Improved Enzymatic Hydrolysis of Pilot Scale Pretreated Rice Straw at High Total Solids Loading

Enzymatic hydrolysis at high solids loading has the potential to reduce both capital and operational expenditures. Here, pretreatment of rice straw (PRS) with dilute acid was carried out at a pilot scale (250 kg per day) at 162°C for 10 min and 0.35% acid concentration, followed by enzymatic hydrolysis at different total solids loadings. It showed that although the total sugar concentration increased from 48 to 132 g/l, glucan conversion reduced by 27% (84–66.2%) with increasing solids from 5 to 20% in batch mode. Therefore, two different fed-batch approaches were evaluated to improve the glucan conversion by the sequential addition of a substrate and/or enzyme. At 20% solid loadings and a 3 filter paper units/g enzyme dosage, the highest glucan conversion obtained was 66% after 30 h of hydrolysis in batch mode. However, in an optimized fed-batch approach, the glucan yield was improved to 70% by simply dividing the substrate feeding into three batches, that is, 50% at 0 h, 25% each after 4 h, and 8 h of hydrolysis reaction. The addition of surfactant (Ecosurf E6) further improved the conversion to 72% after 30 h. The role of critical factors, that is, inhibitors, enzyme–lignin binding, and viscosity, was investigated during the course of hydrolysis in the batch and fed-batch approaches. This study suggests a sustainable approach for improved hydrolysis at high solids loadings by fine-tuning a simple process

Synergistic Enzyme Cocktail to Enhance Hydrolysis of Steam Exploded Wheat Straw at Pilot Scale

Multiple enzymes are required for efficient hydrolysis of lignocellulosic biomass and no wild type organism is capable of producing all enzymes in desired levels. In this study, steam explosion of wheat straw was carried out at pilot scale and a synthetic enzyme mixture (EnzMix) was developed by partially replacing the cellulase with critical dosages of commercially available accessory enzymes (β-glucosidase, xylanase and laccase) through central composite design. Highest degree of synergism (DS) was observed with β-glucosidase (1.68) followed by xylanase (1.36). Finally, benchmarking of EnzMix (Celluclast, β-glucosidase and xylanase in a protein ratio of 20.40: 38.43: 41.16, respectively) and other leading commercial enzymes was carried out. Interestingly, hydrolysis improved by 75% at 6 h and 30% at 24 h, respectively in comparison of control. By this approach, 25% reduction in enzyme dosage was observed for obtaining the same hydrolysis yield with opitimized enzyme cocktail. Thus, development of enzyme cocktail is an effective and sustainable approach for high hydrolysis efficiency

Development of a β-glucosidase hyperproducing mutant by combined chemical and UV mutagenesis

The extracellular β-glucosidase from microorganisms is generally produced in low levels. Therefore, in this study, a β-glucosidase hyperproducing mutant was developed by multiple exposures of ethyl methyl sulfonate (EMS) and ultraviolet (UV) radiation (both individually and jointly) to Bacillus subtilis strain (PS). The developed mutants were screened, selected and characterized. The mutant, PS-UM1 developed after UV exposure alone, indicated a small increase in β-glucosidase production (718 U/l) in comparison to the wild-type strain, PS (675 U/l). The mutant, PS-CM5 developed after EMS exposure alone, displayed a slightly better production (762 U/l) than both the above strains. However, after exposure of the wild-type strain to both UV and EMS mutagens jointly, a better mutant (PS-CM5-UM3) was developed with 1.2-fold increase in production (806 U/l). Further, optimization of culture conditions by classical “one-variable-at-a-time” approach was done to determine the optimum, pH, temperature and nitrogen sources. The selected mutant (PS-CM5-UM3) produced up to 1,797 U/l enzyme and was found to be stable for ten generations. The β-glucosidase from the selected mutant (PS-CM5-UM3) was concentrated and purified using ammonium sulfate, dialysis and size-exclusion chromatography. The enzyme displayed maximal activity at 60 °C and it was found to be fairly stable at temperatures up to 70 °C for 30 min. Its molecular weight was determined to be around 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)