ldentification of Boseongicola sediminum sp.nov.,a Novel Decabromodiphenyl Ether (BDE-209)-tolerant Strain lsolated from Coastal Sediment in Xiamen,China＊

TANG Chen ,FAN Chun ,GUO Dong Bei ,MA Xiu Juan ,CAI Qing Tao ,CHEN Xiao Xuan ,ZHANG Min,LI Jia Yao,AN Qiu Ying,and ZHAO Ran,#

Polybrominated diphenyl ethers (PBDEs) are a group of aromatic organic bromine compounds,which have been used in a broad array of polymeric materials (plastics,foams,resins,and adhesives) as brominated flame retardants.They are used in commercial and household products,including textiles,electronic equipment,airplanes,and automobiles,especially in China and the USA,because they are inexpensive with excellent flameretardant effects[1].During the past decade,over 14,000 tonnes of PBDEs have entered the market,and have become ubiquitous environmental contaminants[2].Although they have been regulated in a wide variety of countries,decabromodiphenyl ether 209 (BDE-209),one kind of PBDE,is still used legally in most countries,and accounts for > 80% of PBDE usage worldwide,especially in China and the USA[3].Based on previous research,exposure to BDE-209 significantly increases many kinds of health risk for humans.In the 2006 PBDE Project Plan,the US Environmental Protection Agency summarized animal studies of various commercial mixtures and individual congeners,which posed potential concerns about the liver toxicity,thyroid toxicity,developmental toxicity,and developmental neurotoxicity of BDE-209 in adult men.There is also growing evidence that prenatal or early-life exposure to PBDEs impairs neonatal neurodevelopment.Previous studies have also documented the relationships between serum PBDEs and reduced sperm motility and counts.Numerous physicochemical methods have been used to remediate environmental PBDE contamination,but these methods entail many problems,including complex operational procedures,high costs,and low efficiency,which restrict their application.Moreover,some of these methods may create secondary pollutants that are even more harmful to the environment.Although few studies have reported techniques for eliminating PBDE contamination,treating environmental pollutants (such as chromium[4],benzene[5],and anthracene[6]) with microorganisms has been shown to be an effective method,with the advantages of practicability,environmental friendliness,and cost-efficiency.

The BDE-209-tolerant bacterial strain TAWCT132Tis a novel species in the genusBoseongicola,a member of the order Rhodobacterales.The genusBoseongicolacontains only one species at present,Boseongicola aestuarii,which was isolated from a tidal flat sediment at Boseong,South Korea[7].B.aestuariiis Gram negative,aerobic,nonmotile,and pleomorphic (coccoid,ovoid,or rod-shaped).In this study,we determined the specific taxonomic characterization of TAW-CT132T,which was isolated from offshore sediment samples in an area of PBDEscontaminated sea in Tong’an Bay,Xiamen City,PR China and evaluated the efficiency of its degradation of BDE-209.This study expands the microbial resources available for the treatment of BDE-209 contamination,and lays a theoretical foundation for investigating the degradation mechanism/s of TAWCT132T.

Sediment samples were collected on April 8,2013,from the PBDE-contaminated area of sea in Tong’an Bay (118°10′ E and 24°37′ N),where the largest comprehensive industrial base in Xiamen City is located (Supplementary Figure S1,available in www.besjournal.com).All sampling procedures followed strict sterility principles,and the samples were transported and stored under low-temperature conditions (+4 °C) before analysis.For enrichment,50 mL of mineral salts medium (MSM; containing 1.0 g/L (NH4)2SO4,0.8 g/L K2HPO4,0.2 g/L KH2PO4,0.2 g/L MgSO4,0.08 g/L CaSO4,0.005 g/L FeSO4·7H2O,and 0.0033 g/L Na2MoO4·2H2O) was inoculated with about 3 g of sediment sample,and 100 mg/L of BDE-209 was added as the sole carbon source.The enrichment medium was incubated at+28 °C with shaking at 160 rpm for 4 weeks.The enrichment suspension (approximately 150 μL) was then serially diluted to 10?6of the original concentration,spread onto marine agar 2216 solid medium (MA; BD Difco,Franklin Lakes,NJ,USA),and incubated at +28 °C for 3 days.Individual colonies were picked and purified by successive streaking.Boseongicola aestuariiKCTC 32576T(= CECT 8489T),also designatedB.aestuariiBS-W15T(obtained from the Korean Collection for Type Cultures [KCTC,Jeongeup-si,Korea]) was selected as the related type strain,based on the phylogenetic relationships represented by phylogenetic tree topologies.Strains TAW-CT132Tand KCTC 32576Twere maintained on MA plates at +4 °C and in 20% (v/v) glycerol suspensions at ?80 °C for short- and long-term storage,respectively.

Gram staining was performed with Tianhe Microorganism Reagent (Hangzhou,China),according to the manufacturer’s instructions.Catalase and oxidase activities were determined using 3% (v/v) H2O2(Sangon Biotech,China) and 1%(w/v) N,N,N′,N′-tetramethyl-1,4-phenylenediamine(bio-Mérieux,France),respectively.Starch hydrolysis was performed as described by Sooyeon Park[7].The gliding motility and morphological characteristics of the bacterium were examined with light microscopy(Olympus IX70,Tokyo,Japan) and transmission electron microscopy (JEOL JEM-1230,Tokyo,Japan),respectively.The presence of poly-βhydroxybutyrate granules was investigated with epifluorescence microscopy (Olympus BX51,Tokyo,Japan) after staining with Nile blue A.To determine the anaerobic growth conditions,the strain was incubated on MA plate for 2 weeks in the Anoxomat Mark II Anaerobic System (Mart Microbiology,Drachten,The Netherlands).The bacterial growth conditions were measured at different temperatures(5,10,15,20,25,30,35,37,40,45,and 50 °C),different NaCl concentrations (0,0.5%,and 1.0%–20.0% [w/v] in increments of 1.0%),and pH 3.0–12.0 (in increments of 1 pH unit),adjusted with citrate/phosphate (pH 3.0–7.0),Tris/HCl (pH 8.0–9.0),or sodium carbonate/sodium bicarbonate buffer (pH 10.0–11.0) in Marine Broth (MB) medium(BD Difco,Franklin Lakes,NJ,USA),prepared according to the formula,but without NaCl.All pH values were verified after autoclaving.The growth rate was determined with a multimode microplate reader (Molecular Devices M5e,USA) as the optical density measured at a wavelength of 600 nm (OD600)at various temperatures,pHs,and NaCl concentrations after culture for 3 days.API 20NE,API 20E,and API ZYM tests (bio-Mérieux,Saint Vulbas,France) were conducted under the optimal growth conditions,with the single modification that the NaCl concentration was adjusted to 3.0%.All the above-mentioned biochemical tests were performed after incubation on solid MA medium at 28 °C for 3 days.

The genomic DNA of strain TAW-CT132Twas extracted with the Tiangen Bacterial Genomic DNA Extraction Kit (Tiangen Biotech,Beijing,China),according to the manufacturer’s instructions.The 16S rRNA gene was amplified with primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′).16S rRNA gene sequences highly similar to that of TAW-CT132Twere identified with EzBioCloud (https://www.ezbiocloud.net/).After sequences were aligned with CLUSTAL W in the Molecular Evolutionary Genetics Analysis software version 7.0 (MEGA,Temple University,Philadelphia,PA,USA),phylogenetic trees were generated with three algorithms: the neighborjoining (NJ),maximum likelihood (ML),and minimum evolution (ME) algorithms.The evolutionary distances were calculated with Kimura’s twoparameter model with the algorithms cited above,with bootstrap values based on 1,000 replications.The G+C content of the chromosomal DNA was determined with reversed-phase high-performance liquid chromatography (HPLC).The complete genome of TAW-CT132Twas sequenced by Majorbio Biotech Co.,Ltd (Shanghai,China) using the Illumina Hiseq X? Ten platform and assembled with a SOAPdenovo analysis (version 2.04).The average nucleotide identity (ANI) and DNA–DNA hybridization (DDH) values were calculated with the EzBioCloud server (http://www.ezbiocloud.net/tools/ani) and the Genome-to-Genome Distance Calculator (http://ggdc.dsmz.de/distcalc2.php),respectively.The genomic data of the related type strain KCTC 32576Twere downloaded from the GenBank database (accession number GCA_900184815.1).

Biomass of strain TAW-CT132Twas harvested for the chemotaxonomic analyses from cultures grown for 3 days on MA at 28 °C.Respiratory quinones were analyzed with HPLC (Shimadzu SIL-20AP,Shimadzu,Japan).Whole-cell fatty acids were prepared,extracted,saponified,esterified,and then analyzed with gas chromatography (GC; Agilent Technologies 6850,Santa Clara,CA,USA) and identified with the MIDI Sherlock? Microbial Identification System (TSBA 6.0 database).The polar lipids of the two strains were extracted and examined with two-dimensional thin-layer chromatography,after which the plates were sprayed with the appropriate detection reagents.

To explore the maximum BDE-209 tolerance of TAW-CT132T,a 4% bacterial suspension (grown overnight) was added to MSM containing various concentrations of BDE-209 (50–500 mg/L,in increments of 50 mg/L).The maximum tolerated concentration was deemed to be the highest concentration of BDE-209 at which bacterial growth was observed after incubation for 5 days at 28 °C.To evaluate the effects of the initial inoculum concentration,2%–10% (v/v) bacterial suspensions(in increments of 2%) were prepared and added to MSM containing 50 mg/L BDE-209.To examine the degradation efficiency of BDE-209,a 4% bacterial suspension (grown overnight) was added to 50 mL of MSM containing 50 mg/L BDE-209,and the cells cultured at 28 °C with shaking at 160 rpm.The effects of heavy metals on the BDE-209 degradation efficiency of TAW-CT132Twere also investigated.We added 3 mg/L and 6 mg/L Cu2+or Cr6+to MSM containing 50 mg/L BDE-209 to examine their effects on BDE-209 degradation efficiency.The residual amounts of BDE-209 in all the assays were detected after incubation for 1,3,and 5 days,and the degradation rate was calculated with the Formula:

Repeated-measures ANOVA was used to compare the statistical significance of the difference between the experimental and control groups.

BDE-209 was extracted as follows.The liquid sample was first adjusted to pH 2.0 before extraction.The sample was then extracted three times with a 1:1 (v/v) mixture of dichloromethane and n-hexane.The organic extracts were combined and concentrated with a rotary evaporator at +40 °C.The lipid in the flask was reconstituted to 10 mL with dichloromethane and stored at ?20 °C until analysis.

The residual amounts and the metabolic products of BDE-209 were detected with an Agilent 7890 gas chromatograph coupled with an Agilent 7010 triple quadruple mass spectrometer,which was operated in the negative chemical ionization (NCI)mode.An aliquot (1 μL) of sample was automatically injected in the spitless mode at an injection temperature of 300 °C.Chromatographic separation was achieved on a DB-5MS column (15 m length ×0.25 mm internal diameter×0.25 μm film thickness).Helium gas (99.99%) was used as the carrier gas at a flow rate of 2.0 mL/min.The interface and ion source temperatures were set to 300 °C and 200 °C,respectively.The GC oven was programmed as followed: initial temperature of 100 °C (held 1 min),which was increased to 310 °C at 30 °C/min and held 6 min.The target ions of BDE-209 were at m/z 486.5 and 488.5,whereas 488.5 was used as the quantitative ion.

Strain TAW-CT132Tis Gram negative,nonmotile and strictly aerobic.The colonies are yellow–white,circular (0.5 mm in diameter),opaque,and smooth when grown on MA plates with incubation at 28 °C for 3 days (Supplementary Figure S2,available in www.besjournal.com).As shown in Supplementary Figure S3 available in www.besjournal.com,the TAW-CT132Tcells appeared rod-shaped (0.5 μm wide and 1.5 μm long) when viewed under an epifluorescence microscope.Poly-β-hydroxybutyrate granules were detected in the strain.TAW-CT132Tdid not grow on MA plates after incubation in an anaerobic jar for 2 weeks.The strain showed mesophilic growth in a temperature range of 10–45 °C and optimum growth at 35 °C.Growth was observed in the presence of 0–12.0% (w/v) NaCl(optimum,2.0%) and at pH 5.0–10.0 (optimum,pH 6.0–7.0).The detailed phenotypic,physiological,and biochemical properties that distinguish strain TAWCT132Tfrom the closely related type strain are listed in Table 1.

Table 1.Phenotypic characteristics of strain TAWCT132T and related type strain KCTC 32576T,with in the genus Boseongicola

The almost-complete 16S rRNA gene sequence(1,331 bp) of TAW-CT132Thas been deposited in the GenBank database under accession number of MH991824.1.The results of an EzBioCloud similaritybased search showed that TAW-CT132Tshares greatest similarity (95.94%) withMaritimibacterlacisalsiX12M-4T,followed byB.aestuariiKCTC 32576T(95.86%),Litorisediminicola beolgyonensisBB-MW24T(95.46%),M.alkaliphilusHTCC2654T(95.41%),Actibacterium pelagiumJN33T(95.34%),andAliiroseovarius zhejiangensisJB3T(95.04%).Its sequence similarities with other strains were <95.0%.All tree-generating algorithms showed that TAW-CT132Tformed a tight clade withB.aestuariiKCTC 32576T,as shown in Figure 1 and Supplementary Figures S4–S5,available in www.besjournal.com.

The genome size of TAW-CT132Tis 3,451,213 bp,and it encodes 3,452 genes.A total of 43 tRNA and three rRNA genes were identified.The G+C contentis 66.4%,whereas the G+C content of the related type strain KCTC 32576Tis 58.7%.The complete genome sequence of strain TAW-CT132Twas uploaded to the GenBank database under accession number JAAJBD000000000.The ANI value between strains TAW-CT132Tand KCTC 32576Twas 71.09%,which was significantly lower than the threshold of 94%–96% for bacterial species delineation[8].The DDH value between the two strains was 18.10%,which is also clearly below the 70% threshold generally accepted for the delineation of species[9].

TAW-CT132Twas positive for oxidase and catalase,and negative for the hydrolysis of starch and gelatin.In the API ZYM system,it was positive for esterase (C4),esterase lipase (C8),leucine aminopeptidase,valine aminopeptidase,acid phosphatase,β-galactosidase,α-glucosidase,Nacetyl-β-glucosaminidase; weakly positive for lipase(C14); but negative for alkaline phosphatase,cystine aminopeptidase,trypsin,α-chymotrypsin,naphthol-AS-Bl-phosphoamidase,α-galactosidase,βglucuronidase,β-glucosidase,α-mannosidase,and αfucosidase.In the API 20NE system,TAW-CT132Twas positive for β-glucosidase,β-galactosidase; and negative for denitrification,the reduction of nitrate to nitrite,indole production,d-glucose fermentation,arginine dihydrolase,urease,gelatin hydrolysis,and the utilization of d-glucose,l-arabinose,d-mannose,d-mannitol,N-acetyl-glucosamine,d-maltose,potassium gluconate,capric acid,adipic acid,malic acid,trisodium citrate,and phenylacetic acid.In the API 20E system,it was positive for β-galactosidase,acid production from rhamnose; weakly positive for H2S production; and negative for arginine dihydrolase,lysine decarboxylase,ornithine decarboxylase,tryptophan deaminase,utilization of citrate,urease,indole production,the Voges–Proskauer reaction,and acid production from glucose,mannitol,inositol,sorbitol,sucrose,melibiose,and amygdalin.

The predominant respiratory quinone of TAWCT132Tis Q10 (72.4%),which is consistent with the closely related type strain KCTC 32576T.Significant and minor amounts of MK-7 (24.5%) and Q9 (3.1%)are also present in TAW-CT132T.The predominant fatty acids (> 5% of the total fatty acids) of strain TAW-CT132Tare C16:0(30.6%),summed feature 8(19.1%),C18:0(14.3%),C19:0cyclo ω8c (10.3%),C20:4ω 6,9,12,15c (7.3%),and C18:1ω7c 11-methyl (5.7%).The differences in the fatty acid contents of strains TAW-CT132Tand KCTC 32576Tare shown in Supplementary Table S1,available in www.besjournal.com.The polar lipids in TAW-CT132Tare diphosphatidylglycerol,phosphatidylglycerol,phosphatidylcholine,two unidentified phospholipids,and four unidentified polar lipids.By contrast,the major polar lipids in KCTC 32576Tare phosphatidylcholine and phosphatidylglycerol.Minor amounts of polar lipids,including diphosphatidylglycerol,three unidentified phospholipids,one unidentified aminolipid,and one unidentified lipid,were also detected(Supplementary Figure S6,available in www.besjournal.com).

Figure 1.Neighbour-joining tree showed the phylogenetic positions of strain TAW-CT132T and relatives based on nearly complete 16S rRNA gene sequences,Azomonas agilis NCIB 11693 (T) (AB175652) was used as an outgroup.Numbers at nodes are bootstrap values (percentages of 1000 replications); only values 70% are shown.Bar,0.020 nucleotide substitution rate (Knuc) units.

The tolerance test showed that TAW-CT132Ttolerates a maximum of about 300 mg/L BDE-209.The efficiency of BDE-209 degradation increased as the initial bacterial inoculum concentration increased,as shown in Supplementary Figure S7,available in www.besjournal.com.After incubation for 1,3,and 5 days,the degradation rates of BDE-209 were 6.04%,11.38%,and 29.55%,respectively,as shown in Figure 2.The main metabolic products of BDE-209 are hexabromodiphenyl ether,octobrominated diphenyl ether,and nonabromodiphenyl ether,which pose lower ecological risks to the environment than BDE-209[10].When polycyclic aromatic hydrocarbons or C18H38were used as alternative carbon sources,the ability of TAW-CT132Tto degrade BDE-209 declined to different extents,as shown in Supplementarys Figures S8–S9 available in www.besjournal.com,respectively.As shown in Supplementary Figure S10 available in www.besjournal.com,both low and high concentrations of Cu2+tended to increase the efficiency of BDE-209 degradation at the very beginning of the incubation period.However,after degradation for 5 days,BDE-209 biodegradation by TAW-CT132Twas inhibited by the high concentration of Cu2+,whereas the low Cu2+concentration had the opposite effect.These results are consistent with those of Yu Liu et al.[11].Cr6+at both low and high concentrations inhibited BDE-209 biodegradation by TAW-CT132T.It is postulated,but as yet unconfirmed,that this lower BDE-209 degradation efficiency is attributable to the toxicity of Cr6+,which inhibits normal bacterial growth[12].Further in-depth studies are required to verify this conjecture.In conclusion,the efficient degradation of BDE-209 byB.sediminumTAW-CT132Thas important potential utility in the bioremediation of BDE-209 pollution.

Figure 2.The degradation rate of BDE-209 after 1-,3-,and 5-day incubation.The initial concentration of the initial bacterial inoculum was 4%.**meant P < 0.05 and P > 0.01;***meant P < 0.01.

The authors declare that they have no competing financial interests.

The authors are very grateful to WANG Wan Peng and HUANG Zhao Bin at the Third Institute of Oceanography,State Oceanic Administration in Xiamen City,China,and HU Dong at the Institute of Urban Environment,Chinese Academy of Sciences in Xiamen City,China for their technical support.The authors would also like to thank the editors and reviewers for their valuable comments and suggestions on this paper.

&These authors contributed equally to this work.

#Correspondence should be addressed to ZHAO Ran,Associate Professor,PhD,Tel: 86-592-2880637,E-mail:zhaoran@xmu.edu.cn

Biographical notes of the first authors: TANG Chen,male,born in 1990,PhD,majoring in environmental health; FAN Chun,male,born in 1965,PhD,Professor,majoring in environmental health.