Removal performance of nitrogen and endocrine-disrupting pesticides simultaneously in the enhanced biofilm system for polluted source water pretreatment

Bioresource Technology 170 (2014) 549–555

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Removal performance of nitrogen and endocrine-disrupting pesticides simultaneously in the enhanced biofilm system for polluted source water pretreatment Li-Juan Feng a,b, Guang-Feng Yang b, Liang Zhu b,⇑, Xiang-Yang Xu b,c a b c

Department of Environmental Engineering, Zhejiang Ocean University, No. 1 Haida South Road, Zhoushan 316022, PR China Department of Environmental Engineering, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, PR China ZJU-UWA Joint Centre in Integrated Water Management and Protection, No. 866 Yuhangtang Road, Hangzhou 310058, PR China

h i g h l i g h t s +



 No significant change of EDPs removal with increase of NH4 –N and NO3 –N transfer.  Simultaneous denitrification and EDPs removal in anoxic niches via reed addition.  Many species related to the nitrogen and EDPs removal enhanced with reed addition.

a r t i c l e

i n f o

Article history: Received 17 May 2014 Received in revised form 31 July 2014 Accepted 2 August 2014 Available online 9 August 2014 Keywords: Polluted source water Endocrine-disrupting pesticide Nitrogen Reed addition Microbial community

a b s t r a c t The removal performances of nitrogen and trace levels of endocrine-disrupting pesticides (cypermethrin and chlorpyrifos) were studied in the enhanced biofilm pretreatment system at various substrates concentrations and dissolve oxygen (DO) niches. No significant change of EDPs removal occurred with the increased feed of ammonia nitrogen in aerobic batch tests or nitrate in anaerobic batch reactors, but significantly enhanced via reed addition both in aerobic and anaerobic conditions. Simultaneously enhanced denitrification and EDPs removal were achieved in the anoxic niche with reed addition. The results of denaturing gradient gel electrophoresis (DGGE) indicated that new bands appeared, and some bands became more intense with the reed addition. Sequences analysis showed that the dominant species belonged to Methylophilaceae, Hyphomicrobium, Bacillus and Thauera, which were related to the nitrogen or EDPs removals. In addition, the growth of functional heterotrophic microbes may be promoted via reed addition. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The presence of endocrine disrupting compounds (EDCs) in natural waters has the ability to mimic or inhibit the natural action of the endocrine systems in invertebrates and possibly human beings at trace levels (lg l1 or ng l1) (Frye et al., 2012; Hirai et al., 2006; Pawlowski et al., 2004; Silva et al., 2012). EDCs have become a key priority in water quality control over last decade, which has been responded by wide investigations of EDCs removal in wastewater and water treatment works all over the world (Kim et al., 2007; Stasinakis et al., 2008). It is now known that removal efficiencies of EDCs can greatly vary in various processes. Currently biological processes are still popular approaches due to its low maintenance cost and effective contaminants removal. ⇑ Corresponding author. http://dx.doi.org/10.1016/j.biortech.2014.08.004 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

In biological systems, generally aerobic condition favors EDCs removal as compared with that in anaerobic condition (Xue et al., 2010). There may be two reasons for this: one was that most of heterotrophic bacteria that can degrade EDCs adapted to aerobic environment; the other was metabolic nitrifiers due to the enzymatic action of ammonia monooxygenase (AMO) in aerobic conditions (Forrez et al., 2009). Yi and Harper (2007) proved that there was a positively linear relationship between17a-ethynylestradiol reduce rate and the ammonia biotransformation rate. However, EDCs and initial ammonia concentrations in most previous studies were much higher than those in effluent of wastewater treatment plant or natural waters. Gaulke et al. (2008) reported that 17aethynylestradiol removal at low concentrations in activated sludge systems was not attributed to cometabolic degradation, but most possibly due to the effect of heterotrophic bacteria (Gaulke et al., 2008). Still, the information of relationship between EDCs removal

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at trace level and nitrogen removal at low concentration is still limited. No matter the effects of heterotrophic bacteria or the cometabolic degradation by AMO that mainly cause the EDCs removal, it is known that microbial population densities and community structure are the key factors for determining the performances of biological systems. Effects of microbial population densities (MLVSS) on EDC of 17b-estradiol removal was reported by Li et al. (2005), which suggested that higher MLVSS was advantage to the increasing in 17b-estradiol degradation rate (Silva et al., 2012). Thus, it is essential to develop ways to improve bacteria growth. In addition, environmental conditions (e.g. pH, temperature and dissolve oxygen), nutrients level (e.g. carbon, nitrogen and phosphorus) could also significantly affect toxic organics removal, especially for water treatment in oligotrophic environment. Phosphorus addition in drinking water showed that the relative abundance of perchlorate-reducing bacteria (PRB) Dechloromonas and Azospira in the bench-scale reactor increased from 15.2% and 0.6% to 54.2% and 11.7%, respectively (Yoon et al., 2006). Studies on nutrients limiting biofilm development in water treatment process and distribution systems demonstrated a positive relationship between biodegradable carbon source in water and bacterial growth in biofilm (Chandy and Angles, 2001; Vanderkooij, 1992). Pure cultures studies also showed that degradation ability of bacteria was positively influenced by the presence of supplementary carbon sources and other nutrients (Cycon et al., 2009; Watanabe et al., 2012). For example, the degradation of beta-cypermethrin (endocrine-disrupting pesticide) significantly enhanced at the presence of glucose, beef extract and yeast extract, the degradation efficiencies reached to 85.4%, 90.4% and 87.0%, respectively (Chen et al., 2011); it was also reported that the initial degradation rate of bisphenol-A via Pseudomonas monteilii strain N-502 was obviously accelerated with the addition of Ca2+, Mg2+ and folic acid (Masuda et al., 2007). Since the molar ratio of carbon, nitrogen to phosphorus required for bacterial growth is approximately 100:10:1, carbon is the most needed for bacterial growth but always limited in oligotrophic waters. Furthermore, carbon source is also a good electron donor for denitrification. For stimulating the growth of microorganism and promoting the performance, extra carbon source were often used in many studies (Ovez et al., 2006; Park and Yoo, 2009; Warneke et al., 2011). The extra carbon sources often used were soluble carbon source (such as methanol, ethanol, acetic acid, acetate, etc.) and solid carbon source (such as wheat straw, rice husk, reed, Typha latifolia, Elodea canadensis, etc.) (Ovez et al., 2006; Park and Yoo, 2009; Warneke et al., 2011). Previous studies showed that solid carbon source was much cheaper than soluble carbon source (Boley et al., 2000; Soares, 2000). The reed as a representative solid carbon source used in this study, depending on its characteristics of cost-saving, widely distributed and abundant carbon content (Ovez et al., 2006). Up to now, previous studies have reported that nitrate and endocrine-disrupting pesticides (EDPs) could be simultaneously removed via external carbon source addition, e.g. Ethanol (Choi et al., 2006), biodegradable polymer poly (Ginige et al., 2004), wheat straw (Xu et al., 2012) and so on. However, the nitrogen and EDPs in these reported studies were much higher than those occurrences in oligotrophic niche, where it is hard to realize the simultaneous removal of nitrogen and EDPs. What is more, little studies have been focused on the characteristics of bacteria community in simultaneous denitrification and EDPs removal, especially in source water pretreatment systems. Due to the information on the removal of trace EDCs in oligotrophic waters remains limited, the effects of initial ammonia, nitrate and carbon source feeding on the removal performances

of selected endocrine-disrupting pesticides (cypermethrin and chlorpyrifos) were studied in biofilm source water pretreatment system. The aim of this study was to (1) ascertain whether EDPs cometabolic degradation occurred at trace levels by ammonia oxidization effects in oligotrophic environment; (2) examine the fate of cypermethrin and chlorpyrifos at presence of various nitrogen and carbon source. 2. Methods 2.1. Reagents Two EDPs (chlorpyrifos and cypermethrin) were purchased from national standard research center in China and used without further purification; the detail description is shown in Table 1. The inorganic salts used in the synthetic water were NH4Cl, KNO3, KH2PO4, and methanol was used as carbon source. All reagents for solid phase extraction (SPE) and gas chromatography mass analyses were of HPLC grade. Reed leachate was self-made in laboratory. Firstly the wilt reed used was collected from a wetland nearby Zhejiang University in China, which was treated by a cryogenic impact grinder. The fine powder of reed was emerged in purified water and heated at 120 °C for 1 h. The supernatant was collected as reed nutrition, and stored at 4 °C for use. The nutrient components of reed nutrition, such as total organic carbon (TOC), ammonia, nitrite, nitrate, phosphate, total nitrogen and total phosphorus were analyzed (Table 2). 2.2. Experiments design To study the relationship between EDPs and nitrogen removal in oligotrophic environment, the initial ammonia in aerobic reactors (OB1–OB3) and nitrate in anaerobic reactors (AB1–AB3) were both conducted in different levels; the effect of reed addition on EDPs and nitrogen removal were also studied. The detail operation parameters are presented in Table 3. All the experiments were conducted in plexiglass batch reactors with working volume of 4.2 L. There were double for each operation condition. TA-II elastic filler, purchased from Tianyu Environmental Protection Engineering Co., Ltd. (Hangzhou, China), was used as biofilm carrier, and all the batch reactors were filled with the same volumetric carriers. This kind of carrier had a diameter and surface area of 200 mm and 200–300 m2 m3, respectively. The DO in aerobic and anaerobic reactors was at the range of 7.5–8.6 mg l1 and 0.01–0.45 mg l1, respectively. The other operational parameters were shown as follows: water temperature of (25 ± 2) °C, hydraulic retention time (HRT) of 24 h, pH 7.2–7.8. After the steady operation performance was achieved, regular index were analyzed every other day and EDPs were analyzed every week. 2.3. Analytical methods 2.3.1. EDPs analysis Chlorpyrifos and cypermethrin analysis were carried out by gas chromatography tandem mass spectrometer (GC–MS) after solid phase extraction (SPE). Prior to SPE, water sample was filtered through Whatman GF/F glass microfiber membrane filters with pore diameter of 0.45 lm. Conditioning of the ENVI-18 cartridges (Supelclean, 6 cc, 500 mg) with 6 ml dichloromethane followed by 6 ml methanol and then washed with 12 ml pure water. A total of 500 ml water sample was passed through cartridge at the flow rate of 5 ml min1. Then the cartridges were washed with 5 ml pure water and dried under vacuum for 0.5 h. The EDPs extracts were eluted with 20 ml of dichloromethane and tenderly dried under nitrogen gas (Organomation nitrogen evaporators, USA).

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L.-J. Feng et al. / Bioresource Technology 170 (2014) 549–555 Table 1 The characteristics of chlorpyrifos and cypermethrin. Name

Molecular weight

Structural formula

Chlorpyrifos

C10H14O5NSP (291.27)

Cypermethrin

C22H19Cl2NO3 (416.32)

Partition coefficient log Kow 4.7

6.3

O CH O CO CN

CH CCl 2

CH3 CH3

Table 2 The main nutrients of reed solution (mg l1). Nutrients

TOC

DOC

TN

NH+4–N

NO 2 –N

NO 3 –N

TP

DP

Reed nutrition

612.60 ± 2.50

541.26 ± 1.90

20.39 ± 0.20

3.03 ± 0.10

0.23 ± 0.02

16.65 ± 0.10

0.21 ± 0.03

0.17 ± 0.01

Table 3 1 The initial concentrations of NH+4–N, NO ). 3 –N and TOC in the aerobic (OB1–OB3) and anaerobic batch reactors (AB1–AB3) (mg l Batch reactors

OB1 OB2 OB3 AB1 AB2 AB3

Influent NH+4–N

0.08 ± 0.01 4.06 ± 0.16 4.10 ± 0.14 0.07 ± 0.02 0.06 ± 0.03 0.06 ± 0.02

Influent NO 3 –N

0.82 ± 0.02 0.79 ± 0.04 0.80 ± 0.03 0.84 ± 0.03 4.45 ± 0.14 4.51 ± 0.15

Influent TOC

4.06 ± 0.45 4.09 ± 0.47 24.89 ± 0.61 4.21 ± 0.39 4.08 ± 0.47 25.62 ± 0.48

Percentage of different carbon source to the influent TOC Methanol (%)

Reed nutrition (%)

100 100 16 100 100 16

0 0 84 0 0 84

Note: The abbreviations of OB1, OB2 and OB3 referred to the oxic batch reactors; the abbreviations of AB1, AB2 and AB3 referred to the anaerobic batch reactors.

The extract was then diluted with 500 ll acetone for GC–MS analysis. Each sample collected from the reactors for EDPs analysis was analyzed by duplicate. Separation of chlorpyrifos and cypermethrin via a Trace DSQ GC–MS (Thermo Finnigan, USA) was performed in a DB-5 fused silica capillary column (30 m  0.25 mm id, 0.25 lm film thickness). The flow rate of carrier gas (helium) was 1.0 ml min1. The temperature of injector was 250 °C and that of the interface was 280 °C. The initial GC oven temperature was kept at 50 °C for 2 min, and programmed to 150 °C at a rate of 10 °C min1, held for 1 min; then raised to 250 °C at a rate of 5 °C min1, held for 1 min and finally increased to 300 °C at a rate of 10 °C min1 for 10 min.

was used to analysis DGGE profile and microorganisms similarity and bacteria diversity. 2.3.3. Other analyses pH was determined with a digital pH meter (METTLER TOLEDO 320, Switzerland), and DO was measured with a DO meter (YSI Model52, USA). Nitrate, nitrite and ammonia were analyzed according to standard analytical methods (Chinese Standard Methods for the Examination of Water, 2002). Total organic carbon (TOC) was determined by means of a catalyzed combustion TOC analyzer (TOC-V CPH, Shimadzu). 3. Results and discussion

2.3.2. Biofilm analysis The mechanism of simultaneously denitrification and EDPs removal with the addition of reed was explored by analyzing biomass and bacteria community according to the method provided by Xu et al. (2012). Briefly, the fixed biomass was shed off from the carrier surface and stayed in the alkaline solution after ultrasonic treatment. The solution was filtered through 0.45 lm membrane and the membrane with biomass was dried at 105 °C. The total biomass (TB) was obtained by weighting method and the volatile biomass (VB) was further measured by weighting method after incineration in Muffle furnace at 550 °C. A soil DNA kit (OMEGA) was used to extract genomic DNA of biofilm samples, following PCR with universal bacterial primers of GC-P357f (5w0 -CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCAC GGGGGGCCTACGGGAGGCAGCAG-30 ) and P518r (50 -ATTACCGCG GCTGCTGG-30 ) for denaturing gradient gel electrophoresis (DGGE) analysis (Ferris et al., 1996). Quantity One software (Version 4.62)

3.1. Nitrogen and organics removals in aerobic and anoxic niches The ammonia and nitrate removal performances of aerobic batch reactors (OB1, OB2 and OB3) are shown in Fig. 1A and B, respectively. In the control reactor OB1 – without feeding ammonia (<0.1 mg l1), there was no nitrification occurred. In OB2 and OB3, the effluent ammonia and nitrate were 0.09 ± 0.02 mg l1, 4.05 ± 0.04 mg l1 and 0.12 ± 0.03 mg l1, 4.12 ± 0.06 mg l1, respectively. It was indicated that completed nitrification was obtained and none denitrification occurred in the OB2 and OB3 with high DO level. The results also showed that the addition of reed nutrition had no effect on nitrification in OB3. The NH+4–N removal rates in the three aerobic reactors were 0.06 g m3 d1 (OB1), 3.97 g m3 d1 (OB2) and 3.96 g m3 d1 (OB3). It was further proved that ammonia could be efficiently removed in aerobic biofilm system for source water treatment, and it was mainly contributed to the effects of ammonia oxidized bacteria enriched on

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B

Influent NH4+-N

5

Effluent NH4+-N

NO2--N concentration (mg l-1)

NH4+-N concentration (mg l-1)

A

4 3 2 1 0

OB1

OB2

OB3

AB1

TOC concentration (mg l-1)

C 20

AB2

Fig. 1.

(A),

Influent NO2--N Effluent NO2--N

5 4 3 2 1 0

AB3

OB1

OB2

OB3

AB1

AB2

AB3

Influent TOC Effluent TOC

18 16 14 12 10 8 6 4 2 0

OB1 NH+4–N

6

NO 3 –N

OB2

OB3

AB1

AB2

AB3

(B) and TOC (C) removal performances in the six batch biofilm reactors.

the elastic filler that has been proved in the previous study (Xu et al., 2012). As external carbon source addition was applied in the study, organics removal performances in the reactors OB1–OB3 were observed (Fig. 1C). The effluent TOC in OB1 and OB2 were both approximately 1 mg l1. The effluent TOC in OB3 was obviously increased up to 7.17 ± 0.14 mg l1 due to the reed addition, but it was lower than that in the AB3 (8.06 ± 0.72) mg l1. Compared with anaerobic reactors, higher organics removal efficiency was obtained in the aerobic systems. For the anaerobic batch reactors (AB1, AB2 and AB3), the nitrate in the tap water was well removed at the influent TOC of 4.21 ± 0.39 mg l1 (Fig. 1). However, the nitrate removal efficiency (28.5%) was obviously decreased when the influent nitrate increased up to 4.45 ± 0.14 mg l1, which was mainly contributed to the deficiency of carbon source as elector donors. The complete denitrification was obtained when the influent TOC was supplemented up to 24.62 ± 0.48 mg l1 by reed nutrition addition. However, the effluent TOC was also increased from 1.21 ± 0.16 mg l1 (AB1) to 8.06 ± 0.72 mg l1 (AB3). The NO 3 –N removal rates in the three anaerobic reactors were 0.81 g m3 d1 (AB1), 1.5 g m3 d1 (AB2) and 4.45 g m3 d1 (AB3). It was proved that biological denitrification highly depended on the availability of carbon source. Thus, external carbon source addition has become more and more popular during nitrogen removal in wastewater or water with low C/N ratio (Calderer et al., 2010; Warneke et al., 2011). 3.2. The removal performances of trace levels of EDPs in various NH+4–N and NO 3 –N conditions Previous studies suggested that the nitrification was an importance driving force for EDCs removal in wastewater treatment (Clara et al., 2005; Svenson et al., 2003). In order to investigate whether cometabolic degradation occurred at trace levels of EDPs removal by AMO in oligotrophic environment, aerobic reactor OB1 was operated with no NH+4–N feed (<0.1 mg l1) and OB2 with NH+4–N environmentally relevant concentration (4.06 ± 0.16 mg l1) (Table 2). With the increase of NH+4–N removal

rates from 0.06 g m3 d1 (OB1) to 3.97 g m3 d1 (OB2), slight increase cypermethrin (from 76.8 ± 5.8% to 80.0 ± 2.7%) and chlorpyrifos (from 63.0 ± 3.6% to 68.4 ± 0.8%) removal efficiencies were demonstrated in the reactor OB2. However, the statistical analysis by Duncan’s multiple range tests showed that no significant differences of cypermethrin and chlorpyrifos removal occurred by increasing NH+4–N level (Table 4). It might be suggested that the relationship between nitrification and EDPs removal was not significant connected. Thus, cypermethrin and chlorpyrifos removal might be not due to the cometabolic degradation of ammonia oxidizing bacteria, but most likely attributed to the heterotrophic bacteria. In the recent years, a great interest has raised in the use of AMO for cometabolic degradation of EDCs. Yi and Harper (2007) also proved that there was a positive linear relationship between degradation rate of 17a-ethynylestradiol and ammonia biotransformation rate. However, the initial EDCs and ammonia concentrations in most previous studies (wastewater) were much greater than those observed in natural waters. Gaulke et al. (2008) reported that 17a-ethynylestradiol removal at trace concentrations in activated sludge systems is not attributed to cometabolic degradation, but most possibly attributed to the effects of heterotrophic bacteria. The similar phenomenon was also observed in the present study. Thus, enhancing heterotrophic bacteria activity was

Table 4 Statistical analysis of cypermethrin and chlorpyrifos removal efficiencies by Duncan’s multiple range test. Reactors

Cypermethrin (%)

Chlorpyrifos (%)

OB1 OB2 OB3 AB1 AB2 AB3

76.8 ± 5.8b 80.0 ± 2.7b 85.0 ± 0.3c 63.0 ± 3.0a 65.0 ± 1.3a 77.9 ± 1.6b

63.0 ± 3.6c 68.4 ± 0.8c 75.1 ± 3.9d 28.8 ± 4.2a 32.9 ± 5.7a 46.9 ± 8.0b

Note: a and b were letter markers. The average removal efficiencies of cypermethrin and chlorpyrifos marked different letters had the significant difference (P < 0.05).

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3.3. The removal performances of trace levels of EDPs under different TOC levels via reed addition The components of reed nutrition are presented in Table 2. The  3 concentrations of NH+4–N, NO 2 –N, NO3 –N, PO4 –P and TOC leached 1 from the reed were (3.03 ± 0.10) mg l , (0.23 ± 0.02) mg l1, (16.65 ± 0.10) mg l1, (0.17 ± 0.01) mg l1 and (612.60 ± 2.50) mg l1, respectively. Remarkably, the major nutrients of reed nutrition were organics. Batch reactors OB3 and AB3 were both fed with 140 ml reed nutrition. It was easily obtained that NH+4–N,  3 NO 2 –N, NO3 –N, PO4 –P and TOC by reed nutrition addition in batch reactors were approximately increased by 0.10 mg l1, 0.008 mg l1, 0.56 mg l1, 0.006 mg l1 and 20.52 mg l1, respectively. Thus, the contribution of nitrogen and phosphorus compounds caused by adding reed nutrition was scare in batch reactors. In the batch test to determine the effect of initial organics on EDPs removal, the initial TOC was varied approximately from 4.0 to 24.6 mg l1 both in aerobic and anaerobic conditions (Table 3). It was shown that cypermethrin and chlorpyrifos removal efficiencies were both increased due to reed addition, and Duncan’s multiple range test demonstrated that the differences were significant (Fig. 2). Therefore, EDPs could be well removed with extra carbon source addition. The highest of removal efficiencies of cypermethrin and chlorpyrifos were both found in OB3, with 84.9% and 75.1%, respectively, but the denitrification efficiency was rather low in OB3 due to the high level of DO. Whereas, simultaneously enhanced denitrification and EDPs removal were achieved in reactors AB3 with reed addition. In the recent years, denitrification and EDPs removal has become more and more important in natural waters due to the intense use of fertilizers and pesticides in agriculture. Due to cometabolic degrading EDPs at trace levels by

R cypermethrin (%) R chlorpyrifos (%)

Effluent cypermethrin Effluent chlorpyrifos

100

1200 1100 1000 900 800 700 600 500 400 300 200 100

0

90 80 70 60 50 40 30 20

EDPs removal rate (%)

EDPs concentration (ng l -1)

suggested to be an effective way for the removal of trace levels of EDPs in oligotrophic environment. To determine whether the removal performance of trace EDPs is enhanced by increasing initial NO 3 –N at anaerobic conditions in oligotrophic environment, anaerobic reactor AB1 fed 0.84 ± 0.03 mg l1 NO (tap water) and AB2 fed 3 –N 4.45 ± 0.14 mg l1 of NO 3 –N (environmentally relevant concentration) (Table 2). With the increase of NO 3 –N removal rates from 0.81 g m3 d1 (AB1) to 1.50 g m3 d1 (AB2), the obvious increase of TOC utilization efficiency for denitrification was also observed 1 from 0.25 to 0.53 mg NO TOC. However, Duncan’s multi3 –N mg ple range tests demonstrated that cypermethrin (from 63.0 ± 3.0% to 65.0 ± 1.3%) and chlorpyrifos (from 28.8 ± 4.2% to 32.9 ± 5.7%) removal efficiencies had no significant differences between AB1 and AB2 (Table 4). Therefore, the increase in initial ammonia and nitrate levels up to environmentally relevant concentration, the removal performance of cypermethrin and chlorpyrifos were both not significantly increased. Also, it was not wise to further increase nitrogen level because the adverse effect of nitrogen caused, e.g. Eutrophication (Camargo and Alonso, 2006). As shown in Table 3, cypermethrin and chlorpyrifos removal efficiencies in anaerobic condition were much lower than those in aerobic reactors, and the Duncan’s multiple range test also showed significant difference between them. Because cometabolic degradation of EDPs by AMO was not expected in this study, the significantly declined removal efficiencies of EDPs in anaerobic condition most possibly due to the heterotrophic bacteria in aerobic reactor. Previous studies also showed that mechanical aeration has been proved to be very effective in reducing EDPs concentrations. Jilani (2008) reported that more than 85% cypermethrin was degraded by Pseudomonas strain (IES-Ps-1) (initial concentration of 80 mg l1) when dissolved oxygen (DO) was above 8 mg l1, and significantly decreased with the decline of DO level.

10

OB1

OB2

OB3

AB1

AB2

AB3

0

Fig. 2. Cypermethrin and chlorpyrifos removal performances in the batch biofilm reactors.

AMO failed in the study, it was suggested that simultaneously removal of nitrogen and EDPs at low concentrations in natural waters might rely on heterotrophic bacteria in biological systems. There were several studies also reported the important role of carbon source in nitrogen and pesticides simultaneously removal. There were ethanol (Choi et al., 2006), biodegradable polymer poly (Ginige et al., 2004), wheat straw (Xu et al., 2012) and so on. 3.4. Biofilm analysis of simultaneously denitrification and EDPs removal in oligotrophic environment It was suggested that simultaneously denitrification and EDPs removal were effective with external carbon source addition in anaerobic condition, probably by way of cometabolic pathways with NO 3 –N transformed to N2 (Park and Yoo, 2009). In the study, biofilm characteristics of AB2 without reed addition and AB3 with reed addition were studied. TB and VB of biofilm on the elastic filler in AB3 (17776.5 and 5760.9 g m3 carrier, respectively) were much higher than those in AB2 (5220.7 and 611.4 g m3 carrier, respectively). It was directly proved that the addition of reed nutrition

Fig. 3. DGGE profile of the total bacteria in anaerobic batch reactors AB2 (without reed addition) and AB3 (with reed addition).

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Table 5 Sequencing results of bacterial DGGE bands of 16S rRNA. Band

Closet match

Phylogenetic group

Similarity (%)

Origin

T1

Uncultured Methylophilaceae bacterium [JQ994355.1] Uncultured Methylophilaceae bacterium [JQ994352.1] Uncultured Methylophilus sp. [GU472577.1] Uncultured Methylophilaceae bacterium [HQ706467.1] Uncultured Methylophilaceae bacterium [FN679078.1] Uncultured Methylophilaceae bacterium [JQ771999.1] Bacillus sp. [JX994094.1] Uncultured Methylophilaceae bacterium [EU642168.1] Uncultured Methylophilaceae bacterium [GU472950.1] Uncultured bacterium [GU766819.1]

b-Proteobacteria

91

Denitrifying bacterium in rice floating-beds waters

b-Proteobacteria

98

Denitrifying bacterium in rice floating-beds waters

b-Proteobacteria b-Proteobacteria

98 98

Low-sulfate lake Subsurface uranium mine

b-Proteobacteria

98

Freshwater sediments

b-Proteobacteria

99

PAHs-contaminated soil

Firmicutes b-Proteobacteria

100 98

PAHs-contaminated site Lake Michigan

b-Proteobacteria

100

Different potato cultivars in two fields

Bacteria

99

Hyphomicrobium sp. [HQ694744.1] Uncultured Methylophilaceae bacterium [EU640146.1] Uncultured Methylovorus sp. [JN601497.1] Uncultured Thauera sp. [HQ658792.1] Hyphomicrobium sp. [HQ694746.1] Uncultured Firmicutes bacterium

a-Proteobacteria b-Proteobacteria

100 98

A membrane filtration system for a drinking water treatment plant Denitrifying bacteria from anode and cathode from MFC Lake Michigan

b-proteobacteria b-Proteobacteria a-Proteobacteria Firmicutes

99 99 100 95

TCP photobiodegradation system Municipal wastewater treatment plants Denitrifying bacteria from microbial Fuel Cells Agricultural systems

T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16

could obviously improve the growth of heterotrophic bacteria. Previous studies have shown a positive relationship between biodegradable carbon source in water and bacterial growth in biofilm (Chandy and Angles, 2001). Simultaneously enhancing denitrification and EDPs removal in AB3 were possibly due to this heterotrophic bacteria growth by reed addition. Srinandan et al. (2012) proved that various carbon sources (e.g. acetate, glucose and methanol) significantly affected the microbial community. To determine the specific dynamic of heterotrophic bacteria at the condition of reed addition, biofilm bacteria community in the reactors AB2 and AB3 were analyzed using specific PCR, DGGE and sequencing of 16S rRNA genes (Fig. 3). DGGE band patterns were examined with the Dice index of similarity and Shannon diversity index. The Dice similarity index of bacteria community of the reactors AB2 and AB3 was low (59.1% similarity). The Shannon index in AB2 (1.78) was much higher than that in AB3 (2.63). It was shown that increase organics could significantly enriched microbial community and improve bacterial growth in oligotrophic environment. To ascertain the dynamic of dominant bacteria of biofilm in reactors AB2 and AB3, sixteen DGGE bands were excised and sequenced (Fig. 3), and the results are shown in Table 5. Specifically, ten of the excised bands belonged to Methylophilaceae bacterium; two were related to Hyphomicrobium; the rest were associated with Thauera, Bacillus and undefined species. Many bands in the DGGE gel appeared and some became more intense with reed addition. The intensity of bands T1 to T10 obviously increased and most of them belonged to Methylophilaceae bacterium except for band T7 (Bacillus sp.). Bands T4, T12 and T16 appeared as dominant bacteria in AB3, which were related to Methylophilaceae bacterium and Firmicutes bacterium. There was methanol fed in both reactors of AB2 and AB3. Previous studies showed that Methylophilaceae were frequent detected as denitrifiers in methanol-fed biological systems (Ginige et al., 2004; Srinandan et al., 2012), which was consistent with the present study. Furthermore, Methylophilus sp. has been isolated for biodegrading N-methyl carbamate pesticide (Aldicarb) to aldicarb oxime and methylamine (Arica et al., 1999). Due to the methyl also occurred in cypermethrin and chlorpyrifos (Table 1), ten of the excised Methylophilaceae bacterium were probably responsible for the

removal of cypermethrin and chlorpyrifos. The other dominant species Hyphomicrobium (Auclair et al., 2012), Thauera (Thomsen et al., 2007) and Bacillus (Verbaendert et al., 2011) were frequently detected as denitrifiers in biological systems. It was further proved that reed addition could improve bacterial growth which was associated with nitrogen and EDPs removal. Therefore, reed addition was an attractive approach to achieve simultaneously enhancing denitrification and EDPs removal in anaerobic condition. 4. Conclusions EDCs removal by AMO was reported in some wastewater treatment systems, but no significant removal of EDPs at trace levels in oligotrophic waters biofilm pretreatment system with increase of ammonia. However, significantly enhanced EDPs removal occurred both in aerobic and anaerobic conditions with reed addition, and simultaneously enhanced denitrification and EDPs removal were achieved in anaerobic condition. Microbial analysis demonstrated that many species related to the nitrogen and EDPs removal appeared and some species became more intense with reed addition. Thus, heterotrophic bacteria growth by reed addition seemed to be an attractive approach to achieve simultaneously denitrification and EDPs removal. Acknowledgements The work was funded by the National Key Technologies Research and Development Program of China (No. 2012BAJ25B07), the National Natural Science Foundation of China (No. 51078327/ 51008269) and the Research Project of Zhejiang Ocean University (No. 21105012313). References Arica, M.Y., Halicigil, C., Alaeddinoglu, G., Denizli, A., 1999. Affinity interaction of hydroxypyruvate reductase from Methylophilus spp. with Cibacron blue F3GAderived poly(HEMA EGDMA) microspheres: partial purification and characterization. Process Biochem. 34 (4), 375–381. Auclair, J., Parent, S., Villemur, R., 2012. Functional diversity in the denitrifying biofilm of the methanol-fed marine denitrification system at the montreal biodome. Microb. Ecol. 63 (4), 726–735.

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