The Use of Novel Enzyme Accelerant Technology in Reducing Costs and Increasing Yields in Ethanol Production.

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The Use of Novel Enzyme Accelerant Technology in Reducing Costs and Increasing Yields in Ethanol Production. Ken Matthews. Business Development Manager Eka Chemicals Inc. 1

Pulp, Paper and More.

The Use of Novel Accelerant Technology in Reducing Costs and Increasing Yields in Ethanol Production. Enzymatic Accelerants: 1.1 Corn-Ethanol: reduced enzyme costs, better ethanol yields. 1.2 Cellulosic Sludge-glucose/ethanol: reduced enzyme costs, improved conversion rates. 1.3 Advanced Bio-fuel conversions: increased enzymatic hydrolysis efficiencies of 2 nd generation and advanced biofuels/renewable raw materials from herbaceous and waste materials.

Introduction to the Concept. The Paper Industry is spending a lot of money getting rid of what could be a valuable resource. The creation of bio-materials from wood is a difficult, complex and costly challenge. The conversion of cellulose to sugar and then ethanol is relatively easy. This enzyme accelerant technology addresses the second point to lower the barriers to economic feasibility. The industry is not ready yet, but the accelerant technology is.

The Use of Novel Accelerant Technology in Reducing Costs and Increasing Yields in Ethanol Production. Enzyme Accelerant: Corn Ethanol. Speeds up the enzymatic conversion process. Reduces the amount of enzyme required. Increases the yield of sugars from the hydrolysis. Increases the amount of ethanol per bushel.

1st Generation Bio-Ethanol Validation. Glucose/ppm 120 100 80 60 40 20 0 EAT - Enzyme Accelerant Technology 0 10 20 30 40 50 60 70 Time (Minutes) 0.12% α Amylase 0.12% α Amylase + 150 ppm EAT 0.09% a Amylase 0.09% a Amylase + 150 ppm EAT Adding the Enzyme Accelerant increases amount of glucose by 10% reduces production time by half

1st Generation Bio-Ethanol Validation. Glucose/ppm 120 100 80 60 40 20 0 EAT - Enzyme Accelerant Technology 0 10 20 30 40 50 60 70 Time (Minutes) 0.12% α Amylase 0.12% α Amylase + 150 ppm EAT 0.09% a Amylase 0.09% a Amylase + 150 ppm EAT Adding the Enzyme Accelerant increases amount of glucose by 10% reduces production time by half a 25% cut still sees an increase in glucose by 13% can cut enzyme by 25% and production time still reduced by half

1st Generation Bio-Ethanol Validation. Glucose/ppm 120 100 80 60 40 20 0 EAT - Enzyme Accelerant Technology 0 10 20 30 40 50 60 70 Time (Minutes) 0.12% α Amylase 0.12% α Amylase + 150 ppm EAT 0.09% a Amylase 0.09% a Amylase + 150 ppm EAT Adding the Enzyme Accelerant increases amount of glucose by 10% reduces production time by half a 25% cut still sees an increase in glucose by 13% can cut enzyme by 25% and production time still reduced by half can cut enzyme by 25% and make the same amount of glucose

Sugar Conversion at Lower Amylase Loadings. Brix Level 23 22 21 Final Brix Level No Enzyme Accelerant Enzyme Accelerant at 150 ppm Amylase dose can be cut by 50% with accelerant Or achieve an increase in Brix Level by 9% 20 19 0.12 0.09 0.06 0.03 Amylase Loading (%)

Increase in Sugar Yield with Different Accelerants. Brix Level 23 22 21 20 19 18 Different accelerants can be used to give the best result Accelerant A & B give a 2% increase Accelerant C give a 8% increase 17 16 15 1 2 3 4 5 6 7 8 9 Time (minutes) Control Accelerant - A Accelerant - B Accelerant - C

Increased Rate of Hydrolysis. Glucose (g/l) 100 80 60 40 Glucose Levels After Hydrolysis with 150 ppm of Accelerant Control Accelerant A Accelerant B Accelerant C Different accelerants can be used to give the best result Accelerant A & C give a 24 to 33% increase Accelerant B give a 48% increase Cut hydrolysis time by 33 to 50% 20 2 4 6 8 10 12 14 Time (Minutes)

Laboratory Set Up.

Enzyme Mechanism.

Comparison of Conventional Hydrolysis and Accelerated Hydrolysis. Variable Reference / Control With Accelerator % Change Alpha Amylase g/100g 0.12 0.08-33.3 Gluco Amylase g/100g 0.008 0.006-25.0 Accelerant ppm Nil 150 Conversion Eff % 94 97 +3.2 Fermentation Eff % 95 95 No Change Final Sugar deg Brix 23.7 24.38 +2.9 Final EtOH g/l 115.5 118.1 +2.2 DDGS Prod #/bushel 16.6 16.0-4.1

The Use of Novel Accelerant Technology in Reducing Costs and Increasing Yields in Ethanol Production. Cellulose Based Enzyme Hydrolysis. Enzyme cost/gallon in corn-ethanol ~ $0.04/gall Enzyme cost/gallon in cellulosic-ethanol ~ $0.40 to $1.50/gall What effect can the accelerant technology have on the this cost?

Enzymatic Accelerant with Cellulose Sludge. Glucose mg/l Hydrolysis of Cellulosic Sludge to Glucose 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 204 216 228 16000 14000 12000 10000 8000 6000 Enzyme Accelerant Technology gave a 25% increase in sugars. Hydrolysis time was cut from 180hr to 120hr, (a 33% reduction). 4000 2000 0 Control Control Smoothed Enzyme Accelerant Enzyme Accelerant Smoothed Time (Hours)

Bleached Hardwood Pulp. Glucose mg/l 24000 22000 20000 18000 16000 14000 12000 Bleached Hardwood Kraft at 5% Consistency with constant stirring using 0.16% Novozyme Cellic Ctec (84 FPU/ml) Control 100 ppm Enzyme Accelerant added at start of treatment Enzyme Accelerant Technology gave a 27% increase in sugars. Hydrolysis time is cut from 140hr to ~90hrs (a 36% reduction). 10000 8000 6000 0 20 40 60 80 100 120 140 160 Time (Hours)

Bleached Softwood Pulp. BSWD fiber was screened & the long fraction collected. The accelerant had a progressively negative effect on the long fiber. Inference: the accelerant benefit decreases as fines decrease. Which is why the best effects are seen with cellulosic sludge samples. Rate of EH Fines>>Rate of EH HWD>Rate of EH SFTW What solutions might there be?

Effect of Sequenced Addition of Accelerant. Glucose mg/l 10000 8000 6000 4000 2000 0 Softwood at 2% Consistency at 50 o C with stirring about 235 rpm 0.1% Novozyme Cellulase Accelerant added at start Accelerant added after 1 hour Hours vs 2 Hr delay Hours vs 4 hr delay Hours vs 6 hr delay 4 Hr 2 Hr 6 Hr 0 Hr 1 Hr 0 5 10 15 20 25 30 Adding Enzyme Accelerant Technology later in the hydrolysis step At 2 Hr ~ 33% inc At 4 Hr ~ 58% inc At 6 Hr ~ 25% inc By adding Enzyme Accelerant 2hr to 4hrs after enzyme addition the production time could be cut by ~40% Time (Hours)

Effect of Agitation Speed on Accelerant. Glucose mg/l 3500 3000 2500 2000 1500 1000 500 0 Softwood at 2% Consistency at 50 o C with stirring varied 0.1% Novozyme Cellulase & 250 ppm Enzyme Accelerant 356 rpm >450 rpm 245 rpm 137 rpm 137 rpm 245 rpm 356 rpm Average of 460 and 554 rpm 0 2 4 6 8 10 12 14 Based on varying the agitation with Enzyme Accelerant Technology glucose production could +33% at 245 rpm +100% at 356 rpm +50% at >450 rpm By selecting the right agitation speed, production time could by cut by 66% to 80% Time (Hours)

Potential Mechanism for Accelerant Performance. The accelerant has polyelectrolytic properties. Cationic material is better than anionic. A higher charge does not appear to have a benefit over lower charge. A higher molecular weight does not offer any advantages. The product format seems to have some influence on performance. It is believed that a patching mechanism is in play due to the above statements.

Further Challenges. Dead-Loads: Inorganic fillers and lignin. Inhibitors: Furfurals, lactic and acetic acids, micro-biological contaminants. Cost: Capital, resources. Will: Change, risk, uncertainty.

Current Status of Project. Looking for industry partners for demonstration in the heritage industry and ultimately a channel partner for the business. Running validation studies with Bio-Process Innovation investigating the accelerant effects with cellulosic bio-materials with a cross-section of different, industrially relevant feed-stocks. Eka is testing some oxidative chemistries (core-competency) on pretreatment of agro-waste with the intention of widening our scope within the bio-refinery business development concept.

Thank You for your attention.