No abstract

Biofloc technology has shown positive effects in aquaculture, especially on the growth performance of cultured animals. The aims of this study were to evaluate the effects of adding different probiotic strains in a biofloc system on the growth performance and disease resistance of red hybrid tilapia (Oreochromis spp.). Three different probiotics (Lysinibacillus fusiformis SPS11, Bacillus amyloliquefaciens L9, and Enterococcus hirae LAB3), commercial probiotics (MG1) and a mixed probiotics (MP) combining all three strains were used in this study. The in vitro assay results showed that the mixed probiotic (MP) was able to inhibit the growth of Streptococcus agalactiae and Streptococcus iniae significantly compared to the single and commercial probiotic. The efficacy of MP was further tested in in vivo tilapia culture challenged with S. agalactiae. The best specific growth rate (3.73 ± 0.23% day−1) and feed conversion ratio (0.76 ± 0.04) were recorded in the group of biofloc with addition of MP. After being challenged with S. agalactiae, the group of biofloc with MP had significantly higher survival (83 ± 1.43%) compared to the other groups. Furthermore, the nitrogen concentration (NO2-N and NH4-N) was significantly lower in all the biofloc groups compared to the control. Hence, the addition of probiotics was able to provide beneficial effects to red hybrid tilapia culture in the biofloc system.

Supplementation with mixed probiotic in aquaculture has been proven to benefit the hosts as disease resistance tool. In this study, a mixed probiotic which consisted of three isolated strains (Lysinibacillus fusiformis strain SPS11, A2, and Bacillus megaterium strain I24) was formulated for the in vitro assays against Vibrio alginolyticus and in vivo preliminary study towards Artemia nauplii. These strains showed antagonism activities against V. alginolyticus in in vitro assay. An increase in biofilm formation of this mixed probiotic was observed which indicated that the strains could work synergistically with each other to confer benefits to the hosts. Enrichment of Artemia nauplii with the formulated mixed probiotic was done to investigate its role in enhancing resistance against the V. alginolyticus. Artemia nauplii were cultured in two different concentrations of mixed probiotic (106 and 108 CFU mL-1) and challenged via immersion method. The mixed probiotic at both concentrations resulted in significantly higher survival of Artemia compared to the challenged group with no probiont added (106 CFU mL-1, 65.00 ± 0.00 % and 108 CFU mL-1, 77.50 ± 3.53 %). Significant reduction of Vibrio loads was observed in Artemia and its culture water supplemented with mixed probiotic at 108 CFU mL-1 whereas there was no reduction of Vibrio at 106 CFU mL-1. This study suggests that the usage of formulated mixed probiotic at high concentration (108 CFU mL-1) as opposed to single-strain probiotic can confer protection against V. alginolyticus infection towards Artemia.

Compatibility of each strain in a multi-strain probiotic (MSP), along with its properties, becomes a strong base for its formulation. In this study, single-strain probiotics (SSPs) and multi-strain probiotics (MSPs) were evaluated in vitro for strain compatibility, microbial antagonism, biofilm formation capacity, and stress tolerance. Bacillus amyloliquefaciens L11, Enterococcus hirae LAB3, and Lysinibacillus fusiformis SPS11 were chosen as MSP1 candidates because they showed much stronger antagonism to Aeromonas hydrophila and Streptococcus agalactiae than a single probiotic. MSP 2 candidates were Lysinibacillus fusiformis strains SPS11, A1, and Lysinibacillus sphaericus strain NAS32 because the inhibition zone produced by MSP 2 against Vibrio harveyi and Vibrio parahaemolyticus was much higher than that produced by its constituent SSPs. MSP1 in the co-culture assay reduced (p < 0.05) A. hydrophila count from 9.89 ± 0.1 CFU mL−1 to 2.14 ± 0.2 CFU mL−1. The biofilm formation of both MSPs were significantly higher (p < 0.05) than its constituent SSPs and the pathogens. The SSPs in both MSPs generally showed resistance to high temperatures (80, 90, and 100 °C) and a wide range of pH (2 to 9). This in vitro assessment study demonstrates that MSP1 and 2 have the potential to be further explored as multi-strain probiotics on selected aquatic species.

Probiotics have been increasingly considered an alternative to antibiotics in combating disease outbreaks. Combined probiotics have been studied to possibly harbor synergistic effects that could provide better protection for aquatic species. Three potential probiotics, which had shown in vitro antagonism towards Aeromonas hydrophila in this study, were Bacillus amyloliquefaciens (L9, isolated from the blue swimming crab), Lysinibacillus fusiformis (A2, isolated from a microalga), and Enterococcus hirae (LAB3, isolated from the Asian seabass) were combined into a probiotic mixture. The probiotic mixture produced significantly higher biofilm (P < 0.05) (2.441 ± 0.346) than A. hydrophila (0.578 ± 0.124) during 24-h and showed a continuous increase in production at 48-h and 72-h time intervals, respectively. Furthermore, no hemolytic action was observed when the probiotic mixture was streaked on sheep blood agar (5%), whereas A. hydrophila presented α-hemolysis. The lowest concentration of the probiotic mixture (107 CFU mL-1) significantly inhibited (P < 0.05) the growth of A. hydrophila at 106 CFU mL-1 after 24 h of incubation, where bacterial count in the treatment was 6.595 ± 0.218 CFU mL-1, which was significantly lower (P < 0.05) than the control (7.247 ± 0.061 CFU) mL-1. Significant reduction (P < 0.05) in Aeromonas count from 7.532 ± 0.026 CFU mL-1 to 6.883 ± 0.015 CFU mL-1 was observed at 12 hours of co-incubation. Hence, this research suggests that the probiotic mixture of L9, A2, and LAB3 potentially confers protection against A. hydrophila infection due to their characteristics meeting the criteria of probiotics.