Effective restoration of partial nitritation and anammox biofilm process by short-term hydroxylamine dosing: Mechanism and microbial interaction
Graphical abstract
Introduction
One-stage partial nitritation and anammox (PN-A) process, known as a promising alternative for biological nitrogen removal from wastewater, is becoming the hottest in the last decade (Lackner et al., 2014, Li et al., 2021, Perez et al., 2020). Compared with conventional nitrogen removal processes, the PN-A process exhibits superiority, such as 62.5% less oxygen consumption, 100% less external carbon source demand, 90% less surplus sludge production and zero carbon emission (Lackner et al., 2014, Sui et al., 2020, Wang et al., 2015), making it energy-positive and environment-friendly wastewater treatment technology. Presently, as less land occupation, more simple operation and less substrate inhibition, more than 80% of the existing full-scale anammox-based installations are one-stage PN-A (Lackner et al., 2014). The one-stage PN-A process includes two autotrophic pathways, where ammonia oxidation bacteria (AOB) oxidize NH4+ to NO2– with oxygen, and anaerobic ammonium oxidation bacteria (AnAOB) convert the remaining NH4+ and NO2– generated by AOB to dinitrogen gas (N2). The hinges for application of PN-A process are maintaining efficient activity of AOB and AnAOB, keeping stable synergy between AOB and AnAOB, and suppressing unexpected nitrite oxidation bacteria (NOB) simultaneously (Sui et al., 2020, Wang et al., 2015).
Dissolved oxygen (DO) control is crucial for PN-A process, because DO not only acts as an indispensable electron acceptor for both AOB and undesirable NOB, but also reversibly inhibits AnAOB (Li et al., 2018). Therefore, limited DO concentrations and intermittent aeration are the most commonly used strategies to suppress NOB, due to lower DO affinity and longer recovery time from non-aeration phase for NOB than AOB (Lackner et al., 2014, Li et al., 2018). However, according to a survey of full-scale PN-A installations for treating side-stream wastewater, almost 30% of the surveyed plants experienced NH4+ accumulation due to AOB deterioration, and 50% of the surveyed plants experienced NO3– accumulation due to NOB build-up, lasting for several weeks generally even under DO control (Lackner et al., 2014). Accordingly, the frequent accumulation of nitrogen pollutants remains problem in PN-A systems for treating side-stream wastewater, let alone for the treatment of mainstream wastewater. As well known, the prerequisite to enhance the performance of one-stage PN-A process with NH4+ accumulation is to promote aeration rate, which will also risk NOB build-up as oxygen input increased (Qiu et al., 2020). Thus, except for oxygen control strategy, the study of effective restoration strategies is imperative for NOB inhibition as well as maintainence and enhancement of AOB and AnAOB.
Hydroxylamine (NH2OH) is known as a highly reactive compound as intermediate or side metabolite of AOB and AnAOB (Soler-Jofra et al., 2021, Zekker et al., 2012), as well as a strong inhibitor for NOB (Harper et al., 2009, Li et al., 2019b). Excellent nitritation performance could be achieved in 5 days by dosing 5 ∼ 10 mg N·L-1 NH2OH at high DO concentrations of 3 ∼ 5 mg·L-1 (Li et al., 2019b, Xu et al., 2012). Sui and his coworkers restored PN-A process through the addition of 2 mg N·L-1 NH2OH for 104 days at limited DO concentrations in an SBR, promoting nitrogen removal efficiency from 0% to 60% (Sui et al., 2020). The PN-A process deteriorated by NO3– build-up was rapidly restored in-situ through adding 20 mg NH2OH·L-1, and was maintained stable combining SRT control in an SBR (Wang et al., 2015). As reviewed, NH2OH is efficient in inhibiting NOB, not damaging but promoting AOB activity (Soler-Jofra et al., 2021). However, most of the studies concluded that inhibition of NOB by NH2OH was reversible (Li et al., 2019b, Soler-Jofra et al., 2021, Zhao et al., 2021). Therefore, stable partial nitritation could be maintained only if a proper operation strategy was implemented after stopping hydroxylamine dosing (Li et al., 2019a, Wang et al., 2015). As reported, adding NH2OH to a nitrification granular system led to disaggregation of granular structure (Harper et al., 2009). To our best knowledge, anammox based processes, especially one-stage PN-A processes, heavily rely on aggregate structure, i.e. granule, biofilm (Lackner et al., 2014, Li et al., 2018). Consequently, this definitely prevented any attempt at restoring PN-A biofilm process by NH2OH dosing (Soler-Jofra et al., 2021). Interestingly, batch tests indicated that external NH2OH could boost AnAOB activity in a biofilm system and showed no adverse effect on anammox biofilm morphology (Zekker et al., 2012). Recently, Soler-Jofra et al. proposed that long-term NH2OH addition showed no negative impact on AnAOB community (Soler-Jofra et al., 2020). Therefore, NH2OH addition is worth a shot in solving deterioration of PN-A biofilm process.
Recently, it was suggested that NH4+ is oxidized to NH2OH by ammonia monooxygenase (AMO), NH2OH to nitric oxide (NO) by hydroxylamine oxidoreductase (HAO) and NO to nitrite by uncharacterized enzyme (Caranto & Lancastera, 2018). Sui et al postulated that the essence of NOB repression might be NO production stimulated by NH2OH addition (Sui et al., 2020), since NO was reported to hamper NOB significantly (Courtens et al., 2015). Zhao et al observed that NO and nitrous oxide (N2O) increased dramatically with NH2OH addition of 5 mg N·L-1 in a nitrification sludge system and proposed that NOB might be essentially inhibited by NO (Zhao et al., 2021). In anammox metabolic process, nitrite is firstly converted to NO by nitrite oxidase (NIR) enzyme and then hydrazine (N2H4) is synthesized from NO and NH4+ catalyzed by hydrazine synthase (HZS), N2H4 is further converts to N2 by hydrazine dehydrogenase (HDH) (Kartal et al., 2013). Surprisingly, hydroxylamine oxidase (HOX), which is proposed to convert hydroxylamine to NO, is one of the most highly expressed enzymes in AnAOB (Hu et al., 2019). There are earlier references about hydroxylamine dosing and anammox activities. Batch tests with NH2OH addition demonstrated NH2OH anammox metabolism (Zekker et al., 2012). Recently, it is suggested that co-metabolisation of other substrates (NH4+ and NO2–) with NH2OH impacts the AnAOB metabolism, resulting less stoichiometric nitrate, and thermodynamic analysis showed that NO is unlikely to be the intermediate of NH2OH metabolism in anammox (Soler-Jofra et al., 2020). Besides, external NO and NH2OH have been both indicated to boost AnAOB or to facilitate its recovery from substrate inhibition (Zekker et al., 2012, Zekker et al., 2015) and to be metabolized by AnAOB (Hu et al., 2019, Soler-Jofra et al., 2020). Notably, the AnAOB in above studies is all referred to Candidatus Kuenenia. However, NO have been scarcely detected with NH2OH addition and vice versa. Overall, the impact mechanism of NH2OH addition on AnAOB, especially the potential role of NO as intermediate in NH2OH anammox pathway still needs to be clarified experimentally.
Therefore, the purpose of this study was to study short-term NH2OH dosing for restoring PN-A process in a sequencing batch biofilm reactor (SBBR) from NO3– build-up resulting from over aeration. Besides, batch tests were conducted to evaluate the activities of AOB, NOB and AnAOB, and SEM detections were conducted to analyze the biofilm morphology with NH2OH dosing. Additionally, NO and N2O changes were detected during typical cycles with and without NH2OH dosing to further uncover the metabolism interaction between AOB and AnAOB via NH2OH and NO.
Section snippets
Experiment set-up and synthetic wastewater
A lab-scale reactor made of polymethyl methacrylate with a working volume of 6 L (L) was used in this study, and the height-diameter was 40 cm to 15 cm. 7 pieces of ring-shaped biofilm carriers (10 cm in diameter) were regularly strung and fixed in the middle of the reactor. The reactor was jacketed with opaque plastic cloth to avoid light effect. The aeration rate was controlled at 0.1 L·min−1 with a rotameter while the DO concentration was 1.2 ± 0.4 mg·L-1 during aeration phase. The
Nitrogen removal performance during the whole experiment
The performance of the PN-A process was shown in Fig. 1. In stage I (day 1 to day 10), the reactor was operated under the same conditions as before and gave an average TNRE of 88.5 ± 1.5% with stable pH of 8.2 ± 0.2 in the effluent. The concentration of FA in influent was 7.2 ± 0.3 mg·L−1. In stage II, the TNRE declined to 67.0 ± 1.8% with NH4+-N residual of about 23.5% and the situation was scarcely improved as experiment went on, which was attributed to shorter reaction time and lower AOB
Conclusions
This study firstly investigated the feasibility of short-term NH2OH dosing combining with aeration control on restoration of PN-A biofilm process from NO3– build-up. Meanwhile, the impact mechanism of NH2OH dosing on the PN-A process was studied. The PN-A process could be restored in 5 days via NH2OH dosing, reducing ΔNO3--N/ΔNH4+-N from 28.5% to less than 11%. NH2OH dosing may essentially repress NOB via the toxicity of NO originated from NH2OH oxidation. NH2OH dosing reduced stoichiometric
CRediT authorship contribution statement
Ting Huang: Conceptualization, Project administration, Writing – original draft, Writing – review & editing. Jianqiang Zhao: Supervision, Funding acquisition, Writing – review & editing. Bo Hu: Writing – review & editing. Junkai Zhao: Investigation, Methodology. Chunbo Yuan: Investigation, Methodology.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (no. 51778057) and the Key Research and Development Program of Ningxia Hui Autonomous Region (no. 2019BFG02031).
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