Regional analysis of potential polychlorinated biphenyl degrading bacterial strains from China

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Environmental

Microbiology

Regional

analysis

of

potential

polychlorinated

biphenyl

degrading

bacterial

strains

from

China

Jianjun

Shuai

_,

_Xurun

_Yu

_,

_Jing

_Zhang

_,

_Ai-sheng

_Xiong

_,

_Fei

_Xiong

a_{JiangsuKeyLaboratoryofCropGeneticsandPhysiology/Co-InnovationCenterforModernProductionTechnologyofGrainCrops,}

YangzhouUniversity,Yangzhou,China

b_{StateKeyLaboratoryofCropGeneticsandGermplasmEnhancement,CollegeofHorticulture,NanjingAgriculturalUniversity,}

Weigang,Nanjing,China

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Articlehistory:

Received30January2014 Accepted28December2014 Availableonline22April2016 AssociateEditor:LaraDurãesSette

Keywords: Polychlorinatedbiphenyls Biphenyl Biodegradation Pseudomonassp. HPLC

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Polychlorinatedbiphenyls(PCBs),thechlorinatedderivativesofbiphenyl,areoneofthe mostprevalent,highlytoxicandpersistentgroupsofcontaminantsintheenvironment. TheobjectiveofthisstudywastoinvestigatethebiodegradationofPCBsinnortheastern (HeilongjiangProvince),northern(ShanxiProvince)andeasternChina(Shanghai munici-pality).Fromtheseareas,ninesoilsampleswerescreenedforPCB-degradingbacteriausing afunctionalcomplementaritymethod.Thegenomic16SrDNAlocuswasamplifiedandthe productsweresequencedtoidentifythebacterialgenera.SevenPseudomonasstrainswere selectedtocomparethecapacityofbacteriafromdifferentregionstodegradebiphenylby HPLC.Comparedtothebiphenylcontentincontrolsof100%,thebiphenylcontentwent downto3.7%forstrainP9-324,36.3%forP2-11,and20.0%fortheotherfivestrains.These resultsindicatethatalongerprocessingtimeledtomoredegradationofbiphenyl. PCB-degradingbacterialstrainsaredistributeddifferentlyindifferentregionsofChina.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Polychlorinatedbiphenyls(PCBs),thechlorinatedderivatives ofbiphenyl,arehighly toxicenvironmentalpollutantsthat havebecomeubiquitousthroughouttheenvironmentandthe foodwebbecause oftheirlowdegradability.Therefore, the removalof PCBsfrom soiland sedimentis a high priority inseveralindustrial countries.1 _{PCBmoleculesconsistofa}

∗ _{Corresponding author at: Jiangsu Key Laboratory of Crop Genetics and Physiology/Co-Innovation Center forModern Production} TechnologyofGrainCrops,YangzhouUniversity,Yangzhou225009,China.

E-mail:feixiong@yzu.edu.cn(F.Xiong).

biphenyl nucleuscarrying 1–10chlorines,which cancreate >200possiblecongenersthatdifferinchlorinenumber and position.Theless-chlorinatedcongenersareusuallylesstoxic than the more-chlorinated congeners.2 _{Theremediationof}

PCBcontaminationintheenvironmenthasbecomeextremely important.

ThemicrobialdegradationofPCBsisoneofthemost cost-effective and energy-efficient methods for removing them

http://dx.doi.org/10.1016/j.bjm.2014.12.001

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

from the environment. Many PCB-degrading bacteria have been isolated, such as Gram-negative strains belonging to thegeneraPseudomonas,Alcaligenes,Achromobacter, Burkholde-ria,Acinetobacter,Comamonas,SphingomonasandRalstonia,and Gram-positivestrains ofthe generaArthrobacter, Corynebac-terium, Rhodococcus and Bacillus.3 _{The genes encoding the}

enzymesforbiphenyldegradationareclusteredandtermed

bph gene clusters.4 _{The enzymes can be divided into four}

types, including biphenyl dioxygenase (BphA), dihydrodiol dehydrogenase (BphB), 2,3-dihydroxybiphenyl dioxygenase (BphC)andhydrolase(BphD).5–11

WepreviouslyinvestigatedthedistributionofPCB-utilizing bacteriainShanghai.12_{Inthisstudy,weinvestigatedthe}

dis-tributionofPCB-utilizingbacteriafromotherdifferentareas of China. Moreover, we chose several Pseudomonas strains fromdifferentsoilsamplesandmeasuredtheirdegradation ofbiphenylusingHPLC.

Materials

and

methods

Chemicalsandmedia

PCB(2,2,3,3-tetrachlorobiphenyl,CASNo.38444-93-8)(99.5% purity)andbiphenyl(CASNo.92-52-4)(99.5%purity)were pur-chasedfromtheJ&KChemicalCoLtd(Shanghai,China)and ShanghaiChemicalAgentCoLtd(Shanghai,China), respec-tively. Theother chemicals used inthis study were ofthe highestpurity available. Mineral salt medium (MM)13

con-tained (g): NH4NO3 1.0, KCl 0.7, KH2PO4 2.0, NaHPO4 3.0, MgSO4·7H2O 0.7, CaCl2 0.02 and NaCl 1.0 in1Lof double-distilledwateratpH7.0.SolidMMwasMMcontainingagar at16g/L.Afterautoclaving,tetrachlorobiphenylwasaddedas thesolecarbonsource.

IsolationofPCB-degradingbacterialstrains

Samples 1–3 were collected from Heilongjiang Province in northeastChina:Sample1fromachemicalfactory;Sample2 fromabotanicalgarden;andSample3fromapharmaceutical factory.Samples4and5werecollectedfromShanxiProvince innorthernChina:Sample4fromtheFenRiverandSample5 fromapapermill.Samples6–9werecollectedfromShanghai MunicipalityineasternChina:Sample6fromanexperimental field;Sample7fromariverbednotfarfromanoldchemical factory;Sample8fromanoldchemicalfactory;andSample9 fromariverbedneararesidentialarea(Fig.1).

For each sample, 10.0gof soilwas putinto asterilized mortarwith50mLofsterilewater,crushedwithasterilized pestle,andthenleftfor5min.Asample(1–5mL)oftheclear supernatantliquidwasaddedtoanErlenmeyerflaskthat con-tained100mLofMMwith0.01%tetrachlorobiphenylasthe solecarbonsource.Then,theErlenmeyerflaskwasincubated for3dat28◦Cindarknesswithshakingat150rpm.A1mL aliquotwastransferredtoanother100mLoffreshMM con-taining0.01% tetrachlorobiphenyland incubated underthe sameconditions.Thisprocesswasrepeatedthreetimes.Of thesuspension,20␮LwasplatedontosolidMMcontaining 0.01%tetrachlorobiphenylasthesolecarbonsourceand incu-batedfor3dat28◦C.

Heilongjiang

Shanxi

Shanghai

Fig.1–GeographicallocationsofthesoilsamplesinChina.

Identificationandanalysisof16SrDNAsequences

Genomic DNA was extracted from the PCB-utilizing bac-terial strains.14 _{The 16S rDNA locus was amplified using}

two primers (5-ACG GCTACCTTGTTACGACTTC-3 and 5 -AGAGTTTGATCCTGGCTCAG-3) and Ex-taq polymerase with genomicDNAasthetemplate,separatedby1.5%agarosegel electrophoresis,andclonedintothepMD-18vector(TaKaRa, Dalian,China)forenzymaticandsequencingidentification. ThePCRprotocolwas94◦Cfor10min,then35cyclesof94◦C for30s,50◦Cfor30s,72◦Cfor2min,andafinalextension at72◦Cfor10minina50␮Lreactionvolume.ThePCR prod-uctswereseparatedby1.5%agarosegelelectrophoresisand quantifiedusingaModelGelDoc1000analyzer(Bio-Rad, Her-cules,CA).Phylogeneticanalysiswasbasedonthe16SrDNA byMEGA5softwareandtheneighbor-joining(NJ)methodwith thefollowingparameters:Poissoncorrection,pairwise dele-tion,andbootstrap(1000replicates).15

HPLCanalyticalassayofbiphenyldegradation

Toverify andcomparethe extentofdegradation,wechose seven Pseudomonasstrains: P1-5, P2-11,P3-24, P4-38, P5-31, P7-245 and P9-324 from sevensoilsamples. Thedetails of detectingbiphenyldegradationbyHPLCwereasfollows:each ofthesevenstrains(100␮L)andacontrol(nostrain)were cul-turedinanErlenmeyerflask(500mL)containing100mLofLB mediumat28◦Cinarotaryshakerat120rpmovernight.The next day,1mLofbiphenylsolution(50mg/mL,dissolvedin methylalcohol)wasaddedintoeachErlenmeyerflaskandthe culturewascontinued.After2and4d,500␮Lofeach solu-tion wasplacedintoanEppendorftubeandthen 500␮Lof etherwasadded.Thetubesweredepositedonanoscillatorfor 5–10minsothatthebiphenylwasfullyextractedbytheether. Thesupernatantofthetwotubeswaswithdrawnandmixed togetherinanewtube,500␮Lofmethylalcoholwasadded andthemixturewasreadyforrotaryevaporation.Theether wasfullyevaporatedandthebiphenylwaskeptinthemethyl

Stenotropltomonas sp. 0 Shanghai 2 1 1 1 1 0 19 ₉ 10 1 1 7 9 1 1 1 1 1 1 111 8 4 19 _{5 3} 25 ₅ 14 _{5 3 2 1 1} Shanxi Heilongjiang 10 20 30 40 Number 50 60 70

Pseudomonas sp. Bacillus sp. Ralstonia sp. Acinetobacter sp.

Exiguobacterium sp. Sphingobacterium sp. Buttiauxella sp. Rhodococcus sp. Streptomyces sp. Cytophaga sp. Achromobacter sp. Alcaligenes sp. Pantoea,sp. Wautersia sp. Serratia sp. Aeromonas sp. Klebsiella sp. Cupriavidus sp. Enterobacter sp. Citrobacter sp. Xanthomonas sp.

Fig.2–TheoveralldistributionofPCB-degradingbacterialstrainsindifferentareasofChina.

alcoholtokeep itfrom adhering to thewallof the round-bottomedbottle.12

BeforeHPLCanalysis,eachsamplewasmembrane-filtered. A10-␮Lsample ofthe resulting solution was subjected to reverse-phaseHPLC(1100series,Agilent Co,USA).The sep-arationcolumnfortheHPLC(4.6mm×150mm×5␮m)was filledwithKromail100-5C18.Themobilephasecontained ace-tonitrile/water(70:30,v/v)andtheflowratewas1.0mL/min. Biphenylwasdetectedatawavelengthof254nm.4_Asolution

ofpurebiphenyl(100␮L/mL)wasusedtodeterminethepeak retentiontime.

Results

IsolationandclassificationofPCB-degradingbacteria From all the soilsamples, a total of 22 PCB-utilizing bac-terialstrainsweredetected,includingthefollowinggenera:

Stenotrophomonas, Pseudomonas, Bacillus, Ralstonia, Acineto-bacter, Cupriavidus, Wautersia, Achromobacter, Rhodococcus, Exiguobacterium,Enterobacter,Serratia,Alcaligenes,Streptomyces, Sphingobacterium, Citrobacter, Aeromonas, Pantoea, Cytophaga, Buttiauxella, Xanthomonas and Klebsiella (Fig. 2). There were 66 strains identified from Shanghai, more than 60 from Heilongjiang,and 45from Shanxi.Furthermore,thestrains from Shanghai encompassed 15 categories,more than the other two regions. Some bacterial strains were found in all three regions, e.g., Stenotrophomonas, Pseudomonas and

Rhodococcus. However,somebacteria only existed ina par-ticular soil, e.g., Achromobacter was only in soil samples fromHeilongjiang;Exiguobacterium,Serratia andStreptomyces

were only in soil samples from Shanxi; Sphingobacterium, Citrobacter, Aeromonas, Pantoea, Cytophaga, Buttiauxella, Xan-thomonas and Klebsiella were only in soil samples from Shanghai.

Phylogeneticreconstructionoftheselectedstrains

Amultiplealignmentanalysisusingfull16SrDNAsequences wasusedtostudythephylogeneticrelationshipsamongthe sevenPseudomonasstrainsselectedfromdifferentsoilsamples andotherPseudomonasstrains.StrainsP7-245andP9-324were foundtobelongtoonebranch,andP3-24andP4-38belongtoa separatebranch,witheachoftheseshowingtheclosest rela-tionship(Fig.3).Moreover,strainsP1-5andP5-31occupy,large sub-branches ofthetwoabove-mentioned branches.Strain P2-11isinadifferentbranchfromtheothersixPseudomonas

strains.

Degradationofbiphenyl

HPLC was used to detect the efficiencyof the seven Pseu-domonasstrainsatbiphenyldegradation.Whenpurebiphenyl was subjected to HPLC, the highest peak appeared at a retention timeof≈5.8min,indicatingthe pointofbiphenyl detection.12 _{The peak’sarea is directly proportional to the}

sampleconcentration.Inourassay,therewasonlyonelarge peak.

After 2d, assay results showed that each strain could degrade biphenyl with the degradation rate ranging from 5.9%(strainP6-245)to65.3%(strainP4-38)(Table1).After4d, strainP9-324haddegradedthemaximumamountofbiphenyl (96.3%),whereastheotherstrainsalldegradedamounts vary-ing from63.7%(strainP2-11)to83.6%(strainP4-38)(Fig.4). However,theamountofbiphenylinthecontrolshadchanged littlebyeither2or4d.

Discussion

PCBsrefertoalargegroupofchlorinatedbiphenylswith209 possible congeners.16 _{Due to their hydrophobic properties,}

P1-5 Pseudomonas putida strain KF703 Pseudomonas sp. NPO-JL-67 Pseudomonas putida strain P16 Pseudomonas putida strain RS-4 Pseudomonas putida strain P19

Pseudomonas putida strain AK5 Pseudomonas putida strain P1 Pseudomonas putida strain ZWL73 Pseudomonas fulva strain OS-10 Pseudomonas sp. WSCIII Pseudomonas sp. AV2A P7-245 P9-324 P3-24 P4-38 P5-31 P2-11 S2-15 Pseudomonas sp. Pseudomonas sp. PDS-7 Pseudomonas sp. D25 Pseudomonas sp. DJF1 Pseudomonas sp. BCNU171 Pseudomonas sp. ND9 Pseudomonas sp. ML2 Pseudomonas plecoglossicida stain S21 Pseudomonas plecoglossicida

strain R4

Fig.3–PhylogeneticanalysisofPseudomonassp.strainsasrevealedby16SrDNAsequencecomparisons.

PCBstendtobecomeabsorbedbynaturalorganicmatterin soil,sedimentsandsludges.PCBsareaseriousenvironmental problemduetotheirtoxicityandpersistenceinthe environ-ment.Naturalbiodegradation ofPCBsincontaminated soil andsedimentoccursatlowrates,17_{andvariousattemptsto}

acceleratebiodegradationhavebeenmade.18–21

Despite active research in developed nations spanning threedecades,extensiveregulatoryaction,and aneffective banon their productionsincethe late 1970s,PCBsremain a focus of research and environmental attention.22 _China

Table1–Comparisonofthedegradationofbiphenylfor controlsandPseudomonassp.strains.

Sample Biphenylcontent(%)

0d 2d 4d Control 100 97.6 92.3 P1-5 100 40.8 23.7 P2-11 100 77.4 36.3 P3-24 100 36.6 21.1 P4-38 100 68.4 16.5 P5-31 100 59.1 19.9 P7-245 100 94.1 23.4 P9-324 100 69.1 3.7

is adeveloping country inwhich pollution prevention and

controlis mostlikelylesseffective comparedtodeveloped countries.Moreover,PCBsarewidespreadintheenvironment

and are found globally as a result of ocean currents and

atmosphericdeposition.23,24 _{Thus,it isurgenttostudyPCB}

contaminationand biodegradationinChinaandacross the globe.

ThePCB-biodegrading bacterialstrains in Shanghai, the biggestindustrialmetropolisinChina,havebeen character-izedindetail.12_{Theobjectiveofthisstudywastoinvestigate}

mau P9-324 VWD1 A, VWD1 A, VWD1 A, 254 nm 254 nm 254 nm 1200 0d 2d 4d 1000 800 600 400 200 0 0 1 2 3 4 5 6 7 min

Fig.4–HPLCresultsofstrainP9-324.Theprocessingtime fromtoptobottom(d):0,2and4.

and compare the PCB-degrading bacteria in northeastern (HeilongjiangProvince),northern(ShanxiProvince)and east-ern China (Shanghai Municipality). Thus, research on PCB contaminationand biodegradation inlarge-scale regions is urgently needed. Three regions were selected to investi-gatePCB-degradingbacteria.HeilongjiangProvince,locatedin northeastChina,isamainagricultural(riceandcorn produc-tion)region.ShanxiProvince,locatedinnorthernChina,isa mainenergy(coalproduction)region.Shanghaihasdeveloped rapidlyinrecentdecadesandalongwithgrowthofthe econ-omy,environmentalpollutionisasubjectworthyofconcern. Soilsampleswerecollectedfromnortheastern,northernand easternChinatoinvestigatethebiodegradationofPCBs.Seven categoriesofstrainswereidentifiedinthesoilsamplesfrom HeilongjiangandShanxiProvincesbutnotinthesoilsamples ofShanghai.

Here,wechoseninesoilsamplesfromnortheastern, north-ernandeastern China,basedon areaswhereindustry has quicklydeveloped and were thus more likely tohave pol-lutedtheenvironment.Therewere>2samplinglocationsin each region to make the results more representative. The PCB-degradingbacterialstrains werevaried,withstrainsof generaStenotrophomonas,PseudomonasandRhodococcuspresent ineach region; thesethree PCB-utilizinggenera havebeen reportedinpreviousstudies.25_{Incontrast,somebacteriawere}

foundto exist onlyin a singleregion, possibly due tothe differentgeographicallocations.Thus,thedistributionof PCB-degradingbacterialstrainsmaybecloselylinkedtogeographic location,andthestateofdevelopmentintheregionmayalso playaroleintheevolutionaryselection.

Accordingtothephylogenetictree(Fig.3),therelationships amongthesevenPseudomonasstrainswerecloserthanwith theothers.HPLCconfirmedthatthesevenPseudomonasstrains coulddegradebiphenyl.Thesamplingresultsat2dshowed thatallbacteriahadstartedtodegradebiphenyl.By4d,the degradationofbiphenylbyallstrainswasnotthesame.Strain P9-324haddegradednearly96.0%ofthebiphenylcompared withthecontrol,andstrainP2-11haddegraded63.7%,which waslessthantheothersixPseudomonasstrains.Inaddition tostrainsP9-324andP2-11,theotherfivePseudomonasstrains degradedapproximately80.0%ofthebiphenyl.Theseresults arerelativelyconsistentwiththephylogenetictree.Strain P2-11camefromSample2,whichwasfromabotanicalgarden inHeilongjiang.Botanicalgardensarewellprotectedandless susceptibletooutsidecontamination;thisbalanced ecosys-temmaycausetheresistanceofbacteriatheretoberelatively weakerthaninotherplaces,e.g.,strainP2-112degradedonly 63.7%ofthebiphenyl.StrainP9-324isolatedfromSample9 wasfrom a riverbednear residentialquarters andhad the strongestabilitytodegradebiphenylofapproximately96.3%. Itismostlikelythattheriverwas seriouslypollutedmany yearsago,andthesestrainsremainedinthemudandretained theirstrongabilitytodegradebiphenyl.

Inthepresentstudy,differentareasinChinawerescreened forthepresenceofPCB-degradingbacteriaduetointerestin geographicalinfluences.Wecombinedmolecularbiologyand chemistrytogetherintheexperiments.Ourfutureworkwill includesearchingforthesourcesofenvironmentalpollution, solvingtheproblemsofpollutionbybiodegradationand com-biningmulti-disciplinarymethodstopreventpollution.

Conflict

of

interest

Theauthorsdeclarenoconflictsofinterest

Acknowledgments

TheresearchwassupportedbyAProjectFundedbythePriority AcademicProgramDevelopmentofJiangsuHigherEducation Institutions(PAPD).

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