BARC Now,k"" 135 Foood,,', D,y Sp""" "'"0 2002

RadionuclideBiomass

Biosorption by Bacterial

Pinaki Sac and S. F. D'SouzaD"i""

Abstract

Introduction

Radionuclide and heavy metal pollution byvarious industrial activities is of significantenvironmental concern (Barkay andSchaefer, 2001; D'Souza, 1999a; D'Souza etal 2001a; D'Souza, 2002a), Microbialbioremediation of such toxic compounds areincreasingly being considered as a potentialalternative with high capacity and efficientmetal removal from diluted effluent with

almost no secondary waste generation,(Taxier et ai, 1999 and Lloyd and Macaskie2000), Although considerable studies aremade on microbial removal of heavy metals,radionuclides in this regard remained little

explored. Among the several microbialprocesses that determine the environmentalfate of metallic toxicants viz, reductive or

enzymatic precipitation, solubilization, etc.,biosorptive accumulation of heavy metalsand radionuclides is of recent interest (Gadd2000, Hu et al. 1996, Dhami et a/1998, Sar

et al 1999, Sar and D'Souza 2001 a, b).Bioremediation of radionuclides, heavymetals and organic waste has been a majorrecent activity in the authours laboratory(D'Souza, 1999a; Bhainsa and D'Souza1999; Joshi and D'Souza, 1999; Sar andD'Souza 2001a,b ; Bhainsa and D'Souza,2001a,b; Sar et ai, 2001a,b; Sangurdekar eta/2001, Melo and D'Souza, 2001; D'Souza,et ai, 2001a,b; Kazy, et ai, 2001;Shanmugam et ai, 2001; Sar and D'Souza2002; Kazy et al 2002; Panchapakesan etal,2002; Irani et ai, 2002). Compared to theconventional treatment methods, these

biomass based systems are more acceptablein being cost effective, with high efficiency of

detoxification of even very dilute effluentsand minimizing the disposable sludgevolume. It also offers the flexibility fordeveloping non-destructive desorptiontechniques for biomass regeneration and/orquantitative metal recovery.

BARCNew,""" 136 Found,,', Day Special '"ne 2002

Metal accumulation by microorganisms iseither of energy driven active bioaccu-mulation or metabolism Independent passivebiosorption. The latter process of microbialmetal removal by purely physico- chemicalprocesses seems more appropriate forbioremediation with cation sequestrationmainly regulated by the characteristics of themicroorganism, the targeted metal and thesolution microenvironment. Since, it is thechemical composition of the cell wall andother surface materials responsible for cationsequestration, cell viability or othermetabolic activities that do not interfere withsuch characteristics effectively have noimpact on biosorption. In some cases,pregrowth conditions, particularly, thegrowth medium ingredients also showsignificant influence on biosorptive metaluptake.

Although,' biosorptive uptake of severalheavy metals is well documented, suchstudies on radionuclide sorption arerelatively less. Particularly, with respect touranium, a variety of living and non-livingbiosorbents composed of fungi and bacteriahave been reported to bind the cationconsiderably, whereas reports on thoriumsorption are not impressive. Previous studieson microbial metal sorption by our grouphave identified the strains of Pseudomonasas a potent accumulator of metals andradionuclides (Sar et allgg8, Sar et a/1999,Kazy et al. 1999, Sar et al. 2001a, Sar andD'Souza 2001a, Sar and D'Souza 2002, Kazyet al 2002). The present study wasundertaken to evaluate the uranium (VI) andthorium (IV) biosorptlon capacity of aPseudomonas soil isolate. Equilibriumsorption behavior of the lyophilized biomasswas characterized employing the Freundlichand Langmuir adsorption isotherm models.The effect of solution pH on the chemistry ofbinding sites of both biosorbent types andalso the metallic species in solution wasassessed. Effect of bacterial culture age andpresence of energy sources or metabolicinhibitor on uranium uptake was alsostudied. In view of a multimetalliccomposition of realistic waste, U and Th

sorption in presence of other interferingcations and anions was investigated.

Materials and Methods

Microorganism, growth medium andculture conditions

Pseudomonas sp., a garden isolate wasgrown and maintained in Tris-minimalmedium (Kazy et a/.. 1999). Midexponentialphase cells were collected by centrifugation(12000 x g, 30 mini, washed thoroughlywith distilled water, dried and used forbiosorption experiments.

Uranium andexperiments

All biosorption experiments were done usingdry Pseudomonas biomass. Nitrate salts ofUranium and Thorium was used (UO,(NO,)"6 H,O or Th (NO,)" 5 H,O, Merck,Germany). Other experimental details weresame as described previously (Sar andD'Souza 2001, Sar and D'Souza 2002).Dissolved uranium and thorium wasdetermined either by the Arsenazo IIImethod or by inductively coupled plasmaatomic spectrometry (ICP-AES).

thorium biosorption

The biosorption equilibrium uptake (q, mgmetal g" biomass dry wt.) for each samplewas calculated according to the massbalance on metal ion expressed as :

q ~ V(Co- C,) / M (1)

Where V is the sample volume (I), Co, theinitial metal ion concentration (mg I"), Ceothe equilibrium or final metal concentration(mg r') and M, the biomass dry weight (g).Adsorption isotherm data were also fitted tothe classical Freundlich and Langmuirisotherm equations (De Rome and Gadd1987).The linearized form of isotherm equationsused are:

Freundlich Equation: Logq ~ Log k + l/nLog C, (2)

Where q is the equilibrium metal uptakecapacity and Ceo the residual metalconcentration at equilibrium. The constant 'k'

HARCNo",'o"" 137 Foond,,', Day Spoda' "'"0 2002

is a measure of adsorption capacity and'l/n', the intensity of adsorption.

LangmuirEquation, l/q ~ l/qm" bx l/Ce +l/qm" (3)

The constant 'qm,/ represents the maximumspecific metal uptake and 'b' the ratio of theadsorption/desorption rates related toenergy of adsorption through Arrheniusequation.

Time course of metal biosorption

For sorption kinetic studies, bacterialbiomass (0.5 mg ml") was mixed withuranium or thorium solution (100 mg 1"),samples withdrawn at timed intervals werecentrifuged and dissolved U/Th wasestimated.

Effect of pH on biosorption

The effect of solution pH on U and Thsorption was studied by adjusting the initialpH of the contact solution (100 mg Th 1")over the range pH 2.0-8.0. For pHadjustment, 1.0 M NaOH or 1.0 M HNO, wasused.

Interference of cations and anions on

uranium and thorium biosorption

Uranium and thorium sorption Insimultaneous presence of other interferingions was tested in bimetallic combinations,by adding equimolar concentrations ofuranium or thorium (430 ~M Th or 420 ~MU; equivalent to 100 mg U or Th 1") and testcation or anion. Details are same asdescribed earlier (Sar and D'Souza 2001a,Sar and D'Souza 2002)

Results and Discussion

Selection of uranium accumulatingstrain

Initial study on selecting a good uraniumaccumulating strain was done using four soilisolates, three belonging to Pseudomonas spand one to Bacillus coagulans. All thesebacterial strains were selected based on their

superior metal tolerance capacity. APseudomonas sp. 2 which showed optimal

biosorption was selected for further study onbiosorption of uranium (VI) and thorium(IV).

Effect of growth medium, culture age,energy source and metabolic inhibitor onuranium biosorption

Metal and radionuclide biosorption bymicrobes is strongly influenced by thenature, availability and arrangements ofvarious cellular binding ligands sequesteringcationic metallic species. Among the othersfactors, growth medium ingredients are oftenfound to regulate the synthesis of thesemetal binding moieties. In the present studyPseudomonas cells were pre-grown insynthetic minimal- and peptone containingenriched- medium and their U sorption wascompared. At low uranium concentration(100 mg I-'), comparable metal sorption wasobserved by both enriched (63 mg gO' drywt.) and minimal (60 mg gO' dry wt.)medium grown cells. However, improved(1.9-fold) uranium loading was found forminimal medium grown cells (245 mg gO' drywt.) at higher uranium concentration (1000mg I-'). Although Chang e1 a/. 1995 reported

a significant enhancement of copperadsorption by growing the culture in peptonecontaining medium, the present datacorroborate very well with U sorption by P.aeruginosa CSU strain (Hu e1 al. 1996). The

latter investigators also showed an improveduranium sorption following the use ofsynthetic defined medium instead of nutrientbroth.

To elucidate the role of bacterial culture age'on uranium sorption, cells collected before,during and after mid exponential growthphase was compared for their Uaccumulation capacity. Although in certaincase (Friis and Myers-Keith, 1986) cultureage has shown a strong effect on bacterialmetal uptake, the present Pseudomonasbiomass did not show any significantdifference in metal loading for the cells ofdifferent growth phase.

Metal accumulation by bacteria could be a

metabolism -dependent active uptake or an-independent passive biosorption. In thepresent investigation, such energy

BARCN,w,I,"" 138 Found,,', Day Spedallss., 2002

dependency of U uptake by Pseudomonasbiomass was tested by adding glucose (ascarbon/energy source) or sodium azide (asmetabolic inhibitor) in uranium uptakesolution. Metal removal was also comparedusing live, lyophilized and autoclaved cells.Noticeably, no significant difference in Uremoval was observed, suggesting themetabolic independency of the test biomassin sequestering uranium.

Time course of uranium and thorium

sorption

The kinetics of uranium and thorium sorption

by lyophilized Pseudomonas biomass isshown in Fig 1. For both the radionuclides,the test biomass exhibited a rapid cationuptake with more than 90% of equilibriumwas reached within one (for Th) or ten (forU) minutes and the process saturates after 2(for U) or 4 (for Th) hours. The rapid cationuptake has been suggested as beingessential for any good biosorbent as it allowsshort solution-sorbent contact time andwould result in the use of much shallower

contact beds of sorbent materials in column

application (Tsezos and Volesky 1982,Volesky 1990, Andres et al. 1993, Hu et al.1996).

Effect of pH on uranium and thoriumbiosorption

Initial soiution pH is the most criticalparameter for metal sorption as it influencesboth the bacterial surface chemistry as wellas the solution chemistry of soluble metalions. Uranium and thorium sorption bylyophilized Pseudomonas biomass wasstudied at a range of pH 2-8 and pH 2-6,respectively. As shown in Fig 2, initialsolution pH significantly affected theequilibrium U and Th loading capacity. Overthe range tested, extreme acid condition (pH2.0) did not favour sorption of both thecations. As the pH increased, sorption of Uand Th also increased and the maximumloading for thorium and uranium wasattained at pH 4.0 and pH 5.0, respectively.

*1

41

F/g 2 .Effectof pH on U ( ') ,nd Th (.) ,o.pt/on(100 mg I') by P"udomon" b/om",

An increase in pH beyond the optimumcaused decline in sorption of respectivecations. The reduced sorption at low pHcould be attributed to (i) the hydrolysis ofbiomass metal binding groups resulting in anincreased competition by H30., and (ii) theincreased solubility and consequent reducedadsorptivity of thorium ions (Beas andMesmer 1976). Furthermore, compared tothe Th'. and Th(OH),h ions formed at lowpH that have been identifiedas a poorsorbate (Tsezos and Volesky, 1982), thehigher uptake at pH 4.0 could be correlated

BARCN,w",U" 139 Found,,'s D,y Sp,d,llssn, 2002

to the predominance of [Th,(OH),]'. andother polymerized species possessing agreater binding affinity thus facilitating fasterand enhanced metal sorption (Gadd andWhite 1989). For uranium, the observedtrend with regard to pH may be explained byan increasing binding affinity of monovalenturanyl species (UO,OW, (UO,)o(OHh.)formed at higher pH (pH 4.0-5.0) over thedivalent (UO".) at low pH (pH 2.0).

Biosorption Isotherm

The biosorptive U and Th uptake byPseudomonas biomass was quantitativelyevaluated by equilibrium sorption isothermsover a concentration range of 0-1200 mg r'(Fig 3). Representative isotherm curves forboth the cations exhibited very efficientmetal binding even at low residualconcentration and a high saturation loadingat equilibrium. The maximum sorptionvalues obtained are 541 mg uranium g" drywt. and 430 mg thorium g" dry wt. atequilibrium concentration of 359 mg U I"and 885 mg Th r', respectively. Suchimpressive U and Th binding by the testbiomass significantly surpasses the economicthreshold level (15% dry wt. basis) forpractically usable biosorbents and so also theprevious values on Th [R. arrhizus (185 mgg" dry wt.) (Tsezos & Volesky 1981) or P.chrysogenum (3~8 mg g" dry wt.) (Gadd &White 1989)] and U [R.. arrhizus and

Penicillium chrysogenum (both 180 mg g")(Tsezos & Volesky 1981) P. aeruginosa CSU(110 mg g") (Hu et al. 1996), and M.smegmatis (44.5 mg g") (Andreas et al.1993)] uptake

Table 1 : Freundlich and Langmuirconstants for uranium andthorium sorption byPseudomonas biomass

The relationship between equilibrium metaluptake capacity (q) and residual metal ionconcentration (Ce) was further describedusing the model equation of Freundlich and

Langmuir. Although linearized sorptionisotherm for both the metals showed a

reasonably good fit to both the models, themaximum correlation coefficient (r) wasobtained with Langmuir equation. Values of

respective sorption constants and correlationcoefficients (r) are presented in Table 1. Thebetter fitting of Langmuir model suggest amonolayerd U and Th binding on to thebiomass with homogeneous surface energyand no interaction between sorbed metals

(Langmuir 1918). The asymptotic maximumadsorption capacity as predicted by theLangmuir constant 'qmax' gives a very highvalue for U and Th while a desirable high

affinity of the biomass for test metals areevident from the low values of otherconstant 'b'.

Effect of interfering ions on uranium andthorium biosorption

Uranium and thorium sorption byPseudomonas biomass was studied inpresence of equimolar amount of severalcompeting ions (Table 2). Selection of suchions are based on their likely occurrence inrealistic effluent interfering biosorption of Uand Th. Among the series of cations tested,a significant antagonism in U sorption was

Uranium ThoriumFreundlich k 199.00 159.20

l/n 0.206 0.176r 0.931 0.973

Langmuir qmax 555.5 476.19B 0.0027 0.0009r 0.997 0.998

BARC N'wsl"", 140 Found,,', Day Sp,dalls,n, 2002

Table 2 : Effect of interfering ions on Uand Th biosorption byPseudomonas

Init,,' UIThbwmos,

100 mg dm", pH 3,5,

offered only by thorium (IV), iron (II andIII), aluminium (III) and copper (II) whilemetals like cadmium (II), lead (II) siiver (II)and anions like chloride, phosphate andsulphate had no effect, The order of

inhibition to uranium binding by the cationswas Fe" >Th" >FeH >Cu" >AIH, Iron

(III), the cation considered as the most

potent competitor of uranium in bindingbiosorptive sites (Hu et ai, 1996), alsocaused a severe decline (80 %) in Uloading, Such inhibition to U sorption byFe", is a common phenomenon e,9, p,aeruginosa CSU (Hu et ai, 1996), R.. arrhizusand 5, levoris (Byerley et ai, 1987),Although AI" and AgH have also been found

to inhibit U adsorption by p, aeruginosa CSUand R, arrhizus, the Present Pseudomonasbiomass remained insensitive with respect toAgH, while AI" was significantlyantagonistic, Noticeably, in case of thorium,except iron (III), no other test cation showed

an inhibition more than 20%, Th sorptionwas insensitive to the presence of Na', CaH,

CdH, PbH, CO/', 50s" and CI, while theorder of inhibition by other cations isUO,"<CoH,<Ni2'<AI"<AgH<CuH, The

observed insignificant interference (only 8%inhibition) of U, on thorium binding can beattributed to a higher oxidation state of Thalong with additional bonding parameters(Andres et al.. 1993). Although, uranium andthorium biosorption by the present biomass

was fairly efficient in the presence of a rangeof cations, the role of Fe (III) in inhibiting Uand Th binding imposes serious limitations inwastewater treatment by this biosorbent.

Ideally, iron shouid be removed by pHadjustment or other methods prior tobiosorption.

(s)

(b)

(e)

Mechanism of biosorption and use ofimmobilized microbes in continuouscolumn reactors

Some studies have been undertaken to studythe mechanism of biosorption. Transmissionelectron microscopy of metal loaded cells

Percentage of sorptionUranium Thorium

Control 100 100

(onlyUorTh)Cations:

Na' 100 100

Ag" 98, 84"K' 98 86"Ca2' 98 100PbH 98 100Cd2, 96 100Cu2' 78" 83"AI" 82" 85"FeH 45" 57"Fe" 20" 60"Th" 37"

UO," 92Anions:

CI' 100 100

PO," 100

SO,' 100 100

COo' 74" 100',.".

BARCNe"""'" 141 Foond,,', Day Sped,,1 "'ne 2002

Conclusionrevealed an intracellular metal sequestration(Fig.4) with X-ray diffraction patternascertaining their phosphide nature. IRspectroscopy and NMR data suggests therole of cellular phosphoryl groups inradionuclide binding. For continuous use in acolumn the biomass was immobilized usingvarious techniques (D'Souza 1999b; D'Souza2001b; D'Souza 2002b) includingentrapment In radiation polymerizedacrylamide beads (Fig. 5). Scanning electronmicrograph of immobilized bacterial biomass(Fig.6) indicated no cellular damage/distortion during the immobilization process.The biomass could be used for repeated

sorption -desorption cycles in continuouscolumn operation. More than 90% ofbiomass bound U and Th was recoveredthrough elution using carbonates.

Fig 5' Immobilized Pseud,m,na, cell, a, bl, bead,

Fig 6 , Scanning electmn micmooopy ,r ImmobilizedPseud,mona, cell,

The overall study using the Pseudomonasstrain suggests the present biomass as apotential candidate for developing biosorbentfor uranium and thorium removal forwastewater remediation, however, a priorreduction of iron content is necessary. Aclear insight on the biomass U and Thbinding mechanism(s) and othertechnological parameters (currentlyunderway) will substantially improve itsfeasibility in process application.

Acknowledgements

The authors thank Dr (Ms) A. M. Samuel,Director, Bio-Medical Group, BARC, for herkeen interest in this work. Special thanks arealso given to Director, CIRCOT, Mumbai,Dr T Das, AIIMS, New Delhi and Mr. K.N.Harindran, UED, BARC for extending theirtechnical help in SEM, TEM and ICP analyses,respectively. Pinaki Sar acknowledges thefinancial assistance made by Board ofResearch in Nuclear Sciences, Department ofAtomic Energy, Govt. of India, in the form ofK.5. Krishnan post doctoral fellowship.

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BARC New,'ett" 144 Fouud,,', Day Spedall"ue 2002

About the authors ...

Dr Pinak' Sar obtamed hi, Ph.D. in Botany toe thi, theoi" "Invewgation on "'eudomona> aewgino,aa> bioaccumulato, of nickel" fmm Banaea, Hindu Unive"ity. He Wa> the ,eeipient of 0, K.S. Kn,hnanR"ea"h A«ociate, 2'" batch and wo,ked in the acea of eadionutlide bio,emediation at BARC Cu,cently,he is a fatUity membee at the Bida In,titote of Tethnology and Science. He ha, to hi, teedit >evenpape" in intemationally ceputed joumals and live pape" in intemational and national confecentepmeeeding'. Hi, field of ce>ea"h intece,t i, on metal/eadionue/ide and o,ganit pollutant biocemediation.

0, S.F. D'Souza g,aduated fmm the 15'" batch ofthe T,aining School (Biology &tU"ently the Head of the Nue/ea, Ageicultuce and Bioteehnology DMsion of BARC Hi,intecest has been in the field of En'yme and Miteobial Bioteehnology with"immobilized cell, foe u,e in biopmce«ing, bio>enso" and biocemediation. He ha> t,scientific pape" and invited ceviews in ,eputed Intemational/Natmnalmembe,/expect at National Scientifit committe,,; membee of the editoeialand has been to deli,., talks/key note lettuces, chai, scienWic >e«ion, at ",iousfowm,. He has cont,ibuted significantly to "ience education a> an invited

National wo,kshops and shoet teem cou"e, a> well as UGC Refce,he, cou"es, as an invit,Ph.D., M.St. examinee (Univ/IIT) and a> a membee of the baaed of studie, and ce>ea"hguided a numbee of ,tudent> fa, Ph.D. and M.St. He"his ,ignfficant cont,ibution, to the field of MicmbiologyScience (1993), Fellow of the A«ociation of Food Scientist>Aeademy of Science (2001).