Ind. Eng. Chem. Res. 2004, 43, 7865-7869
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Biosorptive Decolorization of Reactive Orange 16 Using the Waste Biomass of Corynebacterium glutamicum S. W. Won,† S. B. Choi,† B. W. Chung,† D. Park,‡ J. M. Park,‡ and Y.-S. Yun*,† Division of Environmental and Chemical Engineering and Research Center of Industrial Technology, Chonbuk National University, Chonbuk 561-756, Korea, and Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea
The protonated waste biomass of Corynebacterium glutamicum discharged from an industrial lysine fermentation plant was used for the removal of Reactive Orange 16 (RO16). The maximum sorption capacities of the biomass were as high as 186.6 ( 7.1 and 154.8 ( 2.8 mg g-1 at pH 1 and 2, respectively, which are comparable to those of commercial sorbents such as activated carbons and ion-exchange resins. As the solution pH decreased, the dye uptake increased and, under neutral conditions, it was negligible. Because the RO16 molecule has two negatively charged sulfonate groups, it is likely that the dye cannot be bound to a negatively charged carboxyl and/or phosphonate sites of the biomass. Instead, positively charged amine-occurring protein molecules seem to be dye binding sites. The uptake of RO16 was not significantly affected by a high concentration of salts, and the biomass could be repeatedly reused up to eight times per sorption/desorption cycle, reflecting that the biomass wastes deserve to be a potential regenerable biosorbent for dye removal. Introduction Textile industries consume large volumes of water and chemicals for the wet processing of textiles. The presence of very low concentrations of dyes in effluent discharged from these industries is highly visible and undesirable.1 Because of their chemical structure, dyes are resistant to fading when exposed to light, water, and many chemicals.2,3 According to the survey of the Ecological and Toxicological Association of the Dyestuffs Manufacturing Industry, over 90% of dyes have LD50 values greater than 2000 mg kg-1.4 There are several ways in which colorants cause problems in waters:5 (1) Depending on the exposure time and dye concentration, dyes can have acute and/or chronic effects on exposed organisms. (2) Although the visibility of dyes in rivers depends on their color and extinction coefficient and on the clarity of the water, they are inherently highly visible. This means that even minor releases of effluents may cause abnormal coloration of surface water, which captures the attention of both the public and the authorities. (3) When the aesthetic problem is neglected, the greatest environmental concern with dyes is their absorption and reflection of sunlight entering the water. This interferes with the growth of bacteria to levels sufficient to biologically degrade impurities in the water and start the food chain. Accordingly, government regulations on dye discharge are becoming increasingly stringent. There are many structural varieties of dyes that fall into either the cationic, nonionic, or anionic type. Anionic dyes are the direct, acid, and reactive dyes.6 Brightly colored, water-soluble reactive, and acid dyes are the most problematic because they tend to pass * To whom correspondence should be addressed. Tel.: +82-63-270-2308. Fax: +82-63-270-2306. E-mail: ysyun@ chonbuk.ac.kr. † Chonbuk National University. ‡ Pohang University of Science and Technology.
through conventional treatment systems unaffected.7 Municipal aerobic treatment systems, dependent on biological activity, were found to be inefficient in the removal of these dyes.8 Various physical, chemical, and biological methods have been used for the treatment of dye-containing wastewater. Some chemical oxidation by the Fenton reagent, ozone, UV plus H2O2, or NaOCl results in aromatic ring cleavage and may generate chemical sludge or byproducts that are likely to be more toxic.9 Aerobic biological treatment is known to be ineffective for dye removal, but anaerobic bioremediation allows water-soluble dyes to be decolorized.10 Physical adsorption technology has gained favor recently because it has a high efficiency in the removal of highly stable dyes and is economically feasible when compared to other methods.11 This is the most commonly used method of dye removal12 and is very effective for adsorbing cationic, mordant, and acid dyes and, to a slightly lesser extent, dispersed, direct, vat, pigment, and reactive dyes.13 However, activated carbons are expensive and not easily regenerated.9 Although ion-exchange resins can be regenerated easily, the high cost hinders their wide application for the treatment of dye-bearing wastewater. Therefore, low-cost sorbents able to bind dye molecules and easily regenerated have been extensively searched and tested.5,9 In this study, the waste biomass of Corynebacterium glutamicum was used and evaluated as a biosorbent for the treatment of an anionic reactive dye Reactive Orange 16 (RO16). This biomass is generated in a great quantity from the full-scale fermentation process for amino acid production. Amino acid fermentation industries have been troubled with a huge amount of biological solid waste, which is mainly composed of the biomass of C. glutamicum. Although this fermentation byproduct is potentially recyclable, until now most of it has been dumped at sea. Therefore, it can be seen that the feasibility for reuse of the solid waste as a value-added biosorbent deserves to be assessed.
10.1021/ie049559o CCC: $27.50 © 2004 American Chemical Society Published on Web 10/30/2004
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Figure 1. Chemical structure of RO16.
Materials and Methods Materials. The fermentation wastes (C. glutamicum biomass) were obtained in a dried powder form from a lysine fermentation industry (BASF-Korea, Kunsan, Korea). The protonated biomass was prepared by treating the raw biomass with a 1 N HNO3 solution for 24 h, thereby replacing the natural mix of ionic species with protons. The acid-treated biomass, designated as protonated biomass, was washed with deionized distilled water several times and thereafter dried at 60 °C in an oven for 24 h. The resulting dried C. glutamicum biomass was stored in a desiccator and used as a biosorbent in the sorption experiments. All chemicals used in this study were of analytical grade. RO16 was purchased from Sigma-Aldrich Korea Ltd. (CI 17757, Yongin, Korea). As shown in Figure 1, RO16 has two sulfonate groups that have negative charges in an aqueous solution. The general characteristics of RO16 are molar mass ) 617.54, color index number ) 17 757, and maximum light absorption at λmax ) 494 nm. Sorption Dynamic Experiments. For the sorption dynamic experiment, the biomass (10 g/L) was suspended with deionized distilled water (40 mL) and the solution pH was adjusted at pH 3.0 using 1 M HNO3. Thereafter, the dye solution (1000 mg of RO16/L) whose pH was also adjusted at pH 3.0 was added to the biomass suspension so that the initial RO16 concentration became 500 mg/L. The slurry composed of the biomass and dye solution was agitated at 160 rpm. The water used in this experiment was initially bubbled with CO2 and the dynamic experiment was carried out at a room temperature of 20 ( 2 °C in a N2 atmosphere in order to avoid the effect of atmospheric CO2 on the solution pH. During the experiment, the solution pH was kept constant at 3.0 by adding 1 M HNO3 and samples were intermittently removed from the vessels. The total volume of withdrawn samples did not exceed 2% of the working volume. Here, absorption spectra of intermittently taken samples were measured from 350 to 600 nm using a spectrophotometer. pH Edge Experiments. The pH edge experiments were carried out: an equilibrium relationship between the dye uptake and the final pH, which is helpful to understand the pH dependence of biosorption.14 The pH edge experiments were carried out with 500 mg/L of initial RO16 concentration and 10 g/L of biomass. The pH was intentionally altered by means of adding 1 N NaOH or 1 N HNO3 into the bottles. The suspension was agitated at 160 rpm in a shaker at a room temperature of 20 ( 2 °C. After the system reached the equilibrium state (20-24 h), the final pH was measured and the samples were taken and centrifuged for liquidsolid separation. The supernatant portion was used for the analysis of the concentration of residual RO16 after proper dilution.
Isotherm Experiments. To evaluate the sorption capacity of the biomass, biosorption isotherms of RO16 were obtained at different solution pHs and NaCl concentrations. The isotherm experiments were carried out with 0.4 g of the biomass in 40 mL of working solution volume. While the pH edge experiments were performed with the same initial concentration of RO16, in the isotherm experiment the initial concentration was changed from 0 to 5000 mg/L, which resulted in different final dye concentrations after sorption equilibrium was achieved. Following the addition of biomass into the dyecontaining solutions, the solution pH was controlled at a desired value using 1 N HNO3 because the pH tended to increase during the RO16 binding to the biomass. Other conditions were the same as those used in the pH edge experiment. Measurements of Dye Uptake. The dissolved dye concentration of samples was analyzed using a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan) at 494 nm where the maximum absorption peak existed. Before an analysis of the RO16 concentration, samples were centrifuged at 3000 rpm for solid-liquid separation. In order for the change of the working volume (up to 5%) by added HNO3 solutions to be considered, the dye uptake (q) was calculated from the mass balance as follows:
q)
V0C0 - VfCf M
(1)
where V0 and Vf are the initial and final (initial plus added acid solution) volumes, respectively. C0 and Cf are the initial and final concentrations of RO16, respectively. M stands for the weight of biomass used. Desorption and Repeated Reuse Experiments. To evaluate the desorption efficiency, the RO16-loaded biomass was centrifuged at 3000 rpm and the supernatant was removed. Thereafter, the settled biomass was resuspended with 40 mL of deionized distilled water, and the pH of the suspension was adjusted at pH 7, where the uptake of RO16 was found to be negligible in the pH edge experiments. The suspension was shaken at 160 rpm for 24 h to allow dye to be released from the biomass. Thereafter, the desorbed dye was analyzed, and the desorption efficiency was calculated as follows:
desorption efficiency (%) ) released RO16 (mg) × 100 (2) initially sorbed RO16 (mg) After desorption, the biomass was again reused for subsequent sorption experiments. The sorption/desorption cycle was performed up to eight times to evaluate the possibility of repeated reuse of the biosorbent. The sorption efficiency of each cycle was calculated as a percentage of the uptake at the first sorption. Fourier Transform Infrared Analysis. Infrared spectra of the protonated biomass of C. glutamicum were obtained using a Fourier transform infrared spectrometer (FT/IR-300E, Jasco). Results and Discussion Biosorption Dynamics of RO16. The time required for the batch sorption system to reach the equilibrium state was evaluated under a pH-stat condition (Figure 2). The dye RO16 has the maximum absorption peak
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Figure 2. Dynamics of the light absorption spectra of wastewater under pH-stat condition at pH 1. The times from the top to bottom lines are 0, 0.5, 1, 5, and 10 h, respectively.
Figure 3. pH edge of RO16 biosorption at different biomass concentrations. Biomass concentration: 10 g/L (2); 5 g/L (O).
at 494 nm and the second peak at 387 nm. As soon as the RO16 solution was contacted with the protonated biomass, the peaks rapidly declined, and after 10 h, the peaks did not change significantly. Therefore, the contact time to allow the sorption system to reach an equilibrium state was less than 10 h. To give a sufficient time to equilibrate, 20-24 h of contact time was used in the following equilibrium sorption experiments such as pH edge and isotherm. As can be seen in Figure 2, the shape of absorption spectra did not alter during biosorption, implying that RO16 was sorbed without any change in its original chemical structure. pH Edge and Binding Mechanism. As the pH was decreased, the uptake of RO16 increased, and at a pH between 6 and 8, the uptake was negligible (Figure 3). As the pH was increased to more than 9, the uptake slightly increased. The dye uptake decreased when the biomass concentration was increased because the final concentration of RO16 was lower for the high biomass concentration. Two sulfonate groups of RO16 are easily dissociated and have negative charges in the aquatic environment. Therefore, the negative sites of the biomass such as carboxyl and phosphonate groups may not play a role in RO16 binding, whereas amine groups (-NH2) mainly found in protein molecules in the biomass15 can be protonated as a form of -NH3+. Such positively charged
Figure 4. Fourier transform infrared absorption spectra of the protonated C. glutamicum biomass.
groups are likely the binding sites for negatively charged RO16. Other biosorbents such as fungal biomass16 and chitosan17,18 have also been reported to uptake anionic dyes, and the binding sites were suggested to be the protonated amine. Therefore, it can be postulated that the solution pH decreases, the binding sites (positively charged amine) increase, and thereby the uptake of RO16 increases. However, the pKa values of amine groups in the biological molecules are known to be in the range between 8 and 11.15 Considering the pKa value, the amine groups may be fully protonated at a pH of less than approximately 7. However, the RO16 uptake around pH 7 was not significant (Figure 3). This is likely because of repulsion between negative RO16 and negative carboxyl and phosphonate groups which are redundant in the microbial15 and seaweed19 biomass. Because the pKa of carboxyl groups is around 4,15,19 most of the groups have a negative charge at pH 7, which repulse the negatively charged RO16 molecules. As the pH is increased up to more than 7, the negative charges in the biomass increased because other functional groups such as phosphonate (pKa ) 6.1-6.8) get negatively ionized.15,19 These negatively charged groups of the biomass are likely to further hinder electrostatically the access of negative RO16 to the biomass. To confirm the existence of amine, carboxyl, and phosphonate groups in the C. glutamicum biomass, the FTIR study was carried out. As shown in Figure 4, the FTIR spectrum displays a number of absorption peaks, indicating the complex nature of the biomass examined. The absorption peaks around 3500-3000, 1538, and 1384 cm-1 are indicative of the existence of amine groups. The spectrum also displays the absorption peaks at 3600-3200, 1652, and 1233 cm-1, corresponding to carboxyl groups. The phosphonate groups show some characteristic absorption peaks around 1157 cm-1 (PdO stretching) and 1078 cm-1 (P-OH stretching). Therefore, it can be noted that the FTIR spectrum of the C. glutamicum biomass supports the presence of amine groups, which is likely responsible for the binding of RO16, and carboxyl and phosphonate groups, which may electrostatically inhibit the binding of anionic RO16 in the range of pH 4-7. The reason the uptake of RO16 slightly increased at pH > 9 was not clearly explained at this moment. It is presumed that, at an elevated pH, a certain precipitate like calcium hydroxide entrapped some of the dye molecules from the solution phase. Another possibility
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Figure 5. Isotherms of RO16 biosorption at different solution pHs. The lines were produced by the Langmuir model. Experimental data: pH 1 (O); pH 2 (2); pH 3 (4); pH 4 (b); pH 6 (0).
Figure 6. Effect of the salt concentration on the uptake of RO16 at different initial concentrations of RO16. The solution pH was controlled at pH 1. Initial RO16 concentration: 200 ppm (O); 1000 ppm (2); 3000 ppm (4).
Table 1. Estimated Parameters of the Langmuir Modela parameter
pH 1
pH 2
pH 3
pH 4
pH 6
qmax (mg/g) 186 (7.1) 154.8 (2.8) 128.6 (4.5) 64.0 (2.2) NFb K (mg/L) 90.8 (13.2) 25.8 (2.3) 41.5 (7.5) 30.9 (4.5) NFb R2 0.983 0.994 0.984 0.990 NFb a
Standard errors are in parentheses. b Cannot be fitted.
is that the secondary amine group (-NH-) of RO16 may have a positive charge under highly alkaline conditions, and the charged secondary amine is able to bind to the negative sites of the biomass such as carboxyl and phosphonate groups. Isotherm of RO16 Biosorption. The uptake of RO16 increased with an increase in equilibrium concentrations and eventually reached a certain saturated value depending on the pH (Figure 5). Although empirical models such as the Langmuir equation cannot provide any mechanistic understanding of the sorption phenomena, the latter may be conveniently used to estimate the maximum uptake of dye from experimental data. The Langmuir parameters were estimated using the nonlinear regression method20 and are summarized in Table 1. As the pH decreased, the RO16 uptake increased, and at pH 1, the maximum uptake was estimated to be 186.6 ( 7.1 mg/g, which is comparable to that of commercially available sorbents. Macroporous polymeric resins were reported to uptake various dyes, with maximum uptakes ranging from 100 to 400 mg/ g.21 Activated carbons showed a large variation of the dye uptake performance (50-560 mg/g) depending on the types of activated carbons and dyes.18 Although a chemically cross-linked chitosan bead was reported to have an outstanding uptake of dyes (1900-2500 mg/ g),18 there was no information on whether this type of sorbent could be regenerated and repeatedly reused. Effect of the Salt Concentration. In general, reactive dyes are applied to fabric in a high salt concentration in order to lower the dye solubility.21 NaCl is mainly used as a salt to enhance the bath dye exhaustion. Therefore, unfixed dye in wastewater is accompanied by a large amount of salts that likely interfere with dye biosorption. The effect of the salt concentration in synthetic wastewater on the uptake of RO16 was investigated (Figure 6). At low levels of the RO16 concentration, the effect of the salt concentration on the dye uptake was
negligible. As the initial concentration of RO16 was increased up to 3000 ppm, the uptake did not significantly decrease. This reflects that Cl- ions do not compete with a sulfonate group of RO16 molecules for amine sites of biomass. In addition, it can be noted that an elevated ionic strength with NaCl does not electrostatically interfere with the binding of RO16 to the biomass significantly. From a practical point of view, these results imply that the waste biomass of C. glutamicum can be used for the removal of RO16 from salt-containing wastewaters. Repeated Reuse. To be a good sorbent for dye removal, the dye-loaded sorbent should be regenerated; otherwise, the wastes have to be disposed of, and fresh sorbents should be used. In this study, after the protonated biomass of C. gluatmium was used for RO16 sorption, RO16 was eluted from the dye-loaded biomass by adjusting the solution pH at 7, where the dye uptake was minimal (Figure 3). Figure 7A shows the results of repeated reuse experiments. As the sorption/desorption cycle continued, the sorption and desorption efficiencies gradually decreased, and at the eighth cycle, the sorption capacity was 57.4 ( 10.0% of the capacity in the first use. The main reason the efficiency decreased according to repeated reuse was not because of deactivation of the binding sites but because of the loss of biomass itself during the sorption/desorption cycle. After it was reused eight times, the weight of the biomass was 0.203 ( 0.016 g, which corresponded to 50.8% of the initially applied weight. Accordingly, 0.197 ( 0.016 g of the biomass was lost during centrifugation/resuspension steps of 8 times the sorption/desorption cycle. With an assumption that the biomass loss at each cycle was the same, the real weight of the biomass used in each cycle could be calculated. Considering the biomass loss, the sorption/ desorption efficiencies were replotted in Figure 7B. As a result, even up to the eighth cycle, the sorption/ desorption efficiencies remained almost 100%. It is expected that the biomass loss in the packed column, which is a typical sorption process, is not as significant as batchwise cyclic reuse experiments performed in this work because the shear in the batch reactor used here was higher than that expected for column. Considering that commercial sorbents such as activated carbons are hardly regenerated,9 this waste biomass has great
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Literature Cited
Figure 7. Efficiencies of sorption and desorption of RO16 in repeated reuse experiments. The sorption was carried out at pH 1 and the desorption at pH 7. Black bars represent the sorption efficiency and white ones the desorption efficiency. A: Sorption/ desorption efficiencies when neglecting the biomass loss. B: Sorption/desorption efficiencies when compensating for the biomass loss.
potential as a reusable dye sorbent. Besides, it should be noted that the biomass can be regenerated easily by adjusting the solution pH to a neutral condition. In other types of biomasses, the desorption efficiency ranged from 30 to 80%, although relatively expensive chemicals such as methanol, ethanol, and organic surfactants (i.e., Tween) were used for regeneration.16 Therefore, the biomass wastes of C. glutamicum would be able to be repeatedly reused if the biomass loss was minimized. Acknowledgment This work was financially supported by KOSEF through AEBRC at POSTECH and partially by Grant R08-2003-000-10987-0 from the Basic Research Program of KOSEF.
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Received for review May 24, 2004 Revised manuscript received September 17, 2004 Accepted September 21, 2004 IE049559O
