Exploration of Marine Microbes for Bioethanol Production from Lignocellulosic Substrates in Seawater Medium

Abstract

The growing demand for petroleum-based fuels in the aftermath of rapid growth of world population and climate change concerns has augmented the need for renewable bio-energy resources with minimal greenhouse gas emissions. However, the operational hazard of the transition towards a bio-based economy and running large-scale biomass processing has led to an unprecedented surge in fresh water consumption that renders biofuel a high water footprint product. In the current study, seawater is used as a reaction medium for pretreatment, saccharification and fermentation seeking the reduced consumption of fresh water in bioprocesses. The first objective addresses the feasibility of optimized model of microwaveassisted NaOH pretreatment of lignocellulosic biomass using seawater medium. Response surface methodology (RSM) based Box Behnken design (BBD) was employed to model, predict and validate cellulose release and reducing sugar yield for rice straw which was further validated for sugarcane bagasse and kans grass respectively. The optimized pretreatment conditions (8.50% substrate loading, 1.94% NaOH, 4.09 min and 160 W) in rice straw resulted in a cellulose release of 65.43% and reducing sugar yield of 0.554 g/g. The second objective deals with establishing a seawater-based model of eco-friendly mode of biodelignification by exploiting the efficiency of marine ligninolytic bacterial strains isolated from seawater habitat. The isolated halotolerant strain was identified and named Shewanella chilikensis LDB and used for the biopretreatment of lignocellulosic substrates in seawater medium. Estimation the residue lignin upon biopretreatment revealed a reduction in lignin content from 29.15% to 20.28% in sugarcane bagasse, whereas in case of rice straw and kans grass the observed lignin removal was 23.70% to 16.42% and 25.33% to 22.58% respectively. Further, a similar approach was devised in the third objective, where isolation and screening of a potent marine cellulolytic bacteria Bacillus haynesii CDB3 was carried out and the bacterial cellulase was used for further studies. The crude cellulase was found to be halotolerant, thermostable and exhibited a maximum residual activity of 1.15 U/ml at pH 5 and 45 °C. The bacterial cellulase was employed for the saccharification of alkali pretreated biomass (under conditions optimized in the first objective) in seawater medium which resulted in a sugar release of 3.355 mg/ml for sugarcane bagasse and 3.049 mg/ml and 2.599 mg/ml for rice straw and kans grass respectively. The final objective addressed the feasibility of the sequential pretreatment (cotreatment: microwave-NaOH + ligninolytic LDB1 strain) of lignocellulosic biomass followed by saccharification using crude cellulase from marine cellulolytic CDB3 strain. The lignin content in the cotreated substrates greatly diminished from 10.45% in alkali pretreated rice straw to 6.37% upon cotreatment. Likewise, alkali pretreated sugarcane bagasse contained 13.46% lignin which upon cotreatment reduced to 7.20%, whereas for kans grass the lignin gradually declined from 16.43% to 11.02%. Saccharification of the cotreated substrates using crude cellulase resulted in a reducing sugar release of 6.12 mg/ml for rice straw, 6.98 mg/ml for sugarcane bagasse and 4.65 mg/ml for kans grass respectively. The saccharified hydrolysates were subsequently subjected to fermentation using marine yeast AZ65 strain and the ethanol concentration was estimated by potassium dichromate method. The concentration of ethanol upon cotreatment were 2.76 g/L (kans grass), 3.65 g/L (rice straw) and 4.34 g/L (sugarcane bagasse) respectively. The findings of the study demonstrated the rationale of using a combined biological and chemical mode of pretreatment in seawater medium for lignocellulosic biomass conversion into biofuels.