environments to Model and Enhance Biomass Utilization by Cellulolytic Bacteria
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Li, H-F., Knutson, B.L., Nokes, S.E., Lynn, B.C., and M.D. Flythe. 2012. Metabolic Control of Clostridium thermocellum via Inhibition of Hydrogenase Activity and the Glucose Transport Rate. Appl Microbiol Biotechnol. Feb: 93(4):1777-84. doi 10.1007/s00253-011-3812-3.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Sukumara, S. and J. Seay (2014): A Novel Model for Evaluating the Viability of Strategies for Biorefining Processes from Various Stakeholder Perspectives: Case Study on Marginal Land Utilization, Computer Aided Chemical Engineering.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Joshi, S. Mixed Surfactant Systems: Thermodynamics and Applications in Metal Oxide Imprinting
• Type: Journal Articles Status: Published Year Published: 2015 Citation: S. Nagpure, S. Das, R.K. Garlapalli, J. Strzalka and S.E. Rankin (2015) In Situ GISAXS Investigation of Low-Temperature Aging in Oriented Surfactant-Mesostructured Titania Thin Films. Journal of Physical Chemistry C, 119, 2297022984.
• Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Garlapalli, Ravinder K. Interfacial Engineering of Biomass Hydrolysis by Cellulase Enzymes and Mechanistic Modeling of Hydrolysis of Cellulose Substrates. Ph.D. Dissertation, University of Kentucky, 2015.
• Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Das, Saikat. Fundamental Studies of Surfactant Templated Metal Oxide Materials Synthesis and Transformation for Adsorption and Energy Applications. Ph.D. Dissertation, University of Kentucky, 2015.

Progress 07/01/14 to 06/30/15

Outputs
Target Audience:Scientific community interested in biomass conversion, post-doctoral scholars assisting with project. Graduate students interested in biomass conversion, and biomass logistics. Undergraduate student workers. Undergraduate and graduate students who take BAE 503 Fundamentals of Biorenewable Resources and BAE 504 Biofuels Production and Processing. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided training for 10 undergraduate research assistants (NCSU, UK and ORNL) in Biosystems and Agricultural Engineering, Chemistry, Microbiology, Agronomy, and Chemical Engineering. We have graduated 11 Master of Science students with projects related to this project, with five more students to finish this coming year. Ten PhD have completed degrees withprojects related to this project, with four PhD students still in the pipeline. Two post-doctoral scholars also received training related to this grant during this year. In addition, we have given numerous tours to school groups where we discussed the objectives of the project. How have the results been disseminated to communities of interest?Our results have been disseminated to the scientific community through conference presentations at venues which include the AIChE International Meeting and the ASABE International meeting. We have published 6 refereed journal articles this year, and two extension publications. Master's theses and PhD dissertations are published and accessible to the scientific community. What do you plan to do during the next reporting period to accomplish the goals?Year 5 will concentrate on graduating the remaining students,writing manuscripts and collecting any data needed to complete the data sets for manuscripts.

Impacts
What was accomplished under these goals? Objective A (Technical Area 1): Develop a reliable biomass feedstock supply system using agricultural residues . Agronomic data on corn stover was collected investigating hybrid, seeding rate, row width, and this year we collected data on the effects of irrigation and nitrogen application rate. Switchgrass agronomic studies; three sites continue to bemonitored fornitrogen response, cutting height vs time of cutting, mineral content. Field scale yields are 4-7 tons (plots yield 6-8 tons). Single pass harvesting worked well, except for moisture issues in the biomass. When the grain is ready to harvest, the biomass is usually at a higher moisture content than desired for baling. Crushing stalks increased density 10-15% when off the baler, however no increase in density was seen when the crusher was mounted on the baler. Equipment project on hold given the current lack of interest in biofuels nationally. Bale storage - round bales net wrapped: treatments covered, covered outside, stored inside, outstide on pallets, outside on ground. Very little differences in bale weight adjusted for moisture. Net wrap maintains the integrity of the bales. May cut bales this fall and see if quality varies based on location relative to the ground. Large rectangular bale storage encountered difficulty obtaining consistent data when measuring moisture in the bales. A heat and mass balance mathematical model was developed that predicts moisture migration in large rectangular bales. Geographical information study conducted to evaluate cost in dollars per liter crude biobutanol of transporting crude biobutanol to decentralized refineries versus a centralized refinery. For hauling distances between 8 and 80 km, the cost for transporting to decentralized refineries was approximately half to two-thirds that of transporting to a centralized refinery. Our study of the molecular genetics of cellulose biosynthesis using Seteria veridis L. as a model C4 crop, has resulted in a stable overexpressor of Seteria CESA (plant cellulose synthase gene), resulting in increased biomass and expansion capacity (much larger plant). Objective B (Technical Area 2): Develop the technical and economic feasibility of on-farm storage/processing of high density biomass feedstocks . Pretreatment (Phanerochaete chrysosporium, NaOH, Ca2(OH)2, lignin byproduct analysis) Corn stover: 80% moisture content on 2 mm particle sizes showed 33% lignin degradation after 10 day growth of Phanerochaete crysosporium. Lignin Analysis: We recently evaluated the use of Fenton chemistry for delignification of biomass. While we observed statistically significant gains in enzymatic saccharification (>500% of control for switchgrass), we saw no differences in total lignin (acid soluble and acid insoluble) relative to control. These observations prompted us to explore enhanced analytical approaches to identifying changes in the chemical structure of lignin resulting from various pretreatment. Electrospray ionization followed high resolution, accurate mass (HRAM) mass spectrometry and tandem mass spectrometry have been used to study a wide variety of problems ranging from synthetic polymer to biopolymers (proteins and oligonucleotides). Our first mass spectrometry experiments on pretreated biomass highlighted the nearly intractable problem resulting from three monomers, five bonding types, heteropolymers, multiple charge states and multiple oligomers that produced ions at every mass even at high resolution. Tandem mass spectrometry can be used to reduce this complexity but collision induced dissociation rules are not available for these types of molcules. Therefore, we initiated a study to synthesize biologically relevant lignin homo-oligomers, purify these oligomers to single compounds, determine their structures using 2D NMR techniques and then determine their tandem mass spectrometry thus enabling the construction of a mass spectrometry database that could be used to elucidate real world lignins. To accomplish this goal, we synthesized dehydrogenation polymers using monolignols (prepared in house) as starting material in the present of horseradish peroxidase/ hydrogen peroxide. Two different polymerization methods were used. The α,β-diaryl ether linkage enriched DHPs were prepared based on the method described by Francesco De Angelis (Angewandte Chemie International Edition 1999, 38, 1283) with some modification. While β-o-4 linkage enriched DHPs were made according to Cathala, B. Polymer Degradation and Stability 1998, 59 (1-3), 65-69. Each of the isolated trimers was analyzed by 2D NMR (HSQC and HMBC) to fully elucidate the bonding types. The three trimmers were identified as G(4-O-a)G(b-O-4)G trimer, G(b-O-4)G(b-5)G trimer and G(b-O-4)G(b-b)G trimer. Even though the G(b-O-4)G(b-5)G and G(b-O-4)G(b-b)G trimers have the same exact mass, their tandem mass spectra could be used to clearly delineate the isomer. Interestingly, the G(4-O-a)G(b-O-4)G trimer anion cleanly fragmented by loss of monomer analogous to the fragmentation of peptides. These results represent the beginnings of a lignin mass spectral database that could be applied to lignin samples produced from pretreatment protocols. We are currently expanding the numbers of elucidated structures and also studying alternative methods to generate biologically relevant structures. Hydrolysis (C. thermocellum, enzymes) An interfacial model of cellulose thin film hydrolysis has been developed, and has been used to quantify the effect of cellobiose inhibition on T. reesei cellulase system on the molecular level. Work has also progressed in using C. thermocellum in this thin film cellulose hydrolysis system. Data were collected for the temperature response curve of C. thermocellum growth and enzymatic activity as a function of solids concentration in the fermentation. Fermentation C. beijerinckii SA-1 was grown on sucrose (with and without syngas) and on cellobiose (without syngas). When no syngas was present, C. beijerinckii SA-1 preferred sucrose, producing approximately 13 g/L butanol as opposed to approximately 6.5 g/L biobutanol on cellobiose. When syngas was present, C. beijerinckii SA-1 produced about 6.5 g/L biobutanol on sucrose. C. beijerinckii grown on 60 g/L NaOH pretreated, enzymatically hydrolyzed corn stover yielded 11.0 g/L butanol (no syngas present). Separation Linking ABE fermentation and product recovery using experimental data and simulation will enable us to determine the effects of fermentation stream and product stream recycle on the fermentation yield and selectivity. The model will also allow us to identify and optimize feasible separation approaches, with an emphasis on low-energy adsorption and membrane separation processes. Previously fermentations were modeled as steady state (even in batch), relative fixed fractional conversion, irrespective of environmental variables such as substrate concentration, and by neglecting autocatalytic cell production. We have developed a model that uses ASPEN Plus (the industrial standard for the simulation of process unit operations) yet allows for cell growth, dynamic process inputs (substrate concentration, cell biomass), substrate and product inhibition, and recycle streams. Objective C (Technical Area 3): Develop and validate integrated geographic information system (GIS)-based economic and life cycle analysis models to provide strategic guidance to the development of the proposed on-farm processing system. Farm profitability models have been developed for switchgrass and corn stover used as feedstock for bio-butanol production. Areas identified where data needs to be shared between laboratories. LCA model is complete, and will be used to provide numbers for the Comparison of on-farm with a large bio-refinery; comparing machine costs versus "real" economics using custom rates; rerun LCA on different scenarios and different feedstocks.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Sukumara, S., D. Darkwah, and J. Seay. 2015. Process simulations supporting a techno-economic framework to optimize biorefinery supply chains. Computer Aided Chemical Engineering. Accepted February, 2015.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Sukumara, S., J. Amundson, F. Badurdeen, and J. Seay. 2015. A comprehensive techno-economic analysis tool to validate long term viability of emerging biorefining processes. Clean Technologies and Environmental Policy. Accepted March, 2015
• Type: Book Chapters Status: Published Year Published: 2015 Citation: Amundson, J., S. Sukumara, J. Seay, and F. Badurdeen. 2015. Integrated decision support models for design and evaluation of bioenergy supply chains. Book Chapter in "Handbook of Bioenergy". Sandra D. Eksioglu, Steffen Rubennack, and Panos M. Pardalos, Editors. Springer.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Darkway, K, S. Sukumara, B. Knutson, and J. Seay. 2014. Process simulation models linking biomass to biofuel experimental outcomes with supply chain optimization. AIChE Annual Meeting, November, 2014. Atlanta, GA.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Sukumara, Sumesh. 2014. A multidisciplinary techno-economic decision support tool for validating long-term economic viability of biorefining processes. Thesis. Chemical and Materials Engineering, University of Kentucky.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Gunawardhana, Thilina. 2014. Environmental and Economic Feasibility of On-farm Biomass Preprocessing. Department of Biological Systems Engineering, University of Wisconsin.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Mbaneme-Smith, V. and M.S. Chinn. 2015. Consolidated Bioprocessing for Biofuel Production: Recent Advances. Energy and Emission Control Technologies. 3:23-44.
• Type: Other Status: Published Year Published: 2015 Citation: K.R. Caffrey and M.S. Chinn. 2015. Life Cycle Assessment: Description and Methodology. Extension Bulletin AG-795. North Carolina Cooperative Extension.
• Type: Other Status: Published Year Published: 2015 Citation: K.R. Caffrey and M.S. Chinn. 2015. Carbon Accounting: Description and Methodology. Extension Bulletin AG-793. North Carolina Cooperative Extension.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Caffrey, K., M.S. Chinn, M.W. Veal, and M.G. Kay. 2015. Biomass Supply Chain Management in North Carolina (part 2): Biomass Feedstock Logistical Optimization. AIMS Energy-Renewable Energy Systems and Agro-Residue Management. In Review Energy -111.
• Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Caffrey, K., M.S. Chinn, M.W. Veal, and M.G. Kay. 2015. Biomass Supply Chain Management in North Carolina (part 1): Predictive Model for Cropland Converion to Biomass Feedstocks. AIMS Energy-Renewable Energy Systems and Agro-Residue Management. In Review Energy -111.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Caffrey, Kevin. 2014. A Comparison of Alternative Bioenergy Logistical Operations in North Carolina. http://www.lib.ncsu.edu/resolver/1840.16/10219.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Mbaneme, V. and M.S. Chinn. 2015. Conversion of Perennial Bioenergy Crops using Clostridium thermocellum under Synthetic Gas Headspace. Presented at the 2015 ASABE International Meeting held in New Orleans, LA. July 26-30, 2015.

Progress 07/01/13 to 06/30/14

Outputs
Target Audience: Scientific community interested in biomass conversion, post-doctoral scholars assisting with project. Graduate students interested in biomass conversion, and biomass logistics. Undergraduate student workers. Undergraduate students who take BAE 503 Fundamentals of Biorenewable Resources and BAE 504 Biofuels Production and Processing. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project is providing training for 19 undergraduate research assistants (NCSU, UK, and ORNL) in Biosystems and Agricultural Engineering, Chemistry, Microbiology, Agronomy and Chemical Engineering. Fourteen Masters of Science students are working on projects related to this project, at NCSU, University of Wisconsin and UK in the departments of Chemical Engineering, Biosystems and Agricultural Engineering, and Agronomy.Fifteen PhD students are being trained on projects related to this grant, both at NCSU and UK, in the Biosystems and Agricultural Engineering departments, Chemistry, Chemical Engineering, and Plant and Soil Science. Two postoctoral scholars, one at UK in Horticulture, and one at NCSU in Biological and Agricultural Engineering are also receiving training related to the grant. How have the results been disseminated to communities of interest? Our results have been disseminated to the scientific community through conference presentations (16) at venues which include the American Forage and Grassland Council, American Society of Microbiology, Agronomy America-Crop Science Society, American Institute for Chemical Engineering, Institute for Biological Engineering, Clean Technology and Environmental Policy conference, American Society of Agricultural and Biological Engineerins, and the American Society of Material Science Conference on Mass Spectroscopy. We have published ten refereed journal articles this year in a wide variety of journals, including the Journal of Agricutlural and Food Chemistry, Frontiers in Plant Science, BIomass and Bioenergy, Journal of Colloid and Interface Science, Global Change Biology Bioenergy, Biofuels, Bioproducts, and Biorefining, and Bioresource Technology. Three Master's theses and one PhD dissertation were also published and accessible to the scientific community. What do you plan to do during the next reporting period to accomplish the goals? Future Plans Objective A: Testing of the modified baler. Optimization of the ideal transport distance using GIS. Further calibration of TDR sensors will be conducted. A set of moisture-voltage calibration curves will be developed for baled switchgrass. Storage trials will also be conducted in order to validate sensor accuracy with larger bale sizes where more variability is expected. A 3D moisture profile for small rectangular bales will be developed by placing TDR probes throughout bales of switchgrass with the ultimate goal to provide dynamic measurement of the spatial moisture distribution within herbaceous biomass. The resulting data will be converted to moisture data using the previously developed calibration curve. Complete varying cover factor experiments. Determine which parts of the stover are most important to remain on the field while allowing optimal stover removal for further processing. Due to the translational success of moving from Setaria to Sorghum we are well positioned to examine genetic and biotechnological approaches in year 4. Our overarching goal will be to decode a regulatory framework for lignocellulosic biomass biosynthesis in the Sorghum mutant RG and Drawrf1 mutants. These studies will focus on a combination of PCR assisted genetic linkage analysis, characterization of gene function and transcriptional analyses. Continue collecting agronomic data on switchgrass and corn. Objective B Continue testing the effects of: 1) exogenous butyrate and 2) pH need to be tested in the continuous flow system. These variables simulate mobile phase recycle. Product ratios will be manipulated by hydrogenase inhibition. Continue fermentation studies for C. thermocellum and C. beijerinckii investigating the effects of syngas on fermentation properties moving toward perennial grasses as the carbon source and toward a solid substrate cultivation system. Fabricating and testing the lab-scale gasifier and putting it in place to provide gas for fermentation studies Continue investigating the effect of fungal-pretreated biomass on enzymatic hydrolysis and solvent production Systematically evaluate the technical and economical features of various bioprocess scenarios Scale up butanol fermentation from 12 L to 100 L fermentor. Understand the response of metabolic flux in acidogenesis and solventogenesis to different process scenarios, including pretreatment, hydrolysis as well as nutrients supplement. Incorporate an ABE fermentation model, separation, and recycle in an ASPEN simulation of the on farm biomass processing. Preliminary separation units have been developed. Year 4 will focus on the fermentation model, which should be sensitive to glucose concentration and the concentration of solvents and organic acids in the fermentation broth. The simulation model will guide the design of the recycle process. Develop a series adsorption process for the recovery of butanol, ethanol, and acetone based on multiple adsorbent beds. Issues to be addressed are appropriate adsorbents for acetone and balancing high adsorption capacity with ease of recovery. Develop an interfacial model of cellulose thin film hydrolysis and quantify the effect of fermentation process variables and fermentation products on cellulose hydrolysis by purified enzymes and C.thermocellum. The rate of hydrolysis of cellulose in soluble sugars has been identified as a bottleneck in the co-culturing system and success of this research will suggest approaches to increase the rate of sugar production. Objective C: Integrate production data into economic analysis for switchgrass and stover yields: costs, yields, and production practices. Develop model to estimate machinery costs (variable and fixed) for production practices and transportation based on yield for both switchgrass and corn/wheat stover. Develop model to estimate nutrient removal for switchgrass and stover harvests. Estimate mineralization of various soils to determine long-run net nutrient removal (nutrient removal less mineralization) to be included as a long-run cost. Develop an overall model to combine projected revenues of on-farm biomass value and all costs. This model will project returns given various scenarios (range of value for on-farm biomass and changes in costs). We will revise the life cycle assessment model and economic model based on the inputs of the other member groups within the project team. We will use the models to evaluate system improvement designed to make the process more economically viable and sustainable.

Impacts
What was accomplished under these goals? Impact Objective A (Technical Area 1): Develop a reliable biomass feedstock supply system using agricultural residues (corn stover and wheat straw) and energy crops (switchgrass, and miscanthus) with enhanced plant genetics, improved crop management practices to increase yield, reduced environmental impacts, and reduced biomass harvest and transportation costs. Lignocellulosic biomass, even when baled is not dense enough to fully-load a semi-truck. Alternative equipment to preprocess biomass prior to baling was evaluated. The proposed strategy was able to increase bale density by approximately 10%. The system was tested with wheat straw, corn stover, and miscanthus. This equipment redesign will enable biomass to be transported at a reduced cost, enabling lignocellulosic feedstock to be more economically competitive with other more energy-dense feedstocks. Stored lignocellulose is subject to biological degradation if the moisture content of the bales is too high. Large rectangular bales experience significant heat and mass transfer effects when stored in variable climates, such as outdoors or in unheated buildings. Effective quantification of bale moisture content has eluded researchers, and it was unknown whether this was a problem with the sensors or because of inherent variability within a bale. Our experiments have shown the importance of good contact between the sensor and the material. These results will enable us to develop a mechanistically-based heat and mass transfer model for switchgrass bales, which will enable better management decisions in terms of drying and storage of biomass. Energy crop genetic modification research has suffered from not previously having an easily-transformed model C4 plant to study. We have been studying Seteria viridis to potentially fill this role. Our work to date has resulted in our transformed understanding of the mechanism by which the model C4 grass Setaria viridis makes cellulose. We have also successfully mapped a cellulose regulatory feature in Sorghum as well as a lignin modification locus, which we expect will ultimately lead to an energy crop that is more easily hydrolyzed to simple sugars than the current wild types. Objective B (Technical Area 2): Develop the technical and economic feasibility of on-farm storage/processing of high density biomass feedstocks to enhance biomass conversion to value-added products using a fungal biomimetic delignification reaction, and bacterial cellulose hydrolysis followed by bacterial solventogenesis in a modified solid substrate cultivation with recycle. Biomimetic synthetic lignin model compounds were created in our lab and characterized using mass spectrometry (MS) and MS/MS. There are significant structural differences in products resulting from different reaction conditions. During our study of lignin model compounds, we observed significant electrospray response factors for model compounds with a,b- diaryl ether linkages using different ionization conditions. Another study is ongoing to evaluate the effect of chemical pretreatment on switchgrass, wheat straw, corn stover, and miscanthus using calcium hydroxide, potassium hydroxide, and sodium hydroxide at the same hydroxyl concentration. Our motivation is to determine if there is an alternative to sodium hydroxide that would be more compatible with general farm management. The results of this experiment will allow for the evaluation of the optimal alkaline chemical for pretreatment after laboratory experiments are conducted of the enzymatic hydrolysis of the pretreated biomass. Typically enzymatic hydrolysis of lignocellulose involves a cocktail of enzymes, specifically cellulases and β-glucosidases. Enzymatic saccharification of pretreated corn stover was accomplished using only cellulase and the acetone-butanol-ethanol (ABE) fermentation products were equivalent to the treatment using both enzymes. C. beijerinckii was shown to be capable of metabolizing both cellobiose and glucose in the hydrolyzate, thereby saving the cost of β-glucosidases. Pretreated corn stover (PCS) was successfully converted to ABE products using C. beijerinckii without additional nutrients. Pretreated corn stover loadings a 60 g/L (six % (w/v) solids loading of PCS) corresponded to the highest butanol yield of 11.04 g/L with the butanol/ABE yields of 0.18 g/g/0.24 g/g. The maximum overall butanol/ABE yield (i.e., 0.22 g/g corn stover obtained in this work was about twice as high as the overall yield that previously obtained using corn stover (i.e., 0.13g/g corn stover) or any other agricultural lignocellulose of which we are aware. Aspen simulation process models of gas stripping and adsorption were developed using model ABE fermentation product streams as inputs. The experimental adsorption isotherms for ABE fermentation products on activated carbon were used as parameters in the process simulation model. Objective C (Technical Area 3): Develop and validate integrated geographic information system (GIS)-based economic and life cycle analysis models to provide strategic guidance to the development of the proposed on-farm processing system. These models will be used to evaluate different landscape-scale management scenarios and their effect on food and energy production and the environment, including the potential of marginal or abandoned land to be used for biofuel production. Determine the incentives required to increase ecosystem services and biofuel production when they conflict with maximum farm profitability. Implementing the on-farm biomass processing facilities across Kentucky and the US poses a complicated logistical problem. Geographic Information Systems (GIS) was used to combine soil data, yield, crop area planted, actual road networks, county data, and current refinery locations to ascertain the overall impact of differing transportation cutoffs (for biomass transport to butanol bunker processing facilities. The process of combining all of this data for the analysis was automated using the Model Builder application within GIS and solved using Location-allocation feature to minimize the potential number of facilities for specified transport cutoff distances within the state. For instance, increasing the cutoff distance from 5 to 10 miles for the transport between the farm gate to bunker collection point would result in 71-77% fewer bunker facilities being required. Nonetheless, this increased transport distance resulted in the associated fuel cost per ton of biomass being elevated across the state by 100-120%. The relationship between using routing and direct deposit of the crude biobutanol has also been investigated. Depending upon the number of facilities, routing can increase the cost of transport by 5-125%. The development of the Life Cycle Assessment model (LCA) which includes for production of crop residue and energy crop feedstocks, inventory data for densification and on-farm biomass pretreatment and storage technologies and populated the LCA inventory model with preliminary data. We also developed a techno-economic analysis (TEA) to evaluate the economic performance using the same process description, system assumptions, and parameters as used in the LCA model. A sensitivity analysis has been performed to identify the bottlenecks and pinch-points in the process. Using the LCA and TEA, the cost of production for biobutanol is estimated to be $1.92/gallon under the current processing assumptions.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: January 13, 2014 Presented paper at the American Forage and Grassland Council annual meeting in Memphis , TN on completed tissue analysis research
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: M.D. Flythe, N.M. El�a, S.E. Nokes. Clostridium thermocellum as a biological pretreatment for switchgrass (Panicum virgatum) fermentation by Clostridium saccharoperbutylacetonicum in submerged and continuous-flow solid-substrate fermentation. American Society for Microbiology 114th General Meeting, Boston, MA. May 20, 2014
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: N.M. El�a, M.D. Flythe, M.B. Schmal, S.E. Nokes. Switchgrass (Panicum virgatum) fermentation by sequential culture of Clostridium thermocellum and Clostridium beijerinckii: effect of particle size on gas production American Society for Microbiology 113th General Meeting, Denver, CO. May 21, 2013
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Graduate Student, Martin Battaglia, attended the Agronomy Society of America-Crop Science Society of America and Soil Science Society of America International Meetings and presented results of his thesis.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: ] W. Yao , Sue E. Nokes, Michael D. Flythe, Barbara L. Knutson, Bert C. Lynn, Stephen E. Rankin, Mike Montross. 2013. Improvement of biofuel production by fungal pretreatment and periodic flushing system. American Institute of Chemical Engineers Annual Conference , San Francisco, CA, Nov 3-8, 2013.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: ] W. Yao , Sue E. Nokes, Michael D. Flythe, Barbara L. Knutson, Bert C. Lynn, Stephen E. Rankin, Mike Montross. 2014. Improvement of biomass conversion by periodic flushing system. Institute of Biological Engineering 2014 Annual Conference, Lexington, KY, March 6-8, 2014.
• Type: Journal Articles Status: Submitted Year Published: 2014 Citation: Sukumara, S., W. Faulkner, J. Amundson, F. Badurdeen and J. Seay (2014): A Comprehensive Techno-Economic Analysis Tool to Validate Long Term Viability of Emerging Biorefining Processes, Clean Technologies and Environmental Policy. Invited Paper Submitted April 2014.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: K. Caffrey, M. Veal, M. Chinn, M. Poore, M. Kay. 2014. Biomass storage characteristics of bales and ensilage: Switchgrass (Panicum virgatum), Miscanthus (Miscanthus giganteous), Giant Reed (Arundo donax), and Sweet Sorghum (Sorghum bicolor). Presented at the International Meeting of ASABE. July, 2014, Montreal Canada.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: K. Caffrey, M. Chinn, E. Godfrey III, M. Veal. 2014. Determination of Pellet Quality Parameters from Stored Perennial Grass Bales: Switchgrass (Panicum virgatum), Miscanthus (Miscanthus giganteous), and Giant Reed (Arundo donax). Presented at the International Meeting of ASABE. July, 2014, Montreal Canada.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: V. Mbaneme, M. Chinn, M. Veal and J. Bruno-Barcena. 2014. Influence of Synthesis Gas on Cellulose Conversion using Clostridium Thermocellum in a Consolidated Bioprocessing System. Presented at the International Meeting of ASABE. July, 2014, Montreal Canada.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Petti C�, Harman-Ware AE, Shearer A+, Tateno M�, Downie AB, Crocker M, and DeBolt S (2013) Sorghum mutant RG displays antithetic leaf shoot lignin accumulation resulting in improved stem saccharification properties Biotechnology for Biofuels 6. 146.
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Petti C�, Kushwaha R�, Tateno M�, Stork J�, Harman-Ware AE, Crocker M, Awika J and DeBolt S* (2014) Mutagenesis breeding for increased 3-deoxyanthocyanidin accumulation in leaves of Sorghum bicolor (L.) Moench: a source of natural food pigment Journal of Agricultural and Food Chemistry
• Type: Journal Articles Status: Under Review Year Published: 2014 Citation: . Petti C, DeBolt S (2014) Gibberellic acid regulates expansion via cellulose biosynthesis in the C4 grain crop Sorghum bicolor (L.) Moench (in review)
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: A Potential Alternate Synthesis of Thioacidylosis , Dawn Kato and Bert C. Lynn presented at the 62nd ASMS Conference on Mass Spectrometry and Allied Topics, Baltimore, MD June 15-19, 2014.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Elucidation of Synthetic Lignin Oligomers by Tandem Mass Spectrometry, Fan Huang and Bert C. Lynn presented at the 62nd ASMS Conference on Mass Spectrometry and Allied Topics, Baltimore, MD June 15-19, 2014
• Type: Journal Articles Status: Published Year Published: 2014 Citation: W. Yao, S. Nokes. 2014. Phanerochaete chrysosporium pretreatment of biomass to enhance solvent production using solid-substrate Cultivation. Biomass and Bioenergy. 62,100-107.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: W. Yao, S. Nokes. 2014. First Proof of Concept of Sustainable Metabolite Production from High Solids Fermentation of Lignocellulosic Biomass using a Bacterial Co-culture and Cycling Flush System. Bioresource Technology.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Modenbach, A., Nokes, S. Effects of solids loadings in sodium hydroxide pretreatment and enzymatic hydrolysis of corn stover. Presented at the IBE Annual Meeting, Lexington, KY. March 2014.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Modenbach, A., Nokes, S. Towards improving hydrolysis of sodium hydroxide pretreated corn stover at high-solids loadings. Presented at the ASABE Annual International Meeting, Kansas City, MO. July 2013.
• Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Gray, K., Nokes, S., Montross, M., Modenbach, A., Jackson, J. Investigation of alkaline hydrogen peroxide pretreatment for its use in an on-farm butanol bioprocessing facility. Poster presented at the ASABE Annual International Meeting, Kansas City, MO. July 2013
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Caffrey, Kevin R. and Veal, Matthew W., 2013. Conducting and Agricultural Life Cycle Assessment: Challenges and Perspectives. The Scientific World Journal. Volume 2013. Article ID 472431, 13 pages. doi 10.1155/2013/472431.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Joshi, Suvid, Rao, Alexander, Lehmler, Hans-Joachim, Knutson, Barbara L., and Rankin, Stephen E. 2014. Interfacial molecular imprinting of Stober particle surfaces: A simple approach to targeted saccharide adsorption. Journal of Colloid and Interface Science. 428(2014): 101-110.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Modenbach, Alicia A., Nokes, Sue E. 2013. Enzymatic hydrolysis of biomass at high-solids loadings - A review. Biomass and Bioenergy. 56(2013): 1-19.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Montross, Michael D., DeBolt, Seth, and Adams, William C. 2013. Interplay between yield, nitrogen application, and logistics on the potential energetic and greenhouse gass emissions from biomass crops. Global Chang Biology (GCB) Bioenergy. 5:664-673.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Sukumara, Sumesh, Faulkner, William, Amundson, Joseph, Badurdeen, Fazleena, and Seay, Jeffrey. 2013. A multidisciplinary decision support tool for evaluating multiple biorefinery conversion technologies and supply chain performance. Clean Techn Environ Policy. doi: 10.1007/s10098-013-0703-6.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yao, Wanying, and Nokes, Sue E. 2013. The use of co-culturing in solid substrate cultivation and possible solutions to scientific challenges. Biofuels Bioproducts & Biorefining. 7:361-372.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Modenbach, Alicia Abadie. 2013. Sodium hydroxide pretreatment of corn stover and subsequent enzymatic hydrolysis: An investigation of yields, kinetic modeling and glucose recovery. University of Kentucky.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Elia, Noelia M. 2014. Sequential co-culture of anaerobic bacteria on switchgrass in a continuous flow-through reactor for biofuel production. University of Kentucky.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Gray, Mary Kathryn. 2013. Alkaline hydrogen peroxide pretreatment for its use in an on-farm bioprocessing facility. University of Kentucky.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Wooten, Mary K. 2014. Nanofiltration membranes from oriented mesopourous silica thin films. University of Kentucky.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Petti C�, Shearer A+, Tateno M�, Ruwaya M, Nokes S, Brutnell T and DeBolt S* (2013) Comparative feedstock analysis using Setaria viridis L. as a model C4 bioenergy grass and Panicoid crop species Front. Plant Sci. 4:181.

Progress 07/01/12 to 06/30/13

Outputs
Target Audience: Scientific community interested in biomass conversion, post-doctoral scholars assisting with project. Graduate students interested in biomass conversion, and biomass logistics. Undergraduate student workers. Undergraduate studentw who take BAE 503 Fundamentals of Biorenewable Resources and BAE 504 Biofuels Production and Processing. Attendees at the 2012 Farm Machinery Show; Business School Business Plan Team; Changes/Problems: The major reason for changes in the schedule were sub-contract award delays. The delay in receiving money has delayed many studies; likely resulting in the request for a one-year no-cost extension at the end of the project. Under Objective A, single pass baling was abandoned becasue, while the baler worked well, it was too difficult to time the grain harvest so that the biomass was at the proper moisture content for safe storage. We are now focusing on baling windrowed stover, straw, and energy crops. What opportunities for training and professional development has the project provided? The project is providing the opportunity to train four Master of Science students, two in Biosystems and Agricultural Engineering, and one in Chemical and Materials Engineering. The project is also providing the training for six PhD students; Three Chemical and Materials Engineers, one Biosystems and Agricultural Engineer, one Chemistry student, and one Horticultural student. In addition, oneMaster of Sciencestudent comleted her degree this year, and one PhD student, both in Biosystems Engineering. The project is also providing training for two post-doctoral scholars in Biosystems Engineering and one post-doc in Horticulture. At the University of Wisconsin, one graduate student is working on the Life Cycle Analysis, and at NCSU, one graduate student is working on conversion and one on logistics. How have the results been disseminated to communities of interest? August2012. Oral presentation.Yao, W. and Nokes, S.E. Phanerochaete chrysosporium pretreatment of biomass to enhance solvent production using solid-substrate cultivation. at the 2013 ASABE meeting. Dallas TX July 29-Aug1, 2012. August, 2012. Poster presentation. Yao, W. and S.E. Nokes. Phanerochaete chrysosporium pretreatment to enhance solvent production using solid-substrate cultivation. Annual meeting of S-1041: The Science and Engineering for a Biobased Industry and Economy, Washington, DC. Aug 6-8, 2012. September 22, 2012 - presented switchrass research at the Quicksand Field Day in eastern KY (Smith) October, 2012 - Poster presentation. Yao, W. and S.E. Nokes. Performance of bacterial co-culture for metabolite production using various cultivation modes. National Conference of Sun Grant Initiative - Science for Biomass Feedstock Production and Utilization. New Orleans, LA. October 18, 2012 - showed a group of farmers the processing options for switchgrass and potential alternative used at a field day in Braken County (Smith) November 2012 - R. Garlapalli, B.L. Knutson, S.E. Nokes, and S.E. Rankin. Role of Tween-80 in Reduction of Nonproductive Cellulase Binding to Lignin. American Institute of Chemical Engineers Annual Meeting. Pittsburgh, PA. November 2012 - B.L. Knutson, R. Garlapalli and S.E. Rankin. Concentration Effects in Cellulose Thin Film Hydrolysis by Cellulase Enzymes - a QCM Study. American Institute of Chemical Engineers Annual Meeting, Pittsburgh, PA. November 2012 - Sumesh Sukumara and Jeff Seay. Multidisciplinary Optimization Model for Region Specific Sustainable Biorefining. American Institute of Chemical Engineers Annual Meeting, Pittsburgh, PA. February, 2013 - 3-D model of the proposed on-farm biomass processing system was on display at the National Farm Machinery show, Lousville, KY. May 8, 2013 - Presented the switchgrass research to the UK Forage Workersp; an interest group from the UK Colege of Agricutlure . June 9-13, 2013. Kato, Dawn and Bert C. Lynn. Development of Capillary Electrophoresis-Mass Spectrometry for Analysis of Fenton Chemistry Biomass Pretreatment for Biofuels Productionpresented at the 61st ASMS Conference on Mass spectrometry and Allied Topics. Minneapolis, MN. June 25, 2013 - preliminary results were presented to a switchgrass field day in Braken Couny June, 2013. ASM: American Society for Microbiology 113th general meeting. Noeli Elia, Michael Flythe, Micah Schmal, and Gloria Gellin. Poster Presentation. Switchgrass (Panicum virgatum) fermentation by sequential culture of Clostridium thermocellum and Clostridium beijerinckii. July, 2013. Poster presentation at the 2013 ASABE meeting in Kansas City comparing NaOH and Alkaline Hydrogen Peroxide pretreatments. Kathryn Gray et. al. What do you plan to do during the next reporting period to accomplish the goals? A.2 Energy crop development and lignocellulose The current plan for year 3 is to optimize the Setaria transformation protocol for the introduction of RNAi constructs targeting lignin biosynthesis genes. Our targets are phenylpropanoid pathway components caffeic acid o-methyltransferase (COMT) as well as the cinnamyl alcohol dehydrogenase (CAD) genes. The goal of the RNAi lines are to silence or knockout the genes in order to observe the effect on lignin biosynthesis and the digestibility of the resulting transgenic Setaria. By using RNA1, we will knock down rather than knock out the transcripts and in turn we will recover a range of ‘knock down’ transcript loads in the T1 rescues (aiming between 0-90%). These studies will provide a rational basis for the degree to which lignin biosynthesis can be knocked down (suppressed), before plant performance is severely impacted in the Panicoideae model. We anticipate that this goal may carry over into year four, due to the transformation protocols remaining challenging. During this time, ethyl methane sulfonate chemically mutagenized populations of Setaria are being generated that will be screened for aberrant lignification and cell wall defects. These will provide a critical tool for forward genetics studies into the genes underscoring biomass traits. A.3 Establishment of best management practices One field study in Hardin County and two sites at Spindletop farm in Lexington wer established spring, 2013. All three sites as no-tillabe, and 2 of the 3 sites are on sloping fields that are likely to experience drought-like conditions in July and August. The second location in Lexington is irrigated. B.1 Pretreatment Establish the sterilization capabilities of AHP. Develop detailed mass sectrometry based lignin assays to evaluate Fenton chemistry. B.2 Fermentation SSC with continuous media flow will be conducted with C. beijerinckii testing three flowrates while continuously removing end products. Use proteomics and mass spectrometry to understand protein expression changes in C. beijerinkii during solventogenesis. Develop analytical methods to support process development. Use Clostridium cellulolyticum instead of Clostridium thermocellum for cellulose hydrolysis to perform simultaneous saccharification and fermentation. Simulate the repeated flusing process in the bale scale. Scale-up butanol fermentation from 5L to 14L to 100 L using glucose or biomass. B.3 Adsorption studies of fermentaitno products on commercial adsorbents will be interpreted in terms of species selectivity (butanol:ethanol for example) to design a parallel series of adsorption columns appropriate for butanol recovery. Commercial and custom-synthesized adsorbents will be compared. Fermentation samples "spiked" with butanol will be used to examine the robustness of the system to butanol concentration. Desorption (recovery) of the fermentation product will be explored. The separating of delignification products on adsorbents, potentially potent inhibitors of enzymatic hydrollyis and fermentation, will be examined using hydrophobic classes of adsorbents. The level of inhibition on enzymatic hydrolysis posed by the byproducts of delignification treatments and by the accumulation of fermentation products in the recycle reactor will be quantified using the cellulose thin film (QCM) approach developed in Year 2. Separation techniques based on membraneswill be investigated. C. 1 Economics Include conversion, separation and product concentration in the AspenPlus simulation model. Simulate scenerios to reduce fresh water consummption by recycling waste water. Estimate the energy that can be produced by the residual lignin. Integrate the heat produced from residual lignin into the process simulation model. C.2 Life Cycle Analysis During Year 3 we will develop LCA inventory data for production of cropp resiude and energy crop feedstocks, develop inventory data for densification and on-farm biomass pretreatment and storage technologies, and populate the LCAinventory model with preliminary data. We will use the preliminary LCA to perform sensitivity analyses for the system, which will allow us to identify where the most accurate information is required, and more importantly, the pinch points in the process where performance improvements can improve overall environmental performance of the system. These results will be fed back to the team for developing the year 4 plan of work.

Impacts
What was accomplished under these goals? Harvesting system: For 30% moisture content straw, we increased dry bulk density from 129 to 140 kg/m3 in round bales. In rectangular bales, the dry bulk density increased from 140 to 145 kg/m3. However, there were problems with the control system on the large square baler that limited the pressure we were able to apply. The primary reasonsfor the lower than desired density increases observedwas likely the inconsistent feed rate into the baler between treatments and poor windrow formation. Studies quantifying changes in bale quality during storage are on-going, both in Kentucky and North Carolina. Energy crop development and lignocellulose: The lignocellulosic feedstock composition, cellulose biosynthesis inhibitor (CBI) response and saccharification dynamics of Setaria were compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A (CesA) genes was performed identifying eight candidate sequences. Two-developmental stages, metabolically active young tissue and metabolically plateaued (mature) material were examined and were found to have similar composition to that of sorghum, maize and switchgrass. Best management practices for energy crops: Historical Kentucky agronomic data 2007-2010 which will be used to inform the modelling in objective C. Field soil samples were taken from inside the switchgrass field and from adjacent undisturbed fields to allow comparisons of nutrient and organic matter changes over time. Tissue analysis from switchgrass fertility research was initiated. A series of experiments were initiated in Fayette and Campbell county that include; 1)nitrogen fertilization rates, 2) cutting height in a dual use forage and biomass system, 3) variable rate and timing nitrogen application, and 4) field burning. Research on weed control with herbicides was also initiated and will be expanded in 2014. Corn agronomic studies were also conducted in 2012 at a location in Hardin County, Kentucky and two locations in Lexington, KY. Both the Hardin and Lex 1 site are on eroded soils more prone to drought conditions, while Lex 2 is on a deeper soil less prone to drought. Data on soil fertility, stand counts, growth stage, leaf and soil temperatures, light interception determination, maximum intercepted photosynthetic active radiation, water stress assessment, plant height, and lodging were recorded, as well as corn grain yields. On-Farm Delignification: A study was conducted to compare the white rot fungus Phanerchaete chrysosporium (WRT) with brown rot fungi (BRT) to determine if brown rot fungi efficiently degraded lignin with less loss of cellulose than WRT. WRT exhibited much faster growth and more efficient lignin degradation than BRT on corn stover. In solid substrate cultivation with 5.7% corn stover loading, the treatment pretreated with WRT corresponded to higher yields of solvents and acids in co-culture than those pretreated by BRT. Alkaline hydrogen peroxide (AHP) is a pre-existing method of delignification that potentially provides several advantages in the on-farm high solids environment. At the alkaline pH, powerful hydroxyl radicals are formed; these radicals then attack the lignin structure. AHP removed substantial lignin for four feedstocks (corn stover, wheat straw, switchgrass and miscanthus). Fenton chemistry stoichiometry was evaluated using peroxide consumption as an endpoint and this resulted in the following optimum: 10 g dry biomass, 200 mL of 6% H2O2 and 200 mL 500 mg FeCl2 4H2O (aq). The results support the hypothesis that Fenton chemistry modifies lignin structure making cellulose more bio-available but does not significantly convert lignin into smaller oxidized organic products. Techniques to synthesize lignin thin films, for use in QCM measurements were developed. These thin films will be used to examine the kinetics of pretreatment methods and the effect of pretreatment on the nonspecific binding of cellulases to lignin. On-Farm Production of Biobased Chemicals: Repeated flushing was carried out in 2.5 cm I.D. and 10 cm length glass columns fitted with porous polyethylene discs at the bottom which allowed media to flow through yet retained the solid substrate. The columns were loaded with 5 g of miscanthus (particle size of 5mm) and inoculated with 2 ml of P. chrysosoporium to remove the lignin. After a 5-day fungal pretreatment at 35 oC, the fermentation of Clostridium thermocellum (C.t) ATCC 27405 was held at 65 oC for 2 days to hydrolyze cellulose to sugars. Then the sequential co culture was initiated by inoculating C. beirinjerckii (C.b) ATCC 51743 and held for 2 days for acids and solvents production. C.thermocellum could be maintained alive throughout sequential co culture at 35-37 oC and re-incubation of C.thermocellum at 65 oC increased the availability of glucose by 2.8-4 fold. Re-inoculation of C.thermocellum did not actively improve the sequential co culture. Re-inoculating C. beijerinckii after reincubating C. thermocellum corresponded to the highest amount of total acids (400.43 mM) and total solvents (152.6 mM). Glucose is the limiting factor that determines the metabolite production of the co-culture system. A screening technique for the inhibition of celluose hydrolysis was developed using the mass change of cellulose films in the presence of cellulases, as measured by a quartz crystal microbalance (QCM). In this technique, both the adsortion of the cellulases (increase in film mass) and the hydrolysis of the cellulose thin film (reduction in film mass) can be monitored in real time, allowing for mechanistic modeling. This thin film aproach to measuring inhibition was extended from fungal cellulases to whole cells (C. thermocellum) cellulases for the first time using cellobiose as the inhibitor. C.beijerinckii 824 has the ability to ferment both C5 and C6 sugars for butanol production, which is an added advantage over many other cultures and may improve butanol production in the solventogenesis phase after hydrolysis. We produced as high as 20.8 g/L and 12.33 g/L butanol using 60 g/L glucose and 60 g/L xylose respectively. Approximately 13.8 to 14.2 g/L butanol were produced using 30 g/L corn stover which was hydrolyzed by cellulase and cellobiase. On-Farm Product Recovery/Product Upgrading: The major classes of adsorbents proposed for the recovery of butanol in fermentation processes were identified from literature; zeolites, ion exchange resins, and activated carbon. However literature studies of adsorbents for fermentation products are limited to simple binary or ternary mixtures, with little indication of how adsorption will be altered by complex fermentation mixtures. The ability of zeolite, ion exchange resin (Amberlite), and activated carbon to adsorb fermentation products wastested using fermentation broths produced in the laboratory. Adsorption of glucose, cellobiose, butanol, ethanol, acetone, lactic acid and acetic acid in these mixtures was analyzed. While all the adsorbents were suitable for butanol, each adsorbent offered its own advantages with respect to its selectivity relative to other compounds. Custom silica adsorbents were synthesized, with a goal of controlling the surface functionality of the adsorbents and increasing the specificity of the adsorbents. Profitability, System Modeling and Life Cycle Analysis: The scope of the study is on-farm, from field to farm gate and current time. Current effort is proceeding with focus on analysis of the feedstock supply system, where the intermediate functional unit is kg of crop material on a dry basis. A set of LCA impact categories and performance metrics to be used to assess the on-farm biomass processing system have been identified. The overall process diagram representing the system with all pathways represented is complete.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yao, W. and S.E. Nokes. 2013. The use of co-culturing in solid substrate cultivation and possible solutions to scientific challenges. Biofuels, Bioproducts, and Biorefining. Vol 7(4):361-372.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Petti,C., Shearer, A., Tateno, M., Ruwaya, R., Nokes, S., Brutnel, T. and S. DeBolt. 2013. Comparative feedstock analysis in Setaria viridis L. as a model for C4 bioenergy grassed and panicoid crop species. frontiers in Plant Science. Volume 4 Article 181. 14 p.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Modenbach, A.A., Nokes, S.E. 2013. Enzymatic hydrolysis of biomass at high-solids loadings  A review. Biomass and Bioenergy. 56: 526-544.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Modenbach, A.A. Sodium Hydroxide Pretreatment of Corn Stover and Subsequent Enzymatic Hydrolysis: An Investigation of Yields, Kinetic Modeling and Glucose Recovery. Dissertation. 2013. University of Kentucky, Lexington, KY
• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Montross, M.D., S. DeBolt, and W.C. Adams. 2013. Interplay between yield, nitrogen application, and logistics on the potential energetic and green house gas emissions from biomass crops. GCB Bioenergy. In press.
• Type: Journal Articles Status: Submitted Year Published: 2013 Citation: Yao, W., and S.E. Nokes. Phanerochaete chrysosporium pretreatment of biomass to enhance solvent production using solid-substrate cultivation. Biomass and Bioenergy. Submitted.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Gray, Mary Kathryn. 2013. Alkaline hydrogen peroxide pretreatment for its use in an on-farm bioprocessing facility.

Progress 07/01/11 to 06/30/12

Outputs
OUTPUTS: Demonstrating proof of concept for the innovative and high risk aspects of the proposed approach to on-farm biomass processing was the primary focus of Year One. This approach to the conversion of biomass in a flushed solid substrate cultivation with recycle exhibits clear advantages at the bench scale, and is envisioned as our on-farm storage/processing model. Finally, the framework is prepared for integrating these innovations into the larger goals of the grant, with a focus on harvesting/processing integration, scale up, and analysis of economics and sustainability, with broad participation of all constituents. Our initial results demonstrate the sensitive detection of model lignin degradation compounds when the analytes were infused through a capillary using inlet ionization with voltage into the MS at nanoliter flowrates. These infusion experiments mimic CE flowrates, simulating the hydrodynamics of coupled CE-MS. The analytes are detected, but their separation and detection sensitivity have not been optimized. Broad participation, interest, and feedback from the general public, farmers, manufacturers, equipment dealers, and refiners has been accomplished through outreach activities. Future scientists in this interdisciplinary research area (high school, undergraduate, and graduate students) are being trained. Economic and environmental aspects of this project have been used as case studies in several courses and student group projects. The project has been highlighted at a number of events including meeting with the Kentucky Farm Bureau and Kentucky Legislature. The project occupied an 800 ft2 booth at the National Farm Machinery where a model of the on-farm biomass processing facility was displayed Louisville, KY in February 2012. Approximately 300,000 visitors attend each year from across the world. We estimate that approximately 3,000 people visited the booth and 1,000 asked follow up questions and requested additional information related to the project. There was considerable interest in the project and how agriculture would integrate with renewable energy production. Over 500 copies of extension articles summarizing the proposed project were distributed at the Machinery Show in addition to the people who visited the project website to download the materials. A number of in-class assignments were based on this project. Seniors in Biosystems Engineering calculated the energy lost from the bunker and the several graduate students worked on LCAs of segments of the project. We work closely with CNH America, but we have also received input from state government officials, an oil refiner, and agricultural business leaders (corn ethanol plants, fertilizer manufacturers, and crop consultants) on the potential impact of the project. PARTICIPANTS: Nokes, Sue, PD Montross, Michael, PD Knutson, Barbara, PI Rankin, Stephen, PI Lynn, Bert, PI Debolt, Seth, PI Partner Organizations USDA ARS FAPU, Flythe, Michael, PI North Carolina State University, Chinn, Mari, PI North Carolina State University, Veal, Matt, PI CNH America, Collaborator Training or professional development Yao, Wanying, Post-doctoral fellow Gray, Mary Kathryn, Master of Science student Ruwaya, Mathew, Master of Science student and technician Adams, William, Master of Science student and technician Jackson, Joshua, PhD student TARGET AUDIENCES: Target Audiences Undergraduates and graduate students curriculum development Farmers demonstrations information exchange PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The preliminary results for switchgrass and Miscanthus indicated material could be processed in a manner to reduce compression pressures required. This would translate to less energy required to drive the plunger and less energy being stored in the baled material to produce rebounding forces. Hydraulic conductivities through bales have been quantified and have been shown to be a function of bale density. These values indicate that bale inoculation and product recovery in a flushed bioreactor is feasible, and can be tailored by creating bales of strategic densities using our baling system. Two approaches to delignification were established, the biomimetic Fenton chemistry and fungal pretreatment. Both approaches resulted in fermentable biomass with increased cellulose accessibility. Preliminary data suggest that the fungal pretreatment delignifies whereas the Fenton chemistry likely modifies the structure but does not remove lignin. The two pretreatments therefore present two very different approaches in terms of the subsequent effluents and the LCA. Enzymatic saccharification results showed the treated 5 mm miscanthus, switchgrass and corn stover feedstocks provided a 198%, 172% and 162% increase relative to the untreated feedstocks, respectively. However, acid-insoluble lignin assays showed no change in the amount of acid-insoluble lignin relative to the untreated feedstocks. This suggests that the Fenton pretreatment increased cellulose bioavailability, but did not delignify the lignocellulose. Pretreated lignocellulose produced solvents and mixed organic acids in high solids reactors which were thermally cycled between co-cultivation conditions favoring bacterial cellulase production and those favoring fermentation. Intermittent removal of products by flushing improved the yield of the fermentation products. Endogenous gas production and the energy from burning spent biomass are sufficient to allow for a self-sustaining co-culturing process. The gas produced is sufficient to take the system anaerobic, and we have demonstrated production even in the presence of small amounts of oxygen. Additionally, roughly 1/10th of the spent biomass will be needed to supply the energy required to keep the insulated bunker at the desired temperatures, leaving spent biomass to provide energy for other on-farm processes.

Publications

• Modenbach, A. A. and S.E. Nokes. 2012. The use of high solids loadings in biomass pretreatment, a review. Biotechnology and Bioengineering. Volume 109, Issue 6, June 2012, Pages 1430 through 1442