International Journal of Renewable Energy Development 11 (2) 2022: 423-433 |423 IJRED-ISSN: 2252-4940.Copyright © 2022. The Authors. Published by CBIORE Contents list available at IJRED website Int. Journal of Renewable Energy Development (IJRED) Journal homepage: https://ijred.undip.ac.id Lignocellulosic Bioethanol Production of Napier Grass Using Trichoderma reesei and Saccharomyces cerevisiae Co-Culture Fermentation Thirawat Mueansichai a , Thaneeya Rangseesuriyachai b, , Nuttha Thongchul c,d , Suttichai Assabumrungrat e a Department of Chemical and Materials Engineering, Faculty of Engineering, Rajamangala University of Technology Thanyaburi, Pathumthani 12110, Thailand b Department of Civil Engineering, Faculty of Engineering, Rajamangala University of Technology Thanyaburi, Pathumthani 12110, Thailand c Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand d Research Unit in Bioconversion/Bioseparation for Value-Added Chemical Production, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok 10330, Thailand e Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand Abstract. Bioethanol from agricultural waste is an attractive way to turn waste into added value that will solve the problem of food competition and waste management. Napier grass is a highly productive and effective lignocellulosic biomass, which is an important substrate of the second-generation biofuels. In addition, several processes are required in the production of ethanol from lignocellulosic materials; thus, co-culture fermentation can shorten the production process. This experimental research utilizes Trichoderma reesei and Saccharomyces cerevisiae co-culture fermentation in the bioethanol production of Napier grass using simultaneous saccharification and fermentation technology. To improve ethanol yield, Napier grass was pretreated with 3% (w/w) sodium hydroxide. An orthogonal experimental design was employed to optimize the Napier grass content, mixed crude co-culture loading, and incubation time for maximum bioethanol production. The results showed that pretreatment increased cellulose contents from 52.85% to 82%. The optimal fermentation condition was 15 g Napier grass, 15 g mixed crude co-culture, and 7 days incubation time, which maximizes the bioethanol yield of 16.90 g/L. Furthermore, the fermentation was upscaled 20-fold, and experiments were performed with and without supplemented sugar using laboratory-scale optimal fermentation conditions. The novelty of this research lies in the use of a mixed crude co-culture of T. reesei and S. cerevisiae to produce bioethanol from Napier grass with the maximum bioethanol concentration of 25.02 and 33.24 g/L under unadded and added sugar conditions and to reduce operational step and capital costs. Keywords: Bioethanol; Napier grass; Trichoderma reesei; Saccharomyces cerevisiae Article history: Received: 20 th Nov 2021; Revised: 9 th Jan 2022; Accepted: 16 th Jan 2022; Available online: 25 th January 2022 How to cite this article: Mueansichai, T., Rangseesuriyachai, T., Thongchul, N., and Assabumrungrat, S. (2022) Lignocellulosic Bioethanol Production of Napier Grass Using Trichoderma reesei and Saccharomyces cerevisiae Co-Culture Fermentation. Int. J. Renew. En. Dev., 11(2), 423- 433 https://doi.org/10.14710/ijred.2022.43740 1. Introduction The Sustainable Development Goals (SDGs) are the world’s shared plan to achieve a better and more sustainable future quality of life. Affordable and clean energy is one of the 17 goals for sustainable community development. In addition, the use of clean energy can also achieve the climate action goal to solve the current climate change problem, which is another goal of SDG. Bioethanol is renewable energy made from biomass or agricultural by- products, resulting in a clean emission during combustion. Thailand is abundant in energy plants, including grasses,  Corresponding author: thaneeya.r@en.rmutt.ac.th which can be excellent feedstocks for a variety of high- value products, including bioethanol. Unlike the first- generation ethanol that relies on sugar crops, the second- generation bioethanol utilizes lignocellulosic materials (energy plants) and agricultural wastes to mitigate food insecurity (Restiawaty et al., 2020; Sudiyani et al., 2016; Menegol et al., 2016; Sanford et al., 2017). Napier grass (Pennisetum purpureum) typically grown as animal feed, is considered a lignocellulose material, which has a long lifespan with high crop yields and year- round harvest. It has been known as an energy plant and is a promising alternative for bioethanol production Research Article