Characteristics of the community-structure of A2O processes under different temperature conditions in plateau areas

Background: In this study, laboratory-scale A2O wastewater treatment was performed in Linzhi at an altitude of 3000 meters. Water temperatures were maintained at four operating conditions (25°C, 20°C, 15°C, and 10°C). Sludge in the aerobic tank was assessed by 16SrRNA sequencing and composition analysis. The Phylum, Class, Order, Family, Genus, and Species of the sludge were also confirmed. Results: The bacteria within the sludge showed significant differences at varying temperatures (P<0.05). A significant correlation between the bacteria numbers in anaerobic tanks and anoxic tanks also occurred. Indicators of community richness, community uniformity, community diversity and other areas showed differences. Significant differences in bacteria abundance were also observed and differed to those of previously reported superior community categories and proportions. T-tests were used to identify temperature-sensitive community at each level. Correlation analysis of environmental factors and colony structure further confirmed the association between temperature and colony structure (P<0.05). The removal rates of TP, TN, NH 3 -N, and COD were all affected by the sample community structure. The variety of colony structure include nitrifying bacteria, denitrifying bacteria, phosphorus accumulating bacteria and other bacterial differed, but their proportions were relatively low. Heatmaps were used to identify species sensitive to temperature, TP, TN, NH 3 -N, COD at the species level. Conclusions: Amongst the significantly related common

treatment plants using the A2O process, including Proteus, Bacteroides, Nitrospira and Green Campylobacter as the dominant bacteria. In view of the large differences in microbial colony structures at different water temperatures, numerous studies on water temperature and microbial structure have been performed. Using biological aerated filters, Dou and colleagues (Dou et al., 2016) reported that when the temperature was lower than 18 ℃ (low temperature), the removal rate of COD and NH3-N of the biological aerated filters were less than 60%. The structure of the flora in the tank was simple, and the density of the flora decreased. The activity and quantity of enriched phosphorus accumulating bacteria showed a decreasing trend at increasing temperatures, but when the temperature decreased, the activity of phosphorus accumulating bacteria Acinetobacter enhanced its phosphorus removal effect (Fang et al., 2011). Ma and coworkers (Ma et al., 2020) assessed induced temperature periodic changes. They found that the activity a50nd relative abundance of ammonia oxidizing bacteria, nitrite oxidizing bacteria, and anammox bacteria, were dominated by Candidatus Brocadia at low temperatures, which changed following periodic temperature shocks. The formation of dissolved organic nitrogen was also influenced by both microbial activity and microbial community structure (Liao et al., 2019). The performance and stabilization of biological wastewater treatment systems are also closely related to the microbial community structure, variations in which are driven by temperature . Decreased temperatures result in significant reductions in microbiome diversity, and the alpha diversity of the active community (Paul et al., 2020). Upon analysis of the capacity of nitrogen removal and the spatial distribution of microbial communities at low temperatures, it was shown that low temperatures inhibit nitrogen removal (Paul et al., 2020). It is therefore clear that temperature changes impact microbial structure in wastewater treatment processes and the quality of wastewater treatment water.
Microbial composition and changes in A2O processes under different temperature conditions are typical characteristics of the community structure. This must be explored to fully understand microbial response mechanisms under the influence of plateau environmental factors, and provides a theoretical system for sewage biological treatment systems affected by plateau environmental factors. In this study, we selected the A2O system as a typical sewage treatment process to analyze the influence of temperature conditions. We assessed community structure characteristics and their alterations in anoxic and aerobic regions to explore the microbial response mechanisms influenced by plateau environmental factors.

Methods
The A2O system was selected as a typical sewage treatment process. Microbial structure characteristics were explored to investigate the influence of low temperature at a range of environmental factors. We further explored the performance of experimental scale A2O processes in response to a range of plateau environmental factors..

Experimental setup
Laboratory-scale A2O sewage treatment devices were produced from Plexiglas. Each device had an effective volume of 210 L and was divided into 3 compartments; (1) an anaerobic tank; (2) an anoxic tank; (3) an aerobic tank. The volume ratio of the three regions were 35:58:117, and the effective volume of the sedimentation tank was 39 L. Both the anaerobic and anoxic tank were equipped with a stirring device at a stirring speed of 50 rpm. The aerobic tank was equipped with an aeration head for the oxygen supply. A peristaltic pump was used to control the inlet water, return sludge and nitrification liquid. To ensure constant temperatures in the test, a constant temperature circulator was used to control the water temperature. Each tank wall had a sampling port. The specific device processes are shown in Figure 1. After 35 days of activated sludge culture, the temperature was controlled at 20.0°C, the SV30 was 34%, and the MLSS was 4325 mg/L. We used urban sewage in Linzhi City as the test water, the quality status of which is shown in Table 1.

A2O Operation
A2O processes at each temperature were investigated. The designed inlet flow rate was 10.0 ± 0.1L/s. The aerobic tank had a dissolved oxygen content of 2 mg/L. The hydraulic retention time was 21.0 ± 0.2h, the anaerobic tank residence time: anoxic tank residence time: and aerobic tank residence time were 35:58:117. The reflux ratio of the mixed solution was RI=200%. The reflux ratio of the sludge was R=100%. Both the mixed solution and sludge were continuously refluxed. Changes in dissolved oxygen were achieved through altering the levels of blown aeration. Controls were set to four levels of 25 ℃, 20 ℃, 15 ℃, and 10 ℃. Sampling was performed 72 hours after the temperature reached the designed value.

Results and discussion
Microbiological indicators were compared using SPSS 20.0 and are expressed as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) to assess significant differences. P < 0.05 (Simon et al., 1989) indicated significant differences between samples.
Anaerobic, anoxic and aerobic tank sludge were sampled at each temperature and 16SrRNA sequencing was performed.

Basic information
Taxonomic analysis of the domain, kingdom, phylum, class, order, family, genus and species  Table 2.  Genus  Species  OTU  temp_ana_25  1  1  20  35  97  188  376  533  734  temp_ana_20  1  1  27  52  126  234  477  707  969  temp_ana_15  1  1  29  56  134  241  505  757  1067  temp_ana_10  1  1  30  54  131  239  453  664  898  temp_ano_25  1  1  24  41  103  198  413  607  849  temp_ano_20  1  1  Only a single Domain and Kingdom were investigated through sequencing, and Phylum ∈ (20,30), Class ∈ (35,56), Order ∈ (97,134), Family ∈ (188,242), Genus ∈ (376,505), Species ∈ (533,757) , OTU ∈ (734, 1067), and the corresponding genealogical values were significantly lower than those stated in the literature. At the Phylum level, values were similar to the 24 reported by Sun and colleagues (sun et al., 2019), but were lower than the value of 51 recorded by Tian and coworkers (Tian et al., 2015). At the Genus level, the values were also lower than the Tian study (Tian et al. ., 2015). The value of the test 800 was low. At the OUT level, the values (924～1363) were comparable to Wen and colleagues (Wen et al., 2015) in the SBR process, but Previous studies suggest that the temperature of the Phylum, Class, Order, and Family levels significantly influence the number of species (P<0.05). Compared to previous studies, we observed large differences in the number of microbial species, particularly at the temperature of the plateau, variations in which significantly influenced the number of bacteria at the Phylum, Class, Order, and Family levels. In addition, correlation analysis revealed significant differences in the number of species of phylum, phylum, order, family, genus and species at each temperature (P<0.05). The number of phylum, phylum, order, family, genus and species also differed. A significant correlation between the anaerobic tank and anoxic tank were observed (P<0.05). No significant correlation between the aerobic tank and the above two reactors (P≥0.05) were observed. There were no obvious correlations between the number of genus, order, family, genus and species (P≥0.05).

Alpha diversity analysis
The Alpha diversity comprises the diversity index and the differences between index groups. The commonly used metrics of the index are chao (colony richness), shannoneven (colony uniformity), ace (colony richness), Simpson (colony diversity), and coverage (colony coverage). T tests were performed to detect significant correlations between the two groups. The diversity index of each sample are shown in Table 3. Upon assessment of the four operating conditions: excluding operating conditions of 10℃ and 20℃, there were significant differences in the ace coefficient of community richness between each of the operating conditions (p ≤ 0.05). There were significant differences in the number of species except for at 10℃ and 20℃. The community diversity measured through the Simpson coefficient differed between 10℃ and 20℃, 10℃ and 15℃, and 20℃ and 25℃ (p<0.05); Significance analysis of the community richness chao coefficient revealed significant differences between 10°C and 15°C, 20°C and 25°C, and 15°C and 25°C (P <0.05). The Simpson even coefficient of community uniformity also showed significant differences between 10℃ and 20℃, 10℃ and 20℃, and 20℃ and 25℃ (P<0.05). Under the four groups of working conditions, there are were one or more significant differences in community richness, community diversity or community uniformity, suggesting that temperature significantly influences colony structure. Differences in community richness from the aerobic to anoxic tank, oxygen tank and anaerobic tank, and anoxic tank and anaerobic tank were observed. The community diversity varied in both the anaerobic and anoxic tank, anaerobic tank and aerobic tank, and anoxic tank and aerobic tank. Differences in community uniformity were observed upon comparison of anaerobic and anoxic, anoxic and aerobic, and anaerobic and aerobic tanks.

Community composition analysis
The RDPClassifier was used for classification statistics of the three reactors under each of the operating conditions in the order of Domain, Kingdom, Phylum, Class, Order, Family, and Genus. We further focused on the analysis of fungus function at the Genus level, and analyzed each sample. The composition of the middle population when including community with an abundance greater than 1% of different classification levels showed only a single bacteria at the domain analysis level and no rank_d__Bacteria at the Kingdom analysis level. . The values obtained were low; the abundance of Actinobacteria were 12.33%-25.14% in heterotrophic aerobic phylum, and its change in abundance suggested that the concentration of the anoxic tank exceeded that of the anaerobic tank and oxygen tank. The abundance of Firmicutes was 4.52% to 12.74%. These are denitrifying bacteria that are mainly present in anoxic conditions. Their abundance in the anaerobic tank was higher, and the abundance of Firmicutes was higher than that reported by Beer et al. (2006), but lower than that reported by Wong et al. (2005). The abundance of Chloroflexi was 1.04%～6.26%. These are aerobic sulphur bacteria the abundance of which decreases in anoxic and aerobic tanks as temperature decreases. The abundance of Proteobacteria, Actinobacteria, Firmicutes and Chloroflexi all differed according to temperature (P≤0.05). Of these four dominant bacteria, the Phylum number had a significant effect, but the dominant strains at the gate level showed no significant differences under different reactors.  Gammaproteobacteria, Actinobacteria, Clostridia were the three dominant bacteria that showed significant differences (P≤0.05) under different temperature conditions. In addition, Chloroflexia (0.61-4.61%) and Anaerolineae (0.42 -1.30%) showed changes in abundance at different temperatures (P≤0.05). Whilst temperature has a significant effect on the number of the above five bacterial community, there no significant differences observed as a result of the different reactors (P>0.05).  Figure 4 shows that under the above working conditions, the predominant bacterium order were Betaproteobacteriales, Sphingobacteriales, Chitinophagales, Xanthomonadales, Clostridiales, Microtrichales, Propionibacteriales, Corynebacteriales, Sphingomonadales, Rhizobiales, and Rhodobacterales. A total of 168 Orders were obtained. The abundance of 11 Orders were 71.93-86.24%. In addition, the abundance of seven orders of Thermomicrobiales, Bacteroidales, Micrococcales, Lactobacillales, Caulobacterales, Pseudomonadales, and Solirubrobacterales varied greatly. This accounted for 6.77-13.36% of all bacteria that were sensitive to temperature changes.

Analysis of the characteristics of colony structure at the Order level
The analysis showed that: Microtrichales, Propionibacteriales, Corynebacteriales, Micrococcales, Solirubrobacterales belong to the Actinobacteria phylum. The abundance of Microtrichales and Corynebacteriales were relatively stable, the abundance of Propionibacteriales increased at increasing temperature, and Micrococcales and Solirubrobacterales were relatively unstable at different temperature conditions. Sphingobacteriales, Chitinophagales, and Bacteroidales belong to the Bacteroidetes order. Sphingobacteriales decreased with increasing temperature and were more abundant in the aerobic tank compared to other reactors. Chitinophagales, Bacteroidales are unstable with changes in temperature changes and showed a correlation. Clostridiales, Lactobacillales and Erysipelotrichales belong to the Firmicutes phylum; Betaproteobacteriales, Xanthomonadales, Sphingomonadales, Rhizobiales, Rhodobacterales, Caulobacterales, Pseudomonadales belong to the Proteobacteria phylum. Amongst them, Betaproteobacteriales were more abundant than those of other well-characterised anaerobic bacteria. The proportions of Caulobacterales and Pseudomonadales in anaerobic and anoxic tanks were higher. Betaproteobacteriales tended to increase with increasing temperatures in each reactor and were higher in anaerobic tanks. Xanthomonadales were higher in aerobic tanks and increased with temperature Sphingomonadales and Rhizobiales did not change with temperature or according to the reactor. Rhodobacterales showed an increasing trend with increasing temperature in each reactor. Thermomicrobiales belong to the Chloroflexi door, which was greatly affected by temperature, including at 25℃. All three reactors were lower (less than 1%), the aerobic tank was slightly higher than 1% in the anoxic tank and the anaerobic tank at 15℃.

Analysis of the characteristics of colony structure at the Family level
In addition, Microtrichaceae, Propionibacteriaceae, Nocardiaceae, and Microbacteriaceae belong to Actinobacteria, the abundance of which increased with temperature and were higher in the aerobic tank. AKYH767, Saprospiraceae, and Chitinophagaceae belong to Bacteroidetes, and the abundance of AKYH767 increased with temperature. The aerobic tank showed higher levels of these bacteria than other reactors. The abundance of Saprospiraceae showed an increasing trend with increasing temperature and the aerobic tank was superior to other reactors. The abundance of Chitinophagaceae showed a downward trend with increasing temperature. The reactor was relatively balanced; Peptostreptococcaceae, Clostridiaceae_1, Christensenellaceae, and  Figure 6. Community composition at the genus level. Figure 6 shows that the common dominant bacterial genus were norank_f__AKYH767, norank_f__Saprospiraceae, Ottowia, unclassified_f__Burkholderiaceae, IMCC26207, Novosphingobium, Gordonia, Romboutsia octabacterium. A total of 719 Genus were obtained. The abundance of 8 Genus were 33.33-53.11%, which represent the dominant genus in the plateau environment. The dominant bacteria at the genus level reported in previous studies (Yang et  Betaproteobacteriales as phosphorus-accumulating bacteria. The corresponding proportion in the aerobic tank was significantly higher than other reactors. Norank_f__JG30-KF-CM45 belongs to the Chloroflexi door, which was greatly affected by the temperature conditions, the anoxic tank and the anaerobic tank.

Analysis of the characteristics of colony structure at the Species level
Uncultured_bacterium_g__IMCC26207, metagenome_g__Gordonia, unclassified_f__Propionibacteriaceae, uncultured_bacterium_g__Propioniciclava belong to the Actinobacteria phylum. The abundance of uncultured_bacterium_g__IMCC26207 increased as temperature increased. Unclassified_f__Propionibacteriaceae, uncultured_bacterium_g__Propioniciclava also increased at increasing temperature and were significantly superior to the other two reactors in the anoxic tank. Bacteroidetes_bacterium_OLB10, uncultured_bacterium_g__norank_f__Saprospiraceae_unknown_Bro_derobacterium_genotype_B __ Increased showed an upward trend and were most abundant in the aerobic tank. The abundance of unclassified_g__Ferruginibacter showed a downward trend at increasing temperatures. Uncultured_bacterium_g__Romboutsia, metagenome_g__Trichococcus, unclassified_g__Christensenellaceae_R-7_group belong to Firmicutes_g, culture_rich_g-7 and decreased as temperature increased, at were highest in the anaerobic tank. (0.51-1.92%), uncultured_bacterium_g__Thermomonas (0.00-2.67%), metagenome_g__norank_f__JG30-KF-CM45 (0.17-2.25%). These data suggest that temperature has a significant effect on all these bacteria. The abundance of the dominant bacteria uncultured_bacterium_g__Romboutsia at the species level significantly differed between the reactors (P≤0.05). There were no significant difference between other dominant bacteria in different reactors (P>0.05).

Relationship between colony structure of the sewage treatment system and environmental factors
Correlation analysis of the environmental factors and colony structure were performed through RDA/CCA analysis, which carries out regression analysis on the community structure and environmental factors at different temperature to reflect the relationship between the community structure and environmental factors. The microbial classification level is Species, environment. The factors corresponding to temperature, TN, TP, NH3-N and COD were assessed. Given the purpose of the experiment design, CCA analysis was performed for the anaerobic tank, anoxic tank and aerobic tank. CCA analysis showed that the correlation coefficients r of temperature, TN, TP, NH3-N, and COD were 0.9555, -0.7292, -0.7916, 0.77882, and 0.5565, respectively. Significance analysis showed that the correlation between temperature and the samples was significant (p<0.05), suggesting that environmental factors show a significant relationship to sample colony structure. The removal rate of TP, TN, NH3-N, and COD were affected by the sample community structure. The degree of impact was sequentially weakened. The interpretation degree of RDA1 and RDA2 were 78.47% and 7.86% respectively.
We used correlation Heatmaps to analyze the relationship between different species and environmental variables. To assess the correlation between microorganisms and environmental variables (temperature, TN, TP, NH3-N, and COD) samples were classified according to the top 50 species. Figure 9. Correlation Heatmap. Figure 9 shows that: 1) at the species level, temperature is significantly related to the species

Conclusions
Using 16SrRNA gene sequencing and correlation analysis, the differences in bacterial communities in anaerobic, anoxic and aerobic tanks under four temperature conditions were analyzed based on species number, diversity, composition, and Heatmaps of the sequenced samples.
1) Sequencing analysis revealed that the number of species in the phylum, Class, order, family, genus and species level in the high-in situ samples were lower than those previously reported and varied according to the working conditions of the three reactors. There were significant differences in community number at the phylum, class, order, family, genus and species level. A significant correlation was observed between the number of community at the phylum, Class, order, family, genus and species level (P≤0.05). However, no obvious correlation between temperature and the number of phylum, class, order, family, genus and species of each tank were observed.
2) Alpha diversity analysis showed that the community richness, community uniformity, and community diversity under high in situ temperatures differed to those reported in the literature. T tests showed that, excluding 10 ℃ and 20 ℃ , significant differences exist in the number of community according to working conditions. Community diversity coefficients were also significantly different between 10 ℃ and 20 ℃, 10 ℃ and 15 ℃, and 20 ℃ and 25 ℃. The community richness chao coefficient was significantly different between the three groups of operating conditions at 10°C and 15°C, 20°C and 25°C, and 15°C and 25°C. The Simpsoneven coefficient of community uniformity showed that at 10°C, there were significant differences between the three groups of operating conditions at 20℃, 10℃ and 20℃, 20℃ and 25℃. Significance analysis of the three reactors showed no significant differences in the community numbers in the different reactors.
3) We assessed the community composition of the three reactors under different temperature conditions according to Phylum, Class, Order, Family, Genus, and species. The common dominant community at each level were analyzed and their response to temperature were assessed. 4) Using the CCA chart to further verify that temperature is an important factor that influences the composition of the colony structure, and using the correlation heatmap to analyze the species that are significantly related to temperature, our analysis showed that the abundance of 13 species were related to temperature. In total, 7 species showed a significant correlation; TN significantly correlated were not observed; 4 species correlated with TP, 2 of which showed a high correlation; 1 species significantly correlated with NH3-N; a single species significantly correlated with COD.
Based on these analysis, the A2O species structure in high-in situ correlated with temperature in terms of species number, Alpha diversity, and dominant species composition. The dominant bacteria that were significantly related to temperature at the species level were Bacteroidetes_bacterium_OLB10. The dominant bacteria with an extremely significant correlation were metagenome_g__Gordonia, unclassified_f__Burkholderiaceae, uncultured_bacterium_g__norank_f__Saprospiraceae. The dominant bacteria with significant TP removal rates were Bacteroidetes_bacterium_OLB10. The dominant bacteria with extremely