Deep eutectic solvents(DES) pretreatment of Leucaena leucocephala for production of the bioethanol using Kluyveromyces marxianus UniMAP 1-1

Cellulosic bioethanol production using deep eutectic solvents (DESs) as the pretreatment solvent is a promising step in producing fermentable sugars. It can overcome the drawbacks of using conventional acid and alkaline pretreatment that could be harmful to the environment. Moreover, DESs are capable of solubilizing lignin, opening up the complex biomass matrix, thus allowing enzymatic attack towards the cellulose and hemicellulose structure. However, wide range of DESs combination need to be chosen wisely due to the suitability and compatibility of DESs were strongly depend on the specific biomass and process involved. In addition, non-identical optimum condition between hydrolysis and fermentation have hindered simultaneous saccharification and fermentation (SSF) to be successful in bioethanol production. Hence, this study was conducted to screen the suitable combination of DES mixture to be used in pretreatment of Leucaena leucocephala. In order to effectively pretreat L. leucocephala, several factors that influence the pretreatment and enzymatic hydrolysis need to be optimized before being used in fermentation process, thus the yield of bioethanol can be evaluated. In this work, 30 combination of DESs were synthesized and screened through several criteria such as their physicochemical properties and the stability and compatibility towards L. leucocephala, cellulase enzyme and Kluyveromyces marxianus UniMAP 1-1. After that, the best selected DES was then underwent several pretreatment processes to optimized factors influencing pretreatment process such as solid loading, water addition and enzyme concentration. The optimized conditions were then used in the fermentation process using K. marxianus via SSF and the yield of bioethanol produced was evaluated using high performance liquid chromatography (HPLC). The best screened DES was ChCl:Gly at 1:2 molar ratio with conductivity at 1.86 mS/cm, viscosity at 258.3 cP and pH 4.11. ChCl:Gly (1:2 M) also shows remarkable compatibility and stability with cellulase enzyme by retaining most of cellulase activity at 7.6 FPU during 48 h incubation. It also shows potential compatibility towards L. leucocephala and K. marxianus by showing an optimal adaptation in both conditions. The optimum solid loading was at 10% (w/v), water addition at 25% (v/v) and enzyme concentration at 30 FPU were necessary to obtain the highest glucose possible. The maximum bioethanol yield from this study was at 0.162 g/g with 31.68 % conversion efficiency which showed that this newly isolated K. marxianus can be used in fermentation process. In short, DESs could serve as a great option in biorefineries, not only capable in solubilizing lignin, but also effectively improve subsequent cellulose digestion and glucose yields, thus providing alternative route to achieve consolidated bioprocessing (CBP) strategy.