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

Abstract

Aims: This study aimed to evaluate the chemical oxygen demand (COD) reduction ability of BY8 and optimize 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 optimized 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 optimization 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 optimized 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.