Biofuels & Biochemicals an innovation challenge. From Biomass to Bioproducts. Han de Winde. Leiden University Faculty of Science

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1 From Biomass to Bioproducts Biofuels & Biochemicals an innovation challenge Han de Winde May 14th, 2013 Leiden University Faculty of Science Delft University of Technology Department of Biotechnology >> Focus on sustainability, innovation and international

2 New Dutch Government Policy Ministry of Economic Affairs, Agriculture and Innovation Top Sectors: Agro-Food Chemistry Life Sciences High Tech Systems Water Energy Logistics Horticulture Creative industry Headquarters Netherlands Organisation for Scientific Research

3 Topsector Chemistry By 2050: 1. The Netherlands is the world leader in green chemistry 2. The Netherlands is a global top 3 producer of smart materials High-value, groundbreaking scientific research Topconsortia on knowledge and innovation (TKIs) Process Technology Smart Polymeric Materials Biobased Economy cross sectoral! Nursery for New Chemical Innovations and fundamental science

4 Fundament of TKI BioBased Economy CatchBio Chemocatalysis: Use of organic and inorganic materials BE-Basic Biocatalysis: Use of enzymes and micro-organisms

5 Towards a sustainable bio-based economy TWA Workshop Bio-Based Economy, TU Delft 09 April 2008

6 Potential feedstocks for industrial biotechnology cornstover switchgrass algea? bagasse current feedstocks competing food/feed wheatstraw agricultural residues cheap/abundant elephant grass dedicated crops use of less fertile land 6

7 Biobased Economy TODAY: efficient bio-mass utilization? CO 2 1 e generation biofuels 2 e generation biomaterials agro-emissions (run-offs, N 2 O) biobricks nutrients

8 Bioenergy paradox cascading the pyramid! Farma, Health Added Value Food, Feed Materials, Chemicals Volume Fuel, Fire, Electricity, Energy, Heat Growing the Bioeconomy Banff,

9 Broad technology portfolio required Growing the Bioeconomy Banff,

10 Crop residues and paper waste corn stover bagasse wheat straw paper sludge wood chips paper waste Abundant, NO competition with food production (Hemi)cellulose instead of starch/sucrose Growing the Bioeconomy Banff,

11 Ethanol from Ag-waste: 2 nd generation biotech Hemi-cellulosic plant material Ethanol Pretreatment Cellulose Hydrolysis Sugar Mixture Fermentation Lignin Combustion Heat, electricity Cellulase Growing the Bioeconomy Banff,

12 Sugars in Crop Residues: the Pentose Challenge Corn stover Wheat straw Bagasse Sugars (%) glucose mannose galactose xylose arabinose uronic acids Other (%) lignin

13 Sugar mixtures: glucose, xylose and arabinose ATP (araa) L-ribulose (arab) L-ribulose-5-P ATP glucose L-arabinose (arad) 2 NADPH CO 2 glucose-6-p fructose-6-p ATP D-xylulose-5-P PPP fructose-1,6-bip ATP G-3-P PEP NADH DHAP glycerol NADH D-xylose ATP pyruvate NADH (xyla) D-xylulose ATP (XKS1 / XYL3) CO 2 ethanol 13

14 Further Improvements: Evolution in the lab Generationtime ~ 20 years Generationtime ~ 2 hour evolutionary engineering 14

15 CO 2 profiles glucose-xylose-arabinose (20 cycles) CO2 (%) time (h) 15

16 Evolutionary engineering serial batch cultivation in different sugar mixtures IMS0003 IMS concentration (g l -1 ) glucose Xylose ethanol arabinose SBR concentration (g l -1 ) glucose xylose ethanol arabinose time (h) time (h) Increased specific consumption rates of xylose and arabinose decreased total fermentation time

17 Ethanol Production from Wheat-Straw Hydrolysate ( academic xylose-fermenting strain) Fed-batch Batch ethanol Sugar (g/l) xylose glucose Ethanol (g/l) Ethanol yield: 0.47 g ethanol/g sugar 238 L ethanol/ton dry biomass

18 Is this really applicable??? Inside

19 Crop residues and paper waste corn stover bagasse wheat straw paper sludge wood chips paper waste Abundant, NO competition with food production (Hemi)cellulose instead of starch/sucrose Growing the Bioeconomy Banff,

20 Furaldehydes furfural hydroxymethyl furfural (HMF) Ligno-cellulose feedstock utilization: - Acid-catalyzed dehydration products of pentoses (furfural) or hexoses (HMF) - Diverse toxic effects: oxidative damage (ROS): DNA damage (mutagenic), protein misfolding, aggregation mitochondria, actin structure loss, vacuole fragmentation primary metabolic enzyme inhibition: increased lag phase, reduced fermentation rate synergistic effect with other inhibitors (organic acids, aromatics) Reduced microbial growth and product yield

21 Furaldehyde metabolism Wierckx et al., Microb. Biotechnol. doi: /j x Cupriavidus basilensis HMF14: sugars (glc, xyl, ara) not utilized Ligno-cellulose hydrolysate inhibitors catabolized substrate profile suitable for detoxification biomass sugars furaldehydes org. acids inhibitor-free sugar mix

22 Furaldehyde metabolic pathways of C. basilensis HMF14 o O HmfH o O ATP, CoASH acc ox acc red OH H 2 O furfural furfural dehydrogenase HmfD HmfFG HmfABC TCA cycle HMF HmfH aldehyde dehydrogenase 2-ketoglutarate CoASH HmfE CO 2 H 2 O HmfH 2,5-furandicarboxylic acid H 2 O Growing the Bioeconomy Banff, 2012

23 2,5-Furandicarboxylic acid (FDCA, FDA) Top-12 value-added chemicals from biomass (2004 DOE-NREL report Todd Werpy) Estimated market size $/yr Replacement for terephthalate in polymers Platform chemical Koopman et al. (2010) Biores. Technol. Growing the Bioeconomy Banff, 2012 doi: /j.biortech

24 Whole-cell biocatalyst for FDCA production from HMF Fed-batch cultivation mineral salts medium glycerol feed (metabolically active cells most effective) concentrated HMF feed up to 40 g l -1 FDCA conversion ~100 % (<0.1 % HM-furoic acid) biomass HM-furOH Product recovery acid precipitation + solvent extraction 99.4 % pure 75 % recovery Koopman et al. (2010) Biores. Technol. doi: /j.biortech

25 Conclusions Combination of metabolic and evolutionary engineering is Powerful Tailor-made evolution strategies are applicable to many combinations of microbe strains and substrate mixtures Reverse engineering phenotypes are important challenge towards industrial application! 25 >> Focus on sustainability, innovation and international

26 Fermentation Pretreatment 3rd generation Down stream