Decolorization of Azo Dyes with Enterobacter agglomerans
Immobilized in Different Supports by Using Fluidized Bed Bioreactor
Adnane Moutaouakkil,1Youssef Zeroual,1Fatima Zohra Dzayri,2Mohamed Talbi,2Kangmin Lee,3 Mohamed Blaghen1
1Unit of Bio-industry and Molecular Toxicology, Laboratory of Microbiology, Biotechnology and Environment, Faculty of Sciences Aı¨n Chock, University Hassan II—Aı¨n Chock, Km 8 route d’El Jadida, BP. 5366 Maˆarif, 20100 Casablanca, Morocco
2Laboratory of Analytical Chemistry, Faculty of Sciences Ben M’sik, University Hassan II—Mohammedia, Casablanca, Morocco
3Laboratory of Enzyme Technology, Chonbuk National University, Chonju, Republic of Korea Received: 28 April 2003 / Accepted: 2 June 2003
Abstract. Immobilized cells of Enterobacter agglomerans, able to reduce azo dyes enzymatically, were used as a biocatalyst for the decolorization of synthetic medium containing the toxic azo dye methyl red (MR). This bacterial strain exhibits high ability to completely decolorize 100 mg/L of MR after only 6 h of incubation under aerobic conditions. Cells of E. agglomerans were immobilized in calcium alginate, polyacylamide, cooper beech, and vermiculite, and were used for the decolorization of MR from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when E. agglomerans was entrapped in calcium alginate beads and was of about 3.04 mg MR/g cell/h with a 50% conversion time (t1/2) of about 1.6 h. Moreover, immobilized cells in calcium alginate continuously decolorized MR even after seven repeated experiments without significant loss of activity, while polyacrylamide-, cooper beech-, and vermiculite-immobilized cells retained only 62, 15, and 13%
of their original activity, respectively.
Azo dyes are synthetic organic compounds characterized by the presence of one or more azo bonds (™N¢N™) in association with one or more aromatic systems [1]. These dyes are used widely in the textile, paper, cosmetic and food industries and are also the most common types of synthetic dyes. They have been identified as the most problematic compounds in textile effluents [3]. Aside from their negative aesthetic effects, certain azo dyes have been shown to be toxic [1], and in some cases these compounds are carcinogenic and mutagenic [7]. Purifi- cation of dye wastewater is a matter of great concern, and several physical and chemical treatment methods have been suggested [8, 14, 24], but not widely applied be- cause of the high cost and the secondary pollution that can be generated by the excessive use of chemicals. One interesting approach is to promote the microbiological degradation of these compounds in wastewater treatment systems. Indeed, biological methods are able to provide a more natural and complete clean-up of the pollutants in a more economical way.
Most biological degradations of azo dyes are car- ried out by anaerobic bacteria [3, 18, 19, 28]. Gener- ally, azo dyes are resistant to attack by bacteria under aerobic conditions. Only a few studies reported that bacteria, under aerobic conditions, could degrade azo dyes [4, 5, 11, 13]. In both cases, bacterial degradation of azo dyes is often initiated by an enzymatic biotrans- formation step that involves reductive cleavage of azo bonds with the aid of azoreductase by utilizing both NADH and NADPH as electron donors [27]. The resulting aromatic amines are further degraded by multiple-step bioconversion occurring aerobically or anaerobically [10, 20].
Immobilization of living microorganisms has been described as useful in biological wastewater treatment [6, 12, 26]. Immobilization of bacteria, yeast cells, and fungi has been carried out by various techniques. Conventional immobilization methods are generally classified into three categories: covalent binding, physical adsorption, and entrapment processes [15]. It is widely known that immobilized cells offer many advantages: reusability of
Correspondence to: M. Blaghen; email: blaghen@hotmail.com
Microbiology
An International Journal
© Springer-Verlag New York Inc. 2004
the same biocatalyst, control of reactions, and the non- contamination of products [9].
On the basis of the bacterial abilities to decolorize and degrade azo dyes, and by exploiting advantages that immobilization techniques offer, we aimed to eliminate the toxic azo dye methyl red (MR) from synthetic water, using a bacterial strain that had been previously isolated, identified, and appeared to be able to decolorize and degrade MR and related dyes under aerobic conditions [17]. This bacterial strain has been entrapped in both calcium alginate and polyacrylamide gels, and immobi- lized by physical adsorption on either cooper beech or vermiculite. The MR degradation was studied in a flu- idized bed bioreactor. Degradation rates obtained were compared.
Materials and Methods
Microorganism and culture media. Enterobacter agglomerans was isolated from dye-contaminated sludge collected from an industrial area in Casablanca city (Morocco) [17]. This bacterium expressed azoreductase activity and was able to decolorize a variety of azo dyes via a pathway initiated by azo bond reduction [17]. The bacterial growth was determined by measuring the absorbance at 600 nm and the cell biomass was then estimated using the calibration curve relating cell concentration and absorbance.
Two media were used in this study: one was nutrient broth (NB) (Topley House, Bury, England) used for routine transfer and cell cultivation, and the other was minimum medium (MM) containing 0.6 g/L of K2HPO4, 0.1 g/L of MgSO4, 0.6 g/L of (NH4)2SO4, 0.5 g/L of NaCl, 20 mg/L of CaCl2, 1.1 mg/L of MnSO4, 0.2 mg/L of ZnSO4, 0.2 mg/L of CuSO4and 0.14 mg/L of FeSO4(pH adjusted to 7 with 1 M HCl) used to monitor the decolorization.
Measurement of dye concentration. The azo dye used in this study was methyl red (MR) CI 13020, which was obtained from BDH Chemicals Ltd. Poole (England). The concentration of azo dye in samples was determined after centrifugation at 9,500 g at 4°C for 10
min by measuring the absorbance of the supernatant at 430 nm using a Jenway 6405 UV/Visible spectrophotometer.
Cell immobilization methods.
Entrapment in calcium-alginate gel. 100 mL of sterile sodium alginate solution (2% wt/vol) was mixed, until homogeneous, with 1 g of a bacterial pellet obtained by centrifugation of a bacterial culture (on NB containing 100 mg/L of MR) at 15,000 g for 15 min in a Sigma 3K15 refrigerated centrifuge. The mixture was extruded through a needle (2 mm i.d.) into 150-mMCaCl2, forming beads of 3.0-mm diameter. The beads were allowed to harden in the CaCl2solution at room tempera- ture for 30 min and were rinsed with Tris-HCl buffer (50 mM, pH 7).
Entrapment in polyacrylamide gel. 1 g of a bacterial pellet was mixed with 78 mL of Tris-HCl buffer (50 mM, pH 7), 20 mL acrylamide- bisacrylamide solution (30 – 0.8% wt/vol), and 1 mL ammonium per- sulfate solution (10% wt/vol). The polymerization was initiated by adding 100L of N,N,N’,N’-tetramethyl-ethylenediamine. The poly- acrylamide gel was then divided into particles of 0.5 cm diameter and rinsed with Tris-HCl buffer (50 mM, pH 7).
Immobilization on vermiculite. 5 g of vermiculite was washed with distilled water and placed in a column (1.5 cm i.d.) with 20 mL of bacterial solution (50 mg/mL) in sodium phosphate buffer (0.1 M, pH 7). The solution was recycled for 24 h at room temperature. The supernatant was removed and the active vermiculite washed with the same buffer.
Immobilization on cooper beech. 10 g of fine particles cooper beech were washed with distilled water and placed in a column (1.5 cm i.d.) with 20 mL of bacterial solution (50 mg/mL) in sodium phosphate buffer (0.1M, pH 7). The solution was recycled for 24 h at room temperature. The supernatant was removed, and the active portion was washed with the same buffer.
Decolorization of MR in fluidized bed bioreactor by using free and immobilized cells. The fluidized bed bioreactors are composed of 500-mL conical flasks containing the immobilized bacteria in different supports (calcium alginate, polyacrylamide, cooper beech, or vermic- ulite) suspended in 100 mL of MM with 0.1% (wt/vol) of glucose and 100 mg/L of MR (decolorization medium). The bioreactors were placed in a rotary shaker at 25°C, and the fluidization is assured by a high stirring of 100 rpm. Decolorization rate was followed according to time Fig. 1. Decolorization of methyl red (MR) (-䊐-) by E. agglomerans growing (-E-) in minimum medium (MM) containing 1% (wt/vol) of glucose and 100 mg/L of the dye.
in all bioreactors. The same bioreactor has been used for studying MR decolorization with free cells; thus, 1 g of a bacterial pellet obtained by centrifugation of bacterial culture (on NB containing 100 mg/L of MR) at 15,000 g for 15 min was suspended in 100 mL of MM with 0.1%
(wt/vol) of glucose and 100 mg/L of MR. Decolorization rate was followed according to time in the bioreactor placed in the same con- ditions cited previously. For each experiment, a control test without
bacteria was carried out under the same conditions in order to evaluate the affinity of MR for different supports used (calcium alginate, poly- acrylamide, cooper beech, and vermiculite).
At several time intervals, 1-mL aliquots were collected from bioreactors and centrifuged at 15,000 g for 15 min. The supernatants were analyzed spectrophotometerically at 430 nm to determine the amount of MR.
Fig. 2. Decolorization of methyl red (MR) by E. agglomerans: (A) free cells (-E-); (B) and (C) immobi- lized cells in alginate (-F-), poly- acrylamide (-■-), cooper beech (-Œ-), and vermiculite (-}-).
Repeated-batch operations of decolorization with immobilized cells. The performances of fluidized bed bioreactors used continuously were evaluated by following the decolorization rates for 7 days with a daily renewal of the decolorization medium [0.1% (wt/vol) of glucose and 100 mg/L of MR in MM] in which the immobilized bacteria were suspended. The bioreactors were placed at 25°C in a rotary shaker at 100 rpm. Decolorization rates were followed according to time in all bioreactors. Daily, the immobilized-cell particles were collected, rinsed twice with sterile deionized water, and transferred into a fresh decol- orization medium for a second decolorization experiment.
Results and Discussion
Decolorization of MR by E. agglomerans growing in liquid medium. E. agglomerans was grown aerobically in MM containing 1% (wt/vol) of glucose and 100 mg/L of MR. The culture was incubated at 37°C in a rotary shaker at 100 rpm for 10 h. At several time intervals, aliquots from the culture were sampled, and their cell densities and dye concentrations were measured. The obtained results indicated that this bacterial strain had a higher ability to decolorize and degrade 100 mg/L of the toxic azo dye MR (Fig. 1). The bacterial growth coin- cided with the disappearance of MR. Thus, after only 6 h of incubation, the MR concentration decreased signifi- cantly; about 95% of the orange color of MR was re- moved (Fig. 1), whereas MR was not degraded in a sterile control flask incubated under the same conditions.
This aerobic MR degradation by E. agglomerans was much faster than those reported by other studies for Acetobacter liquifaciens or Klebsiella pneumoniae [22, 25].
Decolorization of MR with free and immobilized cells of E. agglomerans. E. agglomerans was entrapped in both calcium alginate and polyacrylamide gels and was physically adsorbed on cooper beech and vermiculite. At various times, the concentrations of MR were measured
spectrophotometrically at 430 nm for all immobilized cells of E. agglomerans, as well as for free cells.
Figures 2A, 2B, and 2C demonstrate typical re- sidual dye profiles for decolorization with free and immobilized cells of E. agglomerans. The trends for free, calcium alginate-, and vermiculite-immobilized cells were similar, as the dye concentration dropped almost linearly until decolorization was complete, while the profile for polyacrylamide- and cooper beech-immobilized cells exhibited an extra phase (in- dicated by different slopes) during the early stage of decolorization. The different residual dye profile for polyacrylamide- and cooper beech-immobilized cells may correlate with MR adsorption capacities of those immobilization supports (data not shown), since the early drop in the dye concentration (Figs. 2B and 2C) may be owing to a matrix adsorption effect, while the second slope resulted from biotransformation by im- mobilized E. agglomerans cells. For calcium alginate- and vermiculite-immobilized cells, decolorization was primarily contributed by bacterial azo reduction, as the adsorption effect was less important.
The specific decolorization rates determined from Fig. 2 were 4.92, 3.04, 1.27, 0.93, and 0.87 mg MR/g cell/h, respectively, for free cells and immobilized cells in calcium alginate, polyacrylamide, cooper beech, and vermiculite (Table 1). The lower decolorization rate for immobilized cells compared with free cells can be attrib- uted to the mass transfer restriction arising from cell entrapment.
From all immobilized cells, E. agglomerans en- trapped in calcium alginate showed the greatest purifying performance with a specific decolorization rate of 3.04 mg MR/g cell/h and an equilibrium conversion of 98%
(Table 1). All the more that the hydrodynamic behavior and mechanical bead properties of calcium alginate make this polymer a matrix of choice for the utilization in a fluidized bed bioreactor [2, 21]. The immobilization of E.
agglomerans in polyacrylamide gel allows a specific decolorization rate of about 1.27 mg MR/g cell/h (Table 1). This limitation of the decolorization activity is prob- ably due to the existence of an unfavorable microenvi- ronment inside the gel matrix and the presence of resid- ual monomer that leads to a toxicity of bacterial cells [16]. By fixing E. agglomerans on cooper beech or on vermiculite, we eliminate about 94% of MR. The specific decolorization rate obtained by using activated cooper beech is slightly superior to that obtained with activated vermiculite (Table 1). This is owing to the MR affinity for cooper beech, which fixes to it alone more than 30%
of MR, while for the vermiculite, no affinity for MR was observed (data not shown).
Table 1. The specific decolorization rate and the equilibrium conversion of free and immobilized cells of E. agglomerans
Specific decolorization rate
(mg MR/g cell/h)
Equilibrium conversion
(%)
Free cells 4.92 100
Alginate-immobilized cells
3.04 98
Polyacrylamide- immobilized cells
1.27 97
Cooper beech- immobilized cells
0.93 95
Vermiculite- immobilized cells
0.87 94
Repeated-batch decolorization. Repeated-batch experi- ments were performed to examine the reusability of the immobilized cells in azo dye decolorization. After seven cycles, the specific decolorization rate of polyacrylamide-, cooper beech-, and vermiculite-immobilized cells dropped to about 62, 15, and 13%, respectively, while the specific decolorization rate remained over 95% for calcium alg- inate-immobilized cells (Table 2). Thus, calcium alginate- immobilized cells appeared to exhibit better reusability.
Evaluation of t1/2(the time required for 50% color removal) shows that t1/2varied only slightly (1.6 –1.7 h) for calcium alginate-immobilized cells during seven repeated cycles (Table 2). This indicates that calcium alginate, used as an entrapment gel in a continuous fluidized bed bioreactor, offers a great stability of bacterial activity. This stability may be attributed to sweet polymerization conditions of the calcium alginate gel and the direct role that the calcium plays in the cell conservation [23]. However, in the first run of experiments, the t1/2for polyacrylamide-, cooper beech-, and vermiculite-immobilized cells were 3.9, 5.3, and 5.7 h, respectively (Table 2). After seven runs, the t1/2increased for all three types of immobilized cells and reached 6.3, 35.7, and 41.5 h for polyacylamide-, cooper beech-, and vermiculite-immobilized cells, respectively (Table 2).
Conclusion
It has been shown that high-efficiency decolorization of water contaminated by azo dyes with immobilized azo dye-degrading bacteria in a fluidized bed bioreactor is feasible. The obtained results show that this technology allows specific and reproducible decolorization of the azo dye MR with high yield.
The specific decolorization rate obtained by immo- bilizing Enterobacter agglomerans in polyacrylamide is superior to those obtained by fixing this bacterial strain on cooper beech or vermiculite. However, the entrap- ment of E. agglomerans in calcium alginate gel offers the greatest purifying performance as well as the biggest
stability of the bioreactor. Moreover, the hydrodynamic behavior of the beads, in a fluidized bed bioreactor, is very satisfactory. The high specific decolorization rate obtained and the simplicity of the immobilization method mean that alginate would be a suitable immobi- lization matrix for using bacterial strains to remove azo dyes from wastewater on an industrial scale.
ACKNOWLEDGMENTS
The authors thank the Moroccan CNCPRST and the urban community of Casablanca for their support.
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