Characterization of hydrolase-associated COD reduction by Bacillus subtilis BY8.

AIMS: This study aimed to evaluate the chemical oxygen demand (COD) reduction ability of BY8 and optimise its fermentation conditions for aquaculture-derived organic matter reduction.

METHODS AND

RESULTS

BY8 was cultivated in aquaculture feed leachate to assess its growth, COD reduction, and hydrolytic capacity. The genome of BY8 was sequenced and hydrolytic enzyme-related genes were identified. Fermentation conditions were optimised by screening carbon and nitrogen sources and their concentrations. BY8 reduced COD in feed leachate by 45.57% ± 2.47% within 24 h. Clear hydrolysis zones were observed on protease-, amylase-, and cellulase-screening media. Extracellular protease, amylase, and cellulase activities reached 0.68 ± 0.06 U ml-1, 36.31 ± 2.65 U dl-1, and 3.00 ± 0.74 U mg-1 protein at 24 h, respectively. Genome annotation identified 92 non-redundant hydrolase-associated candidate ORFs, including protease/peptidase-, GH13 alpha-glucan/starch-, and cellulose or β-glucan-related candidates. qRT-PCR analysis confirmed transcriptional responses of selected hydrolase-associated genes, with Vpr gene (vpr), the maltogenic α-amylase MdxD gene (mdxD), and the putative FrvX-family M42 peptidase gene (frvX) showing the greatest fold increases at 24 h relative to their respective 0-h levels within the selected functional groups. Fermentation optimisation showed that glucose and soybean meal were the most suitable carbon and nitrogen sources for BY8, with optimal concentrations of 5% and 2%, respectively. Under the optimised small-scale fermentation conditions, the viable cell counts reached 1.08 × 10¹¹ CFU ml-1.

CONCLUSIONS

BY8 showed substantial extracellular hydrolase production and COD reduction in feed leachate, supporting its possible use as a candidate strain for aquaculture organic-load management.