Nitrogen removal performance and microbial ...

Nitrogen removal performance and microbial community of an enhanced multistage A/O biofilm reactor treating low-strength domestic wastewater Han Chen, Ang Li, Qiao Wang, Di Cui, Chongwei Cui & Fang Ma

Biodegradation ISSN 0923-9820 Volume 29 Number 3 Biodegradation (2018) 29:285-299 DOI 10.1007/s10532-018-9829-x

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Author's personal copy Biodegradation (2018) 29:285–299 https://doi.org/10.1007/s10532-018-9829-x

ORIGINAL PAPER

Nitrogen removal performance and microbial community of an enhanced multistage A/O biofilm reactor treating lowstrength domestic wastewater Han Chen . Ang Li

. Qiao Wang . Di Cui . Chongwei Cui . Fang Ma

Received: 13 February 2018 / Accepted: 6 April 2018 / Published online: 7 April 2018 Ó Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract The low-strength domestic wastewater (LSDW) treatment with low chemical oxygen demand (COD) has drawn extensive attention for the poor total nitrogen (TN) removal performance. In the present study, an enhanced multistage anoxic/oxic (A/O) biofilm reactor was designed to improve the TN removal performance of the LSDW treatment. Efficient nitrifying and denitrifying biofilm carriers were cultivated and then filled into the enhanced biofilm reactor as the sole microbial source. Step-feed strategy and internal recycle were adopted to optimize the substrate distribution and the organics utilization. Key operational parameters were optimized to obtain the best nitrogen and organics removal efficiencies. A hydraulic retention time of 8 h, an influent distribution ratio of 2:1 and an internal recycle ratio of 200% were tested as the optimum parameters. The ammonium,

TN and COD removal efficiencies under the optimal operational parameters separately achieved 99.75 ± 0.21, 59.51 ± 1.95 and 85.06 ± 0.79% with an organic loading rate at around 0.36 kg COD/m3 d. The high-throughput sequencing technology confirmed that nitrifying and denitrifying biofilm could maintain functional bacteria in the system during longperiod operation. Proteobacteria and Bacteroidetes were the dominant phyla in all the nitrifying and denitrifying biofilm samples. Nitrosomonadaceae_uncultured and Nitrospira sp. stably existed in nitrifying biofilm as the main nitrifiers, while several heterotrophic genera, such as Thauera sp. and Flavobacterium sp., acted as potential genera responsible for TN removal in denitrifying biofilm. These findings suggested that the enhanced biofilm reactor could be a promising route for the treatment of LSDW with a low COD level.

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10532-018-9829-x) contains supplementary material, which is available to authorized users.

Keywords Nitrogen removal Microbial community Low-strength domestic wastewater Enhanced multistage A/O biofilm reactor Highthroughput sequencing

H. Chen A. Li (&) Q. Wang D. Cui C. Cui F. Ma State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, People’s Republic of China e-mail: [email protected] D. Cui Research Center on Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin 150076, People’s Republic of China

Introduction Biological nitrogen removal (BNR) has been extensively applied in wastewater treatment to mitigate the

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nitrogen contamination for satisfying performance, cost saving and easy implementation (Guo et al. 2007; Li et al. 2016; Wang et al. 2015a, b; Xia et al. 2008). Anoxic and oxic environments created by spatial separation or sequential shift are essential in traditional BNR processes (Ahn 2006; Aslan and Dahab 2008). The low-strength domestic wastewater (LSDW) has drawn much attention in recent years for the huge emission and specific characteristics. The chemical oxygen demand (COD) lower than 200 mg/ L in the LSDW seriously restricts denitrification due to the lack of carbon source and electron donors in the nitrate reduction course (Fernandez-Nava et al. 2010; Liu et al. 2007). As a result, the effluent total nitrogen (TN) of the LSDW treatment can hardly meet the discharge standard, while the deficient organics might cause the biomass loss and system breakdown of the LSDW treatment. Therefore, it is significant to accomplish the efficient TN removal with limited organic matters in the LSDW treatment. Previous researches have explored the nitrogen and organics removal performance of several processes in the LSDW treatment, which include vertical flow constructed wetland (Zhou et al. 2017), anaerobic/ anoxic/oxic process (Fan et al. 2009; Zhang et al. 2017), anoxic/oxic process (Wang and Chen 2016), integrated fixed-film activated sludge process (Bai et al. 2016; Shao et al. 2017), simultaneous nitrification-endogenous denitrification and phosphorus removal system (Wang et al. 2015a, b; 2016), granular sequencing batch reactor (He et al. 2017; Liu et al. 2007; Ni et al. 2009). The influent COD concentration in these researches usually ranges from 150 to 200 mg/L, which accords with the typical COD level of domestic wastewater in China (Zhang et al. 2011). However, the LSDW treatment with a COD concentration lower than 150 mg/L is rarely reported at present. Biofilm processes have been frequently utilized in wastewater treatment. The biofilm system can facilitate the simultaneous nitrification and denitrification and the organics utilization through the oxygen diffusion limitation into the biofilm structure, which finally enhance the TN removal performance (Seifi and Fazaelipoor 2012; Yang et al. 2010). The biomass in the biofilm system can be accumulated to a relatively high concentration (Lo et al. 2010). Moreover, biofilm can protect the bacteria activity against

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adverse conditions (Ghaniyari-Benis et al. 2009; Zeng et al. 2018). It is previously reported that the moving bed biofilm reactor (Cao et al. 2017; Chu and Wang 2011; Wang et al. 2006) and sequencing batch biofilm reactors (Jin et al. 2012; Lim et al. 2012) have been adopted to treat LSDW. Nevertheless, a multistage A/O biofilm process treating LSDW is rarely reported. The multistage A/O biofilm process owns a succession of anoxic and oxic tanks filled with biofilm carriers, which can form the alternate switch of different dissolved oxygen levels for nitrogen transformation. Multiple feeding inlets can improve the utilization of influent organics to promote TN removal without external organics addition. Meanwhile, immobilization carriers in the whole process may enhance the biofilm biomass retention under low influent COD concentrations. However, the treatment performance, parameter effects and microbial compositions of the multistage A/O biofilm reactor treating LSDW are still not clear. Therefore, research on the multistage A/O biofilm reactor treating LSDW is of great significance. The main aim of the present research was to investigate the nitrogen removal performance and microbial community profiles of an enhanced multistage A/O biofilm reactor treating LSDW with a COD concentration of 120 mg/L. The key operational parameters were optimized to obtain the optimum parameters combination and the best treatment performance, while the microbial community compositions and biodiversity variations in the reactor were analyzed by highthroughput sequencing technology.

Methods and materials Biofilm cultivation The cultivation of enhanced biofilm was conducted to provide attached biomass with good nitrification and denitrification abilities for the enhanced multistage A/O biofilm reactor. The seed sludge for enhanced biofilm cultivation was separately taken from an aerobic nitrifying SBR fed with ammonium chloride and sodium bicarbonate, and an anoxic denitrifying SBR fed with potassium nitrate and sodium acetate. Both reactors had been operated for over 30 days to successfully domesticate the nitrifying and denitrifying sludge. The typical operational mode of two

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reactors for enhanced biofilm cultivation included 10 min of feeding, 7 h of aeration for nitrifying sludge (or 7 h of agitation for denitrifying sludge) and 50 min of settling, draining and idling. The concentrations of DO in the nitrifying and denitrifying SBRs were separately controlled at more than 2 mg/L and lower than 0.5 mg/L. The initial mixed liquor suspended solids (MLSS) of both reactors were 6500 mg/L, while the exchange volume ratio of both reactors was 1:3. Polyurethane foams (PUF) were chosen as the biofilm cultivation carriers in this study. The single PUF carrier was an elastic cuboid with a volume of about 9 cm3, a density of nearly 0.03 g/cm3 and a porosity of over 97%. PUF carriers were separately put into the nitrifying and denitrifying SBRs to promote the microbial attachment growth of nitrifying and denitrifying consortia in the porous structure. The configuration and operation of two SBRS for biofilm cultivation were the same as those in sludge domestication phase. Samples of carriers with biofilm were taken to measure the attached biomass, and the biofilm morphology was observed by a scanning electron microscope (SEM) based on a previously reported method (Gonzalez-Martinez et al. 2017). Wastewater composition The feed for enhanced nitrifying biofilm cultivation contained ammonium chloride with the ammonium concentration of about 80 mg/L. The solution of carbon bicarbonate was automatically dosed to keep the pH value of the mixed liquor ranging from 7.0 to 8.0. In the meantime, the nitrate and COD concentrations in the feed for enhanced denitrifying biofilm cultivation were about 40 and 300 mg/L with sodium acetate as the carbon source and potassium nitrate as the nitrogen source. Ingredients including KH2PO4 (100 mg/L), MgSO47H2O (30 mg/L), FeSO47H2O (30 mg/L) and trace element solution (2 mL/L) were dosed into the feeding influent both for the nitrifying and denitrifying biofilm cultivation. The trace element solution was prepared according to a previous report (Ma et al. 2015). The synthetic LSDW treated by the enhanced multistage A/O biofilm reactor was artificially prepared with sodium acetate as the carbon source and ammonium chloride as the nitrogen removal. The influent COD and ammonium concentrations

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separately reached about 120 and 20 mg/L, while the pH value of the wastewater was set as 7.2 without adjustment during the operation period. Ingredients including KH2PO4 (22 mg/L), MgSO47H2O (30 mg/ L), FeSO47H2O (30 mg/L) and trace element solution (2 mL/L) were contained in the synthetic LSDW. Configuration and operation of the enhanced multistage A/O biofilm reactor The schematic diagram of the enhanced multistage A/O biofilm reactor from a vertical view is shown in Fig. 1. The plexiglas-made reactor had two anoxic and four oxic galleries forming a two-stage A/O with an effective volume of 9.6 L. Microporous aeration diffusors were installed in the oxic galleries to keep DO of the liquid ranging from 1.0 to 1.5 mg/L, while DO in the anoxic galleries maintained lower than 0.5 mg/L. Enhanced denitrifying and nitrifying biofilm carriers were separately filled into the anoxic and oxic galleries with a filling ratio of about 30% to work as the sole biomass. A step-feed strategy was adopted to properly distribute the synthetic LSDW into two different anoxic galleries through two feeding inlets and two peristaltic pumps. Based on the step-feed strategy, the influent wastewater was supplied to different treatment galleries to meet the diverse substrate demands of functional bacteria. The stepfeed strategy could optimize the utilization of the limited organics to promote the denitrification efficiency. The water temperature during the whole operation period was 23 ± 2 °C. The mixed liquor reflux was continually pumped to the first anoxic gallery from the terminal of the final oxic gallery. The hydraulic retention time (HRT, calculated as effective volume divided by total influent flux), influent distribution ratio (calculated as influent flux 1# divided by influent flux 2#) and internal recycle ratio (calculated as nitrification reflux divided by influent flux 1#) of the enhanced multistage A/O biofilm reactor were 10 h, 1:1 and 100% in the initialization period, and then varied in the following tests. The operation schedule of the enhanced multistage A/O biofilm reactor is summed up in Table 1. High-throughput sequencing Biofilm samples from the terminal positions of two anoxic galleries (named A1 and A2) and two oxic

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Fig. 1 Schematic diagram of the enhanced multistage A/O biofilm reactor from a vertical view Table 1 Operation schedule of the enhanced multistage A/O biofilm reactor Phase

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galleries (named O1 and O2) after the whole operation of the enhanced multistage A/O biofilm reactor were analyzed to investigate the microbial community composition and diversity variation through the high-throughput sequencing technology. The initial seed sludge from the municipal wastewater treatment plant, denitrifying and nitrifying biofilm samples (named SS, DNB and NB) after biofilm cultivation were taken as contrasts. All samples were stored at - 80 °C for further analysis after careful rinse. The raw sequencing data of all seven samples have been deposited in the NCBI Sequence Read Archive

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Description

Influent distribution optimization Internal recycle optimization

Stabilization

database with the BioProject ID PRJNA432655 and the SRA accession ID SRP132081. The genomic DNA was extracted from the biofilm samples with a DNA Isolation Kit (LifeFeng Co., Ltd, Shanghai, China) according to the manufacturer’s instructions along with the quality verification procedure. The V3–V4 region of the 16S rRNA genes was amplified utilizing primer pair 50 -ACTCCTACGGGAGGCAGCAG-30 and 50 -GGAC0 TACHVGGGTWTCTAAT-3 . The PCR was performed in a GeneAmp 9700 thermocycler (ABI, USA) in triplicate as follows: initial denaturation step


at 95 °C for 2 min; 27 cycles at 95 °C for 30 s; 55 °C for 30 s, and 72 °C for 30 s; 72 °C for 5 min. The triplicate PCR product was mixed and detected with 2% agarose gel electrophoresis. The recovery of the PCR product was carried out with AxyPrep DNA Gel Extraction Kit (AXYGEN, China), and quantified with QuantiFluorTM-ST system. The Illumina MiSeq platform was employed to conduct the high-throughput sequencing of PCR products after Illumina library construction at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The MOTHUR program was utilized to generate operational taxonomic units (OTUs) by the cluster of sequences with a distance limit of 0.03 (Schloss et al. 2011). Microbial Alpha-diversity indices such as Shannon, Simpson, Chao, and abundance-based coverage estimator (ACE) were analyzed in the MOTHUR program. RDP Classifier with a confidence threshold of 70% was used to classify effective sequences into different taxonomy units, while the community composition was analyzed in different taxonomy levels (Wang et al. 2007). Analytical methods Ammonium, nitrite, nitrate and TN were determined by Nessler’s reagent spectrophotometry, N-(1-naphthyl)-ethylene diamine photometry, phenol disulfonic acid photometry and alkaline potassium persulfate photometry, respectively. Biomass concentration was measured by a drying weight method. DO and pH were separately determined with a WTW Oxi730 dissolved oxygen meter and a PHS-3C pH meter. All samples of water quality were thrice tested to calculate the average values with the standard deviations.

Results Biofilm cultivation and reactor initialization The nitrifying and denitrifying sludge taken for enhanced biofilm cultivation had been successfully operated for over 30 days to enrich functional consortia. The removal abilities of the nitrifying and denitrifying sludge during this phase are summarized in Table S1. During nitrifying sludge domestication, the removal rate of ammonium gradually increases from 4.17 mg/(L h) on Day 1

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to 9.14 mg/(L h) on Day 30, and the ammonium removal efficiency shows a similar rising pattern from 41.29% on Day 1 to 91.33% on Day 30. The removal efficiency of TN in the denitrifying sludge SBR is 89.66% with a TN removal rate of 4.51 mg/ (L h) on Day 1, then it keeps more than 99% since Day 10. The removal efficiency of COD maintains more than 92% with a removal rate of about 35 mg/ (L h). The process of biofilm cultivation with PUF carriers and functional sludge lasted for 20 days. The SEM photographs of biofilm samples on Day 20 are displayed in Fig. 2, which exhibits that mature biofilm has stably colonized on the framework of the PUF carriers. The average specific attached biomass of nitrifying and denitrifying carriers reached 18.89 and 23.33 g dry biofilm/L on Day 20, respectively. The biofilm cultivation is successfully achieved by the attached growth of nitrifying and denitrifying sludge on PUF carriers under batch operation conditions, which can provide the attached biomass source for the following experiment. After the filling of enhanced biofilm carriers, the enhanced biofilm reactor was initialized and operated for 20 days. Potential inadequacies of the reactor were investigated by the analysis of the treatment performance. Figure 3 illustrates the removal performance of organic and nitrogen pollutants in the enhanced biofilm reactor. The influent ammonium and COD concentrations are separately about 20 and 120 mg/L in the synthetic LSDW. In the initialization phase, the biofilm system takes 1 week to acclimatize to the hydraulic and substrate conditions of the enhanced biofilm reactor with the increasing efficiencies of ammonium and TN removal during the startup phase. Then the stable removal performance exhibits in the following days. In the first week, the effluent ammonium and nitrate decrease continuously, and maintain at about 0.5 and 10.2 mg/L since Day 8. The nitrite concentrations are below 0.1 mg/L in the whole 20 days. The removal efficiencies of the ammonium and TN keep more than 97 and 45% from Day 8 to Day 20, while the COD removal efficiency is about 80% in the whole phase.

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Hydraulic retention time HRT has been widely proved as a crucial influencing factor on the nitrogen removal performance (Chakraborty and Veeramani 2006; Moussavi et al. 2015). HRT of 10, 8 and 6 h was chosen to investigate the effect on nitrogen removal performance in the enhanced biofilm reactor for 15 days. Figure 4a and b respectively demonstrate the effect of HRT on the ammonium and TN removal performance of the enhanced biofilm reactor. The ammonium removal performance with HRT of 10 h is similar to that of 8 h with the removal efficiency of about 97%. Nevertheless, the ammonium removal efficiency drastically declines to below 90% when the HRT is 6 h (Fig. 4a). Meanwhile the TN removal efficiency decreases along with the increase of HRT, while the highest and lowest TN removal efficiencies appear on Day 3 (HRT of 10 h, 55.23%) and Day 15 (HRT of 6 h, 36.19%), respectively (Fig. 4b). Besides, the COD removal efficiency keeps about 82% in the whole 15 days with little change (data not shown).

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distribution ratios on ammonium and TN removal. e and f: The effect of internal recycle ratios on ammonium and TN removal

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The influent distribution ratio is an important operational parameter when multiple inlets are set in the reactor with a step-feed strategy (Cao et al. 2013; Ge et al. 2012). Appropriate influent distribution ratios in the step-feed reactor can facilitate the efficient utilization of limited organics by denitrifiers in the anoxic areas of biological reactors (Bundy et al. 2017). In this study, the influent distribution ratio between two inlets was respectively set as 1:1, 2:1 and 3:1 for 15 days with all influent distribution ratios. As shown in Fig. 4c, the ammonium removal efficiency keeps more than 90% during the shift of different influent distribution ratios. The best ammonium removal efficiency of 98.57% occurs on Day 10 with the influent distribution ratio of 2:1 (Fig. 4c). The TN removal increases when the influent distribution ratio changes from 1:1 to 2:1, while the best TN removal efficiency of 54.77% occurs on Day 9. Then it drops sharply when the influent distribution ratio increase to 3:1 with the lowest TN removal efficiency of 39.51% on Day 15 (Fig. 4d). The COD removal efficiency maintains about 82% in the whole 15 days (data not shown).

Performance of the enhanced biofilm reactor under optimum parameters After the optimization of operational parameters, HRT of 8 h, influent distribution ratio of 2:1 and internal recycle ratio of 200% were chosen as the optimum operational parameters for the enhanced multistage A/O biofilm reactor treating LSDW. Then the enhanced multistage A/O biofilm reactor was operated for 20 days to obtain the best treatment performance. The influent concentrations, effluent concentrations and removal efficiencies of organics and nitrogen under optimum operational parameters are shown in Fig. 5. After an adaption period under optimum parameters, the effluent ammonium and nitrate concentrations keep lower than 0.10 and 8.30 mg/L in the following days, respectively. During the whole 20-day operation, the effluent COD and nitrite separately remain below 20 and 0.20 mg/L (Fig. 5a). The effluent TN concentration remained lower than 8.50 mg/L, which could stably meet the first grade A standard of Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant of China (GB 18919-2002). The removal efficiencies of

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The mixed liquor internal recycle from the oxic tank to the front anoxic tank is commonly employed to improve the denitrification efficiency in BNR (Jaafari et al. 2017; Tan and Ng 2008). The enhanced biofilm reactor was operated for 20 days with varied internal recycle ratios (0, 100, 200 and 300%). The ammonium removal efficiency maintains over 95% during the optimization of the internal recycle ratio (Fig. 4e), which exhibits that the influence of nitrate reflux on the nitrification performance is limited. Figure 4f displays that the TN removal efficiency of the enhanced biofilm reactor with internal recycle (Day 6–20) is obviously higher than that of the enhanced biofilm reactor without internal recycle (Day 1–5). The best TN removal performance of more than 56% happens with the internal recycle ratio of 200%, while the TN removal performance of lower than 50% are measured with the internal recycle ratios of 100 and 300%. The COD removal efficiency keeps about 83% in the whole 20 days (data not shown).


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ammonium and COD are stable at over 99% and over 83% after the initial adjustment. The efficiency of TN removal overall appears to gradually improve from 43.51% on Day 1 to 62.01% on Day 20 (Fig. 5b). During the whole operation period of 90 days, no obvious biofilm dispersal was observed in the multistage A/O biofilm reactor. Microbial community analysis of the enhanced biofilm reactor Microbial community diversity The average length of valid sequences in 7 samples (SS, DNB, NB, A1, O1, A2 and O2) was 442.26 bp, and the ratio of trimmed sequences with length distribution in the range of 421–460 bp accounted for 99.8%. The diversity and richness estimators of microbial community of different samples are summarized in Table S2. Coverages ranging from 0.993 to 0.997 demonstrate that the sequence libraries can represent the community diversity effectively. All six biofilm samples have lower Shannon indices and higher Simpson indices than those of the initial suspended sludge for nitrifying and denitrifying sludge domestication. The Shannon and Simpson indices are usually adopted to illustrate the microbial diversity variation. The microbial diversity increases with the rise of the Shannon index, and the Simpson index shows a contrary pattern (Luo et al. 2013). Besides, all the denitrifying biofilm samples (DNB, A1 and A2) exhibit lower Shannon indices and higher Simpson indices than those nitrifying biofilm samples (NB, O1 and O2). Microbial community composition Based on the high-throughput sequencing result, 32, 27, 29, 27, 29, 30 and 30 phyla are separately detected in the SS, DNB, NB, A1, O1, A2 and O2 samples. In the meantime, 319, 313, 322, 301, 295, 303 and 305 genera are detected in the SS, DNB, NB, A1, O1, A2 and O2 samples, respectively. The compositions and relative abundances of microbial community at the level of phylum are displayed in Fig. 6a. Proteobacteria is the most dominant phylum accounting for 46.56–75.11% in all seven samples. Several typical phyla, including Bacteroidetes (7.10–19.07%), Acidobacteria (1.02–7.13%), Chloroflexi (1.69–6.27%),

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Saccharibacteria (0.59–4.18%), Chlorobi (0.89–3.18%), Actinobacteria (0.59–4.05%), Firmicutes (0.49–30.45%) and Nitrospirae (0.55–4.12%), are detected with high relative abundances as well. The compositions and relative abundances in different biofilm samples at genus level are shown in Fig. 6b. Several dominant heterotrophic denitrifiers, such as Thauera sp., Flavobacterium sp. and Comamonadaceae_unclassified, are detected to coexist with the autotrophic nitrifiers, which suggests that the potential SND can occur in the interior zone of the oxic nitrifying biofilm. This result is consistent with a previous report of a full-scale Orbal oxidation ditch (Zhou et al. 2012). The heterotrophic bacteria in nitrifying biofilm may protect the autotrophic nitrifiers and strengthen the matrix structure with higher extracellular polymers content around the biofilm (Bassin et al. 2012). Thauera exists as a dominant genus in all samples with the relative abundances of 1.38–21.37%. Meanwhile, genera including Flavobacterium sp. (0.21–7.02%), Comamonadaceae_unclassified (2.23–5.19%), Dechloromonas sp. (0.94–6.72%), Pseudomonas sp. (0.08–5.96%), Zoogloea sp. (0.03–4.64%), Nitrospira sp. (0.28–4.12%), and Nitrosomonadaceae_uncultured (0.23–1.56%) are detected in the samples as well. Whereas, the genus of Sphaerotilus presents high relative abundances (8.31–34.81%) in A1, A2, O1 and O2, whereas the relative abundances are very low in samples of SS, DNB and NB. As the vital participants of BNR, Nitrosomonadaceae_uncultured and Nitrospira sp. separately belong to ammonium oxidizing bacteria and nitrite oxidizing bacteria. The relative abundances of Nitrospira sp. in nitrifying biofilm samples (1.18, 2.01 and 4.12%) are obviously higher than those in denitrifying biofilm samples (0.28, 0.55 and 1.12%). The highest relative abundance of Nitrospira sp. is observed in nitrifying biofilm from O2 gallery (4.12%). The relative abundances of Nitrosomonadaceae_uncultured in oxic galleries (0.68 and 1.54%) are also higher than those in anoxic galleries (0.23 and 0.40%).

Discussion In the initialization phase, the good nitrification performance in the enhanced biofilm reactor is attributed to the filling of nitrifying biofilm carriers

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Author's personal copy 294 Fig. 6 Compositions and relative abundances of microbial community in different biofilm samples. a Phylum level, b genus level


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into oxic galleries with high biomass concentrations of about 5900 mg/L. Meanwhile the removal efficiency of COD undergoes little fluctuation due to the high adaptability and biomass of heterotrophic bacteria in the system. However, the elimination of TN in this stage still needs to be improved, although denitrifying biofilm carriers possess enough biomass concentrations of about 6500 mg/L in each anoxic gallery. The optimization of the distribution and retention of nitrogen and organics in the reactor is possibly a useful approach. Therefore, it is necessary to investigate the effects of operational parameters on the nitrogen removal property in order to acquire the optimum parameters and the best removal performance of the enhanced biofilm reactor. The HRT is certified as a key parameter affecting the nitrogen removal performance. In this study, 8 h is

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30%

the optimum HRT both for the good treatment performance and efficiency treatment capability. The value of HRT decides the contact time of the influent wastewater in pace with the microbial biomass (Ji et al. 2016). Longer HRTs usually induce a better nitrification performance on account of the sufficient biochemical reaction time than shorter ones for slowgrowth autotrophic nitrifiers (Kim et al. 2008). An appropriate extension of HRT improves the nitrate reduction (Wang et al. 2009c). Whereas, an overlong HRT is meaningless for that it may cause the waste of treatment capacity and energy consumption along with little enhancement of performance (Wang et al. 2009a). The long-term deficiency of nutrient substances resulted from the overlong HRT may also destroy the treatment system (Guo et al. 2017). Therefore, the choice of a proper HRT is crucial for


the efficient nitrogen removal in the enhanced multistage A/O biofilm reactor. The influent distribution ratio resulted from the design of multiple inlets can promote the denitrification efficiency. The optimization of organics utilization enhances the nitrogen removal in multistage A/O reactor treating wastewater with low COD concentrations (Zhu et al. 2009). The ammonium removal may be restricted if the subsequent influent flow is superabundant on account that the ammonium in the subsequent influent flow experiences a relatively short nitrification period (Wang et al. 2009b; Zhu et al. 2007). Meanwhile, a high influent distribution ratio caused by the exorbitant flow of the front inlet may lead to the deficiency of carbon source in latter parts and the waste of additional inlets (Ge et al. 2010). Figure 4c and d also demonstrates that the appropriate influent distribution ratio can improve the nitrogen removal performance in the reactor, while 2:1 is the optimum value for the influent distribution ratio in this study. The mixed liquor internal recycle from the oxic tank to the front anoxic tank is commonly employed to improve the denitrification efficiency in BNR (Jaafari et al. 2017; Tan and Ng 2008). In this study, 200% is the optimum internal recycle ratio for the best removal performance especially for the TN removal performance. During the optimization of internal recycle ratio, the influence of nitrate reflux on the nitrification performance is limited which agrees with previous reports that the internal recycle ratio had no significant influence on ammonium removal in a laboratory-scale anaerobic-anoxic-aerobic MBBR and a modified Orbal oxidation ditch (Shi et al. 2010; Zhou et al. 2013). And the result about TN removal is similar to a previous study about the enhancement of BNR by adjusting the internal recycle ratio (Falahti-Marvast and Karimi-Jashni 2015). A proper internal recycle can transfer the nitrate-rich liquid from the oxic tank to the anoxic tank with higher organics concentrations (Chakraborty and Veeramani 2006), which motivates the organics utilization by denitrifiers instead of heterotrophic bacteria without nitrate reduction ability (Nguyen et al. 2016). In this study a low internal recycle ratio of 100% results in a poor enhancement of the TN removal possibly caused by the inadequate supply of nitrate in the nitrate reflux. Then an exorbitant ratio of internal recycle of 300% is found to clearly inhibit the TN removal performance by

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breaking the anoxic condition in denitrification galleries owing to the high DO concentration in nitrate reflux from oxic galleries, which is similar to previous results (Jaafari et al. 2017; Tan and Ng 2008). Moreover, the increase of energy consumption caused by excessive internal recycle is harmful to the further application as well (Bai et al. 2016). In comparison with the initial performance (data from Day 8–20 in the initialization phase), the average TN removal performance of the optimized biofilm reactor (data from Day 7–20 under optimum parameters) exhibits a significant improvement from 48.35 ± 1.15 to 59.51 ± 1.95%. Simultaneously, the average ammonium and COD removal efficiencies slightly rise from 97.49 ± 0.20 to 99.75 ± 0.21% and from 84.09 ± 0.79 to 85.06 ± 0.79% with the organic loading rate at around 0.36 kg COD/m3 d under the optimal conditions. The optimization of key operational parameters effectively promotes the organics and nitrogen removals especially the TN removal performance in the enhanced multistage A/O biofilm. The removal efficiencies in this study are higher than those of a previous research treating LSDW (about 0.6 kg COD/m3 d) with biological aeration filter, which show the removal efficiencies of COD, ammonium and TN at 82.54, 94.83 and 51.85% (Chen et al. 2011). These results manifest that the enhanced multistage A/O biofilm reactor can act as a qualified process for the treatment of LSDW with low COD concentrations, and the optimization of key operational parameters is essential for the improvement of treatment performance. By the analysis of high-throughput sequencing, the microbial diversities of nitrifying biofilm samples are found to be higher than those of denitrifying biofilm samples. The Chao, ACE estimator and OTU values also confirm that the nitrifying biofilm samples have higher richness of the total amount of the bacteria than denitrifying biofilm samples (Table S2). Proteobacteria and Bacteroidetes are the dominant phyla in all the nitrifying and denitrifying biofilm samples in this study, in accordance with many wastewater treatment processes (Hu et al. 2012; Stevens et al. 2005; Zhang et al. 2012). Several members belonging to Proteobacteria and Bacteroidetes have been found to act as aerobic nitrifiers or anoxic denitrifiers (Hu et al. 2012; Mills et al. 2008; Peng and Zhu 2006; You et al. 2009). In the meantime, the sums of relative abundances of Proteobacteria and Bacteroidetes are more than 50%

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in all the samples (Fig. 6a). These evidences suggest that they took part in the removal of organics and nitrogen with high possibility. Moreover, the relative abundance of Nitrospirae phylum is obviously higher in the oxic galleries than that in the anoxic galleries (0.55% of A1 and 1.12% of A2), possibly because of the better oxygen levels for nitrite oxidizing bacteria to grow and metabolize in aerobic conditions (Ye et al. 2011). In addition to Proteobacteria and Bacteroidetes, the phyla Chloroflexi, Firmicutes and Saccharibacteria, and Actinobacteria were reported to participate in the anaerobic fermentation and organics degradation in previous reports (Hu et al. 2012; Kindaichi et al. 2016; Miura et al. 2007; Narihiro et al. 2012; Wan et al. 2011; Yang et al. 2014). According to Fig. 6b, Nitrosomonadaceae_uncultured and Nitrospira sp. could retain in the process stably instead of being washed out after long-period operation. Heterotrophic bacteria including Thauera sp., Zoogloea sp., Flavobacterium sp., Comamonadaceae_unclassified, Pseudomonas sp., and Dechloromonas sp. have been proved to own the abilities of denitrification and organics degradation in many previous studies (Biegert et al. 1996; He et al. 2016; Horn et al. 2005; Huang et al. 2015; Liu et al. 2013; Papirio et al. 2014; Shinoda et al. 2004; Xing et al. 2010). These results indicate that the multistage A/O biofilm process can adapt to the LSDW treatment due to the effective retention of functional nitrifiers and denitrifiers in the system in this study. Nitrosomonadaceae_uncultured,Nitrospira sp., and several dominant heterotrophic genera in the system, such as Thauera sp. and Flavobacterium sp., are likely to be responsible for nitrification and denitrification course on the basis of their potential functions and relative abundances. In conclusion, the nitrogen removal performance and microbial community profile of an enhanced multistage A/O biofilm reactor in the treatment of LSDW was investigated. The optimum operational parameters for best nitrogen removal were HRT of 8 h, influent distribution ratio of 2:1 and internal recycle ratio of 200%. The average ammonium, TN and COD removal efficiency of the reactor separately achieved 99.75 ± 0.21, 59.51 ± 1.95 and 85.06 ± 0.79% with the organic loading rate at 0.36 kg COD/m3 d under the optimal operational parameters. Proteobacteria and Bacteroidetes were the dominant phyla in the all nitrifying and denitrifying biofilm samples, while Nitrosomonadaceae_uncultured,Nitrospira sp., and

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several dominant heterotrophic genera in the system, such as Thauera sp. and Flavobacterium sp., may act as important members for nitrification and denitrification in the biofilm process. Results demonstrated that the enhanced biofilm reactor with nitrifying and denitrifying biofilm carriers showed well organics and nitrogen removal performance in the LSDW treatment. Funding This study was funded by National Natural Science Foundation of China (Nos. 51608154 and 51478140), Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. HC20152601), the Science Research and Technology Development Project of Guangxi (Grant No. Guikehe1599005-2-2), and the HIT Environment and Ecology Innovation Special Funds (Grant No. HSCJ201604). Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

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