Isolation and characterization of potential probiotic bacteria from pustulose ark (Anadara tuberculosa) suitable for shrimp farming

In aquaculture, probiotics have been tested for enhancing the immune system and promoting growth and survival rate of many marine species like shrimp and mollusks. In order to isolate bacteria with a high probiotic potential for marine shellfish aquaculture, homogenates of the gastrointestinal tract from adult mangrove cockle, Anadara tuberculosa, were obtained to perform in vitro and in vivo assays. Isolates were tested in vitro for hemolytic activity, hydrophobicity, tolerance to ammonia nitrogen, salinity and pH as well as for growth kinetics, extracellular enzymatic activity, autoaggregation, coaggregation and molecular identification. Three bacteria with high degree of hydrophobicity (>60% adherence to p-xylene) and four bacteria with medium hydrophobicity, which showed different patterns of attachment to monopolar solvents (chloroform and ethyl acetate) and a high tolerance to ammonia nitrogen (200 mg L^-1), were selected. Six different treatments: T1 (without addition of cultured bacteria); T2 (MAt29, Enterococcus casseliflavus); T3 (MAt35, Citrobacter koseri); T4 (GAtB1, Bacillus subtilis subtilis); T5 (GAt7, Staphylococcus sp.); and T6 (1:1:1:1 mix of strains T2, T3, T4 and T5), were used to evaluate the specific growth rate, and cellular immune response of the shrimp Litopenaeus vannamei. The best specific growth rate was observed for T6 and T4 treatments related to Bacillus subtilis subtilis. A significant difference in total hemocytes count (P < 0.05) was found for T4 treatment with respect to control group. Strains isolated from A. tuberculosa had a beneficial effect on the growth and immune response of L. vannamei, so they have potential use as probiotics in aquaculture of marine shellfish.