Optimization of Laccase Production by Megasporoporia sp. for Bioremoval of Methyl Orange and Cr(VI)

Abstract

Industrial expansion has accelerated the discharge of hazardous pollutants, particularly azo dyes such as methyl orange (MO) and heavy metals such as hexavalent chromium [Cr(VI)], which pose serious environmental and health risks, especially when both contaminants coexist in wastewater. Enzymatic bioremediation offers a sustainable alternative to conventional physicochemical treatments, and this study aimed to optimize laccase production by the white-rot fungus Megasporoporia sp. and evaluate its ability to simultaneously remove MO and Cr(VI). Laccase biosynthesis was optimized using a One-factor-at-a-time (OFAT) approach by systematically varying nutritional and physicochemical factors, including carbon sources, nitrogen sources, copper concentration, pH, temperature, agitation speed, and incubation time. The highest yield was achieved under optimal conditions: glucose or sucrose as the carbon source, a combination of yeast extract and ammonium sulfate as the nitrogen source, 1.5 - 2.0 mM CuSO₄, pH 5.0, incubation at 28 °C for 5 days, and static cultivation. The crude enzyme exhibited strong pollutant removal in the single-pollutant systems, achieving 74.17% MO removal and 98.67% Cr(VI) removal, whereas its performance declined in the dual-pollutant system due to inhibitory interactions between the two contaminants (42.46% MO removal and 87.57% Cr(VI) removal). Phytotoxicity assays using Vigna radiata demonstrated that laccase substantially mitigated toxicity in the single-pollutant treatments, improving germination from 22% to 64% for MO and from 16% to 82% for Cr(VI), whereas only partial recovery was observed in the dual-pollutant system. These findings indicate that Megasporoporia sp. can efficiently produce high laccase yields under mild, low-cost conditions, and that the crude enzyme is highly effective for individual MO and Cr(VI) remediation, although simultaneous removal remains constrained by pollutant interactions. The study highlights the potential of laccase-based bioremediation for wastewater treatment while underscoring the need for further strategies, such as enzyme immobilization, to enhance performance in complex contaminant systems.

HIGHLIGHTS

• Laccase production by Megasporoporia sp. was effectively optimized under the following conditions: Glucose or sucrose as the carbon source, yeast extract combined with ammonium sulfate as the nitrogen source, 1.5 - 2.0 mM CuSO₄, pH 5.0, temperature of 28 °C, 5 days of incubation, and static cultivation.
• In the single-pollutant system, the crude enzyme achieved 74.17% MO removal and 98.67% Cr(VI) removal, demonstrating strong dual catalytic activity toward organic dyes and heavy metals.
• Simultaneous remediation of MO and Cr(VI) was achieved using Megasporoporia sp. laccase, although the removal efficiencies declined due to inhibitory interactions between the pollutants, highlighting mechanistic limitations in the dual-contaminant systems.
• Phytotoxicity evaluation showed substantial detoxification, where laccase treatment increased Vigna radiata germination from 22% to 64% for MO and from 16% to 82% for Cr(VI) in the single-pollutant system. In the dual-pollutant system, germination increased from 8% to 60%, confirming the reduced ecological toxicity of the treated synthetic wastewater.
• The findings support the potential of laccase-based bioremediation as an eco-friendly solution for industrial wastewater containing azo dyes and Cr(VI), and suggest enzyme immobilization as a promising strategy to improve performance under complex pollutant conditions.

GRAPHICAL ABSTRACT