Effects of biomass and environmental factors on nitrogen removal performance and community structure of an anammox immobilized filler

Journal Pre-proofs Effects of biomass and environmental factors on nitrogen removal performance and community structure of an anammox immobilized filler XiaoTong Wang, Hong Yang PII: DOI: Reference:

S0048-9697(19)35250-7 https://doi.org/10.1016/j.scitotenv.2019.135258 STOTEN 135258

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Science of the Total Environment

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23 September 2019 25 October 2019 27 October 2019

Please cite this article as: X. Wang, H. Yang, Effects of biomass and environmental factors on nitrogen removal performance and community structure of an anammox immobilized filler, Science of the Total Environment (2019), doi: https://doi.org/10.1016/j.scitotenv.2019.135258

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Effects of biomass and environmental factors on nitrogen removal performance and community structure of an anammox immobilized filler

Effects of biomass and environmental factors on nitrogen removal performance and community structure of an anammox immobilized filler XiaoTong Wang, Hong Yang* Key Laboratory of Beijing for Water Quality Science and Water Environmental Recovery Engineering，College of Architectural Engineering，Beijing University of Technology，Beijing 100124，China

Abstract In order to reduce the loss of anaerobic ammonia oxidation (anammox) sludge and stabilize the reaction microenvironment, polyvinyl alcohol - polypropylene (PVA-PP) was used to encapsulate anammox bacteria on a filler. The influence of different inoculation amounts (2, 4, 6 and 8%) on the overall nitrogen removal process was first compared and then the anammox characteristics of the immobilized filler under the influence of different environmental factors were evaluated through batch experiments. The results show that the biomass only affected the growth rate of the activity during the logarithmic phase, while the total nitrogen removal rate (NRR) tended to be similar after 99 d of culture. The NRR reached 1.83 kg·(m3·d)-1 on day 140, which was 9.4 times that of suspended sludge before encapsulation, and the structure of embedding filler was complete without shedding. Scanning electron microscopy (SEM) showed

that the internal porous network structure formed channels and a large number of anammox bacteria were observed around. Microbial community analysis of the 16S rDNA gene showed that the diversity was maintained in the entrapped carrier. Furthermore, the effective enrichment of the anammox functional bacteria Candidatus Kuenenia (AF375995.1) increased from 11.06% to 32.55%. The PVA-PP immobilized filler fit well with the biological nitrogen removal kinetic model and could also achieve coupling of anammox and denitrification. The inhibition effect of the organic carbon source interference and starvation on anammox bacteria was significantly weakened.

Keywords:

Anaerobic ammonia oxidation (anammox); Polyvinyl alcohol-polypropylene

(PVA-PP); Immobilized filler; Nitrogen removal; Microbial community structure

1.Introduction In the last few decades, many new nitrogen transformation processes have been identified in nature, such as heterotrophic nitification (Domenic et al., 1984; Mark et al., 1985), aerobic denitrification (Robertson et al., 1990) and anaerobic ammonia oxidation (anammox) (Mulder et al., 1995). Among these new research directions, anammox technologies have a good application prospect because of their many advantages, including a simple process, low energy requirements and lack of secondary pollution. Studies have shown that anammox bacteria are active at high cell concentrations (Zhang et al., 2004). Therefore, obtaining a sufficient number of anammox bacteria is important for achieving and maintaining stable anammox. However, due to the low cell yield coefficient, the accumulation of biomass is very slow and the microbial activity is easily inhibited by environmental factors (Hu et al., 2002). Furthermore, it is very difficult to enrich and cultivate

anammox bacteria (Wang et al., 2012; Tang et al., 2013). The use of microbial encapsulating immobilization technologies to maintain biomass for the anammox reaction system is a positive solution to the problem (Lu et al., 1996; Qiao et al., 2010). Compared to the use of free microorganisms, entrapped and immobilization technologies enhance the retention of biomass using simple solid-liquid separation to prevent scouring (Chen et al., 1996; Hsia et al., 2008). This promotes a higher microbial density in a fixed area, thus improving the reaction speed and reducing or eliminating the occurrence of side reactions (Mao and Wang, 2013). Cell immobilization technologies have been successfully applied to anammox bacteria (Ali et al., 2014, Ali et al., 2015b; Isaka et al., 2007, 2008, 2013; Zhang et al., 2017). Polyvinyl alcohol (PVA) has been widely used for cell immobilization (Hsia et al., 2008; Chou et al., 2012) because it is relatively inexpensive, has good tensile strength and is harmless to microorganisms. Polyvinyl alcohol - sodium alginate (PVA-SA) gel beads have often been used for anammox enrichment (Ali et al.，2015a; Chen and Lin, 1994; Bae et al.，2017). Zhang et al. (Zhang et al.，2017) showed that anammox bacteria can grow rapidly in PVA (6% w/w) -SA (2% w/w) gel beads, with full retention of the new cells. However, during the preparation of PVA-SA, the dense layer formed on the surface caused accumulation of gas generated by microorganisms, resulting in decreased permeability and a tendency for gel expansion (Chen et al., 1996; Chen and Houng, 1997). Moreover, PVA-SA gel beads are not durable and they break after being used at 37℃ for 1-2 months (Zhang et al., 2017). Therefore, maintaining the long-term structural integrity of the immobilized carrier is a bottleneck for its further industrial application. The biomass concentration and microbial community succession of encapsulated anammox bacteria are also worthy of attention. Ali et al. (Ali et al., 2015b) determined that the minimum concentration of anammox

biomass seeding needed to achieve rapid start-up of the anammox process was 0.33 g of volatile suspended solids (VSS) ·L-1. Kyungjin (Kyungjin et al., 2018) suggested that, compared with the provenance type, the operating condition (high nitrogen loading rate) is an important factor for determining the final ecological niche of the encapsulated anammox community. However, the effects of different biomass ratios on the enrichment and stabilization process of entrapped anammox bacteria, as well as the changes associated with long-term operation of immobilized carriers, are not clear. In this study, the improved method of polyvinyl alcohol-boric acid crosslinking was adopted (Yang et al., 2014) with the addition of a polypropylene carrier to improve the stability of the filler in order to cultivate encapsulated anammox bacteria with initially activity. The main objectives of this study were to (1) compare nitrogen removal of the anammox immobilized fillers with different biomass from the start-up stage to the stable stage, and study the differences in physiological activity of entrapped anammox sludge and suspended growth state; (2) verify the long-term stability of the PVA-PP entrapped anammox system using high-throughput sequencing and real-time quantitative qPCR to analyze the niche changes of bacteria in the reaction system at the beginning and end of immobilization; and (3) evaluate the processing capacity of immobilized fillers under different environmental factors.

2. Materials and methods 2.1. Seed anammox sludge Anammox sludge (MLSS: 10,035 mg·L-1, VSS/SS: 80%) was obtained from a laboratory-scale anaerobic sequential batch bioreactor. The NRR was 0.58 kg·N·(m3·d)-1 and the removal efficiencies of NH4+–N and NO2 － –N were above 90%. Part of the anammox sludge was

collected and washed three times in KHCO3 buffer (0.1 M; pH 7.4) to remove impurities. Partially diluted sludge (MLSS: 3,035 mg·L-1, VSS/SS: 79%) was placed in a 500 mL conical flask for comparison. The other part of the sludge was centrifuged (3 min, 2500 rpm) to prepare gel encapsulation.

2.2. Immobilization technique The materials used to prepare the immobilized fillers included calcium carbonate (CaCO3); powdered activated carbon (less than 120 mesh); anhydrous sodium sulfate (Na2SO4); boric acid (H3BO4) and PVA (degree of polymerization 2200, degree of alcoholysis 20 ～ 99%). All the above materials were of analytical purity. PVA powder was dissolved in 95℃ water and mixed to form a 20% (w/w) PVA solution. After cooling to 37℃, the PVA solution was mixed with anammox sludge with 95% water content after concentration. The mixture was divided into four groups with the sludge concentration (dry weight) accounting for 2, 4, 6 and 8% (w/w) of the total mixture, respectively. Then, CaCO3 (18.32 g·L-1) and powdered activated carbon (38.30 g·L-1) were added (Guan and Yang, 2017). The encapsulating solution was evenly coated on the cylindrical polypropylene mesh tube (length 50 cm, diameter 1.0 cm) and then placed into a saturated boric acid solution to complete the cross-linking. Finally, the encapsulated mesh tube was cut into small cylinders with a growth degree of 1 cm.

2.3. Experimental design Reactor configuration. A conical flask with volume of 0.5 L was used in the experiment. The immobilized fillers with biomass of 2% (E1), 4% (E2), 6% (E3) and 8% (E4) were placed into four groups

respectively. Approximately 420 mL of inorganic synthetic wastewater was added to reach the volume filling ratio of 20% (Fig. 1a) and then the reactor was cultured in constant temperature shock box. Three sets of parallel experiments were set for each biomass quantity and all experimental data were averaged. The retained suspended sludge (S1) was cultured synchronously under the same conditions (Fig. 1b). The initial sludge concentration was controlled to be consistent with E2.

(a) Immobilized system (b) Contrast test of free sludge Fig. 1 Device for anammox restoration culture device Feeding media. The synthetic inorganic wastewater used in this study provided ammonium and nitrite sources in the form of (NH4)2SO4 and NaNO2, respectively. Inorganic carbon was provided via NaHCO3 (0.6 g·L-1). The synthetic wastewater also included the following compounds: KH2PO4 0.025 g·L-1, CaCl2·2H2O 0.15 g·L-1, MgSO4·7H2O 0.3 g·L-1, FeSO4·7H2O 0.01 g·L-1 and FeCl3 0.008 g·L-1. Trace element solutions Ⅰ and Ⅱ (mL·L-1) were prepared according to Strous et al. (Strous et al., 1997). Initially, the dosing scheme for suspended sludge and immobilized filler was the same and then the water inlet load was increased over a gradient. It has been suggested that the PVA gel carrier can effectively retain biomass, which is conducive to the successful initiation of anammox

and the maintenance of a high NRR (Quan et al., 2011). In order to further improve the nitrogen removal capacity of the encapsulated anammox filler, the HRT of E2 system was shortened to 11 h and 5.5 h at days 100-120. The specific operating parameters are shown in Table 1. At the beginning of the cultivation, the dissolved oxygen (DO) concentration of the feed solution was lowered to <1.0 mg·L -1 by aerating with high purity nitrogen (99%) before adding it to the reactor. Later, the feed solution was made with tap water without special oxygen removal. The pH of the reactor was maintained at 7.4-8.5 by adjusting with 1% H2SO4. Table 1 Operation parameters of the anammox reactor during the enrichment process Period

t (d)

Sample

HRT (h)

Temperatur

NH4+–N (mg·L -1)

NO2-–N (mg·L -1)

e (℃) Ⅰ

1-100

22

32±1

100-330

100-330

E2

11

32±1

250

300

E2

5

32±1

250

300

E1， E2， E3， E4， S1

Ⅱ

101-11 0

Ⅲ

111-12 0

Ⅰ: Restoration culture stage of immobilized anammox filler and suspended sludge; Ⅱ,Ⅲ: Further improving nitrogen removal efficiency of E2 by shortening HRT

2.4. Analytical methods NH4+–N, NO2 － –N, NO3–N, TN, MLSS and MLVSS were determined according to standard methods (D.E. et al., 2005). The COD was measured with a 5B-3F type COD rapid measuring instrument (Lianhua technology); the pH was measured using a PHS-3C portable pH meter; DO was measured with portable dissolved oxygen meter (Hach, USA); and a mercury thermometer was used to measure temperature. Sample morphology was observed with su8020 SEM (Hitachi,

Japan) and the sample pretreatment was according to the method reported by Qiao (Qiao et al., 2010).

2.5. Biological nitrogen removal kinetics Previous studies have shown that anammox activity was reduced or even inactivated under high concentrations of nitrite. For example, anammox activity decreased by half when the influent NO2 － –N concentration was higher than 350 mg·L-1 (Dapena et al., 2007). However, Kimura (Kimura et al., 2010) concluded through batch experiments that anammox activity decreased by 35% when the concentration of NO2－–N in PEG-fixed gel was higher than 274 mg·L-1. The same results were also confirmed by Magri (Magri et al., 2012) in PVA cryogels, where nitrite concentrations up to 400 mg·L-1 did not inhibit anammox bacteria during continuous operation. Therefore, compared to other forms of anammox, PVA encapsulated carriers improve nitrite resistance of the anammox process. When the matrix concentration is inhibited, the Haldane model is used to describe the kinetics of microbial degradation of a substrate matrix (Gee et al., 1990). The Haldane mathematical equation is expressed as follows: V=

𝑉𝑚𝑎𝑥 𝐾𝑚

𝑆 1+ + 𝐾𝑖 𝑆

𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 1

where V is the reaction rate; S is the substrate concentration; Vmax is the maximum reaction rate; Km is the semi-saturation constant; and Ki is the semi-inhibition constant. The kinetics of the encapsulated anammox filler was studied using the Haldane model. E2 and synthetic wastewater were mixed in a serum bottle with an effective volume of 500 mL to achieve a filling rate of 20% (3200 mg SS·L-1). The concentrations of NH4+–N and NO2 － –N in the synthetic wastewater corresponded to the required concentrations. Each test sample was aerated with high purity N2 (99%) to expel DO. The serum bottle was then sealed and placed in a

32℃ constant temperature shaking bed (ZHWY-2102C, China) at a controlled speed of 80 r·min-1. Samples were taken every two hours to plot the matrix concentration degradation curve. All tests were repeated three times and results are presented as the average of 3 trials.

2.6. Batch experiment Studies have shown that acetate (an organic substance) can be utilized by anammox bacteria (Du et al., 2014) and, within a certain range, anammox and denitrifying bacteria can promote synergistic nitrogen removal. To investigate the influence of organic matter on the composition of the immobilized filler bacterial community and the effect of coupling anammox and denitrification on nitrogen removal, a part of E2 filler was taken during the stable period and the COD concentration was controlled by adding sodium acetate. With 100 mg·L-1 as the starting point, the COD concentration of the inlet water was increased to 400 mg·L-1 along a gradient. Each concentration was run for 5 d. In addition, in order to investigate the recovery process of the suspended sludge and immobilization filler after starvation treatment, S1 and E2 were incubated for 15 days at room temperature (20℃) during the stable period and then recovered for 15 days. The recovery test was divided into two stages: Phases Ⅱ (0-5 d) and Ⅲ (6-10 d). Batch experiments were conducted in a conical flask with an effective volume of 500mL for batch culture. The volume filling ratio of the reaction system was 20% (3200 mg SS·L-1), which was placed into a constant temperature shaking bed at 32℃ at a rotating speed of 80 r·min-1. Periodic sampling was performed at intervals of HRT followed by immediate chemical analysis. Specific operational parameters for each treatment are shown in Table 2. Table 2 Operation parameters of batch experiments Item

Time (d)

Sample

HRT

NH4+–N

NO2-–N

COD

(h)

(mg·L-1)

(mg·L-1)

(mg·L-1)

Ⅰ

0-20

E2

11

250

330

100-400

Ⅱ

0-5

E2

11

200

250

-

Ⅲ

6-10

E2

11

250

300

-

I: Effects of organic matter; II,III: Starvation recovery.

2.7 SEM and molecular biological analysis 2.7.1 SEM The immobilized filler was cut into 1-2 mm pieces and washed twice with 0.1 M phosphate buffer (pH 7.4) for 5 min each time. The particles were fixed with 25% pentanediol for 1.5 h and then washed 3 times with PBS. Subsequently, the sample was dehydrated using a rising ethanol gradient (volume fraction: 30% x 2, 50% x 2, 70% x 1, 80% x 1, 90% x 1, and 100% x 2) with dehydration times of 10-15 minutes each. The sample was displaced with isoamyl acetate for 15 min and lyophilized for 24 h. Finally, the sample was observed by plating gold on the surface with an ion sputtering apparatus.

2.7.2 qPCR The anammox immobilized filler was collected from the E2 and E4 reactors at the starting point (day 1) and the end point (day 99). qPCR was performed with the SYBR Green I staining kit (TaKaRa, Biotechnology). After pretreatment with liquid nitrogen milling, DNA was extracted from 200-500 mg samples using the FastDNA Spin Kit for Soil (B518233). The amplification primers were Amx808/Amx1040r (Meng et al., 2010; Hou et al., 2014). The amplification steps were as follows: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95℃ for 30 s, annealing at 57°C for 30 s and extension at 72°C for 30 s; and a final extension at 72°C for 8 min. All reactions were performed according to the formal test conditions, and each reaction was

repeated three times, requiring the amplification efficiency and coefficient of determination R2 to be greater than 95% and 0.99, respectively. The PCR products from the same sample were mixed and detected by 1.5% agarose gel electrophoresis. qPCR reagent was 2X SG Fast qPCR Master Mix (B639271, BBI) and the instrument was LightCycler480 II fluorescence qPCR instrument (Roche, Rotkreuz, Switzerland). The DNA plasmid concentration was determined with an SMA4000 micro spectrophotometer (Merinton Instrument, Inc). The results are expressed as the number of copies of the anammox bacteria per gram of PVA gel (copies·g-1 PVA gel).

2.7.3 Sequencing and bioinformatic analysis Samples S1 were taken from the anammox sludge used in the immobilization experiments. E2a, E2b and E2c samples were obtained from the earlier stage (30 d), stable period (99 d) and COD interference samples (140 d). The bacterial community structure was determined using the Illumina MiSeq sequencing platform (Illumina, San Diego, USA). Primers (341F: CCTACGGGNGGCWGCAG and 805R: GACTACHVGGGTATCTAATCC) were used for amplification and sequencing of the V3-V4 hypervariable region of the bacterial 16S rRNA gene. The bacterial 16S rRNA gene sequences were aligned using the National Center for Biotechnology Information (NCBI) database. For the sequencing data, MEGAN software was used to analyze the 16S rRNA genes of environmental microorganisms and the obtained sequences were classified according to certain thresholds to obtain multiple sequence clustering into operational taxonomic units (OTUs). Diversity analysis was performed according to the OTUs. Finally, the results of the analysis were visualized to make the information easier to interpret.

3. Results and discussion 3.1. Effect of biomass on nitrogen removal of immobilized filler There was a certain adaptation period after the anammox bacteria was entrapped and almost no net removal of TN occurred in the four systems during the first 5 days (Fig. 2(a)). Between days 6 to 15, the treatments began to differentiate; E4 started-up first with gradual decrease in the TN in the effluent. The other three systems also showed the same trend, but the changes were slower. The reaction system with 2% (E1), 4% (E2), 6% (E3) and 8% (E4) biomass restored the nitrogen removal capacity of the original suspended sludge on days 36, 30, 23 and 17, respectively. Linear fitting of TN removal rate curves of the first three NLR gradients (1-55d) of the four reaction systems was shown in Fig.2 (b). In general, the trend line of E4 was the steepest while the trend lines of the other three systems decreased successively. In terms of stages, E1 and E2 rose rapidly in the later stage, E3 remained stable, and E4 fell slightly. The results fully indicated that high anammox biomass could shorten the recovery period of the immobilized filler and allow it to enter the logarithmic phase first, but the limited carrier capacity would make the growth rate of anammox bacteria in the carrier with high encapsulation biomass tend to be saturated. This was also reflected in the final removal effect. Compared with the effect of encapsulated anammox biomass on the recovery period, the final removal effect was not significant. After day 55, the NRR gap of the four systems gradually decreased. On the 83rd day of reactor operation, the NLR and NRR of E4 were 0.687 kg·(m3·d)-1 and 0.613 kg·(m3·d)-1, respectively. The other three reaction systems also showed similar results, with the final removal efficiencies of NH4+–N and NO2 － –N being above 95%. Therefore, it can be seen that the anammox inclusion filler underwent the adaptation period, recovery period, logarithmic phase and stationary phase. The

correlation between the biomass of encapsulated anammox bacteria and each period went from weak to strong and then weak. In other words, the same removal effect can be achieved after long-term operation with less bacteria. This information is of great value for determining the optimal amount of bacteria and for saving anammox sludge. It can also be found from Fig.2 that in the middle and later stages of cultivation, the impact of NLR increase on the reaction system was weakened (Fig. 2). After stable cycle operation, the system maintained the removal effect of the previous NLR stage and even achieved a total effluent nitrogen lower than the gradient of the previous load. This indicated that the stable anammox nitrogen removal process was realized in the PVA-PP immobilized filler.

(a) Nitrogen loading rate, TN effluent and nitrogen removal efficiency

(b) Fitting curve of the TN removal efficiency from days 1 to 55 Fig. 2 Nitrogen removal performance of immobilized filler with different biomass (percent of anammox bacteria: E1, 2%; E2, 4%; E3, 6%; E4, 8%)

3.2. Anammox activity of suspended sludge The nitrogen removal effect of the suspended sludge (S1) with E2 concentration (2600 mg·L-1 VSS) is shown in Fig. 3. It is worth noting that the nitrogen removal capacity of S1 slowly increased compared to that of encapsulated anammox bacteria and the treatment effect fluctuated due to the inability to adapt to the improved NLR. Finally, the NRR was less than 1/8 of E2. Ali (Ali et al., 2015) reported similar results, showing that immobilization in PVA-SA gel is an effective strategy to start an anammox reactor with minimum biomass. Column reactors containing immobilized biomass (0.33 g·VSS-1) started significantly faster than those containing granular biomass (2.5 g·VSS-1).

Fig. 3 Nitrogen loading rate, TN effluent and nitrogen removal efficiency of S1 The sludge concentration has a great influence on the activity of the anammox suspended sludge and the sludge concentration used to start anammox reaction systems is generally 8-20 g·L-1 (Tang et al., 2014; Yan et al., 2017; Zhang et al., 2017). If the concentration is too low, the start-up time will be prolonged and anammox bacteria will be difficult to enrich. However, the PVA-PP immobilized carrier used less biomass to initiate the anammox reaction in a relatively short time and achieved effective TN removal, which is very meaningful for the enrichment of anammox bacteria with strict cultivation conditions and low sludge yield.

3.3. Further improvement of filler activity The nitrogen removal performance of E2 is shown in Fig. 4. The NLR was 2.64 kgN/(m3·d), the NRR was stable at 1.85 kgN·(m3·d)-1 and the average TN removal efficiency was 70%. The removal ratios of △NO2-–N/△NH4+–N and △NO3-–N/△NH4+–N were stable at around 1.17 and 0.22, respectively, which were similar to the theoretical values. That is, the higher NLR promoted further improvement of the reaction system treatment capacity. Furthermore, the specific anammox activity reached at a higher level of 0.36 kg NH4+–N·(gVSS·d) reaction was the main nitrogen conversion pathway of the system.

-1

and the anammox

Fig. 4 Nitrogen removal performance of encapsulated anammox filler under higher load This was different from the results of Kazuichi (Kazuichi et al., 2007) and Hsia (Hsia et al., 2008), who found that using PEG-PVA gel for encapsulating anammox microorganisms to operate reactors could maintain its activity, but could not enhance it. This may be related to several factors, including the immobilized materials, microbial structure or operation mode. In this experiment, the PVA-PP carrier provided a more stable micro-ecological environment for the anammox bacteria, while reducing the external disturbance and inhibition of DO, which is very necessary for the cultivation of anammox bacteria with initially started activity. SEM images of internal and external structures of the immobilized filler under the stable period (99 d) are shown in Fig. 5. It can be clearly seen that the surface of the immobilized filler was rough and rich in voids, facilitating the smooth transport of substrates and products. There were many skeleton structures inside the immobilized filler for bacterial adhesion and growth, as well as channels that could be used for mass transfer and exhaust. Anammox bacteria with a volcanic crater-like surface and a diameter of 0.8-1.2 µm grew around the pore as a channel. It has been shown that the ideal gel carrier should have a macroporous structure to facilitate unimpeded diffusion of solutes and gas release. Furthermore, there should be large holes of 0.1-1 micron in

the gel materials (Lozinsky and Plieve, 1998). In our study, a pore size of 1.0-3.0 μm was observed (Fig. 5(b)). In addition, Wang et al. (Wang et al., 2006) demonstrated that the microporous framework formed through bacterial reproduction provides favorable space and conditions for matrix transfer and cell growth. Therefore, the unique three-dimensional network structure of the PVA carrier creates an appropriate environment for bacterial growth and metabolism. The existence of this porous structure also verifies that the immobilized filler has good mass transfer performance to some extent.

Fig. 5 SEM images of immobilized carrier during the stable period; (a) surface×500 k; (b) surface×2000 k; (c) inside×5000 k; (d) inside×10000 k

3.4. The 16S rDNA gene concentration of anammox bacteria According to Fig.7, at the beginning, the 16S rDNA gene concentrations of the anammox bacteria in E2 and E4 was quantified as 1.54 x 105 and 3.07 x 107 copies·g-1 PVA gel, respectively. This indicates that the absolute inoculation amount of anammox in E4 was 200 times that of E2. According to previous studies, inoculation with a high concentration anammox bacteria is an important factor for accelerating the initiation of the anammox process (Monballiu et al., 2013; Yang and Jin, 2013). Compared to E2, E4 had a high-concentration anammox bacteria and a shorter start-up period (Fig. 2). Interestingly, at the end point, the difference in the concentration of anammox bacteria between the two bioreactors decreased. For example, the 16S rDNA gene concentrations of the anammox bacteria at the end points for E2 and E4 were 4.7 x 107 copies·g-1 PVA gel and 6.07 x 108, respectively (Fig. 6). At this time, the copies of E4 was only 13 times of

E2. Obviously, anammox bacteria were efficiently enriched in the PVA-PP carrier after 99 days of operation, and their "bacterial population density" tended to be at the same level. This could be attributed to the limited space structure inside the filler, and the anammox bacteria enrichment process was the process of filling the filler carrier. Immobilized vectors with low initial biomass had a long start-up time but relatively large growth space, so the copies of anammox gene increased rapidly during culture. The immobilized carrier with a large amount of inoculated bacteria (e.g. E4) started up quickly (Fig. 2), but the growth rate of anammox bacteria was limited due to the space competition of microorganisms in the later stage of culture. Moreover, after 85d, we observed anammox bacteria dissociated from the filler surface in the E4 system (Please see supplementary materials for pictures), which indicated that the carrier capacity tended to be saturated.

Fig. 6 16SrDNA gene concentration of anammox bacteria at the start and end points (E2i: E2 start point; E2e: E2 end point; E4i: E4 start point; E4e: E4 end point) In addition, researchers have found that, under specific culture conditions, improved systematic denitrification efficiency can reflect an increased anammox biomass (Xu et al., 2007). It has been reported that the number of anammox bacteria in PVA-SA gel beads increases with an increased NRR (Bae et al., 2015a). In this study, the 16S rDNA gene concentration of anammox in

the E2 and E4 immobilized filler was prominently increased, reaching a comparable level. This was consistent with similar results with the ultimate ability to remove nitrogen (Fig. 2). The above results indicated that, from the molecular verification, the ANAMMOX carrier with a low biomass after a certain period of operation, the number of anammox bacteria was comparable to encapsulated ANAMMOX with a higher inoculating amount of bacteria.

3.5. Microbial diversity analysis based on MiSeq Illumina sequencing The four DNA samples from the reactor biomass generated a total of 255,480 sequences and a total of 20,156 bacterial OTUs. Table 3 lists the OTU quantity and diversity indices of each sample. After immobilized cultivation, the Simpson index of the E4 system, which reflects the microbial diversity of a sample, increased from 0.05 to 0.07 to 0.12, indicating that the diversity of the bacterial community decreased during the operation process and gradually evolved into a structurally stable, functionally specific community. Consistent with previous studies, the biodiversity of the anammox granular sludge enrichment system decreased after long-term operation (330 d) (Sobotka et al., 2017). Furthermore, Chao1, which estimates the number of OTUs in the community, and the ACE index, which estimates the number of OTUs in the community via population richness index, first showed a small increase and then decreased from S1 to E2a to E2b, indicating that there was a transitional period after the initial sludge sample was encapsulated. Thus, it was hypothesized that the filler could be considered to be a small ecosystem and that the encapsulation changed the bacterial niche. Some bacteria obtained a more favorable space for growth, thus increasing the total number of species. Then, under specific operating conditions, the microorganisms were selectively screened and enriched, with some bacteria possessing selective advantages

outcompeting other bacteria, thereby decreasing the total number of species. Table 3 Variation of sample diversity indices during different periods Sample

ID

Seq num

OUT

num

ACE index

Chao1 index

Simpson

（97%） S1（0d） E2a（30d） E2b（99d） E2c（+COD）

62752

2866

44075.09

20105.93

0.05

83621

3347

54022.82

25011.78

0.07

60612

1938

25220.11

11434.53

0.12

48495

12005

1172800.55

321026.56

0.03

The PVA-PP carrier maintained good microbial community diversity. Before and after immobilization, the dominant taxa (≥1% relative abundance of the suspended sludge and immobilized filler microbial communities) belonged to ten phyla, including Proteobacteria, Planctomycetes, Firmicutes, Chloroflexi and Armatimonadetes (Fig. 7(a)). However, after the operation, the proportion of each phylum changed significantly. For example, Proteobacteria decreased from 57.18% to 42.71%; however, its proportion in the whole microbial system was still the largest, which is consistent with the distribution of bacterial communities in most anammox reactors (Cao et al., 2016). The second most abundant phylum was Planctomycetes; its proportion increased from 12.8% to 33.13%, which is an increase of nearly three times. The anammox bacteria of the denitrifying functional taxa in the reactor belonged to Planctomycetes. The results indicate that the immobilization materials used in this experiment are suitable for the growth of anammox bacteria, which were successfully enriched in the filler.

Fig. 7 Relative abundance of the microbial community in the sample; (a) phylum level; (b) genus level (S1: suspended sludge before entrapprd; E2a: E2 sample on day 30; E2b: E2 sample on day 99) At the genus level, the most significant groups changed from Thermomonas (21.56%) in S1 to Candidate Kuenenia (32.55%) in E2b (Fig.7b). Thermomonas has denitrification function while Candidatus Kuenenia is an anammox functional bacteria. The change of its proportion indicates that anammox bacteria had a competitive advantage. Moreover, the abundance of Bacillus and Pseudomonas were reduced. These genera have been shown to be heterotrophic nitrifying bacteria (Zhao et al., 2010) with simultaneous nitrification-denitrification ability, while the strictly anaerobic heterotrophic nitrifying genus Igvanivibacterium was slightly increased, indicating that a good anaerobic environment was maintained inside the encapsulated carrier. These heterotrophic

bacteria were dynamically stable in the encapsulated carrier and protected Candidatus Kuenenia from inhibition by DO and organic matter. Furthermore, they could also be used as a skeleton support for flocculating microorganisms (Miura et al., 2007; Lu et al., 2011; Kulichevskaya et al., 2010). In addition, it can be seen that a small amount of Nitrosomonas (0.26%; 0.31%; 0.23%) was present in S1, E2a and E2b, respectively. This genus can oxidize ammonium to nitrite, which may have caused the conversion ratio of ΔNO2 － –N / ΔNH4+–N in the reaction system to be less than that of anammox theoretical ratio 1.32.

3.6. Effects of Environmental Factors 3.6.1. Interference of organic carbon source As shown in Fig.8a, at the beginning, the addition of 100 mg·L-1 COD promoted the denitrification reaction. However, this had no effect on the anammox process. Anammox and denitrification removed the nitrogen in coordination and eliminated nitrate as a by-product of anammox, thereby increasing the TN removal efficiency, which was consistent with the findings of Zhou (Zhou et al., 2006). It is obvious that the anammox immobilized filler had a good effect on the treatment of low-concentration organic wastewater. When the COD concentration was approximately 100 mg·L-1, the TN removal efficiency was the highest at 86.3%. However, the removal efficiency of NH4+–N and NO2 － –N decreased significantly with an increased COD concentration on day 10, and the removal efficiency of NH4+–N decreased more significantly, finally falling to about 40%. This was similar to the phenomenon described by Güven (Güven et al., 2005), where a COD concentration below 180 mg·L-1 had no significant effect on anammox reaction, but significant inhibition was observed at COD concentrations higher than 360 mg·L-1. The effluent NO3+–N was gradually depleted via denitrification and the difference between the

nitrogen conversion ratio of the reaction system and the anammox reaction theoretical value increased (Fig. 8c). Although the COD had an adverse effect on anammox, the denitrifying bacteria metabolized COD in the system, resulting in significantly enhanced biochemical activity with the COD removal rate increasing to 64%.

Fig. 8 Interference of organic carbon source on the immobilized system; (a) contribution rate of anammox-denitrification, change in NO3--N concentration and removal efficiency of COD; (b) the removal efficiency of NH4+-N, NO2—N; (c) nitrogen conversion ratio: △NO3--N/△NH4+-N,△NO2--N/△NH4+-N In an anammox reactor with an organic carbon source (Hu et al., 2007), the anammox and denitrifying bacteria could coexist when the ratio of COD to NH4+–N ranged from 0 to 1.57. Yang (Yang et al., 2006) concluded that the addition of a small amount of organic matter (20 mg·L-1) had little effect on the activity of anammox sludge, while a large amount (200 mg·L-1) could

significantly inhibit activity. At the same time, the sludge would exhibit a higher denitrification activity. In this experiment, when the COD concentration was 400 mg·L-1, the contribution of anammox to TN removal in the system was maintained at 58%. This could be because the structure of the immobilized carrier provided a relatively mild internal environment that reduced the interference of COD on anammox bacteria to some extent.

Fig. 9 Influence of COD interference on the structure of encapsulated anammox bacteria; (a) phyla level; (b) genus level High-throughput sequencing was used to investigate structural changes in the flora (Fig. 9), and the proportion of Proteobacteria in E2c increased significantly, becoming an absolute dominant phylum. Proteobacteria was the most important phylum in waste water treatment plants, including most heterotrophic bacteria such as denitrifying bacteria (Li et al., 2019). In addition, the proportion of Firmicutes and Bacteroidetes also increased slightly. Bacteroides and Firmicutes belong to anaerobic isoxia bacteria, which were often found as the dominant bacteria in anaerobic organic environment (Shen et al., 2013). It could be seen that the addition of COD promoted the reproduction of heterotrophic bacteria. On the contrary, the proportion of Planctomycetes belonging to anammox decreased from 33.06% to 4.04%, and the proportion of Chloroflexi, which was bred by anammox metabolites, also decreased. Chloroflexi played a supporting and

skeleton role in the formation of anammox granular sludge. The increased abundance of Chloroflexi was conducive to the formation of anammox granular sludge (Björnsson et al., 2002). After excessive addition of organics, the abundance of Planctomycetes decreased and metabolites decreased, which was not conducive to the formation of aggregates in the immobilized carrier. This result was consistent with the changing rule of Chloroflexi. At the genus level, the proportion of Pseudomonas (25.66%), a denitrifying functional genre, increased, while the proportion of the anammox functional species Candidatus Kuenenia decreased from 33.13% to 3.72%. This indicated that anammox lost its dominant position. The community diversity indices also reflected these changes (Table 3). The Chao1 and ACE indices of E2c were all higher than E2b, proving that the microbial richness in the immobilized filler increased with the addition of COD. Moreover, the Simpson index of E2c decreased from 0.12 to 0.03, indicating an increase in bacterial diversity. It could be concluded that the addition of organic compounds increased the abundance of heterotrophic bacteria and the total number of bacteria on the filler surface, leading to a decrease in the proportion of anammox bacteria. However, anammox bacteria were enriched in PVA-PP carrier. It was inferred that the absolute number of anammox bacteria was maintained. Therefore, the encapsulated filler still exhibited certain anammox activity.

3.6.2. Biological nitrogen removal kinetics The experimental data were nonlinearly fitted with Origin 9.0 software and the kinetic functions of NH4+–N (Fig. 10a) and NO2 － –N (Fig. 10b) were derived. The correlation coefficient constants R2 were 0.995 and 0.992, respectively, indicating good correlations. The maximum ammonium reaction rate of the inclusion filler was 0.43 mg NH4+–N·(mgVSS·d)-1; the ammonium

half-saturation constant was 45.2 mg·L-1; and the ammonium semi-inhibition constant was 3046.3 mg·L-1 (Fig. 10(a)). The actual maximum reaction rate was 0.337 mg NH4+–N·(mgVSS·d)-1, which was only 78.4% of the theoretical maximum reaction rate, indicating that the immobilized filler also had great potential for the anammox reaction that could be further developed. The maximum nitrite reaction rate was 0.73 mg·(mgVSS·d)-1; the nitrite half-saturation constant was 79.4 mg·L-1; and the semi-inhibition constant of nitrite was 932.2 mg·L-1 (Fig. 10(b)). The experimental results were slightly higher than the previously reported inhibitory range of nitrite nitrogen (98.0-720.6 mg·L-1) on the anammox reaction (Chen 2013). This indicates that the immobilized filler had stronger tolerance to nitrite and showed a better nitrogen removal capacity than high-substrate suspended sludge in the environment, so it could survive longer in a high concentration matrix. This is attributed to the buffering and blocking effect of the PVA-PP carrier on the stimulation of toxic substances on anammox bacteria, which may be related to the population structure. Candidatus Kuenenia was identified as the dominant genus by 16S rRNA gene analysis. Studies have shown that Candidatus Kuenenia can tolerate a nitrous oxide concentration of at least 180 mg·L-1, while Candidatus Brocadia can withstand 70 mg·L-1 (Schmidt et at., 2003). Additionally, Candidatus Kuenenia may be a K-counter (low growth rate but good matrix affinity) (Van der Star et al., 2003). Therefore, it was concluded that Candidatus Kuenenia was better able to adapt to the high concentration of influent NO2－–N.

Fig.10 Biological nitrogen removal kinetics of the immobilization filler (a) NH4+ -N fitting equation; (b) NO2－–N fitting equation

3.6.3. Recovery of starvation In Phase Ⅱ, after 5 days of operation, the removal efficiency of NH4+ -N and NO2－–N in the immobilized system increased from 36.7% and 35.2% to 87.1% and 78.6% respectively (Fig. 11). During the second stage, the loads of influent NH4+ -N and NO2－–N continuously increased. After the second 5 d recovery, the removal efficiencies of NH4+ -N and NO2 － –N were above 83% and the effluent NH4+ -N was stable at about 30 mg·L-1, basically reaching the original level. Compared with E2, the S1 active recovery process was slow and the removal rate decreased sharply with the increased load in stage Ⅲ. Ali et al. (Ali et al., 2014) stored suspended anammox sludge in nutrient solution containing 3 mmol·L-1 molybdate for 90 days at room temperature, and the anammox activity was fully restored after 35 days. Ji et al (Ji et al., 2014) stored anammox sludge at 4℃ for 60 days, and recovered 92.9% of the activity after 14 days. When compared with

free anammox bacteria, which is not suitable for storage and has a long recovery cycle, encapsulated anammox bacteria exhibited the advantages of a short recovery time and high treatment efficiency.

Fig. 11 Restoration of starvation inhibition; (a) immobilized filler; (b) free sludge

4. Conclusions In PVA-PP immobilized filler, anammox bacteria showed rapid growth and long-term stable nitrogen removal. Encapsulated biomass only affected the process of start-up and enrichment. Due to the limitation of carrier capacity, the copies of anammox bacteria gene and the nitrogen removal effect of the system became similar after stage culture. This provided a reference for achieving the balance between starting anammox with less bacteria and obtaining good treatment effect quickly. Encapsulated anammox bacteria showed better anammox activity than suspended sludge by

reducing the loss of bacteria. The immobilized filler and anammox nitrogen removal kinetics curve fitted well. Due to the protection of the immobilized materials, a more balanced ecological niche was established inside the carrier. Moreover, the effects of organic carbon source interference and starvation inhibition on the anammox process were significantly weakened. The microbial community in the carrier was stable, and the nitrogen-removing functional bacteria Candida Kuenenia (AF375995.1) was effectively enriched. In addition, the macroporous structure of PVA-PP promoted the transfer of substrates and metabolites. Consequently, entrapped anammox helped to start-up the anammox reaction with less amount of bacteria, which improved the stability of the reaction system, showing good potential for future applications.

Acknowledgements This research was supported by Beijing Municipal Commission of Education under the municipal government of Beijing under the Program “Research on reinforcement and stability of nitrogen removal performance in wastewater treatment based on the new landmark conditions” (Z161100004516015).

References Ali, M., Oshiki, M., Okabe, S., 2014. Simple, rapid and effective preservation and reactivation of anaerobic ammonium oxidizing bacterium “Candidatus Brocadia sinica”. Water Res. 57, 215–222. https://doi.org/10.1016/j.watres.2014.03.036 Ali, M., Oshiki, M., Rathnayake, L., Ishii, S., Satoh, H., Okabe, S., 2015. Rapid and successful start-up of anammox process by immobilizing the minimal quantity of biomass in PVA-SA gel beads. Water Res. 79, 147–157. https://doi.org/10.1016/j.watres.2015.04.024 Björnsson, L., Philip, H., Tyson, G., Blackall, L., 2002. Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal. Microbiology 148, 2309–2318. https://doi.org/10.1099/00221287-148-8-2309

Bae, H., Choi, M., Lee, C., Chung, Y.C., Yoo, Y.J., Lee, S., 2015. Enrichment of ANAMMOX bacteria from conventional activated sludge entrapped in poly(vinyl alcohol)/sodium alginate gel. Chem. Eng. J. 281, 531–540. https://doi.org/10.1016/j.cej.2015.06.111 Bae, H., Choi, M., Chung, Y.C., Lee, S., Yoo, Y., 2017. Core-shell structured poly(vinyl alcohol)/sodium alginate bead for single-stage autotrophic nitrogen removal. Chem. Eng. J. 322.https://doi.org/10.1016/j.cej.2017.03.119 Castignetti, D., Hollocher, T.C., 1984. Heterotrophic nitrification among denitrifiers. Appl. Environ. Microbiol. 47, 620–623. Chen, K.C., Lin, Y.F., 1994. Immobilization of microorganisms with phosphorylated polyvinyl alcohol (PVA) gel. Enzyme Microb. Technol. 16, 79–83.https://doi.org/https://doi.org/10.1016/0141-0229(94)90113-9 Chen, K.C., Chen, S.J., Houng, J.Y., 1996. Improvement of gas permeability of denitrifying PVA gel beads. Enzyme Microb. Technol. 18, 502–506. https://doi.org/10.1016/0141-0229(95)00160-3 Chen, K.C., Houng, J.-Y., 2008. Cell Immobilization with Phosphorylated Polyvinyl Alcohol (PVA) Gel, in: Immobil Enzyme Cell. pp. 207–216. https://doi.org/10.1385/0-89603-386-4:207 Chou, W., Tseng, S., Ho, C., 2012. Anaerobic ammonium oxidation improvement via a novel capsule bioreactor. Environ. Technol. 33, 2105–2110. https://doi.org/10.1080/09593330.2012.660647 Chen, T., 2013. Performance and microbial characteristics of anaerobic ammonia oxidation. Zhejiang University. (in Chinese) ． Cao, S., Du, R., Li, B., Ren, N., Peng, Y., 2016. High-throughput profiling of microbial community structures in an ANAMMOX-UASB reactor treating high-strength wastewater. Appl. Microbiol. Biotechnol. 100, 6457–6467. https://doi.org/10.1007/s00253-016-7427-6 Cho, K., Choi, M., Lee, S., Bae, H., 2018. Negligible seeding source effect on the final ANAMMOX community under steady and high nitrogen loading rate after enrichment using poly(vinyl alcohol) gel carriers. Chemosphere 208, 21–30.https://doi.org/10.1016/j.chemosphere.2018.05.155 Dapena-Mora, A., Fernández, I., Campos, J.L., Mosquera-Corral, A., Méndez, R., Jetten, M.S.M., 2007. Evaluation of activity and inhibition effects on Anammox process by batch tests based on the nitrogen gas production. Enzyme Microb. Technol. 40, 859–865.https://doi.org/10.1016/j.enzmictec.2006.06.018

D. E. Andrew and H. F. M. Ann, Standard Methods for the Examination of Water and Wastewater, APHA (American Public Health Association), AWWA (American Water Works Association), WEF (Water Environment Federation), 21st edn, 2005. Du, R., Peng, Y., Cao, S., Wu, C., Weng, D., Wang, S., He, J., 2014. Advanced nitrogen removal with simultaneous Anammox and denitrification in sequencing batch reactor. Bioresour. Technol. 162, 316–322. https://doi.org/10.1016/j.biortech.2014.03.041 Gee, C., Suidan, M., Pfeffer, J., 1990. Modeling of Nitrification Under Substrate-Inhibiting Conditions. J. Environ. Eng. - J Env. ENG-ASCE 116.https://doi.org/10.1061/(ASCE)0733-9372(1990)116:1(18) Gu, D., Dapena, A., Kartal, B., Schmid, M.C., Maas, B., Pas-schoonen, K. Van De, Sozen, S., Mendez, R., Camp, H.J.M.O. Den, Jetten, M.S.M., Strous, M., Schmidt, I., 2005. Propionate Oxidation by and Methanol Inhibition of Anaerobic Ammonium-Oxidizing Bacteria 71, 1066–1071. https://doi.org/10.1128/AEM.71.2.1066 Guan, Q., Yang, H., 2017. Determination of diffusion coefficient of PVA gel and optimization of its mass transfer performance. Chinese Journal of Environmental Engineering. 11,1375–1382. Hu, Y., Luo, H., Chen, Z., 2002. Study on the enrichment and cultivation of anaerobic ammonium oxidation biomass. J South Chin Univ Technol Nat Sci Ed 30, 160–164. Hu, Y., Liang, H., Zhu, J., Yan, J., 2007. Batch experiments of anaerobic ammonium oxidation process with organic carbon. Journal of South China University of Technology (Natural Science Edition). 116-119 Hsia, T.H., Feng, Y.J., Ho, C.M., Chou, W.P., Tseng, S.K., 2008. PVA-alginate immobilized cells for anaerobic ammonium oxidation (anammox) process. J. Ind. Microbiol. Biotechnol. 35, 721–727. https://doi.org/10.1007/s10295-008-0336-7 Hou, L., Zheng, Y., Liu, M., Gong, J., Zhang, X., Yin, G., You, L., 2013. Anaerobic ammonium oxidation (anammox) bacterial diversity, abundance, and activity in marsh sediments of the Yangtze Estuary. J. Geophys. Res. Biogeosciences 118, 1237–1246.https://doi.org/10.1002/jgrg.20108 Isaka, K., Sumino, T., Tsuneda, S., 2007. High nitrogen removal performance at moderately low temperature utilizing anaerobic ammonium oxidation reactions. J. Biosci. Bioeng. 103, 486–490.https://doi.org/10.1263/jbb.103.486 Isaka, K., Date, Y., Sumino, T., Tsuneda, S., 2007. Ammonium removal performance of anaerobic ammonium-oxidizing bacteria immobilized in polyethylene glycol gel carrier: Anammox bacteria immobilized in gel carrier. Appl. Microbiol. Biotechnol. 76, 1457–1465.https://doi.org/10.1007/s00253-007-1106-6

Isaka, K., Date, Y., Kimura, Y., Sumino, T., Tsuneda, S., 2008. Nitrogen removal performance using anaerobic ammonium oxidation at low temperatures. FEMS Microbiol. Lett. 282, 32–38.https://doi.org/10.1111/j.1574-6968.2008.01095.x Isaka, K., Kimura, Y., Yamamoto, T., Osaka, T., Tsuneda, S., 2013. Complete autotrophic denitrification in a single reactor using nitritation and anammox gel carriers. Bioresour. Technol. 147, 96–101. https://doi.org/10.1016/j.biortech.2013.07.095 Ji, Y.X., Jin, R.C., 2014. Effect of different preservation conditions on the reactivation performance of anammox sludge. Sep. Purif. Technol. 133, 32–39.https://doi.org/10.1016/j.seppur.2014.06.029 Kimura, Y., Isaka, K., Kazama, F., Sumino, T., 2010. Effects of nitrite inhibition on anaerobic ammonium oxidation. Appl. Microbiol. Biotechnol. 86, 359–365.https://doi.org/10.1007/s00253-009-2359-z Kulichevskaya, I.S., Suzina, N.E., Liesack, W., Dedysh, S.N., 2010. Bryobacter aggregatus gen. nov., sp. nov., a peat-inhabiting, aerobic chemo-organotroph from subdivision 3 of the acidobacteria. Int. J. Syst. Evol. Microbiol. 60, 301–306. https://doi.org/10.1099/ijs.0.013250-0 Lu, C.J., Lee, C.-M., Huang, C.Z., 1996. Biodegradation of chlorophenols by immobilized pure-culture microorganisms. Water Sci. Technol. 34, 67–72. https://doi.org/10.1016/S0273-1223(96)00698-1 Lozinsky, V.I., Plieva, F.M., 1998. Poly(vinyl alcohol) cryogels employed as matrices for cell immobilization. 3. Overview of recent research and developments. Enzyme Microb. Technol. 23, 227–242. https://doi.org/10.1016/S0141-0229(98)00036-2 Li, M., Hong, Y., Klotz, M.G., Gu, J.D., 2010. A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments. Appl. Microbiol. Biotechnol. 86, 781–790.https://doi.org/10.1007/s00253-009-2361-5 Lu, H., Sato, Y., Fujimura, R., Nishizawa, T., Kamijo, T., Ohta, H., 2011. Limnobacter litoralis sp. nov., a thiosulfate-oxidizing, heterotrophic bacterium isolated from a volcanic deposit, and emended description of the genus Limnobacter. Int. J. Syst. Evol. Microbiol. 61, 404–407. https://doi.org/10.1099/ijs.0.020206-0 Lü, G., Li, T., Xu, L., Shen, Y., Wu, P., Zhang, T., Samwine, T., 2017. Quick Start-up Performance of Combined ANAMMOX Reactor Based on Different Inoculated Sludge Types. Environmental Sci. 38, 4324–4331. https://doi.org/10.13227/j.hjkx.201704053

Li, B., Wang, Y., Li, J., Yang, L., Li, X., Zhou, Z., Li, Y., Chen, X., Wu, L., 2019. The symbiosis of anaerobic ammonium oxidation bacteria and heterotrophic denitrification bacteria in a size-fractioned single-stage partial nitrification/anammox reactor. Biochem. Eng. J. 151, 107353. https://doi.org/10.1016/j.bej.2019.107353 Mulder, A., van de Graaf, A.A., Robertson, L.A., Kuenen, J.G., 1995. Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol. Ecol. 16, 177–184. https://doi.org/10.1111/j.1574-6941.1995.tb00281.x Miura, Y., Watanabe, Y., Okabe, S., 2007. Significance of Chloroflexi in performance of submerged membrane bioreactors (MBR) treating municipal wastewater. Environ. Sci. Technol. 41, 7787–7794. https://doi.org/10.1021/es071263x Magrí, A., Vanotti, M.B., Szögi, A.A., 2012. Anammox sludge immobilized in polyvinyl alcohol (PVA) cryogel carriers. Bioresour. Technol. 114, 231–240.https://doi.org/10.1016/j.biortech.2012.03.077 Mao, Y., Wang, J., 2013. Immobilization of activated sludge in PVA matrix using innovative methods. Acta Scientiae Circumstantiae 33, 370–376. Monballiu, A., Desmidt, E., Ghyselbrecht, K., De Clippeleir, H., Van Hulle, S.W.H., Verstraete, W., Meesschaert, B., 2013. Enrichment of anaerobic ammonium oxidizing (Anammox) bacteria from OLAND and conventional sludge: Features and limitations. Sep. Purif. Technol. 104, 130–137.https://doi.org/10.1016/j.seppur.2012.10.046 Poth, M., Focht, D.D., 1985. 15N kinetic analysis of N2O production by Nitrosomonas europaea: An examination of nitrifier denitrification. Appl. Environ. Microbiol. 49, 1134–1141. Qiao, X., Liu, Z., Liu, Z., Zeng, Y., Zhang, Z., 2010. Immobilization of activated sludge in poly(ethylene glycol) by UV technology and its application in micro-polluted wastewater. Biochem. Eng. J. 50, 71–76.https://doi.org/10.1016/j.bej.2010.03.007 Quan, L.M., Khanh, D.P., Hira, D., Fujii, T., Furukawa, K., 2011. Reject water treatment by improvement of whole cell anammox entrapment using polyvinyl alcohol/alginate gel. Biodegradation 22, 1155–1167. https://doi.org/10.1007/s10532-011-9471-3 Robertson, L.A., Kuenen, J.G., 1990. Combined heterotrophic nitrification and aerobic denitrification in Thiosphaera pantotropha and other bacteria. Antonie Van Leeuwenhoek 57, 139–152. https://doi.org/10.1007/BF00403948 Strous, M., Van Gerven, E., Kuenen, J.G., Jetten, M., 1997. Effects of aerobic and microaerobic conditions on anaerobic ammonium- oxidizing (anammox) sludge. Appl. Environ. Microbiol. 63, 2446–2448.

Schmidt, I., Sliekers, O., Schmid, M., Bock, E., Fuerst, J., Kuenen, J.G., Jetten, M.S.M., Strous, M., 2003. New concepts of microbial treatment processes for the nitrogen removal in wastewater 27.https://doi.org/10.1016/S0168-6445(03)00039-1 Shen, Z., Zhou, Y., Hu, J., Wang, J., 2013. Denitrification performance and microbial diversity in a packed-bed bioreactor using biodegradable polymer as carbon source and biofilm support. J. Hazard. Mater. 250-251C, 431–438. https://doi.org/10.1016/j.jhazmat.2013.02.026 Sobotka, D., Tuszynska, A., Kowal, P., Ciesielski, S., Czerwionka, K., Makinia, J., 2017. Long-term performance and microbial characteristics of the anammox-enriched granular sludge cultivated in a bench-scale sequencing batch reactor. Biochem. Eng. J. 120, 125–135. https://doi.org/10.1016/j.bej.2017.01.007 Tang, C., Xiong, L., Wang, Y., Zheng, P., 2013. Kinetic Characteristics of High-rate ANAMMOX Granules. Environmental Science. 34, 3544-3551. Tang, X., Peng, Y., Xu, Z., Yang, X., 2014. Fast start-up of anaerobic ammonium oxidation (ANAMMOX) granular reactor treating low-strength wastewater. Journal of Central South University (Science Technol). 45, 4445–4451. Van Der Star, W.R.L., Miclea, A.I., Van Dongen, U.G.J.M., Muyzer, G., Picioreanu, C., Van Loosdrecht, M.C.M., 2008. The membrane bioreactor: A novel tool to grow anammox bacteria as free cells. Biotechnol. Bioeng. 101, 286–294.https://doi.org/10.1002/bit.21891 Walter, W., 1961. APHA Standard Methods for the Examination of Water and Wastewater. Am. J. Public Heal. Nations Heal. 51.https://doi.org/10.2105/AJPH.51.6.940-a Wang, Y., Yang, X., Li, H., Tu, W., 2006. Immobilization of Acidithiobacillus ferrooxidans with complex of PVA and sodium alginate. Polym. Degrad. Stab. 91, 2408–2414.https://doi.org/10.1016/j.polymdegradstab.2006.03.015 Wang, T., Zhang, H., Gao, D., Yang, F., Zhang, G., 2012. Comparison between MBR and SBR on Anammox start-up process from the conventional activated sludge. Bioresour. Technol. 122, 78–82. https://doi.org/10.1016/j.biortech.2012.02.069 Xu, Q., 2007. An Experimental Study on Enrichment and Cultivation of Anammox Microorganisms and Its Influeneing Factor．Suzhou University of Science and Technology. (in Chinese). Yang, Y., Zuo, J., Shen, P., Gu, X., 2006. Influence of temperature, pH value and organic substance on activity of ANAMMOX sludge. Environmental Science. 27, 691-695. Yang, G.F., Jin, R.C., 2013. Reactivation of effluent granular sludge from a high-rate Anammox reactor after storage. Biodegradation 24, 13–32. https://doi.org/10.1007/s10532-012-9554-9

Zhang, S., Wang, T., Zheng, P., 2004. Progresses of Anaerobic Ammonium Oxidation. China Biogas. 2,28-11. Zhou, S., 2006. Stoichiometric analysis of combined reaction of anaerobic ammonia oxidation with denitrification. Journal of South China University of Technology (Natural Science Edition) 34,1-4. Zhao. Y., 2010.Isolation and characterization of three heterotrophic nitrifiers and its application in aquaculture wastewater treatment. Zhejiang University. (in Chinese)． Zhang, L., Narita, Y., Gao, L., Ali, M., Oshiki, M., Okabe, S., 2017. Maximum specific growth rate of anammox bacteria revisited. Water Res. 116, 296–303. https://doi.org/10.1016/j.watres.2017.03.027 Zhang, L., Okabe, S., 2017. Rapid cultivation of free-living planktonic anammox cells. Water Res. 127, 204–210.https://doi.org/10.1016/j.watres.2017.10.029 Zhang, Z., Li, D., Zhang, J., Guo, Y., Li, S., 2017. Effect of Seeding Single/Mixed Sludge on Rapid Start-up of an ANAMMOX Reactor. Environmental Sci.38, 5215-5221. https://doi.org/10.13227/j.hjkx.201705289

(a) Immobilized system (b) Contrast test of free sludge Fig. 1 Device for anammox restoration culture device

(a) Nitrogen loading rate, TN effluent and nitrogen removal efficiency

(b) Fitting curve of the TN removal efficiency from days 1 to 55

Fig. 2 Nitrogen removal performance of immobilized filler with different biomass (percent of anammox bacteria: E1, 2%; E2, 4%; E3, 6%; E4, 8%)

Fig. 3 Nitrogen loading rate, TN effluent and nitrogen removal efficiency of S1

Fig. 4 Nitrogen removal performance of encapsulated anammox filler under higher load

Fig. 5 SEM images of immobilized carrier during the stable period; (a) surface×500 k; (b) surface×2000 k; (c) inside×5000 k; (d) inside×10000 k

Fig. 6 16SrDNA gene concentration of anammox bacteria at the start and end points (E2i: E2 start point; E2e: E2 end point; E4i: E4 start point; E4e: E4 end point)

Fig. 7 Relative abundance of the microbial community in the sample; (a) phylum level; (b) genus level (S1: suspended sludge before entrapprd; E2a: E2 sample on day 30; E2b: E2 sample on day 99)

Fig. 8 Interference of organic carbon source on the immobilized system; (a) contribution rate of anammox-denitrification, change in NO3--N concentration and removal efficiency of COD; (b) the removal efficiency of NH4+-N, NO2—N; (c) nitrogen conversion ratio: △NO3--N/△NH4+-N,△NO2--N/△NH4+-N

Fig. 9 Influence of COD interference on the structure of encapsulated anammox bacteria; (a) phyla level; (b) genus level

Fig.10 Biological nitrogen removal kinetics of the immobilization filler (a) NH4+ -N fitting equation; (b) NO2－–N fitting equation

Fig. 11 Restoration of starvation inhibition; (a) immobilized filler; (b) free sludge

Table 1 Operation parameters of the anammox reactor during the enrichment process Period

t (d)

Sample

HRT (h)

Temperatur

NH4+–N (mg·L -1)

NO2-–N (mg·L -1)

e (℃) Ⅰ

1-100

E1，

22

32±1

100-330

100-330

11

32±1

250

300

E2， E3， E4， S1 Ⅱ

101-11

E2

0 Ⅲ

111-12

E2

5

32±1

250

300

0 Ⅰ: Restoration culture stage of immobilized anammox filler and suspended sludge; Ⅱ,Ⅲ: Further improving nitrogen removal efficiency of E2 by shortening HRT

Table 2 Operation parameters of batch experiments Item

Time

Sample

(d)

HRT

NH4+–N

NO2-–N

COD

(h)

(mg·L-1)

(mg·L-1)

(mg·L-1)

Ⅰ

0-20

E2

11

250

330

100-400

Ⅱ

0-5

E2

11

200

250

-

Ⅲ

6-10

E2

11

250

300

-

I: Effects of organic matter; II,III: Starvation recovery.

Table 3 Variation of sample diversity indices during different periods Sample

ID

Seq num

OUT

num

ACE index

Chao1 index

Simpson

（97%） S1（0d） E2a（30d） E2b（99d） E2c（+COD）

62752

2866

44075.09

20105.93

0.05

83621

3347

54022.82

25011.78

0.07

60612

1938

25220.11

11434.53

0.12

48495

12005

1172800.55

321026.56

0.03

HIGH LIGHTS  PVA-PP immobilized filler can realize enrichment and fast start-up of anammox.  PVA-PP immobilized carrier maintained a good microbial structure.  Copies of anammox gene in different biomass carriers were similar after operation.  Entrapped anammox can be coupled with denitrifying bacteria for nitrogen removal. The entrapped anammox exhibited good nitrogen removal kinetics.

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.