Next generation technologies for biofuels/bioenergy production in Southern Africa

1. Emile van Zyl Department Microbiology University of Stellenbosch Next generation technologies for biofuels/bioenergy production in Southern Africa

2. Next generation technologies 1. Value of biofuels to southern Africa 2. Potential for biofuels productionin South Africa 3. SANERI Chair of Energy Research : Biofuels 4. Microbial technologies for cellulosic ethanol production 5. Thermochemical technologies for biofuels production 6. Integrating biochemical and thermochemical technologies

3. Next generation technologies Valueofbiofuels to southern Africa

4. Biofuels value to southernAfrica Benefits of biofuel production in southern Africa: • Source of foreign exchange savings for oil-deprived countries. • Boosting of agriculture /forestry production and additional markets /revenue for farmers / industry. • Help generate employment and local economic development opportunities in rural areas. • Reduction of GHG emissions and combating climate change. • Contribute to political security, make Africa less dependent on oil and create local wealth.

5. Bioenergy Production Potential in 2050 (Smeets, Faaij, 2004) Future bioenergy potential in Africa Total bioenergy production potential in 2050 based on different agriculture systems [expressed as EJ (1018 J). yr−1; left to right bars – conventional to highly productive agriculture systems].

6. Biomass potential of Africa at large Ratio of the energy content of the biomass on abandoned agriculture lands relative to the current primary energy demand at the country level. The energy content of biomass is assumed to be 20 kJ g−1. Source: Campbell et al. (2008)

7. Next generation technologies Potential for biofuels productionin South Africa

8. South Africa’s potential: Renewable biomass available 1. Residues Agricultural Maize stover 6.7 Mt/a (118 PJ/a) Sugar cane bagasse 3.3 Mt/a (58 PJ/a) Wheat straw 1.6 Mt/a (28 PJ/a) Sunflower stalks 0.6 Mt/a (11 PJ/a) Agricultural subtotal 12.3 Mt/a (214 PJ/a) Forest industry Left in forest 4.0 Mt/a (69 PJ/a) Saw mill residue 0.9 Mt/a (16 PJ/a) Paper & board mill sludge 0.1 Mt/a (2 PJ/a) Forest industry subtotal 5.0 Mt/a (87 PJ/a) 2. Energy crops From 10% of available land 67 Mt/a (1 171 PJ/a) (Marrison and Larson, 1996) 3. Invasive plant species 8.7 Mt (151 PJ) Total, annual basis93 Mt/a (1 622 PJ/a) Lynd et al. 2003. Plant Biomass Conversion to Fuels and Commodity Chemicals in South Africa: A Third Chapter? South African Journal of Science 99: 499 – 507.

9. maize stover bagasse wheat straw Lignocellulose sources paper sludge wood chips

10. Harvesting red grass Which bioenergy crops? Grain sorghum Sweet sorghum – two harvests? Sugar cane(100+ t/ hect?) Invasive plants

11. Chair of Energy Research: Biofuels and other clean alternative fuels Emile van Zyl Johann Gorgens, Marinda Bloom & Hansie Knoetze [Stellenbosch University] & Harro von Blottnitz [University Cape Town] 11

12. CoER : Biofuels (members) Microbiology Proc Eng Chem Eng

13. Biomass Conversion to Biofuels and By-products Primary Biomass Production Biomass Primary Processing Product Distribution & Marketing Biomass Transport Technologies for Cellulose Conversion Cellulosics biofuels production value chain: • The CoER : Biofuels positions itself in the conversion technologies, but acknowledges the importance of establishing the whole value chain.

14. Non-fermentable sugars high energy aromatics Sugarcane bagasse Lignin28% Arabinan2% Hexoses (fermentable) Pentoses (fermentable) Cellulose46% Xylan25% Technologies for Cellulose Conversion Lignocellulose composition

15. Next generation technologies Microbial Technologies for Cellulosic Ethanol Production

16. Alcohol recovery Yeast Distillation & dehydration Sugarcane\ Sugarbeet Sweetsorghum Fermentation Storage tank Sugar Storage tank Crashing Sugar extraction Fuel blending Spent yeast Technologies for Ethanol Production Ethanol production from sugar

17. Alcohol recovery Cellulases Yeast Saccharification Fermentation Distillation & dehydration Agric Res Woody Material Grasses Steam explosion ~200ºC Water Storage tank mixing tank Chipping Grinding Fuel blending Spent material Technologies for Ethanol Production Ethanol production from cellulosics Pre-treatment

18. Pretreatment Hemicellulose Pretreatment for Ethanol Production Pretreatment - produces an enzyme accessible substrate Cellulose Lignin Amorphous Region Crystalline Region Ladisch, 2006

19. Enzymes required for CBP Component Cellulose Glucan 41.6 Xylan 15.9 Cellobiohydrolases Galactan 0.7 Endo-glucanase Glucose β-glucosidase 2.2 Mannan Endo-xylanase Arabinan 0.8 β-xylosidase Acetyl xylan esterase Acetic acid 5 α-glucuronidase Extractives 1.4 α-arabinofuransidase Hemicellulose Xylose Lignin 25.6 Endo-mannanase β-manosidase Manose Ash 0.5 etc. Arabinose Total 93.7 Partially hydrolyzed during feedstock pretreatment Galactose

20. Enzyme system development T. reesei secretome • CBHs are the major constituent of the T. reesei cellulase system • Second most important species are the EGs • Broad diversity of enzymes contributes to highly active system

21. O O Xyl Ara O O O Glu Man Gal T P YFG Technologies for Cellulose Conversion Consolidated BioProcessing (CBP) Glycosyl Hydrolases Ethanol + CO2

22. Technologies for Cellulose Conversion Growth on amorphous cellulose (PASC) [REF] [EG1] [SFI] [CEL5] Den Haan, R., S.H. Rose, L.R. Lynd, and W.H. Van Zyl. 2007. Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae. Met. Eng.9: 87–94.

23. Mascoma Corporation Technical facilities, Lebanon, NH, USA (www.mascoma.com)

24. Leading Investment, Unprecedented Focus on CBP Technical Focus: Overcoming the biomass recalcitrance barrier and enabling the emergence of a cellulosic biofuels industry via pioneering CBP technology integrated with advanced pretreatment Partners in Mascoma’s CBP Organism Development Effort • BioEnergy Science Center, USA • VTT, Finland • Department of Energy, USA • Dartmouth College, USA • University of Stellenbosch, ZA Three Platforms 1. T. saccharolyticum, thermophilic bacterium able to use non-glucose sugars 2. C. thermocellum, thermophilic cellulolytic bacterium 3. Yeast engineered to utilize cellulose and ferment glucose and xylose Multiple chances to succeed near-term & long-term

25. Screen CBH1 for high level expression • Enzyme activity: • 48 hour Avicel hydrolysis • Best enzyme x13 greater than starting point • Enzyme Production: • 94 mg/L CBH1 • ± 2.5% of Total cell protein in minimal medium + - + - + - + - + 97 66 45 30 57 94 29 37 mg/L secreted CBHI EndoH

26. Mascoma Cellulolytic Yeast Cellulase expression in Mascoma Yeast (robust C5/C6 fermenting) Improved host and culture conditions; ~2500X ↑ 50 50 40 40 Improved CBH2; ~1500X ↑ 30 30 Protein Expression (mg/g DCW) 20 20 Improved CBH1; ~400X ↑ 10 10 0 0 CBH2 expressed (Reinikainen et al., 1992) March 2007 March 2008 Oct. 2008 Dec. 2008 Cellulase expression Time-line

27. Enzyme Reduction on Paper Sludge Mascoma CBP technology on 18% w/w paper sludge Appearance at 120 hrs. 60 50 Background strain 40 Ethanol concentration, g/L 30 CBP + 1 mg Xylanase Background + Cellulase + BGL + Xylanase 20 Background +BGL + Xylanase 10 CBP strain 0 0 50 100 Time (hours)

28. Enzyme Reduction on Hardwood Mascoma CBP Strain (robust C5/C6 fermenting yeast) + 22% w/w unwashed Pretreated Hardwood 60 Equivalent performance with 2.5-fold less added enzyme Further reduction likely 50 40 30 Ethanol (g/L) 20 Non-CBP Yeast + 1X commercial cellulases CBP Yeast + 0.4X commercial cellulases 10 Improved CBP Yeast + 0.4X commercial cellulases 0 0 20 40 60 80 100 120 140 160 Fermentation Time (hours)

29. Rome, NY Pilot & Demonstration Plant January 2008 November 2008

30. Next generation technologies Thermochemical Technologies for Biofuel Production

31. Thermochemical technologies for Cellulose Conversion Technologies Lignocellulose thermo-chemical processes Combustion involves the burning of biomass in the presence of O2 with the harvesting of the released energy as primarily heat, or generating steam. Fast Pyrolysis involves the heating of biomass for few seconds to about 500°C in the absence of O2, followed by rapid cooling. The result is the formation of primarily,bio-oil from condensation of vapours during rapid cooling, biogas and solids called char. Gasification is taking place at higher temperatures for longer in the presence of O2, which yields syngas for Fischer-Tropsch synthesis (SASOL).

32. Application of Thermochemical Processes • Stellenbosch focuses for time been on fast and vacuum pyrolysis. • Decentralised or mobile pyrolysis units could substantially reduces transportation costs • Transportation-grade biofuels can be produced : • Bio-oil is a crude product that requires (expensive) upgrading • Biochar and/or bio-oil can be gasified either as the sole feedstock or in a mixture with coal • Production of electricity and/or heating from bio-oils, char and heating gas could be considered for premium markets

33. Current status on Thermochemical Processes Vacuum pyrolysis: • small batch lab unit (±100 g) (15kPa abs and Temp 300-450°C) – operational since 2002. • In SANERI project with Dr J Klaasen from UWC looked at kraalbos and renosterbos. • Looked at some pioneer/intruder plants, sewage sludge and bagasse. • Yields typically 25-35% char, 25-35% bio-oil. Fast Pyrolysis: • Commissioning small bench-scale set-up. • Looking at bagasse, corn cobs and Eucalyptis. • Fast Pyrolysis: < 10% char (high in ash), mostly bio-oil.

34. Next generation technologies Intergrating biochemical and thermochemical technologies

35. Non-Biological Processing Biological Processing Exported Power /Biofuels • • Gasification • • Fast pyrolysis • Power generation • • Synthesis & separation • Pretreatment • Fermentation • Separation Residues Steam Cellulosic Biomass Thermochemically -derived Chemicals (potentially several) Process Power Ethanol Biologically-derived Chemicals (potentially several) Animal feed Technologies for Cellulose Conversion Biomass Biorefinery Concept Other fermentation products

36. Ethanol & CO2 Starch-rich (grains) Animal feed, Lignin-rich residues Co-products* • (e.g.) maize/sorghum Oil Seeds Biodiesel • soya beans • canola/ sun flower Animal feed, Lignin-rich residues Glycerin, (co-products) Cellulosic Ethanol & CO2 Process energy (Electricity &/or Bio-oil/Char) Co-products*, feeds Residues • stover, bagasse Lignin-rich residues • paper sludge Energy Crops • grasses • intruder plants Evolution towards Cellulose Conversion Biofuel Feedstock & Product Options Ethanol & CO2 Sugar-rich Co-products* • sugar cane • sweet sorghum Lignin-rich residues *Coproducts may include industrial or ag. chemicals, sweeteners, neutraceuticals…

37. Ethanol Upgraded bio-oil (70% of residue) BtL (50%) Petrol Diesel South Africa’s potential: Biofuels production 30000 (70% of residue) 25000 20000 15000 Volumes in ML 10000 5000 0 Burned grasses Total Energy crops Maize Invasive plants Forest biowaste Agricult biowaste Strategy target Subtotal Curr fuel Biomass to ethanol = 280 L/ton Maize to Ethanol = 430 L/ton Biomass to upgraded bio-oils = 310 L/ton Biomass to liquid (BtL) = 570 L/ton

38. Future for Africa??

39. Acknowledgements Danie la Grange Riaan den Haan Shaunita Rose Ronél van Rooyen Tania de Villiers Maryna Saayman Johann Görgens John McBride Lee Lynd Alan Froehlich Elena Brevnova Erin Wiswall Haowen Xu Emily Stonehouse Heidi Hau Mark Mellon Kristen Deleaulte Naomi Thorngren Deidre Willies Vineet Rajgarhia Marja Ilmen Merja Penttilä