Chromium Resistant Laccase Producing Bacillus sp. role on Substrate Fermentation and Reactive Blue Degradation

The bacterial strain capable of decolourizing and degrading reactive blue (RB) dye was isolated from a heavy metal-contaminated site. The strain was screened for chromium resistance and laccase production and the positive isolate was selected and identified as Gram-positive B. cereus. Its decolourization ability of reactive blue dye was then tested under different parameters, such as pH, dye concentration and inoculum volume under static conditions. A translucent, large, irregular colony showed an 87% chromium tolerance index and was identified as a Gram-positive, slender rod. The isolate tested positive for laccase on guaiacol agar and exhibited 63%, 58% and 54% RB degradation at 48 hr for concentrations of 50, 100 and 200 ppm, respectively. The maximum observed decolourization was 70-80% with a 5-10% inoculum size after 48 hr of incubation. Decolourization was most efficient at pH 5 (62%), with 10% culture achieving a maximum of 81.53% decolourization. Bagasse substrates were ideal for laccase production, reaching a maximum of 9 units with 4 g of substrate. The activity of purified enzyme increased 3-10 fold compared to the crude enzyme. Optimal conditions for Bacillus subtilis included 100 ppm dye, pH 5, 37°C temperature and 5% inoculum. Purification resulted in decreased total protein concentration but increased enzyme activity. The study confirmed the potential of Bacillus subtilis for azo dye degradation, as thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analyses showed increased fractions with different response factor (Rf) and retention times, indicating dye degradation. Our results recommend screening for the most efficient bacterial strains resistant to chromium for dye decolourization applications. Additionally, B. subtilis utilizing bagasse—a cost-effective substrate—produces extracellular laccase, which promotes bioremediation of RB dye. This is the first report demonstrating high laccase yield from B. subtilis using bagasse agricultural residues. Further improvements in laccase production can be achieved through standardization of fermentation strategies and enzyme kinetics.