Sreyneang Nhim ¹ Sirilak Baramee ² Chakrit Tachaapaikoon ² Patthra Pason ² Khanok Ratanakhanokchai ² Ayaka Uke ³ Ruben Michael Ceballos ⁴ Akihiko Kosugi ³ Rattiya Waeonukul ^2*

• ¹ Enzyme Technology Laboratory, School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok, Thailand
• ² Excellent Center of Enzyme Technology and Microbial Utilization, Pilot Plant Development and Training Institute (PDTI), King Mongkut's University of Technology Thonburi, Bangkok, Thailand
• ³ Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, Ibaraki, Japan
• ⁴ Molecular and Cell Biology Department, University of California, Merced, Merced, California, United States

Maximizing saccharification efficiency of lignocellulose and minimizing the production costs associated with enzyme requirements are crucial for sustainable biofuel production. This study presents a novel semi-fed-batch saccharification method that uses a co-culture of Clostridium thermocellum and Thermobrachium celere strain A9 to efficiently break down high solid-loading lignocellulosic biomass without the need for any external enzymes. This method optimizes saccharification efficiency and enhances glucose production from alkaline-treated rice straw, a representative lignocellulosic biomass. Initially, a co-culture of C. thermocellum and T. celere strain A9 was established with a treated rice straw loading of 150 g/l, supplemented with Tween 20, which enhanced enzymes stability and prevented unproductive binding to lignin, achieving a remarkable glucose concentration of up to 90.8 g/l. Subsequently, an additional 100 g/l of treated rice straw was introduced, resulting in a total glucose concentration of up to 140 g/l, representing 70.1% of the theoretical glucose yield from the 250 g/l treated rice straw load. In contrast, batch saccharification using an initial substrate concentration of 250 g/l of alkaline-treated rice straw without Tween 20 resulted in a glucose concentration of 55.5 g/l, with a theoretical glucose yield of only 27.7%. These results suggest that the semi-fed-batch saccharification method using co-cultivation of C. thermocellum and T. celere strain A9, supplemented with Tween 20 is an efficient microbial method for saccharifying high-concentration biomass. Moreover, this approach effectively manages high solids loading, optimizes efficiency, and reduces the need for external enzymes, thus lowering production costs and simplifying the process for industrial applications.