Brazilian Potential for Biomass Ethanol: Challenge of Using Hexose and Pentose Co- Fermenting Yeast Strains

Journal of Scientific & Industrial Research 918Vol. 67, November 2008, pp.918-926 J SCI IND RES VOL 67 NOVEMBER 2008

Brazilian potential for biomass ethanol: Challenge of using hexose and pentose co- fermenting yeast strains

Boris U Stambuk1*, Elis C A. Eleutherio2, Luz Marina Florez-Pardo3 Ana Maria Souto-Maior4 and Elba P S Bon5 1Laboratório de Biologia Molecular e Biotecnologia de Leveduras, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil 2Laboratório de Investigação de Fatores de Estresse, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil 3Departamento Sistemas de Produccion, Universidad Autonoma de Occidente, Cali, Colombia 4Departamento de Antibióticos, Universidade Federal de Pernambuco, Recife, PE, Brasil 5Laboratório de Tecnologia Enzimática, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

Received 15 July 2008; revised 16 September 2008; accepted 01 October 2008

This paper reviews Brazilian scenario and efforts for deployment of technology to produce bioethanol vis-à-vis recent international advances in the area, including possible use of hexose and pentose co-fermenting yeast strains.

Keywords: Biomass ethanol, Brazilian ethanol production, Ethanologenic yeasts, Hexose-pentose co-fermentation

Introduction ethanol, 109 produce only ethanol and 16 produce only Brazil produces annually 5 x 109 gallons of ethanol sugar. Brazil will produce around 27 billion litre of ethanol from sugarcane grown on 5 million ha. Sugarcane is in 2008, and plans to build 41 new distilleries before 2010, processed in 365 sugar/ethanol producing units, and it and 45 more until 20156. Land use for energy crops as is forecasted that 86 new distilleries will be built before compared to food crops has been opted for renewable 2015. Sugarcane juice can go either directly to fuels with use of biomass polysaccharide part for the fermentation vats for ethanol production, or be used to production of fuel ethanol7-9. A simple mass balance produce sugar (crystal sucrose, and its residue (Fig. 1) for harvested sugarcane indicates that molasses), depending on which product is priced more lignocellulosic-derived fuel ethanol could add up to > 50% favorably. As a consequence, 75 million tonnes of bonus over total amount of ethanol currently produced bagasse are produced annually. In each sugar and/or from sugarcane, considered that the amount of sugars ethanol mill, approx. 85% of bagasse is burned for the found in sugarcane biomass could be recovered and production of steam (heat) and power/electricity fermented at ~90% efficiency4,10-12. generation. Structure of lignocellulosic biomass (LB) is represented by physicochemical interaction of cellulose, with Bioethanol from Sugarcane hemicellulose and lignin13-15. Cellulose is a highly ordered Brazil has been a front-runner to substitute liquid fuel polymer of cellobiose whose length may present 10,000 gasoline by renewable ethanol1-5. State of São Paulo is glycosyl units in cellulose chain that form fibrils. biggest sugarcane producer (68% of total sugarcane Hemicelluloses are shorter (degree of polymerization plantations in Brazil). Out of 365 sugar/ethanol within 100-200), highly branched heteropolymers of producing units in Brazil, 240 produce sugar and xylose, arabinose, mannose, galactose and glucose as well as uronic acids. Depending on predominant sugar type, *Author for correspondence hemicelluloses are referred to as mannans, xylans or E-mail: [email protected] galactans. Pentose and hexose sugars, linked through 1,3, STAMBUK et al: BRAZILIAN BIOETHANOL 919

Fig. 1—Bioethanol production from sugarcane [current technology of sugarcane processing and fuel ethanol production from sucrose (black arrows) is illustrated at the right side of the figure, while the required technological achievements for bioethanol production from sugarcane bagasse and straw (biomass pre-treatment, hydrolysis and pentose fermentation, gray arrows) are shown on the right side of the figure]

1,6 and 1,4 glycosidic bonds and often acetylated, form observed low sugar yields, formation of inhibitors for a loose and very hydrophilic structure that acts as a subsequent ethanol fermentation step, and corrosion of glue between cellulose and lignin. equipments. Besides, acidic pH of sugar hydrolysate requires a neutralization step prior to fermentation. Parallel to the implementation of Brazilian Proálcool Considering detrimental aspects of acid hydrolysis, Program, in 1970s, several research institutions and studies focused on enzyme-based route that involves companies have carried out research for the production biomass pretreatment because LB are structured for of bioethanol, using primarily and municipal cellulosic strength and resistance to biological, physical and solid wastes. The first pilot-scale facility was built in chemical attack. Pretreatments17,18 (steam explosion, 1981 at Fundação de Tecnologia Industrial (FTI, hydrothermolysis or using catalytic amounts of acid), Lorena, SP) to run using the Scholler-Madison process render raw cellulose digestible by cellulases. Sugarcane based on concentrated sulphuric acid to hydrolyze bagasse was pretreated via steam explosion using a 1.6 l Eucalyptus panicutata to produce 500 l ethanol per day. reactor19. These studies paved way for development of Brazilian company Dedini, in Piracicaba (SP), began sugarcane bagasse steam explosion process used to date in 1987 the development of a biomass-to-ethanol in Brazil for the production of cattle feed. production technology (called Dedini Hidrolise Rapida, Presently, “Bioethanol Project: Bioethanol Production DHR), in partnership with Copersucar (presently Centro through Enzymatic Hydrolysis of Sugarcane Biomass”, de Tecnologia Canavieira) and State of São Paulo initiated in 2006, is supported by Brazilian Ministry of Research Supporting Foundation (FAPESP). DHR Science and Technology through its Research and Projects technology for ethanol production is an organosolv Financing agency (FINEP), and is carried out by a hydrolysis single-stage process16 that employs very network of more than 20 institutions, including universities diluted H2SO4 for hydrolysis of cellulose and and research institutes. This project aims to develop a hemicellulose to sugars, and an organic solvent for viable technology for conversion of sugarcane bagasse lignin extraction. DHR’s unique feature is reduced and straw into fuel ethanol, minimizing expansion of hydrolysis reaction time (few min) in a continuous high- sugarcane fields. It is expected that Brazilian sugar mills throughput process, with quick cooling of hydrolysate. will be gradually converted into biorefineries, able to However, all projects that studied acid hydrolysis eventually process all fractions of sugarcane. This 920 J SCI IND RES VOL 67 NOVEMBER 2008

technology will use all already existing facilities in sugar Best yeast strains24,27,28 known for fermenting xylose and alcohol plants including juice/sugars syrup treatment are P. stipitis, P. tannophilus and C. shehatae. Yeast system, fermentation & distillation, cogeneration, P. stipitis is capable of converting xylose and all major wastewater & residues treatment and recycling, lignocellulosic sugars to ethanol (yield, 0.3-0.44 g/g of instrumentation and automatic control systems, substrate). Although this is suitable for some waste management, and commercialisation. Biomass sugar streams, commercial fermentation for fuel ethanol syrups will be blended with molasses or with sugarcane requires higher performance24,28. juice prior to fermentation carried out by S. cerevisiae Over the past 20 years, metabolic engineering has yeast strains. As a result, lower biomass ethanol been used to construct bacterial and yeast strains with production cost is expected compared with plants advantageous traits for lignocellulosic ethanol dedicated to produce biomass ethanol solely. For production26,29-31. Efforts are concentrated on three most enzymatic hydrolysis of pretreated biomass, the project promising microbial platforms23,29-33 (S. cerevisiae, Z. is developing enzyme blends in situ for sugarcane mobilis, and E. coli). Since S. cerevisiae has been used bagasse. for mature industrial technologies of ethanol production To produce ethanol from LB economically, it is essential from cane and grain sugars, several strategies that have to have a biocatalyst able to ferment hexose and pentose been used to engineer this yeast towards pentose sugars under adverse conditions of an industrial fermentation (Fig. 2) include insertion of bacterial and environment. Yeast S. cerevisiae, applied successfully yeast/fungal pentose-utilization genes34-39, selection or in industrial production of ethanol from sugarcane juice overexpression of the own S. cerevisiae xylose utilization and molasses in Brazil, is able to ferment hexoses rapidly pathway40,41, improvement of xylulose consumption and efficiently20, and exhibits high ethanol tolerance, through overexpression of xylulokinase and pentose- GRAS status, and tolerance to low pH. However, S. phosphate pathway enzymes42-47, engineering redox cerevisiae for use in a second generation process cofactor regeneration to reduce by-product formation, (lignocellulose-to-fuel ethanol process) cannot metabolize in particular xylitol48-51, and application of random pentoses, although development of S. cerevisiae pentose- mutagenesis and evolutionary engineering to select for utilizing strains could be integrated into existing ethanol improved traits52-55. plants. Engineering industrial strains that would be able Unfavorable kinetic properties of enzymes with an to co-ferment efficiently all sugars in lignocellulose imperfect match of cofactor, and an inadequate pentose hydrolysates to ethanol is, however, still a challenge for phosphate pathway, apparently limit ability of recombinant molecular biologist and engineers worldwide21,22. yeast to ferment xylose28,55. Despite development of Pentose Fermentation recombinant strains with improved xylose fermentation performance, high products yields and increased product In 1922, Willaman and co-workers showed that concentrations are key targets that have yet to be Fusarium lini could ferment xylose into ethanol. Since achieved. Recent transcriptome and proteome analysis then, many bacteria have been found to metabolize and have indicated that xylose is not perceived as a ferment hexose and pentose sugars, but all produced an fermentable sugar by S. cerevisiae56,57 probably because unwanted mixture of fermentation products. Although, signaling pathways that ensure efficient utilization of homo-ethanol pathway is found in Z. mobilis, substrate hexoses58-60 do not recognize pentoses xylose or range this organism can metabolize (glucose, fructose arabinose. Thus, genetic modifications that could have and sucrose) is very limited23,24. Many yeasts able to greatest impact on economic feasibility of these ferment xylose were also found, including several fermentations should include amplification and Candida, Pichia, Hansenula, Debaromyces, deregulation of rate-limiting reactions61. Schwanniomyces, etc. If xylose was converted to xylulose, several more yeast could ferment it directly to Sugar uptake from medium is major rate limiting step ethanol, including S. cerevisiae24,25. Thus, although many for fermentation of even naturally fermentable sugars yeasts are able to ferment xylose, large-scale utilization (glucose, fructose, maltose, maltotriose) by is hampered by their low tolerance to ethanol and S. cerevisiae62-65. In general, overexpression of cognate hydrolysate inhibitors, requirement for microaerophilic sugar transporters, which can mediate facilitated diffusion conditions, and an inability to ferment xylose at low pH26. or active transport of sugar across plasma membrane STAMBUK et al: BRAZILIAN BIOETHANOL 921

Fig. 2—Pentose fermentation by yeasts [Simplified scheme of xylose and arabinose metabolic pathways present in yeasts and fungi (black lettering), as well as the bacterial arabinose pathway that has been successfully expressed in S. cerevisiae (gray lettering), and their integration into the main glycolytic pathway towards ethanol production. Abbreviations: AR, L- arabinose(aldose) reductase; LAD, L-arabinitol 4-dehydrogenase; LXR, L-xylulose reductase; XR, D-xylose(aldose) reductase; XDH, xilitol dehydrogenase; XI, D-xylose isomerase; XK, D-xylulokinase; AI, L-arabinose isomerase; RK, L- ribulokinase; RPE, L-ribulose-5-P 4-epimerase]

(Fig. 3), improves significantly fermentation performance xylose transporters36,71,74,75, which is in good agreement of cells66-69. Xylose and arabinose uptake across plasma with analysis of mutated and evolved S. cerevisiae cells membrane is also rate-limiting for pentose fermentation showing improved pentose fermentation due to an by S. cerevisiae, specially because this yeast transports increased expression of HXT5, HXT7 and GAL2 xylose and arabinose with very low affinity, compared permeases36,51-54. with uptake of other fermentable sugars34,36,47,70,71. All yeast species that use xylose and/or arabinose have both In P. stipitis, xylose uptake is also considered rate- low- and high-affinity pentose transport activities, limiting step for xylose utilization76. Although genome of covering a wide range of sugar concentrations extending this yeast indicates presence of a large family of sugar to at least 2-3 orders of magnitude. Affinities for xylose transporter genes77, only three sugar permeases are always lower than those for glucose. While most characterized in P. stipitis are high affinity glucose strains present both high-affinity arabinose/xylose-H+ permeases that transport xylose with significantly lower symport and low-affinity facilitated transport of affinity78. This study also revealed first example of sugar sugars72,73 (Fig. 3) in S. cerevisiae, these pentoses are permeases whose expression is regulated by oxygen transported solely by facilitated transport mediated by availability, which may be also present in other yeasts hexose permeases. Expression of any of the major HXT and fungi72,79. Although pentose transport activities are transporters (HXT1-HXT7 and GAL2) allows growth of described in several other yeasts, only other known an hxt-null strain on xylose74,75. Of these transporters, xylose/glucose permease genes were isolated from mid-to-high-affinity glucose permeases (HXT4, HXT5, C. intermedia through functional expression in an HXT7 and GAL2) were more effective arabinose and hxt-null S. cerevisiae strain80. While there are still many 922 J SCI IND RES VOL 67 NOVEMBER 2008

Fig. 3—Sugar transport by yeasts [Illustration of sugar transport mechanisms present in yeasts, including the high capacity sugar facilitated diffusion transporters (which in general have affinity for hexoses in the mM range, and in the M range for pentoses), and the active sugar-H+ permeases (which in general have affinity in the ¼M range for monosaccharides, and for disaccharides in the mM range) driven by the proton motive force (created by the plasma membrane H+-ATPase) that allows the intracellular accumulation of the sugar]

limitations towards functional expression of heterologous process87-89. This process is a fed-batch fermentation active pentose permeases in S. cerevisiae71,81, that uses high cell densities (~10 g yeast/l) allowing very overexpression of a xylose/glucose facilitated diffusion short residence times for fermentation to be completed transporter from P. stipitis is reported to significantly (<8-10 h), and thus up to 3 fermentation runs can be enhance xylose fermentation by a S. cerevisiae strain performed per day, contributing to overall process engineered to metabolize this sugar82. success. Normally, final sugar concentration employed is also high (~180 g/l), with high conversion yields (up to Current Fuel Ethanol Technology in Brazil: 92%) due to high cell density process that limits cell Challenges and Opportunities for Bioethanol growth. Crucial to the performance of this process is Industrial production of fuel ethanol in Brazil uses raw constant recycling of yeast slurry into fermentors, after sugarcane juice and/or molasses as substrate. Sugarcane treatment with H2SO4, a procedure that ensures less juice contains significant amounts of yeast and bacteria, bacterial and wild yeast contaminations. Consequently, depending on different factors mainly related to cane yeast fitness for industrial process includes high tolerance variety and age, time lag between cane harvesting and to fermentative stresses (high temperatures, ethanol and processing, etc.83-86. While bacterial infections can be osmotic pressure, low pH, production interruptions, etc.), controlled by antibiotics and acid treatment of the yeast and yeast should also dominate fermentation tanks during cells, yeast contaminations can have a significant impact whole crop season. Indeed, analysis of yeast population in fermentation performance of distillery. In wine dynamics during fuel ethanol production in Brazil has fermentations, D. bruxellensis, major contaminant in revealed existence of selected yeast strains that tend to industrial fuel ethanol plants in Brazil, can cause dominate a given industrial process90. Actually, some decreased productivity as well as other operational yeast strains (PE-2, CAT-1) are commercially available problems85,86. Thus, to ensure high productivities and as starters for fuel ethanol industry efficiency in industrial process, currently fuel ethanol (www.fermentec.com/en/index.html), showing high plants (> 80%) work with an adaptation of Melle-Boinot alcohol productivity and increased resistance to stressful STAMBUK et al: BRAZILIAN BIOETHANOL 923 conditions found in this industrial process5,87, again 2 Goldemberg J, Ethanol for a sustainable energy future, Science, contributing significantly to overall process economical 315 (2007) 808-810. success. 3 Wheals A E, Basso L C, Alves D M & Amorim H V, Fuel ethanol after 25 years, Trends Biotechnol, 17 (1999) 482-487. 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