Hemicellulose-derived sugars solubilisation of rape straw. Cofermentation of pentoses and hexoses by Escherichia coli

Juan Carlos López-Linares, Cristóbal Cara-Corpas, Encarnación Ruiz-Ramos, Manuel Moya-Vilar, Eulogio Castro-Galiano, Inmaculada Romero-Pulido


Bioconversion of hemicellulose sugars is essential for increasing fuel ethanol yields from lignocellulosic biomass. We report for the first time with rape straw, bioethanol production from hemicellulose sugars. Rape straw was pretreated at mild conditions with sulfuric acid to solubilize the hemicellulose fraction. This pretreatment allows obtaining a prehydrolysate, consisting basically in a solution of monomeric hemicellulosic sugars, with low inhibitor concentrations. The remaining water insoluble solid constitutes a cellulose-enriched, free of extractives material. The influence of temperature (120ºC and 130ºC), acid concentration (2-4% w/v) and pretreatment time (30-180 min) on hemicellulose-derived sugars solubilisation was evaluated. The highest hemicellulosic sugars recovery, 72.3%, was achieved at 130ºC with 2% sulfuric acid and 60 min. At these conditions, a concentrated sugars solution, 52.4 g/L, was obtained after three acid consecutive contacts, with 67% xylose and acetic acid concentration above 4.5 g/L. After a detoxification step by activated charcoal or ion-exchange resin, prehydrolysate was fermented by ethanologenic Escherichia coli. An alcoholic solution of 25 g/L and 86% of theoretical ethanol yield was attained after 144 h when the prehydrolysate was detoxified by ion-exchange resin. The results obtained in the present work show sulfuric acid pretreatment under mild conditions and E. coli as an interesting process to exploit hemicellulosic sugars in rape straw.


agricultural residue; acid pretreatment; xylose; E. coli; bioethanol production

Full Text:



Carvalheiro F, Duarte LC, Lopes S, Parajó JC, Pereira H, Gírio FM, 2005. Evaluation of the detoxification of brewery’s spent grain hydrolysate for xylitol production by Debaryomyces hansenii CCMI 941. Process Biochem 40(3-4): 1215-1223. http://dx.doi.org/10.1016/j.procbio.2004.04.015

Castro E, Díaz MJ, Cara C, Ruiz E, Romero I, Moya M, 2011. Dilute acid pretreatment of rapeseed straw for fermentable sugar generation. Bioresour Technol 102: 1270-1276. http://dx.doi.org/10.1016/j.biortech.2010.08.057

Castro E, Nieves IU, Mullinnix MT, Sagues WJ, Hoffman RW, Fernández-Sandoval MT, Tian Z, Rockwood DL, Tamang B, Ingram LO, 2014. Optimization of dilute-phosphoric-acid steam pretreatment of Eucalyptus benthamii for biofuel production. Appl Energy 125: 76-83. http://dx.doi.org/10.1016/j.apenergy.2014.03.047

Chaabane FB, Marchal R, 2013. Upgrading the hemicellulosic fraction of biomass into biofuel. Oil Gas Sci Technol 68(4): 663-680. http://dx.doi.org/10.2516/ogst/2012093

Chandel AK, Kapoor RK, Singh A, Kuhad RC, 2007. Detoxification of sugarcane bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501. Bioresour Technol 98: 1947-1950. http://dx.doi.org/10.1016/j.biortech.2006.07.047

Díaz-Villanueva MJ, Cara-Corpas C, Ruiz-Ramos E, Romero-Pulido I, Castro-Galiano E, 2012. Olive tree pruning as an agricultural residue for ethanol production. Fermentation of hydrolysates from dilute acid pretreatment. Span J Agric Res 10(3): 643-648. http://dx.doi.org/10.5424/sjar/2012103-2631

Fernández-Sandoval MT, Huerta-Beristain G, Trujillo-Martínez B, Bustos P, González V, Bolivar F, Gosset G, Martínez A, 2012. Laboratory metabolic evolution improves acetate tolerance and growth on acetate of ethanologenic Escherichia coli under non aerated conditions in glucose-mineral medium. Appl Microbiol Biotechnol 96: 1291-1300. http://dx.doi.org/10.1007/s00253-012-4177-y

Geddes CC, Mullinnix MT, Nieves IU, Peterson JJ, Hoffman RW, York SW, Yomano LP, Miller EN, Shanmugan KT, Ingram LO, 2011. Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160. Bioresour Technol 102: 2702-2711. http://dx.doi.org/10.1016/j.biortech.2010.10.143

Gírio FM, Fonseca C, Carvalheiro F, Duarte LC, Marques S, Bogel-Łukasik R, 2010. Hemicelluloses for fuel ethanol: A review. Bioresour Technol 101: 4775-4800. http://dx.doi.org/10.1016/j.biortech.2010.01.088

Jin M, Balan V, Gunawan C, Dale BE, 2012. Quantitatively understanding reduced xylose fermentation performance in AFEX treated corn stover hydrolysate using Saccharomyces cerevisae 424A (LNH-ST) and Escherichia coli K011. Bioresour Technol 111: 294-300. http://dx.doi.org/10.1016/j.biortech.2012.01.154

Jönsson LJ, Alriksson B, Nilvebrant N, 2013. Bioconversion of lignocelluloses: inhibitors and detoxification. Biotechnol Biofuels 6: 16. http://dx.doi.org/10.1186/1754-6834-6-16

Karagöz P, Rocha IV, Özkan M, Angelidaki I, 2012. Alkaline peroxide pretreatment of rapeseed straw for enhancing bioethanol production by same vessel saccharification and co-fermentation. Bioresour Technol 104: 349-357. http://dx.doi.org/10.1016/j.biortech.2011.10.075

Klasson KT, Dien BS, Hector RE, 2013. Simultaneous detoxification, saccharification and ethanol fermentation of weak-acid hydrolyzates. Ind Crop Prod 49: 292-298. http://dx.doi.org/10.1016/j.indcrop.2013.04.059

Lee JM, Venditti RA, Jameel H, Kenealy WR, 2011. Detoxification of woody hydrolyzates with activated carbon for bioconversion to ethanol by the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum. Biomass Bioenergy 35(1): 626-636. http://dx.doi.org/10.1016/j.biombioe.2010.10.021

López-Linares JC, Cara C, Ruiz E, Moya M, Castro E, Romero I, 2013. Hemicellulose solubilisation of rapeseed straw with phosphoric acid. 2nd Iberoamerican Congress on Biorefineries, Jaén (Spain), pp: 69-75. ISBN: 978-84-92876-21-1.

López-Linares JC, Romero I, Cara C, Ruiz E, Castro E, Moya M, 2014. Experimental study on ethanol production from hydrothermal pretreated rapeseed straw by simultaneous saccharification and fermentation. J Chem Technol Biotechnol 89: 104-110. http://dx.doi.org/10.1002/jctb.4110

Luo C, Brink Dl, Blanch H, 2002. Identification of potential fermentation inhibitors in conversion of hybrid poplar hydrolysate to ethanol. Biomass Bioenergy 22: 125-138. http://dx.doi.org/10.1016/S0961-9534(01)00061-7

Martínez A, Grabar TB, Shanmugam KT, Yomano LP, York SW, Ingram LO, 2007. Low salt medium for lactate and ethanol production by recombinant Escherichia coli. B Biotechnol Lett 29: 397-404. http://dx.doi.org/10.1007/s10529-006-9252-y

Nilvebrant NO, Reimann A, Larsson S, Jönsson LJ, 2001. Detoxification of lignocellulose hydrolysates with ion exchange resins. Appl Biochem Biotechnol 91-93: 35-49. http://dx.doi.org/10.1385/ABAB:91-93:1-9:35

NREL, 2007. Standard procedures for biomass compositional analysis. Nat Renew Energ Lab, Golden, CO, USA. Available in http://www.nrel.gov/biomass/analytical_procedures.html. [21 May 2015].

Qi W, Zhang S, Xu Q, Ren Z, Yan Y, 2008. Degradation kinetics of xylose and glucose in hydrolysate containing dilute sulfuric acid. Chinese J Process Eng 8 (6): 1132-1137.

Rasmussen H, Sørensen HR, Meyer AS, 2014. Formation of degradation compounds from lignocellulosic biomass in the biorefinery: sugar reaction mechanisms. Carbohydr Res 385: 45-57. http://dx.doi.org/10.1016/j.carres.2013.08.029

Romero I, López-Linares JC, Delgado Y, Cara C, Castro E, 2015. Ethanol production from rape straw by a two-stage pretreatment under mild conditions. Bioprocess Biosyst Eng, doi: 10.1007/s00449-015-1389-4. http://dx.doi.org/10.1007/s00449-015-1389-4

Saha BC, Nichols NN, Cotta MA, 2011. Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading. Bioresour Technol 102: 10892-10897. http://dx.doi.org/10.1016/j.biortech.2011.09.041

Shen J, Kaur I, Baktash MM, He Z, Ni Y, 2013. A combined process of activated carbon adsorption, ion exchange resin treatment and membrane concentration for recovery of dissolved organics in pre-hydrolysis liquor of the kraft-based dissolving pulp production process. Bioresour Technol 127: 59-65. http://dx.doi.org/10.1016/j.biortech.2012.10.031

Singleton VL, Rossi SA, 1965. Colorimetric of total phenolics with phosphomolibic-phosphotungstic acid reagents. Am J Enol Vitic 16(3): 144-158.

Taherzadeh MJ, Karimi K, 2008. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review. Int J Mol Sci 9: 1621-1651. http://dx.doi.org/10.3390/ijms9091621

Um BH, Karim MN, Henk LL, 2003. Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover. Appl Biochem Biotechnol 105-108: 115-125. http://dx.doi.org/10.1385/ABAB:105:1-3:115

Van Zyl C, Prior BA, du Preez JC, 1991. Acetic acid inhibition of D-xylose fermentation by Pichia stipitis. Enzyme Microb Technol 13: 82-86. http://dx.doi.org/10.1016/0141-0229(91)90193-E

Villarreal MLM, Prata AMR, Felipe MGA, Almeida E, Silva JB, 2006. Detoxification procedures of eucalyptus hemicellulose hydrolysate for xylitol production by Candida guilliermondii. Enzyme Microb Technol 40: 17-24. http://dx.doi.org/10.1016/j.enzmictec.2005.10.032

Wettstein S, Alonso DM, Gürbüz EI, Dumesic J, 2012. A roadmap for conversion of lignocellulosic biomass to chemicals and fuels. Curr Opin Chem Eng 1: 218-224. http://dx.doi.org/10.1016/j.coche.2012.04.002

Wood IP, Elliston A, Collins SR, Wilson D, Bancroft I, Waldron KW, 2014. Steam explosion of oilseed rape straw: Establishing key determinants of saccharification efficiency. Bioresour Technol 162: 175-183. http://dx.doi.org/10.1016/j.biortech.2014.03.115

Yang B, Wyman CE, 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels Bioprod Biorefin 2: 26-40. http://dx.doi.org/10.1002/bbb.49

Zaldivar J, Ingram LO, 1999. Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol Bioeng 66: 203-210. http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0290(1999)66:4%3C203::AID-BIT1%3E3.0.CO;2-%23/abstract

Zhu J, Yang J, Zhu Y, Zhang L, Yong Q, Xu Y, Li X, Yu S, 2014. Cause analysis of the effects of acid-catalyzed steam-exploded corn stover prehydrolyzate on ethanol fermentation by Pichia stipitis CBS 5776. Bioprocess Biosyst Eng 37: 2215-2222. http://dx.doi.org/10.1007/s00449-014-1199-0

DOI: 10.5424/sjar/2015133-7496