Table 1.
Experimental design of bioassay.
Fig 1.
Concentration of nitrogen compounds in water replacement compared to native Bacillus treatment.
The concentration (mg/L) of nitrite (A), nitrate (B) and total ammonia nitrogen – TAN (C) were measured in low salinity culture water of shrimp Litopennaeus vannamei through 15 days. CO represents the control group with 25% water replacement, T1 represents the treatment with 50% water replacement, and T2 the treatment with 25% water replacement combined with native Bacillus strains (Bacillus megaterium and Bacillus paralicheniformis). Water replacements were carried out on days 3, 6, 10, and 13. Data are presented as means of n = 3 independent replicates.
Fig 2.
Relative abundances of the most representative bacteria detected by 16S rRNA gene amplicon sequencing.
A. Data at the family level. B. Data at the genus level. Each bar represents specific conditions (CO, T1, and T2), and each box corresponds to a particular day.
Fig 3.
Comparative analysis of species in CO, T1, and T2 treatments.
A. Venn diagram depicting the total ASVs shared at the species level between bacterial communities throughout the study for each treatment. B. Relative abundance of Bacillus species (> 100 reads) as exclusive species of the T2 treatment.
Fig 4.
Alpha diversity profile of the bacterial community in low salinity culture water of Litopenaeus vannamei.
The results, presented at the genus level, correspond to the communities from the control (CO), T1, and T2 treatments (p-value: 0.002; ANOVA F-value: 8.38).
Fig 5.
Principal Coordinate Analysis (PCoA) plot of Bray-Curtis distance of bacterial communities at the genus level in CO, T1 and T2 conditions, where shape indicates different days and color treatment (PERMANOVA between treatment, P = 0,001***, R2 = 0,328 with 999 permutations).
Fig 6.
Ward’s hierarchical cluster analysis based on the Bray-Curtis dissimilarity matrix using relative abundance data from each sample in CO, T1, and T2 conditions.
The colors represent each treatment, and the numbers indicate the day of sample collection during the bioassay.
Fig 7.
Heatmap showing the distribution of genes involved in nitrogen metabolism on day 10 for CO and T2.
The abundances of the genes are presented in OPM (ORF per million). The OPM values of the genes were calculated collectively based on their orthology from KEGG.
Fig 8.
Proposed nitrogen metabolism pathways in Gram-positive bacteria (Bacillus sp.) based on metagenomic analysis.
The dissimilatory nitrate reduction to ammonium (DNRA) and assimilatory nitrate reduction (ANR) pathways are illustrated. The genes identified in this study are shown in color, while the undetected genes appear in gray.
Fig 9.
Proposed nitrogen metabolism pathways based on metagenomic analysis of Gram-negative bacteria.
This figure illustrates the dissimilatory nitrate reduction to ammonium (DNRA), assimilatory nitrate reduction (ANR), and denitrification pathways. The genes identified in the study are shown in color, while the undetected genes appear in gray.