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Environ Sci Pollut Res DOI 10.1007/s11356-015-5660-7

RECENT SEDIMENTS: ENVIRONMENTAL CHEMISTRY, ECOTOXICOLOGY AND ENGINEERING

Isolation and characterization of two novel psychrotrophic decabromodiphenyl ether-degrading bacteria from river sediments Linqiong Wang 1 & Yi Li 1 & Wenlong Zhang 1 & Lihua Niu 1 & Juan Du 1 & Wei Cai 1 & Jing Wang 1

Received: 2 July 2015 / Accepted: 20 October 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Decabromodiphenyl ether (BDE-209) is a brominated flame retardant and a priority contaminant. Currently, little information is available about its significance in the environment, specifically about its susceptibility to aerobic biotransformation at low temperature. In this work, five phylogenetically diverse BDE-209-degrading bacterial strains were isolated from river sediments of northern China. These strains were distributed among four different genera—Acinetobacter, Pseudomonas, Bacillus and Staphylococcus. All five isolates were capable of growing on BDE-209, among which two isolates show better growth. By detailed morphological, physiological, and biochemical characteristics and 16S rDNA sequence analysis, the two strains were identified and named as Staphylococcus haemolyticus LY1 and Bacillus pumilus LY2. The two bacteria can grow in mineral salt medium containing BDE-209 substrate across the temperatures ranging from 2.5 to 35 °C, with an optimum temperature of 25 °C which could be considered as psychrotrophs accordingly. The degradation experiment showed that more than 70.6 and 85.5 % of 0.5 mg/ L BDE-209 were degraded and the highest mineralization efficiencies of 29.8 and 39.2 % were achieved for 0.5 mg/L

Responsible editor: Philippe Garrigues * Yi Li [email protected] * Wenlong Zhang [email protected] 1

Key Laboratory of Integrated Regulation and Resource Development of Shallow Lakes, Ministry of Education, College of Environment, Hohai University, Xikang Road #1, Nanjing 210098, People’s Republic of China

BDE-209 by S. haemolyticus LY1 and B. pumilus LY2, respectively. To the best of our knowledge, this is the first demonstration for the biodegradation of BDE-209 by two psychrotrophic bacteria isolated from environment. Keywords Aerobic biodegradation . Decabromodiphenyl ether . Psychrotrophic bacteria . Staphylococcus haemolyticus . Bacillus pumilus

Introduction As flame retardants, polybrominated diphenyl ethers (PBDEs) have been used in a variety of commercial productions for more than four decades. These are diphenyl ethers with more than one to ten bromine atoms (Sutton et al. 2015). The widely using commercial products of PBDEs are as mixtures. The PBDEs composed of flame retardant, resins, plastics, and textiles are penta-BDEs (mostly BDE-99 and BDE-47), octa-BDEs (mostly BDE-183, BDE-197, and BDE203), and deca-BDEs (BDE-209) (Alaee et al. 2003; de Wit 2002). Due to their high lipophilicity and low water solubility, PBDE are being detected in the environment and in wildlife and also exposure in a wide variety of environmental media such as air, solid, sewage sludge, and sediment (Leung et al. 2007; Luo et al. 2009; Song et al. 2005). In addition, PBDEs are perceived as toxic chemicals and bioaccumulative which directly influences the ecosystems and human beings (Shi et al. 2013a). Therefore, it is necessary to develop effective technology for the elimination of PBDEs from the environment. A number of methods have been developed to decrease the existence of PBDEs in the environment, including physical-

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chemical ways (burying and incineration), photodegradable method (oxidation), and biological degradation (Keum and Li 2005; Mas et al. 2008; Nose et al. 2007). One of the most attractive methods in organic pollutant degradation was through a biological way because of its low cost, safety, and environmentally benign nature. Few researchers have studied on the biotic transformation of PBDEs; however, BDE-209 could be debrominated into more toxic lower-brominated congeners. Thus, more attention is required in view of environmental fate for degradation of BDE-209. Microbes can degrade PBDEs both under aerobic and anaerobic conditions. A recent study on biotransformation demonstrated that PBDEs were debrominated to lower-brominated congeners by a variety of anaerobic bacteria (He et al. 2006; Robrock et al. 2008). Gerecke et al. found that degradation of BDE-209 into lower-brominated diphenyl ether congeners in anaerobic mesophilic digester sludge took more than 238 days while the degradation efficiency was only 50 % (Gerecke et al. 2006). Few studies have been conducted to investigate the biodegradation of BDE-209 in the presence of other substrates under the aerobic surrounding. An indigenous bacterium Lysinibacillus fusiformis DB-1 was isolated from PBDE-polluted sediment, and it could degrade BDE-209 to lower-brominated PBDEs using lactate, pyruvate, and acetate as carbon sources under aerobic condition (Deng et al. 2011). Biodegradation of BDE-209 by white-rot fungi could be enhanced in liquid culture with addition of appropriate amounts of surfactant (Tween 80 and β-cyclodextrin) (Zhou et al. 2007). Shi et al. demonstrated the degradation of BDE-209 by Pseudomonas aeruginosa under co-metabolic substrates (Shi et al. 2013a). However, few reports described the degradation of BDE-209 as a sole carbon utilizer by aerobic bacteria. Lu et al. reported a metal-resistant bacteria strain Bacillus cereus JP12 utilized BDE-209 as the sole carbon and energy source for growth and the presence of metal ions had a slight stimulating effect on BDE-209 degradation (Lu et al. 2013). To the best of our knowledge, this is the only report about pure bacteria growing on BDE-209 as sole carbon source. The aim of the present study is therefore to isolate BDE-209-degrading bacteria under aerobic conditions from the river sediment of northern China. The bacteria were then identified and their growth and degradation capacity have been tested under different initial BDE209 concentrations. In addition, the effect of temperature on bacterial growth and BDE-209 degradation was also investigated since varied daily and seasonal fluctuations in the environmental temperatures are inevitable during bioremediation. These results will guide us to declare pure cultures for the mechanisms of the contaminant degradation system as well as aid in the development of wastewater treatment process.

Materials and methods Chemicals Standards of 2, 2′, 3, 3′, 4, 4′, 5, 5′, and 6, 6′-decabromodiphenyl ether (BDE-209; 99 % purity) were used as a sole carbon source for the biodegradation obtained from Sigma (St. Louis, MO, USA). BDE-209 was dissolved in acetonitrile to make a stock solution of 1.0 g/L and stored in dark-brown glass containers at 4 °C to prevent photolysis of BDE-209. A standard mixture of eight PBDEs (BDE-CSM) was obtained from Sigma (St. Louis, MO, USA). For the preparation of solutions, ultrapure water obtained from a Milli-Q Millipore system (Milli-Q plus 185) and absolute ethanol was used, obtained from Panreac. Carbon tetrachloride (99.9 %) from Panreac and high-performance liquid chromatography (HPLC)-grade acetonitrile from Lab-Scan, Analytical Sciences, were used in this study. HPLC-grade methanol (Fisher Scientific) and n-hexane 95 % (Lab-Scan, Analytical Sciences) were used for solid phase extraction. Sample collection and medium Fresh sediment was collected from the Songhua River which could be considered as one of the cold streams in China during September of 2013. The surface sediments (0–15 cm) were collected during low tides using a soil core sampler. All sediment samples were taken randomly in the triplicate from an area of around 1 m2 at the center of each selected site. A medium for aerobic culturing was prepared according to a previous report (Robrock et al. 2009). The mineral medium contains 10 g/L NaNO3, 1.7 g/L K2SO4, 0.37 g/L MgSO4· 7H2O, 0.086 g/L CaSO4·2H2O, 5.3 g/L KH2PO4, 10.6 g/L K2HPO4, 5.74 mg/L ZnSO4·7H2O, 3.38 mg/L MnSO4·H2O, 1.24 mg/L H3BO3, and 5 mg/L CuSO4·5H2O. The following ingredients were added after autoclaving: 0.94 mg/L CoMoO4·H2O, 0.2 mg/L FeSO4·7H2O, 0.1 mL/L H2SO4. The mineral salt medium was used as the degradation medium. The separation and preservation of culture medium (the beef extract peptone medium) ingredients are beef 3, peptone 10, and NaCl 5 g/L. The ingredients of bacterial colony in plates are the beef extract peptone medium and agar powder 25 g/L. Media (except BDE-209) and all Erlenmeyer flasks plugged with cotton and pipette tips were autoclaved at 120 °C for 30 min for sterilization before use. Acclimation and isolation of microbial consortiums To promote the rejuvenation of the microbial consortium and the exhaustion of organic substances, a 250-mL conical flask containing 100 mL mineral salt medium (MSM) supplemented by 10 mg/L BDE-209 with 5 g wet weight sediment was placed in the dark on a shaking incubator at 130 rpm and 25 °C for 3 days. Subsequently, a 10 mL aliquot of the

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enrichment culture was incubated to the MSM which was supplemented by 0.5 mg/L BDE-209 (0.5, 1, 2 mg/L) for 5 days at different time intervals to the fresh medium. After repeating it for six times, a mixed microbial consortium was enriched by an increased turbidity and the ability to degrade 2 mg/L of BDE-209. After 1 month of acclimation, the enrichment of the bacteria process was started and the concentrations of BDE-209 were increased gradually (0.5, 1.0, 2.0, 5.0, and 10.0 mg/L) to achieve the purpose of enrichment and incubation using a shaking incubator (130 rpm, 25 °C) in the dark. After 45 days of enrichment procedure, the samples were diluted in inorganic culture media and plated onto solid enrichment media with 0.5 mg/L BDE-209 to obtain single colonies (the plates were incubated at 30 °C for 24 h). A loopful of single colony from each plate was streaked onto other fresh agar plates containing BDE-209 to check for the purity. Individual colonies were isolated, tested for their degradation activity, and stored at −80 °C in 15 % glycerol. Identification of a BDE-209-utilizing bacteria strains The isolated BDE-209-degrading strains were identified according to morphological, cultural, and physiological properties based on those of Bergey’s manual of determinative bacteriology and the 16S rDNA sequence analysis. Genomic DNA was extracted using the Bacterial DNA Kit (Omega BioTek, USA). Primer pair 27F (5′-AGAGTTTGATCMTGGCTCAG3′) and 1492R (5′-TACGGYT ACCTTGTT ACGACTT-3′) was used for PCR amplification of the 16S rDNA. The PCR amplification products were sequenced on an ABI 3730 via an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Biosystems). The results were compared with other published 16S rDNA sequences using the Basic Local Alignment Search Tool (BLAST) program of the National Center for Biotechnology Information (NCBI) database. Growth and degradation of BDE-209 The degradation of BDE-209 was carried out in the mineral medium that contains BDE-209. The BDE-209 solution was prepared as per the previous literature (Leal et al. 2013). BDE209 was dissolved in acetonitrile and the acetonitrile was removed by air-drying in the sterile stage. A mixture of dichloromethane and deionized water (3:7 in volume ratio) was added to the medium and was air-dried in the sterile stage. The final volume has been adjusted during the vaporization of dichloromethane. The BDE-209 degradation was carried out by adjusting the optical density value of cell suspension of 100 mL mineral medium around 0.02–0.025 at 600 nm (OD600) then they were taken in 250-mL Erlenmeyer flasks and cultured for 3 days on a shaking incubator at 25 °C and 130 r/min for desired time in the dark. On the optimal

conditions, different initial concentrations of BDE-209 (0.5, 1.0, 2.0, 5.0, and 10 mg/L) were investigated to analyze the influence of BDE-209 concentration on cell growth and BDE209 biodegradation. Treatments without the inoculants of bacteria acted as control. Two milliliters of initial samples was centrifuged and the supernates were taken for analysis using HPLC–UV. Then, the individual flasks have been removed at various intervals and analyzed. Analytical techniques Cell concentration in the sample was analyzed by measuring the optical density (OD) at 600 nm using UV–vis spectrophotometer (Perkin-Elmer, Lamda 35, USA) with the culture medium as reference. The OD value was then converted to dry cell by an appropriate calibration curve. HPLC analysis (Leal et al. 2013): quantitative analysis of BDE-209 was achieved by HPLC–UV (Shimadzu), using a device which consists of an UV–vis detector SPD–20A, a degasser DGU–20A5, a bomb LC–20AD, and a column oven CTO–10ASVP (T=25 °C). A chromatographic column is Agilent C 18 (250 mm×4.6 mm, Agilent, Model 1100), and an injection loop of 20 μL was used. Cell temperature was maintained at 25 °C. The mobile phase was 97 % acetonitrile and 3 % ultrapure water where the flow rate was 1.0 mL/min. The detection was done at 240 nm wavelength, corresponding to the maximum absorptivity value. Centrifugation was done using Mixtasel centrifuge, class 2.0 (J. P. Selecta, s. a., Spain). UV–vis spectra were obtained with a T90+UV/vis spectrophotometer (PG Instruments Ltd.) using a slit width of 2 nm. UV–vis spectra of BDE-209 solutions have been taken in rectangular quartz cuvettes of 10-cm path length while the spectra of aqueous solutions of HS fractions were taken in cuvettes of 1-cm path length. Solid phase extraction procedure: The concentrated extract had flowed through a Florisil column (1 g, 6 mL), the column was eluted with 15 mL dichloromethane/n-hexane/methanol (50:45:5, v/v/v). Conditioning procedure (to avoid bleeding) was repeated after drying the cartridge under air stream for 30 min. The eluent was dissolved in 1 mL of methanol and filtered through a 0.22-mm membrane filter. The purified extraction solution was divided into two equal parts; 0.5 mL of extraction solution was analyzed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS) for OH-PBDEs, and the remaining 0.5 mL was acetylated by acetic anhydride, which are then extracted using n-hexane and analyzed using GC–MS/MS for lowerbrominated PBDEs. GC–MS analysis: PBDE and bromophenol analysis was carried out on a Bruker 450 GC-320 triple quadrupole mass spectrometer (Fremont, CA, USA) fitted with DB–XLB column (30 m×0.25 mm×0.1 μm) combined with multiplereaction monitor (MRM) mode of tandem mass spectrometry.

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One microliter of injection volume was taken and the injection temperature was set to 340 °C. Helium was used as the carrier gas at a flow rate of 1.5 mL/min. The column temperature was maintained at 50 °C for the first 1 min and raised it at 20 °C/min up to a temperature range of 230 °C, at 6 °C/min up to a temperature range of 285 °C and at 256 °C/min up to a temperature range of 340 °C which have been maintained for 10 min. Mass spectrometer conditions were maintained with an electron impact at 70 eV of electron energy, 100 μA of filament current with multiplier voltage of 1200 V at full scan mode. Ultra performance liquid chromatography (UPLC): quantitative analysis of hydroxylated PBDEs (OH-PBDEs) was achieved by UPLC–MS/MS (Acouity UPLC–Xevo TQS, Waters, USA). Reversed-phase chromatography was performed by UPLC with a BEH C18 column (2.1 mm×50 mm, 1.7 μm) maintained at 40 °C. An aliquot of each sample (10 μL) was injected into the UPLC system with an autosampler at 10 °C while the cell temperature was maintained at 40 °C. Mobile phase-A (acetonitrile) and the mobile phase-B (ultrapure water) were filtered by a 0.22-μm filter membrane. Gradient eluent procedure was carried out with 70 % B (0– 0.8 min), 70–30 % B (0.8–5 min), 30–5 % B (5–7 min), 5 % B (7–9 min), 5–70 % B (9–10 min), and 70 % B (10–13 min) for a period of 13 min. The flow rate was maintained at 1.000 mL/min while the mass spectrometer was operated in negative electrospray ionization mode (ESI−) using MRM. Biodegradation performance measurement BDE-209 biodegradation efficiency and mineralization efficiency were calculated as follows: C 0 −C  100 C0 M TOC0 −M TOC Mineralization efficiency ð%Þ ¼  100 M TOC0 Degradation efficiency ð%Þ ¼

where C0 was the initial BDE-209 concentration, C was the BDE-209 concentration at the time indicated; M TOC0 was the initial total organic carbon concentration produced from total mineralization of the substrate, MTOC was the total organic carbon concentration at the time indicated. Total organic carbon (TOC) was analyzed as described in the literature (Wang et al. 2012). The supernatant liquor sample was first surged by ultrasonic wave (40 kHz, 250 W, 30 min) to make homogeneous TOC concentration in the sample then it was injected to the TOC analyzer (Shimadzu, TOC-VCPH). Data analysis In this study, all experiments were performed in triplicate to get reliable data, and the results are reported as mean ±

standard deviations. The standard deviations for all measurements ranged from 2.0 to 8.5 %.

Results and discussion Isolation and identification of the BDE-209-degrading bacteria As a result of enrichment procedure, five morphologically different strains were isolated and named W1 to W5. Table 1 shows the information of all five strains identified using the 16S rDNA gene sequencing method. The analyses of 16S rDNA sequence of these strains revealed that they were phylogenetically diverse (Fig. 1) and were widely distributed among four different genera—Pseudomonas, Acinetobacter, Bacillus, and Staphylococcus—of two phyla: Proteobacteria and Bacilli. Strains W4 and W5 were selected for further studies in this paper due to its better growth on plates. Bacterial colony morphology of strain W4 on nutrient agar was bright yellow, circular of uniform, rough, and has a radial edge while strain W5 colony morphology was bright yellow, circular of bump, smooth, and has a neat edge. The biochemical characteristics of strain are shown in Table 2. Microscopic observation of the globular cells revealed that the strain W4 was nonmotile by means of no flagellum whereas the strain W5 was motile with flagellum, which was further confirmed by Leifson staining methods and Bergey’s manual of determinative bacteriology (Leifson 1930). Morphological and biochemical characteristics and 16S rDNA base composition revealed that strains W4 and W5 exhibit highest sequence similarity with Staphylococcus haemolyticus (Accession number: KT158714) and Bacillus pumilus (Accession number: KT158714), respectively, according to the BLAST search using the NCBI GenBank. The 16S rRNA gene sequence of W4 shared 99 % of similarity with S. haemolyticus NC 007168.1 while the sequence of W5 shared 99 % of similarity with B. pumilus NC 009848.1 when compared with data available in GenBank. The two novel BDE-209-degrading bacteria W4 and W5 were then named as S. haemolyticus LY1 and B. pumilus LY2. Due to the extreme hydrophobicity and complex chemical structure, BDE-209 has limited bioavailability. Few reports are available revealing bacteria growing on BDE-209 substrate. Lu et al. reported that a BDE-209-degrading bacterium Bacillus cereus JP12 could use BDE-209 as the sole carbon and energy source for growth (Lu et al. 2013). Current studies mostly focusing on the biotransformation of BDE by bacterial community (Zhang et al. 2013). For example, mixed bacterial cultures were isolated from PBDE-contaminated river sediments which were able to transform the initial concentration BDE-209 of 20 mg/L as the sole carbon (Chou et al. 2013). In addition, many studies have investigated the effect of PBDE

Environ Sci Pollut Res Table 1 Taxonomic identity of BDE-209-degrading bacterial isolates

Isolate number

Closest relative based on 16S rDNA gene sequence

Similarity (%)

Class

Accession number

1 2

Acinetobacter sp. W1 Pseudomonas sp. W2

99 99

Proteobacteria Proteobacteria

KT158713 KT158715

3

Pseudomonas sp. W3

99

Proteobacteria

KT158716

4 5

Staphylococcus haemolyticus W4 Bacillus pumilus W5

99 99

Bacilli Bacilli

KT158717 KT158714

on microbial communities using PCR-DGGE biotechnology. Shih et al. studied that BDE-209 was reduced and removed by microbial community where as PBDEs were also degraded in sewage sludge under aerobic condition by indigenous microflora (Shih et al. 2012; Stiborova et al. 2015). In this study, it was found that both bacteria S. haemolyticus LY1 and B. pumilus LY2 were able to grow on substrate BDE-209 as novel BDE-209-degrading strains.

35 °C. Figure 2b reveals the biodegradation rate of 2.0 mg/L BDE-209 at various temperatures ranging from 2.5 to 35 °C. The biodegradation rates were changed with increase in temperature, which showed consistent effect on its cell growth. The maximum biodegradation rates of S. haemolyticus LY1 and B. pumilus LY2 were 35.0 and 60.2 % at 25 °C, respectively. These results are particularly helpful when considering remediation in contaminated areas with apparent annual temperature fluctuations.

Effect of incubation temperature on cell growth and BDE-209 biodegradation

Effect of initial BDE-209 concentration on bacteria growth

Figure 2a showed the cell growth of S. haemolyticus LY1 and B. pumilus LY2 growing on 2.0 mg/L BDE-209 at temperatures ranging from 2.5 to 35 °C. The optimum growth temperature of the bacteria was found to be 25 °C while utilizing BDE-209 as the sole substrate. However, complete growth inhibition of S. haemolyticus LY1 and B. pumilus LY2 occurred at 45 °C. According to the definitions by Morita (Morita 1975), it is indicated that S. haemolyticus LY1 and B. pumilus LY2 are psychrotrophic bacteria and their bioremediation potential of BDE-209-contaminated environment could occur across a wide range of temperatures from 2.5 to

The effect of the initial concentration of BDE-209 on bacteria growth ranging from 0.5 to 10 mg/L was shown in Fig. 3. As illustrated by the curves in Fig. 3a, b, S. haemolyticus LY1 and B. pumilus LY2 grew well on BDE-209 at different concentrations (0.5–10.0 mg/L). However, the growth curves of S. haemolyticus LY1 and B. pumilus LY2 were much varied at 10.0 mg/L BDE-209, which might be due to the distinct characteristics of each bacterium including the expression of catabolic genes and intracellular enzyme (Shi et al. 2013b). From Fig. 3a, it shows that S. haemolyticus LY1 grew well by utilizing BDE-209 as the sole carbon and energy source

Fig. 1 Phylogenetic distribution of BDE-209-utilizing cultures isolated from river sediment. Calculation of the phylogenetic tree was based on the neighborjoining method with bootstrapping. Values the branch points are on 100 bootstrap replications. The scale bar corresponds to two substitutions per 100 nucleotide positions

Environ Sci Pollut Res Table 2 Physiological and biochemical characteristics of bacterial isolates S. haemolyticus LY1 and B. pumilus LY2

Test item

Result

Test item

LY1

LY2

Result LY1

LY2

Cell morphology

Coccus-shaped

Rod-shaped

Spore formation



+

Cell size (μm)

0.8–1.3 μm

0.8–1.5 μm

Ornithine decarboxylase





Motility Flagellum

− −

+ +

Oxidase





Gram stain

+

+

Dihydrolase

− +

+ −

Aerobic growth Acetoin production

+ +

+ +

Benzidine Hemolysis

+ +

− −

Xylitol



+

Salicin

Sucrose Phosphatase

+ −

+ −

L-arabinose

Catalase

− − +

− + +

Coagulase Methyl red test

− −

− −

Glucose Vpes–Proskauer test

+ +

+ +

Nitrate reduction test

+



Arginine dihydrolase

+

+

Hydrolysis of gelatin



+

Indole test





Fig. 2 Effect of temperature on cell growth of S. haemolyticus LY1 and B. pumilus LY2 (a) and degradation rate (b) in 2.0 mg/L BDE-209 substrate after 14 days at 25 °C, pH 7.0, and 130 rpm culture condition

D-xylose

Fig. 3 Time course of S. haemolyticus LY1 (a) and B. pumilus LY2 (b) cell growth. The incubation temperature was 25 °C, and the initial medium pH was 7.0, shaking at 130 rpm. The bacterial growth was monitored by determining the optical density at 600 nm (OD600)

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with the increase in initial substrate concentrations. In the case of 0.5 mg/L BDE-209, the mass of S. haemolyticus LY1 was increased during the first 4 days then reached the highest biomass on the fourth day and was slightly decreased later. It showed a relatively stable value 0.045 of OD600 from 5 to 14 days. However, the highest cell density was reached with a culture turbidity of 0.177 at 10.0 mg/L BDE-209, which was relatively lower than 5.0 mg/L BDE-209 concentration’s OD600. Figure 3b shows that B. pumilus LY2 grew better than S. haemolyticus LY1. The biomass of B. pumilus LY2 increased quickly in the first 2 days; after 3 days of incubation and the bacterial OD600 was achieved, the maximum value of 0.098 at 0.5 mg/L BDE-209 and at the tenth day, bacterial OD600 achieved the maximum values of 0.243 for 10.0 mg/ L BDE-209. Due to its low solubility (Gandhi et al. 2011), BDE-209 crystals could be formed in the medium when the concentrations are at a high value which results in increased saturation level in the solution. It is well known that only the watersoluble fractions are available to microorganisms from hydrophobic organic pollutants (Deziel et al. 1999). Confluent bacterial growth may not only be restricted to the suspension alone but also on the pollution crystal surfaces (Lu et al. 2013). The result of the present study is in complete agreement with the finding of Lu et al. (2013) who demonstrated that the mass of a bacterial cell depends on the initial concentration of BDE-209 in shaken culture. They have also observed that strain JP12 growth occurred only at very high levels of BDE-209 (20 mg/L). Degradation of BDE-209 by bacteria Biodegradation of BDE-209 at different concentrations by S. haemolyticus LY1 and B. pumilus LY2 was shown in Fig. 4. As can be seen, degradation efficiency of BDE-209 was significantly influenced by the initial concentration. Degradation efficiencies of BDE-209 in the biodegradation system were 68.6, 57.7, 43.0, 31.9, and 19.5 % for strain S. haemolyticus LY1 as well as 85.5, 78.1, 70.2, 59.7, and 45.6 %, for strain B. pumilus LY2, respectively. With increase in concentrations of BDE-209 from 0.5 to 10.0 mg/L, the degradation rates were decreased which is similar to the previous report by Zhang et al. (2013). The effect of an initial concentration of BDE-209 on aerobic strains S. haemolyticus LY1 and B. pumilus LY2 was conducted, and the results were shown in Fig. 4a, b. Few differences were found between strains S. haemolyticus LY1 and B. pumilus LY2 during the biodegradation at various concentrations. It was found that the removal efficiency of S. haemolyticus LY1 was lower than that of B. pumilus LY2. From Fig. 4a, it is shown that the degradation rate of BDE-209 gradually increases in time. Although the degradation rate of BDE-209 by S. haemolyticus LY1 was

Fig. 4 Time course of S. haemolyticus LY1 (a) and B. pumilus LY2 (b) of BDE-209 biodegradation efficiency at different initial concentrations (0.5, 1.0, 2.0, 5.0, and 10.0 mg/L) at 25 °C, pH 7.0, and 130 rpm

increased very slowly during the first 2 days, it increased rapidly from day 6 and then tended to stabilize after 8– 14 days. This demonstrated that S. haemolyticus LY1 could be adapted to the BDE-209 environment and use BDE-209 as carbon source for its growth. From Fig. 4b, it was observed that the BDE-209 degradation efficiency of B. pumilus LY2 was higher than that of S. haemolyticus LY1. There was a short adaptive phase and the degradation rate increased quickly, achieving the maximum after 6 days. In both cases, only a short lag phase was observed, suggesting that the bacteria could adapt to the environment quickly at the beginning of the incubation period. However, in this study, the degradation rates by two strains at 10.0 mg/L of BDE-209 concentrations were just 19.5 and 45.6 %, respectively, even after 10 days. The same kind of results were also shown by Shi et al. with 56 % of degradation efficiency toward BDE-209 (from 0.99 to 0.44 mg/L) after 7 days (Shi et al. 2013a). In general, addition of toxicants as the sole carbon source may cause lag phrase followed by enhanced biodegradation (Anwar et al. 2009).

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Mineralization efficiency of BDE-209 During biodegradation processes, organic pollutants cannot be mineralized immediately once the microbiological reactions start, and the formation of intermediates is inevitable. This is especially true for recalcitrant compounds such as PBDEs. Therefore, it would be necessary to verify the mineralization of BDE-209 through intermediates. The metabolism degrees of BDE-209 with S. haemolyticus LY1 and B. pumilus LY2 were investigated in terms of the mineralization efficiency. After TOC analysis of every sample, the result revealed that the mineralization efficiencies increased slightly during the first several days and increased rapidly during the next days while leveled off with further incubation. The mineralization efficiency declined sharply with the increase in BDE209 concentration, which was consistent with the effect of BDE-209 concentrations on its biodegradation. This may be ascribed to the substrate inhibition caused by some more toxic intermediates (Ross et al. 2009). After 12 days of incubation, the highest mineralization efficiency of 29.8 % was achieved

from 0.5 mg/L BDE-209 solution, while only 6.5 % of the carbon content of BDE-209 was mineralized to carbon at a higher initial concentration (10 mg/L). However, B. pumilus LY2 shows highest mineralization efficiency of 39.2 % from 0.5 mg/L of BDE-209 solutions, and 14.7 % of the carbon content of BDE-209 was mineralized to carbon at 10 mg/L concentration of BDE-209. Mineralization efficiency of B. pumilus LY2 was found to be better than S. haemolyticus LY1, which is in accordance with the results of bacterial growth. Analysis of metabolite pathway To achieve a better understanding of its environmental fate, the possible degradation pathways and mechanism of BDE209 including its simultaneous debromination and mineralization by B. pumilus LY2 were identified based on their metabolites using GC–MS/MS and UPLC–MS/MS analysis. Tables 3 and 4 listed the identified products obtained from degradation of BDE-209 by B. pumilus LY2. As it indicates,

Table 3 MS parameters for the analysis of PBDEs and bromophenols in the culture after 14 days incubation at 25 °C of temperature, 130 rpm, and pH of 7.0 with initial concentration of 1 mg/L BDE-209

Intermediates

Chemical structure Br

Parent ion

Daughter

Collision

(m/z)

ion (m/z)

energy (eV)

22.0

483.9(M-Br2)

376.9

25

19.0

565.7(M+2)

405.9

25

14.0

325.9(M)

325.9

25

9.5

407.8(M+2)

247.8

25

5.5

252

142

25

4.5

242

135

25

Trace time(s)

Br O

BDE-153 Br

Br Br

Br

Br

Br O

BDE-99 Br

Br Br

Br

Br O

BDE-47 Br

Br Br O

BDE-28 Br

Br

OH OH

3-Bromocatechol Br

2-Hydroxy-4-bromo-adipic

OH

Br O

acid

O

OH OH

Environ Sci Pollut Res Table 4 MS parameters for the determination of OH-PBDEs in the culture after 14 days of incubation at 25 °C of temperature, 130 rpm with initial concentration of 1 mg/L BDE-209 Intermediates

Chemical structure

OH

OH

2,2′,3′-Trihydroxy-4,4′dibromodiphenyl ether

O

Parent ion (m/z)

Daughter ion (m/z)

Cone voltage (eV)

Collision energy (eV)

5.38

500.5

81

40

22

5.63

500.6

79

40

22

OH

Br

4′-Monohydric-2,2′,4,4′dibromodiphenyl ether

Trace time(s)

Br Br

Br O

Br

OH

Br

the culture of B. pumilus LY2 resulted in several different debrominated congeners. Several OH-BDEs and open-ring products were also detected and listed in Tables 3 and 4. Based on the metabolites identified, one of the most possible mechanisms of BDE-209 degradation by B. pumilus LY2 was proposed in Fig. 5, which outlines three possible metabolic pathways: debromination, hydroxylation, and cleavage of the diphenyl ether bond. In a single debromination procedure, BDE-209 may potentially be debrominated to several different congeners like BDE-153, BDE-99, BDE-47, and BDE-28 by removal of one to three bromine atoms. This study suggested that BDE153 first undergoes meta-debromination by forming BDE-99 and then into a second meta-debromination step to form BDE47 which finally undergoes to a para-debromination step to form BDE-28 (Fig. 5). The hypothesized debromination pathways described above suggests that loss of Br occurs more readily in the para- and meta-position, which was inconsistent Br

Fig. 5 Possible biodegradation pathway of BDE-209 by aerobic bacterium B. pumilus LY2 (involvement of more than one step was indicated by dotted arrow)

with the previous study in which PBDE congeners were exposed to fish (Usenko et al. 2013). Hydroxylation reaction is an important step in the degradation of halo aromatic compounds by organisms. BDE-28 underwent hydroxylation reaction to yield OH-PBDE (2, 2′, 3′-trihydroxy-4, 4′dibromodiphenyl ether). The identified OH-PBDEs as metabolites in PBDE degradation pathway has been studied in microorganisms and plants (Huang et al. 2010; Malmvarn et al. 2008). The intermediate product was then degraded to 3bromocatechol and 2-hydroxy-4-bromo-adipic acid, which could be future oxidized and finally mineralized to produce carbon dioxide and water. A previous study has been reported that dichloro–diphenyl–trichloroethane (DDT) degrades through hydroxylation of the aromatic rings and the cleavage reaction of aliphatic-aryl carbon bond during the biodegradation (Xiao et al. 2011). To the best of our knowledge, this is the first report on the degradation of BDE-209 by S. haemolyticus and B. pumilus

Br

Br

O

Br Br

Br

Br Br

Br

Br

Br

Br Br

Br

Br

O Br

Br

HO

Br

Br O O

Br

Br - + CO2 + H2O OH

OH

Br O

OH OH

Br Br

Br OH

Br

Br

Br

OH O

Br O

Br

Br

OH

Br

O

Br

Environ Sci Pollut Res

which could be adapted to a wide range of temperature. These results may provide objective evidence for the potential application of S. haemolyticus and B. pumilus in the bioremediation of contaminated environments at high levels. However, the work concerning the metabolic mechanism is still preliminary. Future research is needed to identify the enzymatic mechanism of BDE-209 biotransformation by S. haemolyticus LY1 and B. pumilus LY2.

Conclusion Two novel psychrotrophic BDE-209-degrading bacteria were isolated from the northern river sediment of China. The strains can grow on BDE-209 as the sole carbon source, and they were identified as S. haemolyticus LY1 and B. pumilus LY2 using the 16S rDNA gene-sequencing method. The highest mineralization efficiencies of 31.8 and 42.5 % were achieved from 0.5 mg/L BDE-209 solutions by S. haemolyticus LY1 and B. pumilus LY2, respectively. To our best knowledge, this is the first demonstration for the biodegradation of BDE-209 by two psychrotrophic bacteria isolated from river sediment and would likely be meaningful for the bioremediation in BDE-209-contaminated environments. Acknowledgments This study was funded by the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (51421006), National Natural Science Foundation of China (no. 51479066 and 51322901), Research Fund for the innovation team of the Ministry of Education (IRT13061), Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Fundamental Research Funds for the Central Universities (no. 2014B07614).

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