Activated sludge bulking is easily caused in winter, resulting in adverse effects on effluent treatment and management of wastewater treatment plants. In this study, activated sludge samples were collected from different wastewater treatment plants in the northern Xinjiang Uygur Autonomous Region of China in winter. The bacterial community compositions and diversities of activated sludge were analyzed to identify the bacteria that cause bulking of activated sludge. The sequencing generated 30087–55170 effective reads representing 36 phyla, 293 families, and 579 genera in all samples. The dominant phyla present in all activated sludge were Proteobacteria (26.7–48.9%), Bacteroidetes (19.3–37.3%), Chloroflexi (2.9–17.1%), and Acidobacteria (1.5–13.8%). Fifty-five genera including
Activated sludge process has been extensively used in industrial and domestic wastewater treatment because of its high microbial diversity and activity, resulting in the removal of most organic pollutants and nutrients [
The high-throughput sequencing technologies originated several years ago are easier and less expensive for high-throughput sequencing [
The purpose of this study was to analyze bacterial community structures and diversities of activated sludge samples of different wastewater treatment plants in the north of Xinjiang Uygur Autonomous Region of China in winter via Illumina high-throughput sequencing technology. This study facilitates the evaluation of the similarities and differences in bacterial community composition of samples from different geographic locations and understanding the microbial interaction of activated sludge in Xinjiang.
Activated sludge samples were collected from the aeration tanks of four WWTPs located in the north of Xinjiang Uygur Autonomous Region of China in winter. These four WWTPs applied oxidation ditch process. The following 8 samples were collected: ALT1 and ALT2 from ALT WWTP; SHZ1 and SHZ2 from SHZ WWTP; CJ1 and CJ2 from CJ WWTP; HX1 and HX2 from HX WWTP. Sampling date, flow rate, influents, effluents, and operational parameters of the WWTPs are presented in Table
Characteristics of samples and wastewater treatment plants.
Sample | Sampling date | Flow rate | DO | pH | SRT | Tem | SV/% | MLSS | SVI/(mL/g) | BOD5 (mg/L) | | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Influent | Effluent | Influent | Effluent | ||||||||||
ALT1 | 2016.12.1 | 3 | 3.8 | 7.0 | 27 | 9.8 | 35 | 5980 | 59 | 119 | 8 | 33.1 | 1.6 |
ALT2 | 2017.1.7 | 3 | 3.9 | 7.1 | 27 | 8.5 | 33 | 4861 | 69 | 97 | 9 | 29.7 | 2.0 |
SHZ1 | 2016.3.2 | 10 | 4.0 | 7.50 | 23 | 12.8 | 17 | 4186 | 41 | 147 | 39 | 28 | 3.0 |
SHZ2 | 2017.1.7 | 10 | 3.7 | 7.4 | 23 | 12.9 | 19 | 4297 | 44 | 164 | 30 | 27 | 3.2 |
CJ1 | 2016.1.25 | 10 | 1.5 | 6.75 | 30 | 13.6 | 88 | 3355 | 262 | 539 | 27 | 58 | 21.9 |
CJ2 | 2016.3.2 | 10 | 1.4 | 7.01 | 30 | 13.5 | 91 | 4512 | 202 | 533 | 25 | 47 | 20.6 |
HX1 | 2016.1.25 | 10 | 3.6 | 7.20 | 25 | 10.8 | 88 | 4354 | 199 | 276 | 11 | 50 | 5.8 |
HX2 | 2016.3.2 | 10 | 3.5 | 7.30 | 25 | 10.9 | 62 | 3543 | 175 | 276 | 26 | 31 | 5.2 |
The samples for the microbial analysis were stored in the laboratory at −40°C and were sent to Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) for DNA extraction, PCR amplification, and Illumina high-throughput sequencing.
Microbial DNA was extracted from sludge samples collected from four WWTPs using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, US). The final DNA concentration and purification were determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA), whereas DNA quality was checked by 1% agarose gel electrophoresis. The V4-V5 hypervariable regions of the bacteria 16S r RNA gene were amplified with primers 515 F (5′-GTGCCAGCMGCCGCGG-3′) and 907 R (5′-CCGTCAATTCMTTTRAGTTT-3′) by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions were conducted using the following program: 3 min of denaturation at 95°C, 27 cycles of 30 s at 95°C, 30 s for annealing at 55°C, 45 s for elongation at 72°C, and a final extension at 72°C for 10 min. PCR reactions were performed in triplicate of 20
Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols of Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Numbers: SRP113278, SRP125654, and SRP126028).
Data analysis was conducted using the i-Sanger platform (
As shown in Table
Richness and diversity indices of microbial communities for sludge samples.
Sample | Reads | OTUs | Shannon | Ace | Chao | Coverage |
---|---|---|---|---|---|---|
ALT1 | 32390 | 714 | 4.88 | 811 | 802 | 0.992 |
ALT2 | 38394 | 683 | 4.52 | 863 | 908 | 0.989 |
SHZ1 | 53363 | 1152 | 5.93 | 1292 | 1281 | 0.988 |
SHZ2 | 30087 | 1022 | 5.78 | 1200 | 1192 | 0.988 |
CJ1 | 46680 | 852 | 5.09 | 1054 | 1047 | 0.988 |
CJ2 | 55170 | 855 | 5.16 | 1031 | 1024 | 0.989 |
HX1 | 48497 | 829 | 5.13 | 1003 | 994 | 0.989 |
HX2 | 52873 | 755 | 4.76 | 909 | 905 | 0.990 |
As shown in Figure
Diversity of bacterial communities in activated sludge samples. (a) Rarefaction curves and (b) rank abundance curves.
The ALT WWTP had the lowest microbial diversity, which can be because the temperature was lower than other WWTPs. Temperature has a decisive role in the metabolism of microorganisms, and lowering temperature has a significant effect on the reduction of the maximum specific growth [
The difference and similarity of bacterial community of activated sludge samples collected from different WWTPs were analyzed based on OTUs through the Venn diagram (Figure
Overlap of the bacterial communities from four WWTPs based on OTU (3% distance).
A total of 36 phyla were observed in eight samples. As shown in Figure
Percentages of the major phyla in all samples (the sequence percentage is above 1% in at least one sample).
Among Proteobacteria, Betaproteobacteria was the most abundant class (34.4–65.8%). Gammaproteobacteria was the second dominant class, accounting for 16.2 to 39.8%. Alphaproteobacteria and Deltaproteobacteria were the other important classes, comprising 11.0–38.6% and 2.2–9.1%, respectively. Epsilonproteobacteria had at the lowest abundance in the range from 0.02 to 4.3%. This result was consistent with other studies concluding that Betaproteobacteria was the largest class [
Among the 293 families identified, 44 families including Saprospiraceae, Comamonadaceae, Anaerolineaceae, Xanthomonadaceae, Rhodocyclaceae, Flavobacteriaceae, Rhodobacteraceae, Intrasporangiaceae, Caldilineaceae,
Percentages of the major families in all samples (the sequence percentage is above 1% in at least one sample).
Comamonadaceae were in charge of aromatic degrading and denitrifying processes and were the chief families in numerous wastewater treatment plants [
579 genera were shared in all samples, in total. Fifty-five genera, which accounted for 60.6–82.7% of the classified sequences and included the
Heatmap of the first ten dominant genera in each sample.
Filamentous Saprospiraceae and
The possible relationship between microbial communities and environmental variables was analyzed using a constrained ordination of redundancy analysis (RDA). Six variables including influent BOD5, influent ammonia, pH, water temperature, dissolved oxygen (DO), and solid retention time (SRT) were selected, and the ordination biplot was shown in Figure
RDA analysis to investigate the relationship between microbial communities and environmental variables.
Influent BOD5 is the most crucial environmental factor influencing the community compositions. Previous studies have found that the types of feeding substrate have an impact on bacterial diversity [
DO had a significant influence on the bacterial communities. In the context of its influence on microbial activity and the high operating costs of aeration, DO is a key operational parameter in wastewater treatment systems. However, the explicit selection by DO concentration is not completely understood for diverse bacterial lineages. The results showed that DO had an important influence for shaping the compositions of the microbial community of wastewater treatment processes. Several studies have found that DO concentration was a significant structuring factor for bacterial community compositions running at high and low DO concentrations in bioreactors of two laboratory scales [
The water temperature was correlated with variance of the bacterial community, which is in agreement with other studies [
In this study, pH was found to have the least impact on bacterial community structures, which is in disagreement with the other studies [
In conclusion, 36 phyla, 293 families, and 579 genera were found in activated sludge samples from WWTPs of Xinjiang in winter. The number of shared OTUs accounted for 26.6% of the total observed OTUs; it can be concluded that some microorganisms always existed in the activated sludge collected from different WWTPs. The WWTP with low temperature and single wastewater type was found to have the lowest microbial diversity. The WWTP with mixed wastewater type was found to have the highest microbial diversity. Proteobacteria
The authors declare that they have no conflicts of interest.
This work was supported by the National Natural Science Foundation of China (51568061).