INDUSTRIAL BIOTECHNOLOGY. Production hosts for real-life feedstock utilization

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1 Selection of production hosts for real-life feedstock utilization Karl Rumbold INDUSTRIAL BIOTECHNOLOGY Industrial Biotechnology is the application of biotechnology for the processing and production of chemicals, materials and fuels It uses enzymes, micro-organisms and cell lines to make products in sectors such as chemistry, pharma, food and feed, paper and pulp, textiles and energy, materials and polymers Interest from pharma was always present Recent: keen interest from Chemical Industry: Better process economy Better product green company image : climate change Further growth is predicted 0% of sales in 00 The development and production of novel, innovative products and processes in a cost- and eco-efficient manner, using increasingly renewable raw materials The discovery and optimization of strains and biocatalysts TNO Kwaliteit van Leven, 0 october 007

2 INDUSTRIAL BIOTECHNOLOGY AT TNO Feedstock Engineering Fungal Biotechnology Metabolomics TNO Kwaliteit van Leven, 0 october 007 FEEDSTOCK ENGINEERING / BIOPROCESSING Feedstock engineering Bioprocessing I. Sucrose, Starch II. Lignocellulose Carbohydrate Feedstock Enzymes Biofuels Bulk chemicals Fine chemicals TNO Kwaliteit van Leven, 0 october 007

3 Wheat straw Sugar cane bagasse Cheaper Abundant Green Corn stover Wood chips dirty glycerol TNO Kwaliteit van Leven, 0 october 007 PRODUCTION OF FERMETABLE SUGARS - TWO WAYS Thermozyme process Heat Biosulfurol process Sulphuric acid SO Membrane H S oxidator H S gas Sulphuric acid reactor Hydrolysis neutralisation Impregnation Fermentation process Anaerobic wastewater treatment Waste water Lignocellulosic Lignin Sulphate Membrane biomass Recycle water 6 TNO Kwaliteit van Leven, 0 october 007

4 ISSUES WITH REAL-LIFE FEEDSTOCKS Mixture of sugars (C6 and C) Growth Inhibitors (Furfural, acetic acid) ph (high and low) High salt concentrations ( dirty glycerol) LITTLE TO NO KNOWLEGDE ON COMPARATIVE FERMENTATION PERFORMANCE ON REAL-LIFE FEEDSTOCKS 7 TNO Kwaliteit van Leven, 0 october 007 SELECTION OF PRODUCTION HOSTS - CURRENT STATE-OF-THE-ART Ad hoc, arbitrary Experience with certain favorite microorganisms Well characterized microorganism Genetic accessibility (E. coli and S. cerevisiae) plug-bugs - microbial production platforms THERE IS NO TEST STAND AVAILABLE FOR BENCHMARKING MICROORGANISMS 8 TNO Kwaliteit van Leven, 0 october 007

5 SELECTION OF PRODUCTION HOSTS - GOALS Which microorganisms - for which feedstocks BENCHMARKING Carbon source (C, C6, glycerol) Resistance to inhibitors Performance on real-life feedstocks TEST ORGANISMS Escherichia coli Corynebacterium glutamicum Saccharomyces cerevisiae Pichia stipitis Trichoderma reesei Aspergillus niger 9 TNO Kwaliteit van Leven, 0 october 007 Results Fermentation parameters Controlled aerobic fermentation on minimal medium with glucose, xylose or glycerol as only carbon source Xylose is not used by S. cerevisiae C. glutamicum Glycerol is not used by Enormous differences in growth rates, substrate uptake rates, biomass yield, carbon dioxide production and oxygen uptake rates Samples were taken for exometabolome analysis 0 TNO Kwaliteit van Leven, 0 october 007

6 Results Resistance 00 S. cerevisiae P. stipitis E. coli C. glutamicum T. reesei A. niger % CO production rate / specific growth rate 0 0 g/l furfural g/l furfural 0, g/l furfural g/l HMF g/l HMF g/l HMF 0, g/l HMF 0 g/l acetate 0 g/l acetate g/l acetate g/l acetate 80 g/l magnesium sulfate 60 g/l magnesium sulfate 0 g/l magnesium sulfate 0 g/l magnesium sulfate 00 g/l sodium chloride 70 g/l sodium chloride 0 g/l sodium chloride 0 g/l sodium chloride g/l sodium chloride TNO Kwaliteit van Leven, 0 october 007 Results Resistance On-line determination of growth rate / CO production rate Resistance against high salt concentrations Resistance against high acetate concentrations C. glutamicum Resistance against inhibitors HMF > furfural TNO Kwaliteit van Leven, 0 october 007

7 Results Fermentations on real-life feedstocks Shake flask fermentation on minimal medium using feedstock hydrolysates and glycerol as carbon sources Performance indicators: ph Carbon source utilization Biomass yield (g/g) Protein production Fermentation at low ph S. cerevisiae Fermentation at high ph C. glutamicum C6 / C utilization E. coli Glycerol utilization S. cerevisiae Feedstock versatility TNO Kwaliteit van Leven, 0 october 007 Results Metabolite and protein production potential S. cerevisiae. lactic acid. alanine. glutamic acid. pyruvate. lactic acid 6. malic acid 7. citric acid. gluconic acid. oxalic acid. citric acid. mannitol. arabitol 6. xylulose 7. pyruvate E. coli. succinic acid. lactic acid. orotic acid. alpha-ketoglutaric acid. citric acid 6. glutamic acid 7. -aminobenzoic acid. pyruvate. unknown (specific for P. stipitis). butanediol. arabitol or isomer. lactic acid 6. disaccharide 7. glycolic acid. mannitol. malic acid. arabitol or isomer. succinic acid. citric acid 6. disaccharide 7. mannitol C. glutamicum. lactic acid. disaccharide. glutamic acid. succinic acid. glycine 6. alanine 7. orotic acid TNO Kwaliteit van Leven, 0 october 007

8 Bechmarking study thermozyme biosulfurol doubling time C-source uptake rate Biomass yield Mixed sugars fermentation acetic acid resistance HMF resistance furfural resistance NaCl resistance MgSO resistance Protein production low ph Diauxy GLU-XYL Growth on bagasse Growth on wheat straw Growth on corn stover Growth on bagasse Growth on wheat straw Growth on corn stover Growth on wood Growth on dirty glycerol GLU XYL GLY GLU XYL GLY GLU XYL GLY Feedstock versatility Genetic acessibility Adaptation to lignocellulosic substrates (literature) Performance on lignocellulosic substrate S. cerevisiae P. stipitis NO A. niger YES T. reesei YES E. coli YES C. glutamicum Total TNO Kwaliteit van Leven, 0 october 007 Conclusions ALL 6 MICROORGANISMS TESTED CAN GROW ON TESTED FEEDSTOCKS (GLYCEROL) PICHIA AND ASPERGILLUS SCORE HIGHEST IN THE BENCHMARKING STUDY MOST POPULAR S. CEREVISIAE POOR XYLOSE METABOLISM AND RESISTANCE - ORGANISM OF CHOICE? BENCHMARKING MICROORGANISM PERFORMANCE - FUTURE STRATEGY FOR MPP FEEDSTOCK ENGINEERING AND FUNGAL BIOTECHNOLOGY REAL-LIFE FEEDSTOCKS ARE GOOD SUBSTRATES FOR MANY BIOPROCESSES BIOSULFUROL ALSO VERY PROMISING FOR CLOSTRIDIUM AND BACILLUS 6 TNO Kwaliteit van Leven, 0 october 007