Start-up and operational performance of Anammox process in an anaerobic baffled biofilm reactor (ABBR) at a moderate temperature

Accepted Manuscript Start-up and operational performance of Anammox process in an anaerobic baffled biofilm reactor (ABBR) at a moderate temperature Tao Wang, Xian Wang, Luzi Yuan, Zheng Luo, Hengue Kwame Indira PII: DOI: Reference:

S0960-8524(19)30138-5 https://doi.org/10.1016/j.biortech.2019.01.114 BITE 20991

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

23 December 2018 22 January 2019 23 January 2019

Please cite this article as: Wang, T., Wang, X., Yuan, L., Luo, Z., Kwame Indira, H., Start-up and operational performance of Anammox process in an anaerobic baffled biofilm reactor (ABBR) at a moderate temperature, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.01.114

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Start-up and operational performance of Anammox process in an anaerobic baffled biofilm reactor (ABBR) at a moderate temperature *

Tao Wang , Xian Wang, Luzi Yuan, Zheng Luo, Hengue Kwame Indira Department of Environmental Engineering, School of Energy and Environmental Engineering, Hebei University of Technology, Tianjin 300401, PR China * Corresponding author. Tao Wang, School of Energy and Environmental Engineering, Hebei University of Technology, Tianjin 300401, PR China. Tel: +86-22-60435775 E–mail address: [email protected]

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Abstract A lab-scale anaerobic baffled biofilm reactor (ABBR) was used as a novel reactor to start up Anammox process at a moderate temperature around 20°C and an innovative filling module was adopted as support material. Quick start-up of Anammox process from the aerobic activated sludge was achieved after 47 days operation. The max nitrogen loading rate and nitrogen removing rate attained 1.00 kg N m-3 d-1 and 0.90 kg N m-3 d-1 after 161 days operation. Scanning electron microscope photographs showed that the structure as well as the states of the micro-aggregates (microaggregates sticking on a non-woven fiber, entangling non-woven fibers and enwrapped by non-woven fibers) enhanced biomass retention for Anammox bacteria. Microbial community analysis showed that Anammox bacteria were effectively enriched with Candidatus Brocadia, Candidatus Jettenia and Candidatus Kuenenia being the main Anammox species in the mature biofilms. This contributed to the excellent Anammox operation performance at the moderate temperature. Keywords: ABBR; Anammox process; start-up and operation; moderate temperature

1. Introduction Nitrogen pollution in water bodies has become one of the serious environmental problems no matter in developing countries or in developed countries. Ammonium, a common nitrogen species usually present in wastewater, accelerates eutrophication of water bodies causing frequent occurrences of ecocatastrophes such as water bloom or red tide (Le et al., 2010; Mohamed, 2018). Algal toxin produced in the process of water bloom or red tide is enriched in water bodies and aquatic organisms, making its way 2

into food chains. As a result, ammonium in wastewater indirectly threatens food safety and destroy ecological environment indirectly (Mohamed, 2018; Quilliam, 2003). Thus, removing ammonium from wastewater plays a key role in the control of water eutrophication and the protection of aquatic ecological environment. Notably, several kinds of ammonium-rich wastewater, such as fertilizer manufacturing wastewater and leachate from agriculture and landfill, need to be properly treated. Nitrogen removal in municipal, agricultural and industrial wastewaters is routinely carried out by combining two bioprocesses — nitrification and denitrification. As more stringent standards for wastewater discharge have been enforced recently, this traditional nitrogen removal technologies can’t afford to treat the ammonium-rich wastewater (Du et al., 2015; Jin et al., 2014). As novel bioprocesses such as anaerobic ammonium oxidation (ANAMMOX) and partial nitrification (PN) have emerged, combination of PN and ANAMMOX provides a good alterative to the traditional biological process for nitrogen removal (Du et al., 2015; Li et al., 2014; Zhang et al., 2010). PN-ANAMMOX process has many advantages such as achieving high nitrogen removal capability, saving around 50% oxygen supply, requiring no external organic carbon source, producing low excess sludge and reducing greenhouse gas emission (Joss et al., 2009; Zhang et al., 2010). It has therefore been developed into a highly-efficiency and low-consumption autotrophic biological nitrogen removal technology suitable for treatment of the ammonium-rich wastewater. Until now, start-up of Anammox process is still one bottle neck due to the

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extremely slow growth rate and the high environment sensitivity of Anammox bacteria (Ali et al., 2015; Jin et al., 2012a; Ma et al., 2016; Saleem et al., 2018). The relatively long start-up period of Anammox process restricts wide applications and deeply researches of PN-ANAMMOX process (Kuenen, 2008; van der Star et al., 2007). As a result, an increasing number of researches focus on quick start-up of Anammox process. To shorten the start-up period of Anammox process, selection of reactor configuration is important. The selected reactor should adequately retain biomass and easily maintain specific conditions (ecological niche for Anammox bacteria) (Ma et al., 2016). Anaerobic baffled reactor (ABR) is considered as a suitable reactor configuration for Anammox start-up process because of efficient biomass retention, simple condition control and stable long-term operation. Besides, ABR exhibits a certain tolerance to shock loads. Jin et al. (2012) found that a lab-scale ABR for Anammox operation was insensitive to transient hydraulic shocks but sensitive to transient substrate shocks. However, the researchers also found that the first compartment was more susceptible to substrate shocks compared with other compartments (Jin et al., 2012b). Moreover, in an ABR, a spot of biomass can flow out from the former compartment to the latter compartment and may get into the effluent. Especially when sludge bulking appears, wash-out of a considerable quantity of biomass may influence the reactor performance and the effluent quality with respect to pollutant concentration and suspended substance (Jin et al., 2012b). To further shorten Anammox start-up period and enhance Anammox operation performance, ABR is still needed to be improved from the angle of enhancing

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biomass retention for Anammox bacteria. Biofilm formation is expected to significantly reduce wash-out of biomass, to effectively control sludge bulking, and to obviously improve environmental tolerance such as the tolerance to changes of temperature and accumulation of free ammonium or nitrite (Gilbert et al., 2015). Using biofilm instead of sludge floc may be expected to enhance retention of Anammox bacteria and further improve Anammox start-up and operation performance of ABR. Anaerobic baffled biofilm reactor (ABBR) can be modified from ABR by filling carriers. Some kinds of carriers or support material can be filled in the ABR to promote biofilm formation. In this case, ABBR is established. ABBR may be developed as a novel and robust Anammox reactor, which embrace the superiorities of both ABR and biofilm reactor. Until now, there has been no report on employing ABBR to start up and operate Anammox process. For a biofilm reactor, selected carriers or support material influences formation, evolution and renewal of biofilm and thus influences the reactor performance. Similarly, for ABBR considered as a novel type of biofilm reactor, selected carriers or support material is also a key factor influencing the reactor performance. Nonwoven carriers can achieve high retention of Anammox bacteria but can cause difficulties of substrate transferring if Anammox biofilms attached on the carriers are too thick. Honeycomblike carriers has the porous structure. This benefits discharge of nitrogen gas generated from Anammox reaction and improves substrate transferring by slight scouring effect of the discharged nitrogen gas. But compared with nonwoven carriers, honeycomb-like

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carrier has a relative lower retention capacity for Anammox bacteria. Thus, combination of nonwoven carriers and honeycomb-like carriers may be beneficial for formation of Anammox biofilm and operation of Anammox process. In this study, a kind of filling module consisting of nonwoven cloth and honeycomblike carriers is constructed and filled in an ABBR for start-up and operation of Anammox. Although Anammox bacteria favor the temperature range of 30-38 °C, lower ambient temperature is close to the actual temperature at mainstream condition. Moreover, indoor temperatures in laboratories are mostly in the moderate temperature range. Operation of Anammox process at a moderate temperature are expected to save a considerable amount of energy consumption with respect to temperature controlling. This study aims to investigate the feasibility of applying the ABBR to start up and operate Anammox process and the Anammox performance in the reactor filled with the filling modules at the moderate temperature (20±1°C).

2. Material and methods 2.1. Reactor The ABBR employed for Anammox start-up and operation is shown in Fig. 1. The ABBR was a plastic cuboid reactor (380 mm length, 230 mm width, 115 mm height) with a total work volume of 6 L, which had three equal compartments (Compartment 1, 2 and 3). Here a new type of support material was adopted and named as the filling module. Each filling module consists of a piece of nonwoven cloth enwrapped a series of honeycomb-like carriers (Fig. 2). The nonwoven cloth is made of polyester while the

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honeycomb-like carriers are made of high-density polyethylene. Both the nonwoven cloth and the honeycomb-like carriers have the advantage of corrosion resistance and have the relatively high specific surface area. The nonwoven cloth has a thickness of 3.0 mm and each honeycomb-like carrier has a height of 1 cm, a diameter of 2.5 cm and a density of 0.96 g cm-3 that is close to the water density. Each compartment was filled with four filling modules (Fig. 1). The pH in the reactor was adjusted to around 7.8 by adding 1 M of Na2CO3 or HCl, and the DO level was maintained below 0.05 mg/L. The nitrogen gas produced by Anammox reaction was discharged from water sealing bottles via no-return valves. All tubing and connectors were sealed to avoid leakage of oxygen into the reactor, and Anammox bacteria were protected from growth of photosynthetic bacteria or algae by covering the reactor in black-out cloth. 2.2. Seed sludge The conventional aerobic activated sludge collected from a wastewater treatment plant treating domestic and industrial sewage was used as the seed sludge. After thrice rinsed with water, the seed sludge was inoculated in the three compartments of the ABBR with the same quantity of 1 L. Some characteristics of the seed sludge were as follows: mixed-liquor suspended solids (MLSS) 2.36 g/L; mixed-liquor volatile suspended solids (MLVSS) 1.68 g/L; MLVSS/MLSS 71.12%. 2.3. Operation strategy The ABBR applied for Anammox process was maintained at a moderate temperature (20±1°C) in a temperature-controlled room. The reactor was continuously fed with the synthetic wastewater by a peristaltic pump. The synthetic wastewater was prepared

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according to the Anammox nutrient medium, in which the mineral compositions added were shown in Table 1 and 2 (Van de Graaf et al., 1996; Wang et al., 2016). Nitrogen gas was purged to dispel the dissolved oxygen in the synthetic wastewater for maintaining the anaerobic condition. The experiment was divided into two periods: (1) Period 1: start-up period; (2) Period 2: nitrogen loading-enhancing period. Initially, the influent NH4+-N and NO2--N concentrations were both set to 50 mg L-1 and the hydraulic retention time (HRT) was fixed at 1 d. The nitrogen loading rate (NLR) was enhanced by increasing the concentrations of substrates such as NH4+-N and NO2--N. When the accumulated NO2--N was higher than 20 mg L-1, the substrates (NH4+-N and NO2--N) concentrations in the influent were adjusted to restore the reactor performance. 2.4. Chemical analysis Water quality of the influent and effluent was measured every two days to monitor Anammox start-up and operational performance in the ABBR. According to the Standard Methods for the Examinations of Water and Wastewater, NH4+-N, NO2--N and NO3--N in the influent and effluent were determined by spectrophotometry, and MLSS and MLVSS of the seed sludge were measured by weight method (APHA, 2005; Wang et al., 2016). pH and DO were determined with a digital portable pH meter (PHS-3C, Rex) and DO meter (YSI, Model 55, USA), respectively. 2.5. Morphology observation On day 163 when a high nitrogen loading and removal rate were attained, the cultivated biofilm samples were collected from ABBR. Macroscopic appearances of the

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cultivated biofilms attached on the filling modules were observed, while microbial morphology and microstructure of the cultivated biofilms were observed by optical microscopy and scanning electron microscope (SEM). The biofilm samples were fixed with 2.5% glutaraldehyde, rinsed with 0.1 M phosphate buffered saline (PBS) and dehydrated with a series of ethanol solutions with gradient volume percents (30, 50, 70, 90, 95 and 100%) (Jiang et al., 2013). The dewatered samples were subsequently rinsed with tert-butyl alcohol. After desiccated and spaying coated with gold, the samples were observed by an SEM (Nova Nano SEM 450, FEI, USA). 2.6. Microbial community analysis Real-time quantitative PCR (RTQ-PCR) was conducted to quantify Anammox bacteria and eubacteria in the seed sludge and the cultivated biofilm or sludge by determining the 16S rRNA gene copies of Anammox bacteria and eubacteria in triplicate, while High-throughput 16S rRNA gene sequencing was carried out to investigate the genus-level diversity of Anammox bacteria species in the cultivated biofilm and sludge. As previously described (Lakay et al., 2007), DNA was extracted and purified from the cultivated biofilms and sludge. DNA extraction was conducted in an OMEGA kit (E.Z.N.ATM Mag-Bind Soil DNA Kit, USA). Two pairs of primers targeting for Anammox bacteria (AMX809F-AMX1066R) and eubacteria (Eub341FEub 534R) were designed by Software Premier 5.0 and their information was listed in Table 3. The 16S rRNA genes were amplified by polymerase chain reaction (PCR) using AMX809F-AMX1066R and Eub341F-Eub 534R. PCR amplifications were done

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in triplicate for each sample. Anammox bacteria and eubacteria were absolutely quantified by a fluorescence quantitative PCR system (ABI Stepone plus, USA). The high-throughput sequencing related to Anammox bacteria was performed by IIIumina Miseq platform. The sequences were clustered into operational taxonomic units (OTUs) at the similarity threshold of 97% by Mothur software (Li et al., 2017). Constructing a genus cluster tree of Anammox bacteria for various samples provided insight into the main Anammox genera as well as their percentages.

3. Results and discussion 3.1. Start-up period A kind of reactor improved from ABR that named as ABBR was applied to start-up Anammox process at a moderate temperature around 20°C. In the start-up period, influent NH4+-N and NO2--N were both invariable at 50 mg/L and HRT was fixed at 1 d, which corresponded that NLR was stabilized at 0.10 kg N m-3 d-1. Based on variations of three nitrogen species (NH4+-N, NO2--N and NO3--N) in the reactor (Fig. 3), Anammox start-up period was divided into three phases: sludge conversion phase, lagging phase and activity appearance phase. The phase division was similar to those in previous literatures (Bi et al., 2014; Chen et al., 2016; Wang et al., 2016). At the beginning of the start-up period of Anammox process was sludge conversion phase (day 1~21). The characteristics of this phase were accumulation of NH4+-N in the ABBR and removal of a large proportion of NO2--N from the reactor. The phenomenon was also reported in other reactors (Bi et al., 2014; Chen et al., 2016; Wang et al.,

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2016). After the aerobic activated sludge was inoculated in the ABBR for operating Anammox process, endogenous denitrification became the main process at first. Nonadapted bacteria in seed sludge was subjected to cell lysis because of changes of environmental conditions. As a result, decomposition of organic nitrogen such as proteins caused by cell lysis leaded to NH4+-N accumulation, which was reflected by the phenomenon that effluent NH4+-N concentrations were higher than influent NH4+-N concentrations. Some biodegradable organic matters released by cell lysis of nonadapted bacteria could provide organic electron donors for denitrifying bacteria. Denitrifying bacteria utilized the endogenous carbon source and predominated in the reactor. Consequently, along this phase, most of NO2--N was removed with the removal efficiency higher than 75% while effluent NO3--N decreased quickly in the first three days and afterwards was at a relatively low level that was close to 0 mg/L for most time. After sludge conversion phase, Anammox start-up process went into lagging phase (day 21~29). In this phase, effluent NH4+-N concentrations were close to influent NH4+N concentrations and effluent NO2--N concentrations showed a gradually decreasing trend. Even though removal of 3.32 mg/L NH4+-N and 37.62 mg/L NO2--N was observed on day 23, synchronous removal of NH4+-N and NO2--N was not observed for the next days of this phase. The results indicated that Anammox activity didn’t appeared in this phase. The reason could be attributed to the extremely slow growth rate of Anammox bacteria and the competition between Anammox bacteria and endogenous denitrifying bacteria.

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In activity appearance phase (day 29~47), Anammox activity appeared, which was characterized by synchronous and continuous removal of NH4+-N and NO2--N. Anammox activity first appeared on day 29. Tsushima et al. (2007) first discovered Anammox activity in a FBR inoculated with denitrifying sludge on day 40, and Lopez et al. (2008) and Wang et al. (2012) first discovered Anammox activity in a SBR inoculated with mixed activated sludge on day 60 and day 51. The time required for appearance of Anammox activity in this study was obviously shorter than those in several other researches on start-up of Anammox processes from various activated sludge. The quick appearance of Anammox activity might be attributed to highefficiency biomass retention capability of the ABBR. In the phase, Anammox activity gradually increased to drive transition of the main process from endogenous denitrification to Anammox process. From day 39 to day 47, total nitrogen (TN) removal efficiencies were all over 70% and nitrogen removing rate (NRR) were stabilized at the range from 7.22×10-2 to 7.64×10-2 kg N m-3 d-1, signing successful start-up of Anammox process (Fig. 3b). In this study, Anammox process was successfully started up from the aerobic activated sludge in the ABBR after 47 days operation. Interestingly, the Anammox starup period were shorter than those in other researches listed in Table 4, even though this study was done at a moderate temperature (20±1°C) while the other researches were carried out at the high temperature range (30~38°C) favoring Anammox bacteria (Bi et al., 2014; Chen et al., 2012; Kowalski et al., 2018; López et al., 2008; Tsushima et al.,

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2007; Wang et al., 2012). The fast start-up of Anammox process from the conventional activated sludge at a moderate temperature can be explained from the several aspects: (1) the ABBR was a novel and suitable reactor type for enrichment of slowly-growing Anammox bacteria because of its good ability to retain biomass; (2) the filling module consisted of non-woven cloth and honeycomb-like carriers, fibrous structure of nonwoven cloth and many slight indentations of honeycomb-like carriers could benefit attachment of Anammox bacteria and formation of Anammox biofilm so as to enhance the retention of Anammox bacteria; (3) porous structure of honeycomb-like carriers could enable the easy discharge of biogas produced by Anammox reaction to avoid clogging of packing bed; (4) the moderate temperature around 20°C might slow down the process of cell lysis of non-adapted bacteria and accelerate microbial succession from endogenous denitrifying bacteria to Anammox bacteria species that adapted to the lower temperature. 3.2. Nitrogen loading-enhancing period In the nitrogen loading-enhancing period, the initial NH4+-N and NO2--N concentrations were both 50 mg/L and HRT was kept invariable at 1 d. After quick start-up of Anammox process at the moderate temperature (20±1°C) in the ABBR, nitrogen loading was stepwise enhanced by increasing influent NH4+-N and NO2--N concentrations. In this period, one-time fluctuation of Anammox performance occurred in the ABBR but Anammox performance of the reactor was restored after a few days. Based on Anammox performance of the ABBR, the nitrogen loading-enhancing period

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was divided into 5 phases (Fig. 4). In phase 1 (day 47~69), the ratio of influent NO2--N to influent NH4+-N (R(NO2--N inf /NH4

+-N

inf))

was kept at 1.00 to avoid nitrite inhibition for Anammox bacteria and the

influent NH4+-N and NO2--N concentrations were both stepwise increased from 50 mg/L to 200 mg/L by each increment of 50 mg/L, which corresponded that the total nitrogen loading rate (NLR) was increased from 0.10 kg N m-3 d-1 to 0.40 kg N m-3 d-1. As the NLR was stepwise increased, the total nitrogen removal rate (NRR) steadily increased from 0.08 kg N m-3 d-1 to 0.32 kg N m-3 d-1. Even though the TN removal efficiencies fluctuated between 72.63% and 80.61%, no remarkable accumulation of NO2--N was observed in this phase. Fig. 4 showed that the effluent NO2--N concentrations of the ABBR were all below 10 mg/L. Each time the influent NH4+-N and NO2--N concentrations were increased, the removal amounts of NH4+-N and NO2-N increased and the effluent NO2--N concentrations tended to decreased to a relatively low level below 1 mg/L after several days. The results indicated that Anammox process in the ABBR exhibited a good resistance to shock of substrates (NH4+-N and NO2--N) concentrations when the influent NH4+-N and NO2--N were increased from 50 mg/L to 200 mg/L. In phase 2 (day 69~79), R(NO2--N inf /NH4+-N inf) was unchanged and kept at 1.00 and the influent NH4+-N and NO2--N concentrations were both 250 mg/L, which corresponded that the NLR reached 0.50 kg N m-3 d-1. During this phase, the accumulation of NO2--N was observed and the effluent NO2--N concentrations showed

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an increasing trend. The effluent NO2--N concentrations gradually increased from 0.14 mg/L to 23.13 mg/L. The results indicated that instable state of Anammox process emerged. It can be deduced that the threshold concentrations of total nitrogen for the instability of Anammox process were 500 mg/L or the threshold NLR was 0.50 kg N m3

d-1 in the cased that HRT was kept at 1 d and the influent NH4+-N and NO2--N were

both increased to 250 mg/L by 50 mg/L. The reason for the instability of Anammox process might that Anammox bacteria grew and proliferated extremely slowly and the accumulation of Anammox bacteria in the ABBR could not enough to afford to the NLR of 0.50 kg N m-3 d-1 due to limited operation time of Anammox process. Thereafter, the ABBR required some adjustment to restore Anammox performance in next phase. In phase 3 (day 79~109), the influent NH4+-N and NO2--N concentrations were both decreased to 150 mg/L and then were stepwise increased to 250 mg/L again by each increment of 25 mg/L. After the influent NH4+-N and NO2--N concentrations were adjusted to 150 mg/L, the effluent NH4+-N and NO2--N concentrations were both decreased and the effluent NO2--N was decreased from 23.13 mg/L on day 79 to 0.32 mg/L on day 83. As each increment was reduced from 50 mg/L to 25 mg/L in this phase, NLR and NRR both gradually increased and the effluent NO2--N concentration was at low level below 10 mg/L. When NLR was up to 0.50 kg N m-3 d-1, the average NRR reached 0.39 kg N m-3 d-1 corresponding that the TN removal efficiency on average was 77.97%. The results indicated that Anammox performance in the ABBR

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was restored and attained a better level than before. The ABBR exhibited an excellent restoration capability of Anammox performance with proper adjustment for the substrates (NH4+-N and NO2--N) concentrations. In phase 4 (day 109~117), R(NO2--N inf /NH4+-N inf) was still kept at 1.00 and the influent NH4+-N and NO2--N concentrations were increased twice from 250 mg/L to 350 mg/L. In this phase, each increment of the influent NH4+-N concentration was doubled to 50 mg/L compared with the one in the last phase. As a result, the effluent NO2--N concentrations were below 1 mg/L along this phase and the average NRR reached 0.56 kg N m-3 d-1 as the influent NH4+-N and NO2--N concentrations were up to 350 mg/L corresponding that NLR reached 0.70 kg N m-3 d-1. The results indicated that Anammox process in the ABBR can resist shock loading of the substrates (NH4+-N and NO2--N) concentrations to some extent. However, NH4+-N was accumulated and the max effluent NH4+-N was up to 75.43 mg/L on day 117. The operation strategy was required to be further adjusted in the next phase. In phase 5 (day 117~163), the influent NH4+-N concentrations were stepwise increased from 350 to 450 mg/L and R(NO2--N inf /NH4+-N inf) was increased slightly from 1.00 to 1.22 to remove the accumulated NH4+-N for improving the effluent quality of the ABBR. Accumulation of substrates is more likely to appear under the condition of high nitrogen loading rate than it is under the condition of low nitrogen loading rate. The accumulated substrates, especially NO2--N, may inhibit Anammox process as they have the biological toxicity to Anammox bacteria when their concentrations exceed the

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tolerance threshold of Anammox bacteria (Ma et al., 2017; Strous et al., 1999). To avoid the possible inhibition effects of Anammox process under high nitrogen loading operation, each increment of the influent NH4+-N concentration was narrowed to 25 mg/L. By increasing the total nitrogen concentration up to 1000 mg/L (NLR up to 1.00 kg N m-3 d-1), NRR steadily increased. The TN removal efficiency on average was 89.03 % when NLR was attained at 1.00 kg N m-3 d-1. The max NRR reached 0.90 kg N m-3 d-1 after 161 days operation. 3.3. Morphology analysis of Anammox biofilms The macroscopic appearances of Anammox biofilms formed on the filling modules on day 163 were investigated. The biofilms in Compartment 1 were pale red, while the biofilms in Compartment 2 and 3 were both bright red. Uneven surfaces and red granules were observed on the biofilms of Compartment 2 and 3 except on the biofilms of Compartment 1. The closer the compartment was to the influent port, the redder color and the denser structure the biofilms displayed, no matter from top view or from front view of the biofilms. Qin et al. (2017) reported the impact of different substrate concentrations on Anammox sludge properties in two up-flow blanket filter reactors (UBFs) and found that Anammox sludge displayed a bright red color in one UBF with high substrate concentrations while it displayed a russet color in the other UBR with low substrate concentrations. Similarly, in this study, different substrate concentrations for the three compartments of the ABBR caused different appearance characteristics of the Anammox biofilms including their colors and structures. The appearance

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characteristics of Anammox biofilms could indirectly reflected the intrinsic biofilm property, especially the physiological and behavior characteristics of the predominated bacteria in them. The red color is considered as one typical characteristic of Anammox bacteria or mature Anammox biofilms and the behavior of aggregation is another typical characteristic of Anammox bacteria. Previous researches pointed out that the heme C inside Anammox cells played a key role in Anammox metabolism (Bi et al., 2014; Kartal et al., 2012). The heme C made the Anammox bacteria appear to be red. Since that, the redder biofilms meant that more Anammox bacteria were accumulated. Previous researches also demonstrated that Anammox bacteria tended to grow in aggregations and their intrinsic aggregation capacity was important for Anammox granule formation (Gao et al., 2012; Xu et al., 2018; Zhao et al., 2018). The aggregated growth behavior leaded the formation of some red granules on Anammox biofilms in the ABBR. Gao et al. (2012) also observed some red Anammox granules on the surface of Anammox biofilms that were attached on a couple of non-woven filler disks and demonstrated that aggregated growth was a favorite growth way for Anammox bacteria. Based on the appearance differences among the mature biofilms of the three compartments, the distribution trend of biomass attached on the filling module in the ABBR was as follows: Compartment 3 > Compartment 2 > Compartment 1. The biomass attached on the filling module in Compartment 1 was much less than that in Compartment 2 or 3. The three compartments enabled tertiary interception of the extremely slow growing bacteria — Anammox bacteria. By this way, most Anammox

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bacteria were retained in Compartment 1 and 2, and high-efficient biomass retention was realized in the ABBR. The ABBR was suitable for start-up and operation of Anammox process. The deduction was further confirmed by the follow-up characterization of the Anammox biofilms. Since more biomass was observed to attach on the filling module in Compartment 3 compared with that in Compartment 1 and 2, microscopic morphology and structure of the mature Anammox biofilms in Compartment 3 on day 163 were observed by optical microscopy and SEM. Optical microscopy was employed to observe the overall perspectives of an Anammox granule stripped from outer surfaces of non-woven cloth (granule A) and an Anammox granule stripped from pores of a honeycomb-like carrier (granule B). Granule A and B both displayed the irregular red granular structure with the whole external surface covered by a thick layer of colloidal substance. The colloidal substance on surface of Anammox granules was proven to be extracellular polymeric substance and benefited granulation of Anammox sludge (Lin & Wang, 2017; Zhao et al., 2018). SEM was employed for deeper insight into microstructure of the mature Anammox biofilms and granules. It was found that micro-aggregates were widely distributed in the mature biofilms and granules. The micro-aggregates were mainly composed of coccus and short-rod shaped bacteria. Granule A and B both had the cauliflower structure, which was also reported in other researches and was considered to be the typical characteristic of mature Anammox biofilms or sludge (Trigo et al., 2006; Wang et al., 2011; Zeng et al., 2016; Zhu et al., 2018). For the Anammox biofilms on

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non-woven cloth, three different states of micro-aggregate were found: microaggregates sticking on a non-woven fiber, entangling non-woven fibers and enwrapped by non-woven fibers. The three different states could improve the biomass retention capacity of non-woven cloth, which were beneficial for quick startup and stable operation of Anammox process. Interestingly, filamentous bacteria were found on surfaces of one micro-aggregate of granule A and a non-woven fiber. A small quantity of filamentous bacteria could coexist with Anammox bacteria in Anammox consortia and was proven to have no adverse effect on the performance of Anammox reactor (Wang et al., 2011). Furthermore, it was reported that some filamentous bacteria such as Chloroflexis bacteria living on organic matters from growth metabolism of Anammox bacteria played a positive role in Anammox granulation and biofilm formation for their structure contribution (Cho et al., 2011; Kindaichi et al., 2012; Qin et al., 2017). For the filling module used in this study, the honeycomb-like carriers could support non-woven cloth and their porous structure not only could retain extra biomass but also could facilitate the discharge of nitrogen gas generated by Anammox reaction so as to avoid clogging of the filling bed. 3.4. Microbial community analysis To better understand the outstanding Anammox performance of the ABBR at the moderate temperature (20±1°C), the Anammox biofilms in Compartment 2 and 3 and Anammox sludge in Compartment 1 of the ABBR on day 163 were used for microbial community analysis. Considering that the biomass attached on the filling module in

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Compartment 1 was too little to sample, the Anammox sludge was collected from the bottom of Compartment 1. Meanwhile, the Anammox biofilm was collected from the filling modules of Compartment 2 and 3. RTQ-PCR was conducted to quantify Anammox bacteria and eubacteria in the seed sludge, the Anammox sludge and the Anammox biofilms for investigating the enrichment levels of Anammox bacteria. As Fig. 5 describes, the cell densities of Anammox bacteria and the percentages of Anammox bacteria to eubacteria in the Anammox biofilms and the Anammox sludge were both higher than those in the seed sludge. The densities of Anammox bacteria in the seed sludge, the Anammox sludge in Compartment 1 and the Anammox biofilms in Compartment 2 and 3 were 0.10×1010 gene copies/ml sludge, 3.20×1010 gene copies/ml sludge, 22.04×1010 gene copies/ml biofilm and 30.28×1010 gene copies/ml biofilm, respectively. Correspondingly, the percentages of Anammox bacteria to eubacteria in them were 0.23%, 12.62%, 47.76% and 52.73%. The results demonstrated that Anammox bacteria were effectively accumulated in the ABBR at the moderate temperature (20±1°C) and, moreover, the Anammox biofilms in Compartment 2 and 3 both exhibited obviously higher enrichment levels of Anammox bacteria than the Anammox sludge in Compartment 1. High-throughput 16S rRNA gene sequencing was done to investigate the genus-level diversity of Anammox species and the percentage of each Anammox species to total Anammox bacteria. As shown in Fig. 6, there were mainly three known Anammox species categorized to Candidatus Brocadia, Candidatus Jettenia and Candidatus

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Kuenenia respectively and one unclassified Anammox species on matter in Anammox biofilms in Compartment 2 and 3 or Anammox sludge in Compartment 1. Moreover, the dominant Anammox bacteria was Candidatus Brocadia which occupied more than 75% of Anammox bacteria. Wu et al. (2018) reported that Candidatus Brocadia and Candidatus Jettenia were dominant in three innovative reactors combining anaerobic baffled reactor with membrane bioreactors after start-up of the cold Anammox process at 13 °C. He et al. (2018) also reported that Candidatus Kuenenia became the dominant genus of Anammox bacteria in an up-flow anaerobic sludge blanket (UASB) for Anammox operation when the reactor temperature was controlled at 13 °C. The facts indicated that the three genera Candidatus Brocadia, Candidatus Jettenia and Candidatus Kuenenia could adapted to low temperature. Furthermore, in this study, Candidatus Brocadia, Candidatus Jettenia and Candidatus Kuenenia could also adapted the temperature (20±1°C) lower than the favorable temperature for Anammox bacteria (30~38°C), which guaranteed an outstanding Anammox operation performance in the ABBR at the moderate temperature. Besides, the distribution trend of Candidatus Kuenenia in the ABBR: Compartment 3 > Compartment 2 > Compartment 1. The closer the compartment was to the influent port, the higher the percentage of Candidatus Kuenenia to total Anammox bacteria is. van der Star et al. (2008) pointed out that Anammox population shifted from Candidatus Brocadia to Candidatus Kuenenia as the nitrogen loading was enhanced by increasing the substrates (NH4+-N and NO2--N) concentrations. Similarly, the distribution trend of Candidatus Kuenenia in the ABBR

22

could be attributed to different degrees of the population shift in different compartments. The substrates concentration was increased from Compartment 1 to Compartment 3 (as the substrates were subsequently utilized by Anammox bacteria from one compartment to the next in a direction from the influent port to the effluent port), so that the degree of Anammox population shift from Candidatus Brocadia to Candidatus Kuenenia was increased. van der Star et al. (2008) also indicated that Candidatus Kuenenia that was considered as a K strategist had a higher affinity for nitrite than Candidatus Brocadia. Controlling the accumulation of nitrite was necessary to maintain the stability of Anammox process under operation conditions of high nitrogen loading (Ma et al., 2017). Therefore, Candidatus Kuenenia was helpful to improve the stability of Anammox process under high substrates concentrations or a high nitrogen loading in the ABBR as it can consume the inhibiting substrate nitrite timely and effectively. Synergistic function of Candidatus Brocadia, Candidatus Jettenia and Candidatus Kuenenia enabled a high-efficient and stable operation performance of Anammox process in the ABBR at the moderate temperature.

4. Conclusions A lab-scale ABBR was applied for start-up of Anammox process from the aerobic activated sludge at 20±1°C. Anammox process was started up after 47 days operation and the max NLR and NRR attained 1.00 kg N m-3 d-1 and 0.90 kg N m-3 d-1 on day 161. SEM observations showed that structure and three states of micro-aggregates enhanced biomass retention. Anammox bacteria were effectively accumulated and three main

23

Anammox species were found: Candidatus Brocadia, Candidatus Jettenia and Candidatus Kuenenia. High-efficient and stable operation of Anammox process at the moderate temperature was achieved by synergistic function of the three Anammox species.

Acknowledgements The research was financially supported by National Natural Science Foundation of China (31400432) and Hebei Provincial Natural Science Foundation (E2018202246).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version.

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Figure legend Fig. 1 Schematic diagram of the ABBR for start-up and operation of Anammox process. Fig. 2 The filling module applied in the ABBR. Fig. 3 Nitrogen removal performance of the ABBR during the start-up period of Anammox process: (a) Variations of NH4+-N, NO2--N and NO3--N during the start-up period in the ABBR; (b) Variations of NLR, NRR and TN removal efficiencies during the start-up period. Fig. 4 Nitrogen removal performance of the ABBR during the nitrogen loadingenhancing period of Anammox process: (a) Variations of NH4+-N, NO2--N and NO3--N during the nitrogen loading-enhancing period; (b) Variations of NLR, NRR and TN removal efficiencies during the nitrogen loading-enhancing period. Fig. 5 The percentage of Anammox bacteria to eubacteria in the seed sludge, Anammox sludge (C1), Anammox biofilm (C2) and Anammox biofilm (C3). Anammox sludge (C1) represents Anammox sludge collected from the bottom from Compartment 1 of the ABBR while Anammox biofilm (C2) and Anammox biofilm (C3) represent Anammox biofilm collected from one filling module of Compartment 2 and Anammox biofilm collected from one filling module of Compartment 3 respectively. Fig. 6 Genus cluster tree of Anammox bacteria in Anammox sludge (C1), Anammox biofilm (C2) and Anammox biofilm (C3). Anammox sludge (C1), Anammox biofilm (C2) and Anammox biofilm (C3) represent Anammox sludge collected from the bottom from Compartment 1, Anammox biofilm collected from one filling module of

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Compartment 2 and Anammox biofilm collected from one filling module of Compartment 3, respectively.

Table legend Table 1 The compositions of synthetic wastewater. Table 2 The constituents of trace elements solution. Table 3 The applied specific premiers for Anammox bacteria and eubacteria. Table 4 Overall of Anammox start-up processes in various reactors.

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Research highlights 1. An ABBR was used as a novel reactor to start up and operate Anammox process. 2. The ABBR was seeded with the aerobic activated sludge and run around 20°C. 3. Quick start-up of Anammox process was achieved after 47days operation. 4. The max NLR and NRR attained 1.00 kg N m-3 d-1 and 0.90 kg N m-3 d-1 respectively. 5. Macroscopic and microscopic morphology of the mature biofilms was observed. 6. Microbial community analysis of the mature biofilms or sludge was conducted.

Fig. 1

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(a) Front view

(b) Top view Fig. 2

(a) Variations of NH4+-N, NO2--N and NO3--N during the start-up period

34

(b) Variations of NLR, NRR and TN removal efficiencies during the start-up period Fig. 3

(a) Variations of NH4+-N, NO2--N and NO3--N during the nitrogen loading-enhancing period 35

(b) Variations of NLR, NRR and TN removal efficiencies during the nitrogen loading-enhancing period Fig. 4

36

Fig. 5

37

Fig. 6 38

Table 1 The compositions of synthetic wastewater Compound Synthetic medium (NH4)2SO4 Required amount NaNO2 Required amount KHCO3 1.25 g/L KH2PO4 0.025 g/L CaCl2.2H2O

0.3 g/L

MgSO4.7H2O

0.2 g/L

FeSO4 Trace elements solution

0.00625 g/L 1.25 ml/L

Table 2 The constituents of trace elements solution Compound Synthetic medium (g/L) EDTA 15 . ZnSO4 7H2O 0.43 CoCl2.6H2O 0.24 . MnCl2 4H2O 0.99 CuSO4.5H2O 0.25 NaMoO4.2H2O

0.22

NaSeO4.10H2O

0.21

H3BO4 NaWO4.2H2O

0.014 0.050

Table 3 The applied specific premiers for Anammox bacteria and eubacteria Primer Target Target position (5’-3’) Analytical method name organism RQT-PCR / HighAMX809F GCCGTAAACGATGGGCACT Anammox throughput 16S AACGTCTCACGACACGAGC bacteria rRNA gene AMX1066R TG sequencing Eub341F CCTACGGGAGGCAGCAG eubacteria RQT-PCR Eub534R ATTACCGCGGCTGCTGGC

Table 4 Overall of Anammox start-up processes in various reactors 39

Reactor FBR

Temperature (°C) 37

SBR

36 ± 0 .3

Seed sludge Denitrifying sludge

Timea (d) 40

Start-up Reference period (d) 55 Tsushima et al. 2007 78 Lopez et al. 2008 59 Wang et al. 2012 101 Wang et al. 2012 50 Bi et at. 2014 85 Chen et al. 2012 50 Kowalski et al. 2018

Mixed activated 60 sludge MBR 35 Mixed activated 21 sludge SBR 35 Mixed activated 51 sludge c UCR 35±1 Anaerobic activated 27 sludge Amended 30 ± 1 Mixed activated _ b UASB sludge MBBR 33±1 Granular _ deammonifying sludge ABBR 20±1 Aerobic activated 29 47 This study sludge a Time represents the time required for appearance of Anammox activity; Amended UASBb represents an UASB with amendment of bamboo charcoal; UCRc represents upflow column reactor added with 0.09 mM Fe2+.

40