Modulation of Off-Flavor Compounds and Microbial Community Structure in Acrossocheilus fasciatus Aquaculture by Bacillus licheniformis Strain 23

Geosmin (GSM) and 2-methylisoborneol (2-MIB) are the primary off-flavor compounds in freshwater fish and can severely reduce product quality and market value. Biological regulation using probiotics has emerged as a promising eco-friendly strategy for controlling these off-flavors. In this study, Bacillus licheniformis strain 23 was applied to Acrossocheilus fasciatus culture systems through two practical modes: direct water spraying (water quality regulation group, A) and dietary supplementation (feed regulation group, S), with a control group (D) receiving no probiotic. GSM and 2-MIB concentrations in water and fish muscle were measured by HS-SPME-GC-MS, and microbial community structures in water and fish gut were analyzed by high-throughput sequencing of the 16S rRNA gene. Due to the limited number of available ponds, each treatment was applied to a single pond (n = 1 pond per treatment), and the three samples collected per group were spatial subsamples rather than independent biological replicates. Therefore, between-treatment comparisons are presented as descriptive and exploratory. Under the conditions tested, both treatments were associated with reduced GSM and 2-MIB levels in fish muscle and water. After 48 days, GSM and 2-MIB in the feed regulation group decreased to 0.754 μg/kg and 0.103 μg/kg, respectively, representing reductions of 46.8% and 63.2% compared with the control. The water quality regulation group showed a 52.9% reduction in GSM, while its 2-MIB did not differ markedly from that of the feed regulation group on day 48. Both treatments were associated with changes in microbial community structure. Water quality regulation increased species richness in the water and promoted chlorophyta and cyanobacteriota, whereas feed supplementation simplified the water community, with chlorophyta becoming overwhelmingly dominant. In the fish gut, water quality regulation induced marked community restructuring, characterized by a sharp decline in cyanobacteriota and an increase in Planctomycetota, whereas feed supplementation caused only moderate disturbance. Correlation analysis suggested that Geminiocystis PCC-6308 was strongly positively correlated with 2-MIB concentration, whereas several taxa within Pseudomonas and Planctomycetota were negatively correlated with off-flavor compounds. These findings suggest that B. licheniformis strain 23 may be useful for reducing earthy–musty off-flavors in A. fasciatus aquaculture by reshaping microbial communities. However, given the lack of independent pond-level replication, these results should be regarded as preliminary and hypothesis-generating, and further validation in replicated pond experiments is needed.