Understanding the effects of low enzyme dosage and high solid loading on the enzyme inhibition and strategies to improve hydrolysis yields of pilot scale pretreated rice straw

The commercial viability of 2G bioethanol production is dependent on cost-effective and efficient enzymatic hydrolysis of biomass at low enzyme and high dry solid loadings. In the present study, rice straw was subjected to a dilute acid pretreatment in a continuous pilot plant (250 kg/day). It was observed that, the glucan conversion yields were reduced by 26% at high solid loading (20% w/w) with low enzyme dosage of 3 FPU/g w/w of in-house developed enzyme (MRJ16). Thus, multiple approaches were investigated to enhance the glucan conversion yields, a) fed batch approach (splitting biomass or enzyme dosage during process), b) additives supplementation (surfactants, metal ions, non-enzymatic proteins and activated charcoal) and c) enzyme blending with accessory enzymes. Results showed that, the 3-stage fed-batch approach enhanced the hydrolysis yield by 12% in comparison to 14.3% increase with the selected surfactant (0.5% w/w Ecosurf). The selected metal salt (NiCl2, 1 mM) increased the glucan conversion by 13.2%, while, the non-enzymatic proteins (5% w/w yeast hydrolysate and yeast extract) improved the yields to 14.8% and 14.4%, respectively. The activated charcoal at a concentration of 0.5% w/w enhanced the hydrolysis yields by 14.4%. Further, 15.1% increase in sugar yield was obtained by supplementing the in-house produced enzyme (MRJ16) with xylanase from Thermomyces lanuginosus and BGL from Penicillium janthinellum EMS-UV-8. Finally, a unique optimized strategy was developed on the basis of selected approaches and parameters which demonstrated 24.7% higher saccharification in comparison to control. To the best of our knowledge, this is the only study available in literature where; a wide range of strategies were studies on a single feedstock (i.e. rice straw) pretreated at pilot-scale and determine its effect for improved enzymatic hydrolysis. Furthermore, developed process helps us to save (almost 2X) expensive enzyme to achieve same level of glucan conversion (similar to 62.0%) to make the overall process cost effective by using cheap and easily available additives. The current study also provided an in-depth understanding of enzyme inhibition at low enzyme dosages and high solid loadings in an industrially relevant scenario.