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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
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 (25gmL−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 430nmatafinalconcentrationof200gmL−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).
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(200L)inasealedglasstube(13×100mm)with screwcapwhichfilledwithpurenitrogengasat121◦Cfor2h.The hydrolyzedsolutionwasevaporatedtodrynessunder45◦Cand then2-propanol(500L)wasaddedforfurtherevaporationand completeremovaloftrifluoroaceticacid.Thehydrolysatewasused forderivatization.
Afterhydrolysis,theEPSandmonosaccharidestandardswere pre-derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP), a chemicalmarker.Thereactionswereconductedbyadding40L ofPMPsolution(0.5molL−1 in methanol)and40L ofsodium hydroxide solution (0.3molL−1) to each tube. The tubes were agitatedandincubatedat120◦Cfor2h.Themixturewasthen neu-tralizedbyadding40Lofhydrochloricacidsolution(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,and100gmL−1.The retentiontimes(RT)ofthemonosaccharidecompositionsoftheEPS weredeterminedthroughacomparisonwiththechromatograms ofeachrespectivestandard.Thevolumeofeachsampleinjected intothechromatographwas20L.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
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).
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,
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.
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