Isolation of Marine Bacillus sp. with Antagonistic and Organic-Substances-Degrading Activities and Its Potential Application as a Fish Probiotic

Abstract

We report on the isolation and characterization of an acid- and bile-tolerant bacterial strain, Bacillus sp. YB1701 with antibacterial and quorum-quenching activity. Strain YB1701 was isolated from coastal sediment samples and characterized by biochemical tests and 16S rRNA sequencing. In vitro study indicated that strain YB1701 can survive at pH 2.0 for up to 3 h and tolerate bile up to 2.0% concentration even after 12 h of exposure. Strain YB1701 showed antimicrobial activity against fish pathogens Aeromonas hydrophila and Vibrio parahemolyticus using an agar well diffusion assay. The trial test showed dietary supplementation of YB1701 significantly improved the resistance of Carassius auratus gibelio against A. hydrophila challenge. The safety assessment revealed that the isolate Bacillus sp. YB1701 was not cytotoxic to Carassius auratus gibelio or mice and did not exhibit hemolytic activity on rabbit blood agar plate. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that YB1701 was susceptible to selected antibiotics. Under laboratory conditions, the degradation rate of organic waste (predominately fish excrement) for 14 days by YB1701 was 79.69%. Results from the present study suggest that strain YB1701 is a potential probiotic strain and can be used in aquaculture for degrading organic waste and improving disease resistance of fish against bacterial infection. Further study is needed to assess the utility of strain YB1701 on a commercial scale.

Keywords: Bacillus sp.; antagonist; multienzymes; organic substances degrading; probiotic.

Figure 1

Antagonistic, quorum-quenching and extracellular enzymatic activities of selected isolates (NO. 1–7). (A). Proteolytic activity, (B). Cellulolytic activity, (C). Amylolytic activity, (D). Lipolytic activity, (E). Antibacterial activity against A. hydropila YJ-1, (F). Antibacterial activity against Vibrio parahemolyticus DX-1, (G). Quorum-quenching activity against C. violaceum CV12472.

Figure 2

The phylogenetic tree of 16S rRNA gene sequences for Bacillus sp. YB1701 and other closely related Bacillus bacteria. The tree was generated using Neighbor-joining with scale bar equals 0.01 substitutions per nucleotide.

Figure 3

Chemical oxygen demand (COD) concentration changes in wastewater for 14 days. Error bars in Figure 3 and Figure 4 represent standard deviation of the mean (SD) from three replicate experiments (n = 3).

Figure 4

Protective effect of strain YB1701 on C. auratus gibelio under A. hydrophila challenge trial. YB1701 was administered by feeding at the concentration of 5.0 × 10⁶ CFU/g feed.