Fish-Derived Clostridium butyricum LZK-08: Genomic Insights and Probiotic Evaluation in Largemouth Bass.

Abstract

Clostridium butyricum has gained attention as a probiotic in aquaculture due to its ability to improve growth, gut health, and immune function. However, most strains currently used are derived from non-aquatic sources, which may limit their colonization and efficacy in fish. In this study, a novel strain, C. butyricum LZK-08, was isolated from the intestine of grass carp. The strain was identified based on morphological features, 16S rRNA sequencing, and whole-genome analysis. Its probiotic properties were assessed through comprehensive in vitro assays and in vivo trials in largemouth bass. LZK-08 exhibited strong acid (pH 4.0), bile salt (0.3%), and heat (up to 80 °C) tolerance, along with high auto-aggregation (92.3%) and notable surface hydrophobicity. It produced digestive enzymes (amylase, protease, lipase) and short-chain fatty acids, particularly butyrate (523.32 μg/mL). Genomic analysis revealed a 4.71 Mb genome composed of a circular chromosome and plasmid, encoding 4,207 protein-coding genes. Key genes were enriched in carbohydrate and amino acid metabolism, stress response, and adhesion. Feeding trials in largemouth bass showed that dietary supplementation with LZK-08 significantly improved growth parameters, reduced feed conversion ratio, enhanced intestinal antioxidant enzyme and digestive enzyme activities. Histological analysis revealed improved intestinal morphology, including increased villus height and muscle thickness. 16S rRNA sequencing further showed that LZK-08 modulated gut microbiota by enriching beneficial taxa such as Lactobacillus and reducing potentially unfavorable Romboutsia. These findings indicate that C. butyricum LZK-08 is a physiologically robust, and genomically safe probiotic with strong functional attributes, demonstrating high potential for use in sustainable aquaculture.