Exopolysaccharide production from Bacillus velezensis KY471306 using statistical experimental design

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Environmental

Microbiology

Exopolysaccharide

production

from

Bacillus

velezensis

KY471306

using

statistical

experimental

design

Saad

A.M.

Moghannem

∗

,

Mohamed

M.S.

Farag,

Amr

M.

Shehab,

Mohamed

S.

Azab

Al-AzharUniversity,FacultyofScience,BotanyandMicrobiologyDepartment,Cairo,Egypt

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14July2016 Accepted9May2017

Availableonline18January2018 AssociateEditor:SolangeI. Mussatto Keywords: Bacterialexopolysaccharide Plackett–Burman Box–Behnken DEAE-cellulose

a

b

s

t

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c

t

Exopolysaccharide(EPS)biopolymersproducedbymicroorganismsplayacrucialroleinthe environmentsuchashealthandbio-nanotechnologysectors,gellingagentsinfoodand cosmeticindustriesinadditiontobio-flocculantsintheenvironmentalsectorastheyare degradable,nontoxic.ThisstudyfocusesontheimprovementofEPSproductionthrough manipulationofdifferentcultureandenvironmentalconditionsusingresponsesurface methodology(RSM).Plackett–Burmandesignindicated that;molasses,yeastextractand incubationtemperaturearethemosteffectiveparameters.Box–BehnkenRSM indicated that;the optimumconcentrationforeachparameterwas12%(w/v)formolasses,6g/L yeastextractand30◦Cforincubationtemperature.Themostpotentbacterialisolatewas identifiedasBacillus velezensis KY498625. After production,EPSwas extracted,purified usingDEAE-cellulose,identifiedusingFouriertransform infrared(FTIR),gelpermeation chromatography(GPC) andgaschromatography–massspectroscopy(GC–MS).Theresult indicatedthat;ithasmolecularweight 1.14×105_{Dconsistingofglucose,mannoseand} galactose.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Microbialexopolysaccharidesarethemostsignificantgroup of biopolymer.1 _{Depending on their location, EPS} repre-sented either in capsular form or slimy layer.2 _Microbial exopolysaccharideare mainlycomposedofsugar polymers thatareproducedbylargenumberofmicroorganisms includ-ingbacteria,fungiandyeasts.3_{Naturalbiopolymers(bacterial} polysaccharides) are biodegradable and biocompatible in

∗ _{Correspondingauthor.}

E-mail:[email protected](S.A.Moghannem).

comparisonwithharmfulsyntheticpolymers.4_Overthepast fewdecades,thenumberofknownexopolysaccharide(EPS) producedbymicrobialfermentationhasgraduallyincreased. Microbial biopolymers have many application in biotech-nology fields including pharmaceutical, tissue engineering, cosmetics, food, textile, oil recovery, metal mining and metalrecovery.5,6_{TheproductionofmicrobialEPSarehighly} affectedwithnutritionalandenvironmentalconditions.7,8

Comparedwithconventionalmethods,RSMcanbeused todesignexperiments,buildmodels,searchoptimumfactors fordesirableresponses,andevaluatetherelativesignificance ofseveralinfluencefactorseveninthepresenceofcomplex interactions.9_{Theneweraofmicrobialbiopolymerproduction}

https://doi.org/10.1016/j.bjm.2017.05.012

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

onindustrialscaleisdirectedfortheproductionfrom inex-pensivesourceslikeagro-industrialwastes.10_{Molassesisthe} finaleffluentobtainedintheproductionofsugarbyrepeated crystallization.11_{Sugarcanemolassescouldbeabettersource} ofcarbonduetoitshighercontentoftotalsugarsat48.3%. Largescale productionofmicrobialpolysacchariderequires intensiveresearchactivitiesfortheapplicationofinnovative ideasonalargescale.Thisresearchaimedtoenhance EPS productionfrommostpotentbacterialstrainusingpowerful statisticalexperimentaldesignasoneofthemostimportant stepstowardproductionattheindustriallevel.

Material

and

methods

Isolationandpurificationofbacteriaproducing exopolysaccharide(EPS)

Sevensoil sampleswere collected from Al-Bahariya Oasis, Egypt28◦24¢17N28◦52¢25E.Eachsamplewasseriallydiluted from10–1 _to10–7 _{inphosphatebufferedsaline,pH7.2±0.2.} OnemLofeachdilutionwasinoculatedintotubes contain-ing 9mL EPS culture medium [0.2g KH2PO4; 1.5g K2HPO4; 0.2gMgSO4.7H2O;0.1gCaSO4.2H2O;2.0mgFeCl3;0.5gyeast extract,20gsucrose(perliter)].BacteriaproducingEPS (char-acterized by colonies of bacteria that form thick slime or mucoid)wasselectedandpurified.12

DeterminationofEPSyield

Bacterialculturewasmixedwithtwovolumesofcold abso-luteethanol,and centrifugedfor10minat10,000rpm.This stepwasrepeatedtwicetoremoveimpuritiesoflowmolecular weight.Exopolysaccharidewasdissolvedindeionizedwater andquantifiedusingphenol-sulfuricacidmethod.13_All exper-imentwasrepeated intriplicatetocalculatemeanvalueof results.

Preparationofcanemolassesasthecarbonsource

Sugarcanemolasseswasobtainedfromasugarfactoryat (sug-arcanefactoryinKafrElSheikh,Egypt)thathasbeenused ascarbonsource.Molasseswasdilutedwithdistilledwater containingsodiumdi-hydrogenorthophosphate(2.0g/L)with ratio1:1.Thesolutionwasautoclavedat121◦Cfor15minand lefttosettlefor24h.14Theclarified molasseswereusedas solecarbonsourceatdifferentconcentrations1,2,3,4,5,6,7 and10%.

Experimentaldesignsandoptimization

Classicalonefactoratatime:thismethoddependonchangein onefactoratatimewhereotherfactorsremainingconstant.15 Single-factor experimentswere carried out in500mL flask containing100mLmediumat200rpm/minand30◦C(glucose, sucrose,lactose,maltose,solublestarch,fructoseand sugar-canemolasses),sixnitrogensources(yeastextract,peptone, urea,(NH4)2SO4, NH4Cl andKNO3). Statisticalanalysiswas generatedbyMicrosoftexcel2010softwareandplevelsat0.05 wereconsideredassignificant.

OptimizationofEPSusingPlakett–Burman(PB)design

ThePB experimentaldesignwas appliedtoscreenthe sig-nificant variables that influence EPS production.16 _Eleven variablesofmediumcompositionandcultureconditionswere testedatlow(−1)andhigh(+1) levelsasshowninTable1. BasedonPBmatrixdesign,two-levelfactorialdesign,itallows theinvestigationof12variables(n+1).17_{Thisdesignconsisted} ofthreereplicatedcenterpointstoavoiderror.

Y=ˇ0+



ˇixi (1)

whereYisthepredictedresponse,ˇ0isthemodelintercept;ˇi

isthelinearcoefficientandXiistheleveloftheindependent

variable.

EPSproductionwasmeasuredintriplicateand the aver-agevaluewastakenastheresponse.Thevariablessignificant at 95% level (p<0.05) were considered to have signifi-cant effect on EPS production and thus used for further optimization.

Responsesurfacemethodology

Box–Behnkendesign(BBD)withthreefactorsandthree lev-els, including three replicates at the center point, were used forfittingasecond-order responsesurface toprovide a measure ofprocess stability and inherent variability.18,19 Thecenter points and parameterswere selected according to Plackett–Burman design. Fifteen trials for three differ-ent variables including medium consisted of sugarcane molasses, yeast extract and incubation temperature were designed. Three levels, “high (+1)”, “middle (0)” and “low (-1)” as shown in Table 2. Regression analysis was per-formedonthedataobtainedusingsoftwarepackage‘Design Expert’ software (Version 7.0) and confirmed by Microsoft Excel 2010.20 _{The accuracy of polynomial model} equa-tion was expressed by coefficient of determination R2_{. All} experimentswereperformedintriplicates.Thedesignis rep-resentedbyasecond-orderpolynomialregressionmodelas follows: Y=ˇ0+



ˇixi+



ˇ2 iixi+



ˇijxixj+˙ (2)

whereYisthepredictedresponse,ˇ0istheintercept,ˇiisthe

linearcoefficient,ˇij istheinteractivecoefficients,␤iiisthe

quadraticcoefficients,andistheerror,andxiandxjarethe

codedindependentvariables.

Identification

of

bacterial

isolate

Identification of most potent bacterial isolate was based on 16S rDNA sequence analysis.This stepwas carriedout inSigma CompanyforResearch,Cairo,Egypt.Forward and reverseprimersusedforPCRamplificationwere:

27f 5AGAGTTTGATCCTGGCTCAG3 and 1492r 5GTTACCTTGTTACGACTT3.

Table1–Plackett–BurmanexperimentaldesignforscreeningofcultureconditionsaffectingEPSproduction.

RUN X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 EPSg/Lobserved Predicted

1 −1.00 −1.00 −1.00 1.00 −1.00 1.00 1.00 −1.00 1.00 1.00 1.00 5.8±0.21 5.8 2 1.00 −1.00 −1.00 1.00 1.00 1.00 −1.00 −1.00 −1.00 1.00 −1.00 5.42±0.16 5.55 3 1.00 1.00 1.00 −1.00 −1.00 −1.00 1.00 −1.00 1.00 1.00 −1.00 6.48±0.22 6.61 4 −1.00 −1.00 −1.00 1.00 1.00 −1.00 1.00 1.00 1.00 −1.00 −1.00 3.01±0.31 3.14 5 1.00 −1.00 −1.00 −1.00 −1.00 1.00 −1.00 1.00 1.00 −1.00 1.00 7.05±0.28 7.18 6 1.00 1.00 1.00 1.00 −1.00 1.00 1.00 1.00 −1.00 −1.00 −1.00 6.76±0.44 6.76 7 −1.00 1.00 1.00 −1.00 1.00 1.00 1.00 −1.00 −1.00 −1.00 1.00 2.92±0.13 3.05 8 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 −1.00 2.15±0.35 2.15 9 −1.00 1.00 1.00 −1.00 1.00 1.00 −1.00 1.00 1.00 1.00 −1.00 3.69±0.37 3.69 10 −1.00 1.00 1.00 1.00 −1.00 −1.00 −1.00 1.00 −1.00 1.00 1.00 2.44±0.21 2.57 11 1.00 −1.00 −1.00 −1.00 1.00 −1.00 1.00 1.00 −1.00 1.00 1.00 1.96±0.14 1.96 12 1.00 1.00 1.00 1.00 1.00 −1.00 −1.00 −1.00 1.00 −1.00 1.00 4.65±0.65 4.65 13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.84±0.29 4.69 14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5.13±0.22 4.69 15 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.94±0.51 4.69

X1,yeastextract(g/L);X2,molasses(g/L);X3,MgSO4(g/L);X4,K2HPO4(g/L);X5,KH2PO4(g/L);X6,peptone(g/L);X7,incubationtime(h);X8,

inocolumsize(%);X9,incubationtemperature(◦C);X10,pH;X11,shaking(rpm).

Table2–ExperimentaldesignandresultsofBox–Behnkenoptimizationexperiment.

Trials X1(molasses) X2(yeastextract) X3(incubationtemp.) EPS(g/L)

Codedlevel Reallevel(%) Codedlevel Reallevel(g/L) Codedlevel Reallevel(◦C) Observed Predicted

1 0.00 8 1.00 6 −1.00 30 6.92±0.21 7.05 2 1.00 12 −1.00 2 0.00 35 4.55±0.07 4.53 3 0.00 8 1.00 6 1.00 40 7.78±0.51 7.48 4 1.00 12 0.00 4 −1.00 30 6.90±0.09 6.62 5 −1.00 4 0.00 4 1.00 40 5.31±0.02 4.99 6 1.00 12 0.00 4 1.00 40 5.44±0.11 5.59 7 1.00 12 1.00 6 0.00 35 7.16±0.19 7.29 8 0.00 8 −1.00 2 −1.00 30 5.36±0.44 5.65 9 −1.00 4 0.00 4 −1.00 30 4.78±0.66 4.62 10 −1.00 4 −1.00 2 0.00 35 3.97±0.08 3.83 11 0.00 8 −1.00 2 1.00 40 4.69±0.02 4.55 12 0.00 8 0.00 4 0.00 35 5.88±0.11 5.64 13 −1.00 4 1.00 6 0.00 35 5.39±0.09 5.40 14 −1.00 4 0.00 4 1.00 40 4.41±0.58 4.99 15 0.00 8 0.00 4 0.00 35 5.41±0.06 5.64

X1,molasses(%);X2,yeastextract(g/L)andX3,incubationtemperature(h).

PCRsequence was carried out inGATC(Guanin Adenin ThyminCytosine)GermanCompanyusingABI3730X1DNA sequencer. The sequence of closely related strains were retrieved forconstructing the phylogenetic tree to confirm similaritiesofmostpotentstrainwithotherrelatedgroups. Thesequence obtainedwassubjectedtoBLASTsearchand the bacterial specieswere determined. Thepercentages of sequencematchingwerealsoanalyzedandthesequenceswas submittedtoNCBI-Gen Bank andobtainedaccession num-bers.

Purification

of

EPS

by

diethylaminoethyl-cellulose

(DEAE-cellulose)

DEAE-cellulosewas used asanionexchanger Petersonand Sober,21 _{Ion exchange chromatography was performed for} purificationofthe EPSbythe DEAE-Cellulose columnafter

first purification bytrichloroaceticacid.It wasequilibrated with0.05MTris–buffer(pH7.5)tobeusedin chromatogra-phy.ThelyophilizedcrudeEPSsamplewasdissolvedinsmall volume of0.1M NaCl.Thenfractions was initiallypurified byDEAE–cellulosecolumnchromatographyandelutedwith 25mMTris–HCl(pH8.5).Polysaccharidecontainingfractions weredeterminedforthepresenceofthetotalsugarby phenol-sulfuricacidmethod.13_{Thefractionscontainingsugarwere} pooled,concentratedbyultra-filtration,andprecipitatedwith 2volumesofethanol,respectively.

Characterization

and

identification

of

purified

fraction

Thepurifiedfractionwascharacterizedusingthefollowing spectroscopicanalysis,MolecularWeightDistributionAssay bygelpermeationchromatography(GPC)(Agilent1100series), Infrared IR (Matson Satellite 113 spectrometer), gas mass

chromatographyaccordingtoSeviouretal.22_(Shimadzu GC-MS-QP5050ThermoScientificProp).Thepeakswereidentified andcomparewithbywiththestandardmonosaccharide.

Result

Isolationandpurificationofbacteriaproducing exopolysaccharide(EPS)

Themostpotentbacterialisolatesproducing exopolysaccha-ridewereM7(30),E1(52)and9I(55)thathavebeenselected fromsixtyisolate.Theisolate9I(55)wasthemaximum pro-ductivityofabout3mg/mLthathasbeenselectedforfurther studyofresearch.Theselectionwasbasedonbothropystrand formationandquantitativemethodbyphenolsulphuricafter ethanolprecipitationasshowninFig.1a.Thebacterial iso-late9Iwashighestformucoidandropyformationafter24h ofincubationasshowninFig.1b.

Single

factor

optimization

design

and

data

analysis

Ourresultsshowedthattherewasstrongcorrelationbetween bacterialgrowthandexopolysaccharideproductioninculture mediawheretheisolate9I(55)exhibitedlagphaseduring16h ofgrowth(noproductivityofEPS).After18hofincubation;the logphaseofbacterialgrowthwasmaximumuntil40hwere theproductivityofexopolysaccharidestarttoincreasewith increasingbacterialgrowthwhilethemaximumproductivity

ofEPSwasduringStationaryphase(after48h)andstill con-stantuntil80hofincubation(Fig.2).

Toelucidatetheoptimalgrowthmediumcompositionfor maximalEPSproduction,variousfactorswereassessed. Dif-ferent carbon and nitrogen sources were used, the result obtainedfromthis experimentindicatedthat; thebest car-bonsource4.2g/Lwasmolassesat4%w/vwhilethelowest concentrationwas1.88g/Lwithstarch(4%w/v)(Fig.3).The bestnitrogensource4.4g/Lwasyeastextractatconcentration 3g/L(Fig.4).

ScreeningsignificantvariablesusingPlackett–Burman design

Elevenfactorsofmediacomponentswereexaminedwithfifty differenttrials,andmaximumEPSproductionwasobtained fortrialnumber(5),whilethelowestproductionwasobtained fortrialnumber(theregressioncoefficients(tvalue)and con-fidence level are given in Table 3. Themedia components showedbothpositiveandnegativeeffectsonEPSproduction. Statisticalanalysis(tvalues)demonstratedthatmolassesand yeastextractandincubationtemperaturehadsignificant pos-itive influencesonthe EPSproductionwithmaineffectsof 1.57,1.28and1.01respectively(Fig.5)whereasthemedia com-ponentsnamelyKH2PO4(X5),andMgSO4(X3)werefoundto decreasetheEPSproductionattheirhigherlevel.TheANOVA ofPlackett–Burman(PB)designforEPSproductionisshownin

Table3wherethedeterminantcoefficientR2_{ofthefirst-order}

modelwas0.995forEPSproduction.ThesignificantF-values

ofthemodelswere 0.0095indicatingthatthemodelswere significant.

A

B

Fig.1–Thetwoselectioncriteriaforthemostpotentbacterialisolatewere:(A)EPSprecipitationusingethanol70%and(B) istheropystrandformedby9Istrain.

2.5 2 absorbance at 660 EPS (g/1) 1.5 0.5 0 0 8 16 24 32 40 48 56 64 Time (hr) 72 80 88 96 104 112 120 7 6 5 4 3 2 EPS g/1 OD (600 nm) 1 0 1

5 4 3 2 1 0

Glucose Scurose Lactose

Different carbon sources

Maltose Starch Fructose Molasses

Absorbance 600nm

EPS (g/1)

EPS g/1

O

.D (600nm)

5 4 3 2 1 0

Fig.3–EffectofdifferentcarbonsourcesonEPSproduction.

absorbance 600nm EPS 5 4 3 2 1 0

different nitrogen source 5 4 3 O .D (600 nm) 2 1 0 EPS g/1 Yeast e xtract urea amm.sulphate amm.chlor ide potassium nitr ate peptone

Fig.4–EffectofdifferentnitrogensourcesonEPSproduction.

Table3–IdentificationofsignificantvariablesforEPSproductionofstrain(9I)usingPBdesign. EPSproduction

Variables Coefficients Standarderror tStat p-Value

Intercept 4.69 0.12 39.01 3.71 X1 Yeastextract 1.28 0.14 9.13 0.002 X2 Sugarcanemolasses 1.57 0.57 2.75 0.070 X3 MgSO4 −1.18 0.48 −2.42 0.093 X4 K2HPO4 0.58 0.14 4.12 0.025 X5 KH2PO4 −0.49 0.14 −3.47 0.04 X6 Peptone 0.65 0.14 4.61 0.01 X7 Incubationtime −0.13 0.14 −0.95 0.41 X8 Inoculumsize −0.47 0.14 −3.34 0.044 X9 Temperature 1.01 0.14 7.19 0.005 X10 pH −0.32 0.14 −2.30 0.10 X11 Shaking 0.037 0.14 0.26 0.80 Regressionstatistics

MultipleR 0.995 AdjustedRsquare 0.954

Rsquare 0.990 Standarderror 0.360

Responsesurfacemethodology

Basedonaboveresults,threekeyfactorsyeastextract, sugar-canemolassesandincubationtemperatureweresignificantly affectEPSproduction.Thesefactorswereselectedforfurther analysiswithBox–BehnkenDesign(BBD)(Table2).The follow-ingsecond-orderpolynomialequationwas:

Y=5.64+0.64X1+1.08X2−0.16X3+0.29X1X2−0.35X1X3

+0.38X2X3−0.55X1X1+0.17X2X2+0.36X3X3 (3)

where Y represents EPSproduction (g/L);5.64 isthe inter-cept; 0.64, 1.08 and −0.16 are the linear coefficients; 0.29, 0.35and0.38aretheinteractivecoefficients,−0.55,0.17and 0.36 are the quadratic coefficients; and X1, X2 and X3 are theconcentrationsofsugarcanemolasses,yeastextractand incubation temperature,respectively. Thestatistical signifi-canceofEq.(3)wasevaluatedbyF-testandANOVAanalysis

2 1.5 1 -1 -1.5 0.5 -0.5 0 Media component Main eff ect X1 X2 x3 X4 X5 X6 X7 X8 X9 X10 X11

Fig.5–EffectofenvironmentalandmediacompositiononEPSproductionbyPBdesign.

(Table4).Thethree-dimensionalgraphsthatobtainedfromEq.

(3)wereveryreliable,withR2_{valueof0.9401,whichindicated} that99%ofthevariabilityintheresponseexplainedbythis model(Fig.6).TheModelF-valueof8.72impliesthemodel issignificant.Thereisonlya1.40%chancethata“Model

F-Value”thislargecouldoccurduetonoise.Valuesof“Prob>

F”lessthan 0.0500indicatemodelterms are significant.In thiscase,themutualinteractionsbetweeneverytwoofthe threevariablesweresignificant.Bysolvingtheinversematrix usingExpert-Designsoftware,theoptimalconcentrationsof molasses,yeastextractandincubationtimewere12%(w/v), 6g/Land30◦C,respectively.

Molecular

characterization

Basedonsequencealignmentresultsrevealedthat;theisolate belongstobacillusspecieswithmaximumrelationto Bacil-lusvelezensiswith99%sequence similarities.Thesequence was deposited in NCBI and assigned withaccession num-berKY471306.Thephylogenetictreewasconstructedusing the neighbor-joiningtree making algorithmofthe MEGA 4 package(Fig.7).

Purification

of

bacterial

polysaccharide

by

DEAE

Ionexchangechromatographywasperformedforpurification oftheEPSbytheDEAE-Cellulosecolumnafforded61fractions, thefirstrecoveredpolysaccharideinthenineelutingsolution (fractionno.9to22)thatconfirmedthroughphenol-sulphuric acidassay(Fig.8).

Fourier

transform

infrared

spectroscopy

(FTIR)

ThepurifiedEPSpolymerobtainedfromDEAE-cellulose col-umnchromatographywaspreparedforIRanalysis.Thedata chart(Fig.9)showsthepresenceofmanyfunctionalgroups whicharetypicaltobacterialEPS.

Table4–IdentificationofsignificantvariablesforEPS productionofstrain(9I)usingPBdesign.

ANOVA df SS MS F SignificanceF

Regression 9 16.7 1.85 10.08 0.01

Residual 5 0.91 0.18

Total 14 17.6

Coefficients Standarderror tStat

Intercept 5.64 0.30 18.61 X1 0.64 0.14 4.43 X2 1.08 0.15 7.15 X3 −0.16 0.14 −1.13 X1X2 0.29 0.21 1.38 X1X3 −0.35 0.19 −1.76 X2X3 0.38 0.21 1.78 X1X1 −0.55 0.23 −2.33 X2X2 0.17 0.23 0.73 X3X3 0.36 0.23 1.55

Multiple R=0.973; R2 _{(coefficient of determination)=0.947; Adj.}

R2 _{(adjusted coefficient of determination)=0.853; model are}

significant.

Determination

of

molecular

weight

by

gel

permeation

chromatography

(GPC)

Theweight-average(Mw)andnumber-average(Mn) molecu-larweightsandpolydispersity(Mw/Mn)ofthepolysaccharide producedbyB.velezensiswasanalyzedbyGPCandfoundto haveaweightaveragemolecularweight(Mw)of1.29×105_Da,

numberaveragemolecularweight(Mn)of1.14×105_Daanda

sizeaveragemolecularweight(Mz)of1.47×105_Da(Fig.10). TheMolecularWeightandPolydispersityIndex(PDI)(Mw/Mn) of the exopolysaccharide was obtained as 1.13, which is a measure of the distribution of molecular mass in the sample.

A

B

C

Design-Expert® Software Design-Expert® Software EPS EPS 7.78 3.97 X1 = B: yeast extract B: yeast extract = 1.00 X2 = C: temperature Actual factor A: sugarcane molasses = 1.00 EPS 7.78 3.97 X1 = A: sugarcane molasses X2 = C: temperature C: temperature = -1.00 Actual factor Design-Expert® Software EPS 7.78 3.97 X1 = A: sugarcane molasses X2 = B: yeast extract Actual factor 7.9 6.925 5.95 4.975 4 1.00 0.50 0.00 -0.50 -1.00 -1.00 -0.50 0.00 B: yeast extract C: temperature 0.50 1.00 EPS 7.9 7.2 6.5 5.8 5.1 1.00 0.50 0.00 -0.50 -1.00 -1.00 -0.50 0.00 A: sugarcane molasses C: temperature 0.50 1.00 EPS 7.9 6.975 6.05 5.125 4.2 1.00 0.50 0.00 -0.50 -1.00 -1.00 -0.50 0.00 A: sugarcane molasses B: yeast extract 0.50 1.00

Fig.6–ResponsesurfaceplotsofthreevariablesinmediumonEPSproduction.(A)Interactionofmolassesandyeast extract;(B)interactionoftemperatureandyeastextract;(C)interactionofmolassesandtemperature.

Gas–mass

chromatography

(GC–MS)

MonomersfromtherepeatingunitoftheEPSproducedbyB. velezensis,wereanalyzedbyGC–MSgivingonemajorpeakat 13.59minwithsomeminorpeaksat9.73andat5.36min.The MSfragmentation patterns obtainedfrom GCgeneratedby

eachpeak,weresubsequentlyanalysesitshowsthe character-isticsofaldohexosessuchasglucose,galactoseandmannose insugaralditolacetateform.23_{Toidentifyeachpeak,theMS} wasgeneratedandthencomparedwiththeavailableMSof differentmonomers.TheMSfragmentationobtainedforthe peakat13.59min.givesusfragmentionswithm/zratioof55, 74,87,97,115,129,157,199,227and241(Fig.11).

Bacillus idriensis strain SMC 4352-2

Bacillus deserti strain ZLD-8

Bacillus acidicola strain 105-2

Bacillus pumilus strain NBRC 12092

Bacillus pumilus strain ATCC 7061

Bacillus licheniformis strain DSM 13

Bacillus aerius strain 24K

Bacillus velezensis strain AS 91(55)

Bacillus methylotrophicus strain CBMB205

Bacillus subtilis strain 168

Bacillus vallismortis strain DSM 11031

Bacillus axarquiensis strain LMG 22476

Fig.7–Neighbor-joiningtreeof16SrDNAofthelocalisolate9Iwithrespecttocloselyrelatedsequencesavailablein GenBankdatabases.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Fig.8–Estimationofbacterialpolysaccharidebyphenolsulfuricacidmethods.

50 45 40 35 30 25 20 15 10 5 4000 3000 2500 Wave number cm-1 % T ransmittance 3428.76 2950.83 2402.19 1676.94 1589.25 1433.50 1320.39 1181.84 1100.39 461.82 2000 1500 1000 500 3500

Fig.9–FTIRspectroscopyanalysisofBacillusvelezensispolysaccharideextractedshowingthetransmittancetroughat differentwavenumber.

2.5 2.0 1.5 1.0 0.5 7e4 _1e5 _2e5 _3e5 Molar mass [D] W(log M)

Fig.10–GPCchromatogramofpartiallypurifiedexopolysaccharidefromB.velezensis.

A

100 90 80 70 60 50 50 40 40 30 30 20 20 10 10 12 4 3 5 6 7 8 9 10 1 3 5 0 100 90 80 70 60 50 40 30 20 10 0 0 20 30 40 50 Time (min) Time (min) Relativ e Ab undance 10 0

B

Fig.11–(A)Gaschromatographicofstandardsugarareasfollows:1.Galactose;2.Fucose;3.Mannose;4.Xylose;5.Glucose; 6.Arabinose;7.Mannuronicacid;8.Glucuronicacid;9.Galacturonicacid;10.Mannuronicacid.(B)Gaschromatogramsof extracellularpolysaccharidesproducedbyB.velezensis.

Discussion

Exopolysaccharides (EPS) was extracted from B. velezensis

brothmediabyusingethanoltobacterialcultureinratio2:1 respectively.Thefirstscreeningofdifferentbacterialisolates forselectionofmostpotentEPSproducingstrain,the maxi-mumproductivityobtainedwas3g/Lfromisolate9I(55).This resultis agreed withthat obtainedby Reshetnikov et al.24 accordingtodifferentgrowthconditionsusedinthisstudy, EPSproductionbyB.velezensisincreasedduringthe station-aryphaseofgrowth(after32h)andstillconstantuntil80h ofincubation,Thiswasprobablybecauseoftheactionof gly-cohydrolasespossiblyproducedintheculturethatcatalyzed thedegradationofpolysaccharides,resultingindecreasedEPS yieldsafterstationaryphase.25 _{Theseresultsindicatedthat} the maximum productivity of EPS reachedpost stationary phaseof growth.Thisresultis similar tothat obtainedby RobersonandFirestone.26

Theexperimentofsinglefactoroptimizationwasdesigned toidentifythekeyvariableswhichcanaffectEPSproduction keepingotherfactorsconstant.27_{Theresultobtainedfromthis} experimentindicatedthat;EPSproductionwassignificantly affectedbythetypeofcarbonsourceandconcentrationinthe medium.Molasseswasmosteffectiveandeconomicallyfor

largescaleproductionofEPSamongthecarbonsources. Car-bonandnitrogensourcesarewellknownfactorsthataffect the cellularmetabolismand EPSproduction.28 _Thepresent studywascarriedouttostudytheinfluenceofvarious param-etersonelevationofEPSyield.Onefactoratatime,although difficult and labor intensive method, this experiment sup-portedinselectingthecenterpointsfortheoptimizationstudy usingRSM.Thismethodofusingsinglevariablesis disadvan-tageousastheinteractiveeffectsbetweentwofactorscannot bestudied.

Dataobtainedfromsinglefactoroptimizationwasfollowed byPlackett–Burmandesignforscreeningofsignificant vari-ablesthatdependonmulti-variablesatatime.29_Dataanalysis obtainedfrom this modelindicted theuse ofyeastextract asnitrogensourceand sugarcanemolassesduring produc-tion ofpolysaccharidesbyisolatecode(9I). Moreover,yeast extract could provide growth factorssuch asvitamins and aminoacidsthatsupportmuchbacterialgrowth.28

Withthesameconsequence;RSMdependondataobtained from Plackett–Burmanandthe dataanalysisindicatedthat the maximum yield was estimatedto be 7.68g/L, and the actualyieldobtainedwiththeoptimalmediumwas7.88g/L (theaveragevalueoftripleexperiments),whichwasinclose accordance withthemodelprediction.Inaddition,the sole carbon source was used in the fermentation medium for

maximum EPS production, which would be advantage to reduceproductioncostandoperationprocessofEPS.30

IRanalysisforpurifiedEPSshowedacharacteristic absorp-tionbandappearedat1654.11cm−1(Fig.9)andwasassigned tothestretchingvibrationofcarbonylgroup(C O)ina accor-dancewithGrigoriietal.31

Another absorption band at 2941.03cm−1 was intensi-fiedand assigned to the stretching vibrationof methylene group( CH2 ), usuallypresent inhexoses,like glucose or galactoseordeoxyhexoseslikerhamnoseorfucose. Absorp-tion peak at 1063.57cm−1 was assigned to carbohydrate C Ostretchingvibrationsanddominatedbyglycosidic link-age (C O C) stretchingvibration.ThisalsoagreewithHe etal.32

Absorption peak around1540cm−1 corresponding to an aminogroupwasalsoobserveinthespectrumofEPSanda weaksymmetricalstretchingpeaknear1447–1380cm−1, sug-geststhe presenceofcarboxyl groups.). Thecarbohydrates showhighabsorbenciesintheregion1200–950cm−1,thatis withintheso-calledfingerprintregion,wherethepositionand intensityofthebands arespecificforeverypolysaccharide, allowingitpossibleidentification.Theseresultsareharmony withSunetal.33

BasedontheresultofGC-massanalysis,itwasconcluded thatthemajormonosaccharideforpurifiedEPSobtainedfrom

Bacillus.velezensisKY498625revealedthatglucose,galactose andmannose.Cousoetal.34_{reportedthatG.hanseniiLMG1524} produceEPSthatcontainsglucose,mannosethatagreedwith ourEPS.

Conclusion

Two statistical experimental design Plackett–Burman and Box–Behnken were used to improve Exopolysaccharide production from Bacillus. velezensis KY498625. The pro-duction of EPS under optimal conditions obtained from data analysis succeeded to increase productivity from 4.1 to 7.6g/L compared to the initial EPS production. Indus-trial productionof EPS requires cost effectiveand optimal culture medium. The purified EPS has molecular weight 1.29×105_{Da with number average molecular weight (Mn)} 1.14×105_{Dathatconsistsmainlyofglucose,mannoseand} galactose.

Conflicts

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