Nitrogen removal pathway and dynamics of microbial community with the increase of salinity in simultaneous nitrification and denitrification process

Science of the Total Environment 697 (2019) 134047

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Nitrogen removal pathway and dynamics of microbial community with the increase of salinity in simultaneous nitrification and denitrification process Zhengang Xia b, Qun Wang b, Zonglian She a,b,⁎, Mengchun Gao a,b, Yangguo Zhao a,b, Liang Guo a,b, Chunji Jin a,b a b

Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean University of China, 266100 Qingdao, China College of Environmental Science and Engineering, Ocean University of China, 266100 Qingdao, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The SND process was mainly via nitrite not nitrate at salinity of 1.6% and 2.4%. • Elevated salinity led to shift of functional groups and nitrogen removal pathways. • S-sludge and biofilm shared similar microbial community composition in the HSBBR. • Enrichment of salt-adaptable microbes stabilized SND performance under salt stress. • Diverse pathways of nitrogen removal occurred in the HSBBR with and without salt.

a r t i c l e

i n f o

Article history: Received 17 June 2019 Received in revised form 16 August 2019 Accepted 21 August 2019 Available online 22 August 2019 Editor: Paola Verlicchi Keywords: Nitrogen removal pathway Simultaneous nitrification and denitrification Salinity Microbial community Hybrid sequencing batch biofilm reactor

a b s t r a c t In this study, simultaneous nitrification and denitrification (SND) process was successfully established in a hybrid sequencing batch biofilm reactor (HSBBR). High removal efficiency of NH+ 4 -N (98.0±2.4% to 99.8±0.4%) and COD (86.6±4.0% to 91.6±1.8%) was observed in the salinity range of 0.0 to 2.4%. SND via nitrite, replacing SND via nitrate, became the main nitrogen removal pathway at 1.6% and 2.4% salinity. Suspended sludge and biofilm shared similar microbial composition. Dominant genera were substituted by salt-adaptable microbes as salinity increasing. Abundance of autotrophic ammonia-oxidizing bacteria (Nitrosomonas) increased with elevated salinity, while autotrophic nitrite-oxidizing bacteria (Nitrospira) exhibited extreme sensitivity to salinity. The presence of Gemmata demonstrated that heterotrophic nitrification co-existed with autotrophic nitrification in the SND process. Aerobic denitrifiers (Denitratisoma and Thauera) were also identified. Thiothrix, Sedimenticola, Sulfuritalea, Arcobacter (sulfide-based autotrophic denitrifier) and Hydrogenophaga (hydrogen-based autotrophic denitrifier) were detected in both S-sludge and biofilm. The occurrence of ANAMMOX bacteria Pirellula and Planctomyces indicated that ANAMMOX process was another pathway for nitrogen removal. Nitrogen removal in the HSBBR was accomplished via diverse pathways, including traditional autotrophic nitrification/heterotrophic denitrification, heterotrophic nitrification, aerobic and autotrophic denitrification, and ANAMMOX. © 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Key Lab of Marine Environment and Ecology, Ministry of Education, Ocean University of China, 266100 Qingdao, China. E-mail address: [email protected] (Z. She).

https://doi.org/10.1016/j.scitotenv.2019.134047 0048-9697/© 2019 Elsevier B.V. All rights reserved.

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Z. Xia et al. / Science of the Total Environment 697 (2019) 134047

1. Introduction Saline wastewater is consequently discharged nowadays with the development of urbanization, industry and agriculture (Wang et al., 2017). Negative effects of increasing salt content on nutrient removal has been reported in a sequencing batch reactor (SBR) because of the plasmolysis of the activated sludge organisms at high salt contents (Uygur, 2006). Moreover, the effect of salinity on microbes could influence the pathway of nitrogen removal in biological nitrogen removal (BNR) process. Simultaneous nitrification and denitrification (SND) have been reported to be technically feasible and economically favorable as a promising process for nitrogen removal (Zeng et al., 2003). SND processes rely greatly on harmonious and balanced cooperation between nitrifying bacteria and denitrifying bacteria. Differences in sensitivity to salt of the various bacteria may imbalance the equilibrium established in SND reactors, influencing the nitrogen removal performance and pathway. Nitrogen removal pathway can be changed from SND via nitrate to SND via nitrite with the increase of salinity from 1.0% to 2.0% by the inhibition of nitrite oxidation activity (She et al., 2018). Therefore, extensive and in-depth studies on bacterial diversity and the metabolic pathways in SND system under salt stress should be conducted. The influences of salt content on nitrogen removal via SND process have been reported in some studies. SND was established under continuous aeration (6 mg O2 L−1) in a fixed-bed reactor for treating multielectrolyte saline wastewater (Macêdo et al., 2019). The results showed that high removal efficiencies of NH+ 4 -N and total nitrogen (TN) were obtained at 0.3% salinity, while the system was severely affected by salt stress at 1.7% salinity. High simultaneous removal of COD, NH+ 4 -N and TN was also achieved in an sequencing batch biofilm reactor (HSBBR) when influent salinity was in the range of 1.0–2.0%, while nitrogen removal via SND decreased with the further increase of salinity to 2.5% and 3.0% due to the inhibition of ammonium oxidation (She et al., 2018). In a sequencing batch biofilm reactor (SBBR), the increase of salinity from 10 to 30 g NaCl L−1 did not affect total nitrogen (TN) removal through SND process (Wang et al., 2017a, 2017b). Granular sludge could tolerate higher salinity, so nitrogen removal performance above 98% via SND was reported at salinity up to 50 g NaCl L−1 in an aerobic granular sludge reactor (Corsino et al., 2016). Nitrite accumulation was commonly observed in SND process under salt stress, because nitrite oxidizing bacteria (NOB) showed higher sensitivity to salt compared with ammonia oxidizing bacteria (AOB) (Wang et al., 2017a). A salinity of 5.0 g NaCl L−1 could inhibit NOB activity and cause nitrite accumulation, resulting in the achievement of SND via nitrite in a SBR (She et al., 2016). SND via nitrite, instead of SND via nitrate, became the main pathway for nitrogen removal as salinity was increased from 1.0% to 3.0% in HSBBR and SBBR (She et al., 2018; Wang et al., 2017a). Performance and stability of SND process mainly depend on the microbial community diversity and structure, so does the nitrogen removal pathway. Under salt stress, abundant population of AOB and low abundance of NOB was common phenomenon, which led to the conversion of nitrogen removal pathway to SND via nitrite (Huang et al., 2019; Wang et al., 2017b). Denitrifiers could survive actively over a wider range of salinity with high population, therefore, denitrification process was less affected by salt stress (Hong et al., 2013; Wang et al., 2017a). Traditional aerobic nitrification/heterotrophic denitrification was not the only nitrogen removal route in SND process. Some bacteria capable of heterotrophic nitrification-aerobic denitrification, such as Flavobacterium phragmitis, Paracoccus denitrificans, Pseudomonas_stutzeri and Bacillus_cereus, could co-exist with autotrophic nitrifiers and heterotrophic denitrifiers in SND system treating saline wastewater (Chen et al., 2016; Wang et al., 2017b). Stenotrophomonas and Marinobacter (aerobic denitrifier), and Thioalkalispira (autotrophic denitrifier) were also reported to be responsible for SND in biocathode microbial fuel cell treating saline mustard tuber wastewater (Zhang et al., 2019). Thereby, nitrogen removal

pathway could be multiple in SND process under salt stress. However, few reports concentrated on the co-existing functional microbes in response to complex mechanisms of nitrogen removal via SND in saline environment. The evaluation of microbial characterization and analysis of nitrogen removal pathway with the increase of salinity have been limited in SND process, especially in hybrid reactors containing both suspended sludge and biofilm. In this study, SND process had been developed in a hybrid sequencing batch biofilm reactor (HSBBR). HSBBR combined advantages of biofilm, suspended sludge (S-sludge) and sequencing batch reactor and is proved to be a promising system for SND and could withstand high salt concentration (Huang et al., 2019; She et al., 2018). The aims of this research were: 1) to investigate the performance and pathways of nitrogen removal in the SND system with the increasing salinity; 2) to reveal the dynamics of the microbial community from both S-sludge and biofilm in the HSBBR with the increase of salinity, and in this way to provide a comprehensive insight into the key functional microbes involved in nitrogen removal, such as AOB, NOB and denitrifying bacteria at different salinities. 2. Materials and methods 2.1. The HSBBR and operation A lab-scale HSBBR with a working volume of 7 L was set up and seeded with activated sludge collected from a secondary sedimentation tank of a domestic wastewater treatment plant in Qingdao, China. The structure of the HSBBR was reported by Huang et al. (2019). In some previous studies, mixed liquid suspended solid (MLSS) of 1200–2000 mg L−1 had been applied in hybrid bioreactors which combined S-sludge and biofilm (Huang et al., 2017; Lo et al., 2010; Wang et al., 2008). In this experiment, referring to these studies, the MLSS concentration in the HSBBR was kept at approximately 2000 mg L−1 by adjusting the discharge amount of suspended sludge. The HSBBR was operated 3 cycles per day, and each cycle lasted 8 h. The 8-h cycle consisted of: a 15-min feeding phase, a 6.5-h aeration phase, a 1.0-h settling phase, a 10-min decanting phase and a 5-min idle phase. 3.5 L wastewater was fed into the reactor during the feeding phase and the same volume of effluent was discharged in the decanting phase, resulting in a hydraulic retention time (HRT) of 16 h. The temperature was controlled at 25 ± 1 °C with a heater rod in the reactor. Air was continuously introduced into the reactor with a rate of 360 mL min−1 by an air pump during the aeration phase and the same air rate was employed under different salinity conditions. The dissolved oxygen (DO) concentration ranged from 0.9 to 6.2 mg L−1 during the aeration stage. The HSBBR was fed with synthetic wastewater which simulated domestic sewage under different salinities. Sodium acetate and ammonium chloride was used as the carbon source and nitrogen source, respectively. The concentrations of COD and NH+ 4 -N in the synthetic wastewater were about 400 mg L−1 and 40 mg L−1 (COD/N = 10), which is similar to the influent of a wastewater treatment plant in Qingdao as we investigated. The salinity of the wastewater was adjusted by adding seawater crystal, and 1.0% salinity corresponded to 10 g seawater crystal per liter. The main components of the seawater crystal were 2+ (per 10 g): 5.3 g Cl−, 3.3 g Na+, 0.62 g SO2− , 0.1 g K+ and 4 , 0.3 g Mg 0.09 g Ca2+. The experiment was divided into 4 periods: Period I (initial 62 days, without addition of seawater crystal), Period II (from 63 to 95th days), Period III (from 96 to 125th days) and Period IV (from 126 to 155th days), corresponding to the influent salinity of 0.0%, 0.8%, 1.6% and 2.4%, respectively. The pH of the synthetic wastewater was maintained at 7.6–8.0 during the experiment. 2.2. Analytical methods The influent and effluent samples were collected and analyzed every two days. The parameters including COD, NH+ 4 -N, nitrite nitrogen

Z. Xia et al. / Science of the Total Environment 697 (2019) 134047 − (NO− 2 -N), nitrate nitrogen (NO3 -N), MLSS and mixed liquid volatile suspended solids (MLVSS) were measured according to the Standard Methods (APHA, 2005). Total nitrogen (TN) was calculated by the sum − − of NH+ 4 -N, NO2 -N and NO3 -N. The pH was measured by pH probes (PHB-4, China). The temperature and DO were tested using the detector (WTW Company, WTW 330i, Germany). Suspended sludge (S-sludge) and biofilm samples were collected from the reactor at the end of each period for microbial analysis. The genomic DNA of the sample was extracted using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA). High-throughput sequencing based on partial 16S rDNA was used to analyze the microbial community by an Illumina MiSeq platform of Novogene (Beijing, China) in accordance with the report of Huang et al. (2019).

2.3. Calculations The nitrite accumulation rate (NAR) and the SND efficiency (ESND) were calculated by Eq. (1) and (2), respectively.

3

nitrite and nitrate at the end of aeration. h

i − þ þ ESND ¼ 1− NO−  100% x;e −NOx;i = NH4;i −NH4;e

ð2Þ

−1 where NO− ) is the concentration of nitrite and nitrate at the x, e (mg L −1 end of aeration; NO− ) is the concentration of nitrite and nitrate x, i (mg L −1 at the beginning of aeration; NH+ ) is the concentration of am4, i (mg L −1 monium nitrogen at the beginning of aeration; NH+ ) is the 4, e (mg L concentration of ammonium nitrogen at the end of aeration. Due to the small amount of nitrogen removal by the microbial assimilation (0.56–0.62% of the total nitrogen removed in 1 cycle), the nitrogen loss for growth of microbes can be neglected in the present experiment, similar to formal studies (Ge et al., 2010; Huang et al., 2019; She et al., 2016; Third et al., 2003).

3. Results and discussion 3.1. Reactor performance

h


i − −  100% NAR ¼ NO− 2;e = NO2;e þ NO3;e

ð1Þ

−1 −1 where NO− ) and NO− ) are the concentration of 2, e (mg L 3, e (mg L

0.8%

0%

Salinity 500

In Period I without salt addition (0.0% salinity), after fluctuations in the first 45 days, the reactor achieved stable performance in the later stage, with high average removal efficiencies of 89.9±4.2%, 98.0±2.4% and 91.2±4.8% for COD, NH+ 4 -N and TN, respectively, during the stable 1.6%

2.4% 100

(D)

60

Inf. COD Eff. COD COD removal

200

40

100

20

0 50

0 100

40

80

30

60

Inf. NH4+-N + 4

Eff. NH -N

20

40

NH4+-N removal

10

20

NH4+-N removal (%)

COD (mg/L) NH4+-N (mg/L)

300

0 50

0 100

40

80

30

60

Inf. TN Eff. TN TN removal

20

40

10

20

0 15

0 Eff. NO3--N

12

Eff. NO2--N

9 6 3 0 0

15

30

45

60

75 Time (d)

90

105

120

Fig. 1. Removal performance of nitrogen and COD in the HSBBR during the whole experiment.

135

150

TN removal (%)

(C)

TN (mg/L)

(B)

Eff. NO3--N and NO2--N (mg/L)

(A)

COD removal (%)

80

400

Z. Xia et al. / Science of the Total Environment 697 (2019) 134047

NO3--N COD

(B) 25

200

20

150

10

100

5

0

1

2

(C) 25

3

4 5 Time (h) Aeration 6.5 h +

20 Nitrogen (mg/L)

250

15

0 6

7

Settling NO -N

TN

NO -N

5

50

0

0

6

8

1

2

(D) 25

250 200

7

0 0

5

4 5 Time (h)

200

50

100

3

NO3--N

100

10

2

TN

250

10

150

1

NO2--N

150

15

0

Settling

NH4+-N

15

3

4 5 Time (h) Aeration 6.5 h + -N 4

20

COD

0

Aeration 6.5 h

COD

8

2 3

NH4 -N

3.2.1. Effect of salinity on the microbial community At different salinities (0.0, 0.8, 1.6 and 2.4%), the microbial community in S-sludge and biofilm was analyzed using high-throughput sequencing (Table 1). The Good's coverages of all samples exceeded 98%, suggesting that the microbial diversities were well described by the obtained sequence libraries. The richness and diversity of the microorganisms are indicated by the Chao 1, ACE, Shannon, and Simpson indices shown in Table 1. The microbial richness and diversity of the S-sludge and biofilm showed similar shift with the increase of salinity. Generally, the richness at 0.8% salinity was higher than that at 0.0% salinity by the Chao1 and ACE indices. Then the richness decreased with the increase of salinity from 0.8% to 1.6%, while increased again at 2.4% salinity. According to Shannon and Simpson indices, the increase of salinity from 0.0% to 1.6% decreased the microbial diversity, because the salt addition inhibited the reproduction of the salt-unadaptable microorganisms (Wang et al., 2017). When salinity increased to 2.4%, the microbial diversity increased. This might be explained by that salt-tolerant microorganisms could continue to survive and grow at higher salt

COD (mg/L)

TN

3.2. Microbial community

Nitrogen (mg/L)

NO2--N

COD (mg/L)

Nitrogen (mg/L)

20

Settling

NH4+-N

COD (mg/L)

Aeration 6.5 h

(A) 25

nitrite has been established for TN removal at the salinity of 1.6% and 2.4%. Nitrite accumulation and high NAR was also reported in previous studies for saline wastewater treatment due to the suppression of NOB, which resulted in partial nitrification process (Corsino et al., 2016; Wang et al., 2017a). In this study, the existence of NO− 3 -N during aeration at 1.6% salinity implied that SND via nitrate could not be fully eliminated (Fig. 2C). SND via nitrite and nitrate together contributed to the high TN removal and SND efficiency (71.2 ± 5.9%) at 1.6% salinity. When influent salinity was increased to 2.4%, almost no nitrate was detected during the aeration phase (Fig. 2D), implying that the nitrite oxidation was completely inhibited and SND via nitrite was the predominant pathway for the TN removal. In addition, lower TN removal and SND efficiency (66.2 ± 4.4%), and higher effluent NO− 2 -N level at 2.4% salinity indicated that denitrification of nitrite was inhibited somewhat under this condition.

6

7

8

Settling 2 3

NH

NO -N

TN

NO -N

250 200

COD

15

150

10

100

50

5

50

0

0

0 0

1

2

3

4 5 Time (h)

6

7

Fig. 2. Profiles of nitrogen and COD during a cycle at various salinities: (A) 0.0%, (B) 0.8%, (C) 1.6%, (D) 2.4% salinity.

8

COD (mg/L)

stage (Fig. 1). With the salt addition and the increase of salinity in Periods II (0.8% salinity), III (1.6% salinity) and IV (2.4% salinity), no significant changes in COD and NH+ 4 -N removal were observed, having average removal efficiencies of (86.6±4.0) - (91.6±1.8)% and (99.0± 1.5) - (99.8±0.4)% for COD and NH+ 4 -N, respectively. This result demonstrated that the heterotrophic bacteria involved in removal of organic matter and AOB related to ammonium removal were able to endure salt stress and work effectively in the HSBBR. Previous studies also confirmed high removal efficiency of organic matter and ammonium at salt concentration up to 30 g NaCl L−1 (Corsino et al., 2016; Wang et al., 2017a). Taheri et al. (2012) attributed high removal efficiency of organic matter at salt stress to the necessity for microorganisms to adjust their metabolisms to adapt to high osmotic conditions. A gradual acclimation strategy of autotrophic AOB to high salt level can avoid their inhibition (Corsino et al., 2016). TN removal was negatively affected by salt addition in Periods II, III and IV, achieving average removal efficiency of 77.8 ± 2.8%, 82.7 ± 2.1% and 73.9 ± 3.9% at the salinity of 0.8%, 1.6% and 2.4%, respectively, in the stable stage. Nitrate was observed to be main nitrogen component in the effluent at 0.0% and 0.8% salinity, with an average concentration of 2.4 ± 2.2 and 8.6 ± 0.8 mg L−1, respectively (Fig. 1D). As shown in Fig. 2A and B, almost no NO− 2 -N accumulation was observed during the aeration phase in Period I and II, while NO− 3 -N concentration increased progressively with the transformation of ammonium, achieving the highest level of 7.3 ± 3.0 mg L−1 and 10.4 ± 0.7 mg L−1 at the end of the transformation of ammonium. This result indicated that SND via nitrate occurred in the reactor at the salinities of 0.0% and 0.8%, achieving SND efficiency of 65.7 ± 4.4% and 62.0 ± 2.0%, respectively. When salinity was increased to 1.6%, NO− 3 -N in the outflow dropped to 1.8 ± −1 1.3 mg L−1, while effluent NO− 2 -N increased to 4.9±0.9 mg L −1 − − (Fig. 1D). More NO2 -N (10.3 ± 1.2 mg L ) and almost no NO3 -N was detected in the outflow with the further increase of salinity to 2.4% (Fig. 1D). As shown in Fig. 2C and D, obvious nitrite accumulation occurred during aeration under the salinities of 1.6% and 2.4% due to the inhibition of nitrite oxidation, achieving NAR of 80.3±6.4% and 100.0±0.0%, respectively. Combine with the decline of TN concentration during the aeration phase, it could be concluded that the SND via

Nitrogen (mg/L)

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Z. Xia et al. / Science of the Total Environment 697 (2019) 134047

5

Table 1 Diversity and richness of the microbial community. Salinity (%)

Samples

Effective sequences

OTU

Shannon index

Simpson index

Chao1

ACE index

Good's coverage

0.0 0.8 1.6 2.4 0.0 0.8 1.6 2.4

S-sludge

23,286 33,410 38,323 47,787 40,836 46,344 22,715 51,909

990 1013 877 967 1026 1170 861 1060

7.60 6.42 5.16 5.49 7.15 6.77 5.79 6.03

0.99 0.94 0.90 0.93 0.98 0.96 0.91 0.95

968.1 954.0 931.0 951.6 1036.4 1101.8 876.6 990.3

982.3 993.7 922.4 952.5 1084.8 1120.2 867.5 1016.0

0.993 0.992 0.990 0.989 0.989 0.989 0.992 0.989

Biofilm

concentration. Moreover, the community diversity in biofilm were higher than S-sludge with the addition of salt (0.8–2.4% salinity). The richer biodiversity of biofilm compared with S-sludge was also found in other hybrid bioreactors (Huang et al., 2019; Wang et al., 2018). This result could be explained by the micro multi-habitat within the biofilm, namely, a coupling aerobic-anoxic-anaerobic environment which ascribed to the DO gradient in biofilm (Tang et al., 2017, 2014). The unique structure of biofilm was advantageous for resistance to environmental pressure (salt in this study) and benefited growth of various

(A)

bacteria, thereby promoted the biodiversity and pollutants removal (Huang et al., 2019; Wang et al., 2018, 2017a). 3.2.2. Microbial composition at phylum and class levels In the present study, 24 phyla were found in eight samples. As shown by the bar graph of the phylum level (Fig. 3A), the prevailing phyla (relative abundance N1% in at least one sample) were Proteobacteria (44.2–67.0%), Bacteroidetes (17.2–39.7%), Chlorobi (1.7–11.5%), Chloroflexi (2.0–11.1%), Planctomycetes (1.4–3.1%),

100

Relative abundance(%)

80

Other Cyanobacteria Actinobacteria Acidobacteria Planctomycetes Chlorobi Chloroflexi Bacteroidetes Proteobacteria

60

40

20

0 SS 0% SS 0.8% SS 1.6% SS 2.4% BF 0% BF 0.8% BF 1.6% BF 2.4%

(B)

100 90

Relative abundance(%)

80 70 60 50 40 30 20 10 0 SS 0% SS 0.8% SS1.6% SS 2.4% BF 0% BF 0.8% BF 1.6% BF 2.4%

Other Phycisphaerae Caldilineae unidentified_Acidobacteria Holophagae Chlorobia Cytophagia Planctomycetacia Flavobacteriia Deltaproteobacteria Anaerolineae Ardenticatenia Ignavibacteria Betaproteobacteria Alphaproteobacteria Sphingobacteriia Gammaproteobacteria

Fig. 3. Microbial compositions of S-sludge and biofilm at phylum level (A) and class level (B). The phyla and classes with relative abundance b1% were summarized as others.

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Z. Xia et al. / Science of the Total Environment 697 (2019) 134047

Acidobacteria (0.4–3.0%), Cyanobacteria (0.1–2.1%) and Actinobacteria (0.3–1.1%). Among them, Proteobacteria and Bacteroidetes were the top two abundant phyla in both S-sludge and biofilm samples at all salinities. Generally, higher salinity increased the abundance of Proteobacteria, which played an important role in nitrogen removal (Wang et al., 2017). The phylum Bacteroidetes showed higher abundance at 0.8% and 1.6% salinities than at 0.0% and 2.4% salinities. Bacteroidetes are considered as credible degraders of organic matter, especially for high molecular weights substance (Miao et al., 2018; Tang et al., 2017). Li et al. (2018) found that Bacteroidetes mainly existed in hypoxic or anoxic environment and participated denitrification. The high abundance of Bacteroidetes might play an essential role for the SND process in the HSBBR. In this study, 29 classes were detected in all samples. The dominant classes are Sphingobacteriia (11.1–34.5%), Alphaproteobacteria (10.7–34.3%), Betaproteobacteria (6.9–19.6%) and Gammaproteobacteria (10.4–19.4%). Under unsalted condition (0.0% salinity), Gammaproteobacteria and Sphingobacteriia were the top two abundant class in S-sludge and biofilm, respectively. Sphingobacteriia had the highest abundance in biofilm samples at 0.8% and 1.6% salinities, and in S-sludge sample at 0.8% salinity. Previous studies have proved that Gammaproteobacteria played crucial roles in denitrification in saline wastewater treatment (Miao et al., 2015), and Sphingobacteriia is a major kind of microbes involved in sulfur metabolism, COD and nitrogen removal (Tang et al., 2017). As salinity increased from 0.0% to 2.4%, Alphaproteobacteria increased from 14.9% to 34.3% in S-sludge and from 10.7% to 27.7% in biofilm, turned to be the most dominant class at 2.4% salinity. Betaproteobacteria also exhibited an enrichment in both S-sludge and biofilm with the increase of salinity from 0.8% to 2.4% and became the second abundant class at the salinity of 2.4%. This result suggested that Alphaproteobacteria and Betaproteobacteria can tolerate higher salinity, as confirmed in some previous studies (Cortés-Lorenzo et al., 2014; Qian et al., 2010; Zhang et al., 2011).

Notably, Betaproteobacteria is more abundant in biofilm than S-sludge at all salinities, demonstrating that biofilm was more favorable for this class to acclimate under environmental pressure due to the stable environment provided by biofilm (Wang et al., 2017a). This result is consist with the study of Cortés-Lorenzo et al. (2014), who found the same variation trend of Betaproteobacteria in biofilm reactor. 3.2.3. Microbial composition at genus level The microbial community profiles at genus level were demonstrated by a heat-map (Fig. 4), which showed the genera with relative abundance of N0.5% in at least one sample. At 0.0% salinity, the major microbial population (relative abundance above 1.0% in S-sludge or biofilm) were Candidatus_Competibacter, Defluviicoccus, Terrimonas, Denitratisoma, Ottowia, Ohtaekwangia, Thauera, Ferruginibacter, Hyphomicrobium, Roseovarius and Dokdonella. With the increase of salinity, microbial community in both S-sludge and biofilm experienced great succession. The abundance of the genera Candidatus_Competibacter, Defluviicoccus and Terrimonas appeared continuously decrease trend with the elevated salinity. Candidatus_Competibacter was the predominant genus at 0.0% salinity, accounted for 15.6% and 12.2% of total detected bacteria in S-sludge and biofilm, respectively. However, this genus declined to 2.5% and 3.6% in S-sludge and biofilm at 2.4% salinity, respectively. Genera Ottowia and Ferruginibacter, two kinds of denitrifiers, showed lower abundance when they were exposed to salt stress. Ferruginibacter has been proved to be associated with syntrophic denitrification metabolisms, and be related to biofilm formation with Ottowia in MBBR systems (Rodriguez-Sanchez et al., 2018). Genera Denitratisoma, Ohtaekwangia and Roseovarius showed fluctuation change with the increase of salinity from 0.0% to 2.4%, achieved higher abundance at 2.4% salinity (2.1%, 0.8% and 0.8% in S-sludge, 4.0%, 1.0% and 0.7% in biofilm). The fluctuation might be due to the acclimation to the changing environment (salt addition), and the higher abundance at 2.4% salinity

Phylum Proteobacteria Bacteroidetes Nitrospirae Planctomycetes Spirochaetes Chlorobi Chloroflexi

lg (Relative abundance)

-1.0

Phylum SS0%

SS0.8%

SS1.6%

SS2.4%

BF0%

BF0.8% BF1.6%

BF2.4% Genus

Fig. 4. Distribution of microbial community at genus level in S-sludge and biofilm.

-2.0

-3.0

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could be attributed to the high tolerance and halophilic characters of these genera (Li et al., 2016). With salinity increasing from 0.0% to 0.8%, the percentage of Paracoccus, Dechloromonas, Hyphomicrobium, Dokdonella, Nitratireductor, Phycisphaera, Thiothrix and Nitrosomonas in both Ssludge and biofilm increased. As the salinity was further increased to 1.6%, Hyphomicrobium, Dokdonella and Nitratireductor showed a reduction in abundance, demonstrating these genera preferred lower salinity or unsalted environment. Whereas the abundance of Paracoccus, Dechloromonas, Phycisphaera, Thiothrix and Nitrosomonas further increased at 1.6% salinity. Paracoccus and Dechloromonas became the top two abundant microbes at 1.6% salinity. In addition, Paracoccus, Dechloromonas and Thiothrix were the top three dominant genera at 2.4% salinity, their percentage were 13.7%, 10.6% and 10.1% in S-sludge and 13.5%, 10.0% and 9.1% in biofilm respectively. Taken together, the high-throughput sequencing analysis indicated the presence of a diverse metabolism microbial community in the HSBBR system. The interaction of these dominant microbes contributed to the efficient performance and maintained the stability of the HSBBR. Notably, the microbial composition in S-sludge was analogous to biofilm, while the difference of abundance between S-sludge and biofilm was varied with bacteria. Additionally, several kinds of bacteria with good adaptability to high salt stress, such as Phycisphaera and Nitratireductor, became dominant in the reactor as the increase of salinity. The proliferation of halophilic/halotolerant bacteria was beneficial to the stable operation of HSBBR in response to the impact of salt stress. Details of the variation of functional microbes involved in nitrogen conversion will be discussed in the following section. 3.3. Variation of AOB, NOB and DNB 3.3.1. Nitrifiers community The crucial functional groups in charge of nitrogen removal in traditional WWTPs are AOB, NOB and denitrifying bacteria (DNB). AOB and NOB are two core genera which play indispensable roles in nitrification process. Nitrosomonas was the only autotrophic AOB identified in this experiment. As Table 2 shown, the relative abundance of AOB displayed a continuously rise with the elevated salinity. The salinity of 0.8% led to an obvious enrichment of AOB, and the abundance of AOB was relatively constant in the salinity range of 0.8%–1.6%. After the salinity reached 2.4%, the relative abundance of AOB increase to 1.0% and 1.8% in Ssludge and biofilm, respectively. This result demonstrated that AOB were adaptable to the saline environment, and could explain the excellent NH+ 4 -N removal efficiency under high salt stress (Fig. 1B). Notably, the relative abundance of AOB in biofilm were higher than that in Ssludge. This phenomenon indicated that the biofilm could give AOB more optimal condition to be maintained in saline environment. Bassin et al. (2012) also observed a similar AOB variation in an SBR. They reported that the relative abundance of AOB gradually increased when salinity changed from 0 to 10 g NaCl L−1 and maintained stable when salt concentration continuously reached 20 g NaCl L−1. However, Wang et al. (2017) found that salinity of 20 g NaCl L−1 caused a sharp Table 2 Variation of relative abundance for AOB, NOB and DNB. Salinity (%)

Samples

0.0 0.8 1.6 2.4 0.0 0.8 1.6 2.4

S-sludge

Biofilm

Relative abundance (%) AOB

NOB

Autotrophic DNB

Heterotrophic DNB

Total DNB

0.2 0.7 0.7 1.0 0.2 0.9 1.0 1.8

0.8 0.4 0.3 0.1 0.5 0.4 0.1 0.2

0.4 1.2 4.7 10.4 0.3 1.1 2.5 9.2

36.9 37.5 49.6 37.1 32.3 40.7 41.3 40.6

37.3 38.7 54.3 47.5 32.6 41.8 43.8 49.8

7

decline in the ammonia oxidation rate due to the inhibition of AOB in CANON system. The difference of environment and dominant AOB species might lead to this inconsistency. In particular, Nitrosomonas marina (N. marina), species within genus Nitrosomonas, was detected as the predominant AOB species in this experiment. N. marina originates from marine habitats and is obligatory halophilic AOB (Pan et al., 2018; Wu et al., 2013). Several researches have proved the enrichment of N. marina under high salt stress in wastewater treatment process, agreed with the growth trend of relative abundance of AOB in this HSBBR system. Bollmann and Laanbroek (2002) reported that N. marina/aestuarii-like AOB were enriched in the presence of salt, while AOB members of N. ureae/oligotropha lineage developed without salt addition. Cui et al. (2016) reported top two dominant AOB lineages in the halophilic sludge, Nitrosomonas marina and Nitrosomonas europaea, which can survive in saline environment up to 30–85 g NaCl L−1. Halotolerant character of N. marina insured its resistance under salt stress, making great contribution to the stability of this HSBBR. Nitrospira was the only autotrophic NOB genus detected in the HSBBR, which is widely distributed in traditional biological nitrogen removal process. As shown in Table 2, with the salinity increasing from 0.0% to 2.4%, the abundance of Nitrospira decreased from 0.8% to 0.1% in S-sludge and from 0.5% to 0.2% in biofilm, implying obvious inhibition of salt addition on NOB. Higher NOB abundance under 0.0% and 0.8% salinity induced low nitrite accumulation during aeration phase and the complete nitrite oxidation at the end of aeration (Fig. 2A and B). The lower NOB abundance at 1.6% and 2.4% salinities could be major factor resulted in the nitrite accumulation in the aeration phase (Fig. 2C and D), achieving high NAR of 80.3±6.4% and 100.0±0.0%, respectively. This result indicated that partial nitrification was established because of NOB inhibition by salt, and the dominant nitrogen removal pathway had changed from SND via nitrate to SND via nitrite in the HSBBR at 1.6% and 2.4% salinity. Previous studies also found similar results about the adverse effect of salt on NOB populations (She et al., 2018, 2016; Wang et al., 2017). In addition, some lineages of genus Nitrospira are reported to be able to oxidize ammonia directly to nitrate as comammox (complete ammonia oxidation) bacteria (Zhao et al., 2019; M. Zheng et al., 2019). Bacteria possessed capacity of comammox was mainly affiliated within cluster of Nitrospira nitrifican, Nitrospira nitrosa and Nitrospira inopinata, which was confirmed to have encoding genes for ammonia monooxygenase (AMO), hydroxylamine dehydrogenase (HAO) and nitrite oxidoreductase (NXR) (Zhao et al., 2019; M. Zheng et al., 2019). Therefore, comammox bacteria should be taken into account as one significant ammonia oxidizing microorganisms in future studies to evaluate their contribution together with other nitrifying microorganism in biological nitrogen removal system. Besides the traditional autotrophic AOB and NOB, the presence of Paracoccus, Pseudomonas, Pirellula, Planctomyces and Gemmata should also be noticed. Previous studies have confirmed that some species from genera Paracoccus and Pseudomonas are heterotrophic nitrification-aerobic denitrification (HNAD) bacteria (Chen et al., 2016; Wang et al., 2017b; Zheng et al., 2014). Chen et al. (2016) successfully developed SND process using HNAD in a biofilm reactor for the treatment of salted wastewater, and observed Paracoccus denitrificans as one of the pivotal HNAD bacteria. Wang et al. (2017b) reported that autotrophic nitrification/heterotrophic denitrification and heterotrophic nitrification/aerobic denitrification together contributed to nitrogen removal in a SND system with salinity of 30 g L−1, and Pseudomonas_stutzeri was one of the predominant HNAD bacteria. In present study, the species of genera Paracoccus and Pseudomonas were not identified, thereby it could not be ascertained whether these two genera had the capacity of HNAD. Genus Gemmata was reported to possess the capacity of heterotrophic nitrification (Ji et al., 2019; Zhao et al., 2017). In this study, Gemmata was observed in S-sludge at 0.0% salinity and in biofilm at 0.0–2.4% salinity, implying that heterotrophic nitrification might occur. Serval studies also identified Gemmata as anaerobic ammonium oxidizing (ANAMMOX) bacteria (Tian et al., 2015; X.

8

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Zheng et al., 2019). Other two kinds of ANAMMOX bacteria, genera Pirellula and Planctomyces (Guo et al., 2019; Tian et al., 2015; Xu et al., 2018; X. Zheng et al., 2019), were also detected in both S-sludge and + biofilm samples. These genera can utilize NO− 2 -N to oxidize NH4 -N and generate N2 under hypoxic or anaerobic environment. The ANAMMOX bacteria achieved the highest total abundance under 0.0% salinity in both S-sludge (1.2%) and biofilm (0.6%), and generally decreased with the increased salinity. 3.3.2. Denitrifiers community A total of 37 genera with denitrifying capability were detected in the HSBBR system. The relative abundance of dominant DNB (relative abundance ≥ 0.5% in at least one sample) is shown in Fig. 5. Candidatus_Competibacter and Defluviicoccus were the top two abundant DNB at 0.0% salinity, indicating that these two genera took major part in denitrifying. In addition, genera Candidatus_Competibacter and

Defluviicoccus are known as typical glycogen accumulating organisms (GAOs) (Tian et al., 2017). Some GAOs were reported to be capable of reducing nitrate or nitrite using polyhydroxyalkanoates (PHAs) as an internal carbon source (Zhang et al., 2017). In this experiment, the increase of salinity caused the abundance reduction of Candidatus_Competibacter and Defluviicoccus. Genera Paracoccus and Dechloromonas became the two most abundant DNB in both S-sludge and biofilm at 1.6–2.4% salinity. Paracoccus are common denitrifiers (Lu et al., 2014), having genes related to nitrite reduction (Yang et al., 2017). The highest abundance of Paracoccus was found in S-sludge at 1.6% salinity and in biofilm at 2.4% salinity, which was about as 23 and 27 times as in S-sludge and biofilm at 0.0% salinity respectively. High population of Paracoccus under saline environment demonstrated its salt tolerance. Dechloromonas are denitrifying phosphate accumulating organisms (DPAOs), which was reported mainly distributed in anoxic zone of biofilm (Tian et al., 2017). Aerobic denitrifiers were also

(A) 25 SS 0.0% SS 0.8% SS 1.6% SS 2.4%

Relative abundance (%)

20 15 10

4 3 2 1

Ca nd id at

us

_C om pe tib ac D te ef r lu vi ic oc D cu en s itr at iso m a O tto wi a Th a Fe ue rr ra ug in H ib yp ac ho te r m ic ro bi um Pa ra co cc us H al ia n D gi ec um hl or om on as D ok Ph do ae ne od lla ac ty lib ac M es te r or hi zo bi um Ph yc isp ha N er itr a at ire du ct or Th O th i o er th rix ty pe so fD N B

0

(B) 25 BF 0.0% BF 0.8% BF 1.6% BF 2.4%

Relative abundance (%)

20 15 10 5

2 1

Ca nd id at us _C om pe

tib ac te

D ef r lu vi ic oc D cu en s itr at iso m a O tto wi a Th au Fe e rr ra ug in H ib yp ac ho te r m ic ro bi um Pa ra co cc us H al ia n D gi ec um hl or om on as D ok Ph do ae ne od lla ac ty lib ac M es te r or hi zo bi um Ph yc isp ha N er itr a at ire du ct or Th O th i o er th rix ty pe so fD N B

0

Fig. 5. Variation of DNB in S-sludge (A) and biofilm (B) with increasing salinity. The DNB with relative abundance b0.5% were summarized as others.

Z. Xia et al. / Science of the Total Environment 697 (2019) 134047

identified in this HSBBR system, such as Denitratisoma and Thauera, which have been confirmed with the ability of denitrifying in both aerobic and anoxic environment under saline condition (Fu et al., 2018; Wan et al., 2018). Some autotrophic DNB which use inorganic rather than organic electron donors for denitrification were identified in this study. Genus Hydrogenophaga, a facultative hydrogen-based autotrophic denitrifier which could use hydrogen as electric donors (Xing et al., 2018), was found in both S-sludge and biofilm with relative abundance of 0.1–0.2%. Several sulfide-based autotrophic denitrifiers, Thiothrix, Sedimenticola, Sulfuritalea and Arcobacter (Furukawa et al., 2016; Gonzalez-Martinez et al., 2017; Herrmann et al., 2017; Zhao et al., 2017), were also identified in S-sludge and biofilm samples. With the presence of these autotrophs, nitrate and nitrite could be reduced as electron accepters and sulfide was oxidized as electron donors. The seawater crystal added in the synthetic wastewater contains sulfate (about 6.2%), which could be reduced to sulfide by sulfate reducing bacteria (such as Desulfomicrobium, Desulfotignum and Desulfosarcina observed in the HSBBR) and provided the substrate for the sulfide-driven autotrophic denitrification, promoting the proliferation of sulfide-based autotrophic denitrifiers. Notably, Thiothrix exhibited enrichment with the increase of salinity and became the third abundant genus at 2.4% salinity in both S-sludge and biofilm, its abundance increased from 0.0% at 0.0% salinity to 10.1% in S-sludge and 9.1% in biofilm at 2.4% salinity. This genus has strong adaptation to saline environment and is usually present in biomass when there are sources of sulfide (Gonzalez-Martinez et al., 2017; Zhao et al., 2017). Additionally, Thiothrix have been widely reported as filamentous bacteria in wastewater treatment, could tolerate micro-aerobic conditions and influence formation of biofilm with its filamentous structure (Tang et al., 2017; Zhao et al., 2017). Therefore, the denitrification in present study was attributable to both heterotrophic and autotrophic bacteria. The richness and diversity of denitrifier enabled the high nitrogen performance in the reactor although the influent salinity varied. For S-sludge, the total DNB abundance increased when salinity was elevated from 0.0% to 1.6%, while decreased with the further increase of salinity to 2.4% (Table 2). For biofilm, the total DNB abundance increased continuously with the increase of salinity from 0.0% to 2.4%. As Table 2 shown, the DNB abundance accounted for N32% in total detected bacteria during the whole experiment. These results indicated a better tolerance to salt stress of denitrifiers. The highest total abundance for heterotrophic DNB was achieved at 1.6% salinity in both S-sludge (49.6%) and biofilm (41.3%). Whereas the total abundance of autotrophic DNB increased with the increase of salinity, achieving the highest under 2.4% salinity in both Ssludge (10.4%) and biofilm (9.2%). Based on the aforementioned results, the coupling S-sludge and biofilm in this HSBBR system could create favorable multi-habitats for the co-existence of nitrifying and denitrifying bacteria, demonstrating the SND activities in this reactor on microbiology (Li et al., 2018; Zheng et al., 2010). These functional groups insuring the efficient TN removal under both unsalted and saline conditions. Combined with operating performance, the inhibition of autotrophic NOB and high abundance of autotrophic AOB and autotrophic/heterotrophic DNB under 1.6–2.4% salinity interpreted that SND process was mainly via nitrite not nitrate at elevated salinity. The diversified functional groups under saline condition indicated a variety of potential mechanism for SND in the HSBBR. The major metabolism included traditional autotrophic nitrification/heterotrophic denitrification, aerobic denitrification and autotrophic denitrification. The aerobic denitrification might mainly rely on the genus Thauera, while the autotrophic denitrification on Thiothrix and other sulfide-based or hydrogen-based autotrophic denitrifiers. The presence of heterotrophic nitrifying bacteria Gemmata demonstrated that heterotrophic nitrification could also occur in the SND system. ANAMMOX process was another pathway for nitrogen removal under conditions with and without addition of salt.

9

4. Conclusion An HSBBR system was evaluated for simultaneous removal of organic matter and nitrogen via SND process at elevated salinity of 0.0–2.4%. High COD removal efficiency (86.6±4.0 to 91.6±1.8%) was achieved during the whole experiment. Good removal performance on nitrogen through SND via nitrite was successfully achieved at salinities of 1.6% and 2.4%, with TN removal efficiencies of 82.7±2.1% and 73.9 ±3.9%, respectively. 16S rDNA high-throughput sequencing results showed that the increase of salinity had great impact on the composition and population of microbes, and the predominant functional bacteria involved in nitrogen removal in both S-sludge and biofilm, further resulted in diverse nitrogen removal pathways. The autotrophic AOB (Nitrosomonas) and heterotrophic nitrifying bacteria (Gemmata) together contributed to the partial nitrification under the salinity of 1.6–2.4%. The heterotrophic denitrification was the main mechanism for nitrite and nitrate transformation due to the high relative abundance of heterotrophic denitrifiers in the HSBBR. Aerobic and autotrophic denitrification also occurred in the reactor, owing to the presence of aerobic (Denitratisoma and Thauera) and autotrophic denitrifiers (Thiothrix, Sulfuritalea, Sedimenticola, Arcobacter and Hydrogenophaga). The discovery of ANAMMOX bacteria Pirellula and Planctomyces demonstrated that ANAMMOX process was another pathway for nitrogen removal in this study under conditions with and without addition of salt. The proliferation of salt-adaptable/halophilic bacteria for ammonia oxidizing and denitrification stabilized the operation of SND process at the elevated salinity.

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