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ENZYMATIC CONVERSION OF BIOMASS FORFUEL PRODUCTION

Annamma AnilDBT-UICT Centre of Energy Biosciences

Institute of Chemical Technology

Matunga, Mumbai

[email protected]

Training Course And Summer School

On

Next Generation Biofuels

Conducted byInternationalCentrefor

ScienceandHighTechnologyUnitedNationsIndustrial

DevelopmentOrganization

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Needs of human kind:

To find FUEL for transportation

- Alternative Liquid fuel

- Gas Fuel

To develop sustainable energy sources forprimary energy To develop renewable platform chemical source

Renewable Sources of Energy

Different generations of biofuels

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First generation Biofuels : Corn and sugar to ethanol

Chemical transesterification of veg oils

Second Generation Biofuels : Lignocellulose to ethanol

Enzymatic bioconversion of Vegetable oil

Fisher-Tropsch syngas to HC oil

Third generation Biofuels: Energy crops for bio-alcohol

Algal Hydrogen

Algal Oil/Biodiesel

Fourth generation Biofuels : GMO carbon-negative Energy crops

Evolution of Biofuels

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Conditions for better fuel options

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Algal Hydrogen

Algal Oil/Biodiesel


Evolution of Biofuels Loser in Food vs. Fuel

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Algal Hydrogen

Algal Oil/Biodiesel


Evolution of Biofuels Still struggling with technologies !!

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Algal Hydrogen

Algal Oil/Biodiesel



Future : Distant or near !!

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Enzymatic bioconversion of Vegetable oilFisher-Tropsch syngas to HC oil

Third generation Biofuels: Energy crops for bio-alcoholAlgal Hydrogen

Algal Oil/Biodiesel



Challenge : To use renewable sources to produce one ormore of the above in sustainable manner

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SUGAR- BASED BIOFUELS


Second Generation Biofuels : Lignocellulose to ethanolFisher-Tropsch syngas to HC oil



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Basic Concept

Complex macromolecules

Simple molecules

Chemicals

Hydrolyzed andbroken down

Converted

Bio-alcohol, Bio-

hydrogen, Bio-gas,

Bio-oil, Bio-desiel

Bioplastics, etc

Technology Innovation

Components:

- Biological SciencesPlant Biotechnology

Cellular/Molecular Biology

Microbial/Industrial Biotechnology

- Chemical Technology/Sciences

Catalysis

Reaction Engineering

Separations

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MethodsHydrolytic mechanisms

- to liberate free monosaccharides from the lignocellulosicpolysaccharides,

- acids and enzymes facilitate the process

Thermochemical processes- Dehydrate and volatilise both polysaccharides and lignin

- metal and catalysts can alter volatilisation profiles or reform thegases liberated

Each technology currently has its own drawbacks and

advantages

Range of procedures are needed to fully exploit the valuesof very diverse ranges of lignocellulosic feedstock.

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Different forms of carbohydrate macromolecules

Sugar cane juice --- glucose

Corn --- starch

Lignocellulosic matter - Cellulose,Hemicellulose, Lignin and other associatedchemical moieties

Can be

directly used

forfermentation

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Corn --- starch


Hydrolyzed to

glucose beforefermentation

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Corn --- starch


Pretreated,

separated into

individualcomponents

and

hydrolyzedbefore

fermentation

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Architectural arrangement of Lignocellulose

Lignocellulose model showing lignin, cellulose and hemicellulose

SOURCE: USDA Agricultural Research Service

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Biomass

Source

Kg/ton LBM (dry weight basis)

Cellulose Hemicellulose

Lignin

Softwood 450-500 250-350 250-350

Hardwood 400-550 240-400 180-250

SugarcaneBagasse

430 310 230

Straw 300-360 290 150-170

Corn cob 450 350 150

Cotton stalk 310 110 300

Composition of various potential lignocellulosic biomass as a

starting material for bioethanol

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CELLULOSE

HEMICELLULOSE

LIGNIN

Repertoire of enzymes for lignocellulosic hydrolysis

Fungiandv

eryfewbac

teria Lignin peroxidases (LiP),Manganese peroxidases (MnP)

Laccases

Endo-1-4--xylanase(endoxylanase),

acetyl esterase,-glucuronidase,-xylosidase

Endoglucanases

Cellobiohydrolase

-glucosidase

C. Snchez / Biotechnology Advances 27 (2009) 185194

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SACCHARIDE HYDROLYZING ENZYMES

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Cellulose: structural complexity

-(1,4)-D-glucopyranose units in4

C1 conformationFully equatorial conformation

Crosslinked intramolecularand

intermolecularhydrogen bondsof varying levels making it

crystalline or metacrystalline oramorphous

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Enzymatic hydrolysis of cellulose

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Hemicellulose

Xylan, Glucuronoxylan, Arabinoxylan, Glucomannan andXyloglucan.

Angiosperms: Xylan, is a polymer of(1-4)D-xylopyranose.

Gymnosperms : Glucomannans comprised primarily of D-mannosyl and D-glucosyl residues.

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Cellulases and Hemicellulases

Exo--l,4-glucanase hydrolyzes the chain by removing cellobiosemoiety from the non-reducing and reducing ends (solublecellodextrins and amorphous cellulose)

Endo--1,4-glucanase cleaves the chain at random internalpoints (crystalline cellulose)

-glucosidase catalyzes the hydrolysis ofcellobiose andcellodextrins

Enzymes may be non-complexed (extracellular) or complexed in

cellulosomes

Trichoderma. reesei produces at least two exoglucanases (CBHI andCBHII), five endoglucanases (EGI, EGII, EGIII, EGIV, and EGV), and two

glucosidases (BGLI and BGLII (358, 494, 664).

Enzymes may be non-complexed (extracellular) or complexed in

cellulosomes

Trichoderma. reesei produces at least two exoglucanases (CBHI andCBHII), five endoglucanases (EGI, EGII, EGIII, EGIV, and EGV), and two

glucosidases (BGLI and BGLII (358, 494, 664).

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Glycoside Hydrolases

-jelly roll-propeller

(/) 6 + -helix( /) 8

http://www.cazy.org/fam/acc_GH.html

Four clans GH-A, GH-D, GH-H, GH-K (Tim-Barrel fold)

27 different families Retention mechanism

Two catalytic residues - general acid (proton donor) and anucleophile/base residues

Include -glucosidases, Endo1,4 xylanase, Exocellobiohydrolase,Endo-1,4-beta-glucanase and many diverse glycoside bondscleaving enzymes

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Hydrolyzed products D-xylose

Pentose sugars cannot be

metabolized by Saccharomyces

cerevisiae.

Technologies to overcome this

problem are required.

Cellulose

Crystalline

Difficult to hydrolyzeEfficient and low-cost

technologies are

required.

Cellulose

Crystalline

Difficult to hydrolyzeEfficient and low-cost

technologies are

required.

Hydrolyzed product D-glucose

Easily fermented to ethanol with

conventional systems

Hemicellulose

Amorphous

Relatively easy tohydrolyze

Hemicellulose

Amorphous

Relatively easy tohydrolyze

Two major technological barriers

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LIGNIN DEGRADING ENZYMES

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Chemical nature of lignin

Made up of hydroxyphenyl

(H), guaiacyl (G), and syringyl (S)-type phenylpropane units

The phenylpropane units are

connected by different types of

carboncarbon (e.g. 55') and ether

(e.g. -O-4) linkages.

Softwood and hardwood lignins are

mainly of the G and GS types,

respectively, while grasses and

other non-wood lignocellulosic plantsmay contain a significant proportionof H-type lignin.

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Lignin degrading enzymes

Ligninolytic mechanisms in fungi involve secreted fungal

peroxidases

These enzymes and a variety of extracellular systems to

produce H2O2 used for oxidising lignin

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Laccases

Blue copper phenoloxidase

Can oxidise phenolics and non-phenolics

Lignin peroxidases

strong oxidants interacting directly with nonphenolic lignin

structure

inefficiently, and apparently cannot penetrate the small pores in

sound lignocellulose.

Manganese peroxidases

Small, diffusible strong oxidants that can penetrate thesubstrate,

Cleave the principal structures of lignin in low yields.

Lignin degrading enzymes

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Engineered Lignin peroxidase (1LGA) from

Phanerochaete Chrysosporium

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Manganese peroxidase (1MNP) from Phanerochaete

Chrysosporium

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The use of these enzyme are being explored

for better and efficient pretreatment

strategies.

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Lignocellulosic Biomass Fractionation

Recalcitrant part Biodegradable part

EnergyCellulose hemicellulose lignin

hydrolysis topentosans Chemical Feedstocks

hydrolysis tohexoses Chemical Feedstocks

EnzymaticEnzymatic

Bio/conversions

Bio/conversions

PRE-TREATMENTS !!

Energy/Chemicals

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Lignocellulosic Biomass Fractionation

Recalcitrant part Biodegradable part

EnergyCellulose hemicellulose lignin

hydrolysis topentosans Chemical Feedstocks

hydrolysis tohexoses Chemical Feedstocks

EnzymaticEnzymatic

Bio/conversions

Bio/conversions

PRE-TREATMENTS !!

Energy/Chemicals

Recalcitrant lignin, Inhibitors formed,

Hemicelluloses and cellulose occluded by lignin

Pretreatment methods harsh, difficulty indepolymerisation

Natura

lMOGsfew

,inhib

itor

issues,

utilize

fractions

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Enzymatic Saccharification of Biomass components

cellulose and hemicellulose : Need for Mixture of Improved Enzymes

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Enzymes - One of the bottlenecks in the bioethanol technology

Problems :

Low specific activity of enzymes;High cost,

Low heat and pH stability of enzymes;

Product inhibition to the enzymes;

Lack of understanding of enzyme biochemistry and rxn mechanism

Loss of enzyme activity due to irreversible binding of enzyme to substrate

Improved enzymes required through

- Site directed mutagenesis- Directed Evolution

- QSAR methods

- Metabolic Engineering for cheap production

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Lipases for biodesiel

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Biodiesel

Transesterification - the displacement of alcohol from an ester by

another alcohol in a process similar to hydrolysis

Methanol and ethanol are utilized most frequently, especially

methanol because of its low cost and its physical and chemical

advantages.

Fatty acid methyl esters (known as biodiesel fuel) obtained by

transesteritication can be used as an alternative fuel for diesel

engines.

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Transesterification can be achieved using acid- and alkali

catalysis, lipase enzyme, and supercritical fluids

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Enzymes for transesterification

OILS

Transesterification

Separation of

reactionmixture

Purification of glycerol Glycerol

Methyl estersUpperphase

Lowerphase

Microbial lipases Aspergillus niger, Bacillus thermoleoVorans, Candidacylindracea, Candida rugosa, Chromobacterium viscosum, Geotrichum

candidum, Fusarium heterosporum, Fusarium oxysporum, Humicola lanuginose,Mucor miehei, Oospora lactis, Penicillium cyclopium, Penicillium roqueforti,Pseudomonas aeruginosa, Pseudomonas cepacia,Pseudomonasfluorescens,Pseudomonas putida, Rhizomucor miehei and other strains andStaphylococcus hyicus

Animal sources are from pancreatic lipases, and plant lipases are from

papaya latex, oat seed lipase, and castor seed lipase

Soluble,immobilizedor

intracellular

enzymes

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Structural attributes for lipases

Schematic representation of the solvent accessible van der Waals surface of the lid andadjacent residues in the wire frame representation of : HL lipase (1TIB); RM lipase (3TGL

closed and open ; CAL B lipase (1LBS). Active site residues are displayed as CPK models;and lid or neighboring residues are displayed as Van der Waals surface.

M. Petkar et al. / Journal of MolecularCatalysis B: Enzymatic 39 (2006) 8390

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Total enzyme cost = Enzyme cost/kg X Activity X Amount of enzyme

Improvedproduc t i v i t y

Improvedproduct ion

Improvedef f ic iency

Enzymes with higher specific rates, thermostability and reduced

recalcitrance

Prerequisites for any process

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Directions in Cellular/Molecular/Synthetic Biologyfor Second/Third/Fourth Generation Biofuels

Improvement of Enzymes through Structure-Activity Relationships

- Cellulases and Hemicellulases

- Strains capable of CBP (composite bioprocessing) i.e.

combined treatment and SSF

- Lignin degrading enzymes/microbes

- current degradations too slow- no commercial enzyme production

- important but neglected biomass component

- Lipases

- commercial lipase for more efficient oil trans-esterifcation

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PROCESS and ENZYME BIOTECHNOLOGY for

- Cost effective saccharification of lignocellulosic biomass

- LBM fractionation

- New designed Cellulase and Hemicellulase systems

- New enzyme reactor systems

- Cost effective saccharification of algal biomass

- Engineering algal strains for optimal production

- Algal biomass is not wholly cellulosic

- New enzymes like mannanases and glucanases required

- Cost effective enzymes for delignification processes

- Current enzymes incapable of giving sustainable solution/s

- Need to design new enzymes and expression systems

- New approaches to Fermentation system design- Need to increase productivities and control

- Possible with cleaner feedstock/s

REQUIRED : Molecular and Synthetic Biology approaches

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SYNTHETIC and SYSTEMS BIOLOGY for

- Improved expression systems for conversion of plant and

algae biomass derived sugars and other components to

- Ethanol

- Higher alcohols e.g. butanol

- Biodiesel (fatty acid mono-esters)

- Hydrocarbons- Biohydrogen

Genomics for enzyme design and pathway engineering

Proteomics for enzyme design

Metabolomics for optimal fermentation conditions

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Integrated

Science &

Engineering

for

Technology

Development

Integrated

Science &

Engineering

for

Technology

Development

We are at

Institute of Chemical Technology (ICT)Mumbai, INDIA

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DBT-ICT Centre of Energy Biosciences

- Indias first National Bioenergy Research Centre

- Being set up at an initial cost of Rs. 24.8 crore

- Multidisciplinary Centre with emphasis on cutting-edge

technology development and transfer to Indian industry

- Networked with Institutions & Industry in India and abroad

>40 PhD scholars; several Senior Research Scientistsand >10 faculty in different disciplines of modern biological

sciences and bioengineering

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CENTRAL THEME

To Develop Alternative Energy Technologies

- Through intervention of Biological Sciences

- Specific Emphasis on Renewable Liquid Fuel

- Innovation and Translation of Technologies

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ICT Technology forLignocellulosic Ethanol

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Salient Features of the ICT Technology (Patents filed)

1. Novel combination of known fractionation technologies

to obtain homogeneous fractions of

CELLULOSE; HEMICELLULOSE AND LIGNIN

2. Two Step Enzymatic Hydrolysis of cellulose and hemicellulose

3. Two Step combination of Membrane Reactor andColumn Enzyme Reactor to permit reuse of enzymes

over many cycles

4. All steps - pretreatment, fractionation and hydrolysis to

fermentable sugars and finally ethanol operated incontinuous reactor systems

5. Both glucose and xylose converted to ethanol in near theoretical

yields using indigenous mutated strains

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Biomass Fractionation

The Novel combination Technology

- Converts toughest biomass to soft biomass to

CELLULOSE; HEMICELLULOSE AND LIGNIN

- 125C Alkaline Hydrolysis : Mild Conditions

- No un-desirable derivatives

- Products ideal for next step enzyme hydrolysis

- Continuous Process - Easy to control

- Lignin recovered intact for value-added uses

- Complete alkali recovery

- Recovery of acetic acid, silicates, uronates, xylose and arabinose

En matic h drol sis of cell lose/hemicell lose

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Enzymatic hydrolysis of cellulose/hemicellulose

G G G G G

G

G

G G G

G G G G G G G G G G

G

G G

G G

G GGG GG GG

G G

G

G

G G

G

G

G GG GG GG G

G G G

Endo-hydrolases

action

Processive

Exo-hydrolase

action

Glycosidase

Action

INHIBITION

Mono-sugars Glucose and Pentose

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Two Step Enzyme Hydrolysis

- Use Endo- and Exo- Enzymes separate from Glycosidase

- Reaction speeded up 6 times by Division of enzyme action

in two steps

- Complete Hydrolysis of cellulose to glucose and hemicellulose

to pentose (xylose)

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Features/Advantages of the Technology

- Brings down enzyme cost by factor of more than 5

- Brings down energy costs esp. in heat form

- Fractionation produces clean substrates

- Results in longer life of both enzyme reactors and membranes

- Continuous Technology reduces capital cost component

- Both glucose and xylose converted to ethanol

- Ethanol yield per ton of dry biomass >300L

- Estimated cost of production of Ethanol < USD 0.50/L

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Important Events

- Patents filed for- Two Step Enzyme Process

- Combination of membrane + immobilized

reactors process

- Composite reactor technology

- Lignin separation by dual electrolyte process

- MOU signed with India Glycols Limited, Uttrakhand forPilot plant Design and Commissioning

by Dec. 2009 at capacity of 10 ton biomass/day

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Vegetable Oil Biorefinery

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RED PALM OIL

Solvent

Purified carotene

And tocopherol

ExtractionColumn

CaroSep +TocoColumns

PretreatmentResin Column

2-EnzymeColumnReactor

GlycerolCaptureColumn

Glycerol

BIODIESEL

(after simple MeOH/Solvent recovery)

Process Flow Sheet for Recovery of Carotene and Tocopherol fromRed Palm Oil Followed by Immobilized Lipase Catalyzed Productionof Biodiesel coupled with On-line glycerol separation

FFA,

phospholipids

etc

MeOH

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SUMMARY

- No one complete solution for future energy

- Interplay of several sciences and technology disciplines required

- Modern biotechnology to play a major role

- Next generation biotechnology will derive major mileage from energysector than pharma sector and constraints and playing fields

completely different

- Scale of operation to be much larger and diverse than current liquid fueltechnologies

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Thank you

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