The objective of this work was the production of ethanol

1.12 Bioethanol from sugar cane bagasse Urribarrí, Lauris 1 * Ferrer, Alexis 2 Aiello, Cateryna 3 Rivera, Jhoandry 4 Abstract The objective of this work was the production of ethanol by simultaneous saccharification and fermentation (SSF) from sugar cane bagasse treated with ammonia at 1.25 kg/kg dry matter ammonia loading, 30% moisture content, 100 C for 5 min on the ethanol production by thermotolerant Kluyveromyces marxianus CECT 10875. Non treated bagasse was used as a control. The results are compared with separate saccharification and fermentation (SHF). Enzymatic hydrolysis was carried out with 5% w/v dry bagasse using 5 IU/g dm of cellulases (Accellerase 100) at 50 C for 48 h and the fermentation at 42 C for 96 h. The SSF was carried out for 96h at 42 C. The sugars released in the hydrolysis were measured with the 3.5 dinitrosalicylic acid method and the ethanol produced by gas chromatography. The SSF ethanol yields with respect to the theoretical value for the treated material was 71%, 1.85 times higher than that obtained for the untreated material and also considerably higher than SHF (1.64 times higher). Clearly, elimination of feedback inhibition due to sugars by SSF enhanced considerably the sugar yield, and therefore ethanol yield. Sugar cane bagasse can be used for bioetha- 1 Chemistry Department, Science Faculty, University of Zulia, Maracaibo, Venezuela. 2 Zulian Institute of Technological Research, Km. 15 vía La Cañada de Urdaneta, Venezuela. 3 Biochemical Engineering Department, Engineering Faculty, University of Zulia, Maracaibo, Venezuela. 4 Biology Department, Science Faculty, University of Zulia, Maracaibo, Venezuela. * Corresponding author. E-mail: laurisurribarri@gmail.com 95 II Congreso Iberoamericano sobre Biorrefinerías

nol production with a 71% yield by using a mild ammonia treatment, low enzyme doses and a simultaneous saccharification and fermentation. Keywords: Ammonia treatments, Ethanol production, Sugar cane bagasse. Introduction Lignocellulosic materials are the major source of cellulose and hemicellulose in nature and have been recognized as a potential renewable and inexpensive source of to obtain fermentable sugars for fuel ethanol production (second generation biofuels) and other products of high added value, mainly by biotechnological and / or thermochemical processes. However, the complex organization and interaction of the cell wall polymers make it necessary to subject them to physical and chemical pretreatments [DOE, 2005] which minimize structural barriers, increasing their chemical and enzymatic susceptibility. Ammonia processes are an excellent alternative because the volatile agent is easily recovered and process conditions are relatively mild which ensures a minimum degradation of the biomass (Ferrer et al, 2000). Sugar cane bagasse (SCB) is the third largest agro-industrial residue in Venezuela, with an approximate production of 1,580,000 tons per year (FEDEAGRO, 2010) and barely 50% of the volume produced is used. Its high carbohydrate content makes it an attractive substrate for production of bioethanol. Traditionally, SCB is burned to produce a modest amount of energy and as a way to limit the disposal of this waste. The pretreatment of pressurization and depressurization Ammonia (PDA) (Ferrer et al, 2000) which is a treatment with ammonia in liquid phase under pressure, using relatively low temperatures and short times, is available at a pilot plant scale and a 1 ton/h demonstration plant is currently being built. This treatment has been successful in eliminating structural barriers such as high crystallinity of cellulose, high lignin content, high acetylation of hemicellulose and high degree of linkage between these molecules. The aim of this study was to produce bioethanol from ammonia treated SCB by simultaneous saccharification and fermentation. 96 Methodology Substrate SCB was provided by La Pastora, a mill located in Carora, Lara, Venezuela. The sample was dried in a forced convection oven at 60C for 48 hours to reduce its moisture content to 10%, and stored in plastic bags until use. SCB was sieved to a size not greater than 2 mm. 2 nd Iberoamerican Congress on Biorefineries

Ammonia treatments Samples of 350 g of dry substrate were treated with ammonia in a 2 kg pilot plant at 1.25 kg of ammonia / kg dry substrate loading, 30% moisture contents at 100ºC for 5 min, using a pressure of 300 psi (ATB). Subsequently, the treated samples were aerated under a hood for 12 h. Non treated bagasse (NTB) was used as a control. Biomass Analysis The moisture content of the samples was determined by the method described in AOAC 930.15 (2010) for SCB. The contents of hemicellulose, cellulose and lignin were determined using the method of Goering and Van Soest (1970) based on determinations of neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid lignin (ADL). Separate Saccharification and Fermentation For these experiments, the saccharification of untreated (NTB) and ammonia treated (ATB) sugarcane bagasse was performed using Accellerase and β-glucosidase for 48 h. A 5% substrate and 5 IU cellulase/g dm were used. After this, the hydrolysate was filtered and the fermentation was initiated by adding the inoculum of the organism corresponding to a volume equal to 10% of the workload. The sugars released in the hydrolysis were measured with the 3.5 dinitrosalicylic acid method and the ethanol produced by gas chromatography. Simultaneous Saccharification and Fermentation The process was applied to both NTB and ATB. The substrate, the enzyme complex Accellerase and β-glucosidase, and thermotolerant Kluyveromyces marxianus CECT 10875, were added simultaneously. The SSFs were performed in 500 ml Erlenmeyer flasks with 100 ml of the culture medium at 100 rpm for 72 hours in an orbital incubator model Innova 4300 (New Brunswick Scientific, Edison NJ, USA), in duplicate. A 5% substrate and 5 IU cellulase/g dm were used. The medium was inoculated with a 10% volume of the workload to initiate the fermentation. Aliquots were taken at 0, 6, 12, 24, 48 and 72 hours. Sugars and ethanol were measured as in SHF. Results The compositions of NTB and ATB were 49.32% cellulose, 23.96% hemicellulose, 11.05% lignin, 11.08% solubles, and 36.82% cellu- 97 II Congreso Iberoamericano sobre Biorrefinerías

lose, 13.75% hemicellulose, 8.96% lignin, 21.89% solubles, respectively. The treatment solubilized 43% of the hemicellulose and 25% of the cellulose. The enzymatic hydrolysis of NTB and ATB produced 8.5 mg/ ml and 13.03 mg/ml of reducing sugars, respectively, for 48 h of hydrolysis. Figure 1 shows the fermentation of both NTB and ATB hydrolysates. For NTB, as the fermentation proceeds the concentration of ethanol increases whereas the glucose concentration decreases in the medium, until exhausted at 24 h at which time the fermentation ceases. The ethanol yield with respect to the theoretical value obtained from the fermentation of NTB was 26.30%, which is quite low but expected for a recalcitrant material with a high lignin content and intricate fiber structure, which has not undergone prior treatment. Figure 1 Kinetics of the fermentation using Kluyveromyces marxianus CECT 10875. a) Untreated bagasse hydrolysate. b) Treated bagasse hydrolysate 98 For ATB, the system behaved as for NTB, however, glucose was exhausted at 48 h, due to the higher concentration of glucose to start. The total consumption of glucose indicates that the treatment did not produce inhibitory compounds or at least not in concentrations which could inhibit the yeast. The fermentation yield of the ATB hydrolysate was 65% higher than the untreated, however, it is relatively low (43.24%). This suggests the need either to apply more aggressive ammonia treatment conditions, to further simplify the lignocellulosic biomass matrix, or to use SSF. The kinetics of the SSF for the untreated and treated material is shown in Figure 2. During the first phase, there was a steady increase in ethanol concentration while the glucose content remained very low, showing a good performance of the yeast during the fermentation. The glucose concentration increased only slightly at the end of the trials. 2 nd Iberoamerican Congress on Biorefineries

The highest concentrations of ethanol for both NTB and ATB were obtained at 96 hours, although 48 and 72 h may be considered as final times, respectively, since the difference between 48 and 96 h for NTB (3.8%) and between 72 and 96 for ATB (5,1%) were small. Figure 2 Kinetics of SSF for SCB. a) Untreated, b) treated The ethanol yield with respect to the theoretical value was 71.29%, 1.64 times higher than in SHF, clearly indicating the importance of removing the glucose as it is formed. The yield is higher than those reported by Bollók et al. (2000) in SSF experiments (44%) with 5% of substrate (softwoods treated with steam explosion) using strains of K. marxianus, In another study (Ballesteros et al., 2004), using the yeast K. marxianus CECT 10875 in SSF with a substrate concentration of 10%, yields of ethanol with respect to the theoretical were between 60 and 71%, using different lignocellulosic substrates, results that are consistent with those obtained in this work with the same thermotolerant strain but using only one third of the cellulolytic enzymes and 5% substrate. Ethanol yields obtained for the treated material in this work were between 1.8 and 1.9 times higher than those obtained for the untreated material. Conclusions The ammonia treatment considerably increased the ethanol yields compared to untreated, but most important, the use of SSF conditions improved the ethanol yields compared to those obtained by SHF. In the case of ATB, the increase was 1.64 times. The production of bioetha- 99 II Congreso Iberoamericano sobre Biorrefinerías

nol in SSF processes allowed not only to obtain higher yields but also they were achieved in less time, 48h and 72h for the untreated and treated materials, respectively, while the SHF processes for both materials required 24 h more. It is possible to further increase the ethanol yields by optimizing pretreatment and SSF conditions. Acknowledgement Authors thank the contribution of FONACIT (Caracas, Venezuela) and the Swiss Agency for Development and Cooperation (Lausanne, Switzerland) for partly financing this work, and to the CIEMAT (Madrid, Spain) for kindly supplying the yeast strain. References 100 AOAC, Official methods of analysis. (2010). 18th edition online, Association of Official Analytical Chemists. Available in: http://www. eoma.aoac.org. Ballesteros, M., Oliva, J., Negro, M., Manzanares, P., Ballesteros, I. (2004). Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SSF) with Kluyveromyces marxianus CECT 10875. Process Biochemistry, 39 (12),1843-1848. Bollók, M., Réczey, K., Zacchi, G. (2000). Simultaneous saccharification and fermentation of steam-pretreated spruce to ethanol. Applied Biochemistry and Biotechnology, 84/86, 69-80. DOE, U.S. Department of Energy (2005). Breaking the biological barriers to cellulosic ethanol: a join research agenda. A research road map resulting from the biomass to biofuels workshop, December 7-9, Maryland, USA, 206 p. Ferrer, A., Byers, F. M., Sulbarán de Ferrer, B., Dale, B. E., Aiello, C. (2000). Optimizing ammonia pressurization/depressurization processing conditions to enhance enzymatic susceptibility of dwarf elephant grass. Applied Biochemistry and Biotechnology, 84/86, 163-179. FEDEAGRO. Confederación Nacional de Asociaciones de Productores Agropecuarios. (2010). Estadísticas Agrícolas. Venezuela. Goering, H., Van Soest, P. (1970). Forage fiber analyses (apparatus, reagents, procedures, and some applications), Agric. Handbook N 379. ARS-USDA, Washington, D.C. 8 p. 2 nd Iberoamerican Congress on Biorefineries