Bioethanol from Lignocellulose

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

2. Biomass Sources ECONOMICAL Corn Starch Corn Fiber Sugar Cane Paper Switch Grass Wood Chips Stover Cottonwoods ABUNDANT & AVAILABLE

3. 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 thebagasse and stillage can be used for the production of energy (ethanol and biogas) as well as component sugars (glucose, xylose, xylitol)

4. 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.

5. 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 US$1.8 billion/yr. % Ethanol in Gasoline (gasohol) 1977: 4.5% 1979: 15% 1981: 20% 1985: 22% 1998: 24% 1999: 20% 2002: 22% 2005: 25%

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

9. 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

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

12. Escherichia coli • An important vehicle forthe cloning and modification of genes • Ferment hexose and pentose as well with high ethanol yield by recombinant strains • High glycolytic fluxes • Reasonable ethanol tolerance

13. Klebsiella.oxytoca • Wide sugar utilization • Form ethanol through the PFL pathway after being modified • High ethanol yield

14. 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

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

16. 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

17. 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

18. BioEthanol from Bacteria: Klebsiella oxytoca The most promising ethanologenic bacteria are: Escherichia coli Zymomonas mobilis Klebsiella oxytoca

19. 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. (2003)

20. Klebsiella oxytoca: casAB operon Ingram et al. (1999) casA and casB genes allow K. oxytoca to transport and metabolize cellobiose

21. Klebsiella oxytoca: EtOH production EtOH is naturally produced through the Pyruvate Formate Lyase (PFL) pathway (similarly to E. coli) Dien et al. (2003)

22. 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

23. 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)

24. K. oxytoca, E. coli, Z. mobilis Dien et al. (2003)

25. 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

26. 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.

27. 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

29. Strategies Employed

30. 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 xyloseconsumptionand substantial xylitol secretion. • The problem of xylitol excretion is attributed to the cofactor imbalance (NAD+ and NADPH)

31. zwf1 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

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

33. 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.

34. 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)

35. GLN1 GLT1 GDH2 GDH1

36. 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.

37. 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

38. 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

39. Fermentation efficiency production

40. Source Fermentation Alternatives approach in Bioethanol production 10% ?% 20% - ?% Pre-treatment Ethanol Plant improvement: Sucrose content Pathogen response Photoreceptors Aluminum tolerance Microbial improvement: Fixing nitrogen to the plant Phytohormones: Auxin, giberillin and cytokinin. Antagonism against pathogens.

41. 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).

42. 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 • 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

43. 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.

44. 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.

45. 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.

46. 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%.

47. 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