Bioethanol from Lignocellulose

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Bioethanol from Lignocellulose
Group 10 Alessandro Fazio Fen Yang Marcelo
Bertalan Vijaya Krishna Woril Dudley
International collobaration for the production
of Bioethanol
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Biomass Sources
ECONOMICAL
Corn Starch
Corn Fiber
Sugar Cane
Paper
Switch Grass
Wood Chips
Stover
Cottonwoods
ABUNDANT AVAILABLE
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The Products

• Ethanol
• 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)
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The World Ethanol Market

• Total World Ethanol production in 2004 40.92
  billion Litres.
• Global ethanol market will be worth over US16
  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.

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The Brazilian Ethanol Experience

• 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
  US1.8 billion/yr.

Ethanol in Gasoline (gasohol) 1977 4.5 1979
15 1981 20 1985 22 1998 24 1999 20
2002 22 2005 25

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Ethanol cost x Oil cost

• The direct cost of 1 l of gasoline in the USA was
  US0.21 and the cost of 1 l of ethanol was
  US0.34.
• The average cost of sugarcane production in
  Brazil was US180/t of sugar or US0.20/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.

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Criteria for microorganisms

• Broad substrate utilization
• Converting hexose and pentose to ethanol
  efficiently.
• High ethanol yield (gt90 of theoretical) and
  productivity
• High tolerance to acids, ethanol, inhibitors
  and process hardiness.
• Can be robust to simple growth medium

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• However, no natural microorganism displays all of
  the features.
• Metabolic engineering of microorganism is a very
  efficient tool for increasing bioethanol yield.

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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

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Klebsiella.oxytoca

• Wide sugar utilization
• Form ethanol through the PFL pathway after
  being modified
• High ethanol yield

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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

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Bacillus stearothermophilus

• Thermophilic organisms fermenting hexose and
  pentose after being modified
• Avoid the limitation of high concentration of
  ethanol harmful to fermentaion

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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

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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

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BioEthanol from Bacteria Klebsiella oxytoca

• The most promising ethanologenic bacteria are
• Escherichia coli
• Zymomonas mobilis
• Klebsiella oxytoca

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BioEthanol from Bacteria 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 ß-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

SSF conditions 35-37 C pH 5.0-5.4
Dien et al. (2003)
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Klebsiella oxytoca casAB operon
Ingram et al. (1999)
casA and casB genes allow K. oxytoca to transport
and metabolize cellobiose
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Klebsiella oxytoca EtOH production
EtOH is naturally produced through the Pyruvate
Formate Lyase (PFL) pathway (similarly to E. coli)
Dien et al. (2003)
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Klebsiella oxytoca metabolic engineering for
EtOH production
Strategy redirection of metabolism towards EtOH
production through the insertion of pet operon
PDC
ADH
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
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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)
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K. oxytoca, E. coli, Z. mobilis
Dien et al. (2003)
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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

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Bottlenecks in using bacteria for industrial
production of EtOH

• Production of EtOH in large reactors
• Contamination
• GRAS status
• Relevant economic advantages respect to yeasts
  (e.g. reduced need for enzymes)

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

• 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

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Strategies Employed
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• 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)

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First Strategy

• 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

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

• Xylitol NADP ltXRgt D-xylose NADPH H
  (1)
• Xylitol NAD ltXDHgt 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.

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• 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.

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Second Strategy
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.
a) Deletion of GDH1 Reaction 1 is encoded by
GDH1 and reaction 2 is encoded by GDH2
L-Glutamate NAD H2O ltgt 2-Oxoglutarate
NH3 NADH H . (1) L-Glutamate NADP
H2O ltgt 2-Oxoglutarate NH3 NADPH H..(2)
b) Over expression of GDH2 or GS-GOGAT system
(GLT1GLN1) Reaction 1 is encoded by GLT1 and
reaction 2 is encoded by GLN1 (Alternate
pathway) 2 L-Glutamate NAD ltgt L-Glutamine
2-Oxoglutarate NADH (1) ATP L-Glutamate
NH3 ltgt ADP Orthophosphate L-Glutamine
... (2)
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Results

• 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.

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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

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Bioethanol efficiency production

• Sugar cane yields the best energy balance in
  production of ethanol.

Macedo, I. et alii, F.O. Lichts 2004
David Pimentel D. And Tad W. Patzek 2005
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Fermentation efficiency production
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Alternatives approach in Bioethanol production
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20 - ?
?
Ethanol
Pre-treatment
Plant improvement Sucrose content Pathogen
response Photoreceptors Aluminum tolerance
Microbial improvement Fixing nitrogen to the
plant Phytohormones Auxin, giberillin and
cytokinin. Antagonism against pathogens.
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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).

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Ethical and Conclusion

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

million hectares

• Brazils Territory 850.00
• Total Arable Land 320.00
• Cultivated - all crops 60.40
• - with Sugar Cane 5.34
• for ethanol 2.66
• Denmarks Territory 4.3
• Total Arable Land 2.679

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• 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.

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• 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.

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• 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.

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Blend 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.

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• 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