Addition of anaerobic ammonium oxidation bacteria to lower running cost during the membrane bioreactor process treating sewage

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.

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.

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).

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.

Chemical oxygen demand (COD_Cr) 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).

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).

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

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

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.

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.

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.

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.