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orgJournalofMiningWorldExpress(MWE)Volume4,2015
doi:10.14355/mwe.2015.04.005

MicrobiologicalBeneficiationofLowGrade
ManganeseOres:AReview
JaiS.Ghosh1*andArvindD.Agate2
1*

DepartmentofMicrobiology,ShivajiUniversity,Kolhapur416006,India

ExDirector,AgharkarResearchInstitute,Pune411004.India

ghoshjai@gmail.com

Abstract
Manganeseisatransitionelementlikeiron.However,itexistsindifferentvalencieslike2,3,4and7.Ofthese,Mn2+ishighly
water soluble, but is rarely found in natural conditions. Mn4+ is the least water soluble and under different pH and redox
conditionsitcanslowlybecomecolloidalinnaturalwater.Itexistsnaturallyasaverycommonmineralcalledpyrolusiteandis
used for pyrometallurgical processes in ferroalloys industries. However, in order to have such beneficial application, the
manganese content should be more than 80% in the ore. Anything less than this is treated as low grade ore and is usually
dumpedonlandwithcultivablesoil,inordertoreachdeeperforthehighgradeores.Thisishighlyecotoxictotheenvironment
andoveraperiodoftimecanbeverydamagingtoagricultureandthebiodiversitywhichincludeshumanhealthasitfindsits
waytogroundwatertablelikewellsandponds.Astimewentbyandhighgradeorestarteddepleting,minersstartedgoing
deeperintheground,withtheresultofdumpinglowergradeoresoversurfacesoils.Inthisstudy,suchlowgradeoreswere
examinedandfoundtocontainamixtureofMn3+andMn4+oxides.Whensuchoresweresubjectedtomicrobialoxidation,it
wasfoundthatundercertainconditionsofgrowth,ArthrobacterspcouldnotonlyconverttheMn3+toMn4+and67%oftheMn4+
oxides had the ramsdellite crystalline structure, which could be used as a depolarizer in the dry cell batteries. This was
consideredasaveryimportantbiotechnologicaldiscoverywhichnotonlycouldsavethebiodiverseenvironmentbutalsogave
avalueaddedproductforcommercialexploitation.
Keywords
Arthrobacter;Pyrolusite;Beneficiation;Depolarizer

Introduction
It is reported that the existence of manganese was known to the Indians since time immemorial (Fermor, 1909).
They regarded it as a kind of iron ore and used it in iron smelting, for coloring of glasses and enamels and for
makingsurmausedforblackeningofeyebrows(Fermor1909).
EarlySourcesintheWorld
Till1865,whenthefirstferromanganesealloysproductionstarted,manganesewaschieflyusedasadecolorizerin
glass industries and was used to make bleaching powder. The requirement for manganese for such purposes in
EuropewasmetfromsourcesatTavistock,inDevon,LauncestoninCornwell,HarzinGermanyandPiedmontin
Italy (Fermor, 1909). Soon these deposits got exhausted and were replaced by Spanish deposits, which also got
exhausted.However,theferromanganeseindustrywasspreadingfastinEuropeandUSAandtherewasalarge
demand of manganese. The suppliers of manganeseore which emerged at this time weremainlyRussia(815070
tonnesin1900),Turkey(49468tonnesin1899)andBrazil(111000tonnesin1900)(Ghose,1967).
EmergenceofManganeseMinesinIndia
Duringthe1890s,thecollectorofVishakhapatnam,AndhraPradesh,detectedthatsomecontractorswerebreaking
manganeseoreboulderstoprepareballastfortherailwaysandthusthedepositsofKodurweredetectedfirst.The
Collectorformedasyndicateandproduced685tonnesfromthisdistrictandexportedtheentireamountin1892.In
1895Garbhamdepositswerediscoveredandin1898theproductionroseto228951tonnes,ofwhich215181tonnes
wereexported(80%toEnglandand20%toUSA).

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TheRiseandFallofIndianManganeseIndustries
DuetotheincreasingdemandintheInternationalmarket,othercompanieswereformedandnewfindsinMadhya
Pradesh,MysoreandBiharwerediscovered.Theannualproductionreached58500tonnesbytheendof1906and
India was ahead of Russia which produced 53800 tonnes during that year. Soon there was a demand for Indian
manganese ores by many developed nations in the next 15 years and India dominated the market as a major
producing nation and exporter of manganese ore. This bright period for India lasted till 1912when she was
relegated into the second position by Russia. This was obvious because barring Russia and China, almost all
manganeseoreproducingareasintheworldfallincountries,whichhadpracticallylittleornodomesticuseofthis
ore.Hencetheyhadtodependtotally,onexportofthisore.Thiscouldbeoneofthereasonsforslowgrowthof
manganese industry in India. Then the First World War came to Indias rescue, when Russia was completely
paralyzed and India was reinstated to the top by 1916. India maintained the lead during 1919 to 1923, with an
average production of634600 tonnes of manganeseores, whichwas about 44% of the worldsaverage.Then the
export trade witnessed the entry of new competitors from Egypt, Cuba and the Gold Coast (Ghana) (Coggin,
BrownandDey,1955).By1932,RussiahadrecoveredandagaindisplacedIndiafromthetop.However,another
formidable competitor entered the market and it was South Africa. By the end of 1934, South Africa was in the
thirdpositionfollowedbyGhana.Indiamaintainedhersecondpositiontill1958.ThesecondWorldWardidnot
haveaseriousimpactonIndiasmarketshare.LaterRussiawithdrewfromthewesternmarketandIndiashowed
signsofimprovement.Inlate1950s,thoughtherewasincreaseddemandofmanganeseores,Indiasexportdidnot
increase till 1971, when average annual production touched 1.5 million tones. From here onward, there was a
steadydeclineofIndianmanganeseoreindustrytilldate.Forexamplethealltimelowexportwasrecordedin1970
when India exported only 82000 tonnes. Table 1 summarizes the rise and fall of Indian Manganese ore industry
overtheyears.
TABLE1:RISEANDFALLOFINDIANMANGANESEOREINDUSTRY.

Period

Production(Tonnes)

PercentageofWorldproduction

18921898

228,951

18991903

715,060

19041908

2,586,449

19091913

3,621,008

40.7

19141918

2,933,481

34.1

19191923

3,173,147

44.1

19241928

4,841,440

33.6

19291933

2,837,668

19341938

3,942,846

16.0

19391943

3,919,075

19441948

1,840,392

19491953

6,284,601

13.2

19541958

7,767,165

12.2

19591963

7,105,562

9.8

19641968

7,991,703

8.8

19691971

5,028,340

8.4

This declinewasstated to be not solely due to the low production values, but also due to the inferior variety of
manganeseoresthatwerebeingmined.
At the end of Second World War manganese ores were found to have many other uses. The two other two
importantuseswere:
1.

Asadepolarizerindrycellbatteries

2.

Asalixiviuminleachingmineralssuchasuranium.

Besidethesetwousesmanganeseoreswerealsousedtomanufacturechemicalslikeiodineandcertainorganicand

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inorganicchemicals.Previously,itwasusedasadrierinmanufactureofpaintsandvarnishes,duetoitsvirtueof
absorbing oxygen fromair.Highgrade pyrolusite isalso used to manufacture different manganesesaltslike the
manganates,asdecolorizeringlassandceramicindustryandapigmentinglazes.Itwasalsousedinmakinggood
quality black bricks. The addition of manganese ores produces tints varying from purple to brown and black,
whichareusedinpotteryandenameledironwares(Johnstone,1954).
In spite of these limited uses, since the manganese industry in India, was still thriving as export dependent
economy,andaconcertedeffortwasneededtoconcentrateonimprovingthequalityoftheores.Theinformation
(since 1979) from one of the foremost manganese producing province of India, Goa, was that the quantity of
manganesedeposits wasdiminishing fast in the explored mines andsome of the mines were not expected to be
producingafter1015yearshence.Moreover,thequalityofmanganesewassufferingbadlyduetothepresenceof
impuritieslikeiron,silica,phosphorousetc.Thesehadmadethemanganeseorestobeaverylowpayingexport
item or not fit for export. For instance, presence of excess of phosphorous (more than 0.1%) makes the ore
unacceptableforuseinmanufactureofferromanganesealloys,asthealloysdevelopmicrocracksandmakesthe
finishedproductunacceptable.
The classical metallurgical beneficiation processes such as crushing, grinding, sieving, passing over magnetic
platformsorchemicaltreatmentslikefrothfloatationeitherdonotsucceedoriftheydo,onlypartiallydoso.For
instanceforanorefoundinAndhraPradesh,FerroAlloysCorporationofIndia,providedalistoftheirattemptsto
improvetheoreatRaiBahadurShreeramMinesatShreeramnagar(Table2).
TABLE2:EFFECTOFPHYSICALANDCHEMICALTREATMENTSONBENEFICIATIONOFMANGANESEORES(DESHPANDE,1979)

Treatment

Analysisofcomponents(%)
Mn

Fe

SiO2

Originalsample(notreatment)

44.6

6.1

6.6

0.6

Crushingandsieving(3to12and18mm)

44.6

6.1

6.6

0.6

GrindinginRodMilltoobtainconcentrate

58.6

6.1

4.2

0.6

5860

35.8

23

0.6

5860

35.8

23

0.6

Groundto100meshwithDuplexClassifierandpassing
throughimmersionmagneticseperator
Passingthroughabatchoffloatationcellsto(chemically)
removephosphorous.

As is evident from the Table, although there was some slight improvement regarding beneficiation as far as
removalofironandsilicawereconcerned,thequantityofphosphorouswasnotremovedatallasresultofthese
physicalandchemicaltreatments.
Thedifferentbeneficiationprocessesthatcanbetriedonmanganeseoreare:
1.

Removalofexcessiron

2.

Removalofexcessphosphorous

3.

OxidizetheloweroxidestotheMnO2form

4.

Removalofeithermetallicornonmetallicimpuritieswhichotherwisemayinterferewiththeorequality.

Out of these processes, for some, use of microorganisms to beneficiate the ore was attempted or recommended.
Thiswasnotconventionalmethodinthestrictestsenseoftheword.Ratheritwaseffectiveremovalofimpurities
ormicrobialconversion.Againitmustbenotedthatnotalltheseprocessescouldbetriedusingmicroorganisms.
For instance, there existed sketchy information on removal of iron through the use of iron reducing
microorganisms (DeSa, 1978) and rather no information on topic 4 from the above list. Out of the 2 beneficial
processes (topic 2 and topic 3) that have been tried out in our laboratory (Deshpande, 1979; Bhole, 1979), the
microbial conversion of manganese (lower oxides to the higherstate of oxidation byusing microorganisms) was
selected,duetoitstopicalnatureanditseaseofhandlingbysingleperson;eventhoughboththeseprocessesare
beingattemptedunderaCouncilofScientificandIndustrialResearchGovt.ofIndia(CSIR)scheme.Theprocess
wasdiscoveredandillustratedindetailfromthislaboratoryasresultofworkforsevenyearsofwork(Agate,1972;
AgateandDeshpande,1974;Agate,1975;AgateandDeshpande,1977and1978).Thisprocesswasclaimedtobe

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inexpensive, energy conserving and with least pollution potential and had a relevance to the national need.
Although beneficial, this type of a conversion was restricted to monopolize practices in India. Moreover, the
electrolyticprocesscouldonlyworkprofitablyinacountrywithcheapsourceofelectricity.
ManufactureofDryCellBatteries
Properties of manganese dioxide like the chemical composition, the physical specifications, crystal structure and
particlemorphologyetc.,wereimportantfordecidingtherequirementofanoreemployedasdepolarizerforthe
manufactureofdrycellbatteries.Althoughthechemicalanalysisprovidesausefulguideinselectionofanore,the
finaldecisionrestsontheactualresultsofthetestsoncellsmadeunderworksconditions,particularlywithregard
totheirshelflife(Anon,1962).ABritishspecificationofthechemicalcompositionofthetypeoforethatwouldbe
bestsuitedfordrycellbatteriesisthattheoreshouldcontain84%MnO2,withamaximumof2%to5%ironand
carbondioxide.Thisoreisrequiredtobeporousandlumpyandthesizeshouldnotexceed25cmor8inch.Table3
givestheorespecificationsasrequiredbydifferentdrycellindustriesinIndia(Anon,1970):
TABLE3:MANGANESEORESPECIFICATIONSREQUIREDBYDIFFERENTDRYCELLMANUFACTURINGINDUSTRIES(ANON,1970).

Particulars

UnionCarbideIndia
Ltd,India

HaveroIndustries,
India

EstrellaBatteries,
India

GeepFlashLight,
India

MnO2

8487%

8595%

7085%

7085%

Iron

2%(maximum)

1%(maximum)

Copper

0.03%

AsandSb

0.05%

Fe2O3andAl2O3

10%(maximum)

SiO2andinsolubles

1%(maximum)

Acidinsolubles

10%(maximum)

Structureandvariety

Batterygradeore,
gammatype,size
shouldbeinlump
formnotexceeding8
inchinany
dimensions.

Crystalstructure
gammatype

Crystalstructure
gammatype

Crystalstructure
gammatype

It appears that the primary requirement of an ore to be battery grade type is thatit shouldhave more than80%
MnO2andthatitshouldhavecrystalstructuregammatypeasshownschematicallyinFigure1.Itdiffersfromthe
betavarietyinthatthebetavarietyismoretetragonalinstructure,whereasthegammavarietyisorthorhombic
ramsdelliteinstructure(DeWolff,1959).

FIGURE1,CRYSTALLATTICEOFGAMMAVARIETYOFMNO2DUETOMICROTWINNINGANDDEWOLFFDISORDER(SIMON,
MORTONANDGISLASON,2004)

Duetothestructureofthegammavariety,itmakestheMnO2agooddepolarizerwhichisusedindrycellbatteries.
Therefore, the battery grade ores are classified based on the content of gamma variety MnO2 and then be used
suitablyinmanufacturingofdifferentbatteries.
OresofManganese
Manganeseoccursinminutestamountsinalmostallrockslikegranite,limestoneandclay,butitsmerepresenceis
notofanycommercialvalueunlessmanganeserichoresarepresentinsufficientquantitiesofmineablesizeand
shape.

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ThecommerciallyworkedmanganeseoresaretheoxidesandthecommonestonesarePyrolusiteandpsilomelane.
Besidetheseores,theothertypesarebrauniteandjacobsitewhichareusuallytheoxysilicatetypes.Incombination
withthesearethehausmanitetypeoforewhichoccursabundantlyinmanypartsoftheworldincludingwestern
Australia.Thecarbonatetypeoforeistherhodochrosite.InsomepartsofGhanaandSouthAfrica,averyvaluable
oreisfound,whichiscalledasramsdellite.Thisisthepureformofgammavarietyofpyrolusite,idealforusein
any dry battery manufacturing process. Small quantities of these ores are found in some parts of India, such as
DongriBuzurgminesofManganeseOresIndiaLimited(MOIL)inBhandaradistrictofMaharashtra,India.Thelist
oforesofmanganeseispresentedinTable4.
TABLE4:DIFFERENTTYPESOFMANGANESEORES

Ores

Chemicalnature

Manganosite

MnO

Pyrochroite

Mn(OH)2

Manganite

Mn2O3,H2O

Hausmanite

Mn3O4

Polianite,Pyrolusite

MnO2

Taphroite

Mn2(SiO4)

Braunite

3Mn2O3MnSiO2

Rhodonite

(Mn,Fe,Ca,)(SiO2)

Pyromanganite

(Mn,Fe)(SiO2)

Spessarite

Mn3Al2(SiO4)

Psilomelane

BeMn,Mn8O16(OH)4

Albendite

MnS

Rhodochrosite

MnCO3

Jacobsite

MnFeO4

1.

Pyrolusite: It is a black oxide of manganese (MnO2). It occurs as an earthy material or as radiating fibres
(Barnowitz, 1943). It is usually in the beta form of crystal structure. Its harder variety is called Polianite,
whichoccursasneedleshapedcrystalsandisofwidespreadoccurrence.Themanganesecontentisabout
80%orabove.

2.

Psilomelane:AfterPyrolusite,thisisthemostimportantoxidemineralofmanganese.Itusuallyoccursina
massivebotryoidallystalactiteform.Dependingonthequantityofadmixtureitsmanganesequantityvaries
widelyfrom4560%.

3.

Braunite:Itisanoxysilicateofmanganeseusuallyhavingabove60%manganese.

4.

Hausmanite:Itgenerallyoccurswithbrauniteandishigherinmanganesecontent(above60%)Itisbrown
incolorwhenoccurringfreelyorblackishbrownincolorwhenitiswithothertypesofores.

Thesearesomeimportantoresofmanganeseofcommercialimportance.TheotheroresarelistedinTable4.Outof
these ores, the manganese ores of relevance to the present study are the hausmanite and braunite type of ores,
which occurs in southern and central parts of India. Generally a typical Indian Manganese ore is a mixture of
psilomelaneandbraunite.
GeomicrobiologyofManganese
Manganese is present in earths crust at 0.1% level but its distribution is not uniform. The element can exist in
oxidationstatesof0,+2,+3,+4,+6and+7.However,itismostcommonlyfoundin+2and+4states.Rarely+3is
seeninnature.Ofallthese+2isthemostwatersolubleformwhile+3and+4arewaterinsolubleoxides.
Manganese ions are more stable than the ferrous ions under different conditions of pH and Eh. Based on the
equilibriumcomputationmanganeseshouldexistpredominantlyasMn2+belowpH5.5andasMn4+abovepH5.5.
AtEharound0.8VMn2+is100ppminpresenceofHCO3ions.AtEhbelow0.5VMn2+maydominateuptopH7.8to
8.0.

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FIGURE2.THESTABILITYFIELDSOFMANGANESESPECIESINAQUEOUSSOLUTEION(HEM,1963).

Althoughintheory,0.1ppmofMn2+ionsinaqueoussolutionshouldreadilyautooxidizewhenexposedtoairatpH
value above 4.0. However, this will not happen until pH exceeds 8.0. This fact is extremely important when
manganeseoxidationbymicroorganismsisconsidered.
Themostimportgeomicrobiologicalinteractionwithmanganeseis:
1.Reduction;2.Oxidationand3.Precipitation/concentration.
The 2 steps 1 and 2 are enzymatic processes and the last effect is non enzymatic, initiated by mechanical
adsorption.Outoftheseprocessesmicrobialoxidationofmanganeseisdetailedasfollows:
Theenzymaticreductionofmanganesehasbeenthoroughlystudiedwhichinvolvesalotoffundamentalstudies.
Thesearedissimilatoryreductionprocess,todetoxifythemetal.
DissimilatoryReductionofMn(IV)
Tetravalent manganese (Mn4+) and trivalent iron (Fe3+) have been studied in detail with respect to microbial
physiology. Both these ions are good alternative electron acceptors for many microorganisms. There are similar
ionslikenitrate,sulphateetc.whichtooalsoacceptelectronsbesideoxygen.Ithasbeenprovedbygrowingstrict
autotrophs like Acidthiobacillus thiooxidans and Acidthiobacillus ferrooxidans in presence of Mn4+ in 9K medium,
wheretheseorganismshadbeenfoundtocarryoutoxidationofelementalsulfurorsulfidestosulfatesorferrous
toferricionsandreducingMn4+toMn2+(GhoshandImai,1985).Apparently,Mn4+waspreferredoverferricionsto
beusedaselectronacceptor.SimilarlymanyheterotrophicorganismshavealsobeenreportedtouseMn4+.Inone
instance, Bacillus subterraneus has been isolated from an Australian deep thermal aquifer and was found to be
growingwellinpresenceofyeastextractmostlyascarbonsourceandutilizingMn4+aselectronacceptor,naturally
(Kanso, Greene and Patel, 2002). Similarly, Shewanellaoneidensis has been reported to carry out MnO2 reduction
under CO2 stress conditions (Wu et al., 2010). In Indonesia in Matano lake it has been found that anoxygenic
microbialmethaneoxidationtakesplacewhileMn4+reductionoccurs(Croweetal.,2011).Thissortofreductionis
foundtobehighlyessentialintheenvironmentslikebenthicdeposits,wheremicroorganismsneedsuchelectron
acceptors to oxidize organic carbon (which is usually present as either natural or anthropogenic pollutants),
essentialfortheirsurvival(Thamdrup,RosselloMoraandAmann,2000).
It must be remembered that both manganese and iron are toxic to the environment in macro concentrations i.e.
above 1015M level. However, iron can be immobilized and its toxicity can be reduced by converting it to its
sulfideform,whichisnotthecasewithmanganese(StummandMorgan,1981).Infactmanganeserarelyformsits

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sulfidecompound.ThishasbeenfurtherconceptualizedwiththehelpofShewanellaputrefaciensduringhydrogen
andformateoxidation(NealsonandMyers,1992).Overaperiodoftimeifthisreducedmanganeseisnotremoved
byartificialchelation,thenitcansuppressthegrowthoftheorganism.ThishasbeenobservedwiththeMn(IV)
and Fe(III) reducer Geobacter sulfurreducens (Mehta et al., 2005) especially when c type membrane bound
cytochromewasrequiredtobereduced.
The enzymatic oxidation of manganese is studied in case of Arthrobacter sp. (formerly Corynebacterium strain B),
whichwasisolatedbyBromfieldin1956.TheotherorganismsstudiedinthisrespectwereSphaerotilusdiscophorous
(now Leptothrix discophora) (Ali and Stokes, 1977), which is doubted by other workers (Hogan, 1973; Haji and
Makenson, 1976), Citrobacter freundii (Douka, 1977 and 1980) and Metallogenium sp along with the organisms
mentioned such as Leptothrixpseudoochracea and Arthrobactersiderocapsulatus (Dubinina, 1978). Among the fungi
thathavebeenfoundtooxidizeMn(II)toeitherMn(III)orMn(IV)areAcremonium,Penicillium,Cladosporium,Phoma,
Verticellium etc. Most of the time these organisms use polyphenol oxidases like laccase which is a multi copper
oxidasesandcarriesoutoxidationofmanganesebyoneelectrontransfertooxygen(Miyataetal.,2007).
TheOxidationofmanganesebybacteriaproceedsby3differentmechanismsas:
1.

TheoxidationoffreeMn++ionsiscatalyzedbyamanganeseoxidasethatconveyselectronstooxygenbya
cytochromepathway.
Mn+++O2+H2OMnO2+2H+

2.

TheoxidationoffreeMn++ionsmayalsobecatalyzedbycatalaseinareactionwithmetabolicallyproduced
H2O2.
Mn+++H2O2MnO2+2H+

3.

The manganese oxidation is prebound to Mn++++ oxidase that shunts electrons to oxygen bya cytochrome
pathway.
MnMnO3+O2+H2O2H2MnO3

In Marine bacteria a different kind of constitutive Mn++ oxidizing system is detected and detailed studies on
Arthrobacter37byEhrlich(1964)showedthattheMn++isoxidizedbya2stepreaction:
Mn+++H2MnO3MnMnO3+2H+
MnMnO3+O2+2H2O2H2MnO3
This is the system which is responsible for formation of ferromanganese nodules in deep sea sediments. This is
becausethepoorcrystallinestructureofoxidizedMnO2:

FIGURE3.THESTRUCTURALMODELOFBIOGENICMnO2(Miyataetal.,2007).

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It can be seen from the above Figure 3, that due to gaps in the crystalline structure of biogenic MnO2, toxic
elementslikelead(Pb)istrappedandthisisoneofthemechanismsofformingpolymetallicnodulesintheocean
bed.
Intheculturalmanifestationitmaybestressedthatdepositionofmanganeseinanyliquidmediumorincolonies
isnoproofofenzymaticmanganeseoxidation,becausenoenzymaticallyoxidizedcanalsoaccumulateasbrown
manganese oxide in their colonies. In this group many bacteria are included as listed by Ehrlich (1981) which
oxidizesMn++byindirectmeansorbyanunknownmechanism.Suchbacteriaseemedtobepresentinfreshwater
andotherwaterdistributionsystem.Themicroorganismscausingprecipitationofpreformedoxidizedmanganese
through adsorption can do so by production of slime as in the case of budding sheathed bacteria belonging to
generaMetallogenium,Pedomicrobium,HyphomicrobiumandSiderocapsa.
Manganesereducingandoxidizingmicroorganismsplayanimportantroleinmobilizationandimmobilizationof
manganese in soil. Fixed manganese in soil may be concentrated in nodules or in arid environments as desert
varnishes. Conversely manganese reducing microorganisms may mobilize the oxidized or fixed manganese,
releasing it into the water column. This reduced manganese often leads to manganese carbonate formation, a
differentformoffixedmanganese.
Outoftheprocessesmentionedabove,themicrobialoxidationofmanganeseisveryinteresting,particularlythe
bacterial oxidation of manganese yields economically important products like battery grade MnO2. Hence, this
processofbacterialconversionofmanganesewasselectedforfurtherstudies,sincethebasicdataontheprocess
hadaccumulatedinourlaboratoryandhencetheoptimizationoftheprocesswasstudiedhere.
AttemptstoObtainManganeseOxidizingBacteriaUsedforBeneficiationPurposes
BacterialoxidationofmanganeseinsoilwasfirstobservedbyBeijerinckasearlyas1913.Sinceintheearlydays,
the pure culture techniques to study microorganisms were not developed, the role of the microorganism
responsible for bringing about such changes was not clearly understood. One of the first efforts to establish the
importance of systematic study of manganese oxidizing microorganisms in soil was initiated by Bromfield and
Skerman(1950),whentheroleplayedbythiselementinplantgrowthwasspeculatedupon(Ottow,1969).Itwas
reportedthatChromobacteriumandCorynebacteriumsppfromsoilcarriedoutmanganeseoxidation(Bromfieldand
Skerman, 1950). Now various genera of bacteria have been implicated in oxidation of manganese, such as
Arthrobacter, Bacillus (Ehrlich, 1966), Pseudomonas (Dubinina, 1970), Sphhaerotilus (Rouf and Stokes, 1964),
Hyphomicrobium(TylerandMarshall,1967)andCitrobacter(Douka,1980).Theecosystems,whichwerestudiedfor
isolationofmanganeseoxidizingbacteriaincludedsoils(Bromfield,1956),freshwaterpipelinedeposits(Tylerand
Marshall,1967)seawater(Douka,1980)andmanganeseores(Deshpande,1979).
OrganismsbelongingtogeneraArthrobacter,BacillusandHyphomicrobiumsppwereisolatedandtheirmanganese
oxidizing capacities werestudied (Agate,1972;Agate and Deshpande, 1977,1978). It was found that manganese
oxidizing cultures of Hyphomicrobium and Arthrobacter spp could oxidize hausmanite variety of manganese ore
(containingpreponderanceofMn3O4form)tothepyrolusitetype(containingMnO2predominantly)(Deshpande,
1979).ItwasrevealedthatHyphomicrobiumsppcouldconcomitantlyreleasephosphorousasanimpurityinsomeof
theGoamanganeseores,whileprobablyoxidizingthemanganesemoietyfromthecrystallatticestructureofthe
ore (Bhole, 1979). Since both these processes were commercially important the former in the manufacture of
MnO2fordrycellbatteries(bioconversion)andthelatterforremovalofimpuritiesfromtheores(beneficiation),it
wasproposedtooptimizesuchbeneficialactivitiesofbacteriaatanexpandedscaleinthelaboratory,sothatthe
possibleparameterscouldbeworkedoutforextrapolationtolargescaleoperations.
Out of the 2 processes available, the process of microbial conversion of manganese was taken for optimization
purposesduringthesestudies.EventhoughtheprocesswascarriedoutequallyandefficientlybyArthrobacterand
Hyphomicrobium spp (Deshpande, 1979), it was considered worthwhile to develop only the process using
Arthrobacterspforthefollowingreasons:
1.

Hyphomicrobium are temperaturesensitive i.e. theseorganisms will grow only at around 25oC and, hence,
theseorganisms(orprocessesbasedontheseorganisms)willbedifficulttouseinthefield,wherethereare
usuallytemperaturefluctuationsexceeding25oC.

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2.

Thereareveryslowgrowingheterotrophicorganismswhichneedsasolidsurfacetoadhereto,whichother
contaminatingheterotrophicbacteriacanovergrowandhindertheprocessormaketheprocessineffective.

Therefore,theprocessofoxidation/conversionusingArthrobacterspwaspreferredanditwasdecidedtooptimize
the conditions for such a process. Arthrobacter sp are sturdy, fast growing organism and they can tolerate
fluctuatingfieldtemperatures(fromvirtually10oCtoaround40oCinIndia).
Isolation and Identification of Cultures
Asthefirststep,theavailablecultureswerescreenedanditwasfoundthateithertheyhavelosttheirmanganese
oxidizing activity or it is present on a greatly diminished scale. While investigating the reasons for such a
phenomenon, it becomes apparent that even thoughregular culture preservation programdid exist at that time,
theactivitycheckswererarelycarriedout,atthetimeofpassingontheseculturesfromonepersontoanotherin
thelaboratory.Moreover,theculturesweremostlypreservedbyserialtransfersanditwaspossiblethatunderthe
circumstances periodicselections might have takenplace and the culturescould have geneticallyselected outso
that they failed to retain their activity conditions of the tests in this investigation. Rather than attempting to
rejuvenatetheseculturesorfindoutacausefortheirreportedfailure,itwasdecidedtocarryoutfreshisolationof
manganeseoxidizingbacteriaandlaterinstituteaplanforculturepreservationcoupledwithactivitycheck,sothat
thenewculturesdonotsufferthesamefate.
EcoSystemUsed
Theisolationofmanganeseoxidizingbacteriawascarriedoutfromtheecosystemsthatwereusedbeforeviz.soils
andwater.Soilsusedwereprimarilyfromminingregions.Watersamplesusedwerefromwellsandstreamsand
pile deposits on the river flowing through the city. The important environmental factors, which influence the
microfloraoftheseecosystems,viz.thepiledepositswere:(i)thequantityofmanganeseintheflowingwaterand
(ii)thetemperatureeffect.Itwasreportedthattheorganismhadtheabilitytoconcentratemanganesefromsucha
systemwherethemanganesecontentwasusuallylow(Agate,1972).Thetemperatureinsuchasystemwasfound
to be 35oC in summer and about 15oC20oC in winter. On microbiological examination of such deposits during
summer and winter months, it was reported that during the summer months, the Arthrobacter sp were
predominantandduringthewintermonths,threadlikefilamentous,buddingbacteriaviz.theHyphomicrobiumsp
werepredominant(AgateandDeshpande,1977).Therefore,isolationofArthrobacterspwascarriedoutduringthe
summer months, as these organisms were reported to bring about conversion of hausmanite form of the ore to
pyrolusiteform(Deshpande,1979).
Isolation
The isolation protocol that was followed was based on the earlier work of Deshpande (1979) and Bhole (1979).
Brieflyitmayberecordedasfollows:
Using an enrichment culture methodology, the manganese oxidizing organisms were enriched in Bromfields
liquid medium (Bromfield, 1958) containing 0.5% MnSO4, H2O. The medium was modified with the following
composition (KH2PO40.005%; MgSO40.002%; (NH4)2SO40.01%; Yeast Extract0.005% and MnSO40.5%). Since no
acid was expected to be produced, Ca3(PO4)2 was omitted from the original medium along with the readily
assimilable carbon source, sucrose as the organism would utilize sucrose as an alternative energy source and
would stop oxidizing manganese. The yeast extract was sufficient to develop the required biomass for the
manganeseoxidationprocess.
A set of 200 ml of sterile medium were inoculated with different soil and water samples. The flasks were then
incubatedonarotaryshakerat140rpmat28oCfor4days.Thegrowthintheflaskswascheckedanditwasnoted
thatdifferenttypesoforganismspredominantlyconsistedofgrampositivenonsporulatingrods.After4daysof
incubation,1%ofthiswasusedasinoculumandwastransferredtoaseriesofflaskscontainingfreshmediumfor
thesecondenrichment.Thegrowthfromtheseflaskswasreconfirmedtobeofthesamevarietyasbefore.These
were then cultured on plates of modified Bromfields medium solidified with 2.9% agaragar. The plates were
incubated at 28oC for 4 days. Isolated colonies, showing a zone of brown coloration around the colonies were

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picked up, in preference to colonies which remained colorless. The brown coloration indicated manganese
oxidation,whichwasconfirmedbyBenzidinetest(Bromfield,1956),wherein1%solutionofbenzidineHCl100ml
of 2N acetic acid was poured on plates and it gave a dark blue color with oxides of manganese in the brown
coloredzones.ThepureculturesweretakenonslantsofmodifiedBromfieldsagar.
Identifications
Thebacterialisolateswerefurthercharacterizedasperthekey,constructedoutoftheeightheditionofBergeys
ManualofDeterminativeBacteriology(1978)uptothegenericlevel.
PreservationofCultures
Therewasaneedforanongoingculturepreservationprogramtopreservetheseisolatesandasystematicprogram
wasinstitutedasfollows:
1)Maintenance
Thecultureswereroutinelymaintainedbyserialtransfersonagarslants.Earlier,themanganeseoxidizerswere
maintained on Bromfields medium. It was felt that the medium may be too rich for the growth of these
organisms for a prolonged period of time. Hence an attempt was made to slow down their metabolism by
reducing the nutrients and compounding a modified medium, as discussed before. Such a method of
maintaining cultures of manganese oxidizers on this modified medium couple with routine monthly activity
checks,itwasfoundthatthemanganeseoxidizingactivityoftheculturescouldbemaintainedforaperiodof
150dayswithoutappreciabledecrease,insteadof90daysonoriginalBromfieldsmediumasshowninFigure
4a,4b4cand4d.

FIGURE4A.ACTIVITYCHECKAFTER60DAYS,OFCULTURESPRESERVEDONBROMFIELDSMEDIUMANDMODIFIED
BROMFIELDSMEDIUM.ACTIVITYONBROMFIELDSMEDIUM
ANDONMODIFIEDBROMFIELDSMEDIUM.

FIGURE4B.ACTIVITYCHECKAFTER90DAYS,OFCULTURESPRESERVEDONBROMFIELDSMEDIUMANDMODIFIED
BROMFIELDSMEDIUM.ACTIVITYONBROMFIELDSMEDIUM
ANDONMODIFIEDBROMFIELDSMEDIUM.

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FIGURE4C.ACTIVITYCHECKAFTER120DAYS,OFCULTURESPRESERVEDONBROMFIELDSMEDIUMANDMODIFIED
BROMFIELDSMEDIUM.ACTIVITYONBROMFIELDSMEDIUM
ANDONMODIFIEDBROMFIELDSMEDIUM.

FIGURE4D.ACTIVITYCHECKAFTER150DAYS,OFCULTURESPRESERVEDONBROMFIELDSMEDIUMANDMODIFIED
BROMFIELDSMEDIUM.ACTIVITYONBROMFIELDSMEDIUM
ANDONMODIFIEDBROMFIELDSMEDIUM.

2)PreservationbyLyophilization
In addition to this it was also decided to lyophilize the cultures for longer preservation. The cultures were
grownin modified Bromfields liquidmedium for48hrs onarotaryshaker (120 rpm) at28oC. Thecells were
then centrifuged out in cold(4oC)insterile conditions at 22000 x g.and thenwashed by centrifuging3 times
withsterile0.1Nphosphatebuffer(pH6.0).Thecellpelletthusobtainedwasresuspendedin(10%w/v)sterile
skimmed milk. Aliquots of 0.5 ml of this suspension were frozen in shell freezing bath at 80oC, containing
methanolandthendriedundervacuumonaVerTisLyophilizer.Sixvialsofeachculturewassealedandtested
under vacuum for leaks. One vial from each set was opened for viability (in modified Bromfields liquid
medium) purity check and activity in the same medium. The efficacy of conversion of ore containing
hausmanitewastestedbythemethodoutlinedlater(op.cit).TheresultsareshowninFigure5.

FIGURE5,ACTIVITYCHECKOFCULTURESAFTERLYOPHILIZATION.
AND

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3MONTHS;

24MONTHS

6MONTHS;

12MONTHS

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Itcanbeseenfromthefigure5,thattheactivityofthrculturestesteddidnotdecreasesingnificantlyevenafter
aperiodof2years,indicatingthatthepreservationbylyophilizationwasperhapsoneofthebestmethodsof
maintenancewithoutthelossofactivitytoaconsiderableextent.
PreparationofInoculumbyUseofInertCarriers
Eventhoughthecultureslyophilizedbythemethodoutlinedabovewereshowntoretaintheirbeneficialactivity,
there were certain limitations on the amount of cultures generated in this way. The most important limitation
would be to use these cultures on a large heap under the field conditions. Here, a certain amount of inoculum
wouldbenecessary,whichwoulddefinitelybemorethatntheamountoflyphilizedculturesinsmallampoules.
Again opening and rehydrating the lyophilized ampoules or sealed materials in the field would be extremely
difficult. Hence, it was thought that if such materials were to be mixed with an inert carriers, not only would it
solvetheseproblems,butthecarriermaterial,ifchosenjudiciously,wouldalsobeableaffordsomeprotectionto
thecellmassonexposuretofieldconditions.
Aparallelanalogyofsuchpreparationofbacterialculturescouldbetakenwithrespecttonitrogenfixingbacteria.
Intheearlydays,pureculturesofnitrogenfixersinagarmedium,wereusedtospreadonfieldpriortosowing.In
early 1920s, these cultures were replaced by sterile soil and thento peatand lignite(DateandRougheley,1977).
Theapparentadvantageofthesetreatmentresultedinbettersurvival,longershelflife,massofmanufactureand
distribution.Sincetheseweretheexactpoints,whichwehadinmind,thefollowingexperimentswerecarriedout.
TypesofCarriers
The successful inoculum preparationis dependent on selectionof suitable carrier material andits pretreatment,
variousmaterialssuchassoilandoreweretriedforthispurpose.Theoresarequiteanunconventionalsunstrateto
beusedinthisfashion,butthepreviousresultshadindicatedthatAcidthiobacillusferroxidanscultureswhenmixed
with sterile chalcopyrite ores retained their activity for a prolonged period of time (Kulkarni, 1983). This has
apparentjustification,inthesensethatthemicroorganismswerefoundtohavesurvivedprolongedperiodoftime
intheoreitself,asshownbyearlierresultsoncopperandmanganeseores(AgateandDeshpande,1974and1977;
Agate,1980and1981)madeitlogicaltothinkthatthismaterialdevoidofanyexternalnutrientswouldbeableto
sustain the microbial cultutres. There is another advantage and that is the ore from which the cultures were
isolatedorthematerialonwhichthemicroorganismsweregoingtoreact,isaddedascarrier,woulddoawaywith
problemsofthemicroorganismsnotcomingincontactwiththeconcentrationofmaterialswhichwouldbetheir
substrateforbioconversion.
TheculturesofArthrobacter(A9)wasgrowninmodifiedBromfieldsliquidmediumonrotaryshaker120r.p.m.at
28oC for 48 hrs. The cells were centrifuged, washed and the density was adjusted to more tha 109 cells /ml as
confirmedbycountinginNeuberschamber.Thetwocarriersusedweresoilandore.Thecarriersweresterilised
by autoclaving at 121oC for 20 minutes for 3 days successively. These carriers were checked for sterility. The
culturesweremixedwiththecarriersandtheactivitywascheckedfor3months.
Outofthese2carrierstried,orewasfoundtobebetterinthat,itallowedthemicroorganismstonotonlysurvive
butretaintheiractivity.ItisshowninFigure6.
However, when experiments were carried out to mix large amounts of cultures with the ores, it was found that
duringmixingofsuchcultureswithoreormaterialstobeconvertedresultedingrowthofotherorganismsalso.At
timesitwasdifficulttorestarinotherorganismsfromgrowingastheconversionexperimentsutilizednutrientsfor
theheterotrophs.Incorporationofantimicrobialantibioticshadadebilitatingeffectonthebeneficialheterotrophic
bacteria,althoughinlatterpartsoftheexperiment,thefungalgrowthwascheckedusingantifungalantibioticslike
nystatin(50ppm).
Itwasnotknownwhetherthemixingwithcarrierpromotesashocktothecultures,sothatthereisasignificantlag
ingrowthofthedesiredorganismswhichallowedthesetobeovergrownbyotherheterotrophs.Thepossibilityof
contamination due to insufficient sterilization of carrier was ruled out through the use of proper controls.
Therefore,itisconcludedthatunlessabetterchoiceofsubstarteprecludesthepossibilityofcontamination,mixing
withoresmightnotworkinthecaseofmanganeseconvertingbacteria.ThatithadworkedwithAcidthiobacillus

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ferrooxidanscouldbelargelyattributedtothenatureoftheseorganisms(chemolithotrophic),whichautomatically
prevents the heterotrophic bacteria from growing in the acidic inorganic salts medium used for growing and
sprayingtheseorganismsonthematerialtobeleached(Kulkarni,1983).
AlltheseculturesweremaintainedwithaMCMNumberseriesstartingwithB.Thesewillbereferredaccordingly
in the following optimization studies unless otherwise specified. The cultures will be mostly of Arthrobacter and
Bacillusspp.

FIGURE6,ACTIVITYCHECKON2CARRIERSVIZ.SOILANDORE.
SOILAND

60DAYSINSOIL;

60DAYSINORE;

90DAYSIN

90DAYSINORE.

Optimization Studies
One of the beneficial activities of manganese oxidizing bacteria is the bioconversion of manganese from a lower
varietyhausmanitetoabettervarietypredominantlycontainingMnO2form.Thistypeofactivitywasreported
for the first time from this laboratory (Agate and Deshpande, 1974; Agate, 1975), while studying the ecology of
freshwater pile deposits. This is the activity, which is loosely termed as beneficiation as it could be used for
upgrading the quality of manganese minerals present in our country (Agate and Deshpande, 1977). It might be
notedthatsuchapossibilitywashintedatbyDeshpandeandAgate(1978)intheirworkonanotherorganismviz.
theHyphomicrobiumsp.Itwasalsoconfirmedthatmanganeseobtainedaftermicrobialinteraction,contained80%
ofcrystalsofthegammavariety,usedinthemanufactureofdrycellbatteries(Deshpande1979).
As mentioned earlier, thegamma variety ofMnO2usedin the manufacture of dry cell batteries is mined only in
fewcountriesintheworldlikeGhanaandtherehasbeenaconstanteffortinWesternAustraliatoupgradetheir
low grade manganese ore deposits, through biological beneficiation. Since in India the presence of manganese
conversion through electrochemical means is a monopolized process and the alternate means suggested by
NationalMetallurgicalLaboratory,Jamshedpur(India)isenergyintensiveprocess,suchamicrobiologicalprocess
involvingminimumenergyconsumptionneedstobedeveloped.
Thisinvestigationwasdecidedtobelimitedonlytotheprocessofmanganeseoxidation,carriedoutbymanganese
oxidizingbacteriaotherthanHyphomicrobiumsp.Therefore,itwasdecidedtocheckthemanganeseoxidationby
culturesisolatedduringthestudies,viz.Arthrobacter,BacillusandPseudomonasspandthiswascarriedoutbyusing
2methodsasfollows:
QualitativeDetection
ThecoloniesappearingonBromfieldsmediumshowedbrowncolorationaroundthem,whichwasanindication
that soluble manganese ins in the medium have probably got transformed into manganic oxide. This brown
coloration around the colonies was observed, when the plates were incubated at 28oC for 48 hrs. The colonies
themselves did not show the brown coloration. The oxidation of manganese was further confirmed by adding
BenzidineHClreagentontotheplates,whenthebrownzonesgavebluecolorationwiththisreagent.

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During the primary screening only those colonies which showed brown zones around them were isolated and
maintained.
QuantitativeEstimation
Inordertoselectandquantifythemanganeseoxidized,2methodswereused.Theorganismsweregrowninliquid
modifiedBromfieldsmedium.
When using a soluble form of Mn++ such as MnSO4 as substrate, the changes in the redox potential and the
subsequentchangesinpHvalueswererecorded.Asaresult,theredoxpotential,alsotermedasEh,changeswith
thechangesintheoxidativestateofmanganeseandalsowiththesolublemanganeseionconcentration.ThepH
checkwascarriedouttoseethatpHdidnotgotowardsalkalinity,whichleadstoautooxidationofmanganese.
Ifinsolubleformofmanganese(suchashausmaniteore)isusedasasubstrate(Figure3),theoxidizedmanganese
intheorescouldbedeterminedchemicallybyoxalicacidKMnO4titration(Vogel,1978),whereMnO2presentin
the ore oxidizes oxalic acid under hot acidic conditions and the residual oxalic acid is then quantitated using
standardKMnO4solutionunderthesameconditions.
Since the aim of this study was to optimize the manganese oxidizing ability of suitable organism, which could
oxidizetheinferiorvarietyoforetothesuperiorpyrolusitevariety,itwasessentialthattheefficiencyofoxidation
of manganese should be calculated in terms of increased MnO2 and the organism giving the highest conversion
shouldbetakenupforfurtherstudies.
Selection of Cultures
PreparationofInoculum
All the bacterial cultures of bacterial cultures Arthrobacter,Bacillusand Pseudomonas sp were grown on modified
Bromfieldsagarslantsfor48hrsat28oC.Thisgrowthonslantswaswashedandsuspendedinsterile0.85%NaCl
solution. The absorbance of the suspension was adjusted to 0.4 at 550nm for the sake of uniformity. The culture
suspensionswereaddedat1%(v/v)leveltotheore,unlessotherwisespecified.
PreparationofOreSamples
Themanganeseorecontainingapreponderanceofhausmanite(2MnO,MnO2)wasobtainedthroughkindcourtesy
of Mysore Minerals Limited, Dandeli, India. The chemical analysis of this ore was provided by the organization
whichisreproducedbelow:
Manganese4550%
Iron812%
Phosphorous0.020.03%
Itwasfoundthatoutofthemanganesecontent(40%)theMnOcontentwasabout60%.
Theorewasfirstcrushedwitha3H.P.Lynxjawcrusher(M/sLawrenceandMayo,NewDelhi)to0.5cmsizeand
thewithhelpofa2H.P.rollercrusher(M/sMineralsProcessingEquipmentPvt.Ltd.,Mumbai)andtherequired
particlesizewasobtainedaftersievingthroughpropersieves(30meshto200mesh).Thedifferentparticlesized
orewasaddedat10%(w/v)levelforthefollowingexperiments.
Onepercentsuspension(0.4O.D.550)oftheculturewasaddedtoeachofthe500mlErlenmeyersflaskscontaining
theoreat10%(w/v)andthemodifiedBromfieldsliquidmedium.Theflaskswereincubatedat28oConarotary
shaker(120r.p.m)for15days.Attheendoftheincubationperiodthecontentsoftheflaskswerefilteredthrougha
gauzeclothandwerewashedseveraltimeswithwatertoremove,asfaraspossible,allmediumcomponentsand
anyothersolubleimpurities.Thewashedorewasdriedat100oCinahotairoven.Thesamequantityoforefrom
thecontrolexperiment(withouttheinoculum)wasalsodriedinthesamemanner.TheMnO2contentoftheoreto
beconvertedandallsamplesofwashedanddriedoresexposedtobacterialactionwasmeasuredbyaquantitative
methodusingoxalicacidKMnO4titration.

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Thisbasicmethodologywasfollowedforallsubsequentexperiments.Atthetimeofselectionoroptimizationonly
oneparametertobetested,waschangedatatime.
ChoiceoftheMedium
Here3differentmediaweretried,theywere:
i)

Modified Bromfields liquid medium (KH2PO40.005%; MgSO40.002%; (NH4)2SO40.01%; Yeast Extract


0.005%andMnSO40.5%)

ii) 1%peptonewater(Peptone1%;NaCl0.5%),and
iii) Glucoseyeastextractmedium(Glucose1%;YeastExtract1%).
Theresultsofobservationobtainedusing3mediaarerecordedinFigure7.

FIGURE7,PERCENTAGEOFMnO2AFTERTHECULTURESWEREGROWNINPRESENCEOFOREINDIFFERENTMEDIA.
INPRESENCEOFMODIFIEDBROMFIELDSMEDIUMAND

GROWN

1%PEPTONEWATER.

ItcanbeseenfromthisfigurethattherewasnoconversioninGlucoseyeastExtractmediumandtheconversionin
1%peptonewaterwaslessthanthatofmodifiedBromfieldsmedium.Therefore,forallconversionexperiments
onlymodifiedBromfieldsmediumwasused,unlessotherwisespecified.
PulpDensity
In order to optimize the pulp density of the ore in the medium, experiments were carried out using the above
medium,towhichoregroundto50meshsizewasaddedindifferentconcentrationsfrom10%to100%.Itmight
benotedthataslurryconsistingof100gorein100mlmediumgaveathicksuspensionwhichcouldbestirredona
shaker.Concentrationsabovethisdensityposedproblemsduringshakingincubations,asitstartedformingathick
paste.TheresultsoftheexperimenttofindtheoptimumpulpdensityaregiveninFigure8.

FIGURE8,PERCENTAGECONVERSIONTOMnO2AFTERTHECULTURESWEREGROWNINDIFFERENTPULPDENSITYOFORE.

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10%,

50%,

100%AND

150%

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Itwasshownincaseofalltheculturestestedthatupto100%(w/v)pulpdensitytheconversionwasmaximum.It
wastherefore,decidedtokeepthepulpdensityoftheoreat100%levelinallexperimentsunlessotherwisestated.
Itwasevidentthatoutofthe21typesofculturestested,10cultures(8ofArthrobacterand2ofBacillussp)showed
the maximum conversion between 53.3% and 61.59% with Arthrobacter and between 55.43% and 60.25% with
Bacillus sp. Hence, these cultures were selected for further studies for optimization of other parameters. It was
believedthat,iftheconversionrateswerehighenoughthentheseratescouldbeusedforscaleupprocessasthe
efficiently may slightly fall during scaleup. There are other obvious reasons, such as economics of the process,
whichwillbedealtlater.
MetalToxicity
Itwasgatheredfrompreviousreports,thatmercury,cobalt,leadandzincoccurquitefrequentlywithmanganese
ores(KuzinandEgorov,1979).Itwas,therefore,decidedtostudytheeffectsofthesemetalionsbyincorporating
theseinthemediumonmanganeseoxidationusingMnSO4assubstrate.Thiswascarriedoutbymeasusingthe
redox potential and the change in pH as the growth proceeded (as described in quantitative estimation of
manganeseinthischapter).
PreparationofMediumwiththeToxicants
Modified Bromfields medium was used with 0.5% MnSO4, H2O. Four different concentrations (0.001%, 0.005%,
0.01%and0.02%w/v)ofsuchsolublemetalsalts,viz.CoCl2,ZnSO4,Pb(NO3)2andHgCl2wereincorporatedinto
themedium.Twocontrolswerekept,oneinwhichthesemetalsaltswerenotaddedandalsonotinoculatedand
theother,inwhichthereweremetalsaltswerenotaddedbutwerewiththerespectivecultures.Allthe10selected
culturesweretestedformetaltoxicityusingtheabovemediumandthechangeinEhandpHwasrecorded.Itwas
observedthatallthemetalsaltsweretoxicat0.001%level.However,whenthesewerepresentinthenaturalsate
intheorenotoxiceffectswerenoticedduringtheconversionexperiments,asthemanganeseoxidationproceeded
withoutanyhinderances.Thiswaspossiblyduetothefactthatmetalsexistedintheoreininsolubleformsand,
therefore,themetalscouldnotbetoxictothegrowthandsubsequentmanganeseoxidationprocess.
EffectofTemperature
Itwasessentialtofindouttheoptimumtemperatureforconversion.Hence,experimentsin5differentsetswere
carriedoutasdescribedbefore.Theflaskswereincubatedat10oC,20oC,30oCandt35oC.At10oCand20oC,the
incubationwascarriedoutinrefrigeratorandBODincubator.At30oCand35oCtheincubationwascarriedouton
ametabolicshakerwithstirringat150r.p.m.(M/sThermolabEquipments,Mumbai).Itwasnotedthatat10oCand
20oC,therewasneithergrowthoroxidationofmanganese.Thiscouldbedueto2reasons:
i)

Theculturesneededvigorousaerationandhence,theywereunabletogrowandoxidizemanganese.

ii) As noticed in seasonal ecological successions of these organisms, the temperatures were too low for the
growthandsubsequentosidationofmanganese.
Similarly at, temperatures at 35oC and above, no oxidation and growth were observed as the temperature of
incubationwastoohigh.Theresultsofmanganeseoxidationat30oCand35oCarerecordedinFigure9.

FIGURE9,EFFECTOFTEMPERATUREONMANGANESECONVERSION.

AT30oCAND

AT35oC.

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Itwasobservedthattheorganismscarriedoutmaximumbeneficiationat30oC.
AdaptationtoHigherConcentrationOfMn++
Theecosystemsfromwhichtheseisolateswereobtainedweresoil,freshwaterandfreshwaterpiledeposits.Inthe
former 2 systems, the concentration of manganese was low ( in the range of 0.001% to 0.002%), where as in the
latteri.e.freshwaterpiledepositsthemaximumconcentrationofmanganesewas0.01%(w/w).Thisindicatedthat
thecultureswereabletowithstandhigherconcentrationofmanganesewithoutpreviouslycomingincontactwith
it, since they effected conversion in 100% pulp density, as illustrated above. Inorder to exploit this ability of the
organisms further, the organisms were subjected to grow at increasingly higher concentration of manganese i.e.
from3600ppmto54000ppmofMn++inmodifiedBromfieldsmedium.Itisknownthattheorganismswereknown
to grow at 1800 ppm of Mn++ right from the time fof isolation as the recommended concentration of Mn++ in
Bromfieldsmedium.
ModifiedBromfieldsliquidmediumwaspreparedwithmanganeseat36000ppmofMn++.Onehundredmlofthis
medium was dispensed in 250 ml capacity Erlenmeyers flasks and the flasks were autoclaved at 121oC for 20
minutes. These flasks were then inoculated with 1% culturesuspension (0.4absorbance550nm) and the flasks were
incubatedat28oConarotaryshaker(140r.p.m.)for10days.Whenvisibleindicationofgrowth(browncoloration
with turbidity) was observed in the flasks (confirmed microsopically), the cultures were serially transferred to
increasingconcentrationsofMnSO4(from0.5%to15%),correspondingto1800ppmto54000ppmofMn++ions,
andwereincubatedasabove.
Allthe10culturesindicatedvisuallygoodmanganeseoxidationin10daystimein29000ppmofMn++,exceptfor
thecultureofArthrobactersp(B26),whichwasgrowingveryslowlyaftertransferringfrom1800ppmofMn++and
wasnotgrowingatallin29000ppmofMn++.At36000ppmofMn++onlyArthrobactersp(B21,B23andB25)and
Bacillussp(B97)gavegoodgrowthandmanganeseoxidation.Theresultsoffurtheradaptationstudyat54000ppm
ofMn++arerecordedinTable5.
TABLE5:STUDIESONADAPTATIONTOHIGHERCONCENTRATIONOFMN++.

CultureMCMNo.

Mn++concentrationinPPM
3600

10,500

18,000

29,000

36,000

54,000

B20

B21

B22

B23

B97

B24

B25

B98

B26

B27

Note:
+growthandoxidationofmanganese
nogrowthandnooxidationofmanganese
vScantygrowthwithorwithoutoxidationofmanganese

ItcanbeseenfromthistablethatonlyArthrobactersp(B23)gavegoodgrowthandManganeseoxidation(67%)at
54000ppmofMn++.
TestingofManganeseOxidizingActivityoftheCultures
Allthe4culturesi.e.Arthrobacter(B21,B23andB25)andBacillus(B97)wereusedinthisstudy.Thecultureswere
grown in modified Bromfields liquid medium as it was not possible to get a solid agar medium at that
concentrationofMn++(36000ppm).Thewashedsuspensionsofthecultureswerepreparedinfollowingmanner.

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Theorganismsweresubcultured4to5timesintheabovemediumwith36000ppmofMn++(B21,B25andB97)and
with54000ppmofMn++(B23),tillthecultureswereabletogrowin4daystimeinsteadof10days.Such4daysold
cultureswereusedtotestthemanganeseoxidationasdescribedbefore.TheresultsarerecordedinFigure10.

FIGURE10,MANGANESEOXIDIZINGABILITYOFCULTURESAFTERADAPTATIONTOHIGHERCONCENTRATIONSOFMn++IN
MODIFIEDLIQUIDBROMFIELDSMEDIUM.

EffectofShaking/StaticConditionofIncubation
Since the organisms to be tested were strict aerobes and the process of oxidation of manganese was an aerobic
process,itwasessentialtohavegoodaerationforoptimumoxidation.Thiscouldbeachievedin2ways:wecould
providesterileairthroughanaeratortotheflaskswhentheoxidationisbeingcarriedoutortheflaskscouldbe
incubatedonareciprocatoryshaker.
The experiment was carried out in 2 sets of flasks in the way described before. It was found that the shaking
condition of incubation gave 61% conversion with cultures: B21 and B25 and 67% conversion with B23, while
60.25%conversionwasobservedwithB97.Whenthesamecultureswereincubatedatstaticconditionstherewas
noconversionofMn++evenafter15daysofincubationat28oC.
EffectofParticleSizeoftheOre
It was very essential that the effect of particle size on the oxidation of manganese should be determined, as one
must obtain an ore with a particle size which should prove to be cost effective for processing, at the same time
yield optimum oxidation of the particles, in order to make the process economically feasible on a large scale
operation. The other factors that might be playing a crucial role are that the finer the particle size, the toxicants
wouldtendtoformcolloidsinthemediumandhenceinhibittheprocess.Thiscouldeverhinderproperaeration
whichalsomighthampertheprocess.Ontheotherhand,iftheparticlesaretoolargethenonlythesurfaceofthe
particles would get oxidized and the process would not be economically feasible, as the conversion will not be
extensive.Forthispurposetheorewascrushedinthemannerdescribedbeforesoastoconformto:
30meshequivalenttoparticlesize650nm
50meshequivalenttoparticlesize300nm
100meshequivalenttoparticlesizeof150nm
200meshequivalenttoparticlesizeof75nm
Itwasobservedthatpercentageconversionwith30meshsizewasaslowas20%andthatwith100and200mesh
sizewasnil.However,using50meshparticles,itwaspossibletoachieve61%conversionwithB21andB25,with
B23andB97;itwas67%and60.25respectively.Oneofthemostprobablereasonforverysmallparticlesizedore,
thatitcouldbedeathofalargeportionofthebiomassduetoabrasiveeffectoftheparticles.
EffectofpH
ItmaybementionedthatinthecompositionofmodifiedBromfieldsmediumthepHwasaround6topreventall
possibility of autooxidation, as this is reported to take place at and above pH 8.0 (Hem 1963). In this study,

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different pH values below pH 8.0 (5.2, 6.2 and 7.2) were tested for manganese oxidation by the 4 cultures. The
experimentwascarriedoutasmentionedbeforein3setsofmediaatthepHvaluesstatedabove.Theresultsareas
showninFigure11.

FIGURE11.:EFFECTOFpHONMANGANESECONVERSION.

pH5.2;

pH6.2AND

pH7.2.

ItwasobservedthatB21andB97couldmaximallyoxidizemanganeseatpH5.2,whereasB23andB25didsoatpH
6.2,againcoroboratingthefactthatitwasmostlymicrobialoxidationofmanganese.
Oureffeortstooptimizethedifferentparametersforconversionprocesscouldbesummarizedasfollows:
Temperature:30oC
pH:5.2(forB21andB97);6.2(B23andB25)
Mediumforconversion:ModifiedBromfieldsliquidmedium
PulpDensity:100%
Particlesize:300nm
Hence, Arthrobacter (B23) was superior to the other 3 cultures in conversion efficiency from the point of view of
economyanditwasusedinscaleupprocessoutlinedlater.
Eventhough,B23wasbetter,allthe4cultureswerestudiedfurtherbecauseoffollowingreasons:
Inanaturalsysteminthefield,itisquitepossiblethatnosingleculturewoulddothejobaloneandtherefore,itis
alwaysadvisabletotestmorethanoneculture(intheformofconsortium)forsuchpurposes.Infactpermutation
andcombinationexperiments,usingthese4culturesoughttohavebeentriedandifasaresult,oneobservesthe
cumulativeeffectinconversion,itwouldhavebeenideal.However,theseexperimentswerenotcarriedoutdueto
paucityoftime,butthisareaisreferredforfutureexplorationstoworkersinthisfield.
Scale-Up Studies
Since the parameters were optimized for conversion of manganese at laboratory level using the strain of
ArthrobacterB23,itwasdecidedtousethefindingstoscaleuptheprocess,usingavat,ifpossible.
Itwasobservedthattheconventionalscaleuptechniques,employingglasscolumnsorairliftpercolators,would
beoflimiteduseduetotheimpedingcontaminationproblemsasitwasdifficulttogetthecolumnssterilizedand
theminimumprecautiontokeepthecolumnfreefromexcessivecontaminationwouldbeverydifficult.Theearlier
experiments using inverted glass bottles (Deshpande, 1979) were not feasible in this case, as preventing the
contamination was a problem through the use of antibacterial agents. It was decided therefore, to carry out the
experimentsusingvatleachingmethods.
Thescaleupwasfirsteffectedat1.5Kglevelusinga2literflask,at100%pulpdensityandtheexperimentwas
carriedoutintriplicateonashaker(120r.p.m.)at30oC.Theconversionefficiency,asobtainedpreviouslywithout
changinganyoftheparameters,wasagain67%.
ArthrobacterstrainB23wasinoculatedat1%levelinastainlesssteelvat(18gauge,50cminheightandadiameter

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of 38 cm) provided with a lid. The vat could be surface sterilized, before charging it with 14 kg ore in 14 L of
modifiedBromfieldsliquidmedium.Thecontentsofthevatwerestirredperiodicallywiththehelpof2stirrers(at
150r.p.m,)havingvariablespeed(Remiindustries,India).Thelidcouldnotbemodifiedduringthetenureofour
experiment and hence, it was not possible to prevent the entry of other microorganisms from the top, although
adequatecarewastakentotrytopreventsuchoccurrencesbyclosingthetopwithaluminumfoil.Itwasfoundat
the end of the fourth day of incubation that contamination had occurred and it was not possible to control it
thereafter.Thisaffectedtheconversionefficiencyofthecultureinsuchawaythatitwasnotpossibletocontinue
theexperimentbeyondtheseventhday,whenitwasobservedthattheoxidizedmanganesewasgettingreduced
andwasinsolution.Although,thistypeofleachingeffortswasconducivetoexperimentsdescribedbyDeshpande
(1979),inthepresentstudiesithadnovalue..Therefore,thisconversionexperimenthadtobestopped.
Itispossible,infuture,tocarryouttheseexperimentsinsterilefermentertypeofvatorbyemployingsterilization
techniquesduringtheprocessinaclosedvat.Ifusingsuchaconverter,manganeseisoxidizedtoMnO2formata
scaleof500kgto800kgatatime,evenwithareducedefficiencyofconversion(say55%),whichisexpectedwith
suchlargevolume,itwouldstillbecommerciallyfeasible.Furtherpyrometallurgicalprocesseswouldrequireless
energyas50%ofthe80%particleswouldhavealreadygotconverted.
Commercial Feasibility of Such a Process
Thecostwouldbelessthantheoneestimatedifelectrochemicalmethodshadtobedeployedforsuchlowgrade
ores with the 67% conversion obtained with Arthrobacter than the reported one with Hyphomicrobium (58%)
(Deshpande,1979).Itisgrantedthatmanyothervariableslikelabor,capitaloutlay,depreciationetc.arenottaken
intoconsiderationandhencethesespeculationswouldremainidle.However,abeginninghastobemade,hence,
suchattempts.Itwashoped,thatsuchacosteffectiveandenergysavingprocesswouldpavethewayforutilizing
suchmethodsbytheendusersoftechnologyi.e.theminingindustry(asandwhentherewouldbearevivalofthe
manganeseminingindustryinIndia)andthedrycellbatterymanufacturers.
ACKNOWLEDGEMENT

The authors are grateful to the Department of Microbiology and the authorities of Agharkar Research Institute,
Pune,India,forextendingthelaboratoryfacilitiesforcompletionofthisstudy.Theworkwascompletedwithhelp
offundingfromCouncilofScientificandIndustrialResearch,NewDelhi,India(19801983).
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