Bioethanol from Lignocellulose

Group 10:Alessandro FazioFen YangMarcelo BertalanVijaya KrishnaWoril Dudley

International collobaration for the production

of Bioethanol

ABUNDANT & AVAILABLE

ECONOMICAL

Corn Starch

Switch Grass

Biomass Sources

Paper

Sugar Cane

Cottonwoods

Stover

Wood Chips

Corn Fiber

The ProductsEthanol:• Fuel (crops and residues) 68%

– Anhydrous Ethanol, gasoline aditive– Hydroethanol destined for Biofuels

• Beverages (crops) 11% • Perfumes & Pharmacology (crops) 21%

Alternative Products:• Sugar Powder (crops)• Biodegradable Plastic (crops)

– Polyhydroxybutyrate-PHB

*In Sugarcane the bagasse and stillage can be used for the production of energy (ethanol and biogas) as well as component sugars (glucose, xylose, xylitol)

•Total World Ethanol production in 2004: 40.92 billion Litres. •Global ethanol market will be worth over US$16 billion by 2005 •The largest consuming regions are South America and Asia. •In Brazil the sugar-ethanol market trade reaches about $7.5 billion/yr.

The World Ethanol Market

World Ethanol Production

0

5

10

15

20

25

30

35

40

Brazil U.S. China India Others

%

The Brazilian Ethanol Experience

% Ethanol in Gasoline (gasohol)

1977: 4.5%1979: 15%1981: 20%1985: 22%1998: 24%1999: 20% 2002: 22%2005: 25%

% Ethanol in Gasoline (gasohol)

1977: 4.5%1979: 15%1981: 20%1985: 22%1998: 24%1999: 20% 2002: 22%2005: 25%

•Oil price: 1973: $2.50/barrel. 1979: $20.00. 1981: $34.40/barrel

•In 1973 Brazil development of the first car fueled by hydrated ethanol in the world.

•Today there are 9 million vehicles with hydrated ethanol.

•Anhydrous ethanol is utilized in 25% blend with gasoline.

•The production of ethanol reduces petroleum importation. In the last 22 yr, an economy of US$1.8 billion/yr.

•The direct cost of 1 l of gasoline in the USA was US$0.21 and the cost of 1 l of ethanol was US$0.34.

•The average cost of sugarcane production in Brazil was US$180/t of sugar or US$0.20/L of ethanol.

•However, the energy originating from 1 L of ethanol corresponds to 20.5 MJ, and from 1 L of gasoline, 30.5 MJ.

Ethanol cost x Oil cost

Criteria for microorganisms

• Broad substrate utilization

• Converting hexose and pentose to ethanol efficiently.

• High ethanol yield (>90% of theoretical) and productivity

• High tolerance to acids, ethanol, inhibitors and process hardiness.

• Can be robust to simple growth medium

• However, no natural microorganism displays all of the features.

• Metabolic engineering of microorganism is a very efficient tool for increasing bioethanol yield.

Bacteria Escherichia coli

Klebsiella oxytoca

Zymomonas mobilis

Bacillus stearothermophilus

Yeast Saccharomyces cerevisiae

Pachysolen tannophilus

Candida shehatae

Pichia stipitis

Escherichia coli

• An important vehicle for the cloning and modification of genes

• Ferment hexose and pentose as well with high ethanol yield by recombinant strains

• High glycolytic fluxes

• Reasonable ethanol tolerance

Klebsiella.oxytoca

• Wide sugar utilization

• Form ethanol through the PFL pathway after being modified

• High ethanol yield

Zymomonas mobilis

• A gram-negative, natural fermentative bacteria in ethanol production

• The only bacteria which can use Entner-Doudoroff pathway anaerobically

• Unable to ferment pentose but hexose

• Limitation of using lignocellulose

• Relatively easier to receive and maintain foreign genes

• High ethanol yield

Bacillus stearothermophilus

• Thermophilic organisms fermenting hexose and pentose after being modified

• Avoid the limitation of high concentration of ethanol harmful to fermentaion

Saccharomyces cerevisiae • The most common and natural fermentative yeast

for ethanol

• Only convert glucose to ethanol for wild-type

• Limitation of using lignocellulose

• Relative high ethanol yield

• Can be easily modified by metabolic engineering to ferment pentose

Other yeasts

Pachysolen tannophilus, Candida shehatae, and Pichia stipitis

• Ferment xylose

• Low ethanol yields

• High sensitivity to inhibitors, low PH and high concentration of ethanol

BioEthanol from Bacteria: Klebsiella oxytoca

The most promising ethanologenic bacteria are:

•Escherichia coli

•Zymomonas mobilis

•Klebsiella oxytoca

Main features:

• Enteric Bacterium (Gram negative)

• EtOH is formed through the PFL (Pyruvate Formate Lyase) pathway, like in E. coli

• It produces its own β-GLUCOSIDASE and therefore it is able to metabolize dimeric (cellobiose) and trimeric (cellotriose) sugars, besides monomeric (hexoses and pentoses) sugars

• Less enzymes are required for the pre-treatment of cellulose: economic advantage for the solubilization of cellulose

BioEthanol from Bacteria: Klebsiella oxytoca

Dien et al. (2003)

SSF conditions: 35-37 C

pH 5.0-5.4

Klebsiella oxytoca: casAB operon

casA and casB genes allow K. oxytoca to transport and metabolize cellobiose

Ingram et al. (1999)

Klebsiella oxytoca: EtOH production

Dien et al. (2003)

EtOH is naturally produced through the Pyruvate Formate Lyase (PFL) pathway (similarly to E. coli)

Klebsiella oxytoca: metabolic engineering for EtOH production

Strategy: redirection of metabolism towards EtOH production through the insertion of pet operon

Pet operon: Pyruvate decarboxylase (PDC) and Alcohol dehydrogenase (ADH)

Two main strains were produced:

K. Oxytoca M5A1 + plasmids with pet operon = K. Oxytoca M5A1 (pLOI555)

K. Oxytoca M5A1 + chromosomal integration of pdc and adhB from Z. mobilis = K. Oxytoca P2

PDC ADH

Klebsiella oxytoca: metabolic engineering for cellulose hydrolysis

K. Oxytoca P2

+ two extracellular endoglucanase genes (CelZ and CelY) from Erwinia chrysanthemi.

+ out gene for secretion from Erwinia chrysanthemi

= K. oxytoca SZ21 (pCPP2006)

However, the strain fermented poorly cellulose without addition of commercial cellulose

Zhou and Ingram (2000)

K. oxytoca, E. coli, Z. mobilis

Dien et al. (2003)

Possible strategy for the future

• Since casAB operon insertion has been attempted in E.coliKO11, a possible strategy could be the integration of casAB operon and endoglucanase genes in S.cerevisiae genome in order to allow this yeast to solubilize cellulose and, therefore, to reduce the cost of the process

Moreover, industrial acceptance of recombinant bacteria will depend upon the relative success of yeast microbiologists in developing industrially relevant pentose-fermenting Saccharomyces strains.

•Production of EtOH in large reactors

•Contamination

•GRAS status

•Relevant economic advantages respect to yeasts (e.g. reduced need for enzymes)

Bottlenecks in using bacteria for industrial production of EtOH

• Saccharomyces cerevesiae is unable to ferment pentoses. Metabolic engineering can be used to make S.cerevesiae able to ferment xylose, the main component of pentoses.

• The efficiency of the constructed strain depends on its substrate utilization range, to use all the sugars of lignocellulose substrate

• Xylose metabolism involves conversion of xylose to xylulose, whcih after phosphorylation, is metabolized through pentose phosphate pathway

Metabolic Engineering Metabolic Engineering of of

Saccharomyces cerevesiaeSaccharomyces cerevesiae

Strategies Employed

Strategy Result

Insertion of pentose utilization genes XYL1(xylose reductase) and XYL2 (xylitol dehydrogenase) from P.Stipitis

a) Over expression of XYL1+XYL2b) Chromosomal integration of XYL1 and XYL2c) Expressing different ratios of XR and XDH and

over expression of TKL1 and TAL1

a & b) Could grow on xylose but the ethanol yield was less

c) Could produce more ethanol but still was not economically viable. A ratio of 0.06 had higher xylose consumption and lower xylitol formation.

Improvement of xylulose consumption

a) Expressing the gene XKS1 (xylulo kianase) XYL1 and XYL2

b) Expressing the genes XKS1, XYL1 and XYL2 in a multi-copy vector.

c) Chromosomal integration of the above strain.

d) Expressing the genes XKS1, XYL1 and XYL2 in a single-copy vector.

a) Capable of growing on xylose alone. xylitol yield was still high. xylose was fermented with 66% of the theoretical yield. In a mixture of sugars, 90% of the yield was achieved but arabinose was not metabolised.

b) Unstable in non-selective media.

c) Chromosomal integration solved the problem resulting in a stable strain. 70% of the theoretical yield attained in a glucose-xylose mixture.

d) 25% of the theoretical yield was attained in a minimal medium containing glucose and xylose.

• Now S.cerevesiae can ferment xylose efficiently through genetic modifications

• But the expected ethanol cannot be obtained in any case and resulted in a low rate of xylose consumption and substantial xylitol secretion.

• The problem of xylitol excretion is attributed to the cofactor imbalance (NAD+ and NADPH)

• The metabolic strategy applied was to delete the zwf1 gene encoding the glucose-6-phosphate dehydrogenase in the strain with the genes XKS1, XYL1 and XYL2 expressed in a multi-copy vector.

• As it can be seen the main source of NADPH originating form the oxidative part of the pentose phosphate pathway has there by been reduced

First Strategy

• Xylitol + NADP+ <=XR=> D-xylose + NADPH + H+ ……… (1)

• Xylitol + NAD+ <=XDH=> D-xylulose + NADH + H+....…… (2)

• As it can be seen from the reaction (1) that xylose reductase is NADPH dependent and reaction (2) that xylitol dehydrogenase is NAD+ dependent.

• The imbalance leads to more of the first reaction and less second reaction, thus forming a lot of xylitol and less converted to xylulose.

The strategy of redox metabolism to improve the strain for the conversion of xylose to

ethanol

Results of first strategy:

• Significant improvement of ethanol yield. • Reduction of xylitol yield.

Explanation:

• The possible explanation for this is that with the less availability of NADPH, it is using NADH to convert xylose to xylitol releasing NAD+. Inorder to reconvert the NAD+ it is utilising it to form xylulose from xylitol.

a) Deletion of GDH1

Reaction 1 is encoded by GDH1 and reaction 2 is encoded by GDH2

L-Glutamate + NAD+ + H2O <=> 2-Oxoglutarate + NH3 +NADH + H+ …….…… (1) L-Glutamate + NADP+ + H2O <=> 2-Oxoglutarate + NH3+ NADPH + H+……..…(2)

b) Over expression of GDH2 or GS-GOGAT system (GLT1+GLN1)

Reaction 1 is encoded by GLT1 and reaction 2 is encoded by GLN1 (Alternate pathway)

2 L-Glutamate + NAD+ <=> L-Glutamine + 2-Oxoglutarate+ NADH ………… (1)ATP + L-Glutamate + NH3 <=> ADP + Orthophosphate +L-Glutamine ………... (2)

The strategy applied was to modulating the redox metabolism to favour xylose metabolism through metabolic engineering of ammonium assimilation in the strain with the genes XKS1, XYL1 and XYL2 expressed in a multi-copy vector.

Second Strategy

a) Results:• Increased Ethanol yield.• Decreased Glycerol yield. • The specific growth rate reduced dramatically.

b) Results:• Specific growth rate could now be recovered.

Experimental results:• Glycerol decreased in both the cases• The specific growth rate could be recovered in the second

case• But deletion of gdh1 alone reduced the ethanol yield

substantially.

Results

Possible strategies for the future

A future possibility is to find a mutant strain that can ferment both xylose and arabinose, thus utilizing all the pentoses of lignocellulose.

• Insertion of genes for arabinose metabolism and xylose transport will increase the pentose utilization. Genes for arabinose metabolism can be obtained form yeasts such as Candida aurigiensis and for xylose transport from P.stipitis.

• Expression of the genes araA (L-arabinose isomerase), araB (L-ribulokinase), araD (L-ribulose-5phosphate-4-epimerase) from E.coli into the mutant strain of S.cerevesiae for arabinose metabolism

Bioethanol efficiency productionBioethanol efficiency productionSugar cane yields the best energy balance in production of ethanol.

Raw Material Energy Output / Energy Input

Wheat 1.2

Corn 1.3 – 1.8

Sugar Beet 1.9

Sugar Cane (under Brazilian production conditions)

8.3

Macedo, I. et alii, F.O. Lichts 2004

Raw Material Energy Output / Energy Input

Wood 0.47

Switchgrass 0.50

Corn 0.71

David Pimentel D. And Tad W. Patzek 2005

Fermentation efficiency productionFermentation efficiency production

Alternatives approach in Bioethanol production

EthanolFermentationSource Pre-treatment

Plant improvement:Sucrose contentPathogen responsePhotoreceptorsAluminum tolerance

Microbial improvement:Fixing nitrogen to the plantPhytohormones: Auxin, giberillin and cytokinin. Antagonism against pathogens.

10%20% - ?% ?%

Pre-treatment of Lignocellulose for bioethanol fermentation

• It was considered necessary to give a brief overview of this pre-treatment step, since the method employed can have implications for fermentation conditions and the choice of microbe.

• The hydrolysis is usually carried out by the use of enzymes or by chemical treatment.

• Enzymatic Hydrolysis• This is carried out by cellulose enzymes which are highly specific. • Novozymes is launching three new enzymes which make the

production of ethanol from wheat, rye and barley up to 20% • The new enzymes break down components of the grain which

would otherwise result in a thick consistency. This saves producers the amount of water and energy that would otherwise be required to dilute and handle the mash. A thinner mash also makes life easier for the enzymes in the next stage of the process, which break the material down into sugars for fermentation into ethanol (alcohol).

Ethical and Conclusion • Lands used for lignocellulose production for

ethanol production, could be used for edible crops, in helping to alleviate current food shortage

Brazil´s Territory 850.00Total Arable Land 320.00Cultivated - all crops 60.40- with Sugar Cane 5.34-for ethanol 2.66

Denmark´s Territory 4.3Total Arable Land 2.679

million hectares

• From the present statistics, about 57% more energy is required to produce a litre of ethanol than the energy harvested from ethanol using lignocellulose. The poor tropical countries of the world are best suited for the growth of sugar cane, and most of these countries have vast unused lands that could be utilized for this purpose.

• It would therefore be an advantage to all parties to used the vast resources being spent on trying to make something work which might not be economically viable, to helping these countries cultivate sugar cane on a large scale, and then either locating ethanol plants there, or having the harvested cane shipped to the developed countries for the fermentation process. It would provide much needed cash flow for some of these countries.

• Ethanol from sugar cane although more efficient, still consumes more energy than is produced. It therefore means that a lot of the energies being channelled into metabolic engineering for lignocellulose bioethanol production could be used for finding means of improving this process, which represents greater economic viability.

Blend gasoline - urban pollutionBlend gasoline - urban pollution

• Studies have found (Australia) that the use of E10:– Decreased CO emission by 32%;– Decreased HC emission by 12% ;– Decreased toxic emissions of 1-3 butadiene

(19%), benzene (27%), toluene (30%) and xylene (27%);

– Decreased carcinogenic risk by 24%.

• In the USA, wintertime CO emissions have been reduced by 25% to 30%.

• Conclusion:• For bioethanol from lignocellulose to be a

viable alternative to fossil fuel, then the cost of production will have to be reduced.

• The perfect microbe that provides broad substrate utilization, give high ethanol yields and is tolerant to the harsh conditions after chemical pretreatment will have to be engineered

• Reduction in process costs, by integrating process engineering tools with metabolic engineering