1 Lignocellulosic biomass to ethanol-hydrolysis and fermentation

2 Agenda lEnzymatic hydrolysis »Cellulases lWhat is fermentation? »Fermentation inhibitors »Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF)

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

4 Enzyme Function lThere 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 lTrichoderma reesei is an industrially important cellulolytic filamentous fungus. lT. reesei: »present in nearly all soils and other diverse habitats »favored by the presence of high levels of plant roots. Trichoderma reesei

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

7 Pretreated substrate HandsheetMicroplate “Rapid microassay method (1)” Pretreated substrate Flasks

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

9 Equipment 1mL200 mL4L 40L

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

11 Fermentation lDefined as: Cellular metabolism under anaerobic conditions (absence of oxygen) for the production of energy and metabolic intermediates lMany organisms can “ferment” (i.e., grow anaerobically) lNot all produce ethanol as an end-product of fermentation »Butanol »Acetic acid »Propionic acid »Lactic acid

12 Strain selection lChoice of microorganism for ethanol production has traditionally been a Yeast lYeast: »Single cell microorganism »Fungi »Facultative anaerobe lMost common industrial fermenter is Saccharomyces cerevisiae (baker’s or brewer’s yeast) lWhy?

13 Why S. cerevisiae? lHas been selected over thousands of years lHigh ethanol yield and productivity lRelatively simple to culture lG.R.A.S organism lRobust: »High ethanol tolerance »Resistant to inhibitors

14 Fermentation (1)

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

16 Inhibitors l5 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 Corn fibre Hydrolysis Fermentation Steam Explosion (solid +liquid fraction) Corn fibre SSF Steam Explosion (solid +liquid fraction) SHF 50°C, pH hours 30°C, pH 6 12 hours 37°C, pH 5 24 hours

19 Pros and cons of SHF and 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) SHFSSF