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Lignocellulosic biomass to ethanol-hydrolysis and

fermentation

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Agenda

Enzymatic hydrolysis» Cellulases

What is fermentation?» Fermentation inhibitors» Separate Hydrolysis and Fermentation

(SHF) and Simultaneous Saccharification and Fermentation (SSF)

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BiomassPretreatment

Liquid phase

Solid phase

Cellulose

Sugars EthanolFermentation

Ethanol SugarsFermentation

Hydrolysis

LigninRecovery

Bioconversion of biomass to ethanol (hydrolysis)

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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.

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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

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Cellulases

Endoglucanases  (EG) cutting the cellulose chains randomly

Cellobiohydrolyses (CBH) cutting cellobiose units of the ends of the cellulose chains

Binding domain Catalytic domain

7 Pretreated substrate Handsheet Microplate

“Rapid microassay method (1)”

Pretreated substrate Flasks

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“Rapid microassay method (2)”

Handsheets

Microplate

ShakerMicroplate

Reader

HPLC

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Equipment

1mL 200 mL 4L

40L

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BiomassPretreatment

Liquid phase

Solid phase

Cellulose

Sugars EthanolFermentation

Ethanol SugarsFermentation

Hydrolysis

LigninRecovery

Bioconversion of biomass to ethanol (pretreatment)

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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

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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?

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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

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Fermentation (1)

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Fermentation (2)

Conversion factor 0.51

1g/L of glucose: 0.51g/L ethanol (maximum)

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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

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HMFs and Furfurals

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Experimental

Corn

fibre Hydrolysis Fermentation

SteamExplosion(solid +liquid

fraction)

Corn

fibre SSF

SteamExplosion(solid +liquid

fraction)

SHF50°C, pH 4.848 hours

30°C, pH 612 hours

37°C, pH 524 hours

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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)

SHF SSF