2005 OBP Biennial Peer Review Processing Integration Dan Schell Biochemical Platform November 15, 2005

Project Goals and Objectives Investigate integrated processing to reduce risk of industry-led efforts to commercialize biomass refining technology Improve understanding of process chemistry and integrated performance Identify problems and showstopper issues Develop integrated testing methods, tools, and capabilities

Overview Budget USDA/Universities Subcontracts Baylor University Hauser Laboratories Hazen Research Partners Barriers Process integration Pathways Agricultural Residues Energy Crops Work Objective

Platform Fit with Pathways Program Outputs Feedstock R&D Sugars R&D Thermochemical R&D Products R&D (from) Residual Starch Conversion Fiber Conversion Systems-level demonstration and validation by 2009 Corn Wet Mill Improvements (Corn) Element Strategic Goals Corn Dry Mill Improvements (Corn, Grain) Agricultural Residue Processing (Corn Stover, Wheat Straw, Rice Straw) Biomass Fractionation Sugars Production Sustainably supply biomass to biorefineries Low-cost sugars from lignocellulosic biomass Biomass Program Strategic Goal Cost-competitive biorefinery technologies for the nation’s transportation, chemical and power industries Integrated Biorefineries Residual Starch Conversion Fiber Conversion Milled Grain Fractionation Energy Crops (Perennial Grasses, Woody Crops) Pulp and Paper Mill Improvements (Mill Wastes, Wood) New Fractionation Process for hemiicellulose removal Chemical building blocks from lignocellulosic biomass Fuels, chemicals and power from bio-based sugars and chemical building blocks Systems-level demonstration and validation by 2012 Systems-level demonstration and validation by TBD Systems-level demonstration and validation by 2010 Biomass Fractionation Sugars Production

Approach Focus on integrated performance testing using a model feedstock (corn stover) and a baseline process based on thermochemical dilute acid pretreatment followed by enzymatic cellulose hydrolysis Directly addresses process integration barrier Working to understand current performance and demonstrate progress towards the sugar platform cost target, while improving integrated testing capabilities and identifying showstopper issues Measure performance relative to technical targets established by economic analysis

Work Breakdown Structure NREL Academia Industry Earmark

Project Structure Processing Integration Feedstock Variability Analytical Methods Integrated Processing  Test integrated performance  High solids operation  Assess advanced enzymes  Develop and improve methods  Distribute methods  Understand breath and impacts of feedstock variability

Barriers Commercial Success Barriers Price of Sugars from “Cellulosic” Biomass Major General Barriers Feedstock Cost Sugars Composition Sugars Yield Conversion Rate Sugars Quality Capital Investment R&D Technical Barriers Feedstock-Sugars Interface Biomass Pretreatment Enzymatic Hydrolysis Sugars Processing Process Integration

Feedstock Variability Stover Compositional Database Comparison of commercial (FY02) and non-commercial (FY05) corn hybrids The addition of non-commercial hybrids in the sample set has expanded the range of cellulose and xylan compositions

Feedstock Variability Stover Compositional Database Lignin content varies only modestly between commercial and non-commercial hybrids Further data mining is necessary to answer questions such as, “How does carbohydrate content correlate with lignin content and/or other components?” We believe the corn stover database now captures the extent of feedstock compositional variability No additional survey work is planned for corn stover

Understanding Risks Composition used in 2002 Design Report

Analytical Methods Identifying the Problems CelluloseXylanLignin Extractives Other Hemi. Uronic Acid Acetyl Ash Protein Sucrose Corn Stover 6.6% 60.3% 30.7% 3.6% 1.9% 2.4% Pretreated Corn Stover Solids Liquor Furfural Other Xylose Glucose Pretreatment

Improving HPLC-Based Sugar Analysis Implementing Solutions Improved baseline resolution produces more accurate and reproducible compositional data Still need reliable method to measure fructose in hydrolysates BioRad HPX-87P, RI Detector Shodex SP-0810, RI Detector New Old BioRad HPX-87P, RI Detector

Improving Lignin Analysis Current wet chemical methods for lignin determination Behavior-based definition: Lignin = Acid Insoluble Residue Valid assumption for wood Invalid for agricultural residues and herbaceous materials Interferences from protein, carbohydrate degradation products, extractives, and silica Unacceptably high error, ± ~25% Application of method gives inaccurate mass closures Poor correlation with spectra limits ability to develop reliable rapid analysis method

Strategies for Improving Lignin Analysis Investigate alternative analytical methods for measuring lignin such as those now used in the food sciences Improve understanding of the fate of protein and extractives and their effects on lignin measurements Develop functional group-based lignin determination Use this new information to develop more accurate spectroscopic-based rapid analysis methods for lignin

Understanding Extractives Subcontract issued to Baylor University (work began in May 2005) Goal Identify 90% or more of the extractives and develop analytical methods for measuring the concentrations of extractive components  Progress Have identified several major constituents that comprise most of the extractives Currently developing analytical methods for these materials Hour in Water Extraction Dry Weight (%) UnknownSucroseAshProtein

Improving Rapid Analysis Methods Real-time methods are needed to support commercial biorefinery operation Process control and optimization Improved efficiency for in-house and CRADA research projects Reduces cost Increases number of samples/experiments that can be run

On-line Testing and Validation Optics over conveyor weigh belt Spectrometer Controls Direct light spectrometer installed in pilot plant

Moving Toward Deployment Methods have been transferred to Vision ® Software Provides a more flexible and robust platform for using these methods on other instruments

Drive Towards High Solids Operation Pretreatment High solids operation significantly increases sugar concentrations Pretreatment is possible at 30% solids loading without significant yield loss Still need to improve hemicellulosic sugar yields Significant reduction in minimum ethanol selling price (MESP) can be realized through reduced operating and capital costs

Drive Towards High Solids Operation Enzymatic Cellulose Hydrolysis Inhibition by sugars and mass transfer limitations become important issues at high solid concentrations Further cost reductions are possible with high solids enzymatic cellulose saccharification Genencor Spezyme (40 mg/g)

Understanding Process Relevant Performance Recycle Water Studies Performance is significantly affected at modest solids concentrations and recycle water ratios Inhibitors besides acetic acid are responsible for poor performance Hydrolysate liquor conditioned by overliming and then fermented using xylose-utilizing Z. mobilis

Understanding Process Relevant Performance Integrated Performance Testing Partially washed solids recombined with liquor conditioned by overliming  Ethanol yields are low due to unutilized glucose (25% left as mono- and oligo-glucose) and incomplete xylose utilization  Integrated processing illustrates limitations of current ethanologens Cellulose hydrolysis with Genencor Speyzme (40 mg/g, 45  C, pH 4.8) and fermentation with Z. mobilis (35  C, pH 5.0)

Performance Summary

Interim Stage Gate Overview Processing Integration Task Interim Stage Gate Review Stage B project Meeting held September 15, 2004 Reviewers: Rob Anex (Iowa State University) Susan Hennessey (Dupont) Dale Monceaux (Katzen International) Quang Nguyen (Abengoa Bioenergy) Amy Miranda (DOE OBP) Jim Spaeth (DOE GO) Stan Bower (NREL)

Review Meeting Feedback Feedback received in four categories Large view issues Feedstock variability Analytical methods Integrated processing

Large View Issues The answer to many questions raised at the meeting was that the activity resides in a different part of the Biomass Program. However, systems solutions are required and fragmentation of the overall effort into manageable sized projects should not be allowed to silo the Program. There is no capital cost reduction target. NREL has capital cost modeled, but inclusion of depreciation in the MESP is not an adequate reflection of the barrier of raising large capital. Capital reduction should be targeted and tracked.

Feedstock Variability Efforts to piggyback on work being performed by the USDA and others are good and should continue. You not only obtain well-characterized samples at very low cost, but add substantial value to the studies being performed by the researchers that provide the samples. We have continued this effort and over 200 new samples were acquired this year. Need to extend the variability studies to determine the impact of corn stover variability on pretreatability (sugar yields), enzymatic cellulose hydrolysis, and fermentability. There is a theoretical impact based on carbohydrate content; how does it play out in final yields? We acquired several new large lots of corn stover that enable this work to proceed. Need to expand interface to other areas to allow studies of impact of storage on feedstock composition, pretreatability, enzymatic cellulose hydrolysis and fermentability. This work should be accomplished in the Feedstock-Biochemical Interface Area with NREL providing bioconversion processing and analytical support.

Analytical Methods Develop on-line monitoring capability, especially for monitoring the enzymatic saccharification reactor. Enzymatic saccharification is the least understood of all the unit operations. The second area for application of on-line monitoring would be in the fermentation reactor. Moved forward last year with on-line feedstock monitoring Other on-line monitoring development activities planned in future years There is little point in trying to develop process control strategies based on on-line monitoring. There is no clear target for what is being controlled and the control parameters will be process specific. Will not be done Functional group based lignin determinations is an area that should be pursued, as well as work to characterize chemical changes to lignin during and after pretreatment. Work planned in future years, probably via subcontract

Integrated Processing Perform thin studies that indicate problems and generate representative results, that is, determine the problem, skip the solution. In-depth studies that provide solutions are not justified because they will not be generally applicable across a range of processes. Integrated process testing this year identified problems with high recycle water use and with achieving good C5 sugar conversion. Enzymatic saccharification time is too long and needs to be characterized with unwashed materials, that is, with background components (non-sugars) present during enzymatic saccharification. Determine components that are inhibitory to the cellulases (e.g., Maillard reaction products). Perform spiking studies to determine what chemicals inhibit cellulases. Initial performance results in the presence of background sugars were presented. New work going forward in the Pretreatment & Enzymatic Hydrolysis Task. Move forward efforts to characterize waste streams; three years away is too late for those studies to be useful. Also generate real data on thin stillage evaporate. What is in it besides water? Are any of the streams or residues appropriate for putting back on the fields? Initial effort began this year to understand effect of recycle water on process performance.

Integrated Processing Examining the gypsum question is low priority and should not be undertaken. The fact that gypsum is an issue was an important recognition, but the solution will be unique to each process and approaches for handling gypsum are well understood from existing industries. Work in this area was eliminated Advance efforts to understand new feedstocks and new pretreatments. Work began this year in the Pretreatment & Enzymatic Hydrolysis Task Interface Question: What is the root cause of biomass recalcitrance? Generate residue that can be characterized, both compositionally and structurally. This work resides in Targeted Conversion Research Task

Future Work Feedstock Variability Survey of corn stover composition finished Maintain collaborations with USDA and academic institutions performing field studies to advance understanding of the effect of environmental and genetic factors on stover composition, primarily through our expertise with rapid biomass analysis techniques Transition feedstock procurement activities to Idaho National Laboratory (INL) Develop collaborations with INL and other laboratories to explore variability issues for other promising feedstocks (e.g., switchgrass) SWITCHGRASS

Future Work Analytical Methods Improve rapid analysis methods hand-in-hand with improved wet chemical methods Direct Light spectrometer for feedstocks Demonstrate ability to measure stover composition on-line (FY06) Fourier Transform Infrared (FTIR) in-line probe for pretreated slurries and solids (FY07) Near Infrared (NIR) opti-probe for fermentation broths (FY08) Improve wet chemical methods for agricultural and herbaceous materials (FY07 and beyond) Lignin, extractives Automation Distribute and publish new methods as developed (ongoing) 2500 hits on EERE/Biomass web site accessing laboratory analytical methods in last quarter of FY05

Future Work Integrated Processing Determine effect of corn stover compositional and structural variability on pretreatment hemicellulose hydrolysis yields and enzymatic cellulose digestibility (FY06) Investigate integrated performance of new advanced enzyme preparations from Genencor and Novozyme One new preparation will be tested this year (FY06) Other preparations will be tested in outyears (FY07 and beyond) In collaboration with the thermochemical platform, produce representative lignin-rich process residues for thermochemical conversion testing (FY06) In collaboration with the Feedstock-Biochemical Interface, determine effect of wet storage on process performance (FY07 and beyond)

Future Work Integrated Processing Improve hemicellulose conversion yields in dilute acid pretreatment (FY07 and beyond) Collaborative effort across Biochemical Platform Continue to supply process materials to stakeholders, industry, and universities (ongoing) In 2004 and 2005, we supplied over 100kg of raw stover and over 1500 kg (wet) of pretreated stover to 5 industry stakeholders, 12 academic institutions, and 2 government laboratories

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