JournalofMiningWorldExpress(MWE)Volume4,2015www.mwejournal.

org
doi:10.14355/mwe.2015.04.002

BioflotationofEgyptianPhosphateUsing
DesulfvibrioDesulfuricansBacteria
N.A.,AbdelKhalek,K.A.Selim,K.E.Yassin,S.S.Abdallah
CentralMetallurgicalResearch&DevelopmentInstitute,Helwan,Cairo,Egypt,P.O.Box.87HelwanCairoEgypt
naguialy@gmail.com;k2selem1@gmail.com;khaled_yassin@yahoo.com;samahsaleh86@gmail.com
Abstract
The bioflotation and bioflocculation processes concern the mineral response to the bacterium presence, which is essentially
considered as interplay between microorganism and the physicochemical properties of the mineral surface. The adhesion of
microorganisms to minerals results in alteration of surface chemistry of minerals relevant to beneficiation process due to a
consequenceoftheformationofabiofilmonthesurfaceorbiocatalyzedsurfaceoxidationorreductionproducts.Inthispaper,
the amenability of utilization of Desulfvibrio desulfuricans isolated and adapted on surfaces of phosphate ore, as flotation
reagentsforseparatingsilicafromapatiteinthebioflotationofquartzapatitemineralssystemhasbeenstudied.Theeffectof
thisbacterialisolatesonthesurfacepropertiesofthetwosinglemineralshasbeenstudiedthroughzetapotentialandadhesion
measurementsaswellasmicroflotationtests.TheeffectofpHofthemediumonthesurfacepropertiesandflotationbehaviour
of each singlemineral is determined. Flotation of binary mixtures of quartzapatite as well as naturalphosphate orehas also
been performed at differentoperating parameters. Different characterization techniques for both single minerals and bacteria
isolated from their surfaces have been done using XRD, SEM, and contact angle as well as morphological and biochemical
identificationofbacterialisolates.
Keywords
BioFlotation;PhosphateOre;Apatite;Quartz;DesulfovibrioDesulfuricansBacteriaandSurfaceModification

Introduction
Phosphate rocks are vital nonrenewable resources and are essential components in agricultural fertilizers and
phosphorousbased chemicals. About 85% of the phosphate produced is consumed in the fertilizer industry.
Phosphate deposits can be divided into three groups: sedimentary, igneous and biogenetic deposits [1]. In the
recentyears,miningindustryhasbeenfacingseveralproblemsthatinfluencethemineralprocessing,suchasthe
depletionofhighgradeores[2]andenvironmentalregulations[3].Theformeraspectcompelstheminingindustry
to process low grade ores, fine mineral particles and flotation tailings to produce material suitable for a global
market.Thus,ithasbecomeveryimportanttodevelopappropriateandenvironmentallyfriendlytechnologiesable
tocomplementtheconventionaltechniquesusedatthemineralconcentration.Inthiscontext,biobeneficiationis
increasing its role in mineral processing. The main purpose of this procedure is to selectively undertake the
removal of undesirable mineral constituents from an ore, through interaction with microorganisms and/or their
metabolicproducts,thusenrichingit,withrespecttothedesiredvaluableminerals[46].
Bioflotationisbecomingveryattractiveforpresentingagreattechnologicalpotential,environmentalacceptability,
flexibilityinthechoiceofmicroorganismsandespeciallyduetoitsmineralselectivity[2,5]andalsoforprocessing
fineandultrafinemineralparticles.Oneofthemostimportantstepsinmineralbioflotationistheadhesionofthe
bacteria onto the mineral surface [5, 7]. The bacterial adhesion occurs as a net result of attractive and repulsive
forces of the cell and mineral surfaces. The interactions that result in such adhesion include electrostatic
interactions, acidbase interactions, van der Waals forces and hydrophobic interactions, all of which are
determinedbythecellwallandmineralsurfaceproperties.Thesurfacemodificationcanbedirectorindirect;the
direct mechanism involves the adhesion of the bacterial cells to mineral particles, while the indirect mechanism
refers to metabolic products which act like surface activator reagents. Both interactions may allow the mineral
surface to acquire hydrophobic or hydrophilic properties [8, 9]. This paper aims at studying the role of bacterial
isolate of Desulfvibriodesufuricans in bioflotation of apatitequartz system in relation to Egyptian phosphate ore.

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Theroleofbacteriaonthesurfacepropertiesofthetwosinglemineralshasbeenstudiedthroughzetapotential,
adsorption, surface tension and adhesion measurements as well as microflotation tests. Flotation of binary
mixturesofapatitequartzaswellasEgyptiannaturalphosphateorehasalsobeenperformed.
Materials and Methods
Materials
Samplesofsinglemineralsofquartz(SiO2),apatiteCa5(PO4)3(OH,F,Cl)weredeliveredfromWardsCompany,
USA. The purity (99.9 %) of the samples was confirmed using XRF. The 200 mesh fractions were used in
adsorption and flotation studies. Analytical grade HCl and NaOH, from Aldrich, were used for pH regulations.
NaturalphosphateorewascollectedfromNewValley,Egypt.
Methods
1)

MicroorganismGrowingandIsolation

28gmofbacterialmediumconsistingofpeptone,beefextracted,NaClandmycologicalAgarwasdissolvedin
1Lofbidistilledwater.Thesolutionwasautoclavedat120oC,cooledandpouredinPetridishforsolidification.
After that, about 0.5 gm from the autoclaved solid was suspended in 100 ml of bidistilled water; 1 ml of
solutionwaspouredonagarplateandthenincubatedatatemperatureof37Cfor2448hours.Appearing
differentcoloredspotsindicatedthepresenceofdifferenttypesofmicroorganisms.Themicroorganismswere
grown in a bacterial medium without agar and incubated for 24 hr at 37C. The bacterial population can be
determined by measuring the turbidity or the optical density of the bacterial suspension using a UV visible
spectrophotometer(Lambda3B,PerkinElmer).Becausetheturbidityisdirectlyproportionaltothenumberof
cells,thispropertywasusedasanindicatorforbacterialconcentration.
The cells suspended in the suspension to interrupt the passage of light and allow less light to reach the
photoelectric cell and the amount of light transmitted through the suspension was measured as percentage
transmission. The turbidity for cell suspension is measured at a wavelength of 550 m against clear water as
reference,atwhichthe0.01readingisequivalentto106cells/mL.Sixmicrolitersofbacteriawascentrifugedat
16000 rev. /min. and rinsed three times with double distilled water and thereafter, diluted in 100 ml double
distilledwater[8,10and11].Differenttypesofbacteriawereisolated,grown,countedandtheirefficiencywas
screened using a laser particle size analyzer. Based on the later test, one of these types of bacteria (which
showedthebestefficiency)wasselectedtoconductthisstudy.Microbiologicalinvestigationsindicatedthatthis
type of bacterium, Desulfovibrio desulfuricans, has 106 cells/mL. These values were used in calculating the
concentrationofbacteriaintheexperiments.
2)

MeasuringSelectivityofMicroorganismstoMineralSurface

Alaserparticlesizeanalyzer(FRITSCHModelAnalyst22)wasemployedformeasuringsizeanalysisofsingle
mineralsbeforeandaftertreatmentwithbacteria.Fixedvolume10mlofDesulfovibriodesulfuricanssolutionwas
conditionedwithonegramofeachmineralfor30minutesbeforerecordingthechangeinsizedistribution,[8,
and10].
3)

AdhesionMeasurements

Adhesion of Desulfovibrio desulfuricans bacterial isolate onto the mineral surfaces was determined by dry
weightdifferencebeforeandafterconditioningwiththemineralparticles.0.5gramofthegroundmineral(200
mesh)wasaddedto80mlofthecellularsuspensionwithafixedinitialconcentrationofthebacteriasolution,
and conditioned for 30 minutes after adjusting the pH values. An additional time of 5 min. was allowed for
settlingofthemineralparticles,afterwhich20mlofthesupernatantwascollectedinaporcelaincrucibleand
driedonahotplateat4045C.Adhesionstudieswereperformedasafunctionofdifferenceinweightbefore
andafterdrying[8,10].

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4)

ChemicalAnalysis

Routine chemical analysis of samples was conducted using standard methods. Phosphorus content was
determined using spectroscopic technique. Quartz was determined by Gravimetric method. Meanwhile
completechemicalanalysisofsampleswasconductedusingPhilipsXrayfluorescence(XRF)[8,10].
5)

ZetaPotentialMeasurements

AlaserZetaMeterMalvernInstrumentsModelZetaSizer2000wasusedforzetapotentialmeasurements.0.01
gofgroundsamplewasplacedin50mldoubledistilledwaterwithdefiniteconcentrationofthebacteria
suspensionatfixedionicstrengthof2x102MNaCl.NaOHandHCLwereusedaspHmodifiers.The
suspensionwasconditionedfor30minutesduringwhichthepHwasadjusted.Aftershaking,theequilibrium
pHwasrecorded.Itwasthenallowedtosettlefor3min,afterwhich10mlofthesupernatantwastransferred
intoastandardcuvetteforzetapotentialmeasurement.Solutiontemperaturewasmaintainedat(25oC2).
Threemeasurementsweretakenandtheaveragewasreportedasthemeasuredzetapotential[8,10].
6)

AdsorptionStudies

The adsorption density of the bacteria on the mineral surface was determined by adding 1 g dry sample of
quartzorapatitetothebacteriasolutions(50cm3)ina100cm3volumetricflask.Themixturewasshackedfor
10minutesusingashaker(ModelJANKE&KUNKELTypeVx10).ThepHwasadjustedtothedesiredvalues
using HCl and NaOH, after which the samples were centrifuged at 15000 rpm for 15 min to separate
supernatant from the settled fraction. The total organic carbon content (residual concentration) in the
supernatantwasdeterminedusingaPhoenix8000TotalCarbonAnalyzer.Theaverageofthreereadingswas
taken as a measure for the residual concentration of organic carbon. All the experiments were done at room
temperature(25oC2)[8,10and12].
7)

ContactAngleMeasurements

The contact angle measurements were carried out througha GONIOMETERRAMHART using the captive
bubble method. A mineral crystal was carefully cut (dimensions at (0.50.51.0)cm, and then the surface
samplewascautiouslypolishedwithadiamondpastecontainingparticlesof3mand1m.Toremoveany
particles sticking to the polished surface, the samples were cleaned with jetsof distilled water andultrasonic
water bath. The contact angle measurements of the mineral were taken before and after interaction with the
bacterialsuspensionatdifferentpHvalues.Themineralsectionwassuspendedinabacterialsuspensionwith
10mlofthebiomassfor5min.Afterthistime,thesampleswerewashedwith103MNaClsolutiontoremove
thebacterialcellsthatwerenostuck.Thesampleswerevacuumdriedindesiccatorsforapproximately10min.
Finally, the contact angle measurements were carried out. The pH of the solution was adjusted for the same
valuesofthebacterialsuspensionpreviouslyused,usingdilutedHClandNaOHsolutions
8)

SurfaceTensionMeasurements

ThesurfacetensionmeasurementswereperformedusingtheringmethodinaKrussK9digitalTENSIOMETER
with an accuracy of 0.1mNm1. The surface tension of the bacterial isolate suspensions was set at different
bacterialconcentrationsandinfunctionofthepHsuspension,thepHsuspensionswereregulatedwithdiluted
HClandNaOHsolutions.
9)

SurfaceCharacterization

SelectedsampleswereobservedonfracturedsurfaceunderaJSM6400scanningelectronmicroscope(SEM)to
examinethemorphologyofsinglemineralbeforeandaftertreatmentwithDesulfovibriodesulfuricans.
10) MicroFlotationTests
A series of benchscale flotation experiments were conducted using a modified Halimond tube with 150 ml
capacity. In carrying out these flotation experiments one gram (of single minerals or their binary mixture as
well as natural ore) was conditioned at the predetermined optimum conditions of pH 9, concentration of
bacterial isolate suspension of 10 ml (@106 cells/ mL) and conditioning time 10 minutes using a horizontal
shaker. The pH was adjusted with dilute solutions of NaOH and HCl. The flotation was conducted for 5

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minutesatairfollowrateof0.7cm3/min.Bothfloatedandsinkfractionswerecollected,dried,weighted,and
analyzed[8].
Results and Discussions
SelectivityofMicroorganismtoMineralSurface
The change in size distribution of single pure mineral samples, quartz and apatite after its treatment with
Desulfovibriodesulfuricanswastakenasameasurefortheselectivity.Successfuladsorptionofthebacterialisolate
willcause,therefore,adegreeofaggregation(ordispersion)formineralparticlesleadingtoachangeintheirsize
distribution.Thistechniquewassuccessfullyusedtoscreendifferentmicroorganismsforselectiveadhesiononto
apatite,quartzorironoxidesurfaces,[8,10,11,13and14]. TheresultsobtainedinFigures1and2showdifferent
degreesofvariationinthesizedistributionofsamplesaftertheirtreatmentwithbacterialisolate,whichindicates
the largest degree of selectivity of Desulfovibrio desulfuricans for apatite more than quartz that caused varying
degreesofaggregationforapatitewhileleavingthequartzparticlesmoredispersinginthepulp.
100
pure apatite d50 = 16.12 mm
with MO d50 = 26.66 mm

Cummulatitive wt%

80

60

40

20

0
0.1

10

100

Size, m (log)

FIG.2.SIZEDISTRIBUTIONOFQUARTZBEFOREANDAFTER
TREATMENTWITHDESULFOVIBRIODESULFURICANS

FIG.1.SIZEDISTRIBUTIONOFAPATITEBEFOREANDAFTER
TREATMENTWITHDESULFOVIBRIODESULFURICANS

SurfacePropertiesofSingleMineralsandMicroorganism
Thezetapotentialmeasurementsofthebacteriaaloneaswellasforeachsinglemineral(quartzapatite)inabsence
and presence of Desulfovibrio desulfuricans bacterial isolate have been conducted, Figures 35. Figure 3 depicts
thatthisbacterialisolateis,moreorless,hydrophobicinnaturewheretheirzetapotentialvaluesarevariedfrom
about2to6mvonlyovertheentirerangeofpH(pH2.012).Inthemeantime,thevaluesofthezetapotential
forthetwosinglemineralswerefoundtobeaffectedbythepresenceofDesulfovibriodesulfuricans.Thesurfaceof
eachmineralbecomeslessnegativeduetothehydrophobicnatureofthisbacterium.
10

20
apatite
apatite with bacteria

2x10-2M NaCl

10

2
0
1
-2

10

11

12

13

14

Zeta, mv

Zeta Potential, mv

-10

10

12

14

pH

-20

pH
-30

-4
-6

-40

-8

-50

FIG.3.ZETAPOTENTIALOFTHEDESULFOVIBRIO
DESULFURICANS

22

FIG.4.ZETAPOTENTIALOFAPATITEBEFOREANDAFTER
TREATMENTWITHDESULFOVIBRIODESULFURICANS

JournalofMiningWorldExpress(MWE)Volume4,2015www.mwejournal.org

Meanwhile, Figure 4 depicts the zeta potential of apatite as a function of pH before and after interaction with
bacteria.Theseresultsshowthattheisoelectricpoint(IEP)ofapatitecorrespondstopHofabout(4.8).However,
conditioningofapatitewithbacteriaresultedinadisplacementfortheIEPofapatitetoabout(3.7).Moreover,the
valuesofzetapotentialofapatiteafterinteractionwithbacteriabecamelessnegativeovertheentirepHrange.The
hydrophobiceffectofDesulfovibriodesulfuricansisappearedatpHrange(610).Ontheotherhand,conditioning
ofquartz with Desulfovibriodesulfuricans bacterialisolates resulted inaslight change in thezeta potential values,
Figure5.Interestingly,ThevaluesofzetapotentialwereseentobeshiftedtolowervaluesintheentirepHrange
(111),i.e., the surface ofapatite became more hydrophobic andclose to those of bacterial isolate of Desulfovibrio
desulfuricansasshownbeforeinFigure4.
75
70

Adhesion%

65
60
55
50
45

quartz
apatite

40
35
0

FIG.5.ZETAPOTENTIALOFQUARTZBEFOREANDAFTER
TREATMENTWITHDESULFOVIBRIODESULFURICANS

10

12

pH

FIG.6.ADHESIONOFDESULFOVIBRIODESULFURICANSONTO
MINERALSSURFACES

BacteriaAdhesionontoMineralsSurfaces
Figure 6 shows the adhesion of Desulfovibrio desulfuricans bacteria onto the surface of the two single minerals
over a wide range of pH (210). The results showed that, the Desulfovibrio desulfuricans could be adhered onto
bothmineralssurfaceswithahigheraffinitytoapatitesurface.Atthesametime,thehighestvaluesforadhesion
forbothquartzandapatitewereobtainedatpHfrom26followedbyagradualdecreaseinadhesionvaluestill
reachingpH10.
SurfaceTension
ThesurfacetensionofDesulfovibriodesulfuricansbacterialisolatewasconductedatairwaterinterfaceat25C2
and pH 24, Figure 7. The results confirmed a gradual decrease of surface tension values (7050 mN/m) by
increasingthebiomassconcentration(110mL)tillreachingastablestate(1017mL).

FIG.7.SURFACETENSIONOFDESULFOVIBRIODESULFURICANS
SUSPENSIONSATDIFFERENTCONCENTRATIONS

FIG.8.SURFACETENSIONOFDESULFOVIBRIODESULFURICANS
SUSPENSIONSATDIFFERENTPHVALUES

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On the other hand, the lowest values of surface tension of biomass suspensions of Desulfovibrio desulfuricans
bacterial isolate were achieved at pH (14), Figure 8. According to Lambert et al., [15], it was expected that the
bacterialisolatesuspensioncouldformstablehighdensityfoamatthesepHrange.Whilethefoamformedabove
pH5wouldbelessstable.
ContactAngleMeasurements
Contactangleisameasureofstatichydrophobicity.Resultsobtainedthroughthemeasurementofcontactangles
for both single mineral apatite and quartz before and after treatment with Desulfovibrio desulfuricans bacterial
isolateconfirmedthehydrophobiceffectofthistypeofbacteria.AsshowninFigure9,thevaluesofcontactangles
asafunctionofpHareverysmall(~12forapatiteand~8forquartz)whichindicatedthehydrophilicnatureof
singlemineralssurfaces.Aftertheinteractionwithbacterialisolate,thereisasignificantincreaseincontactangle
valuesforbothapatiteandquartz,especiallyinacidicmediumatpH3(~35forapatiteand~15forquartz).The
resultsalsoshowedthat,theDesulfovibriodesulfuricanscouldbeadheredontobothmineralssurfaceswithahigher
affinitytoapatitesurfaceasmentionedabove.Thesurfaceofapatitebecamemorehydrophobicthanthatofquartz
duetotheadhesionofbacteriacellsand/ormetabolicproductsontothesinglemineralssurfaces.
40
apatite + bacterial cell
apatite
quartz + bacterial cell
quartz

Contact angle

30

20

10

0
0

pH

10

FIG.9.CONTACTANGLEOFPOLISHEDMINERALSCRYSTALSECTIONSBEFOREANDAFTERINTERACTIONWITHDESULFOVIBRIO
DESULFURICANSBACTERIAATACONCENTRATION~107CELL/ML

ScanningElectronMicroscope(SEM)
SEM was used to reveal the morphology and particle size of single minerals before and after treatment with
Desulfovibriodesulfuricans.SEMmicroimagesindicatedthatbacterialisolatecouldbeadheredontobothminerals
but with higher affinity towards apatite as the amount adhered onto apatite surface is higher than that adhered
ontoquartzsurface,Figures10and11.
BioFlotationofBinaryMixtures
Figure12showstheeffectofpHonadsorptionofDesulfovibriodesulfuricansontoquartzandapatitesurfaces.The
results indicate that the bacterial isolate could be adsorbed onto both minerals surfaces with different degrees
whereithasahigheraffinitytoapatitesurface.Also,itisnoticedthattheadsorptiondensityincreasesatpHfrom
15 and starts to decrease till reaching pH 11 with the apatite single mineral. On the other hand, the adsorption
densityincreasesatpHfrom13andstartstodecreasewithincreasingofpHtillreachingpH11incaseofquartz.
Theresultsofadsorptionareinagreementwiththeresultsoffloatability,Figure13.Theresultsindicatedthatthe
better selectivity could be achieved at pH 3 in which a highest floatability for apatite (~48% floated) and lowest
floatability for quartz (~10.21% floated) was obtained after treatment with Desulfovibrio desulfuricans bacterial
isolate. The amenability of applying Desulfovibrio desulfuricans to be used as the sole flotation reagent, to
selectively separate silica from apatitein their binary mixtures was studied. The results indicated clearly that on
usingthistypeofbacterialisolate,aconcentrateassaying98%P2O5and2%SiO2witharecoveryof70%couldbe

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obtained from a mixture containing 90 % by weight apatite and 10% by weight silica. The experiments are
performedatpH3.0using 1x107cell/mlofbacterialisolate.Onapplyingthesameconditionsonflotationofthe
naturalphosphaterock,aconcentratecontaining30%P2O5and10%SiO2with68%recoverywasobtainedfroma
feedcontaining20.52%P2O5and23.51%SiO2.

FIG.10.SEMOFTHESINGLEAPATITE(A)AND(B)AFTER
TREATMENTWITHDESULFOVIBRIODESULFURICANS

FIG.12.EFFECTOFPHONADSORPTIONOFDESULFOVIBRIO
DESULFURICANSONTOAPATITEANDQUARTZ

FIG.11.SEMOFSINGLEQUARTZ(A)AND(B)AFTERTREATMENT
WITHDESULFOVIBRIODESULFURICANS

FIG.13.FLOATABILITYOFSINGLEMINERALSAFTER
TREATMENTWITHDESULFOVIBRIODESULFURICANS

Conclusions
The results showed a strong interaction between Desulfovibrio desulfuricans bacterial isolate and minerals
surfaces, especially with apatite. Adhesion, adsorption, zeta potential measurements showed a better affinity of
Desulfovibriodesulfuricanstoapatitemineralsurfaceratherthanquartzsurface.
Theresultsofzetapotentialshowedthattheisoelectricpoints(IEP)forapatitemineral(atpH4.8)isdisplacedto
lower values (at pH 3.7) after interaction with the bacterial isolates, i.e., the IEP became very close to that of
Desulfovibrio desulfuricans in which the hydrophobic effect of Desulfovibrio desulfuricans is appeared at pH
range(610).
Higherbacterialaffinitytoapatitemineralsurfaceincomparisontosilicasurfaceisreadilyevidentfromtheresults
ofadhesion,adsorption,contactangle,surfacetensionandfloatability%.
Onusingthistypeofbacterialisolate,aconcentrateassaying98%P2O5and2%SiO2witharecoveryof70%could
be obtained from a mixture containing 90 % by weight apatite and 10% by weight silica. The experiments are
performedatpH3.0using1x107cell/mlofbacterialisolate.
On applying the same conditions on flotation of the natural phosphate rock, a concentrate containing 30 % P2O5
and10%SiO2with68%recoverywasobtainedfromafeedcontaining20.52%P2O5and23.51%SiO2
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