Use of combined UV and chemical mutagenesis treatment of Aspergillus terreus D34 for hyper-production of cellulose-degrading enzymes and enzymatic hydrolysis of mild-alkali pretreated rice straw

Abstract

Background

Microbial production of cellulose-degrading enzymes could be significantly improved using traditional mutagenesis treatment. Development of high-titre cellulase producing mutants drastically reduces the costs involved in cellulase production and downstream processing in commercial-scale enzyme production. Here, we have evaluated the efficacy of different Aspergillus terreus D34 mutants for hyper-production of improved cellulase enzymes utilizing locally available lignocellulosic biomass residues as growth substrates in solid state fermentation conditions. Further, enzymatic hydrolysis of mild-alkali pre-treated rice straw was performed using the improved cellulases.

Results

A 4.9-fold higher β-glucosidase activity was obtained from ethyl methyl sulphonate (EMS) treated mutant strain (EMS2) when grown on mixed rice straw/sugarcane bagasse (RSBG) biomass growth substrate. Similarly with the EMS2 mutant and BG-grown culture extract a 1.1-fold higher xylanase activity was observed. Irrespective of the growth substrates and the mutant strains, the maximum cellulase (FPase, carboxymethyl cellulase, avicelase, β-glucosidase) and xylanase activities (U mL⁻¹) were 2.34, 39.8, 2.46, 19.9 and 655, respectively. Further, external supplementation of 20% bovine serum albumin (BSA), 3% tween 80 and 20% polyethylene glycol (PEG) 6000 to the crude enzyme extract increased the FPase activity nearly 4.0-, 2.8- and 2.2-fold. Addition of 0.05% sodium benzoate marginally increased the stability of cellulase enzyme and retained more than 60% of the initial activity after 96 h incubation at 37°C. While at 4°C, no loss in enzyme activity was observed even after prolonged incubation period (up to 90 days). Further, maximum reducing sugars of 0.842 g g⁻¹ at a rate of 0.25 mM g⁻¹ h⁻¹ at 10% biomass loading of mild-alkali pretreated rice straw was produced using the BG-grown culture extract of EMS2 mutant strain.

Conclusion

The extracellular protein production and corresponding cellulase activities of A. terreus D34 were significantly enhanced after combined UV and chemical mutagenesis treatments. In the present study, besides accelerating the rate of cellulase production, we have also demonstrated production of high reducing sugars by enzymatic saccharification of pretreated lignocellulosic biomass using hyper-produced cellulase enzymes. Due to high enzyme activity of the cellulase enzymes produced from the mutant strains, the volume of enzyme loadings in enzymatic hydrolysis could be reduced up to 7-fold. These studies clearly show the potential of the developed hyper-cellulase producing mutants in decreasing the overall process economics of cellulosic ethanol technology.

Keywords

A. terreus / Cellulases / Mutagenesis / Lignocellulosic biomass / Enzymatic hydrolysis

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Funding

ICAR/ACRIP-RES(VVN/RES/DRET-LBT/2014/4)