Consolidated bioprocessing (CBP) in bioethanol production involves the combination of four essential biological procedures in a single bioreactor, using a mixture of organisms with favourable cellulolytic ability without the addition of exogenous enzymes. However, the main disadvantage of this process is the complexity to optimise all factors considering both enzymes and microbial activity at the same time. Hence, this study aimed to optimise suitable culture conditions for both organisms to work efficiently. Six single factors that are considered crucial for bioethanol production were tested in one-factor-at-a-time (OFAT) analysis and analysed using Response Surface Methodology (RSM) software for Aspergillus niger B2484 and Trichoderma asperellum B1581 strains. The formulation of a new consortia setting was developed based on the average of two settings generated from RSM testing several combinations of consortia concentrations (5:1, 2:4, 3:3, 4:2, and 1:5). The combination of 5:1 Aspergillus niger B2484 and Trichoderma asperellum B1581 produced the most ethanol with 1.03 g/L, more than A. niger B2484, alone with 0.34 g/L of ethanol, indicating the potential of the combination of A. niger B2484 and T. asperellum B1581 co-culture for bioethanol production in CBP.

• Akintunde, A. M., Ajala, S. O., & Betiku, E. (2015). Optimization of Bauhinia monandra seed oil extraction via artificial neural network and response surface methodology: A potential biofuel candidate. Industrial Crops and Products, 67, 387-394. doi: https://doi.org/10.1016/j.indcrop.2015.01.056

• Artifon, W., Bonatto, C., Bordin, E. R., Bazoti, S. F., Dervanoski, A., Alves, S. L., & Treichel, H. (2018). Bioethanol production from hydrolyzed lignocellulosic after detoxification via adsorption with activated carbon and dried air stripping. Frontiers in Bioengineering and Biotechnology, 6, 1-6. doi: https://doi.org/10.3389/fbioe.2018.00107

• Azhar, S. H. M., Abdulla, R., Jambo, S. A., Marbawi, H., Gansau, J. A., Faik, A. A. M., & Rodrigues, K. F. (2017). Yeasts in sustainable bioethanol production: A review. Biochemistry and Biophysics Reports, 10, 52-61. doi: https://doi.org/10.1016/j.bbrep.2017.03.003

• Behera, S. K., Meena, H., Chakraborty, S., & Meikap, B. C. (2018). Application of response surface methodology (RSM) for optimization of leaching parameters for ash reduction from low-grade coal. International Journal of Mining Science and Technology, 28(4), 621-629. doi: https://doi.org/10.1016/j.ijmst.2018.04.014

• Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008). Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 76(5), 965-977. doi: https://doi.org/10.1016/j.talanta.2008.05.019

• Bhaumik, R., Mondal, N. K., Chattoraj, S., & Datta, J. K. (2013). Application of response surface methodology for optimization of fluoride removal mechanism by newly developed biomaterial. American Journal of Analytical Chemistry, 4(8), 404-419. doi: 10.4236/ajac.2013.48051

• Biswas, G., Kumari, M., Adhikari, K., & Dutta, S. (2017). Application of response surface methodology for optimization of biosorption of fluoride from groundwater using Shorea robusta flower petal. Applied Water Science, 7, 4673-4690. doi: https://doi.org/10.1007/s13201-017-0630-5

• Bradáčová, K., Florea, A. S., Bar-Tal, A., Minz, D., Yermiyahu, U., Shawahna, R., … & Poşta, G. (2019). Microbial consortia versus single-strain inoculants: An advantage in PGPM-assisted tomato production? Agronomy, 9(2), 1-23. doi: https://doi.org/10.3390/agronomy9020105

• Cheng, J. R., & Zhu, M. J. (2013). A novel co-culture strategy for lignocellulosic bioenergy production: A systematic review. International Journal of Modern Biology and Medicine, 1(3), 166-193.

• Chin, K. L., & H’ng, P. S. (2013). A real story of bioethanol from biomass: Malaysia perspective. In M. D. Matovic (Ed.), Biomass now: Sustainable growth and use (pp. 329-346). Rijeka, Croatia: InTech Open Access Publisher. doi: http://dx.doi.org/10.5772/51198

• Correa, S. J., Jaramillo, A. C., Merino, R. A., & Hormaza, A. (2018). Evaluation of individual fungal species and their co-culture for degrading a binary mixture of dyes under solid-state fermentation. Biotechnology, Agronomy and Society and Environment, 22(4), 242-251. doi: 10.25518/1780-4507.16675

• Cui, Y., Dong, X., Tong, J., & Liu, S. (2015). Degradation of lignocellulosic components in un-pretreated vinegar residue using an artificially constructed fungal consortium. BioResources, 10(2), 3434-3450.

• Cutzu, R., & Bardi, L. (2017). Production of bioethanol from agricultural wastes using residual thermal energy of a cogeneration plant in the distillation phase. Fermentation, 3(2), 1-8. doi: https://doi.org/10.3390/fermentation3020024

• Czitrom, V. (1999). One-factor-at-a-time versus designed experiments. American Statistician, 53(2), 126-131.

• Dasgupta, D., Suman, S. K., Pandey, D., Ghosh, D., Khan, R., Agrawal, D., … & Adhikari, D. K. (2013). Design and optimization of ethanol production from bagasse pith hydrolysate by a thermotolerant yeast Kluyveromyces sp. IIPE453 using response surface methodology. SpringerPlus, 2, 1-10. doi: https://doi.org/10.1186/2193-1801-2-159

• Du, R., Yan, J., Li, S., Zhang, L., Zhang, S., Li, J., … & Qi, P. (2015). Cellulosic ethanol production by natural bacterial consortia is enhanced by Pseudoxanthomonas taiwanensis. Biotechnology for Biofuels, 8, 1-10. doi: https://doi.org/10.1186/s13068-014-0186-7

• Grewal, J., Tiwari, R., & Khare, S. K. (2020). Secretome analysis and bioprospecting of lignocellulolytic fungal consortium for valorization of waste cottonseed cake by hydrolase production and simultaneous gossypol degradation. Waste and Biomass Valorization, 11, 2533-2548. doi: https://doi.org/10.1007/s12649-019-00620-1

• Hamilton, D. F. (2015). Interpreting regression models in clinical outcome studies. Bone and Joint Research, 4(9), 152-153. doi: https://doi.org/10.1302/2046-3758.49.2000571

• Hamouda, H. I., Nassar, H. N., Madian, H. R., Abu-Amr, S. S., & El-Gendy, N. S. (2015). Response surface optimization of bioethanol production from sugarcane molasses by Pichia veronae strain hsc-22. Biotechnology Research International, 2015, 1-10. doi: https://doi.org/10.1155/2015/905792

• Hasunuma, T., & Kondo, A. (2012). Development of yeast cell factories for consolidated bioprocessing of lignocellulose to bioethanol through cell surface engineering. Biotechnology Advances, 30(6), 1207-1218. doi: https://doi.org/10.1016/j.biotechadv.2011.10.011

• Horisawa, S., Inoue, A., & Yamanaka, Y. (2019). Direct ethanol production from lignocellulosic materials by mixed culture of wood rot fungi Schizophyllum commune, Bjerkandera adusta and Fomitopsis palustris. Fermentation, 5(1), 1-8. doi: https://doi.org/10.3390/fermentation5010021

• Huang, J., Chen, D., Wei, Y., Wang, Q., Li, Z., Chen, Y., & Huang, R. (2014). Direct ethanol production from lignocellulosic sugars and sugarcane bagasse by a recombinant Trichoderma reesei strain HJ48. The Scientific World Journal, 2014, 1-9. doi: https://doi.org/10.1155/2014/798683

• Ire, F. S., Ezebuiro, V., & Ogugbue, C. J. (2016). Production of bioethanol by bacterial co culture from agro waste impacted soil through simultaneous saccharification and co fermentation of steam exploded bagasse. Bioresources and Bioprocessing, 3, 1-12. doi: https://doi.org/10.1186/s40643-016-0104-x

• Ivory, R., Delaney, E., Mangan, D., & McCleary, B. V. (2020). Determination of ethanol concentration in kombucha beverages: Single-laboratory validation of an enzymatic method, first action method 2019.08. Journal of AOAC International, qsaa122, 1-25. doi: doi.org/10.1093/jaoacint/qsaa122 doi: https://doi.org/10.1093/jaoacint/qsaa122

• Ja’afaru, M. I. (2013). Screening of fungi isolated from environmental samples for xylanase and cellulase production. International Scholarly Research Notices, 2013, 1-7.

• Jawed, K., Yazdani, S. S., & Koffas, M. A. G. (2019). Advances in the development and application of microbial consortia for metabolic engineering. Metabolic Engineering Communications, 9, 1-10. doi: https://doi.org/10.1016/j.mec.2019.e00095

• Jena, N., & Satpathy, S. (2017). Production of ethanol by Trichoderma spp. in solidstate fermentation of sugarcane molacess. International Journal of Engineering Mathematics, 6(6), 281-288.

• Jiang, L. L., Zhou, J. J., Quan, C. S., & Xiu, Z. L. (2017). Advances in industrial microbiome based on microbial consortium for biorefinery. Bioresources and Bioprocessing, 4(1), 1-10. doi: https://doi.org/10.1186/s40643-017-0141-0

• Jiang, Y., Wu, R., Zhou, J., He, A., Xu, J., Xin, F., … & Dong, W. (2019). Recent advances of biofuels and biochemicals production from sustainable resources using co cultivation systems. Biotechnology for Biofuels, 12, 1-12. doi: https://doi.org/10.1186/s13068-019-1495-7

• Kanagasabai, M., Maruthai, K., & Thangavelu, V. (2019). Simultaneous saccharification and fermentation and factors influencing ethanol production in SSF process. In Y. Yun (Ed.), Alcohol fuels - Current technologies and future prospect (pp. 1-15). London, UK: InTech Open Access Publisher. doi: 10.5772/intechopen.86480

• Kaneko, S., Mizuno, R., Maehara, T., & Ichinose, H. (2012). Consolidated bioprocessing ethanol production by using a mushroom. In M. A. P. Lima (Ed.), Bioethanol (pp. 191-208). Rijeka, Croatia: InTech Open Access Publisher.

• Kanmani, P., Karthik, S., Aravind, J., & Kumaresan, K. (2013). The use of response surface methodology as a statistical tool for media optimization in lipase production from the dairy effluent isolate Fusarium solani. International Scholarly Research Notices, 2013, 1-8. doi: https://doi.org/10.5402/2013/528708

• Kartini, A. M., & Dhokhikah, Y. (2018). Bioethanol production from sugarcane molasses with simultaneous saccharification and fermentation (SSF) method using Saccaromyces cerevisiae-Pichia stipites consortium. IOP Conference Series: Earth and Environmental Science, 207(1), 1-9.

• Kolasa, M., Ahring, B. K., Lübeck, P. S., & Lübeck, M. (2014). Co-cultivation of Trichoderma reesei RutC30 with three black Aspergillus strains facilitates efficient hydrolysis of pretreated wheat straw and shows promises for on-site enzyme production. Bioresource Technology, 169, 143-148. doi: https://doi.org/10.1016/j.biortech.2014.06.082

• Liu, D., Zhang, R., Yang, X., Wu, H., Xu, D., Tang, Z., & Shen, Q. (2011). Thermostable cellulase production of Aspergillus fumigatus Z5 under solid-state fermentation and its application in degradation of agricultural wastes. International Biodeterioration and Biodegradation, 65(5), 717-725. doi: https://doi.org/10.1016/j.ibiod.2011.04.005

• Liu, J., Wang, J., Leung, C., & Gao, F. (2018). A multi-parameter optimization model for the evaluation of shale gas recovery enhancement. Energies, 11(3), 1-29. doi: https://doi.org/10.3390/en11030654

• Mauch, F., Mauch-Mani, B., & Boller, T. (1988). Antifungal hydrolases in pea tissue and inhibition of fungal growth by combinations of chitinase and β-1,3-glucanase. Plant Physiology, 88, 936-942. doi: https://doi.org/10.1104/pp.88.3.936

• Mutreja, R., Das, D., Goyal, D., & Goyal, A. (2011). Bioconversion of agricultural waste to ethanol by SSF using recombinant cellulase from Clostridium thermocellum. Enzyme Research, 2011, 1-6.

• Naik, S. N., Goud, V. V., Rout, P. K., & Dalai, A. K. (2010). Production of first and second generation biofuels: A comprehensive review. Renewable and Sustainable Energy Reviews, 14(2), 578-597. doi: https://doi.org/10.1016/j.rser.2009.10.003

• Pambi, R. L. L., & Musonge, P. (2016). Application of response surface methodology (RSM) in the treatment of final effluent from the sugar industry using Chitosan. In C. A. Brebbia (Ed.), WIT Transactions on Ecology and the Environment (Vol. 209 pp. 209-219). Southampton, UK: WIT Press.

• Park, J. Y., Shiroma, R., Al-Haq, M. I., Zhang, Y., Ike, M., Arai-Sanoh, Y., … & Tokuyasu, K. (2010). A novel lime pretreatment for subsequent bioethanol production from rice straw - Calcium capturing by carbonation (CaCCO) process. Bioresource Technology, 101(17), 6805-6811. doi: https://doi.org/10.1016/j.biortech.2010.03.098

• Ray, R. C., & Behera, S. S. (2017). Solid state fermentation for production of microbial cellulases. In G. Brahmachari, A. L. Demain & J. L. Adrio (Eds.), Biotechnology of microbial enzymes: Production, biocatalysis, and industrial applications (pp. 43-79). Amsterdam, Netherland: Academic Press. doi: https://doi.org/10.1016/B978-0-12-803725-6.00003-0

• Safa, Z. J., Aminzadeh, S., Zamani, M., & Motallebi, M. (2017). Significant increase in cyanide degradation by Bacillus sp. M01 PTCC 1908 with response surface methodology optimization. AMB Express, 7, 1-9. doi: https://doi.org/10.1186/s13568-017-0502-2

• Sarkar, N., Ghosh, S. K., Bannerjee, S., & Aikat, K. (2012). Bioethanol production from agricultural wastes: An overview. Renewable Energy, 37(1), 19-27. doi: https://doi.org/10.1016/j.renene.2011.06.045

• Satyakala, K., Alladi, A., & Thakur, K. D. (2017). Effect of physiological parameters on growth of Aspergillus niger and Trichoderma harzianum. Indian Journal of Pure and Applied Biosciences, 5(4), 1808-1812.

• Saunders, L. J., Russell, R. A., & Crabb, D. P. (2012). The coefficient of determination: What determines a useful R2 statistic? Investigative Ophthalmology and Visual Science, 53, 6830-6832. doi: https://doi.org/10.1167/iovs.12-10598

• Selim, K. A., El-Ghwas, D. E., Easa, S. M., & Hassan, M. I. A. (2018). Bioethanol a microbial biofuel metabolite: New insights of yeasts metabolic engineering. Fermentation, 4(1), 1-27. doi: https://doi.org/10.3390/fermentation4010016

• Shah, S. R., Ishmael, U. C., Palliah, J. V., Asras, M. F. F., & Ahmad, S. S. N. W. (2016). Optimization of the enzymatic saccharification process of empty fruit bunch pretreated with laccase enzyme. Bioresources, 11(2), 5138-5154.

• Shaw, R., Festing, M. F. W., Peers, I., & Furlong, L. (2002). Use of factorial designs to optimize animal experiments and reduce animal use. ILAR Journal, 43(4), 223-232. doi: https://doi.org/10.1093/ilar.43.4.223

• Shong, J., Diaz, M. R. J., & Collins, C. H. (2012). Towards synthetic microbial consortia for bioprocessing. Current Opinion in Biotechnology, 23(5), 798-802. doi: https://doi.org/10.1016/j.copbio.2012.02.001

• Singh, D. P., & Trivedi, R. K. (2013). Acid and alkaline pretreatment of lignocellulosic biomass to produce ethanol as biofuel. International Journal of ChemTech Research, 5(2), 727-734.

• Suhag, M., & Singh, J. (2014). Recent Advances in fermentation of lignocellulosic biomass hydrolysate to ethanol. Journal of Advances in Science and Technology, 7(13), 1-8.

• Syazwanee, M. M. F., Izzati, M. N. A., Azwady, A. N., & Muskhazli, M. (2019). Screening of lignocellulolytic fungi for hydrolyzation of lignocellulosic materials in paddy straw for bioethanol production. Malaysian Journal of Microbiology, 15(4), 379-386. doi: http://dx.doi.org/10.21161/mjm.180250

• Syazwanee, M. M. F., Shaziera, A. N., Izzati, M. N. A., Azwady, A. N., & Muskhazli, M. (2018). Improvement of delignification, desilication and cellulosic content availability in paddy straw via physico-chemical pretreatments. Annual Research and Review in Biology, 26(6), 1-11. doi: https://doi.org/10.9734/ARRB/2018/40947

• Tesfaw, A., & Assefa, F. (2014). Co-culture: A great promising method in single cell protein production. Biotechnology and Molecular Biology Reviews, 9(2), 12-20. doi: https://doi.org/10.5897/BMBR2014.0223

• Wahid, Z., & Nadir, N. (2013). Improvement of one factor at a time through design of experiments. World Applied Sciences Journal, 21, 56-61. doi: 10.5829/idosi.wasj.2013.21.mae.99919

• Wongwilaiwalin, S., Rattanachomsri, U., Laothanachareon, T., Eurwilaichitr, L., Igarashi, Y., & Champreda, V. (2010). Analysis of a thermophilic lignocellulose degrading microbial consortium and multi-species lignocellulolytic enzyme system. Enzyme and Microbial Technology, 47(6), 283-290. doi: https://doi.org/10.1016/j.enzmictec.2010.07.013