Major operational parameters of full-scale A2O processes
Operational parameters of A2O processes in the four WWTPs are presented in Table 1. The A2O processes at TH1 were operated at the average flow rate of 218,433 m3 d‒1. The average flow rate was 7,673 m3 d-1 for TH2. The average flow rates at SK1 and SK2 WWTPs were 220,655 and 497,174 m3 d‒1, respectively. Flow rates from high to low are SK2 > SK2 ~ TH1 > TH1. Compared with TH1 (30 mg L‒1), TH2 (198 mg L‒1), and SK1 (75 mg L‒1), high BOD values (260 mg L‒1) were found at SK2 because the WWTP received industrial wastewaters as well (approximately 25% of total volume). TH1 received wastewater with very low BOD. It is because that TH1 treated wastewater collected from a combined sewer system with domestic sewage being diluted by stormwater. Infiltration and inflow are able to enter this combined sewer system. Meanwhile, the high temperature inside the sewer lines could promote the degradation of BOD. Finally, the wide practice of septic tanks installation in the residential houses could remove BOD before wastewater entering the sewer lines.
All operational parameters of DO, SRT, and HRT can be found in Table 1. Comparison these operational parameters at TH1, TH2, SK 1, and SK2, low DO level (0.9±0.2 mg-O2 L-1), longer SRT of 30 day, and HRT (8 hr.) were found at TH1 and high DO level (4±0.5 mg-O2 L-1), shorter SRT of 17 day and HRT (3.6 hr.) were found at SK1. At TH2, quite high DO level (2.6±0.2 mg-O2 L-1), SRT of 19 day, and longer HRT (15.4 hr.) were found at TH2 and quite high DO level (3±0.3 mg-O2 L-1), quite longer SRT of 26 day, and longer HRT (10.1 hr.) were found at SK2. Longer SRT of ≥26 day with DO level (≥ 0.9±0.2 mg-O2 L-1) could be suggested to use as operation parameters when treating low COD/N ratio wastewaters at TH1 or COD/N ratio of 4.2 at SK2.
Performances of full-scale A2O processes WWTPs
COD, BOD, and nitrogen removals efficiencies in the full-scale A2O processes WWTPs are shown in Table 2. COD, BOD, NH4+-N, TN, and TP removals efficiencies were 67%, 83%, 95%, 49%, and 35% respectively, at TH1. These values were 98%, 92%, 91%, 86%, and 96% respectively, at TH2. They were 96%, 89%, 99%, 70%, and 95% respectively, at SK1, and were 99%, 96%, 98%, 82%, and 98% respectively, at SK2.
The average nitrogen concentration and removal efficiencies in each month are shown in Figure 2. A2O processes are designed for biological N and P removals and suitable for municipal WWTP . However, total nitrogen (TN) removal efficiency at TH1 was quite low (only 49%) compared to other plants. The low TN removal performance could be explained by the very low COD/TN ratio (3.7) in the wastewater received at TH1. The low nitrogen removal efficiencies were also reported by Liu et al.  for WWTPs treating wastewater of relatively low COD/TN ratios. It was reported that denitrification process could not significantly occur due to the insufficient carbon source for denitrification process with wastewater having relatively low COD/TN ratios of lower than 4. On the contrary, efficient TN removal (86%) was reported for TH2 which received wastewater with COD/TN ratio of 8.4.
For WWTPs treating low COD/TN ratio (≤ 4) wastewater, maintaining longer SRT (≥ 60 day) is recommended to overcome the low TN removal efficiency  as the longer SRT would increase nitrifying bacteria abundancy. Meanwhile, long SRT could also enhance NH4+ removal by increasing nitrification activity . The effects of SRT on NH4+ removal were reported by . The effluent NH4+ concentrations in activated sludge process were reported for SRTs at 5 day (2.6±2.3 mg N L‒1), 10 day (0.04±0.01 mg N L‒1), 20 day (0.03±0.007 mg N L‒1), and 40 day (0.02±0.003 mg N L‒1), corresponding to NH4+-N removal efficiencies of 94.5%, 99.9%, 99.9%, and 99.9%, respectively. The effluent NH4+ concentration at TH2 plant was 4.8 mg N L‒1 and was the highest among the WWTPs studied due to the highest NH4+ concentration in the raw water (55.4 mg N L‒1). To further enhance the removal of NH4+-N, a long SRT of > 19 day should be recommended.
The results of TP removal confirm that the A2O processes at three WWTPs (BOD/TP ratio of > 20) was able to remove P concentration well (more than 96% of TP removal efficiencies) except TH1 which has lowest BOD/TP ratio of 13 and achieved only 35% of TP removal. To increase phosphorus removal efficiency at TH1 (in case there is low BOD:TP ratio), the chemical treatment by using alum as coagulant would be recommended.
Nitrogen-cycling microbial abundances and predominated existing would be relative with various environmental factors such as dissolved oxygen level, SRT, temperature, pH, ammonium loading rates (ALRs), etc., and will be discussed in the following section.
Nitrogen-cycling microbial abundances and communities
Abundances and communities of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaeal (AOA)
Autotrophic nitrifying bacteria responsible for ammonia oxidation process are detected at WWTP TH1 and belonged to two orders: Nitrosomonadales (affiliated to Nitrosomonas sp. Nitrosospira sp., Nitrosococcus sp., and Thiobacillus sp.) and Rhodocyclales (affiliated to Azospira sp., Thauera sp., and Zoogloea sp.) as shown in Table 3. Zhang et al.  reported that in full-scale municipal WWTPs, the most important genera of AOB were Nitrosomonas and Nitrosospira. Furthermore, they mentioned that Nitrosomonas were the most dominated ones. Consistently, in the full-scale A2O WWTPs, Nitrosomonas sp. are the most dominant AOB, especially in the WWTPs TH2, SK1, and SK2 which were operated at high DO levels. By contrast, the microbial community of Nitrosospira sp. was found at the TH1 because this WWTP was operated under long SRT, a favor condition for the growth of Nitrosospira sp. (see Table 3). Although the abundance of Nitrosospira sp. is less than that of Nitrosomonas sp., the existence of Nitrosospira sp. might be suitable a factor for satisfying good nitrification process when the conditions are not optimal for growth of nitrifying bacteria .
Figure 3A shows the abundance of AOA-amoA genes at WWTP TH1, which is the highest among the WWTPs full-scale A2O process investigated, and the significance of each tank at TH1 shown the high mean difference of letter grouping in blue clustered column, (p < 0.05), see Table S4. The lower DO level, high temperature, and longer SRT (> 30 day) operated in this plant would significantly promote the growth of AOA . Gao et al.  studied the effects of DO levels on the growth of AOB-amoA and AOA-amoA, showing the former is much more abundance under high DO levels of 1.9–3.5 mg-O2 L‒1. Phanwilai et al.  analyzed the abundance of microorganisms in the step-feed aerobic tanks of a municipal wastewater treatment plant, reporting that AOA-amoA was the most abundance genes in the tank with low DO levels (0.9±0.5 mg-O2 L‒1) while AOB-amoA gene was higher than AOA-amoA genes in the tank with high DO level (1.8±0.5 mg-O2 L‒1). In this work, the result of AOB and AOA abundance at WWTPs TH2, SK1, and SK2, which are operated at high DO levels of 2.6–4 mg-O2 L‒1, is inline with the results by Gao et al.  and Phanwilai et al.  (see Figure 3A). Other factors such as the high NH4+ loading rate could also increase AOB abundance. The predominated AOB-amoA gene over AOA-amoA gene at TH2, SK1, and SK2 compared to TH1 could be attributed to the higher NH4+ loading rates in these plants (see Table 2), and the significance of the gene (p < 0.05) shown difference of letter grouping in orange clustered bar chart (see Table S4). The typical design DO level for a nitrogen-removal process of around 2 mg-O2 L‒1 was recommended by .
Although abundance of AOA was not found three WWTPs TH2, SK2, and SK3, AOA and AOB would collaborate and offer possible advantage in ammonia oxidation rates at lower ammonia concentration at TH1. It is postulated that in the practical operation, it is desire to maintain low DO level in an aerobic tank to reduce energy and sustaining SRT range base on characteristics of each full-scale WWTP, the abundance of AOA might be possible group of microorganisms to collaborate with AOB for nitrification process. However, in further research on suitable DO level and SRT range would be investigated to find the optimum conditions of growth AOA that could collaborate with AOB.
Abundances and communities of nitrite-oxidizing bacteria (NOB)
Figure 3B shows that Nitrobacter sp. was more abundance than Nitrospira sp. at WWTP TH1. The DO levels (0.7 to 1.1 mg-O2 L‒1) at TH1 are the lowest among the WWTPs investigated (DO concentration from 2.4 to 4.5 mg-O2 L‒1 for the other three WWTPs), and the temperature range at TH1 is moderate high at 27.7–28.1ºC. The low DO condition is favorable for the growth of Nitrobacter. Huang et al.  reported that DO concentration of > 1.0 mg-O2 L‒1 was suitable condition for the growth of Nitrobacter while DO concentration of < 1.0 mg-O2 L‒1 was optimum condition of Nitrospira. Similarly, Park et al.  suggested that at the low operational DO concentration of 0.5–0.6 mg-O2 L‒1, Nitrospira was selectively enriched over Nitrobacter in the activated sludge from a small-scale SBR. Furthermore, Liu and Wang  investigated the nitrification performance of activated sludge with the long-term effect of low DO concentration, finding that higher abundance of Nitrospira (1012) than abundance of Nitrobacter (1010.4) under the condition of 0.16 mg-O2 L‒1.
The optimal temperature ranges for Nitrobacter and Nitrospira growth are still ambiguous. Huang et al.  concluded that Nitrobacter was favorable species under the temperature ranges of 24–25ºC while Nitrospira dominated at relatively high temperature range of 29‒30ºC. On the contrary, Alawi et al.  indicated that lower temperature range of 10‒20ºC was the optimum condition for Nitrospira growth. Roots et al.  mentioned that Nitrospira increased from 3.1 to 53% under the DO level of 0.2–1.0 mg-O2 L‒1 with 99 d of SRT and NH4+ 0–14 mg-N/L. Qian et al.  found decreasing of Nitrospira from 0.44% to 0.04% by DO level of 0.8–1.5 mg-O2 L‒1 with SRTs between 33 and 56 d and NH4+ 105 mg-N/L. While Sun et al.  set a short SRT of 15 d with DO concentration at 1.0 and 2.0 mg-O2 L‒1 that increased 1.81 and 2.99%, respectively. Under the longer SRT (30 d) with the DO level (0.7–1.1 mg-O2 L‒1) at TH1 presented low abundance of Nitrospira than Nitrobacter, while the three plants with the shorter SRT (17 to 26 d) and higher DO level of 2.4 – 4.5 mg-O2 L‒1 presented high abundance of Nitrobacter than Nitrospira. However, the point of SRT could not be one major effect on Nitrospira but other factors: DO, temperature, ammonium influent, pH, HRT, FA. ALR could also be the significant factors affecting the competition between Nitrospira and Nitrobacter .
At WWTPs TH2, SK1, and SK2, Nitrospira was more abundance than Nitrobacter. These plants were operated at relatively DO of 2.6 to 4 mg-O2 L‒1, HRT of 3.6 to 15.4 hr., and SRT of 17 to 26 d. These operation parameters along with ammonium loading rate (ALR, NH4+-N /m3-d) were important factors affecting Nitrospira growth but less extent on Nitrobacter growth.
Meanwhile, Nitrobacter is more sensitive to the free ammonia (FA) concentration compared to Nitrospira . Mehrani et al.  reported that FA was a major inhibitor on NOB activity. FA concentrations at these three WWTPs (0.25 mg-N L‒1 for TH2, 0.32 mg-N L‒1 for SK1, and 0.29 mg-N L‒1 for SK2) were higher than that at TH1 (0.17 mg-N L‒1). It could be postulated that FA concentration was inhibitor to decrease abundance of Nitrobacter in these three WWTPs.
In this work, only qPCR technique was used to identify both Nitrobacter and Nitrosipra. The specific primers to detect nitrifying bacteria population via the DGGE technique were not used. As Nitrosipra are able to complete oxidation of NH4+ direct to NO3- without into NO2- (complete ammonia oxidizer, comammox process), the specific primers to detect nitrifying bacteria population for Nitrobacter and Nitrosipra are recommended in the further research. In practical, if the information of Nitrosipra in full-scale WWTP is reliable, a new approach of comammox process would be applied for increasing BNR in the future.
Abundances and communities of denitrifying bacteria (DNB)
Three coding genes of nitrite (nirK or nirS) and nitrous oxide (nosZ) reductases were evaluated for the abundance of denitrifying bacteria from these four full-scale WWTPs. As indicated in Figure 3C, higher abundance of nosZ-type denitrifiers was found at TH1 among WWTPs investigated due to the low COD/TN ratio of ≤ 3.7 in TH1 (see Table S4). The effects of COD/TN ratio on the abundance of nosZ-type denitrifiers were consistent with the results reported by Yuan et al.  who reported that the nosZ-type denitrifiers was two orders of magnitude more at the influent COD/TN ratio of 4.6 (1.29 × 108 copies) compared to that at COD/TN ratio of 8.4 (1.31 × 106 copies) at the Beijing municipal WWTP in China.
The average number of DNB copy presenting at TH1 shows that nosZ-type denitrifiers in anoxic and anaerobic tanks were most dominated. Wang et al.  found that the abundance of nosZ was a good indicator for rechecking anoxic and anaerobic conditions, having more oxygen concentration those conditions. Base on this result, it can be concluded that the DO level in the anoxic and anaerobic tanks of TH1 were quite high, and denitrifying bacteria could not use NO3‒ electron acceptor for denitrification process, resulting in poor denitrification efficiency at TH1 in anoxic condition. As shown in Table 2, DO level in anoxic tank at TH1 was 0.3±0.1 mg-O2 L‒1. It should be noted that the low denitrification efficiency at TH1 could also attributed to the low COD/TN ratio in the receiving water.
Tallec et al.  and Jia et al.  indicated that low DO concentration in WWTPs favors nitrous oxide (N2O) production during nitrification/denitrification process. High abundance of nosZ gene in denitrifies was also found in aerobic tank of WWTP TH1. Henry et al.  indicated that nosZ-type denitrifiers could be responsible in N2O production. It could be postulated that the BNR process at TH1 could produce higher N2O gas among WWTPs investigated due to the low DO level of this plant (0.9±0.2 mg-O2 L‒1).
On the other hand, the high abundance of nirS-type denitrifiers and less abundance of nosZ-type denitrifiers were found in anaerobic and anoxic tanks at TH2, SK1, and SK2 due to high DO centration (2.6‒4.5 mg-O2 L‒1) operated at the A2O process. Meanwhile, nirS-type denitrifiers were higher than the nirK-type denitrifiers at all full-scale WWTPs. Complete denitrification is possible with nirS-type denitrifiers . Che et al.  found a predominance of nirS-type level over nirK-type of all eight full-scale municipal WWTPs in different cities of China. Based regression analysis, Zhang et al.  suggested that the abundance of nirK-type denitrifiers was correlated with temperature and nirS-type denitrifiers was linearly correlated with temperature and ammonium concentration.
Both heterotrophic and autotrophic communities of denitrifying bacteria were found as indicated in Table 3. Heterotrophic denitrifying bacteria (Ilumatobacter sp., Comamonas sp., Rhodoferax sp., Terrimonas sp., Niabella sp., Sediminibacterium sp., Tistrella sp., Oryzobacter sp.) are normally found in WWTPs [31, 32] Autotrophic denitrifying bacteria belonging to Arcobacter (affiliated Arcobacter suis) relate to pathogenic bacteria that were found in high abundance in the municipal full-scale biological N and P removals processes . Kristensen et al.  reported that pathogenic Arcobacter bacteria was not found in WWTPs with longer SRT (25‒35 day) because they could be able to pass through both anoxic and aerobic tanks. In this work, Arcobacter suis was only found at SK1. Other filamentous autotrophic denitrifying bacteria were commonly found in wastewater worldwide and were presented. Chloroflexi plays a role in sludge flocculation and is more commonly found in WWTPs designed to remove nutrients, and most appearance with a long SRT operation and expose the biomass to anaerobic conditions . Haliscomenobacter sp. were filamentous bacteria and satisfied being in phosphorus concentrations . Their filamentous bacteria were found and achieved to remove phosphorus in A2O processes.