Surface modiﬁcation of the Corynebacterium glutamicum biomass to increase carboxyl binding site for basic dye molecules

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Contents lists available atScienceDirect

Biochemical Engineering Journal

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b e j

Surface modiﬁcation of the Corynebacterium glutamicum biomass to increase carboxyl binding site for basic dye molecules

Juan Mao

^a

, Sung Wook Won

^b

, Sun Beom Choi

^b

, Min Woo Lee

^c

, Yeoung-Sang Yun

^a,b,∗

aDepartment of Bioprocess Engineering, Chonbuk National University, Jeonbuk 561-756, South Korea

bDivision of Environmental and Chemical Engineering and Research Institute Technology, Chonbuk National University, Jeonbuk 561-756, South Korea

cDepartment of Chemical Engineering, School of Environmental Science and Engineering, Pohang University of Science and Technology, Pohang 790-784, South Korea

a r t i c l e i n f o

Article history:

Received 29 November 2008 Received in revised form 6 April 2009 Accepted 7 April 2009

Keywords:

Biosorption

Corynebacterium glutamicum Citric acid

Modiﬁcation Basic dye

a b s t r a c t

The objective of this study was to develop a surface-modified biosorbent with enhanced sorption capacity of cationic dye Basic Blue 3 (BB 3). The biomass ofCorynebacterium glutamicumwas modified using citric acid (CA). The optimal modification conditions were the mixing of the biomass with 0.8 M citric acid solution at a 1:10 ratio, and heating at 120^◦C for 3.5 h. Hydroxyl groups on the biomass surface underwent an esterification with citric acid, forming carboxyl groups which were available for cationic dye binding.

From the Fourier transform infrared analysis, it was confirmed that the CA-modified biomass possessed a large amount of surface carboxyl functional groups comparing the raw biomass. The sorption capacity of BB 3 onto CA-modified biomass was 2.02 times higher than that onto the raw biomass. Furthermore, BB 3 sorbed on the CA-modified biomass was easily eluted by shifting the solution pH, making repeated sorption/desorption cycle (up to 4 times) possible without significant performance decrease. Therefore, the method developed for amplifying the carboxyl sites on the biomass surface may be a useful modification tool for the creation of a high-performance and regenerable biosorbent for the cationic dyes.

1. Introduction

Synthetic dyestuffs are used extensively in paper printing, leather tanning and textile industries [1]. There are more than 100,000 commercially available dyes, most of which are difﬁcult to be decolorized due to their complex structure and synthetic origin [2]. The extensive use of dyes often poses pollution problems in the form of colored wastewater discharged into environmental water bodies. They do not only affect aesthetic merit, but also reduce light penetration and photosynthesis. In addition, some dyes are toxic, mutagenic or carcinogenic[3].

In general, five main methods exist for reducing the color in textile effluent streams: adsorption, oxidation–ozonation, biologi- cal treatment, coagulation–flocculation and membrane processes.

Currently, the adsorption process is one of the most effective and attractive processes for the treatment of these dye-bearing wastewaters. The most commonly used adsorption agent in industry is activated carbon, which has also been extensively studied for the removal of dyes[4–7]. However, the high operation costs

∗ Corresponding author at: Department of Bioprocess Engineering, Chonbuk National University, Jeonbuk 561-756, South Korea. Tel.: +82 63 270 2308;

fax: +82 63 270 2306.

E-mail address:[email protected](Y.-S. Yun).

and problems associated with regeneration hamper the large-scale application of activated carbon. Therefore, a number of other non- conventional sorbents have been evaluated for the treatment of wastewaters. Natural materials, biosorbents and waste materials from industry and agriculture represent potentially more economical alternative sorbents[8], and many have been tested for the removal of dyes, e.g. apple pomace and wheat straw[9], coir pith [10], straw, corncobs and barley husks[11].

In recent years, attention has been focused on the utilization of native or modified biomass as sorbents. Generally, the sorption capacity of a crude biosorbent is low, but chemical modification can greatly improve the sorption capacity of these biomaterials. Wing [12]developed a method to thermochemically modify corn fiber using citric acid. When heated, citric acid will dehydrate to yield a reactive anhydride, which can react with the hydroxyl groups of cel- lulose to form an ester linkage. The introduced free carboxyl groups of citric acid increase the net negatively charged on the corn fiber;

thereby, increasing its binding potential for cationic contaminants.

Basic dyes are the brightest class of soluble dyes used in the textile industry[13]. Their tinctorial value is very high, with less than 1 ppm of the dye producing an obvious coloration. Therefore, the color-bearing efﬂuents require a treatment that will remove the color/dye in an economical fashion and to be prescribed concentration levels prior to their discharged into water bodies. Considering both discharge volume and efﬂuent combustion, the wastewater 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.bej.2009.04.004

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Fig. 1.Molecular structure of BB 3.

from the textile industry is rated the most polluting of all industrial sectors[14]. Therefore, there is a deﬁnite need for a suitable and cost effective dye/color-removal technology that works under the above circumstances.

The biomass of Corynebacterium glutamicum is generated in large quantities during the full-scale fermentation process for the production of mono sodium glutamate (MSG). Although this fermentation byproduct is potentially recyclable, until now most has been dumped at sea. Related research[15]has been reported about the use ofC. glutamicumfor dye treatment. There is much room for further enhancement of sorption capacity of the biomass by proper chemical modification for increasing the binding sites. Thus, in this study,C. glutamicumwas thermochemically modified using citric acid as an esterifying agent. Two newly introduced free carboxyl groups of the esterifiedC. glutamicumwere examined as a biosorbent for the removal of Basic Blue 3 (BB 3), as a model cationic dye, because it is one of popularly used basic dyestuffs.

2. Materials and methods 2.1. Materials

All chemicals used in this study were of analytical grade and pur- chased from Sigma–Aldrich Korea Ltd. BB 3, a model basic dye, was used as the adsorbate. As shown inFig. 1, BB 3 is positively charged in an aqueous solution.Table 1summarizes the main characteristics of BB 3.

TheC. glutamicumbiomass was obtained in powder form from a MSG fermentation industry (Deasang, Gunsan, Korea). The pow- dered biomass, regarded as raw biomass, was dried in an oven at 60^◦C for 24 h, then stored in a desiccator and used as the biosorbent in the sorption experiments.

2.2. Surface modiﬁcation

PowderedC. glutamicumbiomass was mixed with various concentrations of citric acid (CA) at a ratio of 5.0 g biomass to 50 mL CA solution and stirred for 2 h at room temperature (25±2^◦C).

The acid/biomass suspension was dehydrated at 60^◦C for 24 h in a forced air oven. The oven temperature was then raised to the desired temperature (60–140^◦C) for various reaction times (0.5–4 h). The CA-modiﬁed biomass was removed and allowed to cool, and then washed several times with distilled water until the pH of the distilled water became constant. The washed biomass was ﬁnally dried in an oven at 60^◦C for 24 h and preserved in a desiccator as the biosorbent for future use.

Table 1

Main characteristics of BB 3.

Name BB 3

Chemical formula C20H26ClN3O

Formula weight 359.89

Color index number 51104

max(nm) 654

3 sorption capacities of the raw and CA-modified biomasses. The pH edge experiment is an equilibrium plot of dye uptake versus the final pH. Conversely, the isotherm experiment represents the equilibrium relationship between the dye uptake by sorbent and the final dye concentration in the aqueous phase[16].

In the pH edge experiments, the solution pH was adjusted and controlled at the desired values, ranging from about 3–10, using 1 M HNO₃and 1 M NaOH. Biomass (0.1 g) was added to 50 mL falcon tubes, including 40 mL BB 3 solution (250 mg/L), and agitated in the shaker at 160 rpm for 24 h at 25±2^◦C, which was sufﬁcient to attain equilibrium. After 24 h, the ﬁnal pH values of the working solutions were measured, the samples were centrifuged and the remaining concentration in the supernatant solution were analyzed at 654 nm, the maximum absorption peak, using a UV spectrophotometer (UV-2550, Shimadzu, Kyoto, Japan), after appropriate dilution with distilled water.

In the sorption isotherm experiments, different initial concentrations (0–2330 mg/L) of BB 3 were prepared in a series of 50 mL falcon tubes, containing 40 mL of the dye solution. Following the addition of biomass (0.4 g) into each tube, the suspension was agitated in a shaker at 160 rpm and 25±2^◦C for 24 h, with the pH value of the working solution maintaining at the desired value using 1 M HNO₃and 1 M NaOH. After equilibrium being attained, the concentrations in the samples were analyzed, as mentioned above.

Isotherm and pH edge experiments were carried out in dupli- cate, and the reported values were average values of two data sets.

In one-point checking experiments for optimization of modiﬁca- tion reaction conditions, the uptake of raw biomass was performed in triplicate.

The dye uptake (q) was calculated from the mass balance, as followed below:

q= V0C0−VfCf

M (1)

whereV₀is the initial volume andV_fthe ﬁnal (initial plus added acid or alkali solution) volume.C₀andC_fare the initial and ﬁnal BB 3 concentrations, respectively andMthe mass of biomass used.

2.4. Fourier transform infrared spectroscopy analysis

The raw and CA-modiﬁed biomasses were analyzed using a Fourier transform infrared spectrometer (FTIR-8900, ABB Bomem, Quebec, Canada) to investigate the nature of the functional groups presenting on the biomass surface. For the FTIR study, 0.3 g of biomass was placed into 30 mL distilled water, with the suspension maintained at pH at 9±0.5 using 1 M HNO₃and 1 M NaOH for 24 h. The resulted deprotonated biomass was prepared as KBr disc for FTIR analysis within the range 400–4000 cm⁻¹.

2.5. Sorption/desorption experiments

To determine the reusability of the modiﬁed biomass, sorption/desorption experiments were conducted. The BB 3-loaded biomass was centrifuged at 3000 rpm, the supernatant was removed, the settled biomass subsequently resuspended with 30 mL of distilled water, and the pH of the suspensions adjusted to the desired values, i.e. the pH values where the binding of BB 3 to each of the biomass was lowest. The working solution was stirred at 160 rpm for 24 h at 25±2^◦C to allow the dye molecules to be released from the biomass. Each sample was carried out in triplicate to conﬁrm the accuracy of the trials.

After desorption, the biomass was reused for subsequent sorption experiments. The sorption/desorption cycles were continued

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Fig. 2.Effect of temperature on the sorption performance.

for four cycles to evaluate the feasibility of repeated biosorbent reused.

The sorption efficiency of each cycle was calculated as a per- centage of the uptake of BB 3 from the aqueous solution, and the desorption efficiency was calculated from the amount of dye adsorbed onto the biomass and the final dye concentration in the desorption medium. Therefore, the sorption and desorption efficiency were calculated from the following equations:

Sorption efﬁciency(%)

= Sorbed BB 3

BB 3 in the initial aqueous solution×100% (2)

Desorption efﬁciency(%)= Released BB 3

Initially sorbed BB 3×100% (3) 3. Results and discussion

3.1. Optimization of modiﬁcation conditions

Citric acid will dehydrate to yield a reactive anhydride when heated. When biomass is present in the reaction mixture, the anhydride can react with the hydroxyl groups on the biomass surface to form a biomass–citrate adduct[17].

To determine the optimum modiﬁcation conditions, i.e. concentration of chemical reagent, reaction temperature and time, a series of experiments were conducted. The raw biomass was ﬁrst exposed to 1 M CA solution, at various temperatures (25, 60, 80, 100, 110, 120, 130 and 140^◦C) for 2 h.Fig. 2shows that the uptake of BB 3 increased from 31.99 to 45.79 mg/g when the temperature was increased from 25 to 130^◦C. This is likely because the added heat is used to form the condensation product, citric acid anhydride, which combines with hydroxyl groups of the biomass to form an ester linkage as reported previously[12]. The reaction can be expressed as follows:

(4) where RB–OH represents the hydroxyl groups on the surface of the raw biomass.

Fig. 3.Effect of reaction time on the sorption performance.

However, with further increase in temperature to 140^◦C the BB 3 uptake decreased. This may have been due to the very high temperature increasing the degree of cross-linking, which would hamper the adsorption of BB 3 cations. This aspect may be expressed as follows[12]:

(5) Therefore, according to the experimental results, the optimum heating temperature was ﬁxed at 120^◦C for further studies.

As shown inFig. 3, the uptake of BB 3 increased with increasing of the heating time from 0.5 to 3 h. When the heating time was pro- longed for 4 h, a dye uptake of 45.92 mg/g was maintained, nearly the same as that for 3.5 h. Therefore, maintaining a heating of up to 3.5 h could be the optimum choice for modiﬁcation.

The performance of CA-modiﬁed biosorbent was studied at different CA-concentration at 120^◦C for 3.5 h (Fig. 4). A maximum BB 3 uptake was obtained when the raw biomass was reacted with 0.8 M CA. However, with a CA concentration greater than 0.8 M, the BB 3

Fig. 4.Effect of the concentration of CA on the sorption performance.

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Fig. 5.Fourier transform infrared absorption spectra of the raw biomass (a) and CA-modiﬁed biomass (b).

adsorption capacity decreased. This was because in case that very high CA concentration used may bring steric hindrance and make against the adsorption of BB 3[12]. Therefore, a CA concentration of 0.8 M was selected as the optimum concentration for modiﬁcation, and used for further studies.

3.2. FTIR analysis

For a detail investigation of the intensity and nature of the functional groups on the biomass surface, FTIR spectra of the raw biomass and CA-modiﬁed biomass were analyzed.Fig. 5displays the infrared spectra of these two types of biomass in aqueous solution at pH 9.0±0.5. As shown inFig. 5a, the broad band ranging from 3800 to 2500 cm⁻¹ should be due to the overlapping of –OH in carboxyl group[18,19]. A medium strength adsorption peak at 1411 cm⁻¹ could be assigned to the symmetrical stretching of the carboxylate anion[20]. After citric acid was introduced on the biomass surface (Fig. 5b), the broad overlapping shifted from 3325 to 3309 cm⁻¹, which might be attributed to that the hydroxyl groups on the raw biomass surface took esteriﬁcation reaction with citric acid. And the peak evidence at 1411 cm⁻¹, which associated with C O stretching vibration, markedly changed to 1404 cm⁻¹. Also new peak at 1729 cm⁻¹was observed, which can be assigned to C O stretching vibration of carboxylic acid[29].

It reﬂected that the carboxyl binding sites on the biomass surface were increased when the raw biomass was modiﬁed with CA.

3.3. Effect of pH

The solution pH is an important controlling parameter in the adsorption process[21]. The effect of pH on BB 3 uptake was inves- tigated over the pH range of 3–10. The sorption capacity of BB 3 increased with increasing pH (Fig. 6). The carboxyl groups in biolog- ical polymers have pK_Hvalues ranging from 3.5 to 5.0[22]. At pH <3, the non-ionic form of carboxyl groups, –COOH, would be present;

therefore, the sorption capacity of BB 3 should be negligible. With a pH greater than 3, the carboxyl groups (–COOH) would change to carboxylate anions (–COO⁻), and basic dye (BB 3) released positively charged dye ions in aqueous solution, which would exhibit electrostatic attraction towards the positively charged biomass surface.

As shown in Fig. 6, at pH >5, the BB 3 uptakes onto CA- modiﬁed biomass signiﬁcantly increased comparing that onto the raw biomass. It is because that CA was introduced onto the surface

Fig. 6.The effect of pH on the biosorption of BB 3.

of the biomass, the carboxyl binding sites increased accordingly.

These additional negatively charged groups are likely to electro- statically bind further cationic dye molecules. It was noted that the uptake of CA-modiﬁed biomass was not signiﬁcantly altered beyond pH 6, with the uptake of BB 3 maintaining at around 21.66–23.08 mg/g. This indicated the fact that at pH greater than 6, almost all cationic BB 3 ions have been adsorbed on the binding sites on the biomass surface.

3.4. Adsorption isotherms

An adsorption isotherm is the basic requirement in the design of any sorption system, as it expresses the relation between the mass of dye adsorbed per unit mass of adsorbent and the liquid phase dye concentration. The adsorption isotherms of the dye onto the raw and CA-modiﬁed biomass were evaluated by varying the BB 3 initial concentration within the range 0–2330 mg/L, with a constant biomass dosage of 10 g/L. The uptake of BB 3 increased with increasing equilibrium concentration, and eventually reached to a constant saturated value (Fig. 7).

Although the Langmuir equation cannot provide any mechanis- tic understanding of the sorption phenomena, the Langmuir model may be conveniently used to estimate the maximum uptake of dye from experimental data. The Langmuir biosorption isotherm assumes that biosorption takes place at speciﬁc homogeneous sites

Fig. 7. Isotherms of BB 3 biosorption onto the raw biomass and CA-modiﬁed biomass. The lines were predicted according to the Langmuir model.

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Table 2

Langmuir model parameters.

Biomass qmax(mg/g) b(L/mg) R²

Raw biomass 25.0±0.9 0.010±0.002 0.982

CA-modiﬁed biomass 50.4±0.7 0.013±0.001 0.998

within the adsorbent[23], and has been successfully applied to many sorption process of monolayer biosorption. The Langmuir isotherm model is given as:q_e=q_maxC_e/(1/b+C_e), whereq_eis the adsorbed dye (mg/g),qmax the maximum uptake (mg/g), b the adsorption equilibrium constant (L/mg), reﬂecting the afﬁnity of the biomass towards BB 3, andCethe equilibrium concentration of dye molecules in the solution (mg/L).

The Langmuir parameters were estimated using the nonlin- ear regression method [24]. The model parameters are given in Table 2. As shown inTable 2, the values ofqmaxwere 25.0±0.9 and 50.4±0.7 mg/g for the raw and CA-modified biomass, respectively. Comparing these values, it was noted that the adsorption capacity ofC. glutamicumincreased 2.02 times when treated with citric acid. In addition, the coefficient brepresents the affinity between the sorbent and sorbate. As shown in Table 2, the b value of CA-modified biomass is almost similar to that of the raw biomass, indicating the sorption affinity of the raw biomass towards BB 3 did not changed a lot, after chemical modification.

The shape of an isotherm can be used to predict whether a sorption system is ‘favorable’ or ‘unfavorable’ in batch processes [25]. The essential characteristics of the Langmuir isotherm can be expressed in term of either a dimensionless constant separation factor or equilibrium parameter,RL[26].

RL= 1 1+bC0

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whereC0is the initial dye concentration (mg/L), andbthe Lang- muir constant (L/mg). The parameter ofR_Lindicates the shape of the isotherm as either unfavorable (R_L> 1), linear (R_L= 1), favorable (0 <RL< 1) or irreversible (RL= 0). All values ofRL, calculated from equation(6), were maintained within the range of 0–1 at all initial dye concentrations, which conﬁrmed the favorable uptake of the BB 3 process. In addition, the higherRLvalues at lower dye concentration showed that the biosorption was more favorable at lower dye concentration (data were not showed here).

3.5. Sorption/desorption

The regeneration of a biosorbent is likely to be a key factor in assessing its potential for commercial application[27]. In this study, both of the raw and CA-modiﬁed biomass had been used for the sorption of BB 3. The dye was eluted from the dye-loaded biomass by adjusting the solution pH to 3, where the BB 3 uptake was minimal (Fig. 6).

Fig. 8 shows the results of the repeated reuse experiments using the raw and CA-modiﬁed biomass, respectively. The sorption/desorption experiments cycle was performed up to 4 times.

The observed sorption/desorption efficiencies of CA-modified biomass remained very high, more than 89.59%; a bit higher than that of the raw biomass (>84.67%). Besides, it is noted that the biomass can be regenerated easily in a simple method, which is only by shifting the solution pH. Considering the commercial sorbents, such as activated carbons which are not very easy to be regenerated [28], the CA-modified biomass has great potential as a reusable dye sorbent.

Fig. 8.Repeated reuse sorption/desorption cycles. The black and white bars represent the sorption and desorption efﬁciencies, respectively.

4. Conclusions

The biomass ofC. glutamicumwas thermochemically modified using citric acid under optimal conditions of 0.8 M CA, 120^◦C and 3.5 h. The FTIR results confirmed the esterification between the citric acid and hydroxyl groups on the biomass surface. As a result of modification with CA, the negatively charged sites available for cationic dye binding were increased. The sorption of dye molecules onto the raw and CA-modified biomass was well described using the Langmuir sorption isotherm. According to the Langmuir model, the adsorption capacity of the CA-modified biomass was enhanced 2.02 times. At the same time, sorption/desorption experiments showed that it was possible to elute BB 3 from the dye-laden biomass, with regeneration of the biosorbent. Therefore, the waste biomass ofC.

glutamicum, with higher dye sorption capacity following modiﬁca- tion with CA, could act as a low-cost biosorbent for the treatment of dye-bearing wastewaters.

Acknowledgements

This work was supported by the Korea Science and Engineer- ing Foundation (KOSEF) NRL Program grant funded by the Korea government (MEST) (No. R0A-2008-000-20117-0) and in part, by Ministry of Environment as “The Eco-technopia 21 project” and KOSEF through AEBRC at POSTECH.

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