Production of Fungal Laccase Using Orange Peelings as Substrate by Submerged Static Fermentation

Abstract

Aims: Laccases are diphenol oxidases that have numerous applications in biotechnological processes. In this work, the production of fungal laccase using organic and inorganic feed substrates in submerged static fermentation was investigated. Study Design: One-factor-at-a-time strategy was adopted to optimize the cultural parameters for enhanced laccase production. Place and Duration of Study: Department of Microbiology, Faculty of Science, University of Port Harcourt, Nigeria, between October 2014 and November 2015. Methodology: A total of nine fungal isolates were obtained from wood decaying sites of University of Port Harcourt forest areas and subjected to laccase screening with 2,2-Azinobis-3-ethyl(benzthiazoline-6-sulphonate) (ABTS). The influence of medium components using the basal medium at pH 5.0 as base was evaluated and these cultural parameters include carbon sources (glycerol, rice bran, glucose and ground orange peelings), nitrogen sources (yeast extract, potassium nitrate, peptone and ammonium chloride), metal ions (copper sulfate and manganese sulfate) and inducer compounds (ABTS, Tween 80 and soya oil). Time course study was conducted with the unoptimized and optimized cultural medium. Results: Out of nine cultures tested, seven were found to be laccase-positive with isolates CF-1 and CF-2 being the best potential cultures. Isolate CF-1 which had the highest laccase activity was identified as Pleurotus ostreatus and was chosen for further studies. Ground orange peelings (0.1% w/v) and NH4Cl (0.1% w/v) were the most suitable carbon and nitrogen source for laccase production by the fungus. Maximum laccase production was obtained with Cu2+ at a concentration of 0.05%w/v among other metal ions. Soya oil at concentration of 0.05% (v/v) was the best inducer of the enzyme. The highest laccase production was achieved at an Initial pH 4.5. Under optimal culture medium, the maximum laccase activity was determined to be 7.21 U ml-1 on the 7th day of cultivation; which was approximately three times higher than that in basal medium (2.5 U ml-1). The results obtained indicate that the extracellular laccase production is dependent on various cultural parameters. Conclusion: One-factor-at-a-time strategy adopted in this study proved that the optimum conditions enhanced laccase production by three folds using orange peelings. The results obtained are very interesting since orange peelings are common agricultural wastes in several countries and imply that their re-utilization in the production of enzymes would help solve pollution problems caused by their improper disposal. In addition, Pleurotus ostreatus having shown promise for laccase production using low-cost lignocellulosic substrates could be suggested as a prospective candidate for higher laccase production for several biotechnological applications.