Research progress of nitrogen removal by anaerobic ammoxidation combined process

. Anaerobic ammonia oxidation (ANAMMOX) is an efficient and low-fare biological nitrogen removal process. Nevertheless, there are still some challenges in mainstream request due to the limitations of substrate type and nitrate accumulation. At the present, anaerobic ammox combined process has been widely studied to solve the above issue. In this paper, the anammox combined processes developed in recent years are reviewed, and the process principles, pros and cons, influencing factors, process scalability, key bottlenecks in application and relevant work of the investigation group are discussed. Finally, the evolution of complex anammox process in urban domestic wastewater treatment was prospected.


Anaerobic ammonia oxidation, anammox
Excessive nitrogen input into water is an important factor causing eutrophication of water body, nitrogen removal is one of the basic objectives of sewage treatment plant.Traditional biological nitrogen removal process needs to consume a lot of energy and carbon sources, while releasing a lot of greenhouse gas nitrous oxide, which is inconsistent with the goal of high efficiency, low consumption and clean.Under Anaerobic conditions, Anaerobic ammonia oxidizing bacteria (AnAOB) use NH4 + -N as an electron donor and NO2 --N as an electron acceptor, eventually converting nitrogen to N2.Compared with the traditional nitrogen removal process, the anammox process is an autotrophic nitrogen removal process that does not require supplementary organic carbon sources and does not require aeration.
At present, the common combined processes for treating urban sewage based on Anammox include PNA and PDA (Fig. 1), and the research direction is mostly the influence of substrate type, functional bacteria and temperature on the treatment effect.The main forms of nitrogen in urban domestic sewage are organic nitrogen, NH4 + -N and a small amount of NO2 --N and NO3 -demand of anammox [1] .In addition, since AnAOB produces nitrates in the anabolic process (Formula 1), the theoretical nitrogen removal efficiency of anammox process can only reach 88%.Lackner's research shows that more than 50% of abnormal PN/A processes are caused by NOB over propagation.At present, the Anammox process has a solid research foundation under medium and low temperature environment, and the pilot study has achieved phased success [2] .However, the low temperature environment, the limitations of substrate types and the problems of nitrate and nitrogen accumulation restrict the wide application of anammox process.To solve the above problems, the combined process around anammox came into being and effectively solved these two problems.

Anaerobic ammox combined nitrogen removal process
Focusing on the two important issues, the researchers further improved and supplemented the anammox process by means of process combination and conducted an expanded study.Including Partial nitrification and anaerobic ammox (PNA) process, which has been widely studied and applied, this combined process effectively solves the substrate problem of anammox.On the other hand, anammox is also combined with short-range denitrification and methane-type denitrification processes, which effectively solve the problem of nitrate and nitrogen accumulation in anammox.Several common ANAMMOX combination processes are reviewed below.

Partial nitrification anammox, (PNA)
In 1998, the Dutch Jetten team first tried the PNA process research, and in 2002, the Netherlands Rotterdam sewage plant carried out large-scale application.The PNA process can save nearly 60% of oxygen supply and 100% of external carbon sources, and the sludge production is reduced by 80% [3] .The PNA process is suitable for the treatment of wastewater with high concentration of ammonia nitrogen under intermediate temperature condition, such as anaerobic digestion supernatant, so it is considered to be a mature process for the treatment of side-flow wastewater.At present, PNA process has also been applied [4] in the treatment of industrial wastewater with high ammonia nitrogen under higher temperature conditions.In 2012, Shen started anammox in the PNA treatment process of monosodium glutamate wastewater, and used actual sewage to culture and enrich AnAOB, which proved that granular sludge can significantly improve the biomass concentration of anammox in the reactor, providing a new idea for the engineering application of PNA process.However, when PNA process is applied in mainstream wastewater treatment engineering, there are still some problems [5] to be solved.Low temperature not only affects the growth and metabolism of functional microorganisms, but when the temperature is < 20 ℃, the growth rate of Nitrite-oxidizing bacteria (NOB) begins to exceed that of Ammonia-oxidizing bacteria (AOB).And the lower the temperature, the greater the difference, making it difficult to inhibit NOB at low temperatures.Qiu et [6] al summarized that when the temperature is about 25 ℃, Nitrogen removal efficiency (NRE) can reach 51 %-81 %; however, when the temperature is less than 15 ℃, NRE even drops to 25 % with unstable operation and poor effluent quality.Therefore, when the PNA process is operated at low temperature, the inhibition of NOB and the stable supply of NO2 --N are both challenges for its application in mainstream wastewater treatment.Currently, NOB [7] is usually inhibited by Dissolved oxygen (DO), dissolved oxygen (DO), sludge residence time and ammonia nitrogen, or a combination of various strategies.
PNA process can only remove the restriction of substrate type of anammox process, but with the increasingly strict wastewater discharge standards, PNA process still has the problem of effluent nitrogen substandard.To solve this problem, Yang Fenglin et al proposed in 2009 to couple the denitrification process with PNA to generate the simultaneous nitrification anammox and denitrification (SNAD) process.The mechanism is as follows: AOB partially oxidizes NH4 + -N into NO2 --N, and under the action of AnAOB, NH4 + -N and NO2 --N are converted into N2 and NO3 --N, and then the denitrifying bacteria uses organic matter as electron donor to transform NO3 --N into NO2 --N.Under the condition of sufficient electron donor, the final product of denitrification is only N2.This process not only has the advantages of PNA process, but also because it is carried out in one reactor, it occupies a small area and has a low cost of the capital construction.At present, SNAD is suitable for the treatment of low C/N and ammonia nitrogen wastewater, such as in the digestion of aquaculture wastewater, landfill leachate and photoelectric industry wastewater has a good treatment effect. [8]Some scholars conducted in-depth research.Du [9] used Integrated axed-bio wastewater lm activated sludge (IFAS) to run SNAD process to treat urban domestic sewage with high ammonia nitrogen, and the NRE reached 92.8 %.Meanwhile, the removal rate of Chemical oxygen demand (COD) was 78.8 %.Xu use anaerobic hydrolysi [10] and acidification coupled SNAD to strengthen biological phosphorus removal, adopt Upflow anaerobic sludge blanket (UASB)+IFAS reactor, and control total nitrogen, TN, Total phosphorus (TP) and COD removal rate reached 93.70 %, 97.71 %and 79.93 %, respectively.However, it should be pointed out that the overly complex microbial community structure and interspecific relationship in the system will bring challenges to the coordinated operation and long-term stable operation of the process.

Partial denitrification anammox, (PDA)
DO control is crucial in the SNAD process.Excessive DO will lead to the oxidation of NO2 --N to NO3 --N.Therefore, if NO2 --N can be directly generated in an anaerobic environment, it is not only economical but also more stable.In 2006, Sumino proposed PDA process, using short-range denitrification process to limit denitrification to NO2 --N conversion NO3 --N stage and combining anammox process, which not only has the advantages of SNAD but also simplifies the reaction process, and the whole process does not need aeration.Studies have shown that in the alternating anaerobic anoxic environment, short-range denitrifying bacteria lacking NO2 --N reductase dominate, and the ratio of NO2 --N conversion to NO3 --N can reach more than 80% [11] when organic matter is used as electron donor.It has been reported that with organic matter in domestic sewage as the electron donor, the NO3 --N conversion rate of short-range denitrification process is 88.0% [12] , and denitrifiers are more inclined to use NO-N as electron acceptor [13] , that is, nitrite production in PDA process is morestable than PNA.In addition, some studies have suggested that PDA can reduce aeration by 50% and organic matter dosage by 80% in the treatment [14] of urban sewage.Compared with the PNA process, the PDA process is no longer affected by NOB, followed by the PDA theory of nitrogen removal rate of up to 100%, and effectively reduce the emissions of greenhouse gases NO2, in addition to the PDA process on the temperature requirements are not high PNA, so it can also be applied in high latitudes [15] .However, suitable C/N is the key to achieve short-range denitrification [16] .When C/N is 4, AnAOB is inhibited, and when C/N is 2, AnAOB and denitrification bacteria can work well together.Ma Bin [17] adopted sequencing batch reactor (SBR), and the influent COD/TN remained at 2.28 with a TN removal rate of up to 92.6%.Some researchers run the PDA process through a reactor combined with an anammox carrier and a segmental anaerobic aerobic system to treat domestic wastewater with COD/TIN (total inorganic nitrogen) of around 2.5, and the nitrogen removal efficiency reaches 77.8%±4.3% [18].Ma, the use of biofilm reactor to treat C/N of 2.6, TN concentration of 60.5 mg/L of sewage, the final NRE reached 80 %±4 %.It can be seen that the PDA process plays a role in the treatment of wastewater with low C/N.

Denitrifying type methane oxidationanaerobic ammonia oxidation process
NO3 --N/NO2 --N dependent Denitrifying Anaerobic Methane Oxidation (DAMO) process has the ability to remove NO3 --N/NO2 --N and CH4 at the same time, and the final product is only N2 and CO2.Is a potential green biological nitrogen removal technology.Zhu demonstrated in 2011 that the combined process of DAMO and anammox is feasible, and when the ratio of NO2 --N to NH4 + -N is greater than 1.32, ammonia nitrogen and methanecan be removed simultaneously.Ettwig confirmed that DAMO bacteria can reduce NO2 --N to nitric oxide (NO), then convert to N2 and O2, and finally use O2 to oxidize CH4 to CO2 (Formula 2).Haroon subsequently confirmed that DAMO archaea oxidizes methane through reverse met factor genesis and use the electrons provided by methane for the reduction of NO2 --N to NO-N (Formula 3).Because DAMO archaea can reduce the nitrate and nitrogen produced by AnAOB to NO2 --N, on the other hand, due to the presence of DAMO bacteria, NO2 --N can be consumed, so NH4 + -N and NO2 --N do not need to be added strictly according to thestoichiometric ratio of anammox, which reduces the restriction of substrate type to a certain extent.However, the slow growth rate of DAMO functional bacteria is an important limiting factor of this combined process.The author's research group improved the performance of DAMO process through the medium with improved mineral nutrient concentration and pH control, and also studied the effects of growth factors and trace metal elements on the activity of DAMO microorganisms When Fe(Ⅱ) concentration is 20 μmol/L, it can promote the activity of DAMO.In addition, the research group also enhanced the CH4 mass transfer by optimizing the reactor configuration, and proposed to retain biomass by changing the biological aggregation form such as membrane components.The mass transfer efficiency of CH4 is also an important limiting factor for this combined process.At present, with the development of Membrane materials, the bubble-free breathable fiber membrane in Membrane biofilm reactor (MBfR) significantly improves CH4 mass transfer, and the membrane as a carrier for microbial attachment ensures the retention of microorganisms.
Some researchers found that MBfR significantly improved the activity of DAMO archaea and AnAOB, and the nitrogen removal rate reached 6.1 kg/(m 3 • d).Cai combined AnAOB and DAMO microorganisms, and selected MBfR, NO2 --N and NH4 + -N for nitrogen removal rates of 330 mg/(L• d) and 268 mg/(L• d), respectively.Xie coupled the anaerobic ammoxidation and DAMO processes based on MBfR and selected permeable hollow fiber membrane to transport methane with a nitrogen removal rate of 280 mg/(m 3 • d).Through stoichiometric calculation, the contribution of AnAOB and DAMO bacteria to the removal of NO2 --N was 90 % and 10 %, respectively.When the ratio of NO2 --N to NH4 + -N varied in the range of 1.22--1.55,the removal rates of both NH4 + -N and NO2 --N could reach 96%.The above cases indicate that the coupling of denitrifying methane oxidation and anaerobic ammox process has strong potential in wastewater nitrogen removal.Peng selected two membrane granular sludge reactors with different operating conditions for modeling, and proposed that shorter hydraulic residence time, such as 4.8h, smaller particle size and lower NO2 --N/NH4 + -N, could reduce the operating cost of denit rifying methane oxidation-anaerobic ammox process.

Coupling of anaerobic ammoxidation and new denitrification process
In 2006, Mulder proposed a Sulfide driven partial denitrification and anammox (SPDA) technology.Sulfur oxidizing bacteria (SOB) are used to reduce NO2 --N to NO3 --N by oxidizing sulfide, thus providing an available substrate for AnAOB.Due to the low biomass yield (0.15-0.57g biomass/gNO3 --N) and growth rate (0.04-0.27/h) of SOB, this process does not compete with AnAOB.With sulfur elemental (S) and S2O2 3-as electron donors, SOB can provide stable NO2 --N for AnAOB and reduce the inhibition of AnAOB by additional organic matter.Although the sulfide has an inhibitory effect on the activity of AnAOB, working together with the SOB creates a low toxic environment for AnAOB.Li et al and Chen et al successfully started the SPDA process with sulfur elemental or thiosulfate as electron donor.Qin studied the SPDA process and found that the sulfide was completely oxidized to sulfate in the system.Deng used a UASB reactor to run the SPDA process and remove sulfide and nitrogen at the same time.The nitrogen load was maintained at 0.36 kg/(m 3 • d), and the final NRE reached 79.6%±3.0%.It can be seen that SPDA is feasible in wastewater treatment.
In 2006, Sawayama proved that in the process of Iron reducing ammonium oxidation (Feammox), Fe(Ⅲ) reducing bacteria could use Fe(Ⅲ) to oxidize ammonium ions to NO2 -, NO3 -and N2.This concept was later introduced into sewage treatment.In 2018, it was reported that NH4 + could be significantly reduced by adding trivalent Iron oxides to anaerobic digesters.It was analyzed that Feammox could reduce Fe(Ⅲ) by reducing bacteria, IRB) play a key role.The reaction process can be explained by equation (4-6), which is favorable for iron reduction under acidic conditions.On the other hand, nitrate-dependent iron oxidation (NDFO) can oxidize Fe(Ⅱ) to Fe(Ⅲ) by NO3 -and produce N2 (formula 7), while non-biological denitrification exists to convert Fe(Ⅱ) and NO2 -to N2 and Fe(Ⅲ) (formula 8).Then Feammox, NDFO and non-biological denitrification are coupled together, Fe(Ⅱ) and Fe(Ⅲ) can be recycled, and anaerobic ammoox idation can also be realized without AnAOB.Can also be Feammox, NDFO and anammox process combined, the use of trivalent iron to oxidize ammonia nitrogen to nitrous nitrogen, to solve the problem of anammox substrate type limitation, while the use of iron bivalent anammox accumulation of nitrate nitrogen reduction, to solve the problem of nitrate nitrogen accumulation.

Discussion
Although anaerobic ammox process is already a mature technology, it still faces some challenges in the mainstream application.For example, seasonal temperature changes put higher requirements on the growth and activity of anaerobic ammox bacteria, and more and more stringent nitrogen emission requirements require the anaerobic ammox process to develop in the direction of deep nitrogen removal.
Strengthening the activity of functional flora of anammox process by various means is still a hot research topic in the water treatment industry.At present, external electric and magnetic fields are used to strengthen AnAOB activity, and chemical substances are added to strengthen AnAOB operation.All of the above promoting good effect on anammox.In addition, some studies have suggested that the physiological properties of anammox are controlled by quorum sensing, and Nacyl homoserine lactone signaling molecules have significance of positive regulation on the anammox process.Therefore, strengthening of exogenous signaling molecules and synthesis of engineered bacteria can be used as a means to strengthen anammox and improve its applicability.When the anammox process was enhanced by regulating the concentration of trace metals, the performance of the anammox process could be effectively improved by enhancing the electron transfer between cells, and it was found that the addition of iron carrier could increase the nitrogen removal rate of anammox process by about 50 % (the data was not published).

Conclusion
The reactor configuration and operation were optimized by our research group in conclusion, the anammox process can effectively remove nitrogen from lowconcentration nitrogen-containing wastewater (<10 mg/L), and it is expected to realize the application of anammox process in mainstream wastewater.However, the problem of substrate type restriction and nitrate accumulation can not be completely eliminated by strengthening the ANAMMOX process.Therefore, the combined ANAMMOX process brings new opportunities for sewage treatment, and more process expansion combinations can become the carrier to explore the sustainable development of sewage treatment technology in China.Wu use Anaerobic baffled reactor (ABR)+complete mixed anaerobic reaction (completely mixed anaerobic reaction, CSTR developed a Denitrifying phosphorus removal (DPR) coupled PDA energy saving process, TIN and total inorganic phosphorus removal rate as high as 97.57% and 95.66%.Du used the two-stage nitrifying coupled PDA process, and the NRE reached 93.7%.Ji used the synergistic partial denitrification anammox, and in situ fermentation, SPDAF (Synergistic partial denitrification, anammox, and in situ fermentation, SPDAF) process to treat the actual domestic sewage adding nitrate.Under the condition of no external carbon source and aeration, the NRE could reach 93.1 %.It can be seen that how to make up for each other's defects and cooperate with each other through process expansion and combination in practice is the future research direction of sustainable development of sewage treatment technology.However, it should be noted that the combined process brings more complex microbial community composition and process parameters, so it is still challenging to avoid the competitive disadvantage of AnAOB and realize the long-term stable operation of the combined process.If the microbial community system of the anammox combined process can be constructed by establishing intercellular communication and stable and controllable ANAMMOX combined process.It is expected to achieve the design goal of anammox combined process operation and achieve long-term stable operation.

Fig 1
Fig 1 PNA and PDA Nitrogen removal pathway