Construction and Optimisation of Pentose-fermenting Yeast Strains for Bioethanol Production Prof. Dr. Eckhard Boles Institute for Molecular Biosciences Goethe-University Frankfurt/Main 1 Hier wird Wissen Wirklichkeit
Bio-refinery our contribution: yeast design fermentation CO 2 2 Hier wird Wissen Wirklichkeit
Today s technology: Sugar from cane or beet Starch from cereals 3 Hier wird Wissen Wirklichkeit
Conflicts: is this bioethanol really eco-friendly? 4 Hier wird Wissen Wirklichkeit
The alternative solution: Ethanol from lignocellulosic biomass 5 Hier wird Wissen Wirklichkeit
6 Hier wird Wissen Wirklichkeit
NILE New Improvements for Ligno-cellulosic Ethanol 2005-2009 An Integrated Project supported by the 6th RTD Framework Programme of the EC General objectives To prepare the large-scale production of bioethanol from ligno-cellulosic feedstocks, i.e. a cost effective process for production of bioethanol from lignocellulose = To be ready for the next steps (> 2010) of the European directive on biofuels 7 Hier wird Wissen Wirklichkeit
Background Ethanol from ligno-cellulosics = best biofuel in terms of CO 2 emission reduction necessary for diversification of raw materials necessary for low cost ethanol time table = after 2010 research having an impact on the medium to long term 8 Hier wird Wissen Wirklichkeit
Bioethanol from LCB: main problems still to be solved How to further decrease the cost of enzymatic hydrolysis? Can the pentose-rich hemicelluloses be efficiently converted to ethanol? What quality of lignin can be obtained and how to optimise the value of this product? What about scale-up and process integration (to reduce energy demand)? How to obtain representative and reliable data for cost estimation, environmental and socioeconomic impacts? 9 Hier wird Wissen Wirklichkeit
Project structure WP7: Project management WP1 Enzymatic hydrolysis WP4 Socio-economic and environmental impacts & development strategy WP3 Process technology WP6 Dissemination and training WP2 Ethanol production WP5 Evaluation of lignocellulosic ethanol for automotive applications 10 Hier wird Wissen Wirklichkeit
NILE consortium 21 partners (11 countries) 7 industrial companies Enzyme producers (Roal Oy, Saf-Isis) Ethanol producer and distributor (Sekab) Oil company (Enitecnologie) + SMEs (Etek, Direvo, Granit) 5 research Centres IFP (Coordinator) CRFiat, VTT, INRA, CNRS 7 universities (Lund, ICL, ETH, Lisbon, Frankfurt, IWC, Weizmann) 2 associations (BAFF, EUREC Agency) Support and contribution of the European Commission (FP6): 7.7 M Starting date: October, 2005 11 Hier wird Wissen Wirklichkeit
WP2 ethanol production Objective To carry out an efficient conversion of hydrolyzed material (including pentoses) to ethanol at a high yield and rate Main tasks selection and design of suitable host yeast strains genetic engineering of strains to allow efficient utilisation of all sugars development of control strategies for fermentation of LCB hydrolyzates (including SSF) Fermentation of LCB hydrolyzates 12 Hier wird Wissen Wirklichkeit
Cellulose (33-51 %) LIGNOCELLULOSE (WOOD) Lignin (21-32 %) Extractives (1-5 %) Hemicellulose (19-34 %) HEXOSES HO HO CH 2 O CH 2 O CH 2 HO O HO CH 3 O β-d-glucopyranose β-d-mannopyranose α-d-galactopyranose α-l-rhamnopyranose FERMENTATION INHIBITORS PHENOLICS R R EXTRACTIVES Terpenoids etc. R FURALDEHYDES HO CH 2 5-hydroxymethyl- 2-furaldehyde (HMF) O O CHO 2-furaldehyde (furfural) CHO ETHANOL ALIPHATIC ACIDS CH 3 CO acetic acid HCO formic acid CH 3 COCH 2 CH 2 CO levulinic acid PENTOSES HO H 2 C URONIC ACIDS O CO O O H 3 CO CO HO O β-dxylopyranose α-l-arabinofuranose 4 O-methylα-D-glucuronopyranose α-d-galacturonopyranose 13 Hier wird Wissen Wirklichkeit
Grass: Corn stover: Wheat bran: 16% xylan, 5% arabinan 19% xylan, 3% arabinan 19% xylan, 15% arabinan 14 Hier wird Wissen Wirklichkeit
Pentose Catabolic pathways 15 Hier wird Wissen Wirklichkeit
Inverse metabolic engineering Yeast strains C5 genes Recombinant strains mutagenesis & selection Improved rec. strains Transformation Beneficial genes microarray analyses molecular characterization
Glucose Arabinose Epimerase D-Xylulose -5-P Isomerase Kinase L-Ribulose L-Ribulose-5-P Xylulose Kinase Permease Xylitol Dehydrogenase Xylose Permease Reductase Ethanol 17 Hier wird Wissen Wirklichkeit
Arabinose fermenting S. cerevisiae Kinase Isomerase L-Ribulose L - Arabinose Permease Gal2 galactose/arabinose permease araa L-arabinose isomerase arab L-ribulokinase arad L-ribulose-5-p 4-epimerase 4-Epi - merase L-Ribulose-5-P D-Xylulose-5-P Ethanol 18 Hier wird Wissen Wirklichkeit
Arabinose OD 600nm 12 10 8 6 4 2 0 0 200 400 600 800 1000 g Et/g dw 1,25 1 0,75 0,5 0,25 0 mut glc wt glc mut ara wt ara 0 2 4 6 8 t (h ) t (h) 19 Hier wird Wissen Wirklichkeit
Arabinose fermenting S. cerevisiae E. coli Isomerase: no activity B. subtilis Isomerase: active Isomerase L - Arabinose Gal2 Permease g Et/ g dw 0,6 0,4 0,2 with permease w/o permease 4-Epimerase L-Ribulose L -Ribulose -5 Kinase 0 0 2 4 6 8 t (h) D-Xylulose-5-P Transaldolase Ethanol 20 Hier wird Wissen Wirklichkeit
Arabinose fermentation under oxygen-limited conditions % w/v 2 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 0 10 20 30 40 50 Arabinose Arabitol / Xylitol Glycerol Et Fermentation performance of JBY25-4M in a synthetic arabinose stream. The fermentations were started at high cell densities (9.2 g dw/l). hours specific arabinose consumption rate: ~0.03 g arabinose / g cell dw x hour specific ethanol production rate: 0.01 g ethanol / g arabinose x hour ethanol yield: theoretically: 0.64 % w/v determined: 0.62 % w/v (= 97%) 21 Hier wird Wissen Wirklichkeit
Construction of an arabinose/xylose co-consuming S. cerevisiae strain BWY2 BsaraA EcaraB EcaraD PsXYL1 PsXYL2 ScXKS1 22 Hier wird Wissen Wirklichkeit
BWY2 BWY2 2%glc,2%xyl (fermenter/anaerobic) amount g/l 25 20 15 10 5 0 0 50 100 150 glucose xylose xylitol glycerol acetate etoh corr time h - but problems to ferment arabinose 23 Hier wird Wissen Wirklichkeit
We have: prototype yeast strains fermenting glucose and arabinose (strains and technology patented worldwide; Forskarpatent/Lund) prototype yeast strains fermenting glucose and xylose (patented by Forskarpatent/Lund) in progress: yeast strains co-fermenting glucose, xylose and arabinose knowledge about the bottlenecks and how to solve them What are we doing: to transfer the derived knowledge to the construction of stable industrial yeast strains What do we need: more support and collaborations with industry 24 Hier wird Wissen Wirklichkeit