Isolation, characterization, and assessment of Bacillus rugosus potential as a new probiotic for aquaculture applications

Abstract

Aquaculture is an important component of the world food supply and a significant source of protein. However, this industry faces numerous problems. Including poor fish feed digestion and uneconomic nutrient utilization. This can result in unsatisfactory growth rates and poor stock performance. Utilizing probiotics, which are beneficial microbes that can enhance digestive systems and general fish health, is one possible way to address these issues. This study was designed to identify and evaluate a novel strain of Bacillus as a promising probiotic. The strain of Bacillus rugosus that was examined and coded NM007 showed promising probiotic characteristics that could help fish digest and utilize their feed more efficiently, reduce feed waste, and improve their digestive systems. B. rugosus NM007 exhibited the ability to produce digestive enzymes like protease, amylase, and lipase, which are the main digestive enzymes. It showed strong auto-aggregation activity and co-aggregation activity with Aeromonas sp. and Streptococcus sp. It also demonstrated tolerance to the presence of bile salt, acidic pH, and salinity up to 60 ppt. The sensitivity analysis towards antibiotics, hemolytic activity and the safety assessment on Nile tilapia fish (Oreochromis niloticus) confirmed the safety of this isolate. Based on the findings of this investigation and the isolate's characterization, Bacillus rugosus NM007 could serve as a new promising probiotic bacterium for aquaculture.

Keywords: Bacillus rugosus; Aqua feed; Gut health; Probiotic; Sustainability; Tilapia.

Figure 1

Ratio on enzymes activity of the isolates, calculated by dividing the substrate hydrolysis zone diameter by the colony diameter for each isolate and respective enzyme at 37 ℃ under aerobic stagnant condition. The columns (mean ± SEM, n  = 6).

Figure 2

Oil immersion (100X) magnification of strain NM007 Gram-stained slide.

Figure 3

Phylogenetic relationship between Bacillus rugosus NM007 and the closely related taxa, the tree was drawn to scales using Hasegawa-Kishino-Yano model and using Maximum Likelihood method. All positions with less than 95% site coverage were eliminated using MEGA-X.

Figure 4

Co-aggregation reaction pattern change of B. rugosus NM007 over time (24 h incubation period) in presence of the fish pathogens Streptococcus (a) and Aeromonas (b) strains at 37 ℃ under aerobic stagnant condition. The columns (mean ± SEM, n  = 6) with different letters are significantly different (p  < 0.001, one-way ANOVA).

Figure 5

Auto-aggregation pattern of the B. rugosus NM007 strain over time (24 h incubation period) at 37 ℃ under aerobic and stagnant condition. The columns (mean ± SEM) with different letters are significantly different ( p  < 0.001, one-way ANOVA) The columns (mean ± SEM, n  = 6).

Figure 6

Pathogenicity test: No clinical signs on fish or in internal organs, A: Control fish that injected with PBS, B: injected fish with bacterial strain B. rugosus.