664

Biotechnol. Prog. 2001, 17, 664668

Adsorption Step in the Biological Degradation of a Textile Dye

Aitor Aretxaga, S lvia Romero, Montserrat Sarra ` , and Teresa Vicent*
Departament dEnginyeria Qu mica, Escola Te ` cnica Superior dEnginyeria, Universitat Auto ` noma de Barcelona, 08193 Bellaterra, Spain

This research documents the removal of the dye Gris Lanaset G from aqueous solutions by fungal pellets. Adsorption of the dye by dead biomass pellets of Trametes versicolor was determined and compared with dye removal by enzymatic degradation. Six kinetic equations were fitted to the experimental adsorption data obtained. The results indicate that kinetics such as the Elovich equation, which considers that the rate-controlling step is the diffusion of the dye molecules, show the best fit. Nonlinear Langmuir and Freundlich equations were also fitted into the adsorption data, and it can be concluded that the adsorption equilibrium can be interpreted by the Langmuir isotherm. Adsorption plays an important role in the process of the elimination of color from textile wastewater, although not all of the elimination is due to this physical process when the microorganism is active. The removal of color (around 90%) with active microorganisms is greater than that obtained with the adsorption process.

Introduction
Some specific effluents from industrial production processes may be difficult to purify by traditional wastewater treatment technology, as a result of the complexity of some of their components. These specific components, which are not easily degradable, are commonly toxic, so their discharge can cause serious problems to the environment, and achieving legal purification levels is often very difficult. Wastewater from textile industries creates a great problem of pollution due to the dyes contained therein. Synthetic dyes have been used increasingly in the textile and dyeing industries because of their ease and costeffectiveness in synthesis, firmness, high stability to light, temperature, detergent and microbic attack, and variety in color compared with natural dyes. These chemicals are used for desizing, scouring, dyeing, printing, and finishing. Often, dyes are recalcitrant organic molecules that cause strong color in the wastewater. They contribute to organic load and toxicity of the wastewater (1). Moreover, textile industry effluents can pose serious problems either to the environment, if they are discharged directly to surface waters, or to wastewater treatment plants, which are often incapable of obtaining satisfactory color elimination with current conventional biological treatment processes, so the combination of different processes is necessary. Color is the first contaminant to be recognized, and environmental regulation in most European countries (EU directive 91/271) has made it mandatory to decolorize the dye wastewater prior to discharge. Color elimination in wastewater is, today, the principal problem concerning the textile industry. Adsorption with activated carbon is the treatment that eliminates color with the highest efficiency, but it is a very expensive process. The need for economically viable industrial and wastewater processes that protect the environment and public health has led to research into processes using alternative adsorbents (including rice grain, chitin, crab meal, and different polymers) and also some biological
10.1021/bp010056c CCC: $20.00

treatment of textile wastewater. Some recent experiments have studied the viability of using activated sludge from wastewater treatment plants as adsorbent, obtaining remarkable results, with an effective elimination of toxic compounds such as heavy metals and dyes (2-4). Also the biomass of a variety of microorganisms, including bacteria, fungi, and algae, is capable of adsorbing and accumulating very diverse compounds. As a result of the potential use of microorganisms as a bioadsorbent, research on biosorption has attracted the attention of many researchers in recent years (5-9). Recently, biological processes have received more attention as they are cost-effective and environmentally friendly. Results show that high color elimination can be obtained biologically (10). Color elimination processes with active microorganisms have two different simultaneous steps: an adsorption on the surface of the organisms and a degradation of dyes by enzymes produced by these organisms (11-13). It is important to know the role these steps play in concrete conditions, in which the microorganisms can be living or nonliving. Decolorization may be defined as the removal of color by changing the chromophoric group to a nonchromophoric one, whereas biodegradation is the breakdown of the substrate (dye) molecule by the biological process (14). As a result of the adsorption capacity of the microorganisms, in all biological treatments two processes will take place: adsorption and degradation. This paper evaluates the removal of color with living microorganisms, and then the study focuses on the first step, the physical adsorption process. The work has been carried out using pellets of the fungus Trametes versicolor and the dye Gris Lanaset G.

Materials and Methods

Microorganism. Trametes versicolor was obtained from ATCC# 42530. The fungus was maintained on 2% malt agar slants at 23 C until use. Subcultures were routinely made.

2001 American Chemical Society and American Institute of Chemical Engineers Published on Web 07/13/2001

Biotechnol. Prog., 2001, Vol. 17, No. 4

665
Table 1. Equations Used to Fit Different Kinetic Models to the Data for Dye Adsorptiona kinetic model zero order first order second order two constant rate parabolic diffusion Elovich equation equation Xt ) X0 + Kvt Xt ) X0 exp(Kvt) Xt ) 1/((1/X0) - Kvt) ln(Xt) ) ln(X0) + Kvt Xt ) X0 + Kv(t)1/2 Xt ) X0 + Kv ln t

Culture Methods. A mycelia suspension of T. versicolor was obtained by inoculation of four 1-cm diameter plugs from the growing zone of fungi on malt agar (2%) in a 500-mL Erlenmeyer flask. Flasks were incubated at 23 C and agitated (135 rpm, r ) 25 mm). After 4-5 days, a dense mycelia mass was formed. This was ground with a Polytron homogenizer. The resulting mycelia suspension was stored in sterilized saline solution (0.85% NaCl) at 4 C. This suspension was used to produce pellets by inoculating 1 mL of the suspension in 300 mL of malt extract medium 2% in a 1-L Erlenmeyer flask. Erlenmeyer flasks were shaken (135 rpm, r ) 25 mm) at 23 C for 5 days. Pellets formed by this process were stored at 4 C in sterilized saline solution (0.85% NaCl) Reactor Medium Composition. The culture medium contained per liter 8 g of glucose, 1.9 g of NH4Cl, 11 mL of a supplemented medium (15), 100 mL of 2,2-dimethylsuccinate buffer, and 0.15 g dye. The pH of the solution was adjusted to 4.5 with NaOH 0.5 M. It was sterilized at 120 C for 20 min. The culture medium was inoculated with an amount of pellets equivalent to 0.8 g/L dry weight. Dye. The dye used in this work is Gris Lanaset G, which is in fact a commercial mixture of several metal complex dyes, and was purchased from Ciba (ref 080173.5). This dye was chosen for our study because it is used in a nearby textile industry as a major dye in the production process.

aX , adsorbed dye (mg/g); X , initial adsorption (mg/g); K , kinetic t 0 v constant; t, time (h).

Analytical Methods
Color Determination. Spectrophotometric measurements were carried out at the visible maximum absorbance (590 nm). The dye was quantified using standard curves of absorbance versus concentration. The amount of dye adsorbed could then be calculated by the difference between the added dye and the dye remaining in solution. Enzyme Assays. Laccase activity was measured by using a modified version of the method of Paszczynski et al. (16) for the determination of manganese peroxidase. 2,6-Dimetoxiphenol (DMP) is oxidized by laccase, even in the absence of a cofactor. On the other hand, oxidation by manganese peroxidase requires the presence of H2O2 as cofactor, as well as Mn2+. This avoids potential interference from manganese peroxidase and other peroxidases with the enzyme activity determination. The reaction mixture used to determine laccase activity consisted of 66.6 mM sodium malonate at pH 4.5, 1.3 mM DMP, and up to 600 L of sample in an overall volume of 1 mL. Absorbance changes at 468 nm and 30 C were monitored for 2 min. The activity unit (AU) was defined in terms of the number of micromoles of DMP converted per liter and minute. Metabolic Degradation Experiments. A total of 34 g of biomass was suspended in a reactor with 500 mL of reactor medium. Oxygen was supplied by an air pulsing flow. The electrovalve was controlled by a cycling timer, and the pulsing frequency was defined as the inverse of the sum of the electrovalve opening and shutting times. The opening time was 1 s, the shutting time was 5 s, and the air flow was 8.2 L/h. The air pulse facilitated working in fluidized conditions. The bioreactor was equipped with a probe for oxygen concentration measurements. Temperature was maintained at 25 C, and a pH controller maintained the pH equal to 4.5. Batch Adsorption Isotherm Experiments. Adsorption of Gris Lanaset G by the pellets of T. versicolor was examined as follows. Pellets of fungus were suspended in 250 mL of solution containing dye in the concentra-

tions 38, 55, 93.5, 135, 156, 183, and 193 mg/L and sodium azide (1 g/L), a toxic compound that inactivated the microorganisms. The suspension was stirred at 250 rpm and 25 C until equilibrium was reached. Samples of the solution were taken at different times during the process and then centrifuged before colorimetrical analysis. The amount of dye and microorganism changed in all of the experiments, and in consequence the relation between the masses of dye and fungus also varied. Adsorption Kinetics. The data of adsorbed dye at various time intervals were fitted into six different kinetic equations (zero-, first-, and second-order equations and the parabolic diffusion, two constant, and Elovich-type equations). The equations are listed in Table 1. Desorption Experiment. After an adsorption experiment, with an initial dye concentration of 150 mg/L, the pellets were removed from the solution and placed in 250 mL of water. Absorbance at 590 nm was periodically measured.

Results and Discussion

Metabolic Degradation Experiments. An experiment in which microorganisms were alive was compared with experiments in which adsorption was the only factor responsible for color removal. The evolution of certain parameters in the reactor was measured throughout the process of degradation, such as glucose concentration, laccase activity, pO2, and the absorbance of the solution at 590 nm. Figure 1 shows the timecourse of some of these parameters from the moment that the dye was added to the reactor until the termination of the process. Microorganisms consume glucose for their metabolism. Consequently, the concentration of glucose decreased until it reached zero. The oxygen remained at over 70% of saturation. The production of the laccase enzyme started after about 3 h and reached a peak of 80 activity units after 26 h. The dye was added when activity was detected. The color, the parameter that indicates the concentration of the dye in the water, decreased about 75% in the first 2 h, and the color removal continued up to 90%. This water color reduction was due to two simultaneous effects: the physical process of adsorption on the fungus surface and the enzymatic degradation caused by the activity of the laccase enzyme produced by the fungus. Adsorption Kinetics. Several experiments with different amounts of dye were carried out. The contact time necessary for the system of nonliving fungal pellets and dye to reach the equilibrium is an important parameter for research and possible commercial applications for the process. Figure 2 shows the elimination of color versus time of adsorption in three runs. It was shown that in the first 60 min of contact between dye and pellets, high removals were obtained (about 60%) and that the rate of elimination of color decreased until equilibrium was reached.

666

Biotechnol. Prog., 2001, Vol. 17, No. 4

Figure 1. Levels of dye concentration (b and dotted line), glucose concentration (4 and dashed line), and laccase activity (9 and solid line) in the reactor during the degradation process.

Figure 3. Adsorption kinetics of dye to pellets. Lines show the fits of three kinetic equations, two constant rate (dotted line), parabolic diffusion (dashed line), and Elovich equation (solid line), to experimental data corresponding to two different initial concentrations in solution: 183 (2) and 38 mg/L (b).

Figure 2. Adsorption kinetic data of three runs of dye adsorption in pellets, with initial dye concentrations of 184 (( and solid line), 158 (b and dashed line), and 38 mg/L (3 and dotted line).
Table 2. Parameters of Fitted Kinetic Models and Statistics for Five Different Initial Concentrations (C0)
kinetic model two constant rate C0 (mg/L) 183 156 135 55 38 183 156 135 55 38 183 156 135 55 38 X0 ( SD Kv ( SD r2 0.946 0.893 0.792 0.835 0.916 0.893 0.819 0.731 0.812 0.769 0.973 0.944 0.903 0.955 0.958

parabolic diffusion

Elovich equation

2.129 (1.284 0.506 ( 0.046 2.016 ( 1.412 0.530 ( 0.070 9.902 ( 1.389 0.268 ( 0.074 3.677 (1.349 0.295 ( 0.067 1.591 ( 1.206 0.378 ( 0.042 7.732 ( 5.525 1.740 ( 0.231 10.040 ( 6.520 1.618 ( 0.291 20.959 ( 6.363 0.982 ( 0.322 7.923 ( 2.295 0.529 ( 0.130 4.495 (1.492 0.393 ( 0.079 -28.665 ( 4.675 13.750 ( 0.86 -21.725 ( 5.926 12.946 ( 1.21 2.179 (7.358 8.036 (1.66 -2.870 ( 2.063 4.207 ( 0.46 -1.538 ( 0.933 2.835 ( 0.21

The results of fitting the kinetic equations (Table 1) to the observed data are shown in Table 2, where C0 is the initial dye concentration, Xt is the dye adsorption at a time t, X0 is the initial adsorption, and Kv is the kinetic constant. For the zero-order kinetic equation, the reaction rate is only dependent on the initial concentration of adsorbable dye. First-order kinetics indicate that the process of adsorption occurs at a rate proportional to dye concentration, which is particularly suitable for low concentrations. Second-order kinetics are thought to derive from sorption processes in which the rate-controlling step is an exchange reaction. The poorness of fit for

these three models indicates that they should not be considered. On the contrary, kinetics such as parabolic diffusion, which consider that the rate-controlling step is the diffusion of the dye molecules, either to the interior of pellets or from the solution to the surface, show better fit. Nevertheless, the best fit for all the concentrations under study was shown by the Elovich equation. This kinetic model describes a diffusion step controlling the adsorption process. Figure 3 shows two examples of fit. Adsorption/Desorption. When a process of adsorption is studied, it is very important to ascertain if it is a reversible process or not, studying desorption from the surface of hifae. Figure 4 shows that the process under study was practically irreversible, since a very small percentage of the dye adsorbed can be desorbed in the studied conditions. The surface of the pellets keeps its intense blue color when the desorption process reaches its equilibrium. Adsorption Isotherms of Dye on the Pellets of T. versicolor. Eight experiments, with different quantities of adsorbent and solute, were carried out, so that the adsorption isotherm could be evaluated. Nonlinear Langmuir and Freundlich equations were fitted into the experimental adsorption data using the least squares parameter estimation method. Parameters of Langmuir (X/M) ) KbCe/(1 + KCe) and Freundlich equation (X/ M) ) K(Ce)1/n, where X denotes mass of dye adsorbed (mg), M represents the mass of adsorbent (g), Ce is the equilibrium concentration of dye (mg/L), b is the maximum adsorption capacity, and K is a constant that can be calculated. The values of the parameters of these two equations, together with the corresponding correlation coefficients (r2) are listed in Table 3. Very effective fits of the Langmuir equation to the experimental data were obtained, though values of the determination coefficient corresponding to the Freundlich equation were also impressive. It can be concluded that the adsorption equilibrium of Gris Lanaset G on the pellets can be interpreted by the Langmuir isotherm. Adsorption versus Degradation. By comparing the color elimination obtained by the physical process of adsorption by inactive microorganisms and by the experiments with metabolic degradation (Figure 5), it can be concluded that the removal of color in the reactor with living microorganisms is higher than that obtained with the adsorption process. Moreover, there is also a significant difference in the cause of the color elimination. In the adsorption processes, the dye changes phase from the

Biotechnol. Prog., 2001, Vol. 17, No. 4

667

Figure 4. Evolution of the absorbance during the adsorption (b) and desorption (4) of dye, with an initial dye concentration of 150 mg/L.

Figure 6. Tubes in the image contain (A) initial solution of dye; (B) initial pellets; (C) solution after the adsorption process; (D) pellets after the adsorption process; (E) solution after the adsorption + degradation processes together; and (F) pellets after the adsorption + degradation processes together.

Figure 5. Elimination of color due to adsorption (( and dashed line) and degradation and adsorption together (b and solid line).
Table 3. Fitting Constants and Coefficients of Correlation (r2) for the Fits of the Freundlich and Langmuir Equations to the Experimental Data for Dye Adsorption by Pellets Langmuir isoterm b ) 6.1425 K ) 0.0268 r2 ) 0.9961 Freundlich isoterm K ) 0.2326 1/n ) 0.6148 r2 ) 0.9499

Figure 7. Absorbance data at visible wavelengths throughout an experiment of adsorption, at 1, 16, 60, 240, and 1440 min (from top to bottom).

solution but becomes adsorbed on the surface of the fungus. To the contrary, when the microorganism is living, it can be observed that the dye is rapidly removed from the medium, as a result of the physical adsorption process, but later the dye is eliminated both from the solution and the surface of the pellets as a consequence of the enzymatic degradation. This enzymatic degradation is not observed when the microorganisms are not living. Figure 6 shows the significant difference existing between the color of the solution and pellets obtained when adsorption-desorption equilibrium is reached and that obtained after the action of adsorption and degradation processes together. It can be seen that the color of both the solution and the pellets after the adsorption process is darker than the color of the solution and the pellets in which the adsorption and degradation processes worked simultaneously. Maximum Absorbance Wavelength. Data of absorbance at wavelengths between 350 and 650 nm throughout the time of an adsorption experiment are shown graphically in Figure 7. The maximum absorbance peak

is placed at 590 nm, the wavelength at which the colorimetric measurements should be acquired. The shape of the curve remains stationary at the different times. The fact that no changes take place demonstrates that different molecules are not produced and that there is therefore no metabolic degradation of the dye in the solution; however, its concentration decreases because of the adsorption process on the surface of hyphae.

Conclusions
This work studies the process of adsorption of the dye Gris Lanaset G on the surface of the pellets of T. versicolor as a part of a biological treatment process for colored wastewater. The results show that when the fungus is active, the elimination of color is not due only to adsorption. Treatment with the fungus eliminates around 90% of the color, as a result of both the adsorption and enzymatic degradation processes. About 70% of the color can be eliminated with the physical process of adsorption alone, but the dye remains in the solid phase. After fitting several kinetic equations and the nonlinear Langmuir and Freundlich equations to the experimental data, it

668

Biotechnol. Prog., 2001, Vol. 17, No. 4

(6) Sing, C.; Yu, J. Copper Adsorption and Removal from Water by Living Mycelium of White-rot Fungus Phanerochaete chrysosporium. Water Res. 1998, 32, 2746-2752. (7) Lie ` vremont, D.; Seigle-Murandi, F.; Benoit-Guyod, J. Removal of PCNB from Aqueous Solution by a Fungal Adsorption Process. Water Res. 1998, 32, 3601-3606. (8) Matheickal, J. D.; Yu, Q.; Woodburn, G. M. Biosorption of Cadmium(II) from Aqueous Solutions by Pretreated Biomass of Marine Alga Durvillaea potatorum. Water Res. 1999, 33, 335-342. (9) Puranik, P. R.; Paknikar, K. M. Biosorption of Lead, Cadmium and Zinc by Citrobacter strain MCM B-181: Characterization Studies. Biotechnol. Prog. 1999, 15, 228-237. (10) Royer, G.; Yerushalmi, L.; Rouleau, D.; Desrochers, M. Continuous Decolorization of Bleached Kraft Effluents by Coriolus versicolor in the form of Pellets. J. Ind. Microbiol. 1991, 7, 269-278. (11) Tatarko, M.; Bumpus, J. A. Biodegradation of Congo Red by Phanerochaete chrysosporium. Water Res. 1998, 32, 17131717. (12) Cripps, C.; Bumpus, J. A.; Aust, S. T. Biodegradation of Azo and Heterocyclic Dyes by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 1990, 56, 1114-1118. (13) Bumpus, J. A.; Brock, B. J. Biodegradation of Crystal Violet by the White Rot Fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 1988, 54, 1143-1150. (14) Azmi, W.; Sani, R. K.; Banerjee, U. C. Biodegradation of Triphenylmethane Dyes. Enzyme Microb. Technol. 1998, 22, 185-191. (15) Kirk, T. K.; Schultz, E.; Connors, W. J.; Lorenz, L. F.; Zeikis, J. G. Influence of Culture Parameters on Lignin Degradation by Phanerochaete Chrisosporium. Arch. Microbiol. 1978, 117, 227-285. (16) Paszczynski, A.; Crawford, R. L.; Huynh, V. B. Manganese Peroxidase of Phanerochaete chrysosporium: Purification. Methods Enzymol. 1988, 161, 264-270.

can be concluded that the Langmuir isotherm and the Elovich kinetic equation are the ones that best fit the data obtained. The adsorption capacity demonstrated by the T. versicolor pellets is an example of the existence of adsorbent products that are starting to be applied in wastewater treatment as a means of lowering the costs of the adsorption process, in which the use of activated carbon has made the application economically unviable.

Acknowledgment
This work has been supported by CICYT (project Bio97-0760-C02-01). The Department of Chemical Engineering UAB is member of the Centre de Refere ` ncia en Biotecnologia de la Generalitat de Catalunya.

References and Notes

(1) Nigam, P.; Banat, I. M.; Singh, D.; Marchant, R. Microbial Process for the Decolorization of Textile Effluent Containing Azo, Diazo and Reactive Dyes. Process Biochem. 1996, 5, 435442. (2) Mart n, M. J.; Balaguer, M. D.; Rigola, M. Evaluation of Adsortive Capacities of Activated Carbons Obtained from Biological Sludge: Preliminary Studies for Pilot Plant Aplication. In Beneficial Reuse of Water and Biosolids; Water Environment Federation: Alexandria, VA, 1997; pp 41-53. (3) Mart n, M. J.; Balaguer, M. D.; Rigola, M. Valorizacio n de Fangos Biolo gicos Excedentes de EDARs por Transformacio n en Carbo n Activo: Ana lisis Comparativo de la Capacidad de Adsorcio n respecto a Colorantes y Metales Pesados. Tecnol. Agua 1999, 184, 42-48. (4) Lo pez, E.; Soto, B.; Arias, M.; Nu n ez, A.; Rubinos, D.; Barral, M. T. Adsorbent Properties of Red Mud and Its Use for Wastewater Treatment. Water Res. 1998, 32, 1314-1322. (5) Chang, J.; Huang, J. Selective Adsorption/Recovery of Pb, Cu, and Cd with Multiple Fixed Beds Containing Immobilized Bacterial Biomass. Biotechnol. Prog. 1998, 14, 735-741.

Accepted for publication May 23, 2001. BP010056C