Free ammonia affects the nitrification performance and nitrifying community structure in the suspended activated system

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1.School of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, P. R. China; 2.Shandong Tongji Testing Technology Co., Ltd, Yantai 264000, Shandong, P. R. China; 3.Lanzhou Urban Water Supply (Group) Co. LTD, Lanzhou 730070, P. R. China; 4. South China Institute of Enviromental Sciencce, MEE, Guangzhou 510655, P. R. China)

Abstract: Four parallel SBRs were established to treat synthetic wastewater with preset concentrations of free ammonia (FA) (0.5, 5, 10 and 15 mg/L), including S0.5, S5, S10 and S15. The four systems removed ammonia well throughout the experiment (average value of 98.7%). The inhibition of FA by nitrite-oxidizing bacteria (NOB) combined with process control was used to achieve a nitrite pathway in S10 and S15. During the initiation of the nitrite pathway, the accumulation rate (NAR) increased dramatically to 90.3% on day 79 in S10 and to 90.5% on day 139 in S15. For S10 on day 80～250 and S15 on day 140～250, the average NARs were steady at approximately 98.8% and 98.2%, respectively. High-throughput sequencing of the 16S rRNA gene played an ever-increasing role in analyzing the relative abundance and structure of the nitrifying bacteria in these samples. The results showed that the changes in the abundance of AOB and NOB were consistent with our experimental results. FA affected not only the relative abundance of AOB and NOB, but also the activity of NOB. Although AOB and NOB coexisted in the four systems, AOB was still the main nitrifying bacteria. We found that a lower abundance of AOB had a higher microbial utilization capacity of ammonia substrate at 15 mgFA/L.

Keywords: free ammonia (FA); partial nitrification; high-throughput sequencing; ammonia-oxidizing bacteria (AOB); nitrite-oxidizing bacteria (NOB)

(1)

FA has a significant effect on the nitrogen removal performance in the biological nutrient removal (BNR) pathway, because FA can affect the activity of nitrifying bacteria (including ammonia- oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB))[1-4]. The earliest inhibition threshold levels were reported by Anthonisen et al.[1], who demonstrated that AOB and NOB are inhibited by 10～150 mgFA/L and 0.1～1.0 mgFA/L, respectively. Considerable research has focused on the inhibition of AOB and NOB activity[5-8]. These studies mainly explored how to achieve effective partial nitrification during aerobic nitrification using the gap in the inhibitory concentration of FA between AOB and NOB. Nitrification, a two-step microbial process, is the oxidation of ammonia to nitrite by AOB and of nitrite to nitrate by NOB[9-12]. Partial nitrification is usually achieved by using the difference in activity between AOB and NOB, so ammonia is only oxidized to nitrite[13-15]. Over several decades, much research has been performed on stabilizing partial nitrification in BNR systems to remove nitrogen from high ammonia nitrogen wastewater by adjusting the concentration of FA to a specific level that inhibits NOB rather than AOB[16-18].

It is widely acknowledged that the nitrogen removal performance in BNR systems is affected by the activity of nitrifying bacteria[19-20]. From the perspective of the biological inhibition mechanism, the relative abundance and structure of nitrifying bacteria in the sewage treatment system is significantly influenced by the concentration of FA. Recently, Illumina high-throughput sequencing (HTS) technology was used to gain an in-depth understanding of characteristics of the microbial community in the water treatment system. During past years, much literature focused on the relative abundance and the structure of the nitrifying bacteria caused by FA in the BNR process[21-23]. These studies revealed that the relative abundance and structure of nitrifying bacteria are significantly affected at 2.9～50.1 mgFA/L.

Although exploration of the effects of FA on nitrifying bacteria during aerobic nitrification is increasingly successful, there is still a technical problem in that there is no consistent threshold level at which FA inhibits AOB and NOB. Thus, it is essential and urgent to investigate a consistent level at which FA inhibits AOB and NOB in biological systems. Most reported studies focused on the nitrifying bacteria in wastewater treatment bioreactors with a random FA concentration range (the concentration of FA depends on the quality of the wastewater). Few studies investigated nitrifying bacteria in the bioreactor under the precisely controlled concentration of FA. In addition, although previous studies revealed that FA has adverse effects on the microbial activity and stability of sludge, detailed information on the mechanisms by which it accomplished this was limited to various sludge characteristics and the structure of the microbial community caused by the concentration of FA. To eliminate these drawbacks, it is indispensable to systematically assess the variety and structure of nitrifying bacteria of relative abundance under different concentrations of FA during aerobic nitrification.

During our experiment, we filled four parallel sequencing batch reactors (SBRs) with precisely controlled FA concentrations of 0.5, 5, 10 and 15 mg/L. Activated sludge (AS) was used to tame microorganisms exposed to a specific concentration of FA in the SBRs. First, the long-term nitrogen removal performance of the SBRs was investigated. A method was explored to successfully achieve rapid and stable partial nitrification in the SBR at a high concentration of FA. The variety and structure of AOB and NOB of relative abundance in the SBRs with different concentrations of FA and the related mechanisms were examined in detail using high-throughput sequencing of the 16S rRNA gene. The information obtained has implications for the biological mechanism of nitrogen removal and the mechanism by which FA inhibits nitrifying bacteria.

1 Material and methods

1.1 Batch experiments design and operation

Four 4 L parallel SBRs with 0.5, 5, 10 and 15 mg/L, concentrations of FA were operated to enrich the microbial community. The concentration of ammonia, the temperature and the pH were adjusted to obtain different concentrations of FA. Every cycle of the four SBRs consisted of 5 min filling, aerobic reaction, anoxic reaction, settling, 5 min decanting, and an idling period. The aerobic reaction, anoxic reaction, settling and idling period were flexible because of the different initial concentrations of FA.

The nitrification and denitrification were performed by adjusting the ORP, pH and DO. The pH was adjusted by the addition of 0.1 mol/L HCl and 0.4 mol/L NaOH. The temperature control system was used to control the temperature in the SBR. The DO concentrations in the four SBRs were maintained at 1.0～2.5 mg/L by an air compressor during aeration and by continuous stirring at 150 r/min by a mechanical stirrer rotating during the anoxic reaction. Table 1 summarizes the reactor operation.

Table 1 Operational conditions with variable concentrations of FA in four SBRs

Activated sludge with a mixed liquor suspended solid (MLSS) of 3 000 mg/L was collected at a local domestic sewage treatment plant (WWTP) in Lanzhou, Gansu to start up the batch reactors.

The four SBRs were operated for 250 days under the above mentioned concentrations of FA. After sustained long-term steady treatment by the reactors, four acclimated activated sludge samples, S0.5, S5, S10and S15, were obtained. On day 240, 12 samples were collected from the four SBRs (3 samples from each SBR). The collected samples were immediately mixed with absolute ethanol at a 1∶1 volume ratio, and placed in a refrigerator, where they were maintained at -20 ℃ for DNA extraction.

1.2 Synthetic media

Nutrients and trace elements in the synthetic media support microbial growth in the reactors. The synthetic media contains (adapted from Kuai et al.[24]). 115 mg/L NH4Cl, 385 mg/L CH3COONa, 26 mg/L of K2HPO4and KH2PO4, and 2 mL of trace elements solution. 5.07 mg MgSO4·7H2O, 1.26 mg Na2MoO·2H2O, 2.49 mg FeSO4·7H2O, 0.41 mg CoCl2·6H2O, 0.44 mg ZnSO4·7H2O, 0.31 mg MnSO4·4H2O, 0.43 mg CaSO4·2H2O, 0.25 mg CuSO4, 1.88 mg EDTA and 0.25 mg NaCl were contained per liter in the trace elements solution.

1.3 Analytical measurements

1.4 Calculations

The concentration of FA was calculated by Eq.(1).

The ammonia oxidation rate (AOR) was calculated by Eq.(2).

AOR (mgN/gVSS·min)=

(2)

The nitrite oxidation rate (NOR) was calculated by Eq. (3).

NOR (mgN/gVSS·min)=

(3)

The nitrite accumulation rate (NAR) was calculated by Eq. (4).

(4)

1.5 DNA extraction steps, PCR quantification and high-throughput sequencing

First,a 10 mL AS sample fixed with absolute ethanol was centrifuged at 12 000 r/min for 10 min. The obtained sediment was used for subsequent DNA extraction. We followed the manual steps to extract DNA with FastDNATMSpin Kit for Soil (MP Biomedicals, USA). Agarose gel electrophoresis was used to assess the quality of the DNA and a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) was used to measure the concentration of DNA. The extracted DNA samples were stored in the refrigerator at -80 ℃. The V3-V4 hypervariable region of the 16S rRNA gene was targeted for PCR using the 338F (5′-ACTCCTACGGGCAGCA-3′) forward primer and the 806R (5′-GGACTACHVGGGTWTCTAAT-3′) reverse primer. The thermocycler was operated with an initial denaturation at 98 ℃ and 2 min, followed by 25 cycles of denaturation at 98 ℃ for 15 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 30 s, and a final elongation at 72 ℃ for 5 min.

Agencourt AMPure Beads (Beckman Coulter,Indianapolis, IN) and PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA) were used to purify and quantify the PCR amplicons, respectively. The amplicons were combined in equal amounts and double-ended 2×300 bp sequencing was performed using the Illumina MiSeq platform and MiSeq kit V3 from the Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China). QIMME software was used to classify and quantify the effective sequences and the sequence with a similarity threshold higher than 97% was classified as the same operational taxonomic unit (OTU).

2 Result and discussion

2.1 Nitrogen removal performance in SBRs

Fig.1 The influent and effluent concentration of and removal efficiency in SBRs during the

Fig.2 Effect of concentration of FA on AOR and

2.2 Achieving a nitrate pathway in S0.5 and S5 and a nitrate pathway in S10 and S15 for nitrogen removal

2.2.1 Achieving a nitrate pathway in S0.5and S5at a lower concentration of FA

Fig.3 Nitrogen removal performance in SBRs along

The concentrations of FA in S0.5and S5were 0.5 and 5 mg/L, that were both within and also much higher than the inhibition threshold of FA on NOB (begin at 0.1～1.0 mg/L) reported by Anthonisen et al.[1]. However, obvious nitrite accumulation was not found in S0.5and S5(Fig. 3(a)), showing that NOB activity could not be inhibited significantly when the FA is lower than 5 mg/L. Our research fully confirmed the conclusion of Vadivivelu et al.[2], who also found no significant decrease of NOB activity from 0 to 4 mgFA/L, indicating that a low concentration of FA has a small inhibitory effect on NOB.

In addition, nitrite accumulation did not occur in S0.5and S5even though we strictly controlled the process in each SBR cycle within 250 days. Our finding was consistent with Cao et al.[22]. There was no obvious nitrite accumulation by applying process control after 29 cycles at 2.9 and 5.6 mgFA/L, but they also achieved high total nitrogen removal efficiency averaging over 99%. However, Vlaeminck et al.[13]obtained stable nitrite accumulation and high nitrogen removal efficiency when the concentration of FA was higher than 3 mg/L, meaning that NOB suffered from strong suppression.

2.2.2 Initiating the nitrite pathway at a high concentration of FA in S10and S15

The nitrite pathway was successfully established at 10 mgFA/L and 15 mgFA/L, and S10achieved the nitrite pathway faster than S15(Fig. 4). NOB activity was strongly inhibited in S15and S10, while AOB activity was reduced more in S15than S10, leading to the decline of the oxidation rate of the ammonia, and increasing the realization time of the nitrite pathway. Both the continuous ammonia oxidation process and the nitrite accumulation indicated that AOB activity wasn’t greatly affected by 10 and 15 mgFA/L, which confirmed that AOB is more tolerant of FA than NOB.

Fig.4 Nitrogen removal performance in SBRs along

Although several authors analyzed the inhibitory thresholds of FA on AOB and NOB, a consistent inhibitory threshold was not obtained. For example, the first inhibition threshold level of FA on AOB and NOB reported by Anthonisen et al.[1]was 10～15 mg/L and 0.1～1.0 mg/L, respectively. Kim et al.[4]suggested that only NOB was inhibited, while AOB could still oxidize ammonium to nitrite at 14～17 mgFA/L. Van Hulle et al.[27]reported that AOB activity was not inhibited in SHARON reactors at 70-300 mgFA/L.

Vadivelu et al.[2]found that the inhibitory effect of FA on NOB started from 1 mg/L and stopped growing when it was higher than 6 mg/L. In comparison, Vadivelu et al.[3]revealed that the inhibitory effect of FA on AOB started at 16 mg/L. Our research and previous studies showed that FA concentrations at S10and S15can strongly inhibit NOB activity, but have little effect on AOB activity.

2.2.3 Maintaining the nitrite pathway at a high concentration of FA in S10and S15

During the period the nitrite pathway was maintained (S10on day 80～250 and S15on day 140～250), the main product in the nitrification was nitrite (81.2 and 48.3 mg/L), the nitrate concentration was still maintained at the bottom level (1.0 and 1.1 mg/L) and the NARs remained stable at 98.8% for 170 days and 98.2% for 110 days in S10and S15, respectively (Fig. 4). Therefore, we can conclude that 10 mgFA/L and 15 mgFA/L can continue to inhibit NOB activity, and the nitrite conversion process was strongly inhibited.

In this study, the selective inhibition at high concentration of FA combined with process control is a basic method to achieve and stabilize the nitrite pathway in the treatment of synthetic wastewater in the SBR.

2.3 Confirmation of the dominant nitrifying bacterial population

Nitrifying bacteria plays a crucial role in nitrogen removal in the SBR. Therefore, we analyzed the relative abundance and structure of the nitrifying bacteria to better understand the microbial role in nitrification using high-throughput sequencing. Considerable studies have reported that five kinds of AOB (Nitrosomonas,Nitrosococcus,Nitrosospira,NitrosolobusandNitrosovibrio) and four kinds of NOB (Nitrococcus,Nitrospira,NitrobacterandNitrospira) were widely found in the sewage treatment system[29-32]. In this work, we found two types of nitrifying bacteria (Nitrosomonas(AOB) andNitrospira(NOB)), which were regarded as the dominant nitrifying bacteria in WWTPs, as reported. A widespread and reliable consensus that NOB is even more sensitive to FA inhibition than AOB was reached and applied in numerous studies[6, 13, 23].

As depicted in Fig.5(a), the relative abundance of AOB and NOB suffered from a significant change by the variation in the concentration of FA, influencing the nitrogen removal. Surprisingly, we found that AOB activity was not inhibited, but enhanced at S10with the increasing concentration of FA. AOB first increased sharply from 0.13% in S0.5to 3.17% in S10and then decreased to 0.60 in S15, but NOB linearly decreased from 6.14% in S0.5to 0.96% in S15and had a significant negative relationship with concentrations of FA (y=-0.3x+5.3,R2=0.72). The reason for the entirely different trend could be that FA can serve as a matrix for AOB to increase its relative abundance when the concentration of FA is lower than 10 mg/L, but FA reached the threshold for inhibiting AOB and made its abundance decrease when the concentration of FA was higher than 10 mg/L. However, the inhibition threshold of NOB was 0.5 mgFA/L. Thus, there was a gradual decline in the abundance of NOB with the increasing concentration of FA. The results were in agreement with AOR and NAR under the four FA treatments and confirmed that NOB was even more sensitive to FA inhibition than AOB.

Furthermore,it was clear that that NOBNitrospirawas the dominant nitrifier (AOB/NOB<1) at FA below 5 mg/L, and led to complete oxidation of ammonia to nitrate. But AOBNitrosomonaswas the predominant nitrifier (AOB/NOB=2.03) at 10 mg FA/L, and resulted in sufficient oxidation of ammonia to nitrite. A stable nitrite pathway was maintained at 15 mg FA/L with higher AOR (0.073 mgN/gVSS·min) than NOR (0.017 mgN/gVSS·min) although the relative abundance of NOBNitrospira(0.95%) was higher than AOBNitrosomonas(0.6%) (AOB/NOB=0.63) (Fig. 5(a) and (b)). In other words, a higher abundance of NOBNitrospirahad lower activity of utilizing the nitrite substrate, indicating that the NOB activity was strongly inhibited in S15. However, AOBNitrosomonasexhibited the opposite trend. For example, the lower abundance had a higher microbial utilization capacity of the ammonia substrate. Thus, it was still able to make the AOR higher than the NOR when the AOB abundance was greater than that of the NOB and remain stable during the ammoxidation process. Similar inhibitory threshold levels of FA on NOB were reported by Sun et al.[32], who obtained a nitrite pathway of over 90% at 16.3 mgFA/L in the UASB-SBR system treating landfill leachate and intense oppression occurred of FA on NOB activity.

Fig.5 Distribution and relative abundance of AOB and NOB, AOB/NOB and NAR in

Based on the results, we can draw the conclusion that FA can affect not only the relative abundance of AOB and NOB, but also the NOB activity. However, AOR decreases with AOB abundance. Thus, we cannot conclude that AOB activity is inhibited at 15 mgFA/L. Moreover, by adjusting the concentration of FA, promoting AOB but suppressing NOB, stable short-range nitrification was successfully achieved.

3 Conclusions

The SBR is an efficient and stable reactor to remove ammonia from synthetic wastewater. Stable partial nitrification was successfully achieved in the SBR at high concentrations of FA (10 and 15 mgFA/L). Although AOB and NOB coexist in the four systems, AOB is still the main nitrifying bacteria. This finding emphasizes the importance of cultivating the appropriate bacteria to achieve short-range nitrification. FA can affect not only the relative abundance of AOB and NOB but also the activity of NOB. Furthermore, we found that a lower abundance of AOB had a higher microbial utilization capacity of ammonia substrate at 15 mgFA/L.

Acknowledgements

The authors would like to acknowledge the financial support from the National Natural Science Foundation of China (Grant No. 51668031).