bioethanolpresentation 204152827 by 5Ceol0j


									            Bioethanol from
Group 10:
Alessandro Fazio     International collobaration
Fen Yang                  for the production
Marcelo Bertalan             of Bioethanol
Vijaya Krishna
Woril Dudley
                     Biomass Sources

        Sugar Cane                Fiber

                                       Wood Chips

                                   ABUNDANT & AVAILABLE
                   The Products
• 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)
                     The World Ethanol Market
•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.
                               World Ethanol Production









                      Brazil   U.S.       China       India   Others
            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.                                               % Ethanol in
•Today there are 9 million vehicles with hydrated ethanol.           (gasohol)

•Anhydrous ethanol is utilized in 25% blend with gasoline.          1977: 4.5%
                                                                    1979: 15%
•The production of ethanol reduces petroleum importation. In the
                                                                    1981: 20%
last 22 yr, an economy of US$1.8 billion/yr.
                                                                    1985: 22%
                                                                    1998: 24%
                                                                    1999: 20%
                                                                    2002: 22%
                                                                    2005: 25%
         Ethanol cost x Oil cost
•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

•However, the energy originating from 1 L of
ethanol corresponds to 20.5 MJ, and from 1 L
of gasoline, 30.5 MJ.
    Criteria for microorganisms
•    Broad substrate utilization

•     Converting hexose and pentose to ethanol

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

•     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
•    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

•    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

 The most promising ethanologenic
           bacteria are:

          •Escherichia coli

        •Zymomonas mobilis

         •Klebsiella oxytoca
       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 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

                                                                 SSF conditions:
                                                                    35-37 C
                                                                   pH 5.0-5.4

                                                   Dien et al.
              Klebsiella oxytoca: casAB operon

                                                                  Ingram et al.

casA and casB genes allow K. oxytoca to transport and metabolize cellobiose
Klebsiella oxytoca: EtOH production

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

 Dien et al. (2003)
       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
      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
= K. oxytoca SZ21 (pCPP2006)

However, the strain fermented poorly
cellulose without addition of commercial

                             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
  Bottlenecks in using bacteria for industrial
             production of EtOH

     •Production of EtOH in large reactors
     •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.
                Metabolic Engineering
               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
                                Strategies Employed
Strategy                                               Result

Insertion of pentose utilization genes XYL1(xylose     a & b) Could grow on xylose but the ethanol yield was
    reductase) and XYL2 (xylitol dehydrogenase) from       less
a) Over expression of XYL1+XYL2                        c) Could produce more ethanol but still was not
b) Chromosomal integration of XYL1 and XYL2                economically viable. A ratio of 0.06 had higher
c) Expressing different ratios of XR and XDH and           xylose consumption and lower xylitol formation.
    over expression of TKL1 and TAL1
Improvement of xylulose consumption                    a)   Capable of growing on xylose alone. xylitol yield
                                                            was still high. xylose was fermented with 66% of
a) Expressing the gene XKS1 (xylulo kianase) XYL1           the theoretical yield. In a mixture of sugars, 90% of
      and XYL2                                              the yield was achieved but arabinose was not
b) Expressing the genes XKS1, XYL1 and XYL2 in a
      multi-copy vector.                               b)   Unstable in non-selective media.

c) Chromosomal integration of the above strain.        c)   Chromosomal integration solved the problem
                                                            resulting in a stable strain. 70% of the theoretical
d) Expressing the genes XKS1, XYL1 and XYL2 in a            yield attained in a glucose-xylose mixture.
      single-copy vector.
                                                       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)
                       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
    The strategy of redox metabolism to improve
      the strain for the conversion of xylose to
• 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.
Results of first strategy:

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


• 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.
                       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 <=> 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)
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
• But deletion of gdh1 alone reduced the ethanol yield
  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

• 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 production
Sugar 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                                     8.3
    (under Brazilian production conditions)
                                                    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 production
  Alternatives approach in Bioethanol production
  ?%          20% - ?%             10%

Source     Pre-treatment      Fermentation        Ethanol

 Plant improvement:        Microbial improvement:
 Sucrose content           Fixing nitrogen to the plant
 Pathogen response         Phytohormones: Auxin, giberillin
 Photoreceptors            and cytokinin.
 Aluminum tolerance        Antagonism against pathogens.
     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
              million hectares
            Brazil´s Territory 850.00
            Total Arable Land 320.00
            Cultivated - all crops 60.40
            - with Sugar Cane 5.34
            -for ethanol 2.66

            Denmark´s Territory 4.3
            Total Arable Land 2.679
• 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 pollution
• Studies have found (Australia) that the use of
  – 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

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