Effects of woody forages on biodiversity and bioactivity of aerobic culturable gut bacteria of tilapia (Oreochromis niloticus)

similarity with their closest NCBI relatives, while the five remaining isolates shared 87% simi- larity with their closest NCBI relatives (Stenotrophomonas sp. tap-10, Accession: EF221778.1). BLAST analyses showed that the 128 bacterial isolates belonged to three phyla, Firmicutes, Actinobacteria and Proteobacteria. Firmicutes accounted for 74.3% of the total isolates, which included five genera and 26 species, followed by Proteobacteria (14.3%, including four genera and five species) and Actinobacteria (11.4%, including three genera and four species). Among the 12 bacterial genera recovered in this study, Bacillus was the most diverse and common, with 18 species and 63 isolates identified. Further phylogenetic analysis was performed on 35 representative isolates (only one isolate was selected from all the isolates in the same species as a representative isolate), which correspondingly showed similarities to 35 different bacterial species (Fig 1). The results showed that SCAU-101, SCAU-102, SCAU-095, SCAU-091 were closely related to Streptomyces albogriseolus, Streptomyces rutgersensis, Rhodococcus sp. and Microbacterium foliorum in the phylum Actinobacteria. SCAU-103, SCAU-100, SCAU-092, SCAU-094, SCAU-093 were closely related to Proteus mirabilis, Pseudomonas fulva, Pseudo- monas sp., Stenotrophomonas maltophilia and Thalassospira sp. in the phylum Proteobacteria. All of the remaining representative bacterial isolates belonged to the phylum Firmicutes. Distribution and comparison of bacteria populations among five experimental groups

In this study, 12 genera, including Bacillus, Fictibacillus, Lysinibacillus, Microbacterium, Pro- teus, Pseudomonas, Rhodococcus, Rummeliibacillus, Staphylococcus, Stenotrophomonas, Strep- tomyces and Thalassospira were recovered from the five tilapia groups (Fig 2). The most bacterial genera were isolated from MOL leaf meal group (nine genera), followed by fermented MOL leaf meal group (eight genera), the control group (seven genera), the FM group (six gen- era), and the fermented FM group (five genera). In addition, three media (LB, MRS and PIYCA) were used to recover bacteria at the same time. And, the bacteria cultured in LB medium had the highest diversity with a total of 70 culturable bacteria belonged to 28 species in 11 genera were recovered. Furthermore, 31 culturable bacteria belonging to 15 species of the 7 genera were isolated from the PIYCA medium. In addition, the MRS medium had the lowest number of culturable bacteria with 27 isolates in 11 species of 4 genera (Table 2). After further analysis of bacterial distribution, it was found that the six most abundant spe- cies were Bacillus baekryungensis, Bacillus marisflavi, Bacillus pumilus, Bacillus methylotrophi- cus, Proteus mirabilis and Pseudomonas taiwanensis, which were isolated from the guts of tilapia from all five experimental groups. These bacteria constitute the core flora of tilapia. Fur- thermore, some unique bacterial communities were present among the five feed groups. For examples, Fictibacillus rigui, Lysinibacillus sphaericusare and Rummeliibacillu spycnus were

only isolated from the MOL leaf meal group, while Microbacterium foliorum and Pseudomonas fulva were unique to the fermented MOL leaf meal group. Bacillus aquimaris and Bacillus megaterium were recovered only from the group of fermented FM (Table 3). The Bray-Curtis analysis (Table 4) showed a 62.50–87.50% dissimilarity of bacterial com- munities between each two groups, except for the FM and fermented FM groups, which had 100% dissimilarity, indicating that the bacterial communities between each two groups dis- played obvious differences. Distribution of aerobic culturable bacteria with antibacterial activity In this study, a total 128 isolates were belonged to 35 different known bacterial species. So, 35 representative isolates (only one isolate was selected from all the isolates in the same species as a representative isolate) were selected for antibacterial activity. All of the 35 representative PLOS ONE Effects of wood forages on gut bacterial biodiversity and bioactivity in tilapia PLOS ONE | https://doi.org/10.1371/journal.pone.0235560 July 2, 2020 6 / 17