Hicks, Kevin  B.

Hotchkiss, Jr., Arland  T.

Johnston, David  B.

English

Engineering Conferences International

3/8/06Bioenergy I -Tomar1Increasing the Yield of Fuel Ethanol from Barley with β-Glucanases and β-GlucosidasesKevin B. Hicks, David B. Johnston, and Arland T. Hotchkiss, Jr.ARS, USDA, Wyndmoor, PA 19038  khicks@arserrc.gov3/8/06Bioenergy I -Tomar2Summary of Presentation? Why Consider Barley for Fuel Ethanol?? Technical Problems with Barley as a Fuel Ethanol Feedstock? How We are Using Enzymes to Solve these Problems3/8/06Bioenergy I -Tomar3In 2005, 92 ethanol plants locatedin 20 states produced a record 3.9 billion gallons!This used ~12% (>12 billion bushels) of the US corn supply!2005 U.S. Fuel Ethanol Production3/8/06Bioenergy I -Tomar4DO WE HAVE ENOUGH CORN TO MAKE A SIGNIFICANT IMPACT IN OUR FUEL SUPPLY?020406080100120140160AMOUNTPRODUCEDAMOUNTNEEDEDLast year we  made ~4 billion gallons of ethanol from 12% of the U.S. corn supply.We need at least 150 billion gallons for automobile fuel!3/8/06Bioenergy I -Tomar5HOW CAN WE INCREASE PRODUCTION OF ETHANOL WITHOUT DEPLETING CORN SUPPLIES?? USE BIOMASS?  ? USE HYDROGEN?  ? USE CANE OR BEET SUGAR?  YES BUT NOT IN THE U.S. UNLESS PRICE SUPPORTS ARE CHANGED.? OTHER GRAINS?  BEST SHORT TERM SOLUTION TO THE PROBLEM AND A GREAT OPPORTUNITY FOR U.S. FARMERS.3/8/06Bioenergy I -Tomar6In 2005, 92 ethanol plants locatedin 20 states produced a record 3.9 billion gallons!This used ~12% (>12 billion bushels) of the US corn supply!2005 U.S. Fuel Ethanol Production3/8/06Bioenergy I -Tomar73/8/06Bioenergy I -Tomar83/8/06Bioenergy I -Tomar9IS THERE ENOUGH BARLEY TO MAKE A SIGNIFICANT AMOUNT OF FUEL ETHANOL?? U.S. Barley Production (All Types)– 5-8 Million Metric Tons =  0.2-0.4 Billion Bushels? Canadian Barley Production (All Types)– ~14 million metric tons =  0.6 Billion Bushels? Using 30-50% of Available Barley in the U.S. and Canada for Fuel Ethanol Could Yield an Additional 0.5-0.8 Billion Gallons of Ethanol.? Significantly More Barley can Be Grown in the U.S. if Markets are there.? Bottom Line:  ~1 Billion Gallons of Ethanol from Barley3/8/06Bioenergy I -Tomar10Number of U.S. Fuel Ethanol Plants that Use Barley as Feedstock = 03/8/06Bioenergy I -Tomar11Technical Issues with Barley as a Fuel Ethanol Feedstock? Abrasive Hull? Low Starch Content (50-55%) Compared to Corn’s (72%)? High Viscosity of Mash due to β-Glucans? β-Glucans in DDGS Limit their Applications to Mainly Ruminants3/8/06Bioenergy I -Tomar12Research Plan to Solve Barley Issue 1? How to Deal with Abrasive Hull?– Use Hulless Barley – Use Pearling and/or Milling Processes to Remove Hull and Create High-Starch Fractions and Low-Starch Coproducts3/8/06Bioenergy I -Tomar13CALLAOPRICETHOROUGHBREDDOYCEBarley Production at Virginia Tech3/8/06Bioenergy I -Tomar14Performance of “Doyce”Doyce SampleStarch β-GlucanOil Protein Test Weight& Yield2002/2003 Crop, 9 East Coast Locations62.28(2.03)3.85(0.09)1.83(0.04)11.39(0.80)55.16 (1.98)~85 bu/acre*2003/2004, 15 East Coast Locations65.58(1.56)4.67(0.33)2.06(.09)9.49(0.83)52.81 (2.63)3/8/06Bioenergy I -Tomar15Research Plan to Solve Barley Issue 3? How to Deal with High Viscosity of Mash due to     β-Glucans– Fractional Milling May Be a Partial Solution by Removing β-Glucan-enriched Fractions Prior to Fermentation– Greatest Gains Will Be Made by Use of β-Glucanase Enzymes that Break down High-Viscosity β-Glucans to Low MWT Oligosaccharides3/8/06Bioenergy I -Tomar163/8/06Bioenergy I -Tomar17Commercially Available β-Glucanases Can Lower Viscosity of Barley Mashes? Commercial Enzyme Preparations Contain Mixtures of Enzymes that Can Partially Degrade Viscous High Molecular Weight β-Glucans, Producing Low Viscosity Oligosaccharides in the Process.3/8/06Bioenergy I -Tomar18Are there Commercially Available β-Glucanases that can Completely Hydrolyze β-Glucan to Glucose?? What Would be the Benefits?– Major Reduction of Viscosity– Additional Glucose Produced Would Be Fermented to Ethanol, Increasing Yields3/8/06Bioenergy I -Tomar19Benefits of Complete Hydrolysis of β-Glucans? Even Greater Reduction of Mash Viscosity ? Elimination of β-Glucan in DDGS (Increasing Value)? Increase in Ethanol Yields:– Barley with 65% Starch and 5% β-Glucan would be Equivalent to Grain having 70% Starch!3/8/06Bioenergy I -Tomar20Next Steps for β-Glucanase StudiesWE’RE LOOKING FOR A FEW GOOD ENZYMES!3/8/06Bioenergy I -Tomar21β-Glucanase Studies – Model System? Commercial Enzymes Evaluated– Alltech: Allglucanase, Heat Stable β-Glucanase from Trichoderma Longibrachiatum and Rhyzozyme, from Rhizopus sp.– GNC Bioferm:  Endo Feed, Feed Enzyme from Aspergillus sp.– Novozyme: Viscozyme L, Multi-Enzyme Mixture from Aspergillus sp. (Obtained from Sigma)– Genencor: GC 220, GC 440 and GC 880, Developmental “Cellulases” from Trichoderma3/8/06Bioenergy I -Tomar22β-Glucanase Studies:Model System? Method of Evaluation of Commercial Enzymes– Reacted Aqueous Solutions (1% w/w) of Soluble Barley β-Glucan with Commercial Enzymes using pH and Temperature as Recommend by the Manufacturer – Used Excess of Enzymes – Measured the Levels of Oligosaccharides (HPAEC-PAD) and Glucose (YSI Glucose Analyzer) Produced Over Time3/8/06Bioenergy I -Tomar23β-Glucanase Studies:Model SystemEnzyme Temperature 0C pH Dosage g./g.βGEndo-Feed 45 5 0.193Multifect-B 45 5 0.73Viscozyme L 45 5 0.71GC 220 45 5 0.34GC 440 55 5 0.35GC 880 55 5 0.35Rhizozyme 50 5 0.12Allglucanase 80 5 0.323/8/06Bioenergy I -Tomar24β-Glucanase Studies: Model System -Results  (1 hr reaction time)Enzyme Glucose Yield g glu/g βG (Mean Value + error)Endo-Feed 0.03 + .009Multifect-B 0.05 + .058Viscozyme L 0.06 + .022GC 220 0.24 + .091GC 440 0.28 + .045GC 880 0.40 + .082Rhizozyme 0.05 + .053Allglucanase 0.06 + .0303/8/06Bioenergy I -Tomar25β-Glucanase Studies:Model System - Results? All Enzymes Dramatically Reduced Viscosity? Several Enzymes Led to Production of Measurable Levels of Glucose? Numerous Fermentations were Conducted but Increased Levels of Ethanol Were Not Statistically Significant.Production of Glucose from β-Glucan by GC 8803/8/06Bioenergy I -Tomar26β-Glucanase Studies:Model System - HPAEC Results3/8/06Bioenergy I -Tomar27Next β-Glucanase Studies:Which Enzymes Are Missing?LaminaribioseBeta 1-3Beta 1-4DP ~ 1000-2000Cellotriosyl-/Cellotetraosyl ratio:3/1Cel6A (CBHII)Cel5A (EGII)CellobioseBARLEYBETA GLUCANCel7A (CBHI)EthanolBG (EC3.2.1.21)Barley Exo IMany Enzymes Are Used by Trichodermasp. to Hydrolyze β-GlucanGraphics Courtesy of Genencor3/8/06Bioenergy I -Tomar28Can We Find the Missing Enzymes?YES!3/8/06Bioenergy I -Tomar29Conversion of Beta-Glucan using Single Enzyme00.10.20.30.40.50.60.70.80 50 100 150Time, minGlucose Concentration, g/L GC880b-glucosidaseLichenaseGC220GC440AllgluanaseRhizozymeViscozyme LBiofremMultifect B3/8/06Bioenergy I -Tomar30Conversion of Beta-Glucan to Glucose using Enzymes + β-G00.20.40.60.811.20 50 100 150Time, minGlucose Concentration, g/Lb-glucosidaseLichenaseGC220 & b-glucosidaseGC440 & b-glucosidaseAllglucanase & b-glucosidase    Rhizozyme & b-glucosidase    Viscozyme L & b-glucosidase    Biofrem & b-glucosidase    Multifect B & b-glucosidase    GCC880 & b-glucosidase3/8/06Bioenergy I -Tomar31Conclusions for β-GlucanaseStudies? We Have Determined the Missing Components of Commercial β-Glucanases? Addition of β-Glucosidase to Even the Cheapest β-Glucanases Should be able to Convert all β-Glucan to Glucose and During Fermentations, – Increase Ethanol Yield– Remove All Beta-Glucans from DDGS3/8/06Bioenergy I -Tomar32Summary of Fermentation Studies Without Beta-Glucanases? Doyce Hulless Barley and a Typical Hulled Barley Appeared to Ferment at Approximately Similar Rates ? Doyce Barley Produced Higher Ethanol Yields– (2.27 versus 1.64 gal/bushel)? Doyce Produced Less DDGS, but the Composition was Higher in Protein (More Valuable) than Hulled Barley DDGS– ~30% versus ~23% Protein? Without use of β-Glucanases, both Doyce and Hulless Barley DDGS Contained Significant Levels of β-Glucans– ~7-8% Beta Glucans in DDGS3/8/06Bioenergy I -Tomar33Next Steps for β-Glucanase Studies? Conduct Fermentation Experiments to Validate Viscosity Decrease, Ethanol Yield Increase and Lack of β-Glucans in DDGS, and Other Process Requirements? Determine Economic Feasibility by Building a 40 MGY Barley Ethanol Plant SuperPro™ Model? Transfer the Technology to Users3/8/06Bioenergy I -Tomar34Acknowledgements? Alltech? Genencor? GNC Bioferm? ERRC Staff – Karen Kohout– Jennifer Thomas– Robyn Moten– Jerry Senske3/8/06Bioenergy I -Tomar35THANKS!

Increasing the Yield of Fuel Ethanol from Barley with Beta-Glucanases and Beta-Glucosidases

https://dc.engconfintl.org/cgi/viewcontent.cgi?article=1033&amp;context=bioenergy_i

Increasing the Yield of Fuel Ethanol from Barley with Beta-Glucanases and Beta-Glucosidases

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