ion of Methylene Blue From Aqueous Solution

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Biosorption of methylene blue from aqueous solution using free andpolysulfone-immobilized Corynebacterium glutamicum: Batch

and column studies

K. Vijayaraghavan, Juan Mao, Yeoung-Sang Yun *

Division of Environmental and Chemical Engineering, Research Institute of Industrial Technology, Chonbuk National University,

Chonbuk 561-756, South Korea

Received 7 May 2007; received in revised form 11 June 2007; accepted 11 June 2007Available online 30 July 2007

Abstract

The amino acid fermentation industry waste, Corynebacterium glutamicum, has been found to possess excellent biosorption capacitytowards methylene blue (MB). Due to practical difficulties in solidliquid separation and biomass regeneration, C. glutamicum wasimmobilized in a polysulfone matrix. The pH edge experiments revealed that neutral or alkaline pH values favored MB biosorption. Iso-therm experiments indicated that C. glutamicum, when in immobilized state, exhibited slightly inferior dye uptake compared to free bio-mass. Also considering the two forms, immobilized biomass took a long time to attain equilibrium. An attempt to identify the diffusionlimitations in immobilized beads was successful, with the WeberMorris model clearly indicating intraparticle as the rate controlling step.Regeneration of the free biomass was not possible as it tended to become damaged under strong acidic conditions. On the other hand,immobilized biomass performed well with 99% desorption of MB from the biosorbent with the aid of 0.1 mol/l HCl. The immobilizedbiomass was also successfully regenerated and reused for three cycles without significant loss in sorption capacity. An up-flow packed

column loaded with immobilized biomass was employed for the removal of MB. The column performed well in the biosorption of MB,exhibiting a delayed and favorable breakthrough curve with MB uptake and % removal of 124 mg/g biomass and 70.1%, respectively. 2007 Elsevier Ltd. All rights reserved.

Keywords: Wastewater treatment; Immobilization; Polysulfone; Packed column; Modeling

1. Introduction

In recent years, several adsorbents have been identifiedas possessing good dye-binding capabilities (Rao and

Rao, 2006; Batzias and Sidiras, 2007; Ozer et al., 2007;Pavan et al., 2007). In particular, biomaterials of microbialorigin have been very effective because of their cell wallconstituents. Important fungal biosorbents include Asper-gillus (Fu and Viraraghavan, 2002), Penicillium (Iscenet al., 2007) and Rhizopus (OMahony et al., 2002; Aksuand Cagatay, 2006; Kumari and Abraham, 2007). Veryfew biosorbents under the class of bacteria have been

reported for the removal of dyes; but of these, Corynebac-terium (Won et al., 2004; Vijayaraghavan and Yun, 2007b)and Streptomyces (Nacera and Aicha, 2006) are notewor-thy. Since these microorganisms are used widely in different

food/pharmaceutical industries, they are generated aswaste and can be acquired free or at low cost from theseindustries.

Microbial biosorbents are basically small particles withlow density, poor mechanical strength and little rigidity.Even though they have merits, such as high biosorptioncapacity, rapid equilibrium attainment, less process costand good particle mass transfer, they often suffer from sev-eral drawbacks. The important demerits include solidliquid separation problem, possible biomass swelling,impossibility of regeneration/reuse and the development

0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.06.008

* Corresponding author. Tel.: +82 63 270 2308; fax: +82 63 270 2306.E-mail address: [email protected] (Y.-S. Yun).

Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 28642871
mailto:[email protected]:[email protected]


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of a high pressure drop when used in column mode. Manyinvestigators have highlighted these limitations (Veglio andBeolchini, 1997; Hu and Reeves, 1997; Vijayaraghavan andYun, 2007a), which often limit the application of microbialbiosorbents to industrial applications. Several establishedtechniques are available that make these biosorbents suit-

able for process applications. Of these, immobilizationtechniques such as entrapment and cross linking have beenfound practical for biosorption applications (Bai andAbraham, 2003; Beolchini et al., 2003). Immobilization ofmicroorganisms in a polymeric matrix exhibits greaterpotential, especially in packed or fluidized bed reactors,with benefits including control of particle size, regenerationand reuse of biomass, easy separation of biomass and efflu-ent, high biomass loading and minimal clogging under con-tinuous flow conditions (Hu and Reeves, 1997).

Corynebacterium glutamicum, used in the present study,is widely employed for the biotechnological production ofamino acids (Hermann, 2003); therefore, a large amount

of biomass is produced as a byproduct. Due to practicaldifficulties in desorption, C. glutamicum was immobilizedin polysulfone matrix and used for the biosorption of meth-ylene blue. Methylene blue was selected as the adsorbate,since it is one of the most important and widely used cat-ionic dyes in the textile and paper industries. Thus, theeffluents emanating from these industries often coloreddue to methylene blue and require proper treatment priorto their discharge. Hence, this study employed polysulf-one-immobilized C. glutamicum for the biosorption ofmethylene blue in both batch and packed column modesof operation.

2. Methods

2.1. Sorbate

Methylene blue (C16H18ClN3S 3H2O) was purchasedfrom SigmaAldrich Korea Ltd. (Yongin, Korea), with apurity of 82% and molecular weight of 373.9.

2.2. Preparation of biosorbent

The fermentation wastes (C. glutamicum biomass) wereobtained in the form of a fine powder from a lysine fermen-tation industry (Deasang, Gunsan, Korea). The biomasswas dried at 60 C for 12 h and subsequently used for bio-sorption experiments. Through potentiometric titration(Won et al., 2005), pHPZC of C. glutamicum was deter-mined as 2.1.

For immobilization of the biomass, a 9% (w/v) solutionof polysulfone was prepared in N,N-dimethyl formamide(DMF) solution. After stirring the mixture for 10 h, theC. glutamicum biomass (14%) was mixed with the polysulf-one slurry, with the resultant slurry dripped in deionizedwater, where beads were formed by a phase inversion pro-cess. The beads were then washed with deionized water,

and placed in a water bath for 18 h to remove any residual

DMF. The resultant beads (12 mm diameter) were thenstored at 4 C, and are designated as PIC in this paper.The pattern of immobilization and other bead character-ization has been reported elsewhere (Vijayaraghavanet al., 2007).

2.3. Batch studies

2.3.1. Biosorption

Batch biosorption experiments were conducted bybringing into contact 0.1 g (dry weight) of raw biomassor 1 g (wet weight) of PIC with 40 ml dye solution, at thedesired pH, in 50 ml plastic bottles (high-density polyethyl-ene), which were maintained on a incubated rotary shakerat 160 rpm and 25 C. The pH of the solution was initiallyadjusted using either 0.1 mol/l HCl or NaOH, which weresubsequently used to control the pH during the experi-ments. After 12 h of contact with the dye solution, the bio-sorbent was separated by centrifugation at 3000 rpm for

5 min. The dye (MB) concentration in the supernatantwas determined using a spectrophotometer (UV-2450, Shi-madzu, Kyoto, Japan) at 660 nm, after appropriate dilu-tion. Kinetic experiments were conducted in the samemanner described above, except the samples were collectedat different time intervals to determine the time at whichbiosorption equilibrium had been attained.

The amount of MB sorbed by the biosorbent was calcu-lated from the differences between the concentrations ofMB added to those detected in the supernatants, usingthe following equation:

Q

V

C0

Cf=M

1

where Q is the MB uptake (mg/g), C0 and Cf the initial andequilibrium MB concentrations in the solution (mg/l),respectively, V the solution volume (l) and M the mass ofbiosorbent (g).

2.3.2. Desorption

The MB-loaded biomass, which was exposed to 250 mg/l of MB at pH 7 and temperature 25 C, was separatedfrom the biosorbent-water slurry by centrifugation. Thebiosorbent was then brought into contact with 20 ml of0.1 mol/l HCl for 3 h on a rotary shaker at 160 rpm. Theremaining procedure was the same as employed in the bio-sorption equilibrium experiments. After desorption, thebiosorbent was washed several times with deionized water,with the regenerated biosorbent reused for the next cycle.These cycles of biosorption followed by elution wererepeated three times, with the biosorbent capacity thenevaluated.

2.4. Column studies

A glass column (1.5 cm ID and 25 cm height) waspacked with a known quantity of biosorbent to yield thedesired bed height. The column was then fed with 50 mg/l

of MB solution in an up-flow mode, at a flow rate of

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1 ml/min using a peristaltic pump. The samples were col-lected at desired time intervals at the exit of the column,using a sample collector arrangement, and the MB concen-trations then analyzed. The operation of the column wasstopped when the effluent MB concentration exceeded97% of the inlet dye concentration. After exhaustion of

the column, desorption was carried out by pumping0.1 mol/l HCl upwards through the column at a flow rateof 2 ml/min. To determine the weight loss after sorptiondesorption cycle, the biosorbent was washed with deionizedwater and dried naturally.

Both, batch and column data modeling were performedby non-linear regression using the Sigma Plot (version 4.0,SPSS, USA) software. The average percentage errorbetween the experimental and predicted values is calculatedusing:

e % PN

i1Qexp;i Qcal;i=Qexp;iN

100 2

where Qexp and Qcal represents experimental and calculateddye uptake values, respectively, and N is the number ofmeasurements.

3. Results and discussion

3.1. Effect of pH and mechanism

In the first series of batch experiments, the influence ofequilibrium pH on the biosorption of MB was examined(Fig. 1). The solution equilibrium pH found to severelyaffect the MB biosorption capacity of C. glutamicum, with

pH values at and above neutral resulted in maximumuptake. The cell wall of Gram-positive bacterium mainlycomprised of peptidoglycan layer connected by amino acidbridges (Mera et al., 1992). Imbedded in the Gram-positivecell wall are polyalcohols called teichoic acids, which givean overall negative charge to the bacterial cell wall due tothe presence of phosphodiester bonds between teichoic acidmonomers (Beveridge et al., 1982). The zero charge poten-tial of the biomass was determined as 2.1 and thus the bio-mass will have a net negative charge above pH 2.1. On theother hand, basic dyes release colored positively charged

dye ions when in solution, which will exhibit electrostaticattraction towards the negatively charged cell surface. Inparticular, the carboxyl groups present in C. glutamicum(Won et al., 2005) were mainly responsible for the biosorp-tion of MB. The pKa value of carboxylic groups usually lieswithin the range of 3.85.0 (Roberts and Caserio, 1977).

Therefore, the carboxyl groups have a negative charge atpHs approximately higher than 5; therefore, will electro-statically bind MB to the bacterial biomass.

The above reaction (Pavan et al., 2007) clearly explainsthe pH effect on the biosorption of MB onto C. glutami-cum. At low pH values, the carboxyl groups will be in theirprotonated form and; thus, the overall charge of the bio-mass will be positive. Under this situation, the occupation

of MB onto carboxyl groups will be difficult and; hence, alow uptake was observed at acidic pH values (Fig. 1).

The results suggest that C. glutamicum possesses anexcellent binding capacity for MB. However, the C. glu-tamicum biomass has been known to cause problems dur-ing column operations due to its poor mechanicalstability, high swelling and solidliquid separation prob-lems (Vijayaraghavan and Yun, 2007a). Therefore in thisresearch, C. glutamicum was immobilized in a polysulfonematrix (Vijayaraghavan et al., 2007) and subsequentlyemployed for biosorption studies. The polysulfone-immo-bilized C. glutamicum (PIC) also exhibited a similar trendas that of the free biomass, with maximum uptakes atpHP 7. Comparing the uptakes, PIC exhibited less uptakecompared to that of the free cells. However, it should alsobe noted that the uptake capacity of PIC was based on thedry weight of the beads. To gain a clear picture of the con-tribution of the biomass in the biosorption of MB, theamount of biomass per gram of dry beads was calculated(Vijayaraghavan and Yun, 2007a) and on the basis of massbalance it was determined as 0.54 g biomass/g dry beads.Subsequently, the MB uptake by PIC was determined, asshown in Fig. 1, which was comparable to that by the freebiomass. Nevertheless, the slight decrease in the uptakemay be attributed to some binding sites not being easily

accessible or blocked due to the immobilization process.Several researchers (Bai and Abraham, 2003; Hu andReeves, 1997) observed that immobilization usuallydecrease the binding capacity of the biomass; however, thisdecrease will be minimal considering the possibility of recy-cling of biosorbent for a number of cycles. Biosorptioncontrol experiments revealed that biomass-free polysulfonebeads exhibited 11.7 mg/g dry beads at pH 7.

3.2. Biosorption isotherms and modeling

The results of the previous section revealed that the bio-

mass of C. glutamicum can be competent for the biosorp-

0

50

100

150

200

0 2 4 6 8 10

Equilibrium pH

Uptake(mg/g)

Free biomassPIC (based on beads)

PIC (based on biomass)

Fig. 1. Effect of pH on the uptake of MB by the free and polysulfone-immobilized C. glutamicum biomasses (C0 = 500 mg/l; tempera-

ture = 25 1 C; agitation speed = 160 rpm).

Dye+

C

O

O- +NH Dye

BiomassC

O-

Biomass

O

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tion of MB at pHP 7; therefore, isotherm experimentswere conducted at pHs 7 and 8 to elucidate the completebiosorption potential of the biomass. Typical biosorptionisotherms were observed for both the free and immobilizedforms ofC. glutamicum (Fig. 2). The biomass ofC. glutam-icum exhibited very high MB uptakes at pHs 7 and 8, with

significant variation in the uptakes observed only at highresidual MB concentrations. The isotherms were steep,indicating the high affinity of the sorbate towards thesorbent.

Modeling of MB isotherm data was attempted using theLangmuir and Toth models, which can be represented asfollows,

Langmuir model Langmuir; 1918 : Q QmaxbCf1 bCf 3

Toth model Toth; 1971 : Q QmaxbTCf1 bTCf1=nT nT

4

where Qmax is the maximum dye uptake (mg/g), b theLangmuir equilibrium constant (l/mg), bT the Toth modelconstant and nT the Toth model exponent. The Langmuiradsorption isotherm has traditionally been used to quantifyand contrast the performance of different biosorbents. Italso served to estimate the maximum dye uptake valueswhere these could not be attained experimentally. The con-stant b represents affinity between a sorbent and sorbate.The Langmuir constant values (Table 1) revealed that thefree biomass obviously performed well. However, the re-corded Langmuir affinity constants were found to havemaxima for the immobilized biomass. For favorable bio-

sorption, high Qmax and a steep initial isotherm slope (i.e.high b) are desirable. The MB biosorption capacity ob-served in this study was superior compared to the resultspublished in the literature (Table 2).

To improve the fitness of the biosorption isotherm data,a three parameter isotherm model, viz. the Toth model,was also used. The Toth isotherm, derived from potentialtheory, has proven to be useful in describing the sorptionin heterogeneous systems, such as phenolic compoundsonto carbon. It assumes an asymmetrical quasi-Gaussian

energy distribution with a widened left-hand side, i.e. mostsites have sorption energy less than the mean value (Hoet al., 2002). As expected, the Toth model described the iso-therm data well with high R2 and low % error vales (Table1). The successful application of the Toth model to thepresent data supports the fact that the surfaces of the bio-sorbent were heterogeneous and contained different func-tional groups.

3.3. Biosorption kinetics and modeling

Fig. 3 shows the plots of MB uptake against time for the

different forms of C. glutamicum. In general, the resultsrevealed that the removal of MB was fast during the initialstages of the contact period, but thereafter became slowernear the equilibrium. This is obvious, in that a large num-ber of vacant binding sites will be available for sorptionduring the initial stage; and after a lapse of time, the occu-pation of the remaining vacant sites will be difficult due tothe repulsive forces between the solute molecules on thesolid and bulk phases. On comparing the two forms of bio-mass, it was clearly visible, as shown in Fig. 3, that the timefor equilibrium to be attained was relatively fast for the freeC. glutamicum compared to the PIC. In the case of free bio-mass, the removal of MB was rapid for the initial 3 h, fol-lowed by the slow attainment of equilibrium at around 5 h.On the contrary, for immobilized biomass; the removal ofMB was slow, only reaching equilibrium after 7 h. This wasnot altogether surprising, since when immobilized, the bio-mass was retained within the interior of the immobilizedmatrix; whereas the free biomass had its binding sites freelyexposed to MB. For immobilization systems, mass transferresistances play a significant role in deciding the rate of bio-sorption. However, for successful immobilization systems,these mass transfer resistances should not influence theoverall biomass biosorption performance. Most impor-tantly, the dye molecules should have access to all possible

binding sites, even at a slower rate.

0

100

200

300

400

0 200 400 600 800 1000 1200 1400 1600 1800

Final MB concentration (mg/l)

MBuptake(mg/g)

pH 7 (Free biomass)

pH 8 (Free biomass)

pH 7 (PIC)

pH 8 (PIC)

Fig. 2. Biosorption isotherms for the free and polysulfone-immobilized

C. glutamicum biomasses (temperature = 25 1 C; agitation speed =

160 rpm). Curves predicted by the Toth model.

Table 1Biosorption isotherm constants for the biosorption of MB by the free andimmobilized C. glutamicum biomasses

Isotherm models Free biomass Polysulfone-immobilized biomass

pH 7 pH 8 pH 7 pH 8

Langmuir

Qmax (mg/g) 336.2 339.2 158.0 (292.4) 166.5 (308.1)b (l/mg) 0.023 0.034 0.045 0.086

R2 0.972 0.987 0.988 0.986

e (%) 9.6 2.9 3.1 4.5

Toth

Qmax (mg/g) 328.1 343.6 168.9 (312.8) 184.8 (342.1)bT (l/mg) 0.020 0.048 0.070 0.211

nT 0.849 1.241 1.423 1.767

R2 0.973 0.992 0.993 0.998

e (%) 8.1 0.2 1.4 0.8

The values in parentheses indicate the uptake based on gram of biomass inthe immobilized bead. R2 = correlation coefficient.

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The experimental biosorption kinetic data were modeledusing pseudo-second order kinetics, which can be repre-sented in a non-linear form, as follows:

qt q2ekt

1 qekt5

where qe is the amount of dye sorbed at equilibrium (mg/g);qt the amount of dye sorbed at time t (mg/g); and k theequilibrium rate constant (g/mg min). The pseudo-secondorder model is based on the sorption capacity onto the so-lid phase, and predicts the sorption behavior over the entirestudy range (Ho and McKay, 1998; Ku et al., 2007). Thecorrelation coefficients were always greater than 0.994,and the predicted curves showed excellent agreement withthe experimental data (Fig. 3). The equilibrium rate con-stant values were recorded as 3.2 104 and 0.8 104 g/mg min for free biomass and PIC, respectively. The pre-dicted equilibrium uptake (qe) values were 185.8 and178.2 mg/g biomass for free biomass and PIC, respectively;which were in good agreement with those foundexperimentally.

In an attempt to visualize the influence of mass transferresistance on the binding of MB to the free biomass andPIC, the kinetic data were analyzed using the equation pro-

posed by Weber and Morris (1963), as follows:

qt kit1=2 6Thus, the intraparticle diffusion constant ki (mg/g min

0.5),can be obtained from the slope of the plot of qt (uptake

at any time, mg/g) versus the square root of time. If thisplot passes through the origin, then intraparticle diffusionis the rate controlling step. Fig. 3 shows the plots ofqt ver-sus t1/2, with multilinearity clearly observed in both thecase of the free and immobilized biomasses, which impliesthe process involves more than one kinetic stage (or sorp-tion rates) (Guo et al., 2003). For instance, the PIC exhib-ited three stages, including an initial linear portion (up top

t < 4.5), a second portion (4.5

ion of Methylene Blue From Aqueous Solution

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