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Evaluation of the biotechnological potential of Rhizobium tropici strains for exopolysaccharide production

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ContentslistsavailableatScienceDirect

Carbohydrate

Polymers

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Evaluation

of

the

biotechnological

potential

of

Rhizobium

tropici

strains

for

exopolysaccharide

production

Tereza

Cristina

Luque

Castellane

a

,

Manoel

Victor

Franco

Lemos

b

,

Eliana

Gertrudes

de

Macedo

Lemos

a,∗

aDepartamentodeTecnologia,UNESP—UnivEstadualPaulista,FaculdadedeCiênciasAgráriaseVeterinárias,Rod.Prof.PauloDonatoCastellanekm5,

CEP14884-900Jaboticabal,SP,Brazil

bDepartamentodeBiologiaAplicadaàAgropecuária,UNESP—UnivEstadualPaulista,FaculdadedeCiênciasAgráriaseVeterinárias,Rod.Prof.Paulo

DonatoCastellanekm5,CEP14884-900Jaboticabal,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22November2013 Receivedinrevisedform16April2014 Accepted20April2014

Availableonline26April2014

Keywords:

Extracellularpolysaccharide Biopolymer

Rhizobiumtropici

Pseudoplastic

a

b

s

t

r

a

c

t

Rhizobiumtropici,amemberoftheRhizobiaceaefamily,hastheabilitytosynthesizeandsecrete extracel-lularpolysaccharides(EPS).RhizobialEPShaveattractedmuchattentionfromthescientificandindustrial communities.RhizobialisolatesandR.tropicimutantsthatproducedhigherlevelsofEPSthanthe wild-typestrainSEMIA4080wereusedinthepresentstudy.Theresultssuggestedaheteropolymerstructure fortheseEPScomposedbyglucoseandgalactoseasprevailingmonomerunit.AllEPSsamplesexhibited atypicalnon-Newtonianandpseudoplasticfluidflow,andtheaqueoussolutionsapparentviscosities increasedinaconcentration-dependentmanner.Theseresultsserveasafoundationforfurther stud-iesaimedatenhancinginterestintheapplicationoftheMUTZC3,JAB1andJAB6strainswithhigh EPSproductionandviscositycanbeexploitedforthelarge-scalecommercialproductionofRhizobial polysaccharides.

©2014TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction

Manyspeciesofbacteriapossesstheabilitytosynthesizeand excrete extracellular polysaccharides (exopolysaccharides, EPS). Oncetransportedtotheextracellularspace,EPSexistaseither sol-ubleorinsolublepolymersandareeitherlooselyattachedtothe cellsurfaceorcompletelyexcretedintotheenvironmentasslime. IthasbeenshownthatbacterialEPSprovideprotectionfrom vari-ousenvironmentalstresses,suchasdesiccation,predation,andthe effectsof antibiotics(Donot,Fontana,Baccou, &Schorr-Galindo, 2012).However,theinterestinEPShasincreasedconsiderablyin recentyearsbecausethesecompoundsarecandidatesformany

Abbreviations:EPS,exopolysaccharide;CDW,celldryweight;RP-HPLC, reverse-phase high-performance liquid chromatography; UV–vis, ultraviolet–visible; Glc, glucose; Gal, galactose; GalA, galacturonic acid; GlcA, glucuronic acid; Man,mannose;Rha,rhamnose;PMP,1-phenyl-3-methyl-5-pyrazolone;EPSWT, exopolysaccharidefromRhizobiumtropiciSEMIA4080;EPSC3,exopolysaccharide fromtheMUTZC3mutantstrain;EPSPA7,exopolysaccharidefromtheMUTPA7 mutantstrain;EPSJ1,exopolysaccharidefromtherhizobialisolateJAB1;EPSJ6, exopolysaccharidefromtherhizobialisolateJAB6.

∗Correspondingauthor.Tel.:+551632092675x217;fax:+551632092675.

E-mailaddresses:teluque@yahoo.com.br(T.C.L.Castellane),

mvictor@fcav.unesp.br(M.V.F.Lemos),egerle@fcav.unesp.br(E.G.d.M.Lemos).

commercialapplicationsinthehealth,bionanotechnology,food, cosmetics,andenvironmentalsectors.

Severalresearchershavediscussedrecentadvancementsinthe understanding of the potential industrial applicability of these bacteriafortheproductionofgumsandtheimportanceofthese compoundsinsoilaggregation.Inaddition,thefunctional prop-ertiesofbacterialexopolysaccharideshavebeendemonstratedin a wide range ofapplications,including foodproducts, pharma-ceuticals,bioemulsifiers(Xie,Hao,Mohamad,Liang,&Wei,2013), bioflocculants (Sathiyanarayanan, Kiran, &Selvin, 2013), chem-ical products (Wang, Ahmed, Feng, Li, & Song, 2008; Shah,

Hasan,Hameed,&Ahmed,2008),thebiosorptionofheavymetals

(Mohamadetal.,2012),andantibiofilmagents(Rendueles,Kaplan,

&Ghigo,2013)inbothindustryandmedicine(Nwodo,Green,&

Okoh,2012;Donotetal.,2012).Intheagriculturesector,the

flu-idityoffungicides,herbicides,andinsecticideshasbeenimproved bytheadditionofxanthan,whichresultsintheuniform suspen-sionofsolidcomponentsinformulations(DeAngelis,2012).Thus, studiesinthisareaareveryimportantfortheidentificationofboth novelbiopolysaccharidesandnewtechniquesforoptimizingtheir production(Bomfetietal.,2011).

Numerous types of exopolysaccharides have already been described(Castellane&Lemos,2007;Monteiroetal.,2012;Mota

etal., 2013;Radchenkovaetal., 2013;Silvi,Barghini, Aquilanti,

http://dx.doi.org/10.1016/j.carbpol.2014.04.066

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Juarez-Jimenez,&Fenice,2013).Theextracellularpolysaccharide, succinoglycan,is producedby Sinorhizobium,Agrobacterium and othersoilbacteria(Simsek,Mert,Campanella,&Reuhs,2009). How-ever,consideringthebiodiversityofthemicrobialworldandthe numberofpaperspublishedeachyeardescribingnewmicrobial exopolysaccharides,itisastonishingtorealizethatonlythreeEPS (i.e.,dextran, xanthan,andgellangums)have beensuccessfully adoptedforindustrialpurposes(Donotetal.,2012;Prajapati,Jani,

Zala,&Khutliwala,2013).

One of the well-known EPS producers are rhizobia, which excrete large amounts of polysaccharides into the rhizosphere and,whengrowninpurecultures(Noel,2009),producecopious amountsofEPS,causingincreasedviscosity.Todate,the synthe-sisofrhizobialEPShasbeenbeststudiedintwospecies,namely Sinorhizobium meliloti and Rhizobium leguminosarum (Marczak,

Dzwierzynska,&Skorupska,2013).Thelatestdataindicatethat

EPSsynthesisinrhizobiaundergoesverycomplexhierarchical reg-ulation,which includestheparticipationofproteinsengagedin quorumsensing and theregulation of motility genes. Previous reportshaveshownthatbiofilmformationandexopolysaccharide productionbybacterialstrainssignificantlycontributetosoil fer-tilityandimproveplantgrowth(Qurashi&Sabri,2012).However, atpresent,theroleofEPSintheseprocessesisnotwell under-stood.Therefore,thereisgreatinterestinelucidatingthepatternsof geneexpressionandthebiochemicalprocessesinvolvedinthe pro-ductionofbacterialextracellularpolysaccharides(Marczaketal.,

2013).

Basedonitssuperiorcharacteristicsasacommonbean( Phaseo-lusvulgarisL.)root-nodulesymbiont,strainPRF81,whichisknown commerciallyasSEMIA4080,isthetype-strainofRhizobium trop-icithat currentlyrecommended(authorized)for theproduction ofcommercialrhizobialinoculant forcommonbeanproduction inBrazil(Hungriaetal.,2000).Becauseofitsabilitytoproduce largequantitiesofpolysaccharides,thisbacteriummayprovetobe anexcellentmodelspeciesforthedevelopmentofbiotechnology products.Industrialbiopolymerproductionwouldoccurunderthe sameconditionsusedfortheindustrialcultivationofrhizobiafor soilinoculationinBrazil.Thus,theproductionofEPSmayrepresent analternativeforthissectorbecausethemarketforinoculum pro-ductioninBrazilishighlydependentontheproductionofsoybean [Glycinemax(L.)Merr.]andcommonbean.InBrazil,theinocula isonlysoldbetweenAugustandDecember,whichistheplanting periodfortheentirecountry.

To date, there is little information on the physicochemical propertiesandrheologicalpropertiesofpurifiedEPSfromR. trop-icistrainsorontheiruseinvariousindustrialapplications.The R. tropici strain SEMIA 4080 combines the advantages of non-pathogenicityand rapidproductivityand hence provedtobe a verypromisingmodelorganismandcellfactoryformicrobialEPS production.However,thisstudyisusefulforthebio-inoculant pro-ducingindustriesinBrazilasbestalternativeactivityduringthe noncropseasonofthecommonbeanandsoybean.Thisstudy inves-tigatedtherheologicalpropertiesoftheEPSfromwild-typestrain ofR.tropiciSEMIA4080,mutantstrains(MUTZC3andMUTPA7) andrhizobialisolates(JAB1andJAB6)todiscoveritspotentialasa soil-stabilizingagentorasarheologicalmodifierofaqueous sys-tems.

2. Materialsandmethods

2.1. Bacterialstrainsandgrowthconditions

Thewild-typestrainofR.tropiciSEMIA4080,mutantstrains (MUTZC3andMUTPA7)andrhizobialisolates(JAB1andJAB6)were usedinthepresentstudy.ARhizobialstraindesignatedJAB1was

isolatedfromthecommonbean(PhaseolusvulgarisL.)and clas-sifiedas R. tropici, whilethe rhizobialisolateddesignatedJAB6 wasisolatedfrompintopeanut(Arachispintoi)and classifiedas Rhizobiumsp.ForroutineRhizobiumgrowth,tryptoneyeast(TY) medium (Beringer, 1974)wasused. Whenrequired, the media wassupplemented withtheantibiotickanamycin.Themutants werecultivatedinYMAmedium(0.4gL−1 yeastextract,10gL−1 mannitol,0.5gL−1 K2HPO4,0.2gL−1 MgSO4,and 0.1gL−1 NaCl, 9gL−1agar,pH7.0)(Vincent,1970)supplementedwithCongoRed (25␮gmL−1)toverifythepurityofeachmutantculture.The cul-tureswereincubatedat30◦Cfor24h.

ForcomparativeanalysesoftheEPSproductionobtainedwith thewild-type,mutantstrainsofR.tropiciandrhizobialisolates, themonosaccharidecompositions,andtherheologicalproperties oftheirEPS,pre-inoculaandbatchexperimentswereperformed usingPGYA,PGYL,PSYA,andPSYLmedia.Thedetailedcontentsof thesecultivationmediaarenotavailablebecausetheformulasare underpatentrestriction(registrationPI0304053-4).

2.2. EPSdetectionandproduction

For phenotypic comparisons, experiments were conducted usingPetridishescontainingsolidPSYAmedium,sucrose(30gL−1), asacarbonsource,withandwithoutafluorescentbrightener28 (calcofluorwhite;Sigma-Aldrich)withanemissionwavelengthof 430nmatafinalconcentrationof200␮gmL−1.Thisfluorescent pigmentisspecifictopolysaccharidesthatcontain␤-1→4or␤ -1→3linkages(Wood, 1980).TheEPSproductionbyeachstrain wasobservedbyilluminatingthedisheswithUVlightat365nm. Themucoidyofthecolonieswasdeterminedvisually.

For the evaluation of EPS production, pre-inocula were ini-tiallyprepared fromcultures cultivatedonsolid PGYAmedium containing glycerol (10gL−1), as a carbon source. After 24h, eachinoculatingstrainwascultivatedin125-mLflasks(20mLof mediumineach)containingPGYLliquidmediumonarotaryshaker at140rpmfor30h,atwhichtimeasuspensionwithanoptical den-sityat600nm(OD600)of0.3wasobtained.Thetemperaturewas maintainedat30◦C.Aliquotsofthecorrespondingcultureswere transferredto 1000-mLErlenmeyerflasks containing500mLof half-liquidPSYLatafinalconcentrationof0.10%(v/v)andincubated for144hat140rpmand29◦C.

2.3. Cellbiomassdetermination

Thegrowthwasmeasuredbasedonthedry weightper vol-umeof theculture.Thecelldryweight(CDW)wasdetermined bycentrifugation(10,000×g,4◦C,50min)followedbydryingtoa constantweightinanovenat60◦Covernight.

2.4. EPSextraction

Cold96%ethanolwasaddedtothesupernatantobtainedfrom thecentrifugationata1:3(v/v)ethanol:supernatantratioto pre-cipitate theEPS(Breedveld, Zevenhuizen, &Zehnder,1990).At thisstage,itwaspossibletoimmediatelyobservetheformation of a precipitate. The mixture was refrigerated at 4◦C for 24h. Aftertherefrigerationperiod,thesampleswerecentrifugedonce again(10,000×g,4◦C,30min)toseparatetheprecipitatefromthe solvent.Theprecipitatewaswashedseveraltimeswithethanol, and theethanol wasevaporated. The solventprecipitationalso achievedapartialpurificationofthepolymerbyeliminatingthe soluble components of theculture media (Castellane &Lemos,

2007;Aranda-Selverioetal.,2010).

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of EPS per liter of culture medium); the results are presented asthemeans±standarderror.Thesampleswerethenprepared forreverse-phasehigh-performanceliquidchromatography (RP-HPLC)andrheologyanalyses.

2.5. DeterminationofEPSmonosaccharidecompositionsusing RP-HPLC

ToassessthemonosaccharidecompositionoftheEPSproduced bythebacterialstrains,eachrawEPSpreparation wasanalyzed byRP-HPLCusingthe1-phenyl-3-methyl-5-pyrazolonemonomer chemicalidentificationmethodologydescribedbyFuandO’Neill

(1995)withmodifications.

TheEPSsamples(1.0mg)werehydrolyzedwith2molL−1 tri-fluoroaceticacid(200␮L)inasealedglasstube(13×100mm)with screwcapwhichfilledwithpurenitrogengasat121◦Cfor2h.The hydrolyzedsolutionwasevaporatedtodrynessunder45◦Cand then2-propanol(500␮L)wasaddedforfurtherevaporationand completeremovaloftrifluoroaceticacid.Thehydrolysatewasused forderivatization.

Afterhydrolysis,theEPSandmonosaccharidestandardswere pre-derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), a chemicalmarker.Thereactionswereconductedbyadding40␮L ofPMPsolution(0.5molL−1 in methanol)and40L ofsodium hydroxide solution (0.3molL−1) to each tube. The tubes were agitatedandincubatedat120◦Cfor2h.Themixturewasthen neu-tralizedbyadding40␮Lofhydrochloricacidsolution(0.3molL−1) at to roomtemperature. For the extractionof monosaccharide derivatives,0.5mLofethyl tert-butyl etherwasadded, andthe tubeswereagitatedfor5seconds.Thelayerswereseparatedby centrifugation(5000×g,4◦C,5min),andtheupperphase(organic layer)wasthenremovedanddiscarded.Thisextractionprocesswas repeatedfivetimes.Theresiduewasdissolvedinwater(1.0mL). Themixturewasfilteredthrough0.45mmMilliporefilter (Waters-MilliporeBedford,MA,USA).

The PMP-labeled monosaccharides were analyzed using the conditions described by Castellane and Lemos (2007) and an HPLC systemequipped with a UV–vis spectrophotometer (Shi-madzu,modelSPD-M10A).Thedetectionwavelengthwas245nm. Themonosaccharidesglucose,mannose,rhamnose,galactose, glu-curonicacid,andgalacturonicacidwereusedasthestandardsat thefollowingconcentrations:12.50,25,50,and100␮gmL−1.The retentiontimes(RT)ofthemonosaccharidecompositionsoftheEPS weredeterminedthroughacomparisonwiththechromatograms ofeachrespectivestandard.Thevolumeofeachsampleinjected intothechromatographwas20␮L.Eachanalysiswasperformed induplicate.

2.6. Rheologicalpropertiesinaqueousmedium

Priortotherheologicalcharacterization,theEPSsampleswere trituratedusing an Inox triturator until a pulverized solid was obtained.Sampleswereresuspendedinpurifiedwaterata tem-peratureof20◦C,atconcentrationsof5and10gL−1.Thesamples werethenstoredforatleast24htoensuretheirfullhydration. Despitetheavailabilityofthesepolysaccharides,itisnecessaryto identifyandcharacterizenewpolysaccharideswithspecific rheo-logicalpropertiesandpotentialapplications.AccordingtoSaude

andJunter(2002),additionalinformationonthechemical

struc-tures and physicochemical characteristics of polysaccharides is requiredfortheiruseinindustry.

Therheologyofthepolymersinaqueoussolutionwasstudied usingacontrolledstressrheometer(RheometricsScientific).The rheologicaltestswereconductedat25◦Cinduplicate.Flowcurves wereobtainedthroughaprogramofup–down–upsteps,and dif-ferentshearstressrangeswereusedforeachsample.Theranges

weredeterminedusingashearratecontrolexperimentinwhich themaximumshearratevaluewas100s−1.

Theconsistencyindex‘K’andtheflowbehaviorindex‘n’were determinedfromthepowerlawmodel(Steffe,1996)givenbythe equation=Kn−1,whereistheapparentviscosity(Pas)andis

theshearrate(1/s).Thevalueof‘n’wasobtainedfromtheslopeof thelog–logplotofviscosityversusshearrate.Thevalueof‘K’was calculatedfromtheinterceptofthesamegraph.

2.7. Dataanalysis

Allofthedeterminationsreportedinthismanuscriptwere per-formedin triplicate,andtheresultsare presentedasthemean values.Resultswereanalyzedbyanalysisofvariance(ANOVA),and meanswerecomparedbyTukey’stest.

3. Resultsanddiscussion

3.1. Phenotypiccomparisonsbetweenthewild-typeandmutant strainsofR.tropici

Inthiswork,wefocusedprimarilyonthechoiceofmediaand growthconditionsthatapromotedhighR.tropiciEPSyieldbecause somebiologicalpolymershaveindustrialvalueinlargequantities. WefoundthatR.tropiciandrhizobialisolatesgrowsandproduces measurableEPSusingsucrose as acarbon sourcetested in liq-uidmedium(PSYL).Thewild-typestrainofR.tropiciSEMIA4080, mutantstrains(MUTZC3andMUTPA7)andtworhizobialisolates (JAB1andJAB6)werecultivatedusingcommercialsucroseasthe solecarbon source.Thiscarbon sourceis inexpensiveand easy toobtainandhasshownsatisfactoryresultsintheproductionof exopolysaccharidesbywild-typestrainsofR.tropici(Castellane&

Lemos,2007).

Bacteria belonging to the Rhizobium genus produce non-negligibleamountsof surfacepolysaccharides.Ourobservations ofR. tropicigrowthandEPSproductionutilizing sucrose asthe solecarbonsourceshowedthatthecoloniesofallofthestrains werelarge,circular,translucent,andmucoidonPSYA(resultsnot shown).Themucoidcoloniesformedlong,viscousfilamentswhen pickedwithaplatinumloop.ThecolonieswerethengrownonPSYA containingthefluorescentbrightener28(calcofluor)andobserved throughUVilluminationat430nm.Weobservedmucoid pheno-typicchangesintheMUTZC3strainsduetoanincreasedproduction ofpolysaccharides;thefluorescentpigmentisspecificto polysac-charides with␤-1→4 and␤-1→3linkages (Wood, 1980), and coloniesofthemutantstrainshowedbrighterfluorescenceunder ultraviolet light. The MUTZC3 mutant strains of R. tropicihave strongercalcofluorfluorescencethanrhizobialisolateJAB1.This indicatesthatMUTZC3andJAB6strainsproducemore calcofluor-fluorescentexopolysaccharidethanrhizobialisolateJAB1onPSYA mediumcontainingcalcofluor(datanotshown).Nodifferencein themucoidphenotypewasobservedbetweenthewild-type,and themutant strain (MUTPA7)of R.tropici.These resultssuggest thatallstrainsproduceEPS,andthestrainsofPhaseolusvulgaris L.exhibitvariationinproductionofEPS.

3.2. Evaluationofexopolysaccharideproduction

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Table1

Evaluationofthedifferencesintheexopolysaccharideproductionandcelldry weightbetweenwild-type(SEMIA4080),mutant(MUTZC3andMUTPA7)strains ofR.tropiciandrhizobialisolates(JAB1andJAB6).

Strain EPS Celldryweight(CDW) EPS/CDW

(gL−1)(mean

±SD)

SEMIA4080 2.52±0.45c 1.14±0.05a 2.21±0.15d

MUTZC3 5.06±0.20a 0.75±0.09c 6.75±0.09a

MUTPA7 3.94±0.41b 0.71±0.12c 5.55±0.25b

JAB1 3.75±0.30b 0.95±0.05b 3.95±0.11c

JAB6 5.52±0.36a 0.78±0.04c 7.08±0.06a

Meanvalues(±standarddeviation)withinthesamecolumnnotsharingacommon

superscriptdiffersignificantly(P<0.05).

yieldsofEPSreportedinRhizobiumspecies,e.g.,R.tropiciCIAT899 exhibitedamaximumEPSyieldof4.08gL−1underoptimized

con-ditionsofLMMdefinedminimalmediumwith2%sucrose(Staudt,

Wolfe,&Shrout,2012).Anotherresearchteamexaminedtwotype

strainsofR.tropici,namelyBR322andBR520,whichwereisolated fromthreenodulesoftheguandubean(Cajanuscajancv.Caqui). Bothstrainswererecommendedfortheproductionofsoil inoc-ulaforbeanstypicallygrowninBrazil.Furthermore,althoughthe strainsweregeneticallyverycloselyrelated,theyexhibited dis-tinctgrowthproductivitiesandcapacitieswithEPSproductionsof 1.13and1.89gL−1,respectively,whencultivatedinYMLmedium

(Fernandes,Rohr,Oliveira,Xavier,&Rumjanek,2009).

However,other knownrhizobia, suchas S.meliloti,are also capableofproducinghighlevelsofEPS,e.g.,S.melilotiSU-47 exhib-itedamaximumEPSyieldof7.8gL−1underoptimizedconditions ofyeastmannitolmedium(Breedveldet al.,1990), whiletheS. melilotistrainFexhibitedamaximumEPSyieldof2.9gL−1under optimizedconditionsofdefinedminimalmediumwithmannitol

(Dudman,1964).ManyresearchersreportthatS.melilotistrainscan

becharacterizedasefficientstrains,betterinbothqualitativeand quantitativeEPS(Mazur,Król,Marczak,&Skorupska,2003;Bomfeti

etal.,2011;Staudtetal.,2012).Asaresult,fromthepresentstudy

itisevidentthatmutantoftheR.tropicistrainMUTZC3andone rhizobialisolate(JAB6)canbeconsideredaspotentialmicrobial cellfactoriesforEPSproduction.TheamountofEPSproducedby thetwostrains(MUTZC3andJAB6)wasnotsignificantly(P>0.05) different.

ThecellularbiomassproductionvaluesoftheJAB1(0.95±0.05), JAB6 (0.78±0.04), MUTZC3 (0.75±0.09gL−1) and MUTPA7 (0.71±0.12gL−1) strains were lower than that of the wild-typestrain (1.14±0.05gL−1).In general,polymerproduction is inverselyproportionaltothebacterialgrowthindex,which sug-gestsaregulatoryrelationshipbetweenthebacterialmetabolism andcatabolisminwhich,uptosomepointonthegrowthcurve,the cellsdonotinvestincarbonskeletonsforgrowth,tothedetriment oftheirmetabolicactivity(FernandesJúnioretal.,2010).

Exopolysaccharideproduction canvary as a function of the growthphaseinsomebacterialspecies(Kumari,Ram,&Mallaiah, 2009). Someexopolysaccharides are produced throughout bac-terial growth, whereas others are only produced in the late logarithmic or stationary phases (Sutherland, 2001). However, althoughEPSyieldsvarywiththebacterialgrowthphase,most studies have shown that the exopolysaccharide composition remainsconstantthroughoutthebatchcycleofgrowth(DeVuyst,

Vanderveken,VandeVen,&Degeest, 1998).However,thelocal

environmental chemistry changes during bacterial growth as metabolitesandintermediatesareconsumedandcreated.Under ourtesting conditions,R.tropicisynthesizedEPSfromtheearly growthphasesthroughthestationaryphase.

WeevaluatedtherelativeefficiencyofEPSproduction,which isgivenbytheratioofthetotalEPStothecellularbiomass.The rhizobialisolateJAB6(7.08)andMUTZC3mutant(6.75)exhibited

Table2

ComparativemonosaccharidecompositionofEPS(%)producedbythewild-type (SEMIA4080),mutant(MUTZC3andMUTPA7)strainsofR.tropiciandrhizobial isolates(JAB1andJAB6)a.

Typeof

exopolysaccharide

Composition(%)

Man Rha GlcA GalA Glc Gal EPSofSEMIA4080(EPSWT) 0.86 2.58 8.6 tr 55.48 32.47 EPSofMUTZC3(EPSC3) 0.74 2.60 tr 2.60 53.53 40.52 EPSofMUTPA7(EPSPA7) 1.15 2.31 tr 2.70 54.63 39.19 EPSofJAB1(EPSJ1) 1.49 2.49 5.97 tr 60.70 29.35 EPSofJAB6(EPSJ6) 2.68 0.60 3.57 tr 54.17 38.99 aMan,mannose,Rha,rhamnose;GlcA,glucuronicacid;GalA,galacturonicacid; Glc,glucose;Gal,galactose;tr=trace.

thehighestEPSproduction efficiency,followedbytheMUTPA7 mutant(5.55),rhizobialisolateJAB1(3.95)andwild-typestrain (2.21).It is commonto find variableEPS productions between bacteria,evenamongbacteriaofthesamegenuscultivatedunder thesameconditions,asshownforRhizobium(Kumarietal.,2009), Xanthomonas (Antunes, Moreira, Vendruscolo, & Vendruscolo,

2003;Rottavaetal.,2009),andSphingomonas(Berwangeretal.,

2007).

3.3. EPSmonomercharacterization

AfterhydrolysisandPMPderivatization,theEPSwere charac-terized,andthemonomercontentswerequantifiedbyHPLC;the resultsaresummarizedinTable2,andtheHPLCchromatograms arepresentedinFig.1.TheanalysisoftheEPSmonosaccharide com-positionshowsthatglucoseandgalactosearethemostabundant monomers,andsmallamountsofmannose,rhamnose,glucuronic acid,andgalacturonicacidarealsopresent(Table2).Many EPS componentsarewater-solublebiopolymerscomposedofawide rangeofmonomersandmaycontainasmanyasninedifferentsugar residuesinrepeatingunits(Castellane&Lemos,2007;Monteiro

etal.,2012;Sutherland,2001).

Interestingly,theEPSproducedbytheMUTZC3andMUTPA7 mutantstrainsofR.tropici,whichareidentifieddesignatedasEPSC3 andEPSPA7,respectively, includedsmallquantitiesofmannose (0.74and1.15%),rhamnose(2.60and2.31%),andgalacturonicacid (2.60and2.70%)andtraceamountsofglucuronicacid.Incontrast, theEPSproducedbythewild-typestrainSEMIA4080,rhizobial isolatesJAB1andJAB6,whichareidentifieddesignatedasEPSWT, EPSJ1andEPSJ6,respectively,includedsmallquantitiesof man-nose(0.86%,1.49%and2.68%),rhamnose(2.58%,2.49%and0.60%), andglucuronicacid(8.6%,5.97%and3.57%)andtraceamountsof galacturonicacid(Table2).Thesedataaresimilartothefindings reportedbyCastellaneandLemos(2007),whofoundthatanEPS obtainedfromthecultivationofR.tropiciSEMIA4077wasprimarily composedofglucoseandgalactosewithtraceamountsofmannose andrhamnose.

Kaci,Heyraud,Barakat,andHeulin(2005)isolatedand

charac-terizedanEPSproducedbyatypestrainofRhizobiumfromarid earth as a polymer of glucose, galactose, and mannuronic acid inthemolarproportionof2:1:1.Ingeneral,EPSsynthesizedby fast-growingrhizobia(e.g.,S.melilotiand R.leguminosarum)are composedofoctasacchariderepeatingunits,inwhichglucoseis adominantsugarcomponent.InR.leguminosarumbv.trifolii,an EPSsubunitiscomposedofsevensugars,noneofwhichis

galac-tose(Amemura,Harada,Abe,&Higashi,1983).InR.leguminosarum

bv.viciae248,theEPSsubunithasanadditionalglucuronicacid

(Canter-Cremers et al., 1991). Low and high molecularweight

fractionsofEPSwerereportedbeproducedbyS.melilotiandR. leguminosarum(Mazuretal.,2003).

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Fig.1.MonosaccharideanalysisoftheEPSsamplesbyHPLCofthePMPderivativesoftheacidhydrolysateoftheEPS:(A)RhizobiumtropiciSEMIA4080,(B)theMUTZC3 mutantstrain,(C)theMUTPA7mutantstrain,(D)rhizobialisolateJAB1and(E)rhizobialisolateJAB6.ThechromatographsoftheEPSfromshowpeaksfor(*)

1-phenyl-3-methyl-5-pyrazoloneresidue,(1)mannose,(2)rhamnose,(3)glucuronicacid,(4)galacturonicacid,(5)glucose,and(6)galactose.

glucuronicacid(8.6%,5.97%and3.57%,respectively),andEPSC3 andEPSPA7showedtracesofglucuronicacid.Staehelinetal.(2006)

alsofound a very small quantityof glucuronic acidin the EPS of theRhizobium sp. type strain NGR234. The presence of acid monosaccharides(glucuronicandgalacturonicacid),eveninlow concentrations,rendersanEPSacidic,anditsaccumulationmakes theheteropolysaccharidehighlyanionic.Therefore,suchEPScan actasion-exchangeresinsandthusconcentratemineralsand

nutri-entsnearthecell(Whitfield,1988).Thepresenceofglucuronicand

pyruvicacidincreasestheionizationofthematerial,thereby pro-motingalterationsinitsmolecularconformationandincreasingits solubility(Diaz,Vendruscolo,&Vendruscolo,2004).

3.4. Rheologicalpropertiesinaqueousmedium

Theflow curvesof theexopolysaccharidesolutions obtained from the wild-type, mutant strains of R. tropici and rhizobial isolates are shown in Fig. 2. As shown in Fig. 2, solutions of thepure exopolysaccharidesEPSWT, EPSC3, EPSPA7,EPSJ1 and EPSJ6showed non-Newtonianbehavior at shear rates between 0.1 and 100s−1. Previous studies evaluating the EPS from rhi-zobial isolates have shown that this type of polymer generally

Fig.2.Flowcurvesofsolutionsoftheexopolysaccharidesfromthewild-typestrain ofRhizobiumtropici(SEMIA4080)andfromthemutant(MUTZC3andMUTPA7) strainsattwodifferentconcentrations.Theseflowcurvesweremeasuredat25◦C.

Thesymbolsrepresentthefollowing: , , ,and䊉forEPSWT,EPSC3,EPSPA7,

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Table3

Coefficients of the power law model for EPS solutions at two different concentrations.

Typeofexopolysaccharide K n

EPSofSEMIA4080(EPSWT)(5gL−1) 0.29±0.02e 0.41±0.03a EPSofSEMIA4080(EPSWT)(10gL−1) 1.71±0.09c 0.22±0.01b EPSofMUTZC3(EPSC3)(5gL−1) 0.30±0.01e 0.40±0.04a

EPSofMUTZC3(EPSC3)(10gL−1) 1.77

±0.12c 0.22±0.01b

EPSofMUTPA7(EPSPA7)(5gL−1) 0.17

±0.01f 0.48±0.08a

EPSofMUTPA7(EPSPA7)(10gL−1) 1.03

±0.02d 0.29±0.02b

EPSofJAB1(EPSJ1)(5gL−1) 2.1

±0.14c 0.25±0.01b

EPSofJAB1(EPSJ1)(10gL−1) 10.2±0.25a 0.16±0.02c EPSofJAB6(EPSJ6)(5gL−1) 1.9±0.09c 0.26±0.04b EPSofJAB6(EPSJ6)(10gL−1) 7.3±0.39b 0.20±0.06b

Meanvalues(±standarddeviation)withinthesamecolumnnotsharingacommon

superscriptdiffersignificantly(P<0.05).

Flowbehaviorindex,n,andconsistencycoefficient,K,obtainedbytheOstwald-de Waelemodel:=Kn−1.

exhibitsnon-Newtonianbehaviorandispseudoplastic(Kacietal.,

2005;Aranda-Selverioetal.,2010).Pseudoplasticorshearthinning

behavior hasbeen reported for other biopolymers with indus-trialapplications,suchasxanthan(Katzbauer,1998),gellangums

(Dreveton,Monot,Ballerini,Lecourtier,&Choplin,1994)and

suc-cinoglycan(Kido,Nakanishi,Norisuye,Kaneda,&Yanaki,2001). Therheologicalbehaviorsofmaterialsmaybedescribedusing models that describe how the surface tension varies with the deformationrate.Themathematicalmodelsthataremost com-monlyutilizedforfoodsystemsaretheOstwald-deWaele(power law), Casson,Herschel–Bulkley, and Mizrahi–Berki models. The firsttwo modelsusemathematical equations withtwo param-eters,whereas the others useequations withthree parameters

(Haminiuk,Sierakowski,Izidoro,&Masson,2006).The

Ostwald-deWaele modelallowedthebest adjustmentsto thesolutions of5and10gL−1 exopolysaccharidesproduced bythewild-type R.tropiciSEMIA4080,themutant(MUTZC3andMUTPA7)strains andrhizobialisolates(JAB1andJAB6)atatemperatureof25◦C. Thepowerlawmodeliseasytouseandisidealforpseudoplastic, relativelymobilefluids,suchasweakgelsandlow-viscosity disper-sions.Thecoefficientsofthismodelforallofthesolutionsanalyzed arepresentedinTable3.Theconsistencycoefficient‘K’describes theoverallrangeofviscositiesacrossthemodeledportionofthe flowcurve.

Thevaluesofboththeconsistencyindex(K)andflow behav-iorindex(n)weresignificantlydependent(P<0.05)onthestrain

(Table3).The‘K’valueindicatedaprogressiveincreaseinviscosity

withanincreaseintheEPSconcentrationforallEPStested.The rhe-ologicalprofilewasveryclosetothatofthebiopolymersproduced bythewild-typeR.tropiciSEMIA4080andtheMUTZC3mutant strainattheconcentrationof5gL−1(Fig.2andTable3).TheEPS producedbytherhizobialisolatesJAB1andJAB6showedhigherK valuesthantheEPSWTandEPSC3,indicatingmoreshear-resistant nature.

AsshowninTable3,thevaluesof‘K’obtainedfortheEPS pro-ducedbytheMUTZC3andSEMIA4080strainswerehigherthan those foundfor theMUTPA7 mutant, which indicatesthat the EPSWTand EPSC3 solutionsatconcentrations of5 and 10gL−1 aremuchmoreviscousthanthepolysaccharideexcretedby the-MUTPA7mutantstrain.Theexponentn(knownasthepowerlaw index)has values between0 and 1 for a shear thinning fluid, whereasmorepseudoplastic productsexhibit lowervaluesof n (closetozero)(Steffe,1996).

Theseresultsobtainedinthisstudyaresimilartothoseobtained

byAranda-Selverioetal.(2010),whoreportedthatEPSsolutions

producedby rhizobiaisolated fromPhaseolusvulgaris,Leucaena leucocephalav.cunnie,Pisumsativum,andA.pintoiexhibit pseu-doplasticbehavior.TheaqueoussolutionsoftheEPSproducedby

threedifferentstrainsofbacteriaoftheRhizobiumgenusbehavedas non-Newtonianfluids.Adecreaseinthesurfacetension accompa-niedbyanincreaseofthesurfaceindexresultsinalowerapparent viscosity,whichmeansthatthesolutionsarepseudoplasticfluids

(Navarro,1997).Therheologicalanalysesdemonstratethatthese

polysaccharidesolutionsexhibitpseudoplasticfluidbehaviorand maythus beutilized asthickening agents withpolyelectrolytic properties.However,despitethispseudoplasticbehavior,the vis-cosityof the solutions of theEPSfrom differentrhizobial type strainsmayvaryeventhoughtheyexhibitthesamesurfaceindexes

(Aranda-Selverioetal.,2010),suggestingthatthesepolymersmay

havedifferentbiotechnologicalapplications.

4. Conclusions

Theexopolysaccharidesproducedbythedifferentmutantsof R.tropicishowedsubtledifferencesintheirmonosaccharide com-positions(primarystructure)comparedwiththewild-typestrain, which may be sufficient to cause alterations in the secondary structuresorconformationsofthemolecules.Thishypothesisis supportedby theresultsof the rheologicalanalysesof each of thestudiedEPS.Allofthebiopolymersproducedbythesestrains demonstratedpseudoplasticbehavior.

Becausetheuseofrhizobiainthecommercialproductionofgum hasnotbeenstudied,rhizobiamaybeconsideredhighly promis-ingunexploredsourcesofmicrobialpolysaccharidesforindustrial applications.Thesebacteriaexhibitgreatmorphological, physio-logical,genetic,andphylogeneticdiversityandcanbeavaluable sourceforthescreeningofstrainswithspecificproperties.Because noneofthe␣-and␤-rhizobiadiscoveredtodatehasbeenshown

tobepathogenic,thisgroupcanbegenerallycharacterizedasan unexploredsourceofmicrobialEPSwithexcellentpotentialforuse inindustrialapplicationsandassoil-stabilizingagents.Thismay representapotentialopportunityforthebio-inoculantproducing industriesinBrazilasbestalternativeactivityduringthenoncrop seasonofthesoybeanandcommonbean.TheMUTZC3mutantand rhizobialisolateJAB6exhibitedthebestEPSproductions.Whilethe EPSproducedbytherhizobialisolatesJAB1andJAB6showedhigher consistencyindexvalues thantheEPSproduced bythemutant strainsMUTZC3,indicatingmoreshear-resistantnature.Therefore, weconcludedthatthesestrainscouldbeexploitedforthe large-scalecommercialproductionofRhizobialpolysaccharides.

Acknowledgment

The authors acknowledge FAPESP (Fundac¸ão de Amparo a Pesquisado EstadodeSãoPaulo)#07/57586-6forthefinancial support.

References

Amemura,A.,Harada,T.,Abe,M.,&Higashi,S.(1983).Structuralstudiesofthe acidicpolysaccharidefromRhizobiumtrifolii4S.CarbohydrateResearch,115, 165–174.

Antunes,A.E.C.,Moreira,A.S.,Vendruscolo,J.L.S.,&Vendruscolo,C.T.(2003). ScreeningofXanthomonascampestrispvprunistrainsaccordingtotheir pro-ductionofxanthananditsviscosityandchemicalcomposition.BrazilianJournal ofFoodTechnology,6,317–322.

Aranda-Selverio,G.,Penna,A.L.B.,Campos-Sás,L.F.,Santos,O.,Vasconcelos,A.F. D.,Silva,M.,Lemos,E.G.M.,Campanharo,J.C.,&Silveira,J.L.M.S.(2010). Propriedadesreológicaseefeitodaadic¸ãodesalnaviscosidadede exopolis-sacarídeosproduzidosporbactériasdogêneroRhizobium.QuímicaNova,33, 895–899.

Beringer,J.E.(1974).AfactortransferinRhizobiumleguminosarum.JournalofGeneral Microbiology,84,188–198.

(7)

Bomfeti,C.A.,Florentino,A.F.,Guimarães,A.P.,Cardoso,P.G.,Guerreiro,M. C.,&Moreira,F.M.S.(2011).Exopolysaccharidesproducedbythesymbiotic nitrogen-fixingbacteriaofleguminosae.RevistaBrasileiradeCiênciadoSolo,35, 657–671.

Breedveld,M.W.,Zevenhuizen,L.P.T.M.,&Zehnder,A.J.B.(1990).Osmotically inducedoligo-andpolysaccharidesynthesisbyRhizobiummelilotiSU-47.Journal ofGeneralMicrobiology,136,2511–2519.

Canter-Cremers,H.C.J.,Stevens,K.,Lugtengberg,B.J.J.,Wijffelman,C.A.,Batley, M.,Redmond,J.W.,Breedveld,M.,&Zevenhuizen,L.P.T.M.(1991).Unusual structureoftheexopolysaccharideofRhizobiumleguminosarumbvviciaestrain 248.CarbohydrateResearch,218,185–200.

Castellane,T.C.L.,&Lemos,E.G.deM.(2007).Composic¸ãodeexopolissacarídeos produzidosporestirpesderizóbioscultivadosemdiferentesfontesdecarbono. PesquisaAgropecuáriaBrasileira,42,1503–1506.

DeVuyst,L.,Vanderveken,F.,VandeVen,S.,&Degeest,B.(1998).Productionby andisolationofexopolysaccharidesfromStreptococcusthermophilusgrownina milkmediumandevidencefortheirgrowth-associatedbiosynthesis.Journalof AppliedMicrobiology,84,1059–1068.

DeAngelis,P.L.(2012).Glycosaminoglycanpolysaccharidebiosynthesisand pro-duction:Todayandtomorrow.AppliedMicrobiologyBiotechnology,94,295–305. Diaz,P.S.,Vendruscolo,C.T.,&Vendruscolo,J.L.S.(2004).Reologiadexantana:Uma revisãosobreainfluênciadeeletrólitosnaviscosidadedesoluc¸õesaquosasde gomasxantana.Semina:CiênciasExataseTecnologia,25,15–28.

Donot,F.,Fontana,A.,Baccou,J.C.,&Schorr-Galindo,S.(2012).Microbial exopolysac-charides: Main examplesof synthesis, excretion genetics and extraction. CarbohydratePolymers,87,951–962.

Dreveton,É.,Monot,F.,Ballerini,D.,Lecourtier,J.,&Choplin,L.(1994).Effectof mixingandmasstransferconditionsongellanproductionbyAuromonaselodea. JournalofFermentationandBioengineering,77,642–649.

Dudman,W.F.(1964).Growthandextracellularpolysaccharideproductionby Rhi-zobiummelilotiindefinedmedium.JournalofBacteriology,88,640–645. Fernandes,P.I.,Rohr,T.G.,Oliveira,P.J.,Xavier,G.R.,&Rumjanek,N.G.(2009).

Polymersascarriersforrhizobialinoculantformulations.PesquisaAgropecuária Brasileira,44,1184–1190.

FernandesJúnior,P.I.,Almeida,J.P.S.,Passos,S.R.,Oliveira,P.J.,Rumjanek,N.G.,& Xavier,G.R.(2010).Produc¸ãoecomportamentoreológicodeexopolissacarídeos sintetizadosporrizóbiosdeisoladosdeguandu.PesquisaAgropecuáriaBrasileira, 45,1465–1470.

Fu,D.,&O’Neill,R.A.(1995).Monosaccharidecompositionanalysisof oligosaccha-ridesandglycoproteinsbyhigh-performanceliquidchromatography.Analytical Biochemistry,227,377–384.

Haminiuk,C.W.I.,Sierakowski,M.R.,Izidoro,D.R.,&Masson,M.L.(2006). Rheo-logicalcharacterizationofblackberrypulp.BrazilianJournalofFoodTechnology, 9,291–296.

Hungria,M.,Andrade,D.S.,Chueire,L.M.O.,Probanza,A.,Guttierrez-Ma˜nero, F.J.,&Megías,M.(2000).Isolationandcharacterizationofnewefficientand competitivebean(PhaseolusvulgarisL.)rhizobiafromBrazil.SoilBiologyand Biochemistry,32,1515–1528.

Kaci,Y.,Heyraud,A.,Barakat,M.,&Heulin,T.(2005).Isolationandidentificationof anEPSproducingRhizobiumstrainfromandsoil(Algeria):Characterizationofits EPSandtheeffectofinoculationonwheatrhizospheresoilstructure.Research inMicrobiology,156,522–531.

Katzbauer,B.(1998).Propertiesandapplicationsofxanthangum.Polymer Degrada-tionandStability,59,81–84.

Kido,S.,Nakanishi,T.,Norisuye,T.,Kaneda,I.,&Yanaki,T.(2001).Ordered con-formationofsuccinoglycaninaqueoussodiumchloride.Biomacromolecules,2, 952–957.

Kumari,B.S.,Ram,M.R.,&Mallaiah,K.V.(2009).Studiesonexopolysaccharide andindoleaceticacidproductionbyRhizobiumstrainsfromIndigofera.African JournalofMicrobiologyResearch,3,10–14.

Marczak,M.,Dzwierzynska,M.,&Skorupska,A.(2013).Homo-andheterotypic interactionsbetweenPssproteinsinvolvedintheexopolysaccharide trans-portsysteminRhizobiumleguminosarumbv.trifolii.BiologicalChemistry,394(4), 541–559.

Mazur,A.,Król,J.,Marczak,M.,&Skorupska,A.(2003).MembranetopologyofPssT thetansmembraneproteincomponentofthetypeIexopolysaccharidetransport systeminRhizobiumleguminosarumbv.trifoliistrainTA1.JournalofBacteriology, 185,2503–2511.

Mohamad,O.A.,Hao,X.,Xie,P.,Hatab,S.,Lin,Y.,&Wei,G.(2012).Biosorption ofcopper(II)fromaqueoussolutionusingnon-livingMesorhizobiumamorphae strainCCNWGS0123.MicrobesandEnvironments,27(3),234–241.

Monteiro,N.K.,Aranda-Selverio,G.,Exposti,D.T.D.,Silva,M.,Lemos,E.G.M., Campanharo,J.C.,&Silveira,J.L.M.S.(2012).Caracterizac¸ãoquímicadosgéis produzidospelasbactériasdiazotróficasRhizobiumtropicieMesorhizobiumsp. QuímicaNova,35(4),705–708.

Mota,R.,Guimarães,R.,Büttel,Z.,Rossi,F.,Colica,G.,Silva,C.J.,Santos,C.,Gales,L., Zille,A.,DePhilippis,R.,Pereira,S.B.,&Tamagnini,P.(2013).Productionand characterizationofextracellularcarbohydratepolymerfromCyanothecesp.CCY 0110.CarbohydratePolymers,92(2),1408–1415.

Navarro,R.F.(1997).Fundamentosdereologiadepolímeros.CaxiasdoSul:Editora daUniversidadedeCaxiasdoSul.

Noel.,K.D.(2009).Rhizobia.InM.Schaechter(Ed.),Encyclopediaofmicrobiology(pp. 261–277).SanDiego,CA:AcademicPress.

Nwodo,U.U.,Green,E.,&Okoh,A.I.(2012).Bacterialexopolysaccharides: Func-tionalityand prospects.International Journal of MolecularSciences, 13(11), 14002–14015.

Prajapati,V.D.,Jani,G.K.,Zala,B.S.,&Khutliwala,T.A.(2013).Aninsightinto theemergingexopolysaccharidegellangumasanovelpolymer.Carbohydrate Polymers,93(2),670–678.

Qurashi,A.W.,&Sabri,A.S.(2012).Bacterialexopolysaccharideandbiofilm forma-tionstimulatechickpeagrowthandsoilaggregationundersaltstress.Brazilian JournalofMicrobiology,43(3),1183–1191.

Radchenkova, N., Vassilev,S., Panchev, I., Anzelmo, G., Tomova, I., Nicolaus, B., Kuncheva, M., Petrov, K., & Kambourova, M. (2013). Production and propertiesoftwonovelexopolysaccharidessynthesizedbyathermophilic Bac-teriumAeribacilluspallidus418.AppliedBiochemistryandBiotechnology,171, 31–43.

Rendueles,O.,Kaplan,J.B.,&Ghigo,J.M.(2013).Antibiofilmpolysaccharides. Envi-ronmentalMicrobiology,15(2),334–346.

Rottava,I.,Batesini,G.,Silva,M.F.,Lerin,L.,Oliveira,D.,Padilha,F.F.,Toniazzo, G.,Mossi,A.,Cansian,R.L.,DiLuccio,M.,&Treichel,H.(2009).Xanthangum productionandrheologicalbehaviorusingdifferentstrainsofXanthomonassp. CarbohydratePolymers,77,65–71.

Sathiyanarayanan,G.,Kiran,G.S.,&Selvin,J.(2013).Synthesisofsilvernanoparticles bypolysaccharidebioflocculantproducedfrommarineBacillussubtilisMSBN17. ColloidsandSurfacesB:Biointerfaces,102,13–20.

Saude,N.,&Junter,G.A.(2002).Productionandmolecularweightcharacteristicsof alginatefromfreeandimmobilized-cellculturesofAzotobacterinelandii.Process Biochemistry,37,895–900.

Shah,A.A.,Hasan,F.,Hameed,A.,&Ahmed,S.(2008).Biologicaldegradationof plastics:Acomprehensivereview.BiotechnologyAdvances,26,246–265. Silvi,S.,Barghini,P.,Aquilanti,A.,Juarez-Jimenez,B.,&Fenice,M.(2013).Physiologic

andmetaboliccharacterizationofanewmarineisolate(BM39)ofPantoeasp. producinghighlevelsofexopolysaccharide.MicrobialCellFactories,12,10. Simsek,S.,Mert,B.,Campanella,O.H.,&Reuhs,B.(2009).Chemicaland

rheologi-calpropertiesofbacterialsuccinoglycanwithdistinctstructuralcharacteristics. CarbohydratePolymers,76,320–324.

Staehelin,C.,Forsberg,L.S.,D’Haeze,W.,Gao,M.,Carlson,R.W.,Xie,Z.,Pellock, B.J.,Jones,K.M.,Walker,G.C.,Streit,W.R.,&Broughton,W.J.(2006). Exo-oligosaccharidesofRhizobiumsp.strainNGR234arerequiredforsymbiosiswith variouslegumes.JournalofBacteriology,188,6168–6178.

Staudt,A.K.,Wolfe,L.G.,&Shrout,J.D.(2012).Variationsinexopolysaccharide productionbyRhizobiumtropici.ArchivesofMicrobiology,194(3),197–206. Steffe,J.F.(1996).Rheologicalmethodsinfoodprocessengineering(2nded.).East

Lansing,MI:FreemanPress.

Sutherland,I.W.(2001).Microbialpolysaccharidesfromgram-negative. Interna-tionalDairyJournal,11,663–674.

Vincent,J.M.(1970).Amanualforthepracticalstudyofrootnodulesbacteria.In IBPhandbook(15thed.).Oxford:BlackwellScientific.

Wang,Y.,Ahmed,Z.,Feng,W.,Li,C.,&Song,S.(2008).Physicochemical prop-erties ofexopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolatedfromTibetkefir.InternationalJournalofBiologicalMacromolecules,43(3), 283–288.

Whitfield,C.(1988).Bacterialextracellularpolysaccharides.CanadianJournalof Microbiology,34,415–420.

Wood,P.J.(1980).Specificityintheinteractionofdirectdyeswithpolysaccharides. CarbohydrateResearch,85(2),271–287.

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