Diversity, community structure, and abundance of nirS-type denitrifying bacteria on suspended particulate matter in coastal high-altitude aquaculture pond water

Abstract

Denitrifying bacteria harboring the nitrate reductase S (nirS) gene convert active nitrogen into molecular nitrogen, and alleviate eutrophication in aquaculture water. Suspended particulate matter (SPM) is an important component of aquaculture water and a carrier for denitrification. SPM with different particle sizes were collected from a coastal high-altitude aquaculture pond in Maoming City, China. Diversity, community structure, abundance of nirS-type denitrifying bacteria on SPM and environmental influencing factors were studied using high-throughput sequencing, fluorescence quantitative PCR, and statistical analysis. Pseudomonas, Halomonas, and Wenzhouxiangella were the dominant genera of nirS-type denitrifying bacteria on SPM from the ponds. Network analysis revealed Pseudomonas and Halomonas as the key genera involved in the interaction of nirS-type denitrifying bacteria on SPM in the ponds. qPCR indicated a trend toward greater nirS gene abundance in progressively larger SPM. Dissolved oxygen, pH, temperature, and SPM particle size were the main environmental factors influencing changes in the nirS-type denitrifying bacterial community on SPM in coastal high-altitude aquaculture pond water. These findings increase our understanding of the microbiology of nitrogen cycle processes in aquaculture ecosystem, and will help optimize aquatic tailwater treatment strategies.

Keywords: NirS gene; Aquaculture ecosystem; Coastal high-altitude aquaculture pond; Denitrifying bacteria; Suspended particulate matter.

Figure 1

Microbial characteristics of the nirS gene on SPM in the high-altitude aquaculture ponds. (a) Neighbor-joining phylogenetic tree of dominant OTUs for the nirS gene (Top 43 OTUs) and the reference sequences from Genbank. Each color block is a genus classification, and the outer ring is the name of each cluster. (b) A cluster division of the neighbor-joining phylogenetic tree. The abscissa in the figure is the sample (The K1-4 is the sampling sites. The number 5, 1, and 0.22 is particle sizes of 5, 1, and 0.22 μm on SPM.). The ordinate is the percentage of each cluster, and different color blocks represent different bacterial classifications.

Figure 2

The heat map of dominant OTUs for the nirS gene (Top 43 OTUs). The abscissa in the figure represents the sample, and the upper tree diagram represents the sample clustering (The K1-4 is the sampling sites. The number 5, 1, and 0.22 is particle sizes of 5, 1, and 0.22 μm on SPM.). The ordinate is dominant OTUs for the nirS gene (Top 43 OTUs), and the left tree diagram represents the clustering of OTU samples. The color square represents the relative abundance of the dominant OTU, and the redder the color, the higher the abundance.

Figure 3

(a) The results of PCoA. (b) The results of NMDS. The black dots in the figure represent the sample name (The K1-4 is the sampling sites. The number 5, 1, and 0.22 is particle sizes of 5, 1, and 0.22 μm on SPM.). The large circle represents the clustering grouping of each point, and G1-3 represents the different clustering groups.

Figure 4

The abundance distribution of the nirS gene denitrifying bacteria in the high-altitude aquaculture ponds. The K1-4 in the figure is the sampling sites. The blue represents SPM with particle size of 5 μm, the green represents SPM with particle size of 1 μm, and the red represents SPM with particle size of 0.22 μm. "*, **, ns" in the figure are the results of the ANOVA (F) analysis of gene abundance on SPM with different particle sizes at each sampling site. “*” indicates a significant difference, that is, P < 0.05; “**” indicates an extremely significant difference, that is, P < 0.01; “ns” indicates no significant difference, that is, P > 0.05.

Figure 5

The network properties of the nirS-type denitrifying bacterial community at the species level in the high-altitude aquaculture ponds. The co-existence network at the species level based on the Spearman’s correlation significant analysis (P < 0.05). The blue circles in the diagram represent the main OTUs (species), and the connection represents the correlation between the two nodes.

Figure 6

Redundancy analysis between the key genera (OTU) and environmental factors. The red arrow in the figure is an environmental parameter, and the length of the environmental factor arrow represents the degree of its influence on the bacterial community. The blue circle represents the Wenzhouxiangella (OTU06, OTU22, OTU34), the green triangle represents the Pseudomonas (OTU13, OTU21, OTU27, OTU30, OTU35, OTU36), and the red diamond represents the Halomonas (OTU20, OTU41).