Optimizing enzyme cocktails and process conditions for production of cellulosic ethanol René Verwaal, Herman Pel, Marco Hensing DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX, Delft, the Netherlands POET-DSM JV Project Liberty • • • • • • First commercial scale cellulosic ethanol plant of POET-DSM Joint Venture Starting capacity 75 million liter ethanol per year from corn residues Shared infrastructure with existing grain ethanol plant in Emmetsburg, Iowa Local corn residues Power for both plants Grand Opening on September 3, 2014 Overview large scale bio-ethanol plant Enzyme screening Enzyme mix optimization Recombinant produced single enzymes DSM thermostable enzyme cocktail for biofuel production Development target: Increase enzyme performance Enzyme cocktail improvement strategies: • Enzyme screening • Classical strain improvement • Genomics tools • Protein engineering Experimental set-up ● Enzyme mix varied in 4 cellulases ● Statistical mixture design: - 55 incubations - same total protein dose - variation in ranges: BG 0.04-0.12 CBH 0.10-0.60 EG 0.18-0.68 ● Conditions wheat straw assay: - pH 4.5 - temperature 65°C - time 20h ● Spiking to base mix ● Compose artificial mixes Enzyme cocktail build around enzymes from thermophilic filamentous fungus Rasamsonia emersonii In 3 steps from corn residues to ethanol ● Identify weak links ● Identify enzymes with highest impact on cellulose degradation Optimized enzyme cocktail Optimized cocktail reduces dosage by >40% Enzyme cocktail is active above 60°C ● High performance in broad temperature range ● Active at acidic pH ● Improved control of contamination in hydrolysis Natural selection of improved cocktails Classical Strain Improvement (CSI) CSI effects enzyme cocktail composition Strong changes in amount and ratio of main cellulases CSI and process optimization Fast feedstock liquefaction Fast viscosity reduction. 20% acid-pretreated wheat straw, 62ºC © 2015 DSM. All rights reserved DSM thermostable enzymes Cost savings! • Fast liquefaction allows high dry matter content up to 25% • CAPEX savings due to lower required capacity of process equipment • Thermostability allows for less energy use for cooling • OPEX savings due to reduced energy costs • Thermostability gives improved control of contaminations • Reduced antibiotic costs • Increased revenues because less sugars are lost