Correlating microbial community structure with operational conditions in biological aerated filter reactor for efficient nitrogen removal of municipal wastewater

Bioresource Technology 250 (2018) 374–381

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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Correlating microbial community structure with operational conditions in biological aerated filter reactor for efficient nitrogen removal of municipal wastewater

T

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Bo Yanga, Jinzhao Wanga, Junfeng Wanga, , Hui Xua, Xinshan Songa, Yuhui Wanga, Fang Lia, Yanbiao Liua, Junhong Baib a College of Environmental Science and Engineering, State Environmental Protection Engineering Center for Pollution Treatment and Control in Textile Industry, Donghua University, Shanghai 201620, PR China b School of Environment, Beijing Normal University, Beijing 100875, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Gas/water ratio Reflux percentage Contaminants removal performance Sequential nitrification and denitrification Microbial community distribution

In this study, the combination of strengthen circulation anaerobic (SCA) and biological aerated filter (BAF) reactor was employed to treat municipal wastewater. Different reflux percentages or gas/water ratios were selected for evaluating the removal performance of contaminants in SCA-BAF system and sequential nitrification and denitrification process in BAF reactor. In general, reflux percentage (200%) and gas/water ratio (3:1) were a relatively suitable operational condition for BAF reactor. The COD, NH3-N, TN concentrations of effluents collected from BAF reactor varied in the ranges of 18–80, 0.2–7.2, 9.1–33.0 mg L−1, respectively. A higher NO3-N concentration in effluents of BAF reactor resulted from the lack of organic carbon resource in wastewater. High throughput sequencing analysis indicated that different nitrification and denitrification bacteria thrived in the BAF reactor. The DO, NO2-N and NO3-N concentrations showed a strong correlation with Nitrospira and Nitrosomonas in bacterial samples outlet (c and e) under gas/water ratio of 3:1.

1. Introduction The strengthen circulation anaerobic (SCA) process has been extensively applied into municipal wastewater treatment due to its higheffective removal of chemical oxygen demand (COD) and biogas energy recycle (Bandara et al., 2012; Lu et al., 2015); however, the COD or NH3-N concentration in effluents of anaerobic reactor usually vary in range of 80–180 or 40–70 mg L−1, respectively (Ozgun et al., 2013). The discharge of such wastewater with a high ammonia concentration directly into environment might arise the water eutrophication in Nature Environment (Coban et al., 2015; Wang et al., 2017). Therefore, the removal of nitrogen for the effluents of SCA reactor prior to discharge is required to after the treatment of SCA reactor. In a biological aerated filter (BAF) reactor, ammonia is easily oxidized to nitrate and nitrite under the aerobic environments (Sun & Sun, 2012). Notably, the combination system of up-flow anaerobic sludge blanket (USAB) and BAF has been successful applied in microcrystalline cellulose or heavy oil wastewater treatment (Ji et al., 2012; Liu et al., 2013). Nevertheless, little is known about the mechanisms of nitrification and denitrification process as affected by spatial distribution of microorganisms in the BAF

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reactor under different operational conditions. It has been reported that the operational conditions of anaerobic bioreactor presented a significant influence on its removal performance of contaminant during the wastewater treatment. In addition, the use of anaerobic treatment for low-strength wastewater has been already indicated under the ambient temperature, hydraulic retention times, and different up-flow velocities (Bandara et al., 2012; Yang et al., 2017). In addition, the bacterial and archaeal communities distributed in the anaerobic reactor were also investigated with findings that the variation of methanogen structure significantly influenced on the pollutant removal performance of bioreactor. It has been reported that there is a competitive relationship between acetoclastic and hydrogenotrophic methanogens (Bandara et al., 2012). The different types of methanogens present a diversity metabolic ability in electrons production, and the increased relative abundance of Dechloromonas in anaerobic environment could enhance the inorganic nitrogen removal (Wang et al., 2016). Therefore, the application of SCA reactor as a pre-treatment technology for treating municipal before the BAF reactor might showed an effect on the bacterial community distribution and contaminant removal of BAF reactor, especially in the process of nitrification and

Corresponding author. E-mail address: [email protected] (J. Wang).

https://doi.org/10.1016/j.biortech.2017.11.065 Received 8 October 2017; Received in revised form 21 November 2017; Accepted 22 November 2017 Available online 23 November 2017 0960-8524/ © 2017 Elsevier Ltd. All rights reserved.

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consisted of two three-phase separators, in addition, a thermostatic water bath was used to heat the external circulating wastewater at a mesophilic temperature of 30 ± 1 °C (Yang et al., 2017). The BAF reactor can be divided into three parts from bottom to the top: supporting layer (height 20 cm; cobblestone 8–30 mm), packing layer (height 160 cm; Fe-C ceramsite 3–5 mm; average porosity 0.37) and separation layer (50 cm). Four continuous pumps were respectively employed to adjust the flow rate of influents and external circulation, and an air compressor was assembled to supply air for the BAF reactor. Five sample ports (a, b, c, d, and e) of the BAF reactor were settled at 20, 60, 100, 140, 180 cm from the bottom to the top.

denitrification. The sequential nitrification and denitrification efficiency in the BAF reactor is tightly related to the microbial diversity and community distribution. In recent years, a high diversity of microbes, including Dechloromonas, Propionivibrio, Thermomonas, Pseudomonas, Flavobacterium and Desulfobulbus, have been described to show a good ability in the inorganic nitrogen removal (Corbella et al., 2015; Wang et al., 2016). In addition, previous study has been demonstrated that the increase of microorganisms (such as Dechloromonas, and Desulfobulbus) with a ability of electrons production in the anaerobic environment might provide electron donors for nitrate and nitrate reduction during the denitrification process (Wang et al., 2016). The direct electron transfer produced by these bacteria is based on the physical contact between c-type cytochromes or pills and the electron acceptors (Holmes et al., 2006; Reguera et al., 2005). Thus, the enhancement of electrochemically active microorganisms (EAM) will favor the removal of inorganic nitrogen (NO3-N and NO2-N) in BAF reactor. In addition, in aquatic environments of BAF reactor, the existence of dissolved oxygen (DO) and oxidation-reduction potential (ORP) gradients will be exploited to stimulate the proliferation of a multitude of microorganisms with respective metabolic activity in spatial distribution. The thrive of these microbes may be beneficial for the process of sequential nitrification and denitrification in BAF reactor. Herein, in this present study, a SCA-BAF system was employed to treat the synthetic municipal wastewater at medium temperature. We hypothesized that: i) the removal performance of contaminant is strongly related to the operational conditions of SCA-BAF reactor; ii) the sequential ammonification, nitrification and denitrification process might be significantly influenced by the DO concentration and bacterial community distribution. To address the above hypotheses, the high throughput sequencing analysis was used to assess the inorganic nitrogen (NO3-N and NO2-N) removal by the biodegradation actions of microorganisms in spatial distribution.

2.2. Raw sludge The seed sludge of SCA and BAF reactor respectively originated from the granular sludge of internal circulation reactor for the treatment of paper-making wastewater (Zhejiang, China) and sewage treatment plant in Shanghai, China. The characteristic [sedimentation velocity, average diameter and mixed liquor suspended solids (MLSS) concentration] of former sludge was similar with the previous study (Yang et al., 2017). The MLSS concentration and settlement value (SV) of the aerobic activated sludge collected from the sewage treatment plant was 3.5 g L−1 and 25%. 2.3. Synthetic wastewater

2. Materials and methods

Synthetic wastewater was prepared from tap water using sucrose, potato starch, powdered milk, beef peptone and beef broth as the carbon source. The COD, total nitrogen (TN), total phosphorus (TP) concentrations of the synthetic wastewater was 450, 50 and 7 mg L−1, respectively. The synthetic wastewater contained (in mg L−1): Sucrose, 200; Potato starch, 100; Powdered milk, 30; Beef peptone, 100; Beef broth, 30; NH4Cl, 100; K2HPO4, 25; CaCl2, 5; FeSO4·7H2O, 6; and MgSO4·7H2O, 6. Alkalinity, as 200 mg L−1 NaHCO3, was introduced to ensure the pH of influents ranged from 7.0 to 8.0.

2.1. SCA-BAF bioreactor

2.4. Experimental methods

The schematic diagram of SCA reactor (effective working volume 70 L; height 200 cm; internal diameter 19 cm; material plexiglass column) and the BAF reactor (height 230 cm; internal diameter 13 cm; material plexiglass column) is showed in Fig. 1. The SCA reactor

The experiments were carried out in four phases, including Inoculated phase (33 days), Period I (28 days; BAF with different reflux ratio: 400%, 300%, 200%, and 100%), Recovery phase (20 days), and Period II (60 days; BAF under different gas/water volume ratio: 4:1, 3:1, Fig. 1. The schematic diagram of a combination system of strengthen circulation anaerobic (SCA) and biological aerated filter (BAF) reactor.

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Table 1 The operation parameters of a combination system of strengthen circulation anaerobic (SCA) and biological aerated filter (BAF) reactor during the all experimental periods. Period

Reactor

HRT (h)

Duration time (d)

Vup (m h−1)

Reflux velocity (L h−1)

Reflux percentage (%)

OLR (kg COD m−3 d−1)

Gas/water ratio

Inoculated phrase

SCA BAF

6.0 2.2

33 33

3.25 0.30

80 /

670 /

1.68 0.70

/ 7:1

Period I

SCA BAF

6.0 2.2

28 7 7 7 7

3.25 1.50 1.20 0.90 0.60

80 16 12 8 4

670 400 300 200 100

1.82 0.64 0.61 0.60 0.60

/ 3:1

Recovery phrase

SCA BAF

5.0 2.2

22 22

3.32 0.90

80 8

570 200

2.12 0.62

/ 3:1

Period II

SCA BAF

5.0 2.2

60 20 20 20

3.32

80

570

0.90

8

200

2.14 0.54 0.48 0.44

/ 4:1 3:1 2:1

Fig. 2. The COD (a), NH3-N (b), and TN (c) concentration of influent and effluent samples from a combination system of strengthen circulation anaerobic (SCA) and biological aerated filter (BAF) reactor.

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COD removal efficiency was stabilized with the reflux percentage increased to 200%. In Period II, the COD concentration of SCA influents, SCA effluents, and BAF effluents varied in the ranges of 411–480, 108–151, and 18–45 mg L−1, respectively. An average COD removal efficiency of 94.1 ± 1.2% was obtained in the SCA-BAF system. In addition, no significant difference was observed in the COD removal efficiency of BAF reactor under different gas/water ratio of 4:1, 3:1, and 2:1 (p < .05). It has been demonstrated that the use of external circulation in anaerobic treatment process could increase the biodegradation ability and growth of microorganisms (Wang et al., 2014). In addition, the added primary three-phase separator in the SCA reactor was benefit for the distribution of a multitude of microorganisms with respective metabolic activity in spatial position, which shows a great potential for the accelerated degradation of refractory organic matter (Gulhane et al., 2017). Hence, during the all experimental phases, a stable removal performance (66–75%) of organic matter was obtained in the SCA reactor. In addition, an added external circulation device in BAF reactor could also enhance the mass transfer between the microorganisms and contaminant (Wu et al., 2015). A relatively high removal of organic matter was observed during the experimental period with a reflux percentage 400%, 300%, and 200%. However, with the decrease of reflux percentage to 100%, the COD concentration of BAF reactor was increased from 18–45 mg L−1 to 60–80 mg L−1. These operational results indicated that the increase of reflux ratio to greater than or equal to 200% in the BAF reactor could reduce the organic loading and the COD concentration of effluents. In addition, no significant difference of COD removal in the BAF reactor under different gas/water ratios demonstrated that the ratio of gas/water maintained at 2:1 was adequate to degrade organic matter under a reflux percentage of 200%. In the literature, the COD removal efficiency could approximately maintain at 65% in BAF when the operational condition of gas/water ratio was 5:1 (Wu et al., 2015). Hence, the using of pre-treated SCA reactor for municipal wastewater treatment may accelerate the biodegradation of organic matter to small molecule compounds, which decrease the requirements of oxygen supply due to their easy degradation characteristic. Data for the NH3-N and TN concentration of influent and effluent samples collected from the SCA and BAF reactor were presented in Fig. 2(b) and (c). In general, the stable removal efficiencies of NH3-N and TN were observed at the end of inoculated phase. The concentration of NH3-N and TN in influents were 32.8 ± 3.7 mg L−1 and 49.7 ± 3.9 mg L−1, respectively. Notably, the organic nitrogen contained in powdered milk, beef peptone and beef broth has been almost completely converted to NH3-N by in the SCA reactor under the anaerobic condition. Hence, the TN removal efficiency of the SCA reactor was only maintained at 7.4%. In BAF reactor, the concentration of NH3N in Period I and II ranged from 0.2 to 7.2 mg L−1. However, a significant decrease of NH3-N removal was obtained in BAF reactor under the operational condition of gas/water ratio of 2:1 and reflux percentage of 200% compared with other operational parameters in Period II (p < .05). In addition, in Period I, the removal efficiency of TN under reflux percentage of 100% was also significantly lower than that of other conditions (p < .05). It is generally accepted that the anaerobic bioreactor shows a lower NH3-N removal efficiency ability during the wastewater treatment (Lijó et al., 2016; Qiu et al., 2013). An increase trend of NH3-N in effluents compared with influents was also found in previous research (Qiu et al., 2013). The main reason resulted in this phenomenon was that the nitrification process was limited to the low DO concentrations in the anaerobic reactor (Vymazal, 2007). Therefore, the quantity of NH3-N has been converted to NO2-N or NO3-N was maintained at a relatively low in this condition (Reino & Carrera, 2016). However, the microorganisms contribute to the conversion process of organic nitrogen to NH3-N can thrive under anaerobic (Adrados et al., 2014), which led to NH3-N was the key component of TN in effluents of SCA reactor. In BAF

and 2:1). At the beginning the inoculated phase, the granular sludge (28 L) and aerobic activated sludge (2 L) was added into the SCA reactor and the BAF reactor, respectively. During the experimental periods, the operational parameters, including duration time, hydraulic retention time (HRT), up-flow velocity, reflux velocity, reflux percentage, organic loading rate (OLR), and gas/water ratio, were presented in Table 1. 2.5. Sampling and analysis methods The influent and effluent samples of SCA and BAF reactor were collected using a conical flask to compare the removal efficiency of COD, NH3-N and TN, in addition, water samples of sampling outlet (a, b, c, d, and e) of the BAF reactor was also collected to assess the fluctuation of DO, ORP, NO3-N, NO2-N, NH3-N and TN. In the Periods I and II, water samples were collected daily and every three days. COD (potassium dichromate method), NO3-N (phenol disulfonic acid luminosity method), NO2-N [N-(1-naphthalene)-diaminoethane method], NH3-N (Nessler’s reagent spectrophotometry) and TN (alkaline potassium persulphate digestion-UV spectrophotometric method) concentration of each sample are analyzed according with the monitoring and analysis methods for water and wastewater (Fourth Edition) of China. 2.6. High throughput sequencing analysis In Period II, the microbial samples (50 g of Fe-C ceramsite) of sample outlets (a, c, and e) in the BAF reactor were collected every five days [selected days were (90, 95, 100) and (110, 115, 120)] under the operational condition of gas/water ratio of 4:1 and 3:1; finally, microbial samples from sample ports (a, c, and e) were mixed to yield to a composite sample for high throughput sequencing analysis. The extracted steps of bacterial DNA were according with the methods in previous reports (Li et al., 2015). During the process of PCR amplification, universal bacterial primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) were used to amplify the V4 and V5 hypervariable regions of the bacteria 16S rRNA. The steps of amplification process and the analysis of pyrosequencing data were similar with the previous studies (Li et al., 2015; Wang et al., 2016). The extraction and amplification of bacterial DNA samples were performed using paired-end sequencing with an Illumina MiSeq PE300 platform (Shanghai Majorbio Bio-Pharm Technology Co. Ltd., China). 2.7. Statistical analysis The data of DO, ORP, COD and nitrogen removal in the BAF reactor under different operational condition (gas/water ratio) were statistically analyzed using SPSS 22.0 software, and p < .05 was considered as the significant level under the one-way analysis of variance (ANOVA) comparison. 3. Results and discussion 3.1. Contaminants removal performance of the SCA-BAF system The COD concentration of influent and effluent samples from the SCA and BAF reactor were presented in Fig. 2(a). In general, a stable COD removal performance (total removal efficiency > 85%) was observed in SCA-BAF system after inoculated phase (20 days). In Period I, the COD concentrations of SCA influents, SCA effluents, and BAF effluents varied in the ranges of 387–490, 133–180, and 25–80 mg L−1, respectively. The average COD removal efficiency in the SCA and BAF reactor in all experimental days was 65.3 ± 2.9% and 71.0 ± 9.7%, respectively. However, a significant reduction of COD removal in the BAF reactor was obtained with the reflux percentage decreased to 100%. During this stage, average COD concentration of effluents from BAF reactor was 70.2 ± 6.9 mg L−1, a corresponding COD removal efficiency ranged from 44.4% to 59.7%. In Recovery phase, a steady 377

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Table 2 The water quality parameters of samples collected from sample ports (a, b, c, d and e) of the biological aerated filter (BAF) reactor. Gas/water ratio

Sample outlet

DO (mg L−1)

ORP (mV)

NH3-N (mg L−1)

NO3-N (mg L−1)

NO2-N (mg L−1)

TN (mg L−1)

4:1

a b c d e

0.8 1.9 2.2 2.6 3.2

(0.3) (0.4) (0.4) (0.5) (0.6)

a a a a a

−22 (15) a 101 (30) a 143 (42) a 151 (50) a 159 (51) a

22.0 (4.0) a 18.3 (3.2) a 9.8 (2.6) a 5.3 (1.7) a 1.5 (0.8) a

1.8 (0.5) a 0.8 (0.4) a 5.3 (1.9) a 9.3 (2.2) a 12.2 (2.7) a

0.8 1.0 1.1 1.3 1.3

(0.4) (0.6) (0.5) (0.6) (0.6)

a a a a a

26.8 22.6 15.6 16.1 15.4

(4.1) (3.5) (2.9) (3.4) (3.0)

a a a a a

3:1

a b c d e

0.6 1.7 2.0 2.3 2.9

(0.2) (0.3) (0.4) (0.5) (0.6)

a a a a a

−160 (30) b 28 (26) b 98 (32) b 126 (35) b 135 (42) b

19.8 (3.2) a 15.8 (2.8) b 11.3 (2.4) a 6.6 (2.1) a 4.3 (1.6) b

1.2 (0.4) a 0.6 (0.3) a 4.1 (1.6) a 8.8 (1.8) a 11.5 (2.5) a

0.7 0.5 1.0 1.2 1.4

(0.3) (0.3) (0.4) (0.5) (0.6)

a b a a a

23.5 18.8 17.6 16.8 17.8

(3.8) (3.2) (2.7) (2.8) (3.2)

a b a a a

2:1

a b c d e

0.6 1.4 1.6 1.9 2.2

(0.2) (0.3) (0.3) (0.4) (0.5)

a b b b b

−254 (50) c 22 (20) b 59 (25) c 93 (36) c 114 (42) c

22.0 (4.2) a 18.1 (3.4) a 13.9 (2.8) b 10.1 (2.9) b 6.9 (1.6) c

6.7 (1.6) b 5.8 (1.4) b 8.8 (2.4) b 12.2 (3.0) b 15.5 (3.4) b

1.7 1.6 1.3 1.8 2.5

(0.6) (0.5) (0.4) (0.5) (0.8)

b c a b b

31.1 25.8 25.1 25.3 24.8

(5.6) (4.2) (3.9) (4.2) (3.7)

b c b b b

Note: 1. Average value is shown with S.E. in brackets; 2. Different letters across treatments mean the significant difference at p < .05 level.

researches (Chen et al., 2009). Although the low C/N has been considered as the limited factor for the denitrification process, the microelectric field environment provided by Fe-C ceramsite may be more efficient for treating wastewater in a low C/N ratio (Zhu et al., 2015). However, in this study, no significantly high removal efficiency of NO3N was obtained in the BAF reactor, the main reason of this phenomenon resulted from a relatively high DO concentration (1.6–2.0 mg L−1) was observed in the sample ports (b, c, d and e). Therefore, the electrons produced by the EAM are prior to combined with oxygen and protons to generate H2O (Villasenor et al., 2013). To achieve a low concentration of NO3-N in effluents, parts of the raw wastewater might be directly pumped into the BAF reactor for the enhancement of C/N ratio of influents.

reactor, the removal of NH3-N was closely related to the gas (oxygen) supply. The gas/water ratio maintained at 4:1 and 3:1 were suitable for the removal of NH3-N to below 3.9 mg L−1; however, oxygen could be a limited factor for the removal of NH3-N when BAF reactor operated under a gas/water ratio of 2:1. With the DO data in Table 2, it also demonstrated that the DO concentration of four sample outlets (a-d) in BAF reactor was maintained below than 2.0 mg L−1. This indicated that the oxidization of NH3-N to NO2-N or NO3-N was limited with the low DO concentration in the BAF reactor.

3.2. Sequential nitrification and denitrification in the BAF reactor To assess the sequential nitrification and denitrification process in BAF reactor, the distribution of NH3-N, NO3-N, NO2-N and TN in spatial was showed in Table 2. As presented in Table 2, the DO concentration in the sample ports (b, c, d and e) of the BAF reactor operated with gas/ water ratio of 2:1 was significantly lower than that of the operational condition of 3:1 and 4:1 (p < .05). Similar findings were also obtained in the ORP value of sample (p < .05). With the gas/water ratio of 4:1 and 3:1, the NH3-N has been almost converted to NO3-N and NO2-N via nitrification function of microorganisms. Hence, the average NH3-N concentration of effluents collected from sample outlet (e) was lower than 4.3 mg L−1. However, an apparent increase of NH3-N concentration in effluents collected from this port was observed when the gas/ water ratio changed to 2:1. In addition, the concentration of NO3-N of sample outlets (a, b, c, d and e) showed an increase trend along with the spatial bottom to the top. Although the NO2-N concentration of effluents show a significant difference among the different sample ports, the maximum average concentration of NO2-N was maintained at only 2.5 mg L−1. Notably, the average TN concentration in samples of sample outlet (e) enhanced to 24.8 mg L−1 with the gas/water decreased to 2:1. It is generally accepted that the DO and carbon resource are the limited factor during the nitrification and denitrification process, respectively (Rahimi et al., 2011; Su et al., 2015). In BAF reactor, the DO concentrations of the samples collected from ports (a, b, c and d) were below 2.0 mg L−1 under the operational condition of gas/water ratio of 2:1, which indicated that the BAF rector under this condition could not provide an aerobic condition for the NH3-N of wastewater completely converted to NO3-N and NO2-N. Hence, a higher concentration of NH3N in effluents was obtained during this stage. Notably, nearly half of NH3-N in the influents of the BAF reactor is aerobic degraded to NO3-N or NO2-N by inflowing gas and water in the bottom. In addition, lower removal efficiencies of NO3-N in BAF reactor might result from the too low concentration of organic matter compared with that of previous

3.3. Biodiversity and distribution of microorganisms in the BAF reactor As presented in Table 3, the effective sequences of the total samples summed up to 152901, and these reads were clustered into a total of 3266 operational taxonomic units (OTUs) with a similarity level of 97%. Compared to the lower outlet (a) and top outlet (e) of samples, the samples collected from outlet (c) was obtained with a higher OTUs number. The biodiversity estimators of ACE and Chao indicated that the richness of bacteria in the middle parts showed a higher value. These findings are similar with the trend of OTUs. In general, the richness of bacteria increased with the decrease of gas/water ratio from 4:1 to 3:1, which might be caused by that parts of the microorganisms have been washed out under the BAF reactor heavily charged with gas. The diversity index of microbial population, Shannon estimator, varied in the ranges of 3.0–3.7 for gas/water ratio 4:1, and 4.2–4.7 for gas/water ratio 3:1. In addition, the coverage of six samples ranged from 0.993 to 0.997, indicating that most of the bacteria in the samples were detected. As shown in Fig. 3, Proteobacteria and Bacteroidetes were the Table 3 The richness and evenness of bacterial samples collected from sample outlets (a, b, c, d and e) of the biological aerated filter (BAF) reactor.

378

Gas/water ratio

Sample outlet

Sequence

OTU

ACE

Chao

Shannon

Coverage

4:1

a c e

21814 27435 20055

421 546 464

587 632 555

597 650 559

3.0 3.7 3.5

0.993 0.996 0.994

3:1

a c e

27170 28162 28265

588 646 601

664 688 652

677 689 658

4.7 4.7 4.2

0.996 0.997 0.997

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Fig. 3. The relative abundance of bacteria at phylum level bacterial samples collected at gas/water ratio of 4:1 and 3:1.

dominant phylum in all sludge samples, accounting for 57.3–95.6% of the total effective reads. These results are similar with the findings in the reports of the component of bacteria in activated sludge collected from municipal wastewater treatment plants (Gao et al., 2016). The other four dominant phyla were Nitrospirae (0.2–22.1%), Chloroflexi (1.2–4.3%), Planctomycetes (0.4–6.4%), Acidobacteria (0.3–5.4%) and Chlorobi (0.6–3.1%), followed by a few phyla with average relative abundance below 1% such as Gemmatimonadetes (0.8%), Cyanobacteria (0.7%), Actinobacteria (0.3%), Fibrobacteres (0.3%), Firmicutes (0.2%), and Spirochaetae (0.1%). In addition, the distribution of bacteria in the BAF reactor at gas/ water ratio of 4:1 and 3:1 was presented in Figs. 4 and 5. In general, the most abundant genera in the activated sludge samples are Flavobacterium, Cloacibacterium, and Nitrospira. Notably, the relative abundance of Flavobacterium in the BAF reactor under gas/water ratio of 4:1 was significant higher that of gas/water ratio of 3:1. In addition, the distribution of this genus decreased along with the BAF reactor from the bottom to top. However, a reverse trend was observed in the BAF reactor under these two operational conditions. Moreover, other seven abundant (average relative abundance > 1%) genera accounted for 20.0–37.4% of the effective sequences, including Aeromonas, Comamonadaceae_unclassified, Nitrosomonas, Thiothrix, Thermomonas, Pseudomonas, and Shewanella. The relative abundance of Thermomonas in the BAF reactor under gas/water ratio of 4:1 was significant higher that of gas/water ratio of 3:1. Additionally, it is interesting to observe that the Nitrosomonas, Nitrospira, and Pseudomonas in the BAF reactor under gas/water ratio of 3:1 was much higher than that of gas/water ratio of 4:1. As shown in Fig. 4, Proteobacteria has been regarded as the dominant phylum in the BAF reactor. This phylum was widely distributed in soils, wastewater and sludge (Gao et al., 2016; Roesch et al., 2007; Wang et al., 2016). Additionally, the phyla of Bacteroidetes, Nitrospirae, Chloroflexi, Planctomycetes, Acidobacteria and Chlorobi were frequently found in bacterial samples of activated sludge (Juretschko et al., 2002). In this present study, it was notable that the biodiversity and structure of bacteria community of sludge samples in BAF reactor was significantly different under different spatial positions and operational conditions. For instance, the relative abundance of Proteobacteria showed an increase trend under the gas/water ratio of 4:1, whereas a decrease trend was observed under the gas/water ratio of 3:1. In addition, the relative abundance of Bacteroidetes was significantly

Fig. 4. The relative abundance of bacteria at genus level of bacterial samples collected at gas/water ratio of 4:1 and 3:1.

improved by the enhancement of gas influent ratio, whereas the relative abundance of Nitrospirae was in the ports (c and e) under gas/water ratio of 3:1 was much higher than that of gas/water ratio of 4:1. Among the Bacteroidetes phylum, a group of chemoheterotrophic microorganisms showed a good ability to degrade refractory organic matter. A potential nitrogen removal bacteria (Flavobacterium) had a dominant advantage in the bottom of the BAF reactor under gas/water ratio of 4:1. This species is a typical genus that can be widely detected in the activated sludge (Adrados et al., 2014; Park et al., 2007). It is reported that the genus of Nitorspirae was a major ammonia- and nitrate-oxidizing bacteria in the activated sludge samples during the wastewater treatment of anaerobic/anoxic-oxic (AAO) process (Kim et al., 2013; Limpiyakorn et al., 2011). In this present study, this genus is more suitable for thriving in an operational condition of gas/water ratio of 3:1. Canonical-correlation analysis (CCA) was employed to evaluate the relationship between the microbial communities (top 10 genera) and operational conditions. As shown in Fig. 6, the DO, NO2-N and NO3-N concentration had strongly correlations with the microbial communities (Nitrospira and Nitrosomonas) of ports (c and e) under the gas/ water ratio of 3:1. These two genera have been respectively reported as the nitrite oxidizing and ammonia-oxidizing genus (Cokro et al., 2017). In addition, NH3-N concentration is strongly linked to the microbial communities (Flavobacterium, Cloacibacterium, Aeromonas, Thermomonas, Shewanella) of ports (a, c, and e) under the gas/water ratio of 4:1. Therefore, the different operational conditions of BAF reactor could form the different diversity and distribution of bacteria community in spatial direction. In general, the BAF reactor could complete the nitrification process of NH3-N converted to NO3-N and NO2-N under the gas/water ratio of 3:1. However, a higher NO3-N and TN concentration 379

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Fig. 5. Heat-map graph of hierarchy cluster for the top 50 genera at gas/water ratio of 4:1 and 3:1. Color intensity in each panel represent the difference and similarity characteristic under different operational condition. Values of red and green color mean the lg(numbers of genus). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

in the effluents of BAF reactor might result from the lack of organic carbon resource in influents. 4. Conclusion In this study, SCA/BAF system was employed to treat municipal wastewater under different operational conditions. The resulted indicated that municipal wastewater can be well treated with BAF reactor under reflux percentage (200%) and gas/water ratio (3:1), although average TN concentration of effluents from BAF reactor was 25.3 mg L−1. High throughput sequencing analysis indicated that different nitrification and denitrification bacteria thrived in the spatial position under gas/water ratio of 4:1 and 3:1, respectively. The NH3-N concentrations are strongly linked to the microbial communities (Flavobacterium, Cloacibacterium, Aeromonas, Thermomonas, Shewanella) of ports (a, c, and e) under gas/water ratio of 4:1.

Fig. 6. Canonical-correlation analysis (CCA) map of bacteria data of environmental factors and operational conditions and top 10 bacteria at genus level [in the legends, 3 and 4 represent the gas/water ratio; (a), (c), and (e) mean the sample ports].

Acknowledgements The authors are grateful to acknowledge the financial support from 380

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the National Nature Science Foundation of China (Grant Nos. 51679041), Project of the Shanghai Science and Technology Committee (Grant No. 17DZ1202204), and National Key Technology Support (Grant Nos. 2015BAB07B09).

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