Bacterial treatment of alkaline cement kiln dust using Bacillus halodurans strain KG1

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ht t p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Bacterial

treatment

of

alkaline

cement

kiln

dust

using

Bacillus

halodurans

strain

KG1

Kunal

a

_,

_Anita

_Rajor

b,∗

_,

_Rafat

_Siddique

c

a_Department_of_{Biotechnology,}_Thapar_University,_Patiala,_Punjab_147004,_India b_School_of_Energy_and_Environment,_Thapar_University,_Patiala,_Punjab_147004,_India c_Department_of_Civil_Engineering,_Thapar_University,_Patiala,_Punjab_147004,_India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received11January2013 Accepted6July2015

AssociateEditor:WelingtonLuizde Araújo

Keywords:

Alkaliphiles Alkalinityreduction

Bacillussp. Cementkilndust

a

b

s

t

r

a

c

t

Thisstudywasconductedtoisolateanacid-producing,alkaliphilicbacteriumtoreducethe alkalinityofcementindustrywaste(cementkilndust).Gram-positiveisolateKG1grewwell atpHvaluesof6–12,temperaturesof28–50◦C,andNaClconcentrationsof0–16%andthus wasfurtherscreenedforitspotentialtoreducethepHofanalkalinemedium.Phenotypic characteristicsoftheKG1isolatewereconsistentwiththoseofthegenusBacillus,andthe highestlevelof16SrRNAgenesequencesimilaritywasfoundwithBacillushaloduransstrain DSM497(94.7%).Onthebasisofitsphenotypiccharacteristicsandgenotypicdistinctiveness fromotherphylogeneticneighborsbelongingtoalkaliphilicBacillusspecies,theisolated strainwasdesignatedB.haloduransstrainKG1,withGenBankaccessionnumberJQ307184 (=NCIM5439).IsolateKG1reducedthealkalinity(by83.64%)andthechloridecontent(by 86.96%)ofcementkilndustandshowedapotentialtobeusedinthecementindustryfora varietyofapplications.

Introduction

Microorganisms havebeen isolated from avarietyof envi-ronmentsbutaremostabundantinmoderateenvironments. Severalenvironmentscanbeconsideredextreme,including environmentswithaloworhighpH,aloworhigh temper-ature,ahighsalinity,thepresenceoforganicsolvents,heavy metals,etc.1_The_{microorganisms}_inhabiting_extreme

environ-mentaretermed“extremophiles”.Extremophileshavegained considerablemomentum inresearchduetotheir abilityto

∗ _{Corresponding}_author._Permanent_address:_School_of_Energy_and_Environment,_Thapar_University,_Patiala,_Punjab_147004,_India.

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

growunderextremeconditionsandtoabateenvironmental pollution.2–4

Alkaliphilicbacteriahavemanypotentialapplicationsin thebiotechnologyduetotheirenzymes,includingproteases, cellulases,andxylanases,whicharestableathighpH(>9.5) andtemperature(>50◦C).5_The_enzymatic_activities_enable_the

bacteriatoadapttoalkalineenvironments,biodegradecertain xenobiotics,produceseveralmetabolites,anddetoxify detri-mentalmetalcompounds.6

Mostofthealkaliphilicbacteriastudiedbelongtothegenus

Bacillus; they may have different characteristics depending

http://dx.doi.org/10.1016/j.bjm.2015.11.001

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on the strain,7 _and _live _in _a _variety _of _habitats _such _as

soda lakes,deserts, and arid soils.8 _At_least₂₆ _alkaliphilic

and alkalitolerant Bacillus species have been identiﬁed to date,9 _which_constitute _the _sixth _rRNA_group _in_the_genus Bacillus.10

Theouter celllayersofthe alkaliphilecanmaintainthe intracellularpHvaluesnearneutralinanalkaline environ-mentatpH10–13.ThepresenceofNa+ _ions_in_the_growth

mediumplaysapivotalroleintheadaptationofalkaliphilic

Bacillus species to high pH values. Various studies have reportedthatBacillusstrainsshowvariationsinNaCl require-mentsduetospecies-speciﬁcdifferencesinhalotolerance.7

Guffantietal.11_observed_that_the_total_membrane_cytochrome

contentwasconsiderablyhigherincellsgrownatpH10.5than inthosegrownatpH7.5,exhibitingthelargestpH-dependent difference.Thegrowth pHalsoresultedindifferentprotein proﬁles.

Mostalkaliphiles have been isolated from neutral envi-ronments,althoughtheircountsarehigherinsamplesfrom alkalineenvironments.1_The_frequency_of_alkaliphiles_in_the

environment,especiallyinordinaryneutralsoilsamples,has beenshowntobe102_–105_per_gram_of_soil,_which_corresponds

to1/10–1/100ofthepopulationofneutrophiles.Forisolation ofalkaliphilic microorganisms invitro, the sample can be enrichedwithdifferentsubstratessuchaspeptones,glucose, bilesalts,casaminoacidsandcasein.12_An_alkaline_medium

hastobe usedbyadjusting pHtoaround 10 withsodium carbonate(Na2CO3)and/orBorax–NaOH,Na2HPO4/NaOH,or

KCl/NaOHbuffersystems,withbufferingcapacitiesoverapH rangeof9–12invariousmedia.

SincepHisanimportantparameterofindustrialwasteand needstobecontrolled,severalenvironmentalauthoritiessuch astheEnvironmentalProtectionAgency(EPA)intheUSand theCentralPollutionControlBoardinIndiahavesetguidelines directingthatthepHoftheefﬂuentorwastedischargedby industriesshouldbeintherangeof6.5–8.5.Variouschemicals suchashydrochloricacid,2_sulfuric_acid,13_phosphoric_acid14

andcarbondioxide15_are_available_to_neutralize_highly_alkaline

industrialwastebyproductsandwastewater.However,these chemicalsaredifﬁculttohandleastheyarehazardousand corrosive,andlargequantitiesofacidwouldberequiredto neutralizealkalinewastes,whichiseconomicallynotfeasible andhasadangerouseffectonthehealthofworkersaswellas onindustrialprocesses.

During the manufacturing of cement clinker, an alka-line waste byproduct known as cement kiln dust (CKD) is generatedandcollectedfromelectrostaticprecipitatorsand baghouses. The chemical composition of CKD may vary, dependingonthetypeofrawmaterialandthecement man-ufacturing process. During clinker production in the kiln, volatile sulfates, alkalis (K2O and Na2O) and chlorides are

preferentiallydrawntowardCKD.In2013,theIndiancement industrygeneratedapproximately42–56millionmetrictons ofcementkilndust,whichisestimatedtoaccountfor15–20% ofclinkerproduction.16_The_major_part_of_it_is_dumped_on_an

openlandaslandﬁllmaterial,because,duetoitshigh alka-linity,CKDisnotreusedinthecementkiln.Gebhardt17_and

MohamedandEl-Gamal18_treated_alkaline_CKD_with_carbon

dioxidegastoremovethealkalinity,butthesemethodsare highlyexpensiveandlaborious.Abetteralternativetotheuse

ofachemicalprocessisbiologicaltreatmentofalkalinewastes usingalkaliphiles,whichgrowwellathighpH.

Thepotentialofalkaliphilicbacteriaforwastewater treat-mentwasreportedbyTanabeetal.19_Ntougias_et_al.20_reported

thatlargebacterialpopulationsexistedinalkalineolivemill wastes,andfacultativealkaliphilicbacteriasuchas Exiguobac-terium sp. were capable of lowering pH of highly alkaline wastewaterfrom 12.0to7.5.6 _These _studies_suggested_that

alkaliphilic bacteria could degradepollutants under highly alkalineconditions,andtheirsigniﬁcantadvantagewasthat theywerenoteasilycontaminatedbyneutrophilic microor-ganisms.However,reportsontheapplicationofalkaliphilic bacteriafortreatmentofsolidalkalinewastesareveryrare. Thus,inthepresentstudyweundertookthetaskof isolat-ingandidentifyingnewalkaliphilicbacteriafromsoil.Oneof theisolates,designatedstrainKG1andbelongingtothegenus

Bacillus,grew wellatveryhighpHvalues(∼11–12)andcan potentiallybeusedforalkalinityremovalforproperdisposal andreuseofcementkilndust.

Materials

and

methods

Isolationofalkaliphilicbacteria

Alkaliphilic bacterial strains were isolated from the rhizo-sphericsoilofacitrusplant(pH8.2),whichisahabitatfor avarietyofmicroorganisms.1,8 _An_enrichment_medium₍₁₀_g

glucose,10gpeptone,5gyeastextract,1gK2HPO4,and15g

bacteriologicalagar per 1000mLofwater, pH10.5adjusted with 1NNaOH)was used formicrobial isolation. Asterile glucosesolution wasaddedtothemediumaseptically.Soil sampleswerecollectedrandomlyfromalocationatThapar University,Patiala,India,and10gweresuspendedin100mL ofsterilizedwater.Thesuspensionwasseriallydiluted,and 100-␮Laliquotswerethenspreadonagarplatesandincubated at35±2◦Cfor48h.Microbialcolonieswereselectedonthe basisofcolonymorphologyandcolor.Singleisolatedcolonies werethenpickedandre-streakedonagarplateswiththesame mediumuntilpurecolonieswereobtained.Theplateswere storedat4◦Cforfurtheruse.

ThepureisolateswerescreenedfortheirtolerancetopH 11 and 12 on a minimal medium(M9), since components oftheenrichmentmediumprecipitateatsuchhighpH val-ues.M9mediumcontained(per1000mL):10gsucrose,2.5g K2HPO4,2.5gKH2PO4,1g(NH4)2HPO4,2gMgSO4·7H2O,0.01g

FeSO4·7H2O,and 0.007gMnSO4·4H2O,andpHwasadjusted

usingKCl–NaOH bufferinsteadofNaOHtoavoid precipita-tionorturbidityofthemedium.Thecoloniesisolatedonthe enrichmentmediumwerethensubculturedandadaptedto M9medium(pH11–12)andstoredat4◦Cforfurtheruse.

Screeningoftheisolatesforreducingthealkalinityof cementkilndust

Screeningoftheisolateswasdoneonthebasisoftheir capa-bility toreduce the pHof the alkalineliquid minimal and enrichmentmedia(pH∼12).Acidproductionbythemicrobial isolateswasmonitoredbydecreaseinpHatregularintervals

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Table1–Chemicalcompositionofcementkilndust

withoutandwithbacterialtreatment.

Chemicalcomposition (compound%) CKDcontrol CKD+strainKG1 CaCO3(C) 21.56(100) 39.40(+82.75) MgO(Mg) 0.69(100) 0.58(−15.94) Al2O3(Al) 2.38(100) 2.19(−7.98) SiO2(Si) 13.17(100) 10.97(−16.70) SO3(S) 1.13(100) 0.82(−27.43) K2O(MAD10feldspar) 1.12(100) 0.59(−47.32) Ca(wollastonite) 55.78(100) 48.19(−13.61) Fe2O3 2.62(100) 2.41(−8.01) CuO 0.89(100) 0.80(−10.11) ZnO 0.66(100) 0.64(−3.03) Equivalentalkali (Na2O+K2O) 0.74(100) 0.39(−47.30) Thevaluesinparenthesesdenotethepercentageofcompound increasedordecreasedcomparedtothecontrol.Thevalueswere statisticalsigniﬁcantatp<0.0001usingtwowayANOVA.

forupto5days(CyberScanpH510pHmeter).Eachtreatment wasdoneintriplicates.

A promising isolate was selected and used for the treatmentofCKD.TheCKDcollectedfromelectrostatic precip-itatorsatacementplantwasﬁnepowderedandgray-blackin color.Thechemicalcompositionofthesamplewasanalyzed byenergydispersiveX-rayspectrometry(EDX;JEOLJSM-6510 LV,USA)(Table1).TheCKDwasmixedwiththeisolated bac-terialstraindesignatedKG1atdifferentODvalues(ODvalue of1.0≈108_cells),_ranging_from_0.6_to_1.0,_and_a_ratio_of_CKD_to

cultureequalto4:1.Thetreatmentmixture(intriplicate)was incubatedat35◦Cfor20days, andsufﬁcientmoisturewas maintainedsothatCKDwasintheformofmoistpowder,not slurryorliquidmixture.Asucrosesolution(10%)wasaddedas acarbonsourceforthegrowthofthebacterialstrainonlyonce duringthetreatment,after5daysofincubation.Aerationwas providedbymanualmixingduringthetreatment.Asacontrol treatment,CKDwasincubatedundersimilarconditions with-outbacteria.After20daysofincubation,samplesweretaken fromthedifferenttreatments,mixedwithwater(1:10)in con-icalﬂasks,shaken(130rpmfor1h)togeneratealeachate,and analyzedforalkalinityandchloridecontent,alongwiththe controltreatment.21_The_pH_values_of_the_leachates_from_the

differenttreatmentswere alsomeasuredusingapHmeter (CyberScanpH510).Toconﬁrmadecreaseinalkalinity,a bac-terialtreatedsamplewasanalyzedbyEDXforachange in thechemicalcompositionandbyX-raydiffraction(XRD)fora changeinalkaliphases(suchasarcanite).Allsampleswere air-driedand groundtoaﬁnepowderbeforeEDX andXRD analyses.

ForEDXanalysis,samplesweremountedonbrassstubs usingcarbontape.TheXRDanalysiswascarried outusing the PANalytical X’Pert Pro system (Netherlands). Random mountedpowderspecimenswerescannedfrom10to60◦2. InterpretationoftheX-raypatternswascarriedoutby match-ingthepeakstothepatternsreportedinthe literatureand byusingstandard JCPDScards,adatabaseofX-raypowder diffractionpatterns maintainedbythe InternationalCenter forDiffractionData.

CharacterizationandidentiﬁcationofstrainKG1

Characterizationofthestrainwasperformedusingstandard biochemicaltests22 _and _16S_rRNA-based_phylogenetic

anal-ysis. Cell morphology was examined under a compound microscope (Nikon E100). Gram stainingwas performed as describedbyCappuccinoandSherman.23_Biochemical_tests,

namely,starchandgelatinhydrolysis,H2Sproduction,methyl

red-Voges Proskauer, citrate utilization, catalase activity, and carbohydrate utilization, were performed according to Aneja.22 _Growth _experiments _at_different_pH _values_(5–12),

NaCl concentrations (0–20%), and temperatures (20–55◦C) were conducted in both enrichment broth and minimal brothmedia.Theefﬁcientacid-producingalkaliphilic bacte-rialstrainwasthenpresumptivelyidentiﬁedasperSneath,24

CowmanandSteel’smanual25_and_Yoon_et_al.26

Sequencing of the 16S rRNA gene was performed by XcelrisLabs Limited,Ahmedabad(India).DNAwas isolated from bacterial strain KG1using the QIAamp DNA Purifica-tion Kit(Qiagen).The16SrRNAgenewasamplifiedbyPCR withuniversalbacterialprimers8F(AGAGTTTGATCCTGG CTC AG) and 1492R (ACG GCT ACC TTG TTA CGA CTT). The amplified PCR product was purified using the Qiagen MinEluteGelExtractionKitaccordingtothemanufacturer’s protocol. Sequencing of the purified 16S rRNA gene prod-uctwasperformedusingtheBigDye®_Terminator_v3.1_Cycle

SequencingKit(AppliedBiosystems,USA)asrecommended by the manufacturer and an ABI 3730xl Genetic Analyzer (Applied Biosystems, USA). The 16S rRNA gene sequence of strain KG1 was compared to the corresponding neigh-borsequencesfromtheGenBankdatabaseusingtheBLAST tool at the National Center for Biotechnology Information server(http://www.ncbi.nlm.nih.gov).Multiplealignmentsof the strain KG1 sequence with those from related Bacil-lus species were performed using the MultAlin program,27

and a phylogenetic tree was constructed by the neighbor-joining method.28 _Evolutionary _distance _matrices _for _the

neighbor joining method were calculated using the algo-rithm ofKimura’s29 _{two-parameter}_model. _The_topology_of

thephylogenetictreewasevaluatedbyperformingabootstrap analysiswith1000replicates.TheGenBank/EMBL/DDBJ acces-sionnumbersforthe16SrRNAgenesequencesofstrainKG1 andrelatedBacillusspeciesareshowninFig.5.

Statisticalanalysis

Statisticalanalysisofthedata(t-testandanalysisofvariance) wasperformedusingtheGraphPadPrism(version5)software, andthesigniﬁcancelevelwassetatp<0.05.

Results

and

discussion

Eightbacterialisolateswereobtainedfromthesoil,ofwhich threeisolates(strainsKG1,KG4andKG5)wereabletogrow atpH12.Amongthethreeisolates,strainKG1showed bet-ter growthinculturebrothathigherpHvalues(11 and12) thantheothertwostrainsaswellastheabilitytotolerateand grewathighpHvaluesbyadjustingitscellmetabolism,which isessentialforsurvival.30Fewstudieshavereportedtheuse

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14 Enrichment medium Minimal medium 12 10 pH 8 6 0.0 2.0 3.0

Time (in days)

4.0 5.0

Fig.1–ReductioninpHofthealkalinegrowthmediaby

BacillushaloduransstrainKG1.

ofbuffersystemssuchasphosphatebuffer11,31 _and

carbon-atebuffer11,30_to_increase_pH_of_the_growth_medium,_but_no

workhasbeenreportedontheuseofKCl–NaOHbufferasa buffersystemforincreasingpHofthemedium.ThepHofthe KCl–NaOHbuffersystemisaround13,soitcanbeusedfor preparingmediawithahighpHvaluesuchas12.

Morphological,biochemical andphylogenetic characteri-zationrevealedthatalltheisolateswererelatedtomembers ofthegenusBacillus,andthustheyweredesignatedasBacillus

sp.strainsKG1,KG4andKG5.StrainKG1wasselectedonthe basisofitspotentialtoneutralizethealkalinemediumand reducealkalinityandchloridelevelsmoreefﬁcientlythanthe othertwostrains.

AcidproductionbybacterialstrainKG1resultedina signif-icant(p<0.0001)reductionofalkalinityinbothminimaland enrichmentmedia(Fig.1).Intheminimalmedium,anoverall changein3.95pHunitswasrecordedafter3dayscomparedto 4thand5thdayoftreatment.AsimilartrendofdecreasingpH during3daysofincubationwasalsoobservedintheenriched medium.ThisreductionofpHsupportedtheresultsofacid productionbyBacillus sp.strain KG1.Sucrose wasthe sole carbonsourceintheminimalmedium.Itwasreportedtobe efﬁciently utilizedbyvarious Bacillus strains, resultingin a decreaseinthepHofthemedium.1_The_change_in_pH_could_be

attributedtotheproductionoforganicacidsinthemediumby enzymes,andtheseresultswereincloseagreementwiththe ﬁndingsofPaavilainenetal.32_Since_the_minimal_medium_is

aneconomicalanddeﬁnedmediumcomparedtoenrichment media,itwasusedforsubculturingoftheisolateinthisstudy. Accordingtotheguidelinesofseveralenvironmental agen-cies,thepHofindustrialwastesshouldbeneutralbeforetheir dischargetotheenvironmentasthisisanimportant parame-tertocontrolsurfaceandgroundwaterpollution.Theremoval ofthe CKD alkalinity using alkaliphilic bacterial strains is aneco-friendlymethodandwasachallengingtaskforthis study,sincenostudyhasbeenperformeduntilnowtotreat alkaline solid industrial wastes with biological organisms.

Fig.2representsthealkalinityreductionofcementkilndust after treatment with strain KG1 at different cell densities (OD0.6–1.0).ThealkalinityofthecontrolCKDleachatewas

1500 100 Alkalinity Chloride 100 16.36 13.04 1.0 0.9 0.8 0.7 0.6 Control mg/L CaCO 3 O.D. of culture 1000 500 0

Fig.2–AlkalinityandchloridereductioninCKDas inﬂuencedbythecelldensityofBacillushaloduransstrain KG1.Thenumbersabovethebarsrepresentthe

percentagesofalkalinityandchloride,respectively,inthe treatedCKDsamplescomparedtotheuntreatedCKD control.Thedifferencesarestatisticallysigniﬁcant(p<0.05) asfoundbythet-testat95%conﬁdencelimits.

1467mg/L,whereasitwasreducedby83.64%aftertreatment withstrainKG1atanODvalueof0.8.Theresultsexhibiteda significant(p<0.05;t-testat95%confidencelimits)reduction inthealkalinityoftheCKDleachatecomparedtothecontrol. AlkalinityandpHaretwodifferentmeasurableparametersof leachateorwateranalysis,andtheyareoftenconfused.The measurementofpHindicatesacidity,alkalinityorneutralityof aleachateorwater,whilethemeasurementofalkalinity pro-videsabufferingcapacity,i.e.,howmuchalkalinityispresent inasampleasalkalinityrangesfromabovepH4.0to14.0,and determinestheformofalkalinitysuchasbicarbonate, carbon-ateorhydratealkalinity.IfpHisabove8.3,thenalkalinityis duetocarbonates,whereasifpHisabove4.3thenthe alka-linityisduetobicarbonates.Inthisstudy,thepHvalueofthe controlCKDwas12.15,andafterbacterialtreatment(atOD 0.8) thepHvaluewasreducedto8.72.AspertheWHO,US EPAandIndianStandardsInstitute(ISI),thepHandalkalinity ofdrinkingwatershouldbeintherangesfrom6.5to8.5and 200to600mg/L,respectively.AftertreatingtheCKDwith bac-teriumKG1(OD0.8),thepHvaluewas8.72,whichisslightly on ahigher side,whereas the alkalinity was240mg/L, i.e., withinthedesirablelimit,accordingtotheWHO,USEPAand ISIdrinkingwaterguidelines.Thus,afterbacterialtreatment CKDcaneasilybeusedinlandfillingorreutilizedindifferent applicationstoreducesurfaceandgroundwaterpollution.

The chloride content in the control CKD leachate was 460mg/L, which is higher than the maximum permissible limit(250mg/L)providedbytheEPAandWHOstandardsfor drinkingwater. TheCKDtreatedwithstrain KG1atOD0.8 registered a signiﬁcant reduction(by 86.96%; p<0.05;t-test

at 95% conﬁdencelimits)in the leachate chloridecontent, compared to the control. Chlorides are generally present in groundwater as sodium chloride or calcium chloride. Chlorides in drinkingwaterare not ofany health concern but high chloride and magnesiumcontents may affect the

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0 2 4 6 8 10 12 14 16 18 20

Full scale 11361 cts cursor: 0.000 Full scale 11361 cts cursor: 0.000

1 mm Electron Image 1 1 mm Electron Image 1

A

Ca Cu C Cu Fe Fe Fe Fe C Ca Ca Zn Al Al Zn Si Si S S Mg Mg O O K K K K Ca

B

keV keV

Fig.3–SEMandEDXanalysisofthecontrolCKD(A)andbacterialstrainKG1-treatedCKD(B).ThereductionintheKpeak heightreﬂectsadecreaseintheK2OcontentinBcomparedtoA.

quality and taste of drinking water. An excessive chloride concentrationincreasesthehardnessofwaterandthelevelof metalcorrosioninwaterdistributionsystems,whichleadsto anincreasedmetalconcentrationingroundwater.Waterwith chlorideinexcessof2000mg/Lisnotrecommendedformany constructionpurposesasitaffectsthesolidityandstrength ofconcrete.Waterwithahighchloridecontentisnotsuitable forirrigationeither,asevapotranspirationtendstoincrease thechloride concentrationand salinityinthe rootzone of plantsandmakesitdifﬁcultforcropstotakeupwaterdueto thedifferenceinosmoticpressurebetweenthewateroutside theplantandwithintheplantcell.Therefore,analysisofthe chloridecontentintheCKDsamplebeforeandafterbacterial treatmentwasnecessarytoknowwhetherthesample(water orleachate)wassuitablefordrinking,construction,irrigation orotherindustrialpurposes.SinceCKDonlycontains inor-ganicmineralsalts,mainlyintheformofoxides(CaO,SiO2,

Al2O3,Na2O,K2O,etc.),acarbonsourceshouldbeaddedfor

thegrowthoftheisolateinCKD,andthewholeenvironment itselfactsasagrowthmediumfortheisolate.

Preliminarystudieswereconductedinthisworktocheck the growthof theisolate inaCKDleachate containing 2% glucosesolutionasacarbonsource.Anincreaseof0.227in thecelldensity(OD)wasobserved,comparedtothecontrol

medium(withoutinoculation),whichrevealedthat2%glucose supportedthegrowthofstrainKG1tosomeextent.The max-imum reductioninalkalinityinCKD+culture(OD0.8) may indicatethatthisistheoptimumcell concentrationforthe treatment.Alowconcentrationofcellsmightaffectthe efﬁ-ciency ofthereaction, whereasahighconcentrationmight generateunfavorableconditionsforthegrowthastherewas limitednutritionforalargenumberofcells.Itappearedthat thetreatmentofCKDwithstrainKG1atacelldensity(OD)of 0.8supportedthealkalinityreduction.

EDX and XRD analysesofthe CKDsample treatedwith abacterialculture(OD0.8)were alsoperformedtoconﬁrm thedecreaseinalkalinity.TheEDXspectrumofCKDrevealed thepresenceofcalcite(CaCO3)andquartz(SiO2)asthemajor

components,alongwithpeaksforAl,Mg,K,S,andO(Fig.3). AnalysisofCKDshowedtheabsenceofNaandClandsmall amountsofFe,Cu,andZn(Table1).

Thus,inthecontrolCKDsamplethealkalinitywasmainly duetothepresenceofK(K2O)andS(SO3).Thecompound’s

percentcompositionofK2OandSO3inthecontrolCKD

treat-mentwas1.12and1.13,respectively.InoculationofCKDwith bacterialstrainKG1resultedinareductionoftheK2Ocontent

to0.59,whichcorrespondedtoa47.32%reductioncompared tothecontrolCKD(nobacterialtreatment),whereasa24.43%

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500 400 300 Intensity 200 100 0 500 400 300 Intensity 200 100 0 10

A

B

Angle 2 - Theta (degrees) Angle 2 - Theta (degrees)

15 20 25 30 35 40 45 50 55 60 10 15 20 25 30 35 40 45 50 55 60 C C S S 3 2 C C C C A C C CO D Ar Sg C C S S 3 2 CH C C Q/A Sg C C C CO D/A

Fig.4–Diffractionpattern,alongwithcrystallinephases,inthecontrolCKD(A)andbacterial-treatedCKD(B).C,calcite (CaCO3);D,dolomite(CaMg(CO3)2);A,anhydrite(CaSO4);CO,calciumoxide(CaO);Sg,syngenite(K2Ca(SO4)2);Ar,arcanite

(K2SO4);C2S,dicalciumsilicate;andC3S,tricalciumsilicate.

reductionintheSO3contentand47.30%equivalentalkaliwas

observedbyEDX.TheEDXpatternofthecontrolCKD com-paredwiththebacterialstrainKG1-treatedCKDisdepictedin

Fig.3.AreductionintheKpeakheight(correspondstoK2O)in

thetreatedCKDwasobserved,comparedtothecontrolCKD, andthisconﬁrmedasigniﬁcant(p<0.0001;two-wayANOVA) reductioninalkalinity.

TheXRD results(Fig. 4) indicatedthat the CKDmainly consisted of calcite (CaCO3), anhydrite (CaSO4), dolomite

(CaMg(CO3)2)andfreelime(CaO).Tracesofcrystallinealkali

sulfatephases,viz.,arcanites(K2SO4),andhydrationproduct

phases, viz.,syngenite (K2(CaSO4)2), ettringiteand calcium

hydroxide(Ca(OH)2),werealsoobserved.Theabsenceofan

arcanitephasepeak(at31◦2)inthebacterial-treatedCKD alsoconﬁrmedalkalinityreductioncomparedtothecontrol (Fig.4).

TheEDXandXRDanalysessupportedtheabsenceof sul-fate and crystalline alkaline sulfate phases in the treated powderedCKDcomparedtothecontrol.Thefreesulfateand K2OpresentinthecontrolCKDwereconvertedafterthe

treat-mentintoboundformssuchasanhydrites,ettringite(calcium aluminumsulfatephase)orsyngenite(potassiumcalcium sul-fatephase),whichreducedthe freesulfate contentinCKD andthusreducedthealkalinity.Theseresultsaresupported bythefactthatmicroorganismsusuallyutilizecarbohydrates throughthefermentationandrespirationprocesses,andthe acidmightbeproducedduetothecarbohydratemetabolism. Bacterialisolatesarehighlydiverseintheirmetabolismand channelavarietyofmaterialsintovariouscentralpathways (glycolysis, Kreb’s cycle, etc.) of cell metabolism for com-pleteoxidation,therebyproducinganarrayoforganicacids.30

Preliminary investigations in this work showed that direct additionoforganicandinorganicacidstoneutralizecement kilndustisnotfeasible,sincelargeamountsoftheseacids wouldbeneeded,whichisnoteconomical.Useofinorganic acids such as hydrochloric acid, sulfuric acid, etc., in the neutralizationprocess canbehazardous andcorrosive and may cause health problems to the workers. Chloride also

increasesthealkalinityofCKD.Reductioninthechloride con-tentalsomarksanimportantparametertostudy,asitshigh concentration in groundwater through leachate affects the tasteofgroundwaterandincreasesitsalkalinity.Excess chlo-ridecausescardiovasculardiseases.Ahighchloridecontent corrodessteelandimpartspermanenthardnessto groundwa-ter.Inagriculture,chlorideincreasessalinityofsoilthrough irrigation. In this study, chloride was reduced by approxi-mately86.96%,whichshowsthatCKDcansafelybereused anddisposedofftolandﬁllafterbacterialtreatment.Thus,the useofabiologicaltoolfortheneutralizationofalkalinesolid wastesisabetteralternativetoconventionalchemical meth-ods.Thegrowthofthebacteriumontheminimalmediumalso showsitseconomiceffectivenessasacheapgrowthmedium, comparedtoready-to-prepareenrichedgrowthmediasuchas nutrientbroth,etc.

Characterizationandidentiﬁcationofthebacterialstrain

Thephenotypiccharacteristics(morphologicaland biochem-ical)ofstrainKG1arepresentedindetailinTable2.Isolate KG1wasGram-positive,rod-shaped,growingaerobically,able tohydrolyzestarchandgelatinandwasalsopositiveinthe catalaseand citratetests.StrainKG1showed characteristic utilization ofthe carbohydrate substrates glucose, sucrose, fructose,maltose,xylose,mannitolandlactose.Itgrewwell atpH6–12,andthegrowthrateatpH8–9wasbetterthanthat atacidicpH.TheNaClconcentrationsof0–16and0–8%were foundsuitableforthegrowthofisolateKG1intheenrichment and minimal media, respectively. Thegrowth temperature rangewas28–50◦C,andtheoptimumgrowthtemperaturewas 33–37◦C.

The16S rRNAgenesequence (1408 bases)ofstrainKG1 wasanalyzedinordertodeterminethephylogeneticposition oftheisolate.Itwascomparedforsequencesimilaritywith sequencesofpreviouslyreportedstrains,andaphylogenetic treewasconstructedbytheneighbor-joiningmethodusing

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Table2–CharacteristicsofBacillushaloduransKG1andsomerelatedBacillusspecies.

Morphological,biochemical andphysiological

characteristics

BacillushaloduransKG1 BacillushaloduransDSM497a _Bacillus_okuhidensisa,b

Gramstaining +ve +ve +ve(inexponentialgrowthphase)and −veinstationerygrowthphase Cellshape Smallrods Rod Rod

Colonyshape Irregular Filamentous Smoothcircular Colonycolor Whiteopaque White Yellowish Growthat: 45◦C + + + 50◦C + + + pH5 + ND ND pH10 + V + pH11 + − + pH12 + ND ND OptimumpH 8–9 9.0 10.5 ToleranceofNaCl 5% + + + 7% + + + 10% + + + 12% + + − 16% + − − 20% − ND −

MethylRedtest − ND ND

Voges–Proskauertest + ND ND Citrateutilizationtest + ND ND Starchhydrolysistest + + +

Gelatintest + + + Catalasetest + ND ND Lipasetest − ND ND Carbohydrateutilization Glucose + + ND Sucrose + + ND Lactose + + + Mannitol + ND + Xylose + + + Fructose + + ND Maltose + + ND

Characteristicsarescoredas:+,positivereaction;−,negativereaction;w,weakreaction;v,variable;ND,nodata.Forallgrowthassays:+,growth; −,nogrowth;w/+,weakgrowth.Superscriptafterthetaxadenotesthereferencefromwherethedatumisobtained.

a _Vargas_et_al.33

b _Li_et_al.34

thesequencesofthecloselyrelatedtaxa,retrievedfromthe GenBankdatabase(Fig.5).

Theresultsofthemorphological,biochemicaland phylo-geneticanalysissuggestedthatstrainKG1wasamemberof thesixthrRNAgroup10_of_the_genus_Bacillus,_which_includes

alkaliphiles,andformedacladewithBacillushaloduransstrain DSM497(AJ302709),withabootstrapvalueof99%.Pairwise sequenceanalysisrevealedthatthehighestsequence similar-itywaswithB.haloduransstrainDSM497(94.7%),followedby

BacillusokuhidensisstrainGTC854(93.9%),whiletheremaining

Bacillusspecieswithvalidlypublishednamesshowedlessthan 94%similarity.

StrainKG1couldbedifferentiatedfromotherphylogenetic neighborsofpreviouslyreportedalkaliphilicBacillus species onthebasisofmorphological,biochemicalandphysiological charactersgiveninTable2.Thegenerallyrecommendedand

acceptedcriteriafordelineatingbacterialspeciesstatethat strainswitha16SrRNAgenesequencedissimilaritygreater than3%orwithDNA-DNAhybridizationrelatednessofless than70%areconsideredtobelongtoseparatespecies.35_It_is

recommendedthatbacterialstrainswithadifferenceinthe 16SrRNAgenesequenceoflessthan3%cannotbeallocated tonewspecieswithoutthesupportofDNA-DNArelatedness studies.In ourstudy, bacterialstrain KG1showeda differ-enceinthe16SrRNAgenesequenceof5.3%withthemost closelyrelatedBacillusspecies(94.7%sequencesimilaritywith

B.haloduransstrainDSM497).Thus,onthebasisof morpho-logical, biochemical,physiologicalandphylogenetic results, itisproposedthatstrainKG1beclassiﬁedasthenoveltype strainofaspecies,B.haloduranssp.strainKG1nov.The16S rRNAsequenceofstrainKG1wassubmittedtoGenBankand assignedaccessionnumberJQ307184.Thecultureisdeposited

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Bacillus wakoensis strain N-1 (AB043851) Bacillus Krulwichiae strain AM31D (AB086897) Bacillus akibal strain 1139 (AB0438558) Bacillus okhensis strain kh10-101 (DQ026060)

Bacillus alkalidiazotrophicus strain MS 6 (EU143680) Bacillus macyae strain JMM-4 (AY032601)

Bacillus pseudofirmus strain DSM 8715 (X76439) Bacillus bogoriensis strain LBB3 (AY376312) Bacillus alcalophilus strain 1 (X76436)

Bacillus hemicelluosilyticus strain C-11 (AB043846)

Bacillus halodurans strain KG1 (JQ307184) Bacillus halodurans strain DSM 497 (AJ301709)

Bacillus okuhidensis strain GTC 854 (AB047684) Bacillus shackletonii strain LMG 18435 (AJ250318) Bacillus acidicola strain 105-2 (AF547209)

Escherichia sp. EC3 (JN987148) 100 0.02 54 82 86 94 65 55 99 98 Bacillus pseudalcaliphilus DSM 8725 (X76449)

Fig.5–Aneighbor-joiningphylogenetictreeshowingtherelationshipsofbacterialstrainKG1andthetypestrainsof closelyrelatedBacillusspecies,basedon16SrRNAgenesequences.TheGenBankaccessionnumbersaregivenin parentheses.Bootstrapvalues(expressedaspercentagesof1000replications)greaterthan50%areshownatthebranch points.Bar,0.02nucleotidesubstitutionspersite.

atNCIM(National CollectionofIndustrialMicroorganisms), NCL(NationalChemicalLaboratory),Pune(India)andassigned depositionnumberNCIM5439.

Conclusions

Cementkilndust,awastebyproduct,isgeneratedduringthe manufacturingofcementclinker.Duringclinkerproductionin akiln,volatilecomponentssuchassulfates,alkalis(K2Oand

Na2O),chlorides,etc.,aredrawntowardCKDandmakeit

alka-line,thusrestrictCKDreuseinacementkiln.Hugeamounts ofCKDaredumpedinlandﬁll,which,duetoleaching,leadsto pollutionofgroundwater.BiologicaltreatmentusingB. halodu-ransstrainKG1fortheremovalofalkalinityandchloridefrom CKDisabetteralternativetochemicalmethodsintermsof costeffectiveness,efﬁciencyandnon-toxicity.

Conﬂicts

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