Sustainable bioconversion of saccharified agro residues into bioethanol by Wickerhamomyces anomalus

  • I. U. Haq
  • A. Tahir
  • A. Nawaz
  • Z. Mustafa
  • H. Mukhtar
  • A.U. Rehman
Keywords: Bioethanol, Pichia anomala, Optimization, Fermentation


Snowballing levels of greenhouse gas emissions and concerns about climate change has led to an ongoing exploration of biofuels. Bioethanol can be obtained from wheat straw and can be readily available as clean fuel for combustion engines. Therefore, Wickerhamomyces anomalus yeast strain IHZ-26 was used to produce bioethanol from sugar solution obtained from enzymatic hydrolysis of  wheat straw. Nineteen different fermentation media were used for this purpose in which carbon source employed was sugar solution obtained from enzymatic hydrolysis of  wheat straw. Out of which, maximum bioethanol yield (1.09 g/L; p <0.05) was observed in ‘C1 Yeast extract, peptone, glucose’ medium.  After optimization of different cultural parameters, surface culture fermentation for 5 days at 25℃ gave maximum results using 2, 1.5 and 2 g of glucose, xylose and ammonium dihydrogen phosphate, respectively. Four hours old inoculum of yeast in a concentration of 3.5% was optimized for maximum bioethanol yield. These optimized parameters resulted in augmented bioethanol production (5.0g/L) by 5.02 folds. This study revelas that W. anomalus IHZ-26 employed was able to covert pentose and hexose sugars simultaneously with efficient ethanol yield.


Acharya, S. and Chaudhary, A. (2012). Alkaline cellulase produced by a newly isolated thermophilic Aneurinibacillus thermoaerophilus WBS2 from hot spring, India. African Journal of Microbiology Research 6(26), 5453-545.

Almeida, J. R., Modig, T., Petersson, A., Hähn‐Hägerdal, B., Lidén, G. and Gorwa‐Grauslund, M.F. (2007). Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. Journal of Chemical Technology and Biotechnology 82(4), 340-349.

Anwar, Z., Gulfraz, M. and Irshad, M. (2014). Agro-industrial lignocellulosic biomass a key to unlock the future bio-energy: a brief review. Journal of Radiation Research and Applied Sciences 7(2), 163-173.

Azhar, S.H.M., Abdulla, R., Jambo, S.A., Marbawi, H., Gansau, J.A., Faik, A.A.M. and K.F. Rodrigues. (2017). Yeasts in sustainable bioethanol production: A Review. Biochemistry and Biophysics Reports 10, 52-61.

Babu, S., Harinikumar, K., Singh, R.K. and Pandey, A. (2014). Optimization of bioethanol production from fruit wastes using isolated microbial strains. International Journal of Advanced Biotechnology and Research 5, 598-604.

Bafrncova, P., Slavikova, I., Patkova, J. and Domeny, Z. (1999). Improvement of very high gravity ethanol fermentation by media supplementation using Saccharomyces cerevisiae. Biotechnology Letters 21(4), 337-341.

Baldwin, E.L., Karki B., Zahler, J.D., Rinehart, M. and Gibbons, W.R. (2019). Submerged vs. Solid‐State Conversion of Soybean Meal into a High Protein Feed Using Aureobasidium pullulans. Journal of the American Oil Chemists' Society 96(9), 989-998.

Berłowska, J., Pielech-Przybylska, K., Balcerek, M., Dziekońska-Kubczak, U., Patelski, P., Dziugan, P. and Kregiel, D. (2016). Simultaneous saccharification and fermentation of sugar beet pulp for efficient bioethanol production. BioMed Research International 2016.

Bokulich, N.A. and Bamforth, C.W. (2013). The microbiology of malting and brewing. Microbiology and Molecular Biology Reviews 77(2), 157-172. https://doi/10.1128/MMBR.00060-12

Cazetta, M.L., Celligoi, M.A.P.C., Buzato, J.B. and Scarmino, I.S. (2007). Fermentation of molasses by Zymomonas mobilis: Effects of temperature and sugar concentration on ethanol production. Bioresource Technology 98(15), 2824-2828.

Chang, Y.H., Chang, K.S., Chen, C.Y., Hsu, C.L., Chang, T.C. and Jang, H. D. (2018). Enhancement of the efficiency of bioethanol production by Saccharomyces cerevisiae via gradually batch-wise and fed-batch increasing the glucose concentration. Fermentation 4(2), 45. https://doi:10.3390/fermentation4020045

Chan-u-tit, P., Laopaiboon, L., Jaisil, P. and Laopaiboon, P. (2013). High level ethanol production by nitrogen and osmoprotectant supplementation under very high gravity fermentation conditions. Energies 6(2), 884-899.

Chingono, T. and Mbohwa, C. (2016). Study on regulations, policies and permits for implementation of bioenergy systems.

Cofré, O., Ramírez, M., Gómez, J.M. and Cantero, D. (2012). Optimization of culture media for ethanol production from glycerol by Escherichia coli. Biomass and Bioenergy 37, 275-281.

Cruz, S.H., Batistote, M. and Ernandes, J. R. (2003). Effect of sugar catabolite repression in correlation with the structural complexity of the nitrogen source on yeast growth and fermentation. Journal of the Institute of Brewing 109(4), 349-355.

Darouneh, E., Alavi, A., Vosoughi, M., Arjmand, M., Seifkordi, A. and Rajabi, R. (2009). Citric acid production: Surface culture versus submerged culture. African Journal of Microbiology Research 3(9), 541-545. http://doi/

Davis, L., Rogers, P., Pearce, J. and Peiris, P. (2006). Evaluation of Zymomonas-based ethanol production from a hydrolysed waste starch stream. Biomass and Bioenergy 30(8-9), 809-814.

Dickinson, B. 2006. Bionutrients tech manual. Sparks, Md.: Becton-Dickinson. Retrieved from

Fadel, M., Keera, A.A., Mouafi, F.E. and Kahil, T. (2013). High level ethanol from sugar cane molasses by a new thermotolerant Saccharomyces cerevisiae strain in industrial scale. Biotechnology Research International 2013, 1-6.

Franca, A. S., Gouvea, B., Torres, C., Lean-dro, S. O., & Oliveira, E. S. (2009). Feasibility of ethanol production from coffee husks. In 13th International Biotechnology Symposium and Exhibition (pp. 269-269).

Gadonneix, P., De Castro, F.B., De Medeiros, N.F., Drouin, R., Jain, C.P., Kim, Y.D. and Naqi, A.A. (2010). Biofuels: Policies, Standards and Technologies. World Energy Council.

Gao, J., Atiyeh, H.K., Phillips, J.R., Wilkins, M.R. and Huhnke, R.L. (2013). Development of low cost medium for ethanol production from syngas by Clostridium ragsdalei. Bioresource Technology 147, 508-515.

Ghosh, T.K. and Prelas, M.A. (2011). Ethanol. In Energy Resources and Systems. Springer Netherlands, pp. 419-493.

Gikonyo, B. (2015). Design and Optimization of Ethanol Production from Bagasse Pith Hydrolysate by a Thermotolerant Yeast Kluyveromyces sp. IIPE453 Using Response Surface Methodology. In Sugarcane as Biofuel Feedstock (pp. 109-128). Apple Academic Press.

Gupta, P., Samant, K. and Sahu, A. (2012). Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential. International Journal of Microbiology 2012, 1-5.

Harde, S.M., Bankar, S.B., Ojamo, H., Granström, T., Singhal, R.S. and Survase, S.A. (2014). Continuous lignocellulosic ethanol production using Coleus forskohlii root hydrolysate. Fuel 126, 77-84.

Hölker, U., Höfer, M. and Lenz, J. (2004). Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Applied Microbiology and Biotechnology 64(2), 175-186.

Júnior, M.M., Batistote, M., Cilli, E.M. and Ernandes, J.R. (2009). Sucrose fermentation by Brazilian ethanol production yeasts in media containing structurally complex nitrogen sources. Journal of the Institute of Brewing 115(3), 191-197.

Jyotsana, K.P., Rao, A.R. and Devaki, K. (2015). Effect of nutritional factors on cellulase production by Streptomyces albaduncus from the gut of earthworm, Eisenia foetida. Pest Management In Horticultural Ecosystems 21(1), 75-80.

Kadam, K.L. and Newman, M.M. (1997). Development of a low-cost fermentation medium for ethanol production from biomass. Applied Microbiology and Biotechnology 47(6), 625-629.

Karagöz, P. and Özkan, M. (2014). Ethanol production from wheat straw by Saccharomyces cerevisiae and Scheffersomyces stipitis co-culture in batch and continuous system. Bioresource Technology 158, 286-293.

Keshav, P.K., Naseeruddin, S. and Rao, L. V. (2016). Improved enzymatic saccharification of steam exploded cotton stalk using alkaline extraction and fermentation of cellulosic sugars into ethanol. Bioresource Technology 214, 363-370.

Kiran, S, Ali, S. and Haq, I.U. 2003. Time course study for yeast invertase production by submerged fermentation. Journal of Biological Sciences 3(11), 984–988.

Kurian, J. K., Ashok, M.K., Banerjee, A. and Kishore, V.V.N. (2010). Bioconversion of hemicellulose hydrolysate of sweet sorghum bagasse to ethanol by using Pichia stipitis NCIM 3497 and Debaryomyces hansenii sp. Bioresources 5(4), 2404-2416.

Laluce, C., Tognolli, J.O., De Oliveira, K.F., Souza, C.S. and Morais, M.R. (2009). Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability. Applied Microbiology and Biotechnology 83(4), 627-637.

Laopaiboon, L., Thanonkeo, P., Jaisil, P. and Laopaiboon, P. (2007). Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology 23(10), 1497-1501.

Lee, Y.J., Choi, Y.R., Lee, S.Y., Park, J.T., Shim, J.H., Park, K.H. and Kim, J.W. (2011). Screening wild yeast strains for alcohol fermentation from various fruits. Mycobiology 39(1), 33-39. https://doi:10.11648/j.ajbio.20160405.11

Li, Z., Wang, D. and Shi, Y.C. (2017). Effects of nitrogen source on ethanol production in very high gravity fermentation of corn starch. Journal of the Taiwan Institute of Chemical Engineers 70, 229-235.

Lin, Y. and Tanaka, S. (2006). Ethanol fermentation from biomass resources: current state and prospects. Applied Microbiology and Biotechnology 69(6), 627-642.

Lin, Y., Zhang, W., Li, C., Sakakibara, K., Tanaka, S. and Kong, H. (2012). Factors affecting ethanol fermentation using Saccharomyces cerevisiae BY4742. Biomass and Bioenergy 47, 395-401.

Liu, K., Atiyeh, H.K., Tanner, R.S., Wilkins, M.R. and Huhnke, R.L. (2012). Fermentative production of ethanol from syngas using novel moderately alkaliphilic strains of Alkalibaculum bacchi. Bioresource Technology 104, 336-341.

Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical chemistry 31(3), 426-428.

Najafpour, G., Younesi, H. and Ismail, K.S.K. (2004). Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae. Bioresource Technology 92(3), 251-260.

Navarro, A.R., Sepúlveda, M.D.C. and Rubio, M.C. (2000). Bio-concentration of vinasse from the alcoholic fermentation of sugar cane molasses. Waste management 20(7), 581-585.

Nawaz, A., Ashfaq, A., Zaidi, S. M. A. M., Munir, M., Haq, I. U., Mukhtar, H., & Tahir, S. F. (2020). Comparison of fermentation and medical potentials of Saccharomyces with Wickerhamomyces genera. Revista Mexicana de Ingeniería Química 19(1), 33-47.

Niladevi, K.N. and Prema, P. (2008). Effect of inducers and process parameters on laccase production by Streptomyces psammoticus and its application in dye decolourization. Bioresource Technology 99(11), 4583-4589.

Olido, E.L.Y. (2018). Xylanase enzyme production by Aspergillus fumigatus in solid state fermentation and submerged fermentation. Revista Mexicana de Ingeniería Química 17(1), 47-61.

Paulova, L., Patakova, P., Branska, B., Rychtera, M. and Melzoch, K. (2015). Lignocellulosic ethanol: technology design and its impact on process efficiency. Biotechnology Advances 33(6), 1091-1107.

Pérez-Carrillo, E., Cortés-Callejas, M.L., Sabillón-Galeas, L.E., Montalvo-Villarreal, J.L., Canizo, J.R., Moreno-Zepeda, M.G. and Serna-Saldivar, S.O. (2011). Detrimental effect of increasing sugar concentrations on ethanol production from maize or decorticated sorghum mashes fermented with Saccharomyces cerevisiae or Zymomonas mobilis. Biotechnology Letters 33(2), 301-307.

Phillips, J.R., Atiyeh, H.K. and Huhnke, R.L. (2014). Method for Design of Production Medium for Fermentation of Synthesis Gas to Ethanol by Acetogenic Bacteria. Biological Engineering Transactions 7(3), 113-128.

Phisalaphong, M., Srirattana, N. and Tanthapanichakoon, W. (2006). Mathematical modeling to investigate temperature effect on kinetic parameters of ethanol fermentation. Biochemical Engineering Journal 28(1), 36-43.

Pinal, L., Ceden, M., Gutie, H. and Alvarez-Jacobs, J. (1997). Fermentation parameters influencing higher alcohol production in the tequila process. Biotechnology Letters 19(1), 45-47.

Plessas, S., Bekatorou, A., Koutinas, A. A., Soupioni, M., Banat, I.M. and Marchant, R. (2007). Use of Saccharomyces cerevisiae cells immobilized on orange peel as biocatalyst for alcoholic fermentation. Bioresource Technology 98(4), 860-865.

Raposo, S., Constantino, A., Rodrigues, F., Rodrigues, B. and Lima-Costa, M. E. (2017). Nitrogen sources screening for ethanol production using carob industrial wastes. Applied Biochemistry and Biotechnology 181(2), 827-843.

Rayabova, O.B., Chmil, O. M. and Sibirny, A.A. (2003). Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha. FEMS Yeast Research 4(2), 157-164.

Rekha, Y.G.G. and Vijayalakshmi, S. (2018). Production and optimization techniques of bioethanol from withered flowers of Allamanda schottii L. by Activated Dry Yeast. Journal of Pure and Applied Microbiology 12(2), 943-952.

Reyes, I., Hernandez-Jaimes, C., Meraz, M. and Rodríguez-Huezo, M.E. (2018). Physicochemical changes of corn starch during lactic acid fermentation with Lactobacillus bulgaricus. Revista Mexicana de Ingeniería Química,17(1), 279-288.

Sanchez, S., Bravo, V., Castro, E., Moya, A.J. and Camacho, F. (1999). Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae. Bioprocess Engineering 21(6), 525-532.

Santos, J., Sousa, M.J. and Leao, C. (2012). Ammonium is toxic for aging yeast cells, inducing death and shortening of the chronological lifespan. PloS one, 7(5).

Satora, P., Tarko, T., Sroka, P. and Blaszczyk, U. (2014). The influence of Wickerhamomyces anomalus killer yeast on the fermentation and chemical composition of apple wines. FEMS Yeast Research 14(5), 729-740.

Shin, S., Chang, D.E. and Pan, J.G. (2009). Acetate consumption activity directly determines the level of acetate accumulation during Escherichia coli W3110 growth. Journal of Microbiology and Biotechnology 19(10), 1127-1134.

Singh, A., Bajar, S. and Bishnoi, N. R. (2014). Enzymatic hydrolysis of microwave alkali pretreated rice husk for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis and their co-culture. Fuel 116, 699-702.

Slininger, P.J., Dien, B.S., Gorsich, S.W. and Liu, Z.L. (2006). Nitrogen source and mineral optimization enhance D-xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124. Applied Microbiology and Biotechnology 72(6), 1285-1296.

Tahir, A., Aftab, M. and Farasat, T. (2010). Effect of cultural conditions on ethanol production by locally isolated Saccharomyces cerevisiae BIO-07. Journal of Applied Pharmaceutical Science 3(2), 72-78.

Temudo, M. F., Kleerebezem, R. and Van-Loosdrecht, M. (2007). Influence of the pH on (open) mixed culture fermentation of glucose: a chemostat study. Biotechnology and Bioengineering 98(1), 69-79.

Temudo, M. F., Muyzer, G., Kleerebezem, R. and Van-Loosdrecht, M. C. (2008). Diversity of microbial communities in open mixed culture fermentations: impact of the pH and carbon source. Applied Microbiology and Biotechnology 80(6), 1121-1130.

Tesfaw, A. and Assefa, F. (2014). Current trends in bioethanol production by Saccharomyces cerevisiae: substrate, inhibitor reduction, growth variables, coculture, and immobilization. International Scholarly Research Notices 2014,

Thenmozhi, R. and Victoria, J. (2013). Optimization and improvement of ethanol production by the incorporation of organic wastes. Pelagia Research Library 4(5), 119-23.

Torija, M.J., Rozes, N., Poblet, M., Guillamón, J.M. and Mas, A. (2003). Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae. International Journal of Food Microbiology 80(1), 47-53.

Unrean, P. and Srienc, F. (2010). Continuous production of ethanol from hexoses and pentoses using immobilized mixed cultures of Escherichia coli strains. Journal of Biotechnology 150(2), 215-223.

Van, M.A.J., Abbott, D.A., Bellissimi, E., VanDen Brink, J., Kuyper, M., Luttik, M.A. and Pronk, J.T. (2006). Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: Current status. Antonie Van Leeuwenhoek 90(4), 391-418.

Vucurovic, V. M., Razmovski, R.N. and Popov, S.D. (2009). Ethanol production using Saccharomyces cerevisiae cells immobilised on corn stem ground tissue. Zbornik Matice Srpske Za Prirodne Nauke (116), 315-322.

Yamada, R., Yamakawa, S. I., Tanaka, T., Ogino, C., Fukuda, H. and Kondo, A. (2011). Direct and efficient ethanol production from high-yielding rice using a Saccharomyces cerevisiae strain that express amylases. Enzyme and Microbial Technology 48(4-5), 393-396.

Yamaoka, C., Kurita, O. and Kubo, T. (2014). Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation. Microbiological Research 169(12), 907-914.

Yu, Z. and Zhang, H. (2003). Ethanol fermentation of acid-hydrolyzed cellulosic pyrolysate with Saccharomyces cerevisiae. Bioresource Technology, 90(1), 95-100.

Zabed, H., Faruq, G., Sahu, J.N., Azirun, M.S., Hashim, R. and Nasrulhaq B.A. (2014). Bioethanol production from fermentable sugar juice. The Scientific World Journal 2014.

Zaldivar, J., Nielsen, J. and Olsson, L. (2001). Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Applied Microbiology and biotechnology 56(1-2), 17-34.

Zhang, Q., Wu, D., Lin, Y., Wang, X., Kong, H. and Tanaka, S. (2015). Substrate and product inhibition on yeast performance in ethanol fermentation. Energy and Fuels 29(2), 1019-1027.

Zhu, J. and Shimizu, K. (2005). Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition. Metabolic Engineering 7(2), 104-115.

How to Cite
Haq, I., Tahir, A., Nawaz, A., Mustafa, Z., Mukhtar, H., & Rehman, A. (2020). Sustainable bioconversion of saccharified agro residues into bioethanol by Wickerhamomyces anomalus. Revista Mexicana De Ingeniería Química, 19(3), 1477-1491.