3.2.1. The Alpha Diversity Analysis of Bacteria
The results of the alpha diversity analysis of the bistriazine ring derivative and KB samples are presented in
Table 3. High-quality bacterial sequences with a notable sum of 1,986,686 were obtained. UPARSE software was used to trim the sequences, with a maximum of 58,522 and a minimum of 41,495 sequences in each sample, while the average sequence number was 50,009. All the high-quality readings were categorized by taxonomy (from door to genus) by default. Alpha diversity analysis showed that all the coverages of the tested samples were above 0.992, indicating that the sequencing coverage of the samples was high. Furthermore, an estimate of the bacterial community diversity was made by the Shannon and Simpson indexes, whereas the Chao index was employed to reflect the bacterial community richness, and the number of bacterial OTUs varied from 692 to 923. The Shannon bacterial community diversity index ranged from 2.757 to 3.835, whereas the Simpson and Chao bacterial community diversity indexes ranged from 0.121 to 0.233 and from 826.161 to 1154.254, respectively.
The Shannon index of the T4 wastewater was high, while the Simpson index was relatively low [
24], indicating that the T4 wastewater had a high diversity of community species. It was reported in our previous study that microorganisms use non-metallic tanning agents as their carbon source, which leads to an increase in species diversity [
16]. As shown in
Table 1, the T4 bistriazine ring derivatives had a lower COD concentration and were more suitable for microbial metabolism and growth. When these derivatives were discharged into the wastewater, after a period of adaptation, the microorganisms might have metabolized the organic matter, resulting in an increase in the community species diversity. The index was the highest [
24] in the KB as no exogenous pollutants and no bistriazine ring derivatives were introduced. Therefore, the impact on the microbial community structure was small. Although the species richness was higher, most of the microbial species showed a lower abundance due to the lack of nutrients.
3.2.2. Effect of Derivatives Containing Different Bistriazine Rings on the Bacterial Microbial Community in the Wastewater
Figure 2a,b illustrate the relative abundance of bistriazine-containing wastewater bacterial communities with respect to phylum and genera.
Figure 2a shows the phylum only with abundance rates higher than 0.01%. A total of six phyla were identified and described:
Firmicutes,
Proteobacteria,
Actinobacteria,
Bacteroidetes,
Chloroflexi, and
Cyanobacteria. The dominance of
Firmicutes and
Proteobacteria was witnessed in T2, T3, T4, and KB wastewater with an average relative abundance of 77.57, 18.58, 58.47, and 32.50% and 41.69, 34.52, 62.30, and 26.76%, respectively. Previous research on the bacterial community composition in wastewater reported the dominance of
Firmicutes and
Proteobacteria. Xu [
12] investigated six sewage treatment plants and studied their bacterial communities. This research proved that the main phyla present in the sewage water were
Cyanobacteria,
Bacteroidetes,
Verrucomicrobia,
Proteobacteria,
Planctomycetes, and
Firmicutes. Wang [
25] found
Proteobacteria,
Firmicutes,
Bacteroidetes, and
Actinobacteria to be the main bacteria in a leather wastewater treatment plant. The abundance of
Actinobacteria was low in T2 and T3 but higher in T4.
Cyanobacteria was almost non-existent in T4, indicating a notable difference in the microbial community composition among the bistriazine ring derivative wastewater samples. Previous reports explained that microorganisms might consume organic matter for growth and reproduction, which can repress the growth of other bacteria [
16,
26] reported that the presence of
Pseudomonas might repress the growth of other bacteria. These results indicate that the addition of bistriazine affects the bacterial community structure in wastewater.
Bacterial genera with a higher abundance (<0.01%) are shown in
Figure 2b. In all, 24 genera were identified, and the type and quantity changed according to the different types of bistriazine ring derivatives. Notably, T4 had virtually no unclassified species, whereas the T2, T3, and KB populations ranged from 22.29 to 43.46%.
In T2, the dominant genera were Clostridium sensu stricto (9.93%), Turicibacter (9.59%), Pseudomonas (4.66%), Intestinibacter (4.43%), and Acinetobacter (3.93%). In T3, the abundance of Turicibacter and Clostridium sensu stricto decreased significantly, while that of Acidocella and Bacillus increased. The dominant bacterial genera were Acidocella (18.33%), Turicibacter (6.48%), Clostridium sensu stricto (6.45%), and Bacillus (3.37%). In T4, the dominant genera changed to Trichococcus (36.26%), Aeromonas (13.02%), Pseudomonas (3.38%), and Tolumonas (3.37%). In KB, Lactococcus (23.58%), Clostridium sensu stricto (4.64%), Turicibacter (4.14%), Raoultella (3.81%), Acinetobacter (3.31%), and Pseudomonas (3.21%) were dominant.
The presence of
Clostridium, Pseudomonas, and
Acidocella had a denitrification effect, whereas the presence of
Aeromonas and
Raoultella dephosphorized the bistriazine wastewater [
16,
27,
28,
29,
30].
Lactococcus removed ammonia–nitrogen more effectively, indicating that the bacteria responsible for denitrification were predominant, whereas the bacteria responsible for dephosphorization and the removal of ammonia–nitrogen were dominant in the blank control wastewater. From the above analysis, we concluded that the bacterial community composition of the bistriazine ring derivative-containing wastewater systems was different from that of the blank control, and that the bacterial community structure changed dramatically. Jayamani [
31] studied hexahydro-1,3,5-trinitro-1,3,5-triazine (generally called RDX) pollutants that affect the groundwater bacterial community structure and found
Pseudomonadaceae and
Acinetobacter to be the dominant bacteria. Livermore [
32] similarly studied the effects of RDX pollutants and found
Pseudomonas to be the dominant bacterial genus.
Figure 3 illustrates the sensitive and tolerant species found in the bistriazine ring derivative wastewater relative to the KB control. The abundance of
Lactococcus,
Acinetobacter,
Raoultella,
Dechloromonas, and
Zoogloea was higher, whereas that of
Trichococcus,
Clostridium sensu stricto,
Turicibacter,
Pseudodomonas,
Acidocella,
Intestinibacter,
Tolumonas, and
Bacillus was lower. This indicates that the wastewater was rich in tolerant bacteria that provided suitable survival conditions and promoted bacterial reproduction and growth to maximize bistriazine degradation. For example, Jayakumar [
33] reported that
Pseudomonas is an effective bacterium for remediating tannery wastewater, and a study by Kapahi [
34] reported that
Bacillus removed heavy metals. A unique microorganism,
Acidocella, also caused degradation. Such sensitive and tolerant bacteria must be investigated further to uncover more bioremediation options.