Reducing energy consumption or running cost associated with the membrane bioreactor (MBR) process is a serious challenge that needs to be addressed in treating sewage. The addition of anaerobic ammonium oxidation bacteria (AnAOB) to a running MBR has the potential to lower the aeration rate, thus decreasing the running cost in treating sewage. The results obtained showed that owing to addition of AnAOB, TN and NH4+-N removal rates increased by 9.8% and 1.13%, respectively, while the aeration rate decreased by 50%. Additionally, high throughput sequencing and isotope experiments showed that both AnAOB and heterotrophic denitrification bacteria could survive simultaneously and play an important role in nitrogen removal, with AnAOB having a significantly greater contribution. It can be concluded that the addition of AnAOB reduced the running cost of MBR in treating sewage.

  • TN and NH4+-N removal rates increased by 9.8% and 1.13% with AnAOB addition;

  • AnAOB addition resulted in a decrease in the aeration rate by 50%;

  • Both AnAOB and HDB play an important role in nitrogen removal;

  • AnAOB contributed more significantly to nitrogen removal than HDB.

Graphical Abstract

Graphical Abstract
Graphical Abstract

Membrane bioreactors (MBR) are effective for wastewater treatment as they have good effluent quality and high-efficiency interception and can ensure high concentrations of biological species within the reactor. However, MBR is prone to membrane fouling (Meng et al. 2009); thus, it becomes necessary for the membrane surfaces to be cleaned to alleviate the membrane fouling and maintain the membrane flux. This causes the MBR process to become very energy-intensive, resulting in an increased cost of sewage treatment. Therefore, reducing energy consumption or operation cost associated with the MBR process is a serious challenge that needs to be addressed.

The anaerobic ammonia oxidation (anammox) process removes nitrogen via the use of anaerobic ammonia-oxidizing bacteria (AnAOB). AnAOB can convert NH4+-N and NO2-N into N2 under anoxic conditions and this reduces the amount of aeration needed in engineering applications and the daily operation cost (Kartal et al. 2010). Nevertheless, AnAOB grows slowly with a doubling time of 2.1–79 days (Laureni et al. 2015; Zhang et al. 2017a), and maintaining its concentration and activity in the reactor remains a challenge. Yet, MBR has the ability to cultivate and cumulate the AnAOB in the reactor (van der Star et al. 2008). Therefore, adding AnAOB into the MBR process is expected to be a win-win result of enriching bacteria and reducing cost simultaneously. Zhao et al. (2018) developed a process that realized the simultaneous partial nitrification, anammox and denitrification (SNAD) process in MBRs and confirmed the coexistence of ammonia-oxidizing bacteria (AOB), AnAOB and heterotrophic denitrification bacteria (HDB). However, to the best of our knowledge, there is no report concerning the addition of AnAOB into MBR during the sewage treatment process. Thus, after addition of AnAOB, the balance between AnAOB and other bacteria is currently unknown.

Based on the above considerations, in this study, the effect of the addition of AnAOB on effluent quality, aeration reduction, and energy saving during the MBR sewage treatment process was investigated. Additionally, to determine the contributions of AnAOB and HDB to nitrogen removal, so as to clarify the nitrogen removal mechanism, high-flux sequencing and the 15N isotope tracer method were used.

Experimental setup

As shown in Figure 1, the MBR process consists of an anoxic tank and an oxygen- limited tank. In this study, the anoxic tank had an effective volume of 5.5 L and external size of 18 cm × 10 cm × 50 cm. The oxygen-limited tank had an effective volume of 4 L and external size of 9 cm × 9 cm × 75 cm. The anoxic tank was connected with the oxygen-limited tank with a pipe. The influent was pumped into the anoxic tank with an influent pump, and then entered the oxygen-limited tank. A recycle pump with twice the flow rate of the influent pump was used to recycle the water from the oxygen-limited tank to the anoxic tank. In the anoxic reactor, an agitator was used for mixing. Additionally, a pH meter (PHB-3, Sanxin Instrument Factory, China) was put in place to monitor pH and temperature changes, and the DO was measured using a portable DO meter (HQ30D, HACH, USA). The DO was controlled below 0.5 mg/L by adjusting the aeration flow.

Figure 1

Schematic of MBR process. (T: temperature online monitoring devices; DO: DO control system; pH: pH online monitoring devices; R: float water level controller; ORP: oxidation reduction potential).

Figure 1

Schematic of MBR process. (T: temperature online monitoring devices; DO: DO control system; pH: pH online monitoring devices; R: float water level controller; ORP: oxidation reduction potential).

Close modal

Activated sludge (AS) and AnAOB were added as the seed sludge. 4 L AS with mixed-liquor suspended solids (MLSS) of 3,500 mg/L was obtained from a SBR tank of a sewage treatment plant in Yanshan, Guilin, P.R. China. AnAOB (600 mL) with an activity of 0.15 kg NH4+-N/(m3·d) was collected from a running expanded granular sludge bed reactor (Jin et al. 2016). The hydraulic retention time was set at 3.5 h according to a previous study (Zhang et al. 2017b). During operation, the sludge produced is negligible. The influent was collected from Yanshan campus of Guilin University of Technology, P.R. China. Sewage mainly includes domestic water from student dormitories, canteen wastewater and teaching area wastewater. The detailed influent parameters can be found in our previously published paper as shown in Table 1 (Zhang et al. 2017c). Influent nitrogen loading rate (NLR) is 0.49 kg N/(m3·d), nitrogen removal rate (NRR) is 0.18 kg N/(m3·d).

Table 1

Wastewater quality characteristics at GUT

ParameterUnitInfluent
COD mg/L 101.2–521.0 
TN mg/L 47.4–122.7 
TP mg/L 3.6–11.3 
NH4+-N mg/L 32.7–82.54 
pH  6.34–7.56 
SS mg/L 37.7–120 
ParameterUnitInfluent
COD mg/L 101.2–521.0 
TN mg/L 47.4–122.7 
TP mg/L 3.6–11.3 
NH4+-N mg/L 32.7–82.54 
pH  6.34–7.56 
SS mg/L 37.7–120 

A flat-sheet ceramic membrane (Weifang Ruicheng Huanbao, China) (Wei et al. 2020) was placed within the anoxic reactor, which had a size of 20 cm × 10 cm. Transmembrane pressure (TMP) was monitored using a programmable logic controller (Weifang Ruicheng Huanbao, China). In order to alleviate the membrane fouling, 1,000 mg/L NaClO was pumped into the flat-sheet ceramic membrane for 2 h every week. When the TMP was above 25 kPa, the flat-sheet ceramic membrane was removed and cleaned with soft brush. During the study, the membrane flux could be maintained at a stable value of 33 L/(m2·h) with the above mentioned strategy.

Analytical methods

Water analysis

Chemical oxygen demand (CODCr) and MLSS were measured according to (Zhang et al. 2017c). Total nitrogen (TN), NH4+, NO2, and NO3 were determined by the methods described by Zhang et al. (2018).

High-throughput sequencing analysis

Samples for high-throughput sequencing analysis were collected from the sludge in the anaerobic and anoxic tanks on day 240. The sludge samples were sealed in a centrifuge tube and stored in a refrigerator at −20 °C. The microbial analysis was performed according to Liu et al. (2020).

15N Isotope experiment

The 15N stable isotope tracer method was used to calculate the contribution rate of nitrogen removal by anammox and denitrification, and to distinguish two different nitrogen removal pathways. The sludge samples were obtained on day 240. It is the most stable time point for reactor operation. Headspace sampling bottles (Labco Exetainer, Lampeter, UK) were used to inoculate the sludge. After centrifugation, the sludge was added into the purified water with a ratio of 1:5, which was blown-off by helium for 20 min. In total, 45 headspace bottles were used for parallel and controlled experiments. Before adding the 15N marker, the headspace bottles were incubated at 25 °C for 48 h to allow for the consumption of residual oxygen and NOx in the sludge. A 1-mL syringe was used to inject 15N solution into the headspace bottles. The headspace bottles were then put into the rotary culture mixer. The 15N concentration was 100 μmol/L. At 0, 2, 4, 6, and 8 h, three parallel headspace bottles were taken from each group, and 200 μL ZnCl2 with a concentration of 7 mol/L was added. A total of 45 samples were obtained. All samples were stored at 4 °C for quantitative determination of 28N2, 29N2 and 30N2 by continuous flow isotope ratio mass spectrometry (MAT253 with Gasbench II and autosampler (GC-PAL), Bremen, Thermo Electron Corporation, Finnigan, Germany) (Thamdrup & Dalsgaard 2002).
formula
(1)
formula
(2)
The labeled target substance (containing 15N marker), mainly 15NO3-N or 15NH4+-N, was added into the solution. The 15N labeled substance was biodegraded into the final product N2 with or without 15N. As shown in Equations (1) and (2), for HDB and AnAOB, N2 would be produced in the form of 28N2, 29N2 and 30N2 if 15NH4+ or 15NO3 was added. Therefore, the headspace bottles were divided into three groups: E-0, E-A, and E-D. Among them, E-0 was the negative control group, and 15NH4+ was added. It was used to test whether the residual oxygen and NOx of the sludge were exhausted. E-A was used as positive group, and 14NO3 and 15NH4+ were added to detect the existence of AnAOB. E-D was the calculating group, and 15NO3 was added. The contribution of nitrogen removal by AnAOB and HDB could be calculated by using the following formulate (Thamdrup & Dalsgaard 2002).
formula
where FN is the ratio of 15N in 15NO3, (NO3)b:(NO3)a is the ratio of 15N before and after adding 15NO3.
The anammox process in groups E-D produced only 28N2 and 29N2. when AnAOBs are consuming: (1) 14NO3 (1-FN) plus 14NH4+ are identified as A28 with 28N2 as a product; (2) 14NH4+ +15NO3 (FN), as A29 with 29N2 product.
formula
where A28, A29:28N2, and 29N2 is the production from AnAOB, μmol/L.
The N2 produced by denitrification is all from NO3, and the N2 produced by denitrification in groups E-D should include 28N2, 29N2 and 30N2. When HDB are using: (1) two 14NO3 (1-FN) are identified as D28 that produce 28N2; (2) 14NO3 (1-FN) plus 15NO3 (FN), as D29 with 29N2 as product; (3) 15NO3 (FN), as D30 with product 30N2.
formula
formula
formula

where D28, D29, D30:28N2, 29N2, 30N2 represent production from HDB, μmol/L.

The respective contribution of denitrification and anammox to nitrogen removal (Rd% and Ra%) can be calculated from the following equations.
formula
formula
formula
formula
formula
formula
formula

where P29, P30:28N2, 29N2 was measured by membrane injection mass spectrometry, μmol/L; DT and AT: represent the total N2 production from HDB and AnAOB.

Reactor performance

The MBR reactor was operated for 240 days. Before adding AnAOB, the MBR was maintained for 60 days as described by Wenjie et al. (Zhang et al. 2017c). On the 60th day, granular activated sludge with high activity and suitable for the quality of wastewater to be treated can be cultivated. At this stage (60 days), the anammox reactor was successfully started, and the average removal efficiencies of TN and NH4+-N were 61.88% and 85.4%. The AnAOB was added on day 61, and the MBR was operated for the following 180 days. During the study, no matter how the influent concentration changed, the treatment performance was stable. In stage I (start-up phase), the concentration range of influent COD is 120.96–154.56 mg/L, the concentration range of influent TN is 70.16–123.25 mg/L, and the concentration range of influent NH4+-N is 42.5–85.59 mg/L. In stage II (stable operation phase), the concentration range of influent COD is 110.88–161.28 mg/L, the concentration range of influent TN is 87.31–117.39 mg/L, and the concentration range of influent NH4+-N is 63.90–85.13 mg/L. The changes of influent COD and nitrogen concentration were not obvious at the whole process of MBR. After stable operation, the effluent COD, NH4+-N, and TN were below 40 mg/L, 4 mg/L, and 15 mg/L, which meets the strict class A of pollutant discharge standard for sewage treatment plants of China (GB18918–2002). As shown in Figure 2, adding AnAOB could significantly improve the nitrogen removal efficiency, and the average removal efficiencies of TN and NH4+-N increased by 9.8% and 1.13%. Moreover, in order to avoid adverse effects on AnAOB, the DO concentration was decreased to below 0.5 mg/L. Owing to a decrease in DO concentration, the aeration rate was reduced by 50%. This indicates that adding AnAOB could save the energy consumption of the blower, implying a significant decrease in the running cost.

Figure 2

Changes in nitrogen removal rate, removal efficiency, and dissolved oxygen content at each stage of the MBR process (Stage I, no added AnAOB from day 1 to day 60; Stage II, added AnAOB from day 61 to day 240).

Figure 2

Changes in nitrogen removal rate, removal efficiency, and dissolved oxygen content at each stage of the MBR process (Stage I, no added AnAOB from day 1 to day 60; Stage II, added AnAOB from day 61 to day 240).

Close modal

15N Stable isotope

As shown in Figure 3, the average nitrogen removal rate of AnAOB was 31.2 μmol N/h, whereas that associated with HDB was 15.6 μmol N/h. As N2 was produced by AnAOB and HDB at different reaction times, the contribution of AnAOB to nitrogen removal in this study was calculated as 60.11–64.93%, whereas that of HDB was 30.17–39.89%. The results show that after the addition of AnAOB, the anammox process was apparent in the MBR, and the anammox process could successfully remove nitrogen. And it has the same high nitrogen removal contribution of anammox as the result of Wang et al. (2020), with 64.7%, but possesses a lower energy consumption in comparison due to the control of DO below 0.5 mg/L in this study.

Figure 3

Variation of 29N2 and 30N2 production with time in groups E-D. (E-D29, 29N2 production from AnAOB with added 15NO3; E-D30, 30N2 production from HDB with added 15NO3).

Figure 3

Variation of 29N2 and 30N2 production with time in groups E-D. (E-D29, 29N2 production from AnAOB with added 15NO3; E-D30, 30N2 production from HDB with added 15NO3).

Close modal

Changes in microbial population in the reactor

As shown in Table 2, the sludges from the anoxic tank and the oxygen-limited tank were similar in microbial diversity. Nitrospira and Nitrosomonas were the detected AOBs. AOBs produce nitrite as an electron acceptor for AnAOB. The AnAOB strain was identified as Candidatus Kuenenia, which was also detected after the addition of AnAOB. Experimental data showed that the AnAOB could survive in the MBR together with HDB, and both of them played important roles in nitrogen removal in this study. Similarly, Lv et al. (2020) also proposed that the coexistence system of AnAOB and heterotrophic bacteria (HB) is important in the application of anammox process.

Table 2

Genus level community structure of sludge samples

SampleStrainsContent (%)SampleStrainsContent (%)
H1 Acinetobacter 17.68 Y1 Acinetobacter 16.74 
Gp 10 3.77 Aridibacter 5.00 
Aridibacter 3.66 Nitrospira 2.65 
Nitrospira 2.29 Gp 10 2.68 
Armatimonadetes-gp5 1.61 Paenisporosrcina 2.01 
Ignavibacterium 1,51 Ignavibacterium 1.8 
Candidatus Kuenenia 1.35 Candidatus Kuenenia 1.77 
Ferruginibacter 1.33 Armatimonadetes-gp5 1.64 
Zavarzinella 1.24 Zavarzinella 1.64 
Thermogutta 1.2 Thermogutta 1.34 
SampleStrainsContent (%)SampleStrainsContent (%)
H1 Acinetobacter 17.68 Y1 Acinetobacter 16.74 
Gp 10 3.77 Aridibacter 5.00 
Aridibacter 3.66 Nitrospira 2.65 
Nitrospira 2.29 Gp 10 2.68 
Armatimonadetes-gp5 1.61 Paenisporosrcina 2.01 
Ignavibacterium 1,51 Ignavibacterium 1.8 
Candidatus Kuenenia 1.35 Candidatus Kuenenia 1.77 
Ferruginibacter 1.33 Armatimonadetes-gp5 1.64 
Zavarzinella 1.24 Zavarzinella 1.64 
Thermogutta 1.2 Thermogutta 1.34 

This study has shown that the addition of AnAOB could be a new step in the MBR sewage treatment. The new process can save running costs and has the potential for significant benefit returns; therefore, future research should focus on optimizing the operating conditions in consideration with environmental variables, such as temperature. Zhou et al. (2021) examined the effect of seasonal temperature fluctuations on the denitrification performance of simultaneous anammox and denitrification (SAD), where low temperatures inhibit the activity of AnAOB and 15 °C is the critical temperature for denitrification and carbon removal. Wang et al. (2021) investigated the denitrification performance of the anaerobic ammonia oxidation process under rapid cooling conditions by macrogenomics and macroproteomics, and the total AnAOB population abundance (20.9% ± 4.9%) and AnAOB protein abundance (75.7% ± 3.3%) remained stable when lowered from 35 °C to 15 °C, but Brocadia was expressed to a lesser extent at lower temperatures, suggesting that metabolism of AnAOB is more responsive to low temperature compared to heterotrophs. The new process applied to actual wastewater at normal or low temperatures is an important developmental direction. The new process can be maintained by increasing the AnAOB populations or the low-temperature acclimation to screen out dominant strains that can adapt to the environment.

By adding AnAOB, TN and NH4+-N removal rates increased by 9.8% and 1.13% in the MBR. The addition of AnAOB resulted in a decrease in the aeration rate by 50%. Thus, energy consumption during the study decreased. The result of isotope experiments and high-throughput sequencing showed that anammox and denitrification occurred simultaneously during the stable stage of the MBR process. The stability of the operation process as well as the high nitrogen removal efficiency shows that the combined system evaluated in this study has significant avenues for future research and potential future applications in sewage water treatment.

This research was supported by the Guangxi Natural Science Foundation [grant number 2019GXNSFFA245017].

Conflict of Interest: The authors declare no conflict of interest.

Wenjie Zhang: conceptualization, funding acquisition, project administration, resources, methodology, supervision. Ronglin Sun: writing – review and editing data curation, investigation, formal analysis, software, validation, visualization, roles/writing – original draft.

All relevant data are included in the paper or its Supplementary Information.

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