Evaluation of the production of exopolysaccharides by two strains of the thermophilic bacterium Rhodothermus marinus

The thermophile Rhodothermus marinus produces extracellular polysaccharides (EPSs) that forms a distinct cellular capsule. Here, the first data on EPS production in strains DSM4252T and MAT493 are reported and compared.

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

Roya R.R Sardaria,∗, Evelina Kulcinskajab,1, Emanuel Y.C Rona, Snædís Björnsdóttirc,

Ólafur H Fri ð jónssonc, Gu ð mundur Óli Hreggvi ð ssonc,d, Eva Nordberg Karlssona

a r t i c l e i n f o

Keywords:

Exopolysaccharide

EPS

Heteropolymer

a b s t r a c t

ThethermophileRhodothermusmarinusproducesextracellularpolysaccharides(EPSs)thatformsa dis-tinctcellularcapsule.Here,thefirstdataonEPSproductioninstrainsDSM4252TandMAT493arereported andcompared.Culturesofbothstrains,supplementedwitheitherglucose,sucrose,lactoseormaltose showedthattheEPSwereproducedbothintheexponentialandstationarygrowthphaseandthat productionintheexponentialphasewasboostedbymaltosesupplementation,whilestationaryphase productionwasboostedbylactose.Thelatterwashigher,resultingin8.8(DSM4252T)and13.7mgEPS/g celldryweight(MAT493)inculturesinmarinebrothsupplementedwith10g/Llactose.TheEPSswere heteropolymericwithanaveragemolecularweightof8×104Daanddifferentmonosaccharides, includ-ingarabinoseandxylose.FT-IRspectroscopyrevealedpresenceofhydroxyl,carboxyl,N-acetyl,amine, andsulfateestergroups,showingthatR.marinusproducesunusualsulfatedEPSwithhigharabinoseand xylosecontent

©2016TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense

(http://creativecommons.org/licenses/by/4.0/)

Extracellular polysaccharides (exopolysaccharides, EPSs) are

themajorpartofextracellularpolymericsubstancesproducedby

microorganisms(Jindal,Singh,&Khattar,2011).Theyexistintwo

mainforms;asacapsuleassociatedwiththecellsurfaceorsecreted

outofthecell,eithertothesurroundingsorremainlooselyattached

tothecellsurface(Tallon,Bressollier,&Urdaci,2003)

Exopolysac-charideshaveimportantecologicalandphysiologicalfunctionsand

playspecialrolesinprotectingthemicroorganismsthatproduce

them.EPSsarebelievedtoprotectcellsagainstantimicrobial

sub-stances,desiccation,bacteriophages,osmoticstress,andantibodies

(Mataetal.,2006;Tallonetal.,2003)

EPSscanbefoundashomopolysaccharidesor

heteropolysac-charides and can be decorated with other residues such as

phosphates, sulfates, N-acetyl-aminosugars, and acetyl groups

(Laws, Gu, & Marshall, 2001) The properties of the EPSs are

influencedbytheircompositionwhichisaffectedbynutrient avail-abilityaswellasbyotherfactorssuchastheirmolecularmassand thelocationoffunctionalgroups

Theunique andcomplex chemical structuresof EPSs,which arenatural polymerswithdifferentfunctional properties,make theminterestingforvariousindustrialapplicationsinfood, phar-maceutical, petroleum and other industries (Castellane, Lemos,

& deMacedo Lemos, 2014), for example by affecting the fluid-ity of active compounds Both prokaryotes (Gram-positive and Gram-negativebacteria)and eukaryotes(fungi,somealgea,and phytoplankton)areknowntoproduceEPSsandnewspeciesare currentlybeingtargetedinthesearchforEPSswithnovel proper-ties.Asaresultofthesescreeningefforts,marinebacteriaarenow widelyacceptedasthesourceofEPSswithuniquepropertiesthat canbeexploitedfornovelbiotechnologicalprocesses(Chi&Fang, 2005)

Rhodothermusmarinusisathermophilic,reddishcolored aer-obic,and Gram-negativebacteriumthat wasfirstisolated from shallowmarinehot springsin Iceland(Alfredsson,Kristjansson, Hjörleifsdotter,&Stetter,1988).Thebacteriumgrows heterotroph-ically and is known to produce highly thermostable enzymes (Nordberg Karlsson, Bartonek-Roxå, & Holst, 1998; Blücher, Nordberg Karlsson, & Holst, 2000) It is of interest as repre-http://dx.doi.org/10.1016/j.carbpol.2016.08.062

sentingthedeepestlineagein thephylumBacteroidetes (Nolan,

Tindall, Pomrenke, Lapidus, & Copeland, 2009) The complete

genomesequence isavailableforthetypestrainandtoolshave

been developed for genetic manipulation of a second strain,

MAT493 (Bjornsdottir, Fridjonsson,Hreggvidsson,& Eggertsson,

2011; Bjornsdottir, Thorbjarnardottir, & Eggertsson, 2005) The

cellsofR.marinushavebeenshowntoformadistinctcapsulewhen

grownoncarbohydrate-richmedium(Alfredssonetal.,1988).To

ourknowledge,however,noinformationisavailableonthe

produc-tionofEPSsbyR.marinus,orontheirphysico-chemicalproperties

TheaimofthisstudywastheevaluationofEPSproductionbythe

R.marinustypestrain(DSM4252T)aswellasbyastrainthatis

amenabletoengineering(MAT493).Theworkinvolvedthe

exam-inationofthekineticsofEPSproductioninbatchculturesusing

shakeflasksundervariednutritionalconditions.Also,theisolation

andcharacterizationofnovelEPSfromthetwoR.marinusstrains

hasopenedpossibilitiesforapplyingEPSsfromR.marinusfor

indus-trialpurposes

2.1 Materials

AllmaterialsandreagentswerepurchasedfromSigma-Aldrich

unlessotherwisespecified

2.2 Bacterialstrains

TheR.marinusstrainsDSM4252TandMAT493obtainedfrom

theMatisculturecollection,wereusedinthepresentstudy

2.3 Cultureconditions

R.marinusDSM4252Twastakenfromthestockcultureand

inoc-ulatedinATCCmedium1599:ThermusEnhancedMedium(ATCC

medium1598)containingagarand1%NaCl.Theplatewas

incu-batedat65◦Cfor24h,afterwhichthecellsweretransferredfrom

theplateintoMarinebroth(Difco2216,USA)(10ml)ina50ml

falcontubeandincubatedinarotaryshakerincubatorat65◦Cand

200rpmfor24h

R.marinusMAT493wasinoculateddirectlyfromastockculture

intoMarinebroth(10ml)ina50mlfalcontubeandincubatedina

rotaryshakerincubatorat65◦Cand200rpmfor24h

Afterthefirstincubation,0.25ml(10%)oftheproducedcell

cul-turesofbothstrainswereseparatelyinoculatedintoMarinebroth

(2.5ml)in50mlfalcontubes,andweregrownfor8h.The

result-ingculturesweresubsequentlyusedasinoculumfortheshakeflask

cultivationswhereEPSproductionwasstudied

ThecellculturesusedforanalysisofEPSproductionweregrown

inmarinebroth(25mlin250mlbaffledshakeflasks)containingthe

sugarsglucose,maltose,lactose,andsucrose,respectively,as

addi-tionalcarbonsourceattheconcentrations1and10g/l.Thecells

wereinoculatedwith2.5ml(10%)oftheinoculumandgrownat

65◦Cand200rpmfor48hinashakingincubator(IKA,KS4000i

control).Samplesweretakenafter0,6,15,24,and48handwere

analysedfor residualcarbon sourceand total producedEPSs A

parallelcontrolexperiment wascarried outusingMarinebroth

withoutcarbonsourcesupplementation

2.4 Determinationofcellbiomass

Bacterialgrowthwasquantitativelydeterminedbymeasuring

celldryweight.Aftercentrifugation,cellswerewashed oncein

waterandthenresuspendedin2mlof0.05MEDTAsodiumsalt

solution(Horn etal.,2013).Themixturewasshakengentlyon

arockingtableat4◦Cfor4htoremove anycapsularEPS.After

this,cellswereharvested bycentrifugation,washedwithwater, transferredtoaluminumweighingpansanddriedinanovenset

at100◦C.Thecelldryweightwasmeasuredperiodicallyuntila constantweightwasreached

2.5 Isolationofexopolysaccharides StrainsDSM4252TandMAT493weregrownasdescribedin sec-tion2.3,andthecapsularEPS(in0.5mlsamples)wereseparatedas describedinsection2.4.Subsequently,thesamplestakenat differ-enttimesfromthecultivationswerecentrifugedat4000rpmfor

30minat4◦C (SigmaPK).TheEPSswereprecipitatedbyadding thethreefold volumeof ethanol (99.5%) tothecell free super-natants.Aftermixingand storingovernightat4◦C,precipitates wereharvested bycentrifugationat4000rpmfor30minat4◦C (Sigma3–16PK)andputinafumehoodtoevaporatethe remain-ingethanol.TheprecipitatesthenweredissolvedinmilliQwater andlyophilized(Labconcofreezedrysystem)toobtainthecrude EPSs

2.6 Purificationandexopolysaccharidefractionation ThepurityandsizefractionsofthecrudeEPSwasexaminedby sizeexclusionchromatographyusingacolumnofHiPrepSephacryl S-200HR(16mm×600mm)(HiPrep,GEhealthcarelifesciences) Eachcrude EPSwasdissolvedin milliQwater (10g/l) andafter filtration(13mm syringe filterw/0.2␮m PTFE membrane) was loadedonthe SephacrylS-200 column and eluted withmilliQ waterattheflowrateof0.3ml/min.Theflowratewascontrolled

byaFPLCpumpingsystem(PharmaciaLKB,Pump-500)and frac-tionsweredetectedusingrefractive-indexmonitoring(ERC-7510, ERMAINC)andcollectedevery10minusingafractioncollector (LKBBromma,2212HELIRAC)

2.7 Determinationofmolecularweight FordeterminationofmolecularweightoftheEPSfractions, stan-darddextrans(1.27,5,12, 50,and80kDa)(Sigma)werepassed through a Sephacryl S-200 column (16mm×600mm) and the retentiontimeofeachdextranwasobtainedfromthe chromato-graphicdata Theretention times ofthe dextranswere plotted againstthelogarithmsoftheirrespectivemolecularweights.The molecularweightsof theproducedEPSs werethendetermined usingtheirretentiontime

2.8 Monosaccharideanalysis TheanalysisofEPSsmonosaccharidecomposition,producedat differenttimesduringcultivation,andafterfractionationbysize exclusionchromatography(Section2.6)wasdoneaccordingthe hydrolysismethoddescribedbySluiter,Hames,Ruiz,andScarlata (2008)withmodifications

Briefly, sulfuric acid (72%) was added to the isolated EPSs and water was added after 30min The samples were heated

at 100◦C for 3h and neutralized after hydrolysis with 0.1M Ba(OH)2·H2O.Aftercentrifugation,themonosaccharidecontentof theEPSsinthesupernatantswasanalysedbyHighPerformance Anion-ExchangeChromatography(HPAEC)(ThermoFisher Scien-tific, Waltham, USA) using a Dionex CarboPacPA-20 analytical columnwhichwascoupledtoaDionexCarboPacPA-20guard col-umn.ThesugarswereseparatedusingNaOH(0.75mM)asamobile phasewiththeflowrateof0.5ml/minandthecolumnwas regen-eratedwith200mMNaOHfor4minwiththesameflowrateatthe endofeachcycle.DetectionwasperformedwithanED40 electro-chemicaldetector

2.9 Analysisoffunctionalgroups

Fourier transformed infrared (FT-IR) spectroscopy was used

todeterminethefunctionalgroupsofthepurifiedEPSs.Infrared

spectra of the purified EPSs fractions were recorded in the

4000–400cm−1 regionusing a FT-IR system (Nicolet is5,

Ther-moFisherScientific)

Thedeterminationswereperformedintwoindependent

repli-catesandarereportedasthemeanwithstandarddeviations

3.1 GrowthandEPSproductionbythetwoR.marinusstrains

Thetwo R marinus strainsexhibiteda distinct differencein

growthbehaviour,withthestrainDSM4252Tformingaggregates

whilestrainMAT493didnot.Aggregationhasbeenobservedfor

manyR.marinusstrains(Bjornsdottiretal.,2005),anditisnow

knownthatbacteriapredominantlylivewithinsurface-attached

biofilmstructuresintheirnaturalhabitats(Davey&O’Toole,2000)

Biofilmsofferseveraladvantages,includingprotectionfrom

dif-ferent stress factors (Jagmann, Henke, &Philipp, 2015; Monier

&Lindow,2003).Bacteriawithinthesestructuresareencasedin

anextracellularmatrix composedofsecretedproteins,

polysac-charides,DNAandothersubstances.Thereasonfortheobserved

differenceinstrainsDSM4252TandMAT493isunknown,asisthe

mechanismofaggregationinR.marinus.Infact,thelackof

aggre-gationwasoneofthereasonsforchoosingstrainMAT493forthe

developmentofmethodsforgeneticmanipulation,whichrequire

theuseofsinglecolonies.Thedifferenceinaggregation,could

how-evernotbeshowntobeimmediatelyrelatedtotheEPSproduction

level(Tables1and2 whichforbothstrainswasinitiatedinthe

exponentialgrowthphase,butwithsomedifferences

Asinitialtrialswiththetypestrain(DSM4252T)showedthat

useofdisaccharidesascarbonsourcesupplementationgenerally

resultedinhigherEPSproductionthantheuseofmonosaccharides

(datanotshown),lactose,maltoseandsucrosewereselectedas

thesupplementationoftheMarinebrothandwasusedforboth

strains In addition,glucose waschosenas a respresentativeof

monosaccharides,andaparallelgrowthexperimentwithout

addi-tionaladdedsugarswasruntomonitorthebackgroundproduction

ofEPS(Figs.1–4).The addedcarbon sourcesweresupplied at

twoconcentrations,1g/L(Figs.1and3)and10g/L(Figs.2and4

respectively

Consumptionofthemonosaccharideglucose,andthe

disaccha-rideslactoseandmaltosewasverifiedforthetypestrain(Fig.1).In

allcases,productionofEPSwasinitiatedintheexponentialgrowth

phaseandwasshowntocontinueinthestationaryphase

Atthe1g/Lsupplementationlevel(Fig.1 theconsumptionrate

ofglucosewasdeterminedto0.13g/l,hduringthefirst6handafter

15hallglucosewasconsumedandthecellmasswas1.2±0.14g/l

After glucose depletion, the change in cell concentration was

marginalduring9h(reaching1.25±0.35g/lafter24h)butthen

increasedto1.68±0.16g/lat48h.Thelateincreaseincell

concen-trationmightbeduetoconsumptionofproducedEPS,asitwas

accompaniedbyadecreaseintheratioofproducedEPSpercelldry

weightinthesameperiodoftime(from7.4×10−5to6.2×10−5),

reachingafinalEPSconcentrationof1.04±0.15␮g/ml(at48h)

Theexperimentsusingmarinebrothwithoutaddedsugar,showed

aslightlylowermaximumcellmass(0.87±0.03g/l,after24h,in

principlemaintainedattheendofthecultivation,0.85±0.07g/l

after48h)andthefinalEPSconcentrationwas0.75±0.24␮g/ml

(withaproductionrateof0.014␮g/ml,hafter48h).Thisindicates

thatthe1g/Lglucose-additionhadasmallboostingeffectonboth

cellmassandEPSproduction

Additionoflactosedidnotstimulatecellgrowth,butresultedin increasedEPSproduction.Theconsumptionrateoflactoseduring thefirst6hwas0.03g/l,hbutincreasedto0.095g/l,h(6–15h)until alllactosewasconsumed(reachingacellmassof0.85±0.21g/lat

24h).After48hthecellmassfinallyreached0.95±0.35g/l,while productionofEPScontinued(0.057␮g/ml,hduringthewhole48h

ofcultivation),reachingafinalconcentrationof2.75±0.13␮g/ml Thecellmassobtainedinmaltosesupplementedcultivations resembledthatoftheglucosesupplementation(1.3±0.28g/lafter

15h,maintainedat24has1.4±0.14g/l,withanoverallmaltose consumptionrateof0.036g/l,hduring24h)(Fig.1).TheEPS con-centrationinthiscasereached3.3±0.73␮g/ml(after48h)which wasthehighestobservedat1g/Lsupplementationlevel,reachinga productionrateof0.22␮g/ml,hbetween6–15h,whichdecreased

to0.031␮g/ml,hinthestationaryphase(24–48h)

Sucrosesupplementedcultivationsweremoredifficultto inter-pret,asthissubstratewasnotconsumedbyR.marinusDSM4252T (sucroseconcentrationwas0.87±0.03g/lafter48hofcultivation) Themaximumcellconcentrationwas1.15±0.21g/landthe con-centrationofproducedEPSwas1.98±0.85␮g/mlafter48h.The aboveresultsshowedthatalthoughtheeffectsoncellmasswere rathersmall,productionofEPSincreaseduponadditionofthe dis-accharideslactose(stationaryphase)andmaltose(primarilyinthe exponentialbutalsointhestationaryphase)

Afurtherincreaseintheconcentrationofaddedsugarsto10g/l resultedinasignificantincreaseintheproductionofEPS(except forsucroseaddedcultures),whichwasmostpronouncedfor cul-tureswithaddedlactose(Fig.2,Table1).Nosignificantincrease

incellmassorgrowthratewasobserved,andcellgrowthceased after15hatallconditionstested,whichmightbetheconsequence

ofeitheroxygenlimitation(whichisdifficulttocontrolinshake flasks) ormore likely thedecrease in pHobserved (decreasing from7.2to5.03, 5.18,and4.84inglucose,lactose,andmaltose medium,respectively).At10g/Lsupplementationlevelitwasalso observedthatmaltosewasgraduallydegradedextracellularlyto glucose(from6hofcultivation)

StrainMAT493grewwithoutvisibleaggregationandconsumed glucose,lactose and maltose withsimilarrates (0.14,0.14, and 0.135g/l.h,respectively)inmediawith1g/lsupplementationof therespectivecarbonsource.In addition,strainMAT493could consumesucrose(whichwasnotthecaseforthetypestrain)with

aconsumptionrateof0.065g/l.h.Allsugarswereconsumedwithin

15hofcultivation

Inaccordancewiththetypestrain,productionofEPSstarted

at the beginning of the cultivation and the concentration

of EPS increased during the exponential growth phase After

24h, the concentration of EPSs was 0.282±0.03, 0.29±0.08, and 0.3±0.18␮g/ml with corresponding cell concentrations of 0.95±0.07,1±0.28,and0.8±0.00g/linthepresenceofglucose, maltoseandsucrose,respectively.Thisshowsthatinthisstrain, theproductionofEPSintheexponentialgrowthphaseinprinciple wasindependentofthecarbonsourcesupplementation

In the stationary phase, the levels of EPS were, however, affecteddifferentlyand dependedoncarbonsource supplemen-tation.Inthemediasupplementedwith1g/Lsucroseandlactose, respectively, the EPS concentrationincreased with time in the stationary phase, in line with the pattern observed in lactose supplemented cultivations of thetype strain.For example, the concentrationofproducedEPSinthemediumcontaininglactose was0.2±0.15␮g/mlafter24h(atacellmassof0.5±0.14g/l)and thenincreased0.46±0.05␮g/mlafter48h.Incultivations supple-mentedwithmaltose,theamountofEPSwasalmostconstantafter

24h,alsoinlinewiththegeneralproductionpatternobservedfor thetypestrain

In both glucose supplemented and unsupplemented cultiva-tions,therewasanapparentdecreaseincellmassinthestationary

Table 1

Type of sugar Sugar concentration (g/l)

Table 2

Type of sugar Sugar concentration (g/l)

0 1 2 3 4

0 0.5 1 1.5 2

Time (h)

0 1 2 3 4

0

0.5

1

1.5

2

Time ( h)

0 1 2 3 4

0 0.5 1 1.5 2

Time (h)

0 1 2 3 4

0

0.5

1

1.5

2

Time (h)

0 1 2 3 4

0 0.5 1 1.5 2

Time (h)

phase.Intheglucosesupplementedmediumthiscoincidedwith

adecreaseinEPSconcentration,indicatingthattheremightbea

degradationoftheEPSbyactiveenzymesproducedbythecells,

whichmaybereleaseduponcelllysis(Mataetal.,2006).No

corre-spondingdecreasewashoweverobservedinthenonsupplemented

cultures(inthatcasethemonitoredEPSwas0.36±0.029␮g/mlat

24hand0.6±0.12␮g/mlat48h)

Anincreaseintheamountofaddedsugarto10g/lresultedin

ahigherrelativeproductionofEPSbystrainMAT493,forallthe

testedcarbonsourcesexceptsucrose(Fig.4;Table2 whichisin

accordancewiththepatternobtainedforDSM 4252T (Table1)

Degradationofthedisaccharidetoitsmonosaccharidecomponents

(after6hofcultivation)wasobservedformaltose(inaccordance

withthedataforthetypestrain)butalsoforsucrose(resultingin detectionofbothglucoseandfructose).TheincreaseinEPS produc-tioninlactoseandmaltosesupplementedcultureswasalsomore pronouncedforMAT493(Table2)thanforDSM4252T,whilecell massproductionwasapproximatelyinthesamerange.ThepH valueintheculturescontainingglucose,lactose,sucroseand mal-tosewasalsoshowntodecreasesignificantly(to4.65,4.39,4.47, and4.54,respectivelyafter48h)andmaybeareasonforstopped growthinshakeflasks

Inconclusion,therelativeefficiencyofEPSproductionwhichis theratioofthetotalEPStocelldryweightafter48h,wasevaluated andshowedthatmarinebrothsupplementedwith10g/llactose resultedinthehighestEPSproductionefficiencyinbothR.marinus

0 2 4 6 8 10 12

0 2 4 6 8 10 12 14

Time (h)

0 2 4 6 8 10 12

0 2 4 6 8 10 12 14

Time (h)

0 2 4 6 8 10 12

0 2 4 6 8 10 12 14

Time (h)

0 2 4 6 8 10 12

0 2 4 6 8 10 12 14

Time (h)

0 1 2 3 4

0

0.5

1

1.5

2

Time (h)

0 1 2 3 4

0 0.5 1 1.5 2

Time (h)

0 1 2 3 4

0 0.5 1 1.5 2

Time (h) 0

1 2 3 4

0

0.5

1

1.5

2

Time (h)

0 1 2 3 4

0 0.5 1 1.5 2

Time (h)

DSM4252T and MAT493followed bymarinebrothcontaining

10g/lmaltose(Tables1and2)UsingthesetwocarbonsourcesDSM

4252TwasshowntoproduceahigheramountofEPS/CDWatlower

concentrationofthecarbonsource,whilestrainMAT493appeared

tobemoredependentontheamountofcarbonsourcesuppliedfor

itsEPSproduction

3.2 Purificationandfractionationoftheexopolysaccharide

Thecrudeexopolysaccharidesobtainedfromthedifferent

sup-plementedculturesofR.marinusDSM4252T andMAT493were

fractionatedusingsizeexclusionchromatographyasdescribedin section2.6.Resultsshowedonemajorpeakforeachsample,which correspondedtoahighmolecularweightfraction.Theretention timeofthemajorpeakinthecrudeEPSsfromR.marinusDSM4252T

inthemediacontainingglucose,lactose,maltose,sucrose,andthe mediumwithoutadditionalsugarswas138.35,138.56,134.14,138, and135.41min,whichcorrespondedtomolecularweightsof73.8, 73.5,80.8,74.4,and78.6kDa,respectively.Also,theretentiontimes

ofthemajorpeakinthecrudeEPSsfromR.marinusMAT493in themediacontainingglucose,lactose,maltose, sucrose,and the medium without additionalsugars was130.07, 133.33, 131.49,

0 2 4 6 8 10 12

0 2 4 6 8 10 12 14

Time (h)

0 2 4 6 8 10 12

0 2 4 6 8 10 12 14

Time (h)

0 2 4 6 8 10 12

0 2 4 6 8 10 12 14

0 2 4 6 8 10 12

0 2 4 6 8 10 12 14

Time (h)

130.01,and128.81mincorrespondingtoamolecularweightof

88.1,82.2,85.5,88.2,and90.5kDa,respectively(Supplementary

data).Generally,theEPSsproducedbymarinebacteriaareoften

linearwithanaveragemolecularweightrangingfrom1×105to

3×105Da(Poli,Anzelmo,&Nicolaus,2010)whichiscompatible

withthemolecularweightofourproducedEPSs

3.3 CharacterizationofEPSmonosaccharidecontent

Afterhydrolysis,themonosaccharidecompositionofthe

puri-fiedEPSswasanalysedbyHPAEC-PADandtheanalysisshowedthat

alltheEPSswereheteropolysaccharides(Table3).Themain

com-ponentsofthepureEPSsfromR.marinusDSM4252Twerexylose,

arabinose,andglucose.Also,therewasamixtureofgalactosewith

glucosamine,andamixtureofmannosewithanaminosugarwhich

mightbeN-acetyl-glucosamineorN-acetyl-galactosamine(data

notshown).Quantificationofthosecomponentswashowevernot

possibleduetooverlappingpeaks

AnalysisofthepureEPSsfromMAT493allowedquantification

ofglucose,arabinose,xylose,andmannose(Table3).Alsointhis

strain,therewasasmallquantityofgalactoseandgalactosamine

InallEPSschromatogramstherewerethreeunidentifiedpeaks

whichneedstobefurtherinvestigatedsincetheidentificationof

themwiththeknownmonosaccharidestandardswasnot

success-ful(Supplementarydata)

ArabinoseandxylosearenotcommonsugarsinbacterialEPSs

(Ahmedetal.,2013;Nicholsetal.,2005).Thus,itcanbeclaimed

thattheEPSsproducedbytheR.marinusstrainsareunique

bac-terialEPSs.Interestingly,theratioofmonosaccharidesalsodiffers

betweenthetwostrains,indicatingthatEPSfromtherespective

strainsmaybeusefulfordifferentpurposes

3.4 Functionalgroupanalysis

InordertoinvestigatethefunctionalgroupsofthepurifiedEPSs

ofR.marinusDSM4252TandR.marinusMAT493FT-IRspectroscopy

wasused(Fig.5 andbandassignmentsweremadeaccordingto

literaturedata

TheIRspectraofthepurifiedEPSsofR.marinusDSM4252Tfrom allmediashowedthesamefunctionalgroups(Fig.5A)and exhib-itedabroadpeakataround3335cm−1 (range3600–3200cm−1) forO Hstretchingvibrationofthepolysaccharide(Kavita,Singh, Mishra, & Jha, 2014) and two weak C H stretching bands at

2924and 2855cm−1.The peakat 2359cm−1 wasattributedto

NH stretchingabsorptionbandandthepeak at1652cm−1 cor-respondedtoa C O stretchingvibration of theN-acetyl group

or protonated carboxylic acid (Ahluwalia & Goyal, 2005; Lillo, Cabello,Cespedes,Caro,&Perez,2014).Also,at1540cm−1apeak wasobservedwhichwasassignedtotheN Hdeformation vibra-tion of an amine group (Lillo et al., 2014) The peak at 1521 wasassignedtothesecondary amidgroup(Ahluwalia&Goyal, 2005).Anotherpeakat1418cm−1couldbeattributedtothe sym-metric stretching of the COO− group (Zhao, Yang, Yang, Jiang,

&Zhang,2007).Thepeakat1217cm−1correspondedtoan

O-S-O group that is an evidence of sulfate esters (Na et al., 2010) andthepeak at1103cm−1 mightbeassigned toO-acetylester linkeduronicacid(Kavitaetal.,2014).Thestrongabsorptionat

1039cm−1 in the range of 1200–1000cm−1 which is anomeric region, was attributed to C O C and C O groups in polysac-charidesandsuggested thatthemonosaccharideintheEPShas pyranosering(Vijayabaskar,Babinastarlin,Shankar,Sivakumar,& Anandapandian,2011).Theweakabsorptionat910and890cm−1 wasassignedtothecoexistanceof␣andßglycosidicbond(Lim

etal.,2005).Thepeakat818cm−1candeterminedtheexact posi-tionof6-sulfateofD-galactoseunit(C6-O-S)(Macieletal.,2008; Prado-Fernández,Rodrı´ıguez-Vázquez,Tojo,&Andrade,2003).The weakabsorption at845cm−1 demonstrated thepresence of 4-sulfateofD-galactose(C4-O-S)(Prado-Fernándezetal.,2003).The peakat770cm−1mightbeattributedtothe(S-F)stretching absorp-tionband(Pretsch,Fernández,Alvarez,2000)andtheabsorption peak at600cm−1 wasattributed tostretchingof alkyl-halides (Kavitaetal.,2014)

According to FT-IR band assignments of the purified EPSs

of R.marinus DSM 4252T from marine brothwith and without addedsugars,theEPScontainssulfatedpolysaccharidesof com-plexstructurecontaininguronicacids.Sulfatedexopolysaccharide derivativesareknowntohaveadvantageousproperties,in

partic-Table 3

ularastherapeuticsubstances.Bestknownareheparin(extracted

fromporcineintestinalmucosaasanticoagulantand

antithrom-boticagentinthepreventionandtreatmentofvenousthrombosis)

andfucoidanfromBrownseaweed,whichhasbeenreportedhaving

arangeofbioactiveproperties.SulfatedEPSfrombacterialorigin are lesswellknown,but mauran, a highlypolyanionic sulfated

beenreportedtohaveantioxidant,antihemolyticand

antithrom-bogenicactivities(Raveendranetal.,2013).Novelpolysaccharides

frombacterialoriginofferanalternativetothewell-knownanimal

varietiesandwillalsoexpandthepotentialrangeofactivitiesand

potencyofEPSderivedhealthpromotingagents

TheFT-IRspectraofthepurifiedEPSsofR.marinus493(Fig.5B)

wasinprinciplesimilartoFT-IRspectraofR.marinusDSM4252T

However,thepeakat910cm−1hadstrongabsorptionwhich

cor-respondedtoaß-glycosidicbondandthepeakat818cm−1was

absent

Inconclusion,bothR.marinusDSM4252TandR.marinusMAT

493producedexopolysaccharides.Differentnutritionalconditions

influencedtheproductionoftheEPSs.ThehighestEPSproduction

efficiencywashoweverforbothstrainsfoundinmarinebroth

sup-plementedbylactosefollowedbyamaltosesupplementedmarine

broth.MonosaccharideanalysisshowedthattheproducedEPSsare

heteropolysaccharidesmainlyconsistingofxyloseandarabinose

TheFT-IRspectrumof theEPSsshowedthepresenceofsulfate

andcarboxylgroupswhichdemonstratedthattheycontainuronic

acids.Italsorevealedthepresenceofaminosugarstogetherwith

acetylgroup.Theunusualfunctionalgroupsandmonosaccharide

compositionmakestheEPSofR.marinusinterestingforfurther

studies,motivatingmoredetailedanalysisofitschemicalstructure

andsuchstudiesareinprogress

Acknowledgment

The authors gratefully acknowledge the EU FP7 program

SEABIOTECHforfinancialsupport

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.carbpol.2016.08

062

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