Granules abrasion cause deterioration of nitritation in a mainstream granular sludge reactor with high loading rate

Chemosphere 243 (2020) 125433

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Granules abrasion cause deterioration of nitritation in a mainstream granular sludge reactor with high loading rate Wenru Liu a, b, *, Fangfang Yin c, Dianhai Yang d a National & Local Joint Engineering Laboratory for Municipal Sewage Resource Utilization Technology, Suzhou University of Science and Technology, Suzhou, 215009, China b School of Environmental Science and Engineering, Suzhou University of Science and Technology, Suzhou, 215009, China c Suzhou Jing Yan Environmental Protection Technology Co. Ltd, Suzhou, 215009, China d State Key Laboratory of Pollution Control and Resources Reuse, Tongji University, Shanghai, 200092, China

h i g h l i g h t s
High-rate nitritation with granular sludge was achieved at mainstream conditions.
Granules abrasion-based deterioration of nitritation was firstly observed.
Detachment of AOB resulted in incomplete stratification of nitrifiers in granules.
Granules abrasion must be well controlled in high-rate granular nitritation system.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 August 2019 Received in revised form 15 November 2019 Accepted 20 November 2019 Available online 21 November 2019

Biomass detachment generally occurred in granular sludge systems. However, little is known about the influence of biomass detachment on the granules performing nitritation. Here, a granular sludge reactor with high loading rates (6.8 ± 0.4 kg N$m3$d1) was achieved at mainstream conditions. Though the low ratio control strategy was maintained, the deterioration of nitritation performance was observed after the further increase of air supply rates to 3.4 ± 0.2 L min1. In parallel with that, the loss of AOB and the proliferation of NOB was observed. Additionally, with the decrease of granules size and biomass concentration, the incomplete stratification of nitrifiers in the granules was confirmed by batch tests. All these results suggested that granules abrasion under the high shear stress conditions caused the detachment of external AOB and hence resulted in the deteriorated stratified structure of nitrifiers, which subsequently contributed to the proliferation of the internal NOB and the deterioration of nitritation. These findings highlight that the granules abrasion should be well controlled in the development of high-rate nitritation process with granular sludge. © 2019 Elsevier Ltd. All rights reserved.

Handling Editor: A Adalberto noyola Keywords: Mainstream Nitritation Granular sludge Abrasion Stratification

1. Introduction Compared with traditional nitrification/denitrification, nitritation coupled with denitritation or anaerobic ammonium oxidation (Anammox) are promising alternatives for achieving efficient nitrogen removal from sewage via nitrite pathway (Winkler and Straka, 2019). The nitritation is the prerequisite for nitrogen removal via nitrite since sewage nitrogen is present mainly as

* Corresponding author. National & Local Joint Engineering Laboratory for Municipal Sewage Resource Utilization Technology, Suzhou University of Science and Technology, Suzhou, 215009, China. E-mail address: [email protected] (W. Liu). https://doi.org/10.1016/j.chemosphere.2019.125433 0045-6535/© 2019 Elsevier Ltd. All rights reserved.

ammonia (NHþ 4 and NH3). The key to achieve nitritation is the selective elimination or inhibition of nitrite oxidizing bacteria (NOB: oxidizing nitrite to nitrate) while retaining the ammonium oxidizing bacteria (AOB: oxidizing ammonium to nitrite). Many approaches have been proposed to realize NOB elimination or inhibition treating sewage or synthetic low-strength wastewater, such as high dissolved oxygen (DO) (Law et al., 2019), aeration duration control (Kouba et al., 2017; Yang et al., 2007), aggressive solids retention time (SRT) in combination with transient anoxia or aerobic starvation (Ge et al., 2014; Liu et al., 2017; Regmi et al., 2014), low ratio between DO and residual ammonium concentration (also named ratio control) (Bian et al., 2017; Isanta et al., 2015; Poot et al., 2016), and side-stream sludge treatment using free

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List of abbreviations Anammox AOB AOR ARE DO FA FISH FNA HRT NAE NOB OTU SAUR SNUR SRT TN TSS VSS

Anaerobic ammonium oxidation Ammonium oxidizing bacteria Ammonium oxidation rate Ammonium removal efficiency Dissolved oxygen Free ammonia Fluorescence in-situ hybridization Free nitrous acid Hydraulic retention time Nitrite accumulation efficiency Nitrite oxidizing bacteria Operational taxonomic units Specific ammonium uptake rate Specific nitrite uptake rate Solids retention time Total nitrogen Total suspended solids Volatile suspended solids

nitrous acid (FNA) and/or free ammonia (FA) (Duan et al., 2019; Jiang et al., 2019; Wang et al., 2014, 2017). Among those technologies, the ratio control generally applied in biofilm and granular sludge systems is increasingly regarded as effective and promising strategy for achieving mainstream nitritation. The advantages of the ratio control strategy are obvious, mainly including the fast start-up of nitritation function, the chronically stable run with high volumetric loading rate (even at low temperatures), and the effluent suitable for a subsequent anammox step (Bian et al., 2017; Isanta et al., 2015; Poot et al., 2016; Reino et al., 2016). Both high biomass concentration and high specific activity can be obtained in the nitritation granular sludge reactor. Recent studies indicated that stratification of nitrifier guilds in granular sludge with AOB-dominated outer shell and NOBoccupied inner layer played an important role in achieving nitritation through ratio control (Picioreanu et al., 2016; Poot et al., 2016; Soler-Jofra et al., 2019). The stratified structure means that the external AOB would be easier to detach from granules under certain operating conditions, whereas the internal NOB are protected against detachment (Poot et al., 2016; Winkler et al., 2012). Therefore, the detachment process would make the SRT of AOB lower than that of NOB. Furthermore, the detachment process would cause the deterioration of the stratification, which in turn influences the overall performances and the stability of the nitritation granular sludge systems. The detachment of biomass from biofilm and granular sludge has been widely investigated (Elenter et al., 2007; Gjaltema et al., 1997c; Paul et al., 2012; Ren et al., 2009). The detachment force is generally attributed to shear stress, which comes from gas or liquid flow and particle-particle collision (Derlon et al., 2008). Particularly for the aerobic granules (including nitrifying granules), enough hydrodynamic shear force is also needed in the formation and performance of compact granules (Liu and Tay, 2002). Analogously, the detachment would occur in the nitritation granules systems especially under the high loading rate conditions. However, till now little attention has been paid to the influence of biomass detachment on the nitritation granular process. The lack of attention could be due to the applied loading rates and shear stress not high enough and the influence of biomass detachment on process performance unremarkable. In our recent study, an aerobic granular sludge reactor

performing nitritation with extremely high loading rate (6.8 kg N$m3$d1) at low temperatures (<20  C) was achieved (Liu et al., 2019). However, a further increase of the air supply rate or DO concentration (that was, the increase of shear stress) resulted in the deterioration of nitritation performance (this study). We hypothesize that the deterioration of nitritation was related closely with the detachment of biomass from the granules surface. Here, we present findings that support the hypothesis. The results of the study could provide valuable insight in the development and operation of highrate and stable nitritation process with granular sludge.

2. Materials and methods 2.1. Reactor operation and monitoring The experiments were carried out in a lab-scale integrated reactor with 1.7 L working volume performing partial nitritation, and a zone with 0.6 L of volume was employed to serve as sedimentation unit (Fig. 1). Before the present study, the reactor had been run for more than four months with effective partial nitritation performance. Inoculum, start-up and stable operation before this study were described in a previous paper. The influent flow rate was maintained at 10 L h1, that corresponded to HRT of 0.17 h. Compressed air was introduced by a fine bubble diffuser placed at the bottom of the reactor. Air flow rates were regulated manually according to the process performance, and concurrently keeping an ammonium to oxygen ratio (below 0.3) in the effluent adequate for NOB inhibition as described previously. Fed was composed of a low-strength synthetic wastewater with the following composition (per liter of tap water): NH4Cl 0.21 g, KH2PO4 0.025 g, CaCl2$2H2O 0.02 g, ZnSO4$7H2O 0.025 g, and 1 mL of trace element solution. The trace elements solution contained (per liter of demineralized water) EDTA 10 g, ZnSO4$7H2O 0.45 g, CoCl2$6H2O 0.25 g, MnCl2$4H2O 1.0 g, CuSO4$5H2O 0.25 g, (NH4)6Mo7O24$4H2O 0.2 g, NiCl2$6H2O 0.2 g, H3BO3 0.015 g and Na2WO4$2H2O 0.05 g, adapted from Poot et al. (2016). The ammonium-N concentration in the feed was around 55 mg N$L1. No organic matter was added into the influent in order to minimize the effect caused by heterotrophic microorganism. The pH in the reactor ranged from 7.0 to 7.5, controlled by a buffer containing NaHCO3. The reactor was run under actual room temperature, and no sludge was deliberately removed from the reactor throughout the operation. The SRT was not controlled throughout the experimentation, and sludge loss only occurred

Fig. 1. Schematic diagram of the reactor set-up.

W. Liu et al. / Chemosphere 243 (2020) 125433

30

8

DO ARE

AOR NAE

120

5

105

20

10

0

7

4

6

3

2

5 0

10

20 30 40 Time (days)

50

60

90 60

ARE, NAR (%)

-1

Temperature

-3

Representative biomass samples were obtained respectively from the granular sludge reactor over time to assess the microbial community through high-throughput sequencing. Each sample contained about 15e20 mL wet granules. Additionally, the suspended solids in the effluent were also collected during the days 20e23 (about 8 mL sludge) for analysis the effluent microbial composition. The following primers were used: forward primer 338 F (50 -ACTCCTACGGGAGGCAGCAG-30 ) and reverse primer 806 R (50 -GGACTACHVGGGTWTCTAAT-30 ). DNA extraction, polymerase chain reaction and Illumina Miseq sequencing protocol are fully described in our previous study (Liu et al., 2018). The Illumina MiSeq reads of the 16 S rRNA amplicon sequencing have been deposited to the NCBI Sequence Read Archive with the accession number of SRR9641092 under the BioProject of PRJNA552422. The obtained high-quality sequences were clustered into operational taxonomic units (OTUs) with a 97% similarity using the MOTHUR program. Sequences were also assigned to corresponding taxonomic ranks via the RDP classifier (80% confidence threshold) using

The reactor was previously operated at room temperatures (2010  C), with HRT of 0.4e0.17 h, for approximately three months performing stable partial nitritation. In the present study partial nitrification at 15-10  C was maintained from day 1e15 with an average loading rate of 6.8 ± 0.4 kg N$m3$d1. Efficient NOB repression was stably maintained under the mainstream conditions. The effluent concentrations of ammonium, nitrite and nitrate in the reactor were 27 ± 3, 24 ± 2 and 0.4 ± 0.3 mg N$L1. Since the effluent of the partial nitritation reactor would be fed into a subsequent anammox process, the nitrite will be always in excess. From day 16 higher effluent nitrite concentration (or nitrite/ ammonium concentration ratio to 1.3) was targeted by increasing the DO concentrations (from 5.9 ± 0.3 to 6.8 ± 0.2 mg O2$L1) or the air supply rates (from 2.8 ± 0.2 to 3.4 ± 0.2 L min1). Afterwards, deterioration of nitritation was observed unexpectedly. As shown  in Fig. 2, the nitrite accumulation efficiency (NAE ¼ NO 2 -N/NOx N  100%) grandually decreased from almost 100% to below 80% within two weeks. Moreover, both the ammonium removal efficiency (ARE) and the ammonium oxidation rates (AORs) respectively decreased to 2.8 kg N$m3$d1 and 37% on day 28. All these results indicated that the further increase of the DO concentration or the air supply rate resulted in the deterioration of the high-rate nitritation granular sludge reactor. The reasons behind the deterioration of the nitritation performance will be described in following parts. From day 28 onwards, the air supply rate was reduced to maintain the DO concentrations near to 6.0 mgO2$L1. After the change, a gradual-recovery of the nitritation performance was observed. The NAE recovered to 98% within two weeks and then the excellent nitritation performance was stably maintained. Interestingly, the value of AREs and AORs were also grandually increased in parallel with the recovery of NAE during the days 30e42. Owing to the slightly warmer temperature after the day 42, the DO concentrations were further reduced to approximately 5.5 mgO2$L1 to limited the AORs ensuring enough residual ammonium concentrations (>20 mg N$L1). Overall, the excellent performance of high-rate nitritation was reestablised.

AOR (kg N·m ·d )

2.3. Microbial analysis

3.1. Reactor performance

-1

Batch-tests were performed in the same (integrated) reactor used for the continuous operation (hence namely in-situ batch tests) to investigate the nitrifying characteristics of the granular sludge along with the reactor performance during continuous operation. Moreover, in-situ batch tests could be easily used for diagnosis of the stratification of nitrifier guilds in granular sludge as promoted by previous study (Soler-Jofra et al., 2019). During each in-situ batch-test, the continuous operation was stopped and an ammonium pulse was added into the reactor. The DO concentration, temperature and pH were controlled according to the value of the reactor continuous operation. Concentrations of TN, ammonium, nitrite and nitrate were measured after certain time intervals. Batch tests were also conducted in some identical reactors with the working volume of 0.5 L (namely ex-situ batch tests) to measure the maximum specific ammonium uptake rate (SAURm) and the maximum specific nitrite uptake rate (SNURm) of the granular sludge. Nitrifying granules obtained from the continuously operated reactor was inoculated to these batch-test reactors. The synthetic wastewater containing low-strength ammonium (25 mg  1 1 NHþ 4 -N$L , for SAURm measurement) or nitrite (15 mg NO2 -N$L , for SNURm measurement) was used as influent to carry out the exsitu batch tests. During each batch test, the DO was maintained above 9 mgO2$L1. The pH was controlled at 7.5 ± 0.2 b y addition of a buffer containing NaHCO3. Temperature was not controlled during each test. At the end of each test, the VSS were determined, and the ammonium and nitrite oxidizing capacity were determined from the slopes of the decreasing ammonium and increasing nitrate profiles, respectively.

3. Results

DO (mg O2·L )

2.2. Batch tests

the SILVA reference databases. The relative abundance of a given phylogenetic group was set as the number of sequences affiliated with that group divided by the total number of sequences per sample.

Temperature (°C)

through washout in the effluent and sampling. Liquid samples were filtered through a 0.45 mm syringe filter before analyses. Concentrations of nitrogenous compounds,   namely, total nitrogen (TN), NHþ 4 -N, NO2 -N, NO3 -N, were analyzed 3e5 times a week according to the standard methods (APHA, 2005). In terms of biomass concentration, total suspended solids (TSS) and volatile suspended solids (VSS), were measured biweekly. Temperature, DO and pH in the bulk liquid was determined by means of a portable multi-parameter meter (HACH, Düsseldorf, Germany). Particle size distributions were measured by a laser particle size analysis system (Malvern MasterSizer Series 3000 h, Malvern instruments Ltd., UK). Every sample was measured in triplicate.

3

45 30

Fig. 2. Profile of ammonium oxidation rate (AOR), ammonium removal efficiency (ARE) and nitrite accumulation efficiency (NAE) of the granular sludge reactor under high DO (DO > 5.0 mg O2$L1) and low temperatures (10e15  C) conditions.

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Note that although the granular sludge reactor was operated under the high DO concentrations, a 10e20% total nitrogen loss was observed during the experimentation with the possibility of biomass assimilation or denitrification/anammox activity taking place in the granules. Regarding the biomass concentration, the obtained MLSS concentrations were 10.1 ± 0.6 (on day 1), 10.3 ± 0.4 (on day 12), 9.4 ± 0.4 (on day 26), 10.5 ± 0.3 (on day 42), and 10.4 ± 0.4 gSS $L1 (on day 55) respectively. In other words, from day 12e26, a decrease of the biomass concentration occurred. But on day 42, the biomass concentration returned to the same level (10.3e10.7 gSS$L1) as before and afterwards was maintained stably.

Kuenenia

Day 10 Day 28 Day 40

Jettenia Brocadia Nitrotoga Nitrospira Nitrosomonas

3.2. Proliferation of NOB in the granules

0.0

SAURm or SNURm (g N·g-1 VSS·d-1)

The deterioration of nitritation or the increased effluent nitrate concentrations indirectly indicated the reactivation and proliferation of the NOB in the granules during the days 16e28. This was further confirmed with the increased values of the SNURm and the relative abundance of NOB populations. As shown in Fig. 3, the value of SNURm reached up to 0.085 g N$g1VSS$d1 on day 28, which nearly increased by ten times compared with that on day 10. Even after the recovery of excellent nitritation, the value of SNURm remained as high as 0.07 g N$g1VSS$d1 on day 40. In good agreement with the observed SNURm, high throughout sequencing results showed a significant proliferation of the NOB, as presented in Fig. 4. The relative abundance of NOB (genus Nitrospira and Nitrotoga) increased from approximately 0.5% on day 10 to over 4.5% on day 28. In particular, the genus Nitrospira was significantly enriched with the relative abundance up to 3.3% of the total reads. All these observations demonstrated the recovery of NOB activity and growth following the increase of air supply rates or DO concentrations during the days 16e28. In other words, the NOB generally located in the inner layers of granular sludge could get oxygen for the nitrite oxidation, though the DO/ammonium concentrations ratio still remained as low as before. Note that, after the reestablishment of excellent nitritation performance, the relative abundance of NOB remained over 4% on day 40, suggesting the need of a long time for complete washout of NOB from the granular sludge.

0.75

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40

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Relative abundance at genus level (%) Fig. 4. Relative abundances of AOB, NOB and anammox bacteria in the granules at genus level.

3.3. Loss of AOB from the granules Note that the AOR and ARE also decreased along with the deterioration of nitritation (Fig. 2). Since the influent composition and the reactor operating conditions (e.g. pH and temperatures) were almost as before, the decrease of AOR and ARE under higher DO concentrations should be attributed to the loss of AOB populations from the reactor. The loss of AOB was also supported by the following experimental evidence: Firstly, large amounts of AOB was observed in the effluent. As presented in Fig. 5, the genus Nitrosomonas dominated the bacterial community in the effluent with a relative abundance of 57.7%. The observed Nitrosomonas in the effluent also suggested that the detachment of AOB from granules surface did occurred. Secondly, the loss of AOB was supported by the decreased SAURm of the granules. As shown in Fig. 3, the value of SAURm decreased by nearly 30% on day 28 compared with the day 10. More directly, the loss of AOB was confirmed by the reduced abundance of AOB in the granules. The relative abundance of AOB (genus Nitrosomonas) reduced from over 60% on day 10 to below 45% on day 28 (Fig. 4). Therefore, in addition with the decrease of biomass concentrations and the reactivation and proliferation of NOB, the loss of AOB did occur unexpectedly after the increase of the DO concentrations. One should be pointed out is that the

Day 10 Day 28 Day 40

0.60 0.45 0.10 0.05 0.00 SAURm

SNURm

Fig. 3. The SAURm and SNURm for the granular sludge.

Nitrosomonas Aquimonas Simplicispira Subgroup_7_norank Limnobacter Subgroup_17_norank Aquicella Unclassified

Brevundimonas Rhodobacter Denitratisoma Thermomonas OPB56_norank TK10_norank Latescibacteria_norank Others

OM190_norank Subgroup_6_norank Blastocatella PHOS-HE36_norank Truepera KD4-96_norank Ignavibacterium

Fig. 5. Bacterial composition in the effluent at genus level during the days 20e23.

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growth of AOB incessantly proceed with the oxidation of ammonium throughout the duration of this study. However, the rate of AOB detachment and washout seemed to be higher than that growth after the increase of the DO concentrations to 6.8 ± 0.2 mgO2$L1. Consequently, the above mentioned decrease of ARE and AOR (Fig. 2) coincided with the loss of AOB from the granular sludge reactor was observed during the days 16e28. With the recovery of nitritation performance after the DO fell back to 6.0 ± 0.2 mgO2$L1, partly increase of the SAURm and the AOB abundance was observed on day 40, suggesting that the growth rate of AOB became higher than that loss after the day 28.

5

incomplete stratification of nitrifiers in the granules. To estimate the extent of stratification of nitrifiers, in-situ batch tests was conducted as proposed by Soler-Jofra et al. (2019). As Poot et al. (2016) and Soler-Jofra et al. (2019) reported that there was a switch from a phase at low nitrate production rate to a phase at high nitrate production rate just after ammonium was depleted for the granules with well stratified nitrifiers, whereas the time course concentration of nitrate is different when both nitrifier guilds are mixed. In concordance with the findings, the performance of the batch-tests performed on day 12 and day 42 clearly suggested the well stratification of nitrifier guilds in the granular sludge (Fig. 7A and C ). For the granules obtained on day 26, however, only a weak

Percentage (%)

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TSS or VSS (g·L-1)

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Fig. 6. Size distribution of the granular sludge.

30 45 Time (min) -

NO2

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Since the rate of AOB detachment was faster than that growth during the days 16e28, the on-going detachment should cause a reduction in the granular sludge size and the biomass concentration. For the purposes, we compared the granular sludge size distribution and the biomass concentration in the reactor on day 12 and day 26. Clearly, the decrease of both TSS and VSS concentrations were observed in Fig. 6B. Additionally, the granules exhibited a reduction in average diameter, with a volume weighted mean of 1013 mm on day 12 and 967 mm on day 26 respectively (Fig. 6A). Aerobic granular sludge performing partial nitritation generally had stratified spatial distribution of nitrifiers (Poot et al., 2016; Soler-Jofra et al., 2019). Since the AOB generally located on the surface layer of the nitritation granules, the reduction of granules size also meant the decreased thickness of the AOB layer. Furthermore, the detachment of external AOB layer would result in the

N-concentrations (mg N.L )

3.4. Incomplete stratification of nitrifiers

15

30 45 Time (min)

60

Fig. 7. In-situ batch-tests performed (A) at day 12 with DO at 6.0 ± 0.2 mg O2$L1, (B) at day 26 with DO at 6.8 ± 0.3 mg O2$L1, and (C) at day 42 with DO at 5.5 ± 0.3 mg O2$L1.

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switch of the time course of nitrate concentrations was observed, with a relatively high nitrate production rate also before ammonium is depleted (Fig. 7B). Consequently, the granules obtained on day 26 had an incomplete stratified structure.

4. Discussion As mentioned above, the deterioration of nitritation in the granular sludge reactor was observed during days 16e28, though the residual ammonium concentration (>25 mg N$L1) and the DO/ ammonium concentration ratio (<0.25) was well controlled. Firstly and obviously, the deterioration of nitritation or the increased effluent nitrate concentrations should not be attributed to the anammox reaction, because only 10e20% total nitrogen loss was observed during the experimentation under such high DO concentrations, suggesting the limited anammox reaction though their relative abundance reached 4e6% (Fig. 4). Note that the operating temperatures during this study were as low as 10e15  C. Previous studies suggested that the residual bulk ammonium concentration or the setpoint of the DO/TAN ratio for achieving and maintaining nitritation was significantly affected by the operating temperatures (Jemaat et al., 2013; Soler-Jofra et al., 2019). Based on these reports, the deterioration of nitritation in the present study was caused possibly by the low temperatures. To be specific, the low temperatures resulted in the given DO concentrations, residual bulk ammonium concentrations or the values of DO/ammonium concentration ratio were not appropriate for nitritation anymore. However, previous studies demonstrated that in case of a granular sludge with stratified nitrifier guilds structure and well DO/ ammonium concentrations ratio control or enough residual ammonium concentration, an transient increase in DO concentration not caused a deterioration of nitritation for the granular sludge system (Bartrolí et al., 2010; Poot et al., 2016). Moreover, the recovery of the nitritation was achieved during the days 30e58 still at temperatures 10e16  C without any changes of the set-point of the residual ammonium concentrations (>20 mg N$L1) and the DO/ ammonium concentration ratios (<0.25). Hence, the deterioration of nitritation in the granular sludge reactor should be associated with other factors rather than the low temperatures or the DO concentration increase. As stated by Soler-Jofra et al. (2019), strategies to repress the NOB activity and minimize the NOB abundance in granular sludge reactors not only focused on the optimization of the DO concentrations and sufficient excess of ammonium (or the low DO/ ammonium concentrations ratio), but also enhanced and maintained the complete stratification of nitrifier guilds in granules. The stratification of nitrifier guilds in granular sludge with AOBdominated outer shell and NOB occupying inner layers had been previously demonstrated (Poot et al., 2016; Soler-Jofra et al., 2019). A complete and dense AOB layer on the granule surface seemed to be a necessary condition to obtain high nitritation in the granular sludge or biofilm reactors (Liu and Yang, 2017; Picioreanu et al., 2016; Soler-Jofra et al., 2019). Under the well control of the residual bulk ammonium concentrations or the DO/ammonium concentration ratios, the external AOB layer would consume the limited oxygen resulting in the asphyxia of internal NOB even at rez et al., 2014). Building on low temperatures (Isanta et al., 2015; Pe these information sources, the observed deterioration of nitritation in the present study should be due to the deteriorative stratification of nitrifier guilds. In fact, in this study the deteriorative stratification of nitrifier guilds was also confirmed by the results obtained from the in-situ batch tests (Fig. 7). The question is what made the deteriorative stratification. The biggest characteristic of the granular sludge reactor is the

extremely high loading rate performance. In other words, the granular sludge reactor was operated under a very short HRT (0.17 h) with high air supply rate (3.4 ± 0.2 L min1) and biomass concentration (10.4 gTSS$L1). Analogously to the detachment of biomass from suspended biofilm in airlift reactors (Chen et al., 2014; Gjaltema et al., 1997a, 1997b, 1997c; Jin et al., 2008), granules abrasion should be the main reason that caused the on-going detachment of biomass from granules surface. According to the Ren et al. (2009) reported, the gas bubble and the granules collision should be the main source for the granules abrasion. The detachment of biomass was consistent with the observed decrease of the particle size (Fig. 6A) and the loss of AOB abundance (Fig. 4), since AOB mainly located on the surface layer (Poot et al., 2016; SolerJofra et al., 2019). The reduced value of ARE, AOR (Fig. 2) and SAURm (Fig. 3) also supported the detachment and washout of AOB from the granules. The biomass detachment from granules surface and the reduction of granules size suggested the decreased thickness of external AOB layer and/or resulted in the incomplete stratification of nitrifiers. The thinner AOB layer or the smaller granular sludge size would cause an increased oxygen flux or oxrez ygen penetration depth into the granules (Jemaat et al., 2013; Pe et al., 2014). Consequently, the thinner or incomplete AOB layer coupling with the less oxygen consumption by the outer AOB made oxygen available for the inner NOB and allowed for the NOB reactivation and regrowth (Joss et al., 2011; Poot et al., 2016; Soler-Jofra et al., 2019). The reactivation and proliferation of NOB was confirmed by the increased value of the SNURm (Fig. 3) and the relative abundance of NOB (Fig. 4). In summary, the deterioration of partial nitritation or the proliferation of NOB resulted from the deteriorated (thinner or incomplete) stratification of nitrifier guilds in the granular sludge. More specially for this study, the granules abrasion under the high shear stress conditions with high biomass concentration and high air supply rate led to the detachment of AOB from granular sludge surface. The detachment of external AOB resulted in the deteriorated stratification of nitrifier guilds in the granular sludge. To the best of our knowledge, this is the first report about the granules abrasion-based deterioration of nitritation. The results from this study indicated that the granules abrasion or the detachment of external AOB layer should be considered for the operation and modeling of high-rate nitritation systems particularly at low temperatures. Note that the operating time of the high-rate nitritation granular sludge reactor at low temperatures was not long enough, and the components of the influent wastewater was simple and didn’t contain organic matter. As is well-known, growth of heterotrophic bacteria will be supported by organic carbon in influent and have impact on the autotrophic granules structure and their microbial community composition. Therefore, further research is needed to get a more comprehensive view of the high-rate nitritation granular sludge system.

5. Conclusions
Nitritation with extremely high loading rate (6.8 ± 0.4 kg N$m3$d1) could be achieved in the granular sludge reactor at mainstream conditions by applying the ratio control.
Further increase of air supply rates (3.4 ± 0.2 L min1) caused the deterioration of nitritation in the granular sludge reactor with high biomass concentrations (>10 gTSS$L1), though the ratio control was maintained.
Granules abrasion under the given conditions caused the detachment of the external AOB and hence resulted in the incomplete stratification of nitrifiers in the granules, so that

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