An efficient process for wastewater treatment to mitigate free nitrous acid generation and its inhibition on biological phosphorus removal

Free nitrous acid (FNA), which is the protonated form of nitrite and inevitably produced during biological nitrogen removal, has been demonstrated to strongly inhibit the activity of polyphosphate accumulating organisms (PAOs). Herein we reported an efficient process for wastewater treatment, i.e., the oxic/anoxic/oxic/extended-idle process to mitigate the generation of FNA and its inhibition on PAOs. The results showed that this new process enriched more PAOs which thereby achieved higher phosphorus removal efficiency than the conventional four-step (i.e., anaerobic/oxic/anoxic/oxic) biological nutrient removal process (41 ± 7% versus 30 ± 5% in abundance of PAOs and 97 ± 0.73% versus 82 ± 1.2% in efficiency of phosphorus removal). It was found that this new process increased pH value but decreased nitrite accumulation, resulting in the decreased FNA generation. Further experiments showed that the new process could alleviate the inhibition of FNA on the metabolisms of PAOs even under the same FNA concentration.

increasing evidences showing that FNA, the protonated form of nitrite, rather than nitrite is the actual inhibitor 5,11,12 . For example, it was reported that FNA could inhibit aerobic phosphorus uptake seriously at a low level of 0.5 3 10 23 mg HNO 2 -N/L 11 , and more than 1.5 3 10 23 mg HNO 2 -N/L could result in the complete loss of aerobic phosphorus uptake 13 . Pijuan et al. 5 showed that the aerobic phosphorus uptake was inhibited by 50% when FNA reached 0.52 3 10 23 mg HNO 2 -N/L. Anoxic phosphorus uptake was also significantly affected by FNA presence 14 , and 0.02 mg HNO 2 -N/L would cause the complete loss of anoxic phosphorus uptake 11 . Furthermore, anaerobic metabolisms of PAOs were also severely affected by FNA, and Ye et al. 8 demonstrated that FNA had an adverse effect on carbon source uptake even at 1.0 3 10 23 mg HNO 2 -N/L. Due to the severe inhibition on PAOs caused by FNA and the massive quantity of wastewaters treated daily, any improvement for reducing FNA generation or mitigating its inhibition on PAOs in current methods should have tangible economic and ecological consequences.
Several strategies, such as activated sludge adaption, pH adjustment, temperature control, and the feed flow and mode optimization, have been recommended to minimize the inhibitory effect of FNA on PAOs 6,15 . Though previous researches have proposed these meaningful methods, the strategy for mitigating the generation of FNA and its inhibition on PAOs from the aspect of modifying wastewater treatment operation regime has never been reported before. In addition, some previously proposed strategies such as pH adjustment and temperature control are rarely or not practically applied in fullscale WWTPs, probably due to the associated costs of adding pH controlling agents or increasing constructions. Thus, the method obtained in terms of wastewater treatment process modification may provide an alternatively practical option for engineers.
Besides the widely accepted anaerobic/oxic (A/O) phosphorus removal regime, PAOs are verified to be also enriched readily in the oxic/extended-idle (O/EI) wastewater treatment regime [16][17][18][19] . The O/EI regime enriches PAOs via some specific metabolic reactions (e.g., a significant idle release of phosphate and a low idle production of PHAs) occurred in the extended-idle phase, which shows a different inducing mechanism from the classical A/O regime. It is also reported that when receiving the same level of nitrate, the transformations of metabolic intermediates (especially the accumulation of nitrite) in the O/EI regime are much lower affected than those in the A/O regime 16 . Thus, one method that might be used to decrease FNA generation or mitigate its inhibition on PAOs from the viewpoint of wastewater treatment regime, we think, is to develop a suitable biological nutrient removal (BNR) process based on the O/EI regime. Although several studies have been performed in terms of the O/EI phosphorus removal regime, the questions as to whether (and how) this regime can achieve good performances of simultaneous nitrogen and phosphorus removal remain unknown. Additionally, it is also unclear whether PAOs cultured in this O/EI based on BNR process can tolerate higher level of disturbances caused by nitrogen removal (e.g., FNA inhibition) than those cultured in the conventional BNR process.
The purpose of this paper is to report this efficient method for significantly mitigating the generation of FNA and its inhibition on PAOs. Firstly, a new BNR process is designed based on the recently exploited O/EI regime, and its feasibility of BNR is evaluated. Since the O/EI regime is a phosphorus removal process with low nitrogen removal performances (around 60%), an anoxic phase is inserted into the oxic phase to enhance nitrogen removal. Therefore, the new BNR process developed here is performed as the oxic/anoxic/ oxic/extended-idle (O/A/O/EI) regime. Then, the performances of BNR and the abundances of PAOs between the new process and conventional four-step (i.e., anaerobic/oxic/anoxic/oxic, defined as A/O/A/O) BNR process are compared. Finally, the reasons for the new process showing higher abundance of PAOs are explored via the analysis of cyclic pH variation, nitrite accumulation, and changes of metabolic intermediates in PAO metabolisms.   Table 1. The above results clearly displayed that by modifying wastewater treatment operation regime the abundance of PAOs and the efficiency of phosphorus removal could be improved.

Results
Phosphorus removal test via chemical precipitation at different pH. Batch tests in the absence of activated sludge microorganisms were performed to figure out the effect of pH on chemical phosphorus removal (Table S1, supporting information). As shown in Table S1, negligible SOP removal via chemical  precipitation was observed. Only 2% of SOP was removed via chemical precipitation even the pH was 8.5, which implied that phosphorus removal in this study was dominated by biological effect.
Comparison of the effect of different FNA levels on PAO metabolisms between the two reactors. The two BNR reactors were developed from different inducing mechanisms of biological phosphorus removal. The different inducing mechanisms might give rise to different metabolic responses generated by PAOs even under the same level of FNA, thus we examined whether the same level of FNA would bring different effects on the metabolisms of PAOs between the two reactors. It can be seen from Table 2 that the metabolisms of PAOs in the conventional A/O/A/O reactor were severely inhibited by the FNA addition. When FNA concentration was 0, the effluent SOP in the A/O/A/O reactor was 0.75 6 0.05 mg/ L. With the increased FNA concentration anaerobic SOP release, PHA-up/VFA ratio, Gly-de/VFA ratio, and Gly-syn were significantly decreased. As a result, effluent SOP concentration was increased largely. Especially when FNA was 0.51 3 10 23 mg/L, 7.05 6 0.09 mg/L of SOP was measured in the effluent, suggesting that only 53 6 0.6% of influent SOP was removed. Also from Table 2

Discussion
The possible mechanisms of O/A/O/EI regime culturing higher PAO abundance. Several parameters can affect significantly the abundance of PAOs. By comparing the operational conditions between the two reactors, dissolved oxygen (DO) and pH might be the effect parameters since they are not constantly controlled during the whole process. Therefore, the cyclic variations of DO and pH between the two reactors during the steady-state operation were first compared, and the data are shown in Figure 3. Except for the anaerobic phase DO concentration in other phases of the A/O/A/ O reactor showed very similar changes with that in the O/A/O/EI reactor. For example, in the experiment of day 80, DO in the O/A/O/ EI reactor kept low levels during the initial period of first oxic phase and then gradually increased to a final concentration of 4.5 mg/L at the end of first oxic phase. During the subsequent anoxic phase, DO decreased rapidly to 0.7 mg/L and kept in the range of 0.4-0.7 mg/L in the remainder of anoxic phase. In the second oxic phase, DO increased gradually to 1.8 mg/L. After that, DO decreased gradually to 0.3 mg/L during the initial 60 min of idle phase and further decreased to 0.2 mg/L during the remainder of idle phase. Similar profiles were also made in other cycle studies. The results indicated that DO was not the main reason for the two reactors showing different PAO abundances.
The profile of pH change in the two reactors, however, exhibited obvious differences. In the O/A/O/EI reactor, pH gradually increased from 8.0 to 8.6 during the initial 60 min of first oxic phase and then decreased slightly during the remaining of this phase. In the following anoxic and oxic phases, a gradual increase to a final pH of 8.6 was observed. During the subsequent idle phase pH decreased gradually to the final pH of 8.2. In the A/O/A/O reactor, pH decreased from 8.0 to 7.4 in the anaerobic phase, and then a gradual increase followed by a slight decrease of pH was measured in the first oxic phase. In the subsequent anoxic phase, pH showed a gradual increase tendency then pH decreased slightly in the following oxic and idle phases. It can be clearly seen that cyclic variation of pH value in the O/A/O/EI reactor (8.0-8.6) was higher than that in the A/O/A/O reactor (7.2- 8.2). The initial higher pH value achieved in the O/A/O/EI reactor might be mainly due to CO 2 expelled from the reactor by air-stripping, while the following pH decline was probably ascribed to nitrification. It was reported that denitrification and phosphorus uptake were the primarily reasons for pH increase in the anoxic phase and oxic phase 20 . During the extended-idle phase, a slight pH decline might be owing to idle SOP release ( Figure 4). There are three forms of phosphorus existed in the activated sludge: metal phosphorus via physical chemistry processes, intracellular polyphosphate inclusion, and bio-phosphorus for bacteria normal growth 21 . It is known that the amount of metal phosphorus in activated sludge is affected by pH, and higher pH value may cause higher chemical phosphorus removal 22 . However, batch tests showed that SOP removal via chemical precipitation was negligible (Table S1, supporting information), which implied SOP removal in this study was primarily due to biological effect. Previous publications showed that a high level of pH could provide a selective advantage to PAOs over other populations such as GAOs 19,23 . Accordingly, the higher level of cyclic pH was one reason for the O/A/O/EI reactor enriching more PAOs. More importantly, the concentration of severe inhibitor to PAOs, i.e., FNA, is reported to closely relevant to pH value 24 . The different cyclic pH variations between the two reactors might cause different levels of FNA generation, thus the amount of FNA production between the two reactors was compared secondly. Besides pH, it is known that FNA concentration is also relevant to temperature and nitrite concentration. Temperature between the two reactors was the same (20 6 0.5uC), and the change of nitrite as well as ammonia,    nitrate, and SOP in the two reactors is shown in Figure 4. In the first oxic phase of O/A/O/EI reactor, SOP release was observed during the initial 30 min before SOP was swiftly taken up probably due to the low level of DO concentration (Fig. 3a). NH  (Table 2). Therefore, it can be understood that the O/A/O/EI reactor enriched more PAOs than the conventional A/O/A/O reactor. Some scientists reported that PAOs could be acclimated high nitrite and FNA concentrations when using nitrite as sole electron acceptor 27 . However, O 2 prior to nitrite was the main electron acceptor in this study, which might be the reason for the inconsistent results.
FNA can inhibit or inactivate the activities of some key enzymes relevant to phosphorus removal. For instance, glyceraldehyde-3phosphate dehydrogenase (GADP) and sulfhydryl (SH)-containing enzymes, which are respectively key enzymes involved in both glycolysis (gluconeogenesis) and the tricarboxylic acid (TCA) cycle, are reported to be heavily inhibited through reaction with FNA ( Figure 5). The transformations of key metabolic intermediates such as glycogen and PHAs are closely related to glycolysis (gluconeogenesis) and the TCA cycle, thus the activity or abundance of PAOs will be reduced when FNA interferes with the pathways of glycolysis (gluconeogenesis) or the TCA cycle. From the ''Methods'' section, it can be found some differences between the two reactors. In the conventional A/O/A/O reactor, acetate is consumed in the anaerobic phase whereas it is taken up aerobically in the new O/A/O/EI reactor. This different metabolic behavior will cause certain metabolic differences, which might be one reason for the O/A/O/EI reactor enriching higher PAOs. Also, this different metabolic behavior might result in different metabolic responses of PAOs to FNA. For example, compared with the conventional A/O/A/O reactor where ATP and NADH 2 for PHAs formation were respectively provided via poly-P hydrolysis and glycogen degradation, the TCA cycle seems to supply both ATP and NADH 2 for PHAs synthesis in the O/A/O/EI reactor since it is generally accepted that the TCA cycle will dominate under aerobic conditions. However, it is still unclear why the O/A/O/EI reactor can alleviate the inhibition of FNA on the metabolisms of PAOs, as the TCA cycle plays an important role in PAO metabolisms of both regimes. Further efforts need to be carried out in future.
Comparison with other strategies for minimizing the inhibitory effect of FNA on PAOs. This paper presents an effective strategy for mitigating the generation of FNA and its inhibition on PAOs. That is, by modifying the wastewater treatment operation regime as the O/A/O/ EI regime the abundance of PAOs and the efficiency of phosphorus removal can be significantly improved. This was experimentally demonstrated via a long-term test in two reactors operated as the new O/A/O/EI regime and the conventional A/O/A/O regime, respectively. The abundance of PAOs cultured in the O/A/O/EI reactor was about 11% higher than those in the conventional A/O/A/ O reactor, which led to 15% of improved phosphorus removal efficiency. Moreover, this wastewater treatment regime based strategy did not decrease but slightly increase the nitrogen removal performance. Considering the huge quantities of wastewater treated daily, this strategy has a significant consequence from an ecological perspective. Compared with other strategies such as pH adjustment and temperature control 6,15 , this wastewater treatment regime based strategy does not require consumption of any additional chemicals and energy, which makes this strategy more economical and practical. This strategy can also integrate with the step-feeding mode easily, a practically effective method for minimizing the inhibitory effects of FNA, to gain a better nutrient removal performance. It was reported that step-feeding modes could greatly reduce the FNA inhibition influence as compared to dump-feeding 28 . By modifying the influent mode of O/A/O/ EI regime, this wastewater treatment regime based strategy can easily combine with the feeding based strategy, which may cause further reduction of FNA inhibitory. Therefore, the strategy presented here might provide a practically promising solution to the ''nitrogen-phosphorus challenge'' faced by WWTPs. Furthermore, the enrichment of PAOs in the O/A/O/EI reactor is driven by the O/EI regime. It was reported that the O/EI regime could achieve very good phosphorus removal readily and steady when using glucose, a substrate usually considered being detrimental for PAO proliferations, as the sole carbon source 30 . Thus, the O/A/O/EI process reported in this paper may provide an ideal technology for BNR removal from carbohydrate-rich wastewaters. Generally, glucose or other carbohydrate compounds in domestic wastewater are at low levels, because it can be readily biofermented to volatile fatty acids in sewer systems. However, in some WWTPs where industrial or agricultural factories discharging carbohydrate-rich wastewaters are located nearby, or in some specific areas where the distance between the wastewater discharge sources and wastewater treatment unit is short (e.g., the highway rest areas, one of our parallel researches), wastewater carbohydrate may maintain at high levels. In these areas, the O/A/O/EI process may have an excellent application perspective, and the batch-scale study presented here may provide a useful reference for designs in future.
It should be noted that although the hydraulic retention time (HRT) between the O/A/O/EI and A/O/A/O reactors operated in this studies was maintained the same, the HRT controlled in the O/ A/O/EI regime may slightly higher than that in the conventional BNR regime, because one cycle of the conventional BNR systems can be shortened to be 6 h via process optimization whereas the O/A/O/EI regime needs a relatively long idle period to enrich PAOs (e.g., 210 min). This characteristic implies that the proposed O/A/O/EI regime will increase the volumes of bioreactors when treating the same amount of wastewater. However, this drawback can be settled via reactor reconfiguration as proposed in Figure S1. Despite that this new strategy was demonstrated using sequencing batch reactors in this study (due to the availability of the equipment), it has also the potential to be applied in a continuous system. For a continuous-flow activated sludge system, an extra reactor for regurgitant sludge rest (3.5 h of the retention time seems to be enough) is required to set up in the side-stream for the enrichment of PAOs, and the construction invest of extra side-stream reactor is low, as compared with other strategies. It should also be emphasized that fullscale tests are required to fully evaluate the feasibility and potential of this strategy though excellent results have already obtained in our laboratory experiments.

Methods
Synthetic wastewater. Synthetic wastewater used throughout these investigations, unless otherwise described, was the same and prepared daily. Acetate was used for the sole carbon source since it was the most common volatile fatty acids present in real domestic wastewaters 29   www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8602 | DOI: 10.1038/srep08602 120 min idle period. For each cycle, certain volume supernatant was discharged from both reactors after the settling phase and was replaced with synthetic wastewater during the initial 5 min of first oxic phase (the O/A/O/EI reactor) and anaerobic phase (the A/O/A/O reactor), respectively. The HRT and sludge retention time (SRT) in the two reactors were controlled at approximately 16 h and 20 d, respectively. During anaerobic phase, the A/O/A/O reactor was mixed with a mechanical stirrer (150 rpm) while during the aerobic phase, air was supplied into both reactors at a flow rate of 15 L/min. The initial pH level in both reactors was controlled at 8.0 by adding 0.5 M HCl or 0.5 M NaOH solutions.
It should be noted that during the idle phase mixture stirring was not conducted in the routine operation of both reactors, but when cyclic tests were carried out, both reactors were mixed with a mechanical stirrer (150 rpm) to facilitate sampling. The mixed liquor samples were taken every 30 min and immediately filtered through a Whatmann GF/C glass microfiber filter (1.2 mm). The sludge sample was used to assay for MLSS, MLVSS, PHAs, and glycogen. The filtrate was used for the analyses of SOP, COD, NH 4 1 -N, and NO x --N.
Phosphorus removal test via chemical precipitation at different pH. Phosphorus can be removed via chemical precipitation when some metal ions such as Ca 21 , Mg 21 are present in wastewater. With the increase of pH the chemical phosphorus precipitation was enhanced 22 . Hence, one batch test was performed without the activated sludge microorganisms to assess the effect of pH on chemical phosphorus precipitation. Firstly, 15 L synthetic wastewater mentioned above was divided evenly into 5 identical reactors with working volumes of 3.0 L each. Then, the reactors were added 0.5 M HCl or 0.5 M NaOH solutions to keep the pH value 6.5, 7.0, 7.5, 8.0, and 8.5, respectively, the other operational conditions were the same as the O/A/O/EI reactor described above expect that there was no sludge microorganisms. Finally, SOP in the supernatant of the 5 reactors was detected after several cycles. Therefore, it is readily to assess the effect of pH on chemical phosphorus precipitation via measuring the SOP concentration in supernatant.
Comparison of the effect of different FNA levels on PAO metabolisms between the two reactors. The A/OA/O reactor is developed from the conventional A/O phosphorus removal regime whereas the O/A/O/EI reactor is developed from the recently exploited O/EI regime, thus it is necessary to investigate whether there are different effects on PAO metabolisms between the two reactors even under the same FNA levels. The following batch experiment was executed to provide such support. Two identical sludge mixtures (2.4 L each) were respectively withdrawn from a WWTP in Changsha, PR China. The mixtures were centrifuged (5000 rpm for 5 min) and washed three times with tap water to remove the residual NH 4 1 -N, NO x --N, SOP, and COD. Then, they were resuspended in tap water with a final volume of 1.2 L each before being evenly divided into six reactors. The two groups of reactors (six each) were respectively operated as the same as the A/OA/O and O/A/O/EI reactors expect for the following differences. Allyl-Nthiourea (a nitrification inhibitor) was added at a concentration of 2 mg/L to each reactor to inhibit the nitrification according to the literature 33 . Temperature was controlled at 20 6 0.5uC and pH was on-line controlled consistently at pre-designed set-point (pH 5 8.0 6 0.1) by a programmable logic controller using 0.5 M HCl solution and 0.5 M NaOH solution. Thus, the FNA concentrations of six reactors for each group were respectively controlled at 0, 0.05 3 10 23 , 0.15 3 10 23 , 0.26 3 10 23 , 0.38 3 10 23 , and 0.51 3 10 23 mg HNO 2 -N/L through controlling nitrite concentration, pH, and temperature. It was reported that the FNA concentration could be calculated by the formula S N-NO2 /(Ka 3 10 pH ) with K a value determined by the formula K a 5 e (22300/(T 1 273)) for a given temperature T (uC) 24 . When effluent SOP concentration among these reactors reached stable, cyclic studies were performed and the data were reported.
The fluorescence in situ hybridization (FISH) with 16s rRNA-targeted oligonucleotide probes was carried out to quantify the abundances of PAOs and GAOs, and the methods were the same as described in the literature 17 . Briefly, sludge samples were taken and fixed in 4% formaldehyde for 20 h at 4uC and then subjected to freezethaw treatment in order to enhance the penetration of oligonucleotide probes. Cell samples were attached to poly-L-lysine coated slides and dehydrated with ethanol. The following hybridization and washing procedures were the same as that in the literature 35 . For quantitative analysis, 20 microscopic fields were analyzed for the hybridization of individual probes using a confocal scanning laser microscope (FV 500) with image database software (VideoTesT Album3.0). The oligonucleotide probes specific for PAOs, GAOs, and total bacteria, which were respectively labeled with 59AMCA, 59Cy3, and 59FITC, were listed in Table S2.