Recent Advances In Fermentation of Lignocellulosic Biomass Hydrolysate to Ethanol

The world’s present economy is greatly dependent on various fossil energy sources such as oil, coal, natural gas, etc., being used for the production of fuel, electricity and other goods (Uihlein et al., 2009). Rising energy consumption, diminution of fossil fuels and increased environmental concerns has shifted the focus of energy generation towards biofuel use. Bioethanol is considered the most potential next generation automotive fuel because it is carbon-neutral and could be produced from renewable resources like lignocellulosic biomass (Kumar et al., 2009). The cost and availability of the feedstock are crucial as it contributes 65–70% to the total ethanol production costs (Balat and Balat, 2009). Lignocellulosic biomass (such as agricultural residues, forestry wastes, waste paper, municipal solid wastes, and energy crops) has been considered as possible raw material for ethanol production due to its renewability, large quantities, low prices (relative to grain or sugar), and potential environmental benefits (Chen, 2011; Talebnia et al., 2010 and Hickert et al., 2013). Lignocellulose is the most plentiful renewable biomass produced from photosynthesis, and its annual production was estimated in 1×1010 MT worldwide (Sanchez and Cardona, 2008). In general, lignocellulosic feed-stocks are divided into three categories: (1) agricultural residues (e.g., crop residues and sugarcane bagasse), (2) forest residues, and (3) herbaceous and woody energy crops (Carriquiry et al., 2011). Usually, lignocellulosic biomass contains 35 - 50% cellulose, 25 - 30% hemicelluloses and 20 - 25% lignin. Cellulose, a polymer of glucose residues connected by β-1, 4 linkages, being the primary structural material of plant cell wall, is the most abundant carbohydrate in nature (Saha et al. , 2006). Hemicellulose is a short, complex carbohydrate structure that consists of different polymers like pentoses (like xylose and arabinose), hexoses (like mannose, glucose and galactose), and sugar acids (Hendriks and Zeeman, 2009). Lignin is the third major component, a complex polymer of phenyl propane (p-coumaryl, coniferyl and sinapyl alcohol), act as cementing agent provides plants with the structural support and an impermeable barrier against microbial attack and oxidative stress (Howard et al., 2003). The biological process for converting lignocellulose to fuel ethanol requires: (1) delignification to liberate cellulose and hemicellulose; (2) depolymerization of carbohydrate polymers to produce free sugars; and (3) fermentation of mixed hexose and pentose sugars to produce ethanol (Balat and Balat, 2009). The key requirements for an economical lignocellulosic ethanol process include: efficient pretreatment methods of lignocelluloses, availability of low-cost hydrolytic enzymes, and use of optimal microbial strains capable of converting hexose and pentose sugars (Chen et al., 2012) to ethanol, at high rates (Chen, 2011). Pretreatment is the first and most important step in cellulose to ethanol technology because it can remove hemicelluloses, lignin and increase the

Physical–chemical pretreatment of lignocellulosic biomass can generate some soluble inhibitory compounds, derived from a partial sugars and lignin degradation, may be toxic to fermenting microorganisms and hinder utilization of sugars

Biological inhibitor abatement is a probable method for eliminating inhibitory compounds from the biomass hydrolysates. In this regard a fungal isolate, Coniochaeta ligniariaNRRL30616, metabolizes furfural and 5-hydroxymethylfurfural (HMF) as well as aromatic and aliphatic acids and aldehydes. NRRL30616 grew in corn stover dilute-acid hydrolysate, and converted furfural to both furfuryl alcohol and furoic acid. Hydrolysate was inoculated withNRRL30616, and the fate of pretreatment side-products was followed in a time-course study. A number of aromatic and aliphatic acids, aldehydes, and phenolic compounds were quantitated by analytical extraction of corn stover hydrolysate, followed by HPLC–UV–MS/MS analysis. Compounds representing all of the classes of inhibitory side-products were removed during the course of fungal growth. Biological abatement of hydrolysates using C. ligniaria improved xylose utilization in subsequent ethanol fermentations (Nichols et al., 2008). With respect to lignocellulosic biomass, one of the detoxification methods, fungal laccase and peroxidase enzymes have been used experimentally to detoxify wood hydrolysates (Martin et al., 2002). Laccase was expressed in recombinant Saccharomyces to increase resistance to phenolic compounds (Larsson et al., 2001). Using laccases enzymes has been explored in which a substantial removal of phenolic compounds by laccases reduced the inhibitory effects of slurry from steam-exploded wheat straw. It led to improve the fermentation performance of thermotolerant yeast strain Kluyveromyces marxianus used, shortening its lag phase and enhancing the ethanol yields, and increase the substrate loadings of saccharification and fermentation broths. According to this study, detoxification by laccases could reduce costs of lignocellulosic ethanol process through the use of partially detoxified whole slurry and increasing higher fermentation rates and ethanol yields (Moreno et al., 2012). In a study, hemicellulose hydrolysate from corncobs, separated by diluted sulfuric acid and sequently detoxed by boiling, overliming and solvent extraction, hydrolysate on xylitol production was investigated. It was found that glucose in hydrolysate promoted growth of Candida tropicalis while acetate at high concentration was inhibitory. The acetate inhibition can be alleviated by adjusting pH to 6 prior to fermentation and a substrate feeding strategy. Under these optimum conditions, a maximal xylitol concentration of 68.4g l−1was obtained after 72h of fermentation, giving a yield of 0.7gg−1 xylose and a productivity of 0.95gl−1 h−1 (Cheng et al., 2009). Alternative methods such as ammonia/sodium hydroxide (NaOH)-neutralization to improve the efficacy of hydrolysate conditioning for ethanol production have been proposed due to no gypsum generated and reduced xylose loss (Pienkos and Zhang, 2009). A new yeast strain of Clavispora NRRL Y-50464 has been reported that is able to utilize cellobiose as sole source of carbon and produce sufficient native β-glucosidase enzyme activity for cellulosic ethanol production using SSF. In addition, this yeast is tolerant to the major inhibitors derived from lignocellulosic biomass pre-treatment such as 2-furaldehyde (furfural) and 5-(hydroxymethyl)-2-furaldehyde (HMF), and converted furfural into furan methanol in less than 12 h and HMF into furan-2,5-dimethanol within 24 h in the presence of 15 mM each of furfural and HMF (Liu et al., 2012).

FERMENTATIVE TECHNIQUES

In the process of ethanol production from lignocellulosic materials, enzymatic hydrolysis and fermentation can be carried out by separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) (Romani et al., 2012). SHF allows the fermentation and hydrolysis to be performed at separate conditions; hence the fermenting organism and the enzymes can be used at independent optimum temperature and pH. However, SHF results in enzyme inhibition by the hydrolysis products, i.e. as the hydrolysis progresses, the sugar concentration in the hydrolysis bioreactor increases which can reduce the efficiency of the cellulase enzymes used (Soderstrom et al., 2005). Compared with separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) is more favoured because in SSF glucose released by the action of cellulase is converted quickly to ethanol by the fermenting microorganism, thus minimizing end-product inhibition to cellulase caused by glucose and cellobiose accumulation (Sassner et al., 2008; Zhao and Xia, 2009 and Jang et al., 2012). In this option, the cellulose hydrolysis and glucose fermentation steps are combined in a single vessel (Ghosh et al., 1982). Since cellulase is inhibited by glucose as it is ethanol concentrations than possible for SHF (Sasikumar and Viruthagiri, 2010). Combining the saccharification and fermentation processes in one vessel is found to be better alternative to separate hydrolysis and fermentation (SHF) in terms of cost (De Bari et al., 2002), perhaps due to reduced process time, lower energy requirement and high bioethanol yields at high solid loading (Ohgren et al., 2007; Nikolic et al., 2009; Lee et al., 2013 and Ofori-Boateng and Lee, 2014). Simultaneous saccharification and fermentation (SSF) is considered an appropriate process that presents significant advantages for conversion of lignocellulosic biomass to ethanol (Olofsson et al., 2008). In spite of the economic advantage of SSF over separate hydrolysis and fermentation (SHF), the critical problem associated with SSF of cellulose is the difference in temperature optima for saccharification (45–50◦C) and fermentation (25–35◦C). Saccharomyces strains are well known as good ethanol producing microorganisms; however they require an operating temperature of 35◦C. Fungal cellulases, which are most frequently applied in the cellulose hydrolysis, have an optimum temperature of 50◦ C. At lower temperatures, the substantially lower hydrolysis rates would be unfavorable in terms of increased processing time. The fermentation efficiency of S. cerevisiae at high temperatures is very low due to increased fluidity in membranes to which the yeast responds by changing its fatty acids composition (Suutari et al., 1990). A possible solution to solve this problem is using thermotolerant yeast strains instead of Saccharomyces, which would allow higher processing temperatures, thus increased rates of the hydrolysis (Kadar et al., 2004). In another study, the thermal treatments made it possible to have one strain, IR2-9a, with greater ethanol yield in SSF process than the control strains (Saccharomyces cerevisiae). With this strain it was possible to convert pretreated lignocellulosic material into ethanol at temperatures closer to the optimal for enzymatic hydrolysis making the SSF process more efficient (Edgardo et al., 2008). Genetic engineering has been employed to develop the various aspects of fermentation from higher yield to better and wide substrate utilization to increased recovery rate (Sarkar et al., 2012). Researchers routinely use HPLC method for monitoring ethanol production during fermentation of biomass pretreatment hydrolysate; ethanol production during simultaneous saccharification and fermentation (SSF); and measuring acetic acid and furans formed during pretreatment (Mohagheghi et al., 2006). In conventional SSF procedures, after pretreatment, cellulolytic enzymes are applied to hydrolyze the cellulose polymers into short oligosaccharides such as cellobiose. Since the commonly used ethanologenic yeast Saccharomyces cerevisiaeis unable to utilize cellobiose, an additional enzyme, β-glucosidase, is cellobiose/xylose co-fermentation strategy provides an opportunity to efficiently utilize lignocellulosic biomass for microbial lipid production. A oleaginous yeast, Lipomyces starkeyi, was shown to consume cellobiose and xylose simultaneously and to produce intracellular lipids from cellobiose, xylose and glucose (Gong et al., 2012). This cellobiose/xylose co-fermentation strategy by passes glucose repression and is expected to improve the economics of lipid production when lignocellulosic biomass is employed as raw material. The reducing sugar produced during hydrolysis were concentrated and used for ethanol production by S. Cerevisiae and S. Stipitis and their co-culture. Highest ethanol production with co-culture was 20.8 g/L and co-culture of S. Cerevisiae and S. Stipitis produced 32% more ethanol than S. Cerevisiae alone and 41% more ethanol than S. Stipitis alone (Singh et al., 2014). Coupling of SSF process with ultrasound can accelerate the production rate of bioethanol at shorter time. In a study, SSF of OPFs was combined with ultrasound irradiation to assess the efficiency of the process on bioethanol yield (Ofori-Boateng and Lee, 2014). Many different types of processes for ethanol fermentation have been proposed, including batch fermentation, continuous fermentation, continuous fermentation with cell recycling, fed-batch cultures and repeated-batch cultures. Batch fermentation process is used extensively to convert sugars to ethanol for the production of beverages and biofuels. As for fed-batch fermentation, the intermittent addition of glucose, without the removal of the fermentation broth, into the fed-batch culture is one of the most common methods for the production of ethanol in the industry (Chang et al., 2012)

Ethanol recovery from fermentation broth is traditionally done by distillation. For lignocellulose-based ethanol production to be economically viable on an industrial scale, the ethanol produced must be above 4% (v/v) in the fermentation broth (Wingren et al., 2003). For most types of lignocellulosic materials, this requires operating at dry mass (DM) concentrations about 15% to achieve sufficiently high cellulose levels (Jorgensen et al., 2007). However, high substrate concentration in the form of fibrous, solid materials poses two problems: (1) the increased concentrations of inhibitors such as acetic acid, furfural, and ethanol hamper the performance of yeast and enzymes and (2) high viscosity results in more power consumption in the fermentor and lowered mixing and heat transfer efficiency (Georgieva et al., 2008). In order to increase the final substrate concentration while avoiding increases in viscosity, fed-batch culture was used in the SSF process (Zhang et al., 2010). To recover low concentrations of ethanol from fermentation, pervaporation may be economically more feasible than distillation (Vane, 2005) as for dilute ethanol streams (less than 5 wt.%), the high energy requirements in distillation (Madson

membrane selectivity, by using hydro-phobic pervaporation before feeding it to distillation. This should reduce the energy load on the distillation. Similarly, the remaining 5 wt. % of water from the top product of distillation can be removed by hydrophilic pervaporation to achieve fuel grade (anhydrous) ethanol (>99.5 wt. %). Here we focus on the ethanol recovery from lignocellulosic fermentation broth by hydrophobic pervaporation (Gaykawad et al., 2013). The breakdown of lignocellulosic biomass by pretreatment and the fermentation of the resulting sugars leads to a variety of by-products mainly divided into carboxylic acids, furans and phenolics (Almeida et al., 2007), which may threat the pervaporation membrane performance.

Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Simultaneous co-fermentation would allow a continuous fermentation process, which is the most effective way to reduce capital expenditures. During past years a lot of research and development has been done in the genetic improvement of strains but still more research have been established to further improve and optimize the fermentation methods and microorganisms for industrial applications.

Almeida, J.R.M., Modig, T., Petersson, A., Hahn-Hagerdal, B., Liden, G., and Gorwa-Grauslund, M.F.: Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J. Chem. Technol. Biotechnol., 2007, 82, pp. 340–349. Balat, M., and Balat, H.: Recent trends in global production and utilization of bio-ethanol fuel. Applied Energy, 2009, 86, pp. 2273–2282. Carriquirya, M.A, Du, X., and Timilsina, G.R.: Second generation biofuels: Economics and policies. Energy Policy, 2011, 39, pp. 4222–4234. Chandrakant, P., and Bisaria, V.S.: Simultaneous bioconversion of cellulose and hemicellulose to ethanol. Crit. Rev. Biotechnol., 1998, 18, pp. 295–331. Chang, Y.H., Chang, K.S., Huang, C.W., Hsu, C.L., and Jang, H.D.: Comparison of batch and fed-batch fermentations using corncob hydrolysate for bioethanol production. Fuel, 2012, 97, pp. 166–173. Chen, W.H., Xu, Y.Y., Hwang, W.S., and Wang, J.B.: Pretreatment of rice straw using an 2012, 102, pp. 10451–10458. Chen, Y.: Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review. Journal of Industrial Microbiology and Biotechnology, 2011, 38, pp. 581–597. Cheng, K.K., Zhang, J.A., Ling, H.Z., Ping, W.X., Huang, W., Ge, J.P., and Xu, J.M.: Optimization of pH and acetic acid concentration for bioconversion of hemicellulose from corncobs to xylitol by Candida tropicalis. Biochemical Engineering Journal, 2009, 43, pp. 203–207. Crespo, C.F., Badshah, M., Alvarez M.T., and Mattiasson, B.: Ethanol production by continuous fermentation of D- (+)-cellobiose, D-(+)-xylose and sugarcane bagasse hydrolysate using the thermoanaerobe Caloramator boliviensis. Bioresource Technology, 2012, 103, pp.186–191. De Bari, I., Viola, E., Barisano, D., Cardinale, M., Nanna, F., Zimbardi, F., et al.: Ethanol production at flash and pilot scale from concentrated slurries of steam-exploded aspen. Ind Eng Chem Res., 2002, 41, pp.1745–53. Dien, B.S., Cotta, M.A., and Jeffries, T. W.: Bacteria engineered for fuel ethanol production: current status. Appl. Microbiol. Biotechnol., 2003, 63 (3), pp. 258–266. Dominguez, H., Nunez, M.J., Chamy, R., and Lema, J.: Determination of kinetic parameters of fermentation processes by a continuous unsteady-state method: application to the alcoholic fermentation of D-xylose by Pichia stipitis. Biotechnol. Bioeng., 1993, 41, pp. 1129–1132. Edgardo, A., Carolina, P., Manuel, R., Juanita, F., and Jaime, B.: Selection of thermotolerant yeast strains Saccharomyces cerevisiae for bioethanol production. Enzyme and Microbial Technology, 2008, 43, pp. 120–123. Eliasson, A., Christensson, C., Wahlbom, C.F., and Hahn-hagerdal, B.: Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1in mineral medium chemostat cultures. Appl Environ Microbiol., 2000, 66, pp.3381–6. Eva, P., and Barbel, H.: Fermentation of lignocellulosic hydrolysates. I. Inhibition and detoxification. Bioresour. Technol, 2000, 74, pp. 17–24. Pervaporation of ethanol from lignocellulosic fermentation broth. Bioresource Technology, 2013, 129, pp.469–476. Georgieva, T.I., Hou, X.R., Hilstrom, T., and Ahring, B.K.: Enzymatic hydrolysis and ethanol fermentation of high dry matter wet-exploded wheat straw at low enzyme loading. Appl. Biochem. Biotech., 2008, 148, pp. 35–44. Ghosh, P., Pamment, N.B., and Martin, W.R.B.: “Simultaneous saccharification and fermentation of cellulose: effect of β-D-glucosidase activity and ethanol inhibition of cellulases”. Enzym. Microb. Technol., 1982, 4, pp. 425-430. Gong, Z., Wang, Q., Shen, H., Hu, C., Jin, G., and Zhao, Z.K.: Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production. Bioresource Technology, 2012, 117, pp. 20–24. Gonzalez, R., Treasure, T., Phillips, R., Jamee, H., Saloni, D., Abt, R., et al.: Converting eucalyptus biomass into ethanol: financial and sensitivity analysis in a co-current dilute acid process Part II. Biomass Bioenerg., 2011, 35, pp.767–72. Hendricks, A.T.W.N., and Zeeman, G.: Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol., 2009, 100, pp. 10–18. Hickert, L.R., Cunha-Pereira, F., Souza-Cruz, P.B., Rosa, C.A., and Ayub, M.A.: Ethanogenic fermentation of co-cultures of Candida shehatae HM 52.2 and Saccharomyces cerevisiae ICV D254 in synthetic medium and rice hull hydrolysate. Bioresource Technology, 2013, 131, pp. 508–514. Howard, R.L., Abotsi, E., Jansen, E. L., and Howard, S.: Lignocellulose biotechnology: issues of bioconversion and enzyme production. Africal J. Biotechnology, 2003, 2, pp. 602-619. Jang, J.S., Cho, Y.K., Jeong, G.T., and Kim, S.K.: Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica. Bioprocess. Biosyst. Eng., 2012, 35, pp. 11–18. Jorgensen, H., Vibe-Pedersen, J., Larsen, J., and Felby, C.: Liquefaction of lignocellulose at high-solids concentrations. Biotechnol. Bioeng., 2007, 96, pp. 862–870. Kadar, Z., Szengyel, Z., and Reczey K.: Simultaneous saccharification and fermentation (SSF) of industrial wastes for the production of ethanol. Industrial Crops and Products, 2004, 20, pp. 103–110. Katahira, S., Mizuike, A., Fukuda, H., and Kondo, A.: Ethanol fermentation from lignocellulosic hydrolysate 2006, 72, pp. 1136–43. Kim, J.H., Block, D.E., and Mills, D.A.: Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass. Appl. Microbiol. Biotechnol., 2010, 88, pp. 1077–1085. Kim, S.R., Park, Y.C., Jin, Y.S., and Seo, J.H.: Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnology Advances, 2013, 31, pp. 851–861. Kumar, A., Singh, L.K., and Ghosh, S.: Bioconversion of lignocellulosic fraction of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to ethanol by Pichia stipitis. Bioresource Technology, 2009, 100, pp. 3293–3297. Kumar, S., Singh, S.P., Mishra, I.M., and Adhikari, D.K.: Recent Advances in Production of Bioethanol from Lignocellulosic Biomass. Chem. Eng. Technol., 2009, 32(4), pp. 517–526. Larsson, S., Cassland, P., and Jonsson, L.J.: Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase. Appl Environ Microbiol, 2001, 67, pp.1163–70. Lee, J.Y., Li, P., Lee, J., Ryu, H.J., and Oh, K.K.: Ethanol production from Saccharina japoni causing an optimized extremely low acid pretreatment followed by simultaneous saccharification and fermentation. Bioresource Technology, 2013, 127, pp. 119–125. Liu, Z.L., Weber, S.A., Cotta, M.A., and Li, S.Z.: A new β-glucosidase producing yeast for lower-cost cellulosic ethanol production from xylose-extracted corncob residues by simultaneous saccharification and fermentation. Bioresource Technology, 2012, 104, pp. 410–416. Lu, X.B., Zhang, Y.M., Yang, J., and Liang, Y.: Enzymatic hydrolysis of corn stover after pretreatment with dilute sulfuric acid. Chem. Eng. Technol., 2007, 30, pp. 938–944. Madson, P., and Lococo, D.: Recovery of volatile products from dilute high-fouling process streams. Appl. Biochem. Biotechnol., 2000, 84–86, pp. 1049–1061.

Meinander, N.Q., Boels, I., and Hahn-Hägerdal, B.: Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without over expression of TALl. Bioresour. Technol., 1999, 68, pp. 79–87. Mohagheghi, A., M. Ruth, M., and. Schell, D.J.: conditioning hemicellulose hydrolysates for fermentation: effects of overliming pH on sugar and ethanol yields. Process Biochemistry, 2006, 41, pp.1806-11. Moreno, A.D., Ibarra, D., Fernández, J.L., and Ballesteros, M.: Different laccase detoxification strategies for ethanol production from lignocellulosic biomass by the thermo-tolerant yeast Kluyveromyces marxianus CECT 10875. Bioresource Technology, 2012, 106, pp. 101–109. Nichols, N.N., Sharma, L.N., Mowery, R.A., Chambliss, C.K., Walsum, G. P., Dien, B.S., and Iten, L.B.: Fungal metabolism of fermentation inhibitors present in corn stover dilute acid hydrolysate. Enzyme and Microbial Technology, 2008, 42, pp. 624–630. Nikolic, S., Mojovic, L., Rakin, M., and Pejin, D.: Bioethanol production from corn meal by simultaneous enzymatic saccharification and fermentation with immobilized cells of Saccharomyces cerevisiae. Fuel, 2009, 88, pp. 1602–7. Ofori-Boateng, C., and Lee, K. T.: Ultrasonic-assisted simultaneous saccharification and fermentation of pretreated oil palm fronds for sustainable bioethanol production, Fuel, 2014, 119, pp. 285-291. Ohgren, K., Bura, R.., Lesnicki, G., Saddler, J., and Zacchi, G.: A comparison between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using steam-pretreatment corn stover. Process Biochem., 2007,42, pp. 834–9. Olofsson, K., Bertilsson, M., and Liden, G.: A short review on SSF- an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnol. Biofuels, 2008, 1, pp. 1–14. Olson, L., and Hahn-Hagerdal, B.: Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb Technol., 1996, 18, pp.312–31. Palmqvist, E., and Hahn-Hägerdal, B.: Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanism of inhibition. Bioresour. Technol., 2000, 74, pp. 25–33. microbial growth and enzymatic hydrolysis rates. Biotechnol. Bioeng., 2007, 96, pp. 250–258. Pienkos, P., and Zhang, M.:. Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates. Cellulose, 2009, 16 (4), pp. 743–762. Romani, A., Garrote, G., and Parajo, J. C.: Bioethanol production from autohydrolyzed Eucalyptus globules by Simultaneous Saccharification and Fermentation operating at high solids loading. Fuel, 2012, 94, pp. 305–312. Saha, S., Roy, R., Sen, S. K., and Ray, A.K.: Characterization of cellulase-producing bacteria from the digestive tract of tilapia, Or Eochromis mossambica (Peters) and grass carp, Ctenopharyngodon idella (Valenciennes). Aquaculture Research, 2006, 37, pp. 380-388. Sanchez, Ó.J., and Cardona, C.A.: Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresour. Technol., 2008, 99, pp. 5270–5295. Sarkar, N., Ghosh, S.K., Bannerjee, S., and Aikat K.: Bioethanol production from agricultural wastes: An overview. Renewable Energy, 2012, 37, pp. 19-27. Sasikumar, E., and Viruthagiri, T.: Simultaneous Saccharification and Fermentation (SSF) of Sugarcane Bagasse - Kinetics and Modeling. International Journal of Chemical and Biological Engineering, 2010, 3(2), pp. 57-64. Sassner, P., Galbe, G., and Zacchi G.: Techno-economic evaluation of bioethanol production from three different lignocellulosic materials. Biomass Bioenerg., 2008, 32, pp.422-30. Singh, A., and Bishnoi, N.R.: Enzymatic hydrolysis optimization of microwave alkali pretreated wheat straw and ethanol production by yeast. Bioresour Technol., 2011.http://dx.doi.org/10.1016/j.biortech.2011.12.084. Singh, A., Bajar, S., and Bishnoi N.R.: Enzymatic hydrolysis of microwave alkali pretreated rice husk for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture. Fuel, 2014, 116, pp. 699–702. Soderstrom, J., Galbe, M., and Zacchi, G.: Separate versus simultaneous saccharification and fermentation of two-step steam pretreated softwood Stulke, J., and Hillen, W.: Carbon catabolite repression in bacteria. Curr. Opin. Microbiol., 1999, 2, pp.195–201. Sukumaran, R.K., Surender, V.J., Sindhu, R., Binod, P., Janu, K.U., Sajna, K.V., Rajasree, K.P., and Pandey, A.: Lignocellulosic ethanol in India: Prospects, challenges and feedstock availability. Bioresource Technology, 2010, 101, pp. 4826–4833. Suutari, K., Liukkonen, K., and Laakso, S.: Temperature adaptation in yeasts: the role of fatty acids. J. General Microbiol., 1990, 136, pp.1469–74. Talebnia, F., Karakashev, D., and Angelidaki, I.: Production of bioethanol from wheat straw: an overview on pretreatment, hydrolysis and fermentation. Bioresource Technology, 2010, 101, pp. 4744–4753. Uihlein, A., and Schbek, L.: Environmental impacts of a lignocellulosic feedstock biorefinery system: an assessment. Biomass and Bioenergy, 2009, 33, pp. 793-802. Vane, L.M.: A review of pervaporation for product recovery from biomass fermentation processes. J. Chem. Technol. Biotechnol., 2005, 80, pp.603–629. Villarreal, M.L.M., Prata, A.M.R., Felipe, M.G.A., and Almeida, J.B.E.S.: Detoxification procedures of eucalyptus hemicellulose hydrolysate for xylitol production by Candida guilliermondii. Enzyme Microb. Technol., 2006, 40, pp. 17–24. Wingren, A., Galbe, M., and Zacchi, G.: Techno-economic evaluation of producing ethanol from softwood: Comparison of SSF and SHF and identification of bottlenecks. Biotechnol. Progr., 2003, 19, pp. 1109–1117. Wright, J.D.: Ethanol from biomass by enzymatic hydrolysis. Chem. Eng. Prog., 1988, 84(8), pp.62 -74. Yao, S., and Mikkelsen, M.J.: Metabolic engineering to improve ethanol production in Thermoanaerobacter mathranii. Appl. Microbiol. Biotechnol., 2010, 88, pp. 199–208. Zaldivar, J., Nielsen, J., and Olsson, L.: Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl. Microbiol. Biotechnol., 2001, 56, pp. 17–34. Zaldivar, J., Roca, C., Le Foll, C., Hahn-Hägerdal, B., and Olsson, L.: Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains. Bioresour. Technol., 2005, 96 (15), pp. 1670–1676. fed-batch simultaneous saccharification and fermentation after combined pretreatment. Bioresource Technology, 2010, 101, pp. 4959–4964. Zhao, J., and Xia, L.: Simultaneous saccharification and fermentation of alkaline-pretreated corn stover to ethanol using a recombinant yeast strain. Fuel Processing Technology, 2009, 90, pp. 1193–1197.