**Background:** The intestinal microbiome of animals is closely linked to host physiology, nutrition, and metabolism, and can confer benefits such as pathogen resistance, immune system development, and improved energy absorption. Marine ascidians, occupying a pivotal position in chordate evolution, harbor unique gut microbiota that may contribute to host environmental adaptation. Previous work by the authors demonstrated that Halocynthia roretzi harbors an indigenous gut microbiome distinct from the surrounding marine environment, with significant seasonal variation. However, most studies on ascidian gut microbes have been limited to species composition, with few bacterial strains cultivated and germplasm resources preserved. The present study aimed to isolate, culture, and functionally characterize gut bacteria from H. roretzi across four seasons to explore their roles in host adaptation.
**Methods:** Adult H. roretzi specimens were collected from an aquaculture farm in Weihai City, Shandong Province, China (37.17713° N, 112.5742° E) in January, April, July, and October 2018. Stools were collected aseptically and stored in sterile glycerol at −80°C. For aerobic culture, samples were inoculated into Marine Agar Zobell 2216 medium or Brain–Heart Infusion Broth and incubated at 20°C for 24 h, then serially diluted and spread on MA, BHI, Yeast Malt Agar, or Ashby's mannitol agar plates for 48–72 h at 20°C. For anaerobic culture (winter samples only), samples were processed in an anaerobic operation box using MA or South Pacific Gyre liquid medium, then spread onto MA or SPG plates and incubated anaerobically at 20°C for 48–72 h. Single colonies were picked for DNA extraction and 16S rDNA sequencing using universal primers B8F and 1510R. Phylogenetic trees were constructed using the neighbor-joining algorithm with 1000 bootstrap replicates. Antibacterial activity of Serratia sp. extracts was examined by zone-of-inhibition experiments against Staphylococcus aureus and Proteus sp.
**Key Results:** A total of 263 bacterial strains were obtained: 165 from aerobic culture and 98 from anaerobic culture. Aerobic isolates belonged to six classes (Gammaproteobacteria, Actinobacteria, Bacilli, Deltaproteobacteria, Clostridia, and Flavobacteriia), 12 orders, 23 families, and 25 genera. The Shannon diversity index was >2.5 in each season. The most abundant genus was Bacillus (71 strains, 43% of aerobic isolates), followed by Serratia (23 strains, 14%) and Lysinibacillus (12 strains, 7%). Ten strains had <97% genetic identity with reference strains, representing potential new species, all belonging to Gammaproteobacteria or Bacilli. Seasonally, 39 strains (9 genera) were obtained in spring, 37 (10 genera) in summer, 30 (8 genera) in autumn, and 59 (11 genera) in winter. Bacillus and Vibrio were present in all four seasons, with Bacillus representing 28%, 35%, 67%, and 46% of culturable microbiome in spring, summer, autumn, and winter, respectively. Serratia had the highest abundance in spring (41%). Anaerobic culture yielded 98 strains from 14 genera, with Bacillus again most abundant (32 strains, 32.65%), followed by Staphylococcus (19.39%). Novel anaerobic strains belonged to Gammaproteobacteria or Clostridia. A Serratia sp. isolate produced red-pigmented colonies at high density, and crude ethanol extracts showed high inhibitory effects against both gram-positive (Staphylococcus aureus) and gram-negative (Proteus sp.) bacteria.
**Clinical Implications:** While this study is primarily a microbiological and ecological investigation rather than a clinical trial, it has implications for natural product discovery and marine biotechnology. The demonstration that gut-derived Serratia sp. produces antibacterial metabolites active against both gram-positive and gram-negative pathogens supports the potential of ascidian gut microorganisms as sources of novel antibiotics. The isolation of numerous Bacillus strains, many known as probiotics in aquatic organisms, suggests potential applications in aquaculture for disease prevention and immune enhancement. Furthermore, the identification of Desulfovibrio with nitrogen-fixing capabilities and other bacteria involved in nitrogen cycling (Proteus with nitrate ammonification, Acinetobacter with denitrification) indicates that the gut microbiome may help H. roretzi cope with nutrient-limited conditions, particularly in winter. The study also notes that H. roretzi has economic value as an edible marine animal and is a source of vanadium compounds with anti-diabetic activity, and its hydrolysates can induce apoptosis of human colon cancer cells, though these are not direct findings of this study. The preservation of 263 cultured strains provides a valuable germplasm resource for future functional studies and potential biomedical applications.