Lignocellulosic biomass to ethanol-hydrolysis and fermentation

1. Lignocellulosic biomass to ethanol-hydrolysis and fermentation

2. Agenda • Enzymatic hydrolysis • Cellulases • What is fermentation? • Fermentation inhibitors • Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF)

3. Bioconversion of biomass to ethanol (hydrolysis) Liquid phase Sugars Ethanol Pretreatment Fermentation Biomass Lignin Recovery Solid phase Cellulose Hydrolysis Fermentation Sugars Ethanol

4. Enzyme Function • There are a large number of fungal enzymes responsible for the breakdown of each wood component. Each enzyme plays specific roles: • Endo-beta-1,4-glucanase acts within the chain, breaking it into smaller units and providing more "ends" for CBH. • Cellobiohydrolase (CBH), acts on the end of the molecule successively cleaving off the disaccharide cellobiose. • Beta-glucosidase (or cellobiase) which cleaves cellobiose to two glucose units.

5. Trichoderma reesei • Trichoderma reesei is an industrially important cellulolytic filamentous fungus. • T. reesei: • present in nearly all soils and other diverse habitats • favored by the presence of high levels of plant roots. Trichoderma reesei

6. Cellulases Binding domain Catalytic domain Endoglucanases  (EG) cutting the cellulose chains randomly Cellobiohydrolyses (CBH) cutting cellobiose units of the ends of the cellulose chains

7. “Rapid microassay method (1)” Pretreated substrate Flasks Pretreated substrate Handsheet Microplate

8. “Rapid microassay method (2)” Microplate Handsheets HPLC Shaker Microplate Reader

9. Equipment 1mL 200 mL 4L 40L

10. Bioconversion of biomass to ethanol (pretreatment) Liquid phase Sugars Ethanol Pretreatment Fermentation Biomass Lignin Recovery Solid phase Cellulose Hydrolysis Fermentation Sugars Ethanol

11. Fermentation • Defined as: Cellular metabolism under anaerobic conditions (absence of oxygen) for the production of energy and metabolic intermediates • Many organisms can “ferment” (i.e., grow anaerobically) • Not all produce ethanol as an end-product of fermentation • Butanol • Acetic acid • Propionic acid • Lactic acid

12. Strain selection • Choice of microorganism for ethanol production has traditionally been a Yeast • Yeast: • Single cell microorganism • Fungi • Facultative anaerobe • Most common industrial fermenter is Saccharomyces cerevisiae (baker’s or brewer’s yeast) • Why?

13. Why S. cerevisiae? • Has been selected over thousands of years • High ethanol yield and productivity • Relatively simple to culture • G.R.A.S organism • Robust: • High ethanol tolerance • Resistant to inhibitors

14. Fermentation (1)

15. Fermentation (2) Conversion factor 0.51 1g/L of glucose: 0.51g/L ethanol (maximum)

16. Inhibitors • 5 groups of inhibitors • Released during pretreatment and hydrolysis • Acetic acid and extractives • By-products of pretreatment and hydrolysis • HMFs and furfurals, formic acid • Lignin degradation products • Aromatic compounds • Fermentation products • Ethanol, acetic acid, glycerol, lactic acid • Metals released from equipment

17. HMFs and Furfurals

18. Experimental Steam Explosion (solid +liquid fraction) Corn fibre Hydrolysis Fermentation SHF 50°C, pH 4.8 48 hours 30°C, pH 6 12 hours Steam Explosion (solid +liquid fraction) Corn fibre SSF 37°C, pH 5 24 hours

19. Pros and cons of SHF and SSF SHF SSF • Pros • Separate temp. for each step (hydrolysis 50°C, fermentation 30°C) • Possibility of yeast and enzyme recovery • Cons • Requires two sets of fermenters • End-product inhibition • Pros • Minimized end-product inhibition • Requires only one set of fermenters • Cons • Difficulties in recovery and yeast and enzyme recycling • Temperature/pH compromise (37°C)