Agitation role (Dissolved Oxygen) in production of laccase from newly identified Ganoderma multistipitatum sp. nov. and its effect on mycelium morphology

Abstract

Background Agitation speed influenced the production rate of laccase. Orbital speed not only influenced the enzyme production, but was also effective to dissolve the oxygen during growth of mycelium, spores, and chlamydospores. Shear effects of speed greatly influenced the morphology of mycelium. Methods Ganoderma multistipitatum was identified by ITS marker. Phylogenetic tree was constructed for species identification. Qualitatively by plate method contained guaiacol indicator, while quantitatively by submerged fermentation and Central Composite Design applied on agitation parameter for maximum laccase potential of this species. The effects of agitation speed on mycelium morphology were observed under compound and scanning electron microscope. Results Statistical optimization of agitation conditions were performed by using response surface methodology to enhance the production of laccase from Ganoderma multistipitatum sp. nov. Maximum laccase yield (19.44 × 105 ± 0.28 U/L) was obtained at 150 rpm grown culture, which was higher than predicted value of laccase production (19.18 × 105 U/L) under aerobic conditions (150 rpm). The 150 rpm provided the continuous flush of oxygen. The DO (dissolved oxygen) was maximum (65%) for “27 h” incubation at 150 rpm during laccase synthesis. The statistical value of laccase production was minimum under anaerobic or nearly static condition of 50 rpm. The predicted (12.78 × 105 U/L) and obtained (12.82 × 105 U/L) yield was low at 50 rpm. Optimization of orbital shaking for aeration conditions were performed by the use of “Response Surface Methodology”. The submerged shaking flasks were utilized as a nutrients growth medium to maximize the production of laccase from G. multistipitatum. The minimum incubation time highly influenced the laccase yield from 7 to 15 days via utilization of less cost-effective medium under a promising and eco-friendly method. The morphological effects of rpm on mycelium were examined under compound and scanning electron microscopy. Higher rpm (200, 230) shear the mycelium, while 150 to 200 rpm exhibited smoother and highly dense branches of mycelia. Conclusion The shear forces of 200 rpm caused the damages of mycelium and cells autolysis with less laccase production. This study concluded that 150 rpm saved the life of mycelium and enhanced the production rate of enzymes.