Optimisation of Laccase Activity From Bacillus atrophaeus Using Response Surface Methodology: A Proof-Of-Concept Dye Decolourisation Study

Abstract

This study investigates the enhancement of laccase activity, a copper-containing enzyme involved in oxidative biodegradation. The enzyme was studied in Bacillus atrophaeus, newly isolated from paper mill wastewater. Initial optimisation of key factors, including carbon and nitrogen sources, incubation time, inoculum size, pH, temperature, and CuSO4 concentration. Subsequently, a systematic refinement of selected parameters was performed through response surface methodology, a statistical optimisation technique. The maximum laccase activity of 0.057 U/mL was achieved under the following conditions: pH 8.0, 35.28°C, 1.5% CuSO₄, 0.5% inoculum size, 3.7 g/L fructose and 1.08 g/L yeast extract. Under these conditions, a 2.51-fold enhancement in enzymatic activity was achieved compared to pre-optimised conditions. The optimised enzyme activity was then tested for its ability to decolourise dyes, specifically Congo red, burazol black and burazol navy. Congo red decolourisation exhibited a 2.95-fold increase after 72 h under optimised conditions, whereas burazol black and burazol navy dyes remained unaffected. These findings underscore the potential of optimised laccase-based methods for efficient dye wastewater treatment. Using response surface methodology, key parameters were fine-tuned to enhance laccase activity and decolourisation efficiency, advancing sustainable bioremediation in environmental biotechnology.