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Journal of Environmental Radioactivity 101 (2010) 969e973

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Journal of Environmental Radioactivity

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Biosorption of uranium by chemically modified Rhodotorula glutinis

Jing Bai a,b,*, Huijun Yao a, Fangli Fan a,b, Maosheng Lin a,b, Lina Zhang a, Huajie Ding a, Fuan Lei a,Xiaolei Wu a, Xiaofei Li a,b, Junsheng Guo a, Zhi Qin a

a Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, PR ChinabGraduate University of Chinese Academy of Sciences, Beijing 100049, PR China

a r t i c l e i n f o

Article history:Received 4 January 2010Received in revised form21 July 2010Accepted 22 July 2010Available online 24 August 2010

Keywords:BiosorptionChemical modificationUraniumFunctional groupsRhodotorula glutinisFTIR analysis

* Corresponding author. Institute of Modern PhSciences, Lanzhou 730000, PR China. Tel.: þ86 931 496

E-mail address: baijing@impcas.ac.cn (J. Bai).

0265-931X/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.jenvrad.2010.07.003

a b s t r a c t

The present paper reports the biosorption of uranium onto chemically modified yeast cells, Rhodotorulaglutinis, in order to study the role played by various functional groups in the cell wall. Esterification of thecarboxyl groups and methylation of the amino groups present in the cells were carried out by methanoland formaldehyde treatment, respectively. The uranium sorption capacity increased 31% for the meth-anol-treated biomass and 11% for the formaldehyde-treated biomass at an initial uranium concentrationof 140 mg/L. The enhancement of uranium sorption capacity was investigated by Fourier transforminfrared (FTIR) spectroscopy analysis, with amino and carboxyl groups were determined to be theimportant functional groups involved in uranium binding. The biosorption isotherms of uranium ontothe raw and chemically modified biomass were also investigated with varying uranium concentrations.Langmuir and Freundlich models were well able to explain the sorption equilibrium data with satis-factory correlation coefficients higher than 0.9.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Uranium is a threatening heavy metal because of its hightoxicity and radioactivity. Excessive amounts of uranium havefound their way into the environment through the activities asso-ciated with the nuclear industry (Barkay and Schaefer, 2001;Benedict et al., 1981). Uranium disposed into the environmentcan reach the top of the food chain and be ingested by humans(Anke et al., 2009), causing kidney or liver damage to humans (Craftet al., 2004; Priest, 2001; Xie et al., 2008). Therefore, it is necessaryto treat wastewater containing uranium in order to prevent itscontamination of the environment. Moreover, with the develop-ment of nuclear industry, uranium will be used more extensively.As a non-renewable resource, the removal and recovery of uraniumis meaningful.

Biosorption, a process, has been increasingly considered asa potential alternative way to remove contaminants from industrialeffluents (Bhainsa and D’Souza, 2001;Wang, 2002). Comparedwiththe conventional methods, biosorption process offers severaladvantages, such as low operating cost, high efficiency in detoxi-fying very dilute effluents and a minimal volume of disposable

ysics, Chinese Academy of9692; fax: þ86 931 4969693.

All rights reserved.

sludge. Studies have already been performed on uranium bio-sorption by various microorganisms viz. fungi, yeast, algae andunicellular bacteria (Kalin et al., 2005; Kazy et al., 2009; Parab et al.,2005). Saccharomyces cerevisiae has been used as a model to assaythe heavy metal resistance of the yeast species due to its wide usein the fermentation industry. Nourbakhsh (Nourbakhsh et al., 1994)studied chromium (VI) removal form industrial wastewater byusing S. cerevisiae. The efficiency of S. cerevisiae for uranium sorp-tion was also tested in pure uranium solution (Kedari et al., 2001)and wastewater from uranium mill (Tykva et al., 2009). However,other yeasts, including Rhodotorula, which have been used in leadand cadmium biosoption (Cho and Kim, 2003; Salinas et al., 2000),have not been studied for uranium sorption. In order to makea better use of the microorganisms and improve their efficiency inuranium biosorption, intensive research is needed, includingdetermination of the cell functional groups involved in uraniumbinding.

Metal ion uptake by biomass is believed to occur throughinteractions with functional groups native to proteins, lipids, andcarbohydrates that make up the cell wall (Chen et al., 2007). Tomaximize the efficiency of the biomass, it is important to identifythe important functional groups responsible for metal binding. Theinformation obtained from these determinations will be useful forfuture attempts at chemically or biosynthetically altering thebiomass to enhance its sorption capacity or selectivity for thespecific metal ions. The identity of the functional groups would also

J. Bai et al. / Journal of Environmental Radioactivity 101 (2010) 969e973970

be helpful for determining the mechanisms responsible for thebinding of targeted metal ions.

Chemical modification of functional groups is a useful techniquein characterizing the functional groups responsible for metalbinding. For instance, Gardeatorresdey et al. (1990) studied Cu(II)binding after esterification of carboxyl groups on an algal speciesand observed the decreased sorption capacity of Cu(II). This sug-gested that carboxyl groups are responsible for Cu(II) binding.Similarly, in order to investigate the significance of amino groups inlead and cadmium sorption, Holan and Volesky (1995) employedformaldehyde modified Ascophyllum nodosum biomass, since theformaldehyde treatment can result in the methylation of aminogroups (Loudon,1984). Bai and Abraham (2002) reported the role ofamino and hydroxyl, as well as carboxyl groups in the sorption of Cr(VI) by chemically modified Rhizopus nigricans biomass. However,chemical modification has not been used to date to study the roleplayed by functional groups in uranium biosorption.

The objective of this study was to investigate the removal ofuranium from aqueous solution by chemically modified R. glutinis.A combination of chemical modifications, metal-binding experi-ments, and infrared spectroscopy was performed to gain insightinto the role played by functional groups in uranium binding.

2. Materials and methods

2.1. Biosorbent

The R. glutinis used in this studywas obtained from the Institute of Microbiology,Chinese Academy of Sciences. The culturemedium contained glucose 20 g/L (Factoryof chemical experiments, Laiyang China), peptone 10 g/L (Hangzhou microbialregent Co. Ltd, China), yeast extract powder 10 g/L (Baorui microbial technology Co.Ltd, China), NaCl 2.5 g/L (Sinopharm chemical reagent Co., Ltd, shanghai, China),KH2PO4 1.0 g/L (Laiyang shuangshuang chemical Co. Ltd, China) and MgSO4$7H2O0.5 g/L (Laiyang shuangshuang chemical Co. Ltd, China). After cultivating the cell inthe sterilized medium mention above at 30 �C for 24 h, 5 mL of the cell suspensionwere added to the same fresh culturemedium of 100mL for further incubation. Cellsat the stationary growth phase were killed by heating them in an oven at 60 �C for 1day. The dead cells were harvested by centrifugation at 5000 rpm for 5 min andwashed several times with distilled water until the biomass looks whitish. Thebiomass obtained was referred as raw biomass in this paper.

2.2. Chemicals

The stock solution of uranium (1.0 g/L) was prepared by using uranyl nitrate(UO2 (NO3)2) purchased from SigmaeAldrich and diluted to appropriate concen-tration with distilled water. The initial pH of the uranium solutions were adjustedwith 0.1 M HCl or 0.1 M NaOH and not monitored during the whole experimentalprocess. 0.05% Arsenazo III solution was prepared by dissolving 0.5 g of the reagentin 1000 mL of distilled water. HCl, NaOH and Arsenazo III were purchased fromsinopharm chemical reagent Co., Ltd, shanghai, China. All other reagents used wereof analytical reagent grade unless otherwise stated.

2.3. Chemical modifications of biomass

Portions of raw biomass were chemically modified in different ways to under-stand the role of functional groups in uranium biosorption. The chemical modifi-cations used to treat the biomass were as follows.

2.3.1. Methanol treatmentThe method described by Kapoor and Viraraghavan (1997) was followed. Briefly,

80 mg of the well washed raw biomass was suspended in 4.8 mL of anhydrousmethanol (CH3OH, Tianjin guangfu fine chemical research institute, China), and480 mL of concentrated hydrochloric acid was added to the suspension. The mixturewas agitated on a rotary shaker at 125 rpm for 6 h. The reaction occurs as follows:

RCOOHþ CH3OH/Hþ

RCOOCH3 þ H2O (1)

The treated cell suspension was then centrifuged and sequentially washed withdistilled water, 0.2 M sodium carbonate, and finally distilled water. The obtainedbiomass was re-suspended in 8 mL distilled water and immediately used in bio-sorption experiment.

2.3.2. Formaldehyde treatmentAccording to Kapoor and Viraraghavan’s (1997), 80mg of biomass was contacted

with 1.6 mL of formaldehyde (HCHO, Shanghai experimental reagent Ltd, China) and3.2 mL of formic acid (HCOOH, Tianjin guangfu fine chemical research institute,China). The reaction mixture was shaken on an agitator at 125 rpm for 6 h. Thistreatment was expected to result in the methylation of amines (Loudon, 1984). Thegeneral reaction occurs as follows:

RCH2NH2 ��!HCOH;HCOOHRCH2NðCH3Þ2þCO2 þ H2O (2)

The biomass obtained was washed and re-suspended as the method used inmethanol treatment. To conveniently measure the same amount of each biomassused in uranium sorption, 80 mg of raw biomass was also re-suspended in 8 mLdistilled water.

2.4. Biosorption studies

Sorption experiments were carried out using raw and chemically modifiedbiomasses to investigate the effect of chemical modifications on biosorption capa-bility. 0.4 mL (80mg/8 mL � 0.4 mL ¼ 4 mg) of raw and chemically treated cellsuspensions were sampled, added to polypropylene centrifuge tubes, and centri-fuged at 5000 rpm for 5min to separate the biomass fromwater. Then 4mL uraniumsolutions of known concentration were mixed with the biomass and shaken ona rotary shaker at 150 rpm for 30 min (Bai et al., 2009) at room temperature (25 �C).The mixtures were centrifuged and the supernatant was assayed spectrophoto-metrically for uranium with Arsenazo III (Khan et al., 2006; Genç et al., 2003). Theamount of adsorbed uranium per unit yeast biomass (mg metal ions/g dry yeastbiomass) was calculated using the following expression,

q ¼ ðC0 � CeÞVm

(3)

where q is the amount of uranium adsorbed onto the unit amount of the biomass(mg/g), C0is the initial uranium concentration (mg/L), Ceis the equilibrium or finaluranium concentration (mg/L), V is the volume of the aqueous phase (L) andm is theamount of the biomass (g).

Previous study revealed that initial pH of 6.0 is the most favorable condition foruranium sorption by R. glutinis and the precipitation of uranyl hydroxides can beavoided at this pH (Bai et al., 2009). Therefore, all the sorption experiments weredone at initial pH 6.0. Control experiments without biomass were carried out todetermine the degree of uranium removal by plastic tube.

Duplicate experiments were performed and mean values were used in theanalysis of data.

2.5. Analytical methods

Uranium concentration was determined on UV-1801 spectrophotometer (Bei-jing rayleigh analytical instrument Co., Ltd, China). 100 mL uranium solution sample,500 mL 0.3 M HCl and 300 mL 0.05% Arsenazo III aqueous solution were added toa glass flask, the final volume of solution was filled up to 10 mL by distilled water,then the absorbance of the mixed solution was analysis at 650 nm. Mixed solutionprepared in the same way but without uranium was used as reference. Uraniumconcentration was calculated from the calibration curve. The detection limits andsensitivity of this method were 7.02 mg/L and 1.01 mg/L, respectively.

The infrared (IR) spectra of raw, chemically modified and uranium bounded R.glutinis cells were obtained using Fourier transform infrared spectrometer (Pelki-nElmer Spectrum GX FTIR, USA). Sample dicks were made by mixing 5 mg of drybiomass with 150 mg of KBr (Sinopharm chemical reagent Co., Ltd, shanghai, China)and pressed them into tablet form. All infrared spectra were recorded over4000e400 cm�1 region with a resolution of 0.2 cm�1.

3. Results and discussion

Fig. 1 shows the FTIR spectra of raw and chemically modifiedcells before uranium sorption. As shown in Fig. 1, the cellular IRspectra were very complex and obvious adsorptions were observedcovering the total range of the wave number. Trough assignmentwas made basing on the references (Drake et al., 1996; Kiefer et al.,1997; Mantsch and Chapman, 1995) and listed in Table 1.

The effects of chemical treatment on the functional groups couldbe evaluated from the difference among the spectrums. The curveb in Fig. 1 shows the IR spectrum of R. glutinis treated with meth-anol. By comparing curve b with curve a, a new shoulder at wavenumber of 1732 cm�1, which was due to the stretching vibration ofester carbonyl, was observed. And the peak at wave number of1068 cme1 in raw biomass (Fig. 1, curve a) was assigned to the CeO

Fig. 1. Fourier transformed IR spectrum of (a) raw biomass, (b) methanol-treatedbiomass, and (c) formaldehyde-treated biomass.

Fig. 2. Uranium biosorption by raw and chemically modified Rhodotorula glutinis. (a)raw biomass; (b) methanol-treated biomass and (c) formaldehyde-treated biomass.(Initial uranium concentration was 140 mg/L).

J. Bai et al. / Journal of Environmental Radioactivity 101 (2010) 969e973 971

stretching vibration of carboxyl groups in the cell. After themethanol treatment, this peak shifted to 1076 cm�1 (Fig. 1, curve b).These all result from the esterification of carboxyl groups presentedon the cell. The curve c in Fig. 1 shows the IR spectrum of biomasstreated with formaldehyde and formic acid. The trough observed at3297 cm�1 in the raw biomass (Fig. 1, curve a) represented eOHgroups of the glucose, eNH stretching of the protein, and acet-amido group of chitin fraction, this peak shifted to 3289 cm�1 afterthe chemical modification. It suggested certain changes have beenhappened to the amino groups present in the biomass. Further-more, the intensity of trough 1456 cm�1 (asymmetric bending ofCH3 or CH2) in the formaldehyde-treated biomass was higher thanthat in the raw biomass, indicating the enhancement of CH3. Theseall demonstrated that formaldehyde treatment caused the meth-ylation of amino groups in the cells. The troughs of amide I andamide II remained nearly unaffected after both chemical treat-ments. It indicated the major components and structure of thebiomaterial remained intact.

3.1. Biosorption of uranium

The results of uranium biosorption by raw and chemicallymodified biomass at the same initial uranium concentration(140 mg/L) were shown in Fig. 2. Both of the chemical treatmentsresulted in increased uranium sorption capacity. The increase was31% for methanol-treated biomass and 11% for formaldehyde-treated biomass. Strandberg et al. (1981) also found increase inuranium sorption capacity by formaldehyde-treated S. cerevisiae,which are consistent with our results. However, Chen et al. (2007)found a dramatic decrease in Cu2þ and Zn2þ binding after acidic

Table 1Trough assignment of the FTIR spectra for raw and chemically modified biomassbefore uranium sorption.

Wave number(cm�1) Assignment

3289e3303 n(OeH), n(NeH)2927 n(CeH)1656 amide I,n(C]O) conjunct ton(NeH)1534 amide II,n(NeH) conjunct ton(CeN)1237 amide III,n(NeH) conjunct ton(CeN)1456 n(CeH) of CH3 or CH2

1065e1076 n(CeO) of carboxyl groups

methanol esterification of the nonliving cells. The effect of chemicalmodification depends on the biomass, modification procedure, andprobably heavy metal ions.

FTIR studies of raw biomass and chemically modified biomassbefore and after uranium binding were carried out and the resultswere shown in Fig. 3. The aim was to find out the reason for theenhanced uranium sorption and simultaneously elucidate the

Fig. 3. Fourier transformed IR spectrum for raw biomass and chemically modifiedbiomass before (1) and after (2) uranium loaded. a, raw biomass; b, methanol-treatedbiomass; c, formaldehyde-treated biomass.

Fig. 4. Uranium biosorption curves of raw biomass and chemically modified biomass.

J. Bai et al. / Journal of Environmental Radioactivity 101 (2010) 969e973972

functional groups involve in metal binding after chemical treat-ment. New peaks at wave numbers of 905e916 cm�1 wereobserved in the uranium treated samples, theses peaks can beassigned to the asymmetric stretching vibration of UO2

2þ (Frostet al., 2006; Tsezos and Volesky, 1982). This peak was stronger inthe chemically modified cells than in the raw cells suggested higheruranium sorption by chemically modified cells.

According to the analysis of the complicated IR spectra, somecharacteristic peaks can be assigned to the involvement of the mainfunctional groups present in the cell. Before uranium sorption, thestrong absorption peaks in between 1065 and 1076 cm�1 ascertainthe presence of carboxyl groups in the yeast polysaccharide struc-ture. After uranium binding, a change of peak positions (1069 cm�1

to 1081 cm�1 for raw biomass, 1075 cm�1 to 1076 cm�1 for meth-anol-treated biomass and 1065 cm�1 to 1069 cm�1 for formalde-hyde-treated biomass) in this region was observed. This suggestedthe involvement of carboxyl groups in uranium sorption. The peakshift of carboxyl group in the methanol-treated biomass was not asobvious as that in the raw and formaldehyde-treated biomass, it wasbecause the carboxyl in the cell walls could form lipids throughmethanol treatment and its effect on the uranium sorption wasblocked (Xie et al., 2008). Reasons for the enhancement of uraniumsorption after methanol treatment may be methanol treatment notonly caused the esterification of the carboxylic acid groups but alsodissolved some soluble components in the cell membrane. Thereforethe permeability of the cell membrane increased (Hu et al., 1996). Inthe previous work of Hu et al. (1996), significant raise of uraniumsorption capacity was also observed after methanol treatment.

In the uranium free spectra of raw and chemically modifiedbiomass, the peaks at 3289 cm�1 to 3303 cm�1 were ascribed to thestretching vibration of NeH and OeH bands. After uranium sorp-tion, these peaks shift form 3297 cm�1 to 3292 cm�1 for rawbiomass, from 3303 cm�1 to 3297 cm�1 for methanol-treatedbiomass and 3289 cm�1 to 3298 cm�1 for formaldehyde-treatedbiomass. These indicated the involvement of amino and hydroxylgroups in uranium sorption. Formaldehyde treatment will causethemethylation of amino groups and reduce the number of positivecharge on the biomass surface (Bai et al., 2009), consequently makeamino groups more convenient to associate with uranium ion.Hence, formaldehyde treatment increased the cell sorptioncapacity of uranium. Tsezos and Volesky (1982) also found thatamino-nitrogen of chitin was the main component for uraniumsorption by Rhizopus arrhizus through coordination or complexeswithmetal ion and did not consider other functional groups such ascarboxyl and phosphate. Treen-Sears et al. (1984) have shown thation exchangewas the principal mechanism for uranium sorption onRhizopus oligosporus. The consistency of the results indicated thatthe biosorption of uranium by R. glutinis was mainly depended onionic interaction and complexion. Amino and carboxyl groups wereimportant functional groups.

3.2. Sorption isotherm analysis

The equilibrium of sorption was an important physicochemicalaspect for evaluating the sorption process as a unit operation. Theisotherm sorption of uranium onto raw biomass and chemicallymodified biomass were carried out with varying uranium concen-trations from 40 to 350 mg/L (Bhainsa and D’Souza, 1999; Li et al.,2004). Fig. 4 demonstrated the uranium biosorption capacities ofraw biomass, formaldehyde- and methanol-treated biomass asa function of the initial uranium concentration in aqueous medium.Uranium biosorption capacities of all the tested biomass increasedwith increasing initial uranium concentrations. The uraniumsorption capacity of raw biomass reached a saturated value at initialuranium concentration of 280 mg/L. The maximum sorption ability

of raw biomass was 149 mg U/g dry biomass. However, saturationwas not reached by the yeast cells modified with methanol andformaldehyde over the concentration range studied. The order ofthe maximum uranium sorption capacity was: methanol-treatedbiomass > formaldehyde-treated biomass > raw biomass.

Several isotherm equations were used for equilibrium modelingthe biosorption systems. The most commonly used ones wereLangmuir (Volesky, 2003) and Freundlich equations (Bayramo�gluet al., 2006). The Langmuir model was based on the assumptionthat maximum sorption occurred when a saturated monolayer ofthe solute molecules were present on the adsorbent surface. Thelinearized mathematical description of this model is:

Ceqe

¼ Ceqmax

þ 1qmaxb

(4)

where Ce is the equilibrium concentration of metal in solution(mg/L), qe is the amount of metal ions adsorbed onto the unitbiomass at equilibrium time (mg/g), qmax (mg/g) and b(L/mg) wereLangmuir constants related to sorption capacity and sorptionenergy, respectively.

The Freundlich equation was an empirical relationship wherebyit is assumed that the sorption energy of ametal binding to a site onan adsorbent depends on whether or not the adjacent sites arealready occupied. This empirical equation took the following form:

qe ¼ KðCeÞ1.

n(5)

Where Kand n are the Freundlich constants, the meaning of Ce andqe are the same as in equation (4). In order to determine how wellthe Freundlich model fitted the experimental data, the plot of logCeversus log qewas employed.

The calculated values of the Langmuir and Freundlichparameters were obtained by the least square method and list inTable 2.

As shown in Table 2, the equilibrium data fitted both theLangmuir and Freundlich expressions with a satisfactory correla-tion coefficient values higher than 0.9. Moreover, the correlationcoefficients of Langmuir isotherm were higher than that ofFreundlich model for raw and methanol-treated biomass. But forthe formaldehyde-treated biomass, Freundlich equation lookedmore appropriate.

The better agreement of the experimental data with the Lang-muir model implies that monolayer adsorption dominates the

Table 2Calculated Langmuir and Freundlich coefficients.

Absorbent Langmuir parameters Freundlich parameters

qmax (mg/g) b(L/mg) R2 K n R2

Raw biomass 187 � 8.68 0.022 � 0.004 0.99 11.7 � 1.60 2.01 � 0.130 0.98Methanol-treated biomass 350 � 39.4 0.041 � 0.012 0.95 21.4 � 6.23 1.60 � 0.249 0.91Formaldehyde-treated biomass 360 � 58.7 0.017 � 0.005 0.90 11.8 � 1.17 1.52 � 0.065 0.99

J. Bai et al. / Journal of Environmental Radioactivity 101 (2010) 969e973 973

sorption process, while better fit with Freundlich equation indi-cates multilayer adsorption occurs (Zhou and Kiff, 1991). Thecalculated maximum uranium sorption capacity was 187 mg/g forraw biomass, 350 mg/g for methanol-treated biomass, and 360 mg/g for formaldehyde-treated biomass. The raw biomass had rela-tively low uranium sorption capacity; it primarily adsorbeduranium in monolayer form. Although methanol-treated biomasshas relative higher uranium sorption capacity, uranium was alsomainly absorbed in the monolayer form. This phenomenon furthersupported our experimental result that methanol treatment dis-solved some soluble component in the cell wall, and more sites canbe used in uranium sorption. Formaldehyde treatment led to thedecrease of positive charge in the biomass surface, the increase ofuranium sorption capacity was due to multilayer sorption. There-fore, the isothermal sorption data of formaldehyde-treated biomassfitted better with Freundlich model.

4. Conclusion

Uranium biosorption was studied in this work by using thechemically modified R. glutinis. Treatment with methanol andhydrochloric acid results in the esterification of the carboxyl groupspresent on the cell and treatment with formaldehyde-formic acidlead to methylation of amino groups. Both of methanol and form-aldehyde treatment slightly increased the uranium sorptioncapacity of the biomass. The enhancement was 31% for methanol-treated biomass and 11% for formaldehyde-treated biomass at aninitial uranium concentration of 140 mg/L. The biosorption ofuranium by R. glutinis was mainly depended on ionic interactionand complexion. Amino and carboxyl groups were determined tobe the important functional groups involved in uranium sorption.In the studied concentration range, the sorption equilibrium data ofboth raw biomass and chemically modified biomass fittedwell withthe Langmuir and Freundlich models, which implied that uraniumsorption by these biomasses was a physical sorption process. Therelative higher sorption capacities of the yeast cells ensured theirpotential use in uranium wastewater treatment.

Acknowledgements

This project was financially supported by the National NaturalScience Foundation of China (No.10575122 and 10705035) andDirector’s Foundation of Institute of Modern Physics, ChineseAcademy of Sciences (No.0812060SZO).

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