h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Rubber
gloves
biodegradation
by
a
consortium,
mixed
culture
and
pure
culture
isolated
from
soil
samples
Chairat
Nawong,
Kamontam
Umsakul
∗,
Natthawan
Sermwittayawong
PrinceofSongklaUniversity,FacultyofScience,DepartmentofMicrobiology,Songkhla,Thailand
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received9March2017 Accepted10July2017
Availableonline3February2018 AssociateEditor:VâniaMelo
Keywords: Degradation Naturalrubber Rubberglove Rhodococcus
a
b
s
t
r
a
c
t
Anincreasingproductionofnaturalrubber(NR)productshasledtomajorchallengesin wastemanagement.Inthisstudy,thedegradationofrubberlatexglovesinamineralsalt medium(MSM)usingabacterialconsortium,amixedcultureoftheselectedbacteriaanda pureculturewerestudied.Thehighest18%weightlossoftherubberglovesweredetected afterincubatedwiththemixedculture.Theincreasedviablecellcountsoverincubation timeindicatedthatcellsusedrubberglovesassolecarbonsourceleadingtothe degra-dationofthepolymer.ThegrowthbehaviorofNR-degradingbacteriaonthelatexgloves surfacewasinvestigatedusingthescanningelectronmicroscope(SEM).Theoccurrence ofthealdehydegroupsinthedegradationproductswasobservedbyFourierTransform InfraredSpectroscopyanalysis.RhodococcuspyridinivoransstrainF5gavethehighestweight lossofrubberglovesamongtheisolatedstrainandposseslatexclearingproteinencoded bylcpgene.Themixedcultureoftheselectedstrainsshowedthepotentialindegrading rubberwithin30daysandisconsideredtobeusedefficientlyforrubberproduct degrada-tion.ThisisthefirstreporttodemonstrateastrongabilitytodegraderubberbyRhodococcus pyridinivorans.
©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
NaturalRubber (NR)isabiopolymerthatisproduced from someplantspecies. Thekey compositionofnaturalrubber latexiscis-1,4-poly(isoprene) thatconsistsofisopreneas a
∗ Correspondingauthor.
E-mail:Kamontam.u@psu.ac.th(K.Umsakul).
monomer. In nature, polyisoprenecanbe divided into two groups, e.g. cis-poly(isoprene) and trans-poly(isoprene). The majorsourceofnaturalrubberwasproducedfromtheHevea brasiliensis.However,notonlyHeveabrasiliensisbutalsomany plantscanproducelatexsuchasFicuselastica,Ficusnitidaand Euphorbia pulcherrima.1 Thelatex from theHevea brasiliensis contains apolymerofcis-1,4-poly(isoprene)unitsandupto 90%ofthedryweightofthelatexiscis-1,4-poly(isoprene)and lessthan10%arenon-rubberconstituentssuchasproteinand carbohydrates.2,3
https://doi.org/10.1016/j.bjm.2017.07.006
1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Global naturalrubber productionincreasedabout 79.4% from 6.8millionmetric tonsin2000to12.2 millionmetric tonsin2013.4 NaturalRubber isutilizeddailyinnumerous applicationssuchasagriculture,transportation,andvarious farmingequipment,inaddition, itisthemajorcomponent inrubbertires.3However,billionsofdiscardedtiresare cur-rentlystockpiledaroundtheworldandtheseareincreasing exponentially and causing important environmental prob-lems.Atpresent,mostrubberwasteiseliminatedbyeither burnedorusedaslandfillsbutthisprocess cancause seri-ous pollution.5 Biodegradation of rubber wastes has been muchofinterest.Rolesofmicrobesindegradationprocessare releasingofextracellularenzymestocleavepolymerchains into smallmoleculesand theseproductsareabsorbed into the cell for use as carbon and energy source. Final pro-cess, CO2, H2O and other metabolic products are released
and they can be used by other living organisms. There-fore, the biodegradation is an alternative way to degrade the rubber waste and could overcome the environmental problems.6–9
However,microbialdegradationofpolyisoprenerubberisa veryslowprocessthattakingmonthsorevenyears.Microbial degradationofcis-1,4-poly(isoprene)rubberiscurrentlybeing intensivelyinvestigated.Rubber-degradingorganismscanbe classifiedintotwodifferentgroups;thefirstgroupis rubber-degradingorganismsproducingclearzonesonnaturalrubber latexagarplates.Membersofthesegroupsgenerallybelong tothemycelium-formingactinomycetessuchasActinoplanes, Streptomyces, Micromonospora and Rhodococcus.10–12 In con-trast, the second group of rubber-degrading organisms is themicroorganisms, whichdonotproduceanyclearzone, therefore, they require the direct contact with the rubber substrates.Theexamplesofthisgrouparenocardioform, acti-nomycetes,i.e.Gordonia sp.,Mycobacterium sp.and Nocardia
sp.10,13–15
Lcp(latex-clearingprotein)encodedbylcpgenehasbeen consideredasakeyenzymeinNRdegradationby clearing-zone-forming gram-positive bacteria. While RoxA (rubber oxygenase) is a key enzyme in NR degradation found in the gram-negative bacterium Xanthomonas sp. strain 35Y. RoxA controlled by roxA gene is an extracellular protein secretedbythis strainduringgrowth onNR.Purified RoxA degradedcis-1,4-poly(isoprene) byoxidative cleavageatthe doublebonds,yielding 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al as the main cleavage product; other minor cleavage products differed only in the number of repetitive iso-preneunits. Invitro experiments also revealed occurrence of two 18 O atoms in the reduced degradation product 12-hydroxy-4,8-dimethyltrideca-4,8-diene-1-ol, thereby dis-closingadioxygenasemechanism.16–18
Mosteffortsonthestudy ofrubberbiodegradation have beendirectedtousingsinglebacterialstrainwhichstillgave poorresults.Itwashypothesizedthatthesynergistic inter-actionamongvariousmicroorganismsshouldacceleratethe rubberdegradationprocess.Sointhisstudy,thedegradation ofrubberlatexglovesinamineralsaltmedium(MSM)bya consortium,amixedcultureoftheselected bacteriaand a pureculturewerecompared.Theweightloss,scanning elec-tronmicroscopy(SEM)oftherubberglovesubstrateandviable cellcountsweredetermined.
Materials
and
methods
Isolationofrubberdegradingmicroorganismsfromsoil
samplesbyenrichmentculturetechnique
The rubber-degrading microorganisms were isolated from soilsamplescollected fromrubbercontaminatedgroundin Songkhlaprovince,Thailand.Soilsamplesweremixedwith pieces ofnatural rubber(NR) glove(0.5×0.5cm) incubated insterilemineralsaltmedium(MSM)at30◦C,150rpm. Min-eralsaltmediumiscomposedof(g/L):Na2HPO49.0,KH2PO4
1.5, (NH4)2NO3 1.0, MgSO4 0.2, CaCl2 0.02,and Fe (III) NH4
0.0012.ThemediumwasadjustedtoaninitialpHof7.0before sterilization.19 Afteramonthofincubation,0.5mLofthose culturebrothsweretransferredintoMSMwithlatexand fur-therincubatedunderthesameconditionsasdescribedabove foranothermonthtoencouragethegrowthofrubber degrad-ingbacteria.Thedevelopedculturebrothwasthenassigned asthenaturalsoilconsortium.Therubberdegradingbacteria werealsoscreenedandisolatedfromthenaturalsoil consor-tiumusingNRlatexagarplates.
ScreeningofNR-degradingbacteria
Thedegradationoftherubberglovesbytheselectedstrains was investigatedthrough plate assay. Latex overlay plates werepreparedbyspreading5mLofsolubilizedlatex concen-trateasathinfilmonMSMagarplatesandthenlefttodry. Alltestedstrainsthatgrewwellonthelatexoverlayagarwere pickedforfurtherstudy.
Rubberglovedegradationinvestigationoftheindividual
testedbacteria,themixedculture,andthenaturalsoil
consortium
Latexglovepieceswerewashedoncewithdistilledwaterprior todryinginanovenat60◦Cuntilobtainedaconstantweight beforebeingusedasacarbonsource.Threeexperimentswere designedtotestthemicrobialactivitiesfordegradingrubber gloves.Thefirstexperimentwastestingeachindividual iso-lated strain.Thesecondexperimentwastesting themixed culturesofhigheffectiveisolatedstrains(mixedcultures)and thethirdexperimenttestedthemixedculturesofallisolated strain(consortium).EachculturewasincubatedinMSM sup-plementedwithdriedlatexglovepieces(0.6%,w/v)asasole carbonsourceat30◦C,150rpmfor4weeks.Eachexperiment was performed in triplicate. The inoculated MSM without anylatexglovepiecesandtheuninoculatedMSMwithlatex glovepieceswereusedasbioticand abioticcontrol experi-ments,respectively.Viablecell countsofeach culturewere determined throughoutthe incubationtime.Theweightof latexgloveswere measuredbefore(W1)and after degrada-tion(W2).Weightlosswascalculatedusingequation:Weight loss=[(W1−W2)/W1]×100.
Scanningelectronmicroscope(SEM)
The surfaces of the NR glove film pieces were analyzed by scanning electron microscopy (SEM) to investigate the
appearancechangesafterthedegradation.Thesampleswere washed withsterilized distilled waterand then were fixed with2% glutaraldehydein 0.1M phosphate-bufferedsaline (PBS;pH7.3).Afterthat,thesampleswerewashedwithPBS anddehydratedingradedethanol(50%,60%,70%,80%,and 90% and absolute ethanol). The dehydrated samples were subjectedtocriticalpointdryingwithliquidCO2 according
tothestandardprocedure.Thesampleswerethenmounted onaluminumspecimenstubsbyusingelectrically conduc-tivecarbon (PLANO,Wetzlar, Germany) and coated with a goldlayer.Micrographswererecordedusingscanningelectron microscope(SEM-JSM5800LV;JEOLLtd.,Tokyo,Japan).
FourierTransformInfrared(FTIR)spectroscopy
TheFTIR (VERTEX70;Bruker,Germany) analysis wasdone todetectthe degradationofNRlatexgloveon thebasisof changesinthefunctionalgroups.Thecorrelationof absorp-tionbandsinthespectrumofanunknowncompoundwith theknownabsorptionfrequencieswasanalyzed.Thepolymer piecesweremixedwithKBrandmadeintoapellet,whichwas fixedtotheFTIRsampleplate.Spectraweretakenintriplicate at400–4000wavenumberscm−1foreachsample.
16SRNAgeneanalysis
Thegenomic DNAofisolate F5was extracted usingPower SoilDNAisolationkit(MOBIOLaboratories,aQIAGEN Com-pany,USA).Molecular geneticidentificationwasperformed byamplificationof16S rRNA genewith bacterialuniversal primers27F(5-AGAGTTTGATCCTGGCTCAG-3)and 1492R (5-CGGCTACCTTGTTACGACTT-3).20ThePCRreactionwascarried outinafinalvolumeof50Lcontaining10mMTris–HCl(pH 8.3),50mMKCl,1.5mMMgCl2,eachdNTPataconcentration
of0.2mM,1.25IUofTaqpolymerase,eachprimerata con-centrationof0.2mMand1LoftheDNAtemplate.PCRwas performedaccordingtothefollowingprogram:2min denatur-ationat95◦C,followedby30cyclesof1mindenaturationat 95◦C;1minannealingat55◦C;1minextensionat72◦C;and afinalextensionstepof10minat72◦C.ThePCRproductwas thenanalyzedandpurifiedusinganagaroseGelDNA Purifi-cation Kit (E.Z.N.A. Gel Extraction Kit; Omegabiotek, USA). The16SrDNAgenewassequenced.Aphylogenetictreewas constructedbymeansofMEGA,version7.0,usinga neighbor-joiningalgorithm,andtheJukes-Cantordistanceestimation methodwithbootstrapanalysesfor1000replicateswas per-formed.
Amplificationofthelcpgene
The coding region of lcp from Rhodococcus
pyri-dinivorans was amplified by using the primers
lcpF (5-ATAATCCGAGCAGGCGCGA-3) and lcpR
(5-TCGGGGATCTCGATGCTGA-3).PCRreactioncontained10mM Tris–HCl(pH 8.3),50mMKCl, 1.5mMMgCl2, each dNTP at
aconcentrationof0.2mM, 1.25IU ofTaqpolymerase, each primer ata concentrationof 0.2mM, and 1L ofthe DNA template. PCR was performed according to the following program:2mindenaturationat95◦C,followedby30cyclesof 1mindenaturationat95◦C;1minannealing at55◦C;1min
extension at72◦C;and a finalextension stepof 10minat 72◦C.
CO2emissiontest
Biodegradation oflatex glove was alsodetermined by CO2
emissiontest.ThereleasedCO2duringthecultivationofcells
inMSMinwhichcontainedrubberglovesasthesolecarbon sourcewastrappedinasolutionof1NBa(OH)2andwas
quan-tifiedbytitratingoftheremainingBa(OH)2with0.1NHCl.21
Results
Isolationofrubber-degradingmicroorganismsfromsoil
samples
Inordertoenrichthegrowthofrubberdegradingbacteria,soil samplewasincubatedwithrubberglovepiecesforamonth. Thedevelopedculturewhichwasassignedasanaturalsoil consortiumwasgrownonNRagartoisolaterubber degrad-ingbacteria.ConsortiumPPdemonstratedthebestdegrading ability causing the rubberweight lossup to 59%. Unfortu-nately,thedegradationabilityofconsortiumPPwasun-stable causingtherubberweightlossvariedfrom30%to59%. There-fore weattempt toconstructastablebacterial community byisolatingrubber-degradingbacteriafromthisconsortium, tenbacterialisolates,F1toF10,obtainedfromanaturalsoil consortiumwerecapableofgrowingonMSMagar contain-ing NR asthe sole carbonsource.Isolate F5was identified asGram-positiveandotherswereGram-negativebacteria.All tenisolatesdidnotproduceclearingzonearoundthecolonies indicatedthattheycoulddegradethelatexconcentratebya directcontactwiththerubbersubstrate.3Thedegradationof rubberglovesweretestedusingeachisolatedstraincompared withthemixedculturesoftop5effectivestrains(F1,F2,F5, F6,F7)andtheconsortiumofall10bacterialisolates(F1–F10).
Weightlossoflatexglovepieces
Theweightlossoflatexglovepiecesafterone-month incu-bation wasaround0.5–9%whenusingindividualcultureof F1–F10andamong10isolates,F5gavethebestweightlossof 9.36%(Fig.1).Thehighestweightlossof18.82%wasobtained fromthemixedcultureoftop5effectivestrains(F1,F2,F5,F6, F7),while18.38%wasobtainedfromtheconsortium(F1–F10) comparedtoonly0.2%oftheabioticcontrol(Fig.1).
Viablecellcounts
Theincreasedviablecellcountsthroughtheendofthe incu-bationtimeinalltestedbacterialgroupsindicatedthatthe bacteriahadtheabilitytodegradenaturalrubberandused rubberglovesasthesolecarbonsource(Fig.2).Inspiteofthe adhesivegrowthbehavior,anincreaseinthenumberofcells suspendedinthemediumduringcultivationoccurred.The bacterialpopulationofthemixedculturesshowedthebiggest increase from 1.2×108CFU/mL to 6.2×109CFU/mL after 1
monthincubationwithrubbergloves,the bacterial popula-tionintheconsortiumhadincreasedfrom1.2×108CFU/mL
25 20 15 10 5 0 % W e ight loss F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 Consor tium Mix ed culture
Abiotic controlBiot ic c
ontrol
Figure1–Percentageoflatexgloveweightlossafter incubationwithindividualisolates,theconsortium,and themixedcultureat30◦Cfor30dayscomparedtothe abioticcontrol. 1.00E+10 1.00E+09 1.00E+08 1.00E+06 1.00E+05 1.00E+04
Viable cell week 0 Viable cell week 4 1.00E+03 1.00E+02 1.00E+01 1.00E+00 Consor tium Mix ed culture Pure culture Abi otic cult ure Biotic c ultu re CFU/mL 1.00E+07
Figure2–Viablecellcountsafterincubationofthe
consortium,themixedcultureandthepurecultureF5with rubberglovesinmineralsaltmedium(MSM)at30◦Cfor30 days.
to4.0×109CFU/mLwhilethepurecultureshowedthe
small-est bacterial population increase from 1.2×108CFU/mL to
1.5×109CFU/mL.Theproductionofbiomassfromrubberalso
impliesthepresenceofoxidativedegradationofrubber poly-mer.
SEM
ThebiodegradationofrubbergloveswasalsoobservedbySEM. After4weeksofincubationwiththeconsortium,mixed cul-tureandF5,thesurfacesofrubbergloveswerecoveredwith bacterialcolonizationcomparedtocontrol(Fig.3a–d). Signif-icantchanges wereirregular crashes,surfaceerosions,and holesinvariable sizesonthe surfaceoflatexglovepieces. Interestingly,isolate F5 (Fig. 3d) cells embedded and colo-nizedintotherubbermatrix.Theadhesivegrowthattachment ofconsortiumandmixedcultureonrubbersurfacescanbe detectedasearlyas7daysofincubationwhilewhenusing singlestrainofF5,bacterialcolonizationoccurredonlyafter 10daysofincubation.
FourierTransformInfraredspectroscopytest
FTIRanalysisoftherubberglovepiecesafterincubatedwith F5showedvariousspectrachangescomparedtothecontrol (Fig.4).Inthetransmittanceareacorrespondingtothecis-1,4 doublebondsinthepolyisoprenechain,arelativedecrease ofthe( CH2)bandat833cm−1 wasobservedforthetested
samplecomparedtothatoftheabioticcontrol.TheFTIRpeak decreasedat1423cm−1inlatexglovepiecestreatedwith iso-lateF-5culture,indicatingthebreakoffunctionalgroupslike C C,methyl and esterbonds. Moreoveranincrease inthe intensity ofthepeak at1634cm−1 showedthe presenceof aldehydes (C O) groupsinthe case oftestpiecesas com-paredtocontrol.TheseindicatedthatF5usedlatexglovesas acarbonsourceduringcultivation.
Molecularidentificationusing16SrRNAgenesequence
comparison
Thesequencingresultof1.470kbnucleotidesof16SrRNAgene ofstrainF5 wasaligned andcomparedwithother bacteria availableintheNCBIGenBank.Thephylogeneticanalysisof the16SrRNAsequenceshowedthatthestrainF5belongsto genusRhodococcussp.having99%similaritieswithRhodococcus pyridinivoransandwassubmittedtoGenBankunderaccession numberKX791436(Fig.5).
Molecularidentificationandphylogeneticanalysisusing
lcpgenesequencecomparison
ThePCRproductoflcpgenewassequencedretrieving1.293kb nucleotides.Thesequenceswerethenalignedandcompared tothedatabaseoftheNCBIGenBank.Thephylogenetic anal-ysisofthelcpsequencesshowedthat99%similaritieswith anlcpgenefromRhodococcuspyridinivorans.Thepresenceof lcpgeneconfirmedthebiodegradationprocessinthisspecies. Furtherinvestigationisrequiredtoconfirmthereaction mech-anismofthisenzyme.
CO2emissiontest
TheCO2 evolvedfrom thebiodegradationwasmeasuredto
comparetheabilitytodegraderubberglovesoftested bacte-ria.Themixedculturereleased14.67%CO2,9.78%fromthe
consortiumand4.89%fromthepurecultureF5(Fig.6).The highestCO2releasewasrelatedtothehighestweightlossof
latexgloveexperimentandconfirmedthebest degradation occurredwhenthemixedculturewasincubatedwithrubber gloves.
Discussions
Thepresenceofrubberwastescurrentlyresultsin environ-mentalpollution.Theabilityofmicroorganisms todegrade rubberwasteshasreceivedconsiderableattention.Recently differentkindsofenvironmentalpollutantswerereportedto beeffectivelydegradedbymicrobialconsortia.23–27The syn-ergetic interaction of microorganisms in this study points out analternative approachtoperformrubberdegradation.
Figure3–Scanningelectronmicrographsofthelatexglovepiecesafterincubatedwithconsortium(b),mixedculture(c), andpurecultureF5(d),for30daysat30◦Ccomparedtotheabioticcontrol(a).
40 50 60 70 80 90 100 0 1000 2000 3000 4000 Tr a n s m itt an c e ( % ) Wave number (cm-1) abiotic control F5 833 1423 1634
Figure4–FTIRspectraoflatexglovepiecesafterincubation withF5inmineralsaltmediumfor1month.Abioticcontrol (thickblackline),sampletreatedwithF5(dotline).
Itwasclearlyshownthatusingmixedcultureandthe con-sortiumresultedinbetterdegradation.Theobviouschanges inrubberappearanceswereobservedandthecolorofrubber piecesbecameorange(Fig.7).However,thedecreaseofrubber degradingpropertyoftheconsortiumaftersuccessive trans-ferinNRmediumwasobserved.Theinstabilityofdegrading abilitymight duetothelossofanybacterialpopulation in theconsortiumduringtransfer.Therubberdegrading prop-ertycouldbere-storedagainafterare-cultivationwithalong incubationtimewhichallowsmicroorganismstoadapt.The efficiencyofconsortiumdegradedrubberglovesinbroth
cul-turewasshownbyweightlossandthedeteriorationofrubber pieces;itmightbeduetothebreakdownoflongesterbonds intolowmolecularweightfragmentsbyoneorganism mak-ingaccesseasierforsomeenzymesproducedbytheother.The rateofrubberdegradationwashighincaseoftreatmentwith microbialconsortiumascomparedtotheindividualstrains. Ourresultsareinconformitywiththosepreviouslyreported andshowthatthesignificantchangesinsurfacemorphology occurduetothemicrobialdegradabilityoftreatedpolymers. Adirectcontactviaabiofilmandtheembeddingofcellsinto rubberglovesmaybeapossiblemechanism, asareportof Gordoniaandsomeotheractinomycetessuggests.19
The FTIR-ATR spectroscopy was applied to detect the changes inthefunctional groupsofthe polymer. TheFTIR spectra of the NR latex gloves recorded in the transmis-sionandATRmodecorrespondedtothecis-1,4doublebonds in the polyisoprene chains in the region of 700–900cm−1. Thedecreased spectraof cis-1,4 doublebonds,methyl and esterbondsinthepolyisoprenechains,andtheappearance of ketone and aldehyde groups from FTIR analysis indi-catedthatanoxidativeattackatthedoublebondsmightbe the firstmetabolicstepsofthe biodegradationprocess.28–30 Aftermicrobialtreatment,FTIRanalysisoftherubbergloves indicatedsomereductionofthemolecularsizesandthe for-mationofnewpeakswiththebreakdownofsomebonds.The increasedspectraofC Ostretchingat1634cm−1,indicated that someofthe unsaturatedbonds(C C) inthe NRwere possiblyconvertedtoC O.
99
Rhodococcus pyridinivorans (NR 121768.1)
Rhodococcus pyridinivorans strain PDB9 (NR 025033.1)
Strain F5 (KX791436)
Gordonia alkanivorans strain 3-8 (KJ571037.1)
Nocardia brasiliensis ATCC 700358 strain HUJEG-1 (NR 074743.1)
Xanthomonas campestris strain ATCC 33913 (NR 074936.1)
100
77
0.020
Figure5–PhylogenetictreeofstrainF5amongtheneighboringspecies.Thephylogenetictreewasconstructedbythe methodofneighborjoining,basedonpairwisecomparisonsof16SrRNAsequenceswithbootstrapanalysesfor1000 replicates. 0 2 4 6 8 10 12 14 16 18
consortium mixed culture pure culture abiotic control
%C
O2
Figure6–PercentageofCO2evolvedduringtheincubation oftheconsortium,themixedcultureandthepureculture F5withrubberglovesat30◦Cfor30dayscomparedtothe abioticcontrol.
Theprimarydegradationreactionofcis-1,4-isopreneisan initial exposure the rubber to bacterial enzymes that can oxidizesomedoublebondsandbreakthelongchain hydrocar-bonlinkagestoproducesmallermoredegradablemolecules. These enzymes are either excreted by clear zone forming rubber degrading bacteria or attached to the bacterial cell wallsinthegroupofbacteria requiredirectgrowth onthe rubber surface.31,32 Tsuchii and his team determined the
degradationproductsofvulcanizedandnon-vulcanized rub-ber using Nocardia sp. 835A.22 The authors concluded that naturalrubberisdegradedbyanoxygenasereaction,andmay alsoinvolvethescissionofisopreneattheunsaturated dou-blebond,leadingtotheformationofaldehydesorketonesat eachendofthesplitisoprenechains.
Tsuchii andTakeda alsodescribed the rubberdegrading activityintheculturemediumusingXanthomonassp.strain 35Ybyattemptingtocleaverubberwithcrudeenzyme,and pointedoutthatthecleavagereactionwasenzyme-catalyzed process.28 Thisevidencerevealed thattheenzymereaction couldbeconsideredtobeatwo-stepreaction.Inthefirststep, thehighmolecularweightrubberpolymerwasdegradedand converted to mediummolecular weight(MW) hydrocarbon intermediates. Theextracellular crudeenzyme from strain 35Yhad ahigh substratespecificityforcis-1,4-polyisoprene compounds and attacked high MW polymer chains rather than lower MW oligomers, 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al was identified as one of the first degradation products.Moreover,Linos andhisteamconfirmedTsuchii’s theories on the rubber cleavage mechanism by a Gordonia strainandfoundthatnotonlyaldehydeandketonegroups weredetectedbutalsothedecreaseinthenumberof cis-1,4-isoprenebondduringtheprocess,inaddition,twodifferent newfunctionalgroupsweredetectedafterbeingtreatedwith bacteria.31Theseresultsindicatedthatanoxidativeenzyme
Figure7–Thelatexglovepiecesafterincubatedwithmixedculture(b),andpurecultureF5(c),for30daysat30◦C comparedtotheabioticcontrol(a).
attackedatthedoublebondinthefirstmetabolicstepofthe biodegradationprocess.31,32Accordingtoalltheevidencesin thebiochemicaldegradationreactionsofpolyisoprenerubber totheidentificationofmetabolites,Bodeand histeam has establishedthe“hypotheticalpathwayofrubberdegradation” presentingthefirststepastheoxidativecleavageatthe iso-prenedoublebonds, withthe formationofcis-1,4-isoprene oligomerssuchas12-oxo-4,8-dimethyltrideca-4,8-diene-1-al and the presence ofketo and aldehyde groups atthe end ofthe splitchains.7These lowmolecularweightoligomers areconsequentlyutilizedbybacteriaandthealdehydegroup is oxidizedto the corresponding carboxylic acid. The acid isactivated to acoenzyme A (CoA)ester and then passes thoughthe-oxidationcycle.7,33 However,further investiga-tionandevidencesonthebiochemicalanalysis,metaboliteor endproductsofrubberarerequired.
Apart from the FTIR and weight loss analysis, carbon dioxide evolution has commonly been used to evaluate biodegradabilityofrubber;microbialdegradationofpolymeric materialcanbeevaluatedbyentrappingCO2evolutionasa
resultofmineralization.Inthepresentstudy,thequantityof CO2releasedinthetestflaskscontainingrubberglovesasthe
onlysourceofcarbonwassignificantlyhigherthanincontrol flaskswithoutrubbergloves,providingtheevidenceof miner-alizationofrubberpolymer.Inmostofthepublishedstudies conducting rubber degradation, Actinomycetes are reported to be the main group of rubber degrading microbes. The latex clearing protein (Lcp) encoded by lcp gene was first reportedinStreptomycessp.K30andalsolaterfoundinother gram-positiverubberdegraderincludingNocardiafarcinicaE1, NocardianovaSH22a,Nocardiasp.NVL3Actinoplanessp.OR16 andGordoniawestfalicaKb1.17,29,30,34–36Toourknowledge,we arethefirstgrouptousebacterialconsortiumtodegrade rub-berandalsothefirststudytodemonstrateanabilitytodegrade rubberby Rhodococcuspyridinivorans. Genus Rhodococcus has beenprovedtobeapromisingoptionforthebioremediation ofmanyxenobioticcompounds.37–40Watcharakuletal.firstly reportedtheabilityofR.rhodochrousinrubberdegradationand alsorevealedsomedifferentpropertiesofLcpproteinofR. rhodochrousRPK1thathas notbeen described previouslyin otherrubber-degradingbacteria.12Thedetectionoflcpgene inR.pyridinivoransF5inthisstudyconfirmedtheabilityofthis strainindegradingrubbergloves.SinceR.pyridinivoransF5did notproduceaclearingzonearoundthecolonyonNR over-layplate,thisresultsupportsthehypothesisofthestudyof Linhetal.thattheLcpitself isnotresponsibleforclearing zoneformation.31Itwillbenecessarytofurtherinvestigate thebiochemicalandbiophysicalpropertiesofthisenzymeto fullyunderstandthevariationoftheenzymepropertiesand toverifytheroleofLcpinrubberdegradation.
Conclusions
Inthepresentstudytheconsortium,mixedcultureandpure culturewereabletodegraderubberglovesinwhichsignificant changescanbedetectedwithin30days.Thedeterminationof theweightloss,theincreaseinviablecellcounts,FTIR,CO2
evolutiontestand the SEMhasconfirmed thatthe natural rubberdegradationprocessoccurred.Thestructureanalysis
from FTIR confirmed that the structureof the rubber was changedaftertreatmentwiththebacteriaandledtothe dete-riorationofthepolymer.RhodococcuspyridinivoransstrainF5 demonstratedthehighestrubberdegradingactivityamongall singlestrainsused.Thepresenceoflcpgeneencodeforlatex degradingenzymeinRhodococcuspyridinivoransF5supportsthe essentialfunctionoflcpinpoly(cis-1,4isoprene)degradation. Mixedcultureoftheselectedstrainsreachedthehighest rub-berdegradationafterone-monthincubationandshowedthe potentialtobeusedefficientlyforrubberdegradation.Further studiesarenecessarytoelucidaterolesofeachstraininthe mixedcultureandconsortium.Theresultsobtainedonbatch biodegradation ofrubberglovesusinga consortiumwillbe usefulinapplicationofcontinuousremovalofrubberwastes. Thishasanadvantagewhentheprocessisappliedinthe con-trolledenvironmentconditions,wherepureculturemightnot beabletoutilizethesecondarymetabolites.
Financial
support
ThisprojectwasfundedbyResearchAssistantscholarships, FacultyofScience,PrinceofSongklaUniversitytheFacultyof Scienceand alsotheResearchgrantfromPrinceofSongkla University.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
Weare really gratefultoDr. BrianHodgson forhis uncon-ditional support and great advices.We are thankful toDr. NicholasHodgsonandMrs.AnnaChatthongforEnglish lan-guagecheckingandediting.
r
e
f
e
r
e
n
c
e
s
1.IsmailAM,MohamedHN,ShoreitMA.DegradationofFicus
elasticarubberlatexbyAspergillusterreus,Aspergillusflavus,
andMyceliophthorathermophile.IntBiodeteriorBiodegradation.
2013;78:82–88.
2.NayanashreeG,ThippeswamyB.Naturalrubberdegradation
bylaccaseandmanganeseperoxidaseenzymesofPenicillium
chrysogenum.IntJEnvironSciTechnol.2015;12:2665–2672.
3.YikmisM,SteinbüchelA.Historicalandrecentachievements
inthefieldofmicrobialdegradationofnaturalandsynthetic
rubber.ApplEnvironMicrobiol.2012;78:4543–4551.
4.InternationalRubberStudyGroup.Statisticalsummaryof
worldrubbersituation.RubberStatisticalBulletin;2016.
5.FattaD,PapadopoulosA,LoizidouM.Astudyonthelandfill
leachateanditsimpactonthegroundwaterqualityofthe
greaterarea.EnvironGeochemHealth.1999;21:175–190.
6.BodeHB,ZeeckA,PlückhahnK,JendrossekD.Physiological
andchemicalinvestigationsintomicrobialdegradationof
syntheticpoly(cis-1,4-isoprene).ApplEnvironMicrobiol.
2000;66:3680–3685.
7.BodeHB,KerkhoffK,JendrossekD.Bacterialdegradationof
naturalandsyntheticrubber.Biomacromolecules.
8. CherianE,JayachandranK.Microbialdegradationofnatural
rubberlatexbyanovelspeciesofBacillussp.SBS25isolated
fromsoil.IntJEnvironRes.2009;3:599–604.
9. HeshamAEL,WangZ,ZhangY,ZhangJ,LvW,YangM.
Isolationandidentificationofayeaststraincapableof
degradingfourandfiveringaromatichydrocarbons.Ann
Microbiol.2006;56:109–112.
10.ChengalroyenM,DabbsE.Thebiodegradationoflatex
rubber:aminireview.JPolymEnviron.2013;21:874–880.
11.GallertC.Degradationoflatexandofnaturalrubberby
StreptomycesstrainLa7.SystApplMicrobiol.2000;23:433–441.
12.WatcharakulS,RötherW,BirkeJ,UmsakulK,HodgsonB,
JendrossekD.Biochemicalandspectroscopic
characterizationofpurifiedLatexClearingProtein(Lcp)from
newlyisolatedrubberdegradingRhodococcusrhodochrous
strainRPK1revealsnovelpropertiesofLcp.BMCMicrobiol.
2016;16:92.
13.LinosA,SteinbüchelA.Microbialdegradationofnaturaland
syntheticrubbersbynovelbacteriabelongingtothegenus
Gordona.KautGummiKunstst.1998;51:498–499.
14.LinosA,BerekaaMM,SteinbüchelA,KimKK,SpröerC,
KroppenstedtRM.Gordoniawestfalicasp.nov.,anovel
rubber-degradingactinomycete.IntJSystEvolMicrobiol.
2002;52:1133–1139.
15.BerekaaMM.Colonizationandmicrobialdegradationof
polyisoprenerubberbynocardioformactinomyceteNocardia
sp.strain-MBR.Biotechnology.2006;5:234–239.
16.BraazR,FischerP,JendrossekD.Noveltypeof
heme-dependentoxygenasecatalyzesoxidativecleavageof
rubber(poly-cis-1,4-isoprene).ApplEnvironMicrobiol.
2004;70:7388–7395.
17.IbrahimEMA,ArenskotterM,LuftmannH,SteinbüchelA.
Identificationofpoly(cis-1,4-isoprene)degradation
intermediatesduringgrowthofmoderatelythermophilic
actinomycetesonrubberandcloningofafunctionallcp
homologuefromNocardiafarcinicastrainE1.ApplEnviron
Microbiol.2006;72:3375–3382.
18.RoseK,SteinbüchelA.Biodegradationofnaturalrubberand
relatedcompounds:recentinsightsintoahardlyunderstood
cataboliccapabilityofmicroorganisms.ApplEnviron
Microbiol.2005;71:2803–2812.
19.JendrossekD,TomasiG,KroppenstedtRM.Bacterial
degradationofnaturalrubber:aprivilegeofactinomycetes?
FemsMicrobiolLett.1997;150:179–188.
20.WeisburgG,BarnsM,PelletierA,LaneJ.16SribosomalDNA
amplificationforphylogeneticstudy.JBacteriol.
1991;173:697–703.
21.ShahA,HasanF,MutiullahZS,HameedA.Degradationof
polyisoprenerubberbynewlyisolatedBacillussp.AF-666
fromthesoil.ApplBiochemMicrobiol.2012;48:37–42.
22.TsuchiiA,SuzukiT,TakedaK.Microbialdegradationof
naturalrubbervulcanisates.ApplEnvironMicrobiol.
1985;50:965–970.
23.KimNK,OhS,LiuWT.Enrichmentandcharacterizationof
microbialconsortiadegradingsolublemicrobialproducts
dischargedfromanaerobicmethanogenicbioreactors.Water
Res.2016;90:395–404.
24.ShahZ,GulzarM,HasanF,ShahAA.Degradationof
polyesterpolyurethanebyanindigenouslydeveloped
consortiumofPseudomonasandBacillusspeciesisolatedfrom
soil.PolymDegradStab.2016;134:349–356.
25.RajamanickamR,KaliyamoorthiK,RamachandranN,
BaskaranD,KrishnanJ.Batchbiodegradationoftolueneby
mixedmicrobialconsortiaanditskinetics.IntBiodeterior
Biodegradation.2017;119:282–288.
26.ZafraG,AbsalónÁE,Anducho-ReyesMÁ,FernandezFJ,
Cortés-EspinosaDV.ConstructionofPAH-degradingmixed
microbialconsortiabyinducedselectioninsoil.Chemosphere.
2017;172:120–126.
27.InesM,SahnounR,GhribiD.Applicationofbacterial biosurfactantsforenhancedremovalandbiodegradationof dieseloilinsoilusinganewlyisolatedconsortium.Process SafEnvironProt.2017,
http://dx.doi.org/10.1016/j.psep.2017.02.002.
28.TsuchiiA,TakedaK.Rubberdegradingenzymefrom
bacterialculture.ApplEnvironMicrobiol.1990;56:269–274.
29.RoseK,TenbergeKB,SteinbüchelA.Identificationand
characterizationofgenesfromStreptomycessp.strainK30
responsibleforclearzoneformationonnaturalrubberlatex
andpoly(cis-1,4-isoprene)rubberdegradation.
Biomacromolecules.2005;6:180–188.
30.LuoQ,HiesslS,PoehleinA,DanielR,SteinbüchelA.Insights
intothemicrobialdegradationandgutta-perchabyanalysis
ofthecompletegenomeofNocardianovaSH22a.ApplEnviron
Microbiol.2014;80:3895–3907.
31.LinosA,ReicheltR,KelleRU,SteinbüchelA.Gram-negative
bacterium,identifiedasPseudomonasaeruginosaAL98,isa
potentdegraderofnaturalrubberandsynthetic
cis-1,4-polyisoprene.FemsMicrobiolLett.2000;182:155–161.
32.LinosA,SteinbüchelA.Biodegradationofnaturalandsynthetic
rubber.Weinheim,Germany:BiopolymersWiley-VCH; 2001:321–359.
33.BodeHB,ZeeckA,CkhahnKP,JendrossekD.Physiological
andchemicalinvestigationsintomicrobialdegradationof
syntheticpoly(cis-1,4isoprene).ApplEnvironMicrobiol.
2000;66:3680–3685.
34.LinhDV,HuongNL,TabataM,etal.Characterizationand
functionalexpressionofarubberdegradationgeneofa
Nocardiadegraderfromarubber-processingfactory.JBiosci Bioeng.2017;123:412–418.
35.BrökerD,DietzD,ArenskötterM,SteinbüchelA.The
genomesofthenon-clearing-zone-formingand
natural-rubber-degradingspeciesGordoniapolyisoprenivorans
andGordoniawestfalicaharborgenesexpressingLcpactivity inStreptomycesstrains.ApplEnvironMicrobiol.
2008;74:2288–2297.
36.ImaiS,IchikawaK,MuramatsuY,KasaiD,MasaiE,Fukuda
M.IsolationandcharacterizationofStreptomyces,
Actinoplanes,andMethylibiumstrainsthatareinvolvedin
degradationofnaturalrubberandsynthetic
poly(cis-1,4-isoprene).EnzymeMicroTechnol.2011;49:
526–531.
37.LarkinMJ,KulakovLA,AllenCCR.Biodegradationby
membersofthegenusRhodococcus:biochemistry,physiology,
andgeneticadaptation.AdvApplMicrobiol.2006;59:1–29.
38.PrettZ,DésiE,LepossaA,KrisztB,KukolyaJ,NagyE.
BiologicaldegradationofaflatoxinB1byaRhodococcus
pyridinivoransstraininby-productofbioethanol.AnimFeed SciTechnol.2017;224:104–114.
39.MartínkováL,UhnákováB,PátekM,NeˇsveraJ,KˇrenV.
BiodegradationpotentialofthegenusRhodococcus.Environ
Int.2009;35:162–177.
40.KunduD,HazraC,ChaudhariA.Enhanceddegradationof
4-nitrotoluenebyRhodococcuspyridinivoransNT2:multivariate
optimizationofprocessparametersandevaluationof
