Journal of Biotechnology 101 (2003) 57 /68 www.elsevier.

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Decolorization of the textile dyes by newly isolated bacterial strains

Kuo-Cheng Chen a,*, Jane-Yii Wu a, Dar-Jen Liou a, Sz-Chwun John Hwang b
a

Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan, ROC b Department of Civil Engineering, Chung Hua University, Hsinchu, Taiwan, ROC

Received 7 January 2002; received in revised form 19 September 2002; accepted 24 September 2002

Abstract Six bacterial strains with the capability of degrading textile dyes were isolated from sludge samples and mud lakes. Aeromonas hydrophila was selected and identified because it exhibited the greatest color removal from various dyes. Although A. hydrophila displayed good growth in aerobic or agitation culture (AGI culture), color removal was the best in anoxic or anaerobic culture (ANA culture). For color removal, the most suitable pH and temperature were pH 5.5 / 10.0 and 20 /35 8C under anoxic culture (ANO culture). More than 90% of RED RBN was reduced in color within 8 days at a dye concentration of 3000 mg l ( 1. This strain could also decolorize the media containing a mixture of dyes within 2 days of incubation. Nitrogen sources such as yeast extract or peptone could enhance strongly the decolorization efficiency. In contrast to a nitrogen source, glucose inhibited decolorization activity because the consumed glucose was converted to organic acids that might decrease the pH of the culture medium, thus inhibiting the cell growth and decolorization activity. Decolorization appeared to proceed primarily by biological degradation. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Aeromonas hydrophila ; Azo dyes; Anthraquinone dyes; Indigo dyes; Microbial decolorization

1. Introduction The first synthetic dye, mauevin, was discovered in 1856. Since then, over 100 000 dyes have been generated worldwide with an annual production of over 7 )/105 metric tones. Synthetic dyes are widely used in textile, paper, food, cosmetics and pharmaceutical industries (Zollinger, 1987; Carliell

* Corresponding author. Tel.: '/886-3-571-6249; fax: '/8863-571-3014. E-mail address: d867608@oz.nthu.edu.tw (K.-C. Chen).

et al., 1995). The inefficiency in dyeing processes has resulted in 10 /15% of unused dyestuff entering the wastewater directly (Zollinger, 1987; Spadarry et al., 1994). Color present in dye effluent gives a straightforward indication of water being polluted, and discharge of this highly colored effluent can damage directly the receiving water. Furthermore, it is difficult to degrade the mixtures of the wastewater from textile industry by conventional biological treatment processes, because their ratio of BOD/COD is less than 0.3 (Chun and Yizhong, 1999). In some cases, traditional biological procedures were combined with physical- or chemical-

0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 3 0 3 - 6

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treatment processes to achieve better decolorization (Vandevivere et al., 1998), but chemical or physical /chemical methods are generally costly, less efficient and of limited applicability, and produce wastes, which are difficult to dispose of. As a viable alternative, biological processes have received increasing interest owing to their cost effectiveness, ability to produce less sludge, and environmental benignity (Banat et al., 1996). Therefore, to develop a practical bioprocess for treating dye-containing wastewater is of great significance. The effectiveness of microbial decolorization depends on the adaptability and the activity of selected microorganisms. Over the past decades, many microorganisms are capable of degrading azo dyes, including bacteria (Zimmerman et al., 1982; Haug et al., 1991; Sani and Banerjee, 1999), fungi (Gold and Alic, 1993; Swamy and Ramsay, 1999; Balan and Monteiro, 2001; Novotny et al., 2001), yeast (Martins et al., 1999), actinomycetes (Zhou and Zimmermann, 1993) and algae (Dilek et al., 1999). Most azo dyes are reduced anaerobically to the corresponding amines with cleavage of azo bonds by bacterial azoreductase, but they are difficult to degrade aerobically (Zimmerman et al., 1982; Banat et al., 1996). Moreover, fungal ligninolytic enzyme system (lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase) might also be involved in the bio-oxidation of dyes (Gold and Alic, 1993). However, the low pH requirement (Swamy and Ramsay, 1999) for an optimum activity of the enzymes and the long hydraulic retention time for complete decolorization (Banat et al., 1996; Swamy and Ramsay, 1999) are the disadvantages of using fungi. In addition, they may inhibit the growth of other useful microorganisms. Thus, large-scale applications of fungal decolorization have been limited. In general, the wastewater from textile industry contains many various dyes. To gain a widespread reception, the azo-degrading bacteria should exhibit decolorizing ability for a wide range of dyes. This study aimed to isolate some bacterial strains, which possessed the ability to decolorize 24 kinds of dyes, including azo, anthraquinone, and indigo dyes. A bacterium displaying the greatest decolorizing ability was chosen for further study to

illustrate the factors influencing its efficiency. In addition, the major cause of the inhibition of glucose on the reduction of azo dye was identified.

2. Materials and methods 2.1. Chemicals Twenty-four dyes were used and chosen from various types (azo, anthraquinone and indigo) of important commercial dyes. Azo, anthraquinone and indigo, containing various substituents such as nitro and solfonic groups, are the major classes of dyes with the greatest variety of colors. Acid Blue 74, Acid Orange 7, Acid Red 106, Direct Yellow 4 and Direct Yellow 12 were purchased from the Sigma Chemical Company, MO, USA. The other dyes (Acid Black 172, Acid Blue 264, Acid Yellow 42, Direct Black 22, Direct Orange 39, Direct Red 224, Direct Red 243, Direct Yellow 86, Reactive Black NR, Reactive Black 5, Reactive Blue 160, Reactive Blue 171, Reactive Blue 198, Reactive Blue 222, Reactive Green 19, Reactive Red 120, Reactive Red 141, Reactive Red 198 and Reactive Yellow 84) were obtained from Everlight Chemical Industrial Co., Taoyuan, Taiwan. All other chemicals were reagent grade. 2.2. Screening of decolorizers Sludge samples were obtained from various sources including the lake-mud in Tsing Hua University (Hsinchu, Taiwan) and the sludge of wastewater treatment plant in Chang Chun Petrochemical Co. (Miaoli, Taiwan). In order to obtain a high-performance bacterial decolorizer, RED RBN, the most commonly used dye, was first chosen as the target for screening azo-degrading bacteria. The mixed bacterial cultures from the sludge samples were acclimated for 3 months, and then served as the stock culture. The bacteriaisolating procedures and the test procedures later used for each dye were carried out in a screening medium (SM medium). The medium contained the following components: yeast extract, 10 g; NaCl, 5.0 g in 1 l of distilled water with 0.1 g (except that described else) of selected dye. Ten milliliter of the

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stock culture was added to a 500 ml conical flask containing 100 ml of SM medium and pH was adjusted to 7.0. The isolates were cultured routinely in an incubator at 30 8C. Next, the broth of the decolorized flask culture was then transferred to a fresh SM medium to screen strains that have color removal ability. The screening procedures in the liquid culture with SM medium were conducted repeatedly until a decolorizing culture appeared. An aliquot (0.1 /1 ml samples) of each supernatant fluid of the isolated cultures was spread on a SM agar medium, and then incubated at 30 8C. Colonies surrounded by a decolorized zone were selected. Isolates were then tested for their color removal ability in a submerged culture. Finally, six promising isolates were selected. 2.3. Dye assays and decolorizing cultures The stock cultures for these isolates were precultured for 20 h at 30 8C by growing a single colony in an anoxic static condition. The same initial cell concentrations of dye-degrading microorganisms were used to decolorize all the dyes. Decolorization in an individual dye solution could be seen visually, and was measured at its maximum adsorption wavelength (lmax) on culture supernatants using a scanning spectrophotometer (UV/vis, Shimadzu, Kyoto, Japan). To ensure that the pH change in dye solution did not influence decolorization, the visible absorption spectra were recorded between pH 4.0 and 11.0 and the pH did not affect spectrum. Biomass concentration was determined by dry cell weight after 24 h drying at 105 8C. All assays were conducted in triplicates with uninoculated controls. 2.4. Analysis of color removal in the medium containing mixture of dyes All 24 dyes, each at a concentration of 50 mg l (1, were dissolved together in SM medium. The mixture of dyes did not have a well-defined peak at the visible absorption spectra. Therefore, the detection of color level was made using the American Dye Manufacturers Institute (ADMI) Tristimulus Filter Method (Eaton et al., 1995).

2.5. Identication of selected azo dye-degrading bacteria Bacterial isolates with the greatest decolorization abilities were first examined by Gram staining, and further identifications were performed by the Culture Collection and Research Center, Food Industry Research and Development Institute (Hsinchu, Taiwan). 2.6. Decolorization at different culture conditions The effects of the various culture conditions such as agitation, aeration, anoxic state and anaerobic state on decolorization of RED RBN were examined owing to their various concentrations of dissolved oxygen (DO). Agitation culture (AGI culture), the only culture at shaking condition, was operated in a rotary incubation shaker running at 200 rpm. All the other cultures were under a static condition with no shaking at all. Anaerobic culture (ANA culture) was bubbled with pure nitrogen only at the beginning until the DO became zero, but anoxic culture (ANO culture) had never been bubbled at all. Aerobic culture (AER culture) was maintained in a continuous aeration condition (airflow rate of 3 l min(1). All the experiments were operated at 30 8C and pH 7.0 under a constant initial dye concentration (RED RBN) of 50 mg l(1. The concentration of cells, RED RBN, and DO were monitored as a function of time. 2.7. Glucose analysis Reducing sugar was analyzed and determined as glucose by the DNS (3, 5-dinitrosalicylic acid) method (Miller, 1959). The color tests were made with 3 ml aliquots of reagent added to 3 ml aliquots of sample in tubes. The reagent contained 1% dinitrosalicylic acid, 0.2% phenol, 0.05% sodium sulfite, and 1% sodium hydroxide. The mixtures were heated for 15 min in a boiling water bath, and then cooled and adjusted to ambient temperature under running tap water. The color intensities were measured in a scanning spectrophotometer (UV/vis, Shimadzu, Kyoto, Japan) at 575 nm.

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Table 1 Decolorization of textile dyes by various bacteria

Type of dyes (C. I. Number) Chemical structure of dye lmax (nm) Color removal (%) at initial concentration (100 mg l ( 1)

DEC1 1 day Azo Acid Orange 7 (15510) Acid Red 106 (18110) Direct Orange 39 (40215) Reactive Red 198 (unpublished) Acid Yellow 42 (22910) Direct Red 224 (unpublished) Direct Red 243 (29315) Direct Yellow 4 (24890) Direct Yellow 12 (24895) Direct Yellow 86 (29325) Reactive Black 5 (20505) Reactive Blue 222 (unpublished) Reactive Red 141 (unpublished) Reactive Red 120 (25810) Direct Black 22 (35435) Acid Black 172 (unpublished) Reactive Blue 160 (unpublished) Reactive Blue 171 (unpublished) Reactive Blue 198 (unpublished) Reactive Black NR (unpublished) Reactive Green 19 (unpublished) Reactive Yellow 84 (unpublished) Anthraquinone Acid Blue 264 (unpublished) Indigoid Acid Blue 74 (73015) 7 days

DEC2 1 day 7 days

DEC3 1 day 7 days

DEC4 1 day 7 days

DEC5 1 day 7 days

DEC6

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1 day

7 days

Monoazo Monoazo Monoazo Monoazo Diazo Diazo Diazo Diazo Diazo Diazo Diazo Diazo Diazo Diazo Polyazo Azo Azo Azo Azo Azo Azo Azo

485 533 414 515 412 520 523 396 395 393 597 613 532 512 482 571 616 608 625 598 630 411

799 /2 /2 709 739 /3 /1 659 159 /1 59 /1 /2 409 469 /2 /3 719 /2 519 469 /2 /2 639 509 /2 419 /1 /2 429 139 /1 /1 659 /1 529 /1 99 819 /2 /1 439 /2 459 459 /2
/2 609

1009 /1 1009 /1 1009 /1 1009 /2 649 /3 669 /3 849 /2 1009 /2 1009 /1 669 /3 959 /2 1009 /2 879 /2 829 /2 699 /2 519 /3 1009 /1 809 /2 209 /2 1009 /2 839 /2 639 /3 809 /1 849 /3

479 /3 529 /2 509 /2 859 /2 69 /1 179 /2 229 /2 249 /2 519 /4 289 /2 679 /3 389 /2 179 /2 169 /2 259 /2 89 /1 609 /2 159 /2 69 /3 789 /3 149 /2 199 /2 279 /2 509 /4

1009 /2 1009 /1 1009 /1 1009 /2 449 /3 499 /3 659 /2 1009 /2 1009 /3 669 /2 539 /4 719 /2 799 /3 669 /2 649 /2 389 /4 1009 /3 699 /2 209 /1 1009 /2 709 /3 629 /2 779 /3 819 /3

389 /1 629 /5 429 /4 869 /3 79 /3 159 /4 139 /1 199 /2 409 /2 209 /1 629 /1 439 /3 139 /1 129 /2 199 /2 99 /1 589 /1 139 /1 59 /1 789 /1 109 /3 159 /2 239 /2 469 /5

1009 /2 1009 /2 1009 /1 1009 /3 459 /1 699 /5 689 /4 839 /3 1009 /2 649 /2 599 /5 709 /4 829 /6 629 /2 659 /3 409 /5 1009 /4 699 /2 229 /1 1009 /2 659 /2 609 /3 689 /2 859 /3

759 /2 859 /6 759 /2 899 /3 119 /2 129 /2 399 /1 609 /2 779 /1 429 /2 689 /3 679 /2 469 /2 409 /4 159 /2 99 /1 679 /2 469 /5 109 /2 839 /3 509 /2 439 /4 469 /3 409 /1

1009 /3 1009 /1 1009 /2 1009 /1 589 /3 569 /2 809 /2 1009 /3 1009 /2 609 /4 859 /2 1009 /1 829 /2 809 /5 459 /2 359 /6 1009 /2 729 /3 109 /1 1009 /2 779 /5 559 /2 679 /2 779 /3

449 /2 459 /5 519 /4 829 /3 39 /2 89 /2 159 /1 229 /2 629 /1 209 /2 669 /3 399 /4 109 /2 119 /2 79 /2 29 /1 659 /2 159 /2 39 /1 779 /5 79 /2 149 /2 259 /2 309 /3

1009 /3 1009 /2 1009 /5 1009 /3 349 /1 579 /2 519 /2 1009 /2 1009 /1 569 /2 559 /5 649 /2 649 /6 599 /2 349 /2 349 /2 1009 /4 629 /2 119 /2 1009 /3 589 /2 509 /5 659 /2 729 /5

279 /2 639 /3 399 /5 829 /5 29 /1 99 /1 129 /3 149 /1 399 /2 69 /1 639 /2 459 /5 59 /2 109 /1 59 /2 0 629 /2 219 /3 39 /2 779 /5 209 /1 119 /2 39 /1 269 /2

1009 /2 1009 /3 1009 /6 1009 /3 69 /1 329 /2 669 /3 1009 /2 1009 /2 369 /6 549 /5 769 /2 399 /3 809 /5 239 /3 0 1009 /4 809 /5 59 /2 1009 /2 709 /3 449 /1 199 /2 709 /3

Anthraquinone 608 Indigoid 609

The names of all the dyes above are recognized by the Color Index.

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3. Results and discussion 3.1. Isolation and identication White and pink colonies surrounded by an almost decolorized zone were isolated and then tested for color removal capability using submerged cultures. Among these colonies, six of them with the highest decolorization ability in SM medium, designated as DEC1-6, were selected for a further study. Decolorization of various dyes by the growing cells of the six isolates were shown in Table 1. Among the 24 dyes, Acid Orange 7, Acid Red 106, Direct Orange 39, Direct Yellow 4, Direct Yellow 12, Reactive Black NR, Reactive Blue 160 and Reactive Red 198 were reduced completely by all the strains, DEC1 /6, while Reactive Blue 198 (5 / 22%) and Acid Black 172 (0 /51%) were reduced only slightly even after 7 days of incubation. The effectiveness of all the six isolates in decolorizing these 24 dyes may depend on the structure and complexity of the dyes, particularly on the nature and position of substituent in the aromatic rings and the resulting interactions with the azo bond (Zimmerman et al., 1982; Sani and Banerjee, 1999). However, no clear relationship can be observed between the position of substituent in the aromatic rings from published structure of dye and the decolorization efficiency using dye-degrading microorganisms in this study, except that most monoazo dyes tested had color removal higher than the diazo dyes and anthraquinone dyes tested under the same initial biomass. The different efficiency may be due to the number of azo groups. Similar observation was obtained on the investigation of the degradability in different structures of azo dyes by Phanerochaete chrysosporium (Podgornik et al., 1999). On the other hand, Table 1 also shows that the decolorization rate of the six isolates were DEC1 /DEC4 /DEC2 X/DEC3 /DEC5 / DEC6 after 1 day of incubation under the same initial cell concentrations. However, if we want to consider an isolate favorable for development of a practical bioprocess for decolorization, the decolorization rate is very important. Therefore, several biochemical and physiological investigations

Table 2 Biochemical and physiological proles of strain DEC1 Characteristics Morphology Motile Gram staining Aerobic growth Anaerobic growth Nitrate reduction Catalase Gas from glucose H2S from cysteine Acetoin from glucose Indole production Acid from glucose Arginine dihydrolase b-galactosidase Cytochrome oxidase Hydrolysis of Esculin Gelation Assimilation of Adipate Arabinose Citrate Gluconate Glucose Malate Maltose Mannitol Mannose N -acetyl-glucosamine Phenyl-acetate Acid from Arabinose Maltose Mannitol Xylose A. hydrophila (DEC1) Rod '/ (/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ '/ (/ '/ '/ (/ '/ (/ '/ '/ '/ '/ '/ '/ '/ (/ '/ '/ '/ '/

were conducted to identify the best strain, DEC1. The strain was identified as Aeromonas hydrophila according to the GN microplate (Biolog, CA, USA), API 20E (BioMerieux SA, Marcy le toile, France), API 50 CHE (BioMerieux) and partial sequencing of 16S rRNA gene. The characterization of strain DEC1 was summarized in Table 2. From phylogenetic analysis based on 16S rRNA sequence, strain DEC1 was also identified as a strain that is most related to A. hydrophila .

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Fig. 2. Effect of pH on color removal of RED RBN by A. hydrophila in SM medium at 30 8C under ANO culture. Initial dye concentration: 100 mg l ( 1. (j) 1 day of cultivation; (m) 2 days of cultivation; (') 3 days of cultivation.

dation by the representative strain DEC1, A . hydrophila .

Fig. 1. Effect of various culture conditions on decolorization by A. hydrophila at 30 8C in SM medium containing 50 mg l ( 1 RED RBN. (j) AER culture (air ow rate of 3 l min 1 ( 1); (m) AGI culture (rotary agitation at 200 rpm); (') ANO culture (no aeration, no agitation); (^) ANA culture (gassing the asks with pure nitrogen before static culture).

3.2. Characteristics of microbial decolorization Bacterial degradation of azo dyes is often an enzymatic reaction linked to anaerobiosis, because it is inhibited by oxygen that is in competition with the azo group as the electron receptor in the oxidation of the reduced electron carrier, i.e. NADH (Wuhrmann et al., 1980; Zimmerman et al., 1982; Banat et al., 1996). Seldom are bacteria able to decolorize azo compounds in the presence of oxygen (Wuhrmann et al., 1980). Although the strong oxygen effect on bacterial decolorization has been proved definitely, the quantitative correlation between DO and color removal has seldom been reported. A. hydrophila was propagated in

In addition, large quantities of RED RBN and Remazol Black B are now used in textile and dyestuff industries in Taiwan. RED RBN and Remazol Black B are denoted as Reactive Red 198 and Reactive Black 5 in Color Index, respectively. Thus, RED RBN and Remazol Black B were chosen as the target dyes for further study on microbial characteristics and the causes of degra-

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SM medium using AER, ANO, ANA, or AGI culture to observe cell growth, DO concentration and decolorization (Fig. 1). In both AGI and AER cultures, the presence of oxygen would normally inhibit the activity of decolorization, resulting in a low efficiency of color removal with A. hydrophila . As a matter of fact, AGI or AER culture was only run for 1.5 days and then switched to anoxic static condition. Color disappeared due to the DO concentration dropping to almost zero. The above results suggest that decolorization of azo dye would not take place at a DO concentration higher than 0.45 mg l (1 and a slight increase in cell mass at the initial stage would enhance the efficiency of color removal (Fig. 1). Therefore, the results indicated that the decolorization by A. hydrophila was very sensitive to DO level. To achieve an effective color removal, agitation and aeration should be avoided. Fig. 2 shows that the suitable pH for decolorization of RED RBN ranged from 5.5 to 10.0 with a sharp change toward both ends of the pH range. At the two extreme pH values (i.e. pH 4.5 and 11.0), a strong negative effect occurred significantly on the growth of bacteria and the stabilization of pH. These results show that decolorization of various types of dyes with A. hydrophila occurred over an extensive range of pH. In other words, they are favorable for developing a practical bioprocess for a dye-containing wastewater. Additionally, when the initial pH of the culture was at 4.5, the cell mats were deeply colored by adsorbed dyes only. The adsorption of dye on the cell surface may be related to the mechanism of charge neutralization. Normally, the dyes tested are negatively charged. In contrast, the cells in solution tend to possess relatively positive charges at lower pH. Thus, the cells may have relatively higher affinity for the dyes. Whether RED RBN was used as a substrate for A. hydrophila , a proper color removal, specific decolorization rate and cell growth under ANO culture was observed in the range of 20 /35 8C (data not shown). The low color removal at a temperature beyond 35 8C may be attributed to the thermal deactivation of the decolorization enzymes and the low biomass. According to the above results, the following decolorization experi-

Fig. 3. Effect of various nitrogen sources on decolorization of RED RBN by A. hydrophila at 30 8C under ANO culture. Initial nitrogen sources concentration: 10 g l ( 1. (I) blank (without any nitrogen); (j) peptone; (m) tryptone; (') monosodium glutamate; (%) meat extract; (") beef extract; (k) urea; (^) yeast extract.

ments using A. hydrophila were performed at 30 8C and pH 7.0 under ANO culture. To determine the maximum RED RBN concentration tolerated by A. hydrophila , experiments with different initial dye concentrations (1000 / 8000 mg l (1) were performed. The decolorization efficiency was above 90% for initial dye concentration less than 3000 mg l (1 after 8 days cultivation, but it decreased with further increase in dye concentration. When the dye concentration was as high as 8000 mg l (1, almost 60% of the dye was removed after 10 days of cultivation (data not shown). This means that an acceptable high color removal can be achieved by the A. hydrophila strain in an extensive range of azo dye concentrations. In addition, a substrate inhibition effect was observed at dye concentrations higher than 3000 mg l(1. Reduction in color removal and cell growth may result from the toxicity of dyes to bacteria through the inhibition of metabolic activities. Azo dyes generally contain one or more sulphonic-acid groups on aromatic rings, which might act as detergents to inhibit the growth of microorganisms (Wuhrmann et al., 1980). On the other hand, it was also reported that dyes were the

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Fig. 4. Time courses of growth and decolorization of mixture dyes by A. hydrophila at 30 8C under static culture (initial pH 7.0). (m), Dye concentration; (k), biomass.

inhibitors for nucleic acid synthesis (Ogawa et al., 1988) or for cell growth (Ogawa et al., 1989). Furthermore, the color removal exhibited a growth-associated pattern (data not shown). The maximum cell growth yield was about 1.2 /1.6 and 0.7 /1.0 g l(1 for dye concentrations between 1000 and 3000 and 4000 /8000 mg l(1, respectively. Our works on the association of growth (kinetic parameters) and decolorization by A. hydrophila are now in progress. Similar results were obtained using Remazol Black B instead of RED RBN. 3.3. Effects of nitrogen sources on decolorization Fig. 3 shows the influence of various organic nitrogen sources on the efficiency of decolorization of RED RBN by A. hydrophila . Decolorization with peptone or yeast extract was very effective, so the dye concentration decreased quickly, resulting in 90% color removal within 2 days of cultivation. In addition to the organic nitrogen sources, the inorganic nitrogen sources such as KNO3, NaNO3, NaNO2, NH4Cl, (NH4)2SO4 were also

selected for decolorization. Similar performances were observed with control flasks (without any nitrogen source) but resulting in around 10 /15% color removal after 6 days cultivation (data not shown). The results clearly indicate that decolorization of RED RBN by A. hydrophila was greatly affected by the addition of various nitrogen sources. The metabolism of yeast extract is considered essential to the regeneration of NADH that acts as the electron donor for the reduction of azo bonds (Carliell et al., 1995). Between these two nitrogen sources, yeast extract was finally chosen as a part of culture medium for further experiments because yeast extract is cheaper than peptone. Similar results were obtained using Remazol Black B instead of RED RBN. It had also been found that increasing yeast extract concentrations (from 0 to 10 g l(1) resulted in higher decolorization rates, and the decolorization rates reached a plateau as yeast extract was higher than 8 g l (1. However, the color removal ( /90%) was not enhanced significantly by the increase in yeast extract from 8 to 10 g l (1 after 1

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Fig. 5. Variation in UV /visible spectra of various dye solutions after decolorizing cultivation with A. hydrophila . ( */) Original dye solution; ( ) decolorized dye solution.

day of incubation (data not shown). Therefore, yeast extract at a concentration of 8 g l (1 as nitrogen source for decolorization of A. hydrophila was added in further experiments. 3.4. Decolorization of mixed dyes Dyes of different structures are often used in the textile processing industry, and, therefore, the effluents from the industry are markedly variable in composition. A nonspecific biological process may be vital for treatment of the textile effluents containing a mixture of dyes. The rate of decolorization was very fast and the color removal was almost 90% within 2 days of cultivation, followed by an insignificant change in decolorization for the next 10 days (Fig. 4). Fig. 4 also displayed a growth-associated pattern on color removal. According to the reports (Knapp and Newby, 1995; Sani and Banerjee, 1999) decolorization of dyes by bacteria can be due to adsorption to microbial cells or to biodegradation. In adsorption, examination of the absorption spectrum will reveal that all peaks decrease approximately in

Fig. 6. Effect of glucose concentration on decolorization of RED RBN by A. hydrophila in SM medium at 30 8C under ANO culture ( initial pH 7.0). Initial glucose concentration, (I) 0 g l ( 1; (j) 0.15 g l ( 1; (m) 1.25 g l ( 1; (") 10.0 g l ( 1.

proportion to each other. If the dye removal is attributed to biodegradation, either the major visible light absorbance peak will completely disappear or a new peak will appear. Dye adsorption can also be judged clearly by inspecting the cell mats. Cell mats become deeply colored because of adsorbing dyes, whereas those retaining their original colors are accompanied by the occurrence of biodegradation. The absorbance peak at 515 nm (A point) disappeared completely after 7 days cultivation (Fig. 5a). As seen in Fig. 5b, there was a significant decrease in color intensity or in the peak absorbance at 306, 370 and 597 nm (Points B, C and D, respectively). Moreover, as the RED RBN and Remazol Black B were removed, the A. hydrophila strain remained colorless. A similar result was also observed in a

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mixture of dyes. Consequently, according to the above results, the color removal by A. hydrophila strain might be largely attributed to biodegradation, and the biosorption onto the bacterial surfaces was not significant. 3.5. Effect of glucose on the degradation of the mixed dyes Glucose has been added to enhance the decolorization performance of biological systems in some studies (Haug et al., 1991; Carliell et al., 1995; Kapdan et al., 2000). However, others reported that glucose inhibited the decolorizing activity (Chung et al., 1978; Knapp and Newby, 1995). The variability may be due to the different microbial characteristics. In this study, various concentrations of glucose (0 /10 g l (1) were first evaluated for decolorization of RED RBN by A. hydrophila under ANO culture (Fig. 6). Fig. 6 clearly indicates that glucose concentration of higher than 0.15 g l(1 inhibited appreciably the azo reduction of azo dye by A. hydrophila . In addition, the color of the cell surface became red, and the color removal, decolorization rate and biomass decreased significantly with increasing glucose concentration. Fig. 6 depicts that after 1 day cultivation, only 1.0 /1.9 g l(1 of glucose was consumed in the medium supplemented with the glucose concentration at a range of 1.25 /10 g l(1, and the pH of the media dropped from 7.0 to 4.7 /5.0, followed by a relatively stable pH value for the next 2 days. Obviously, this low pH range (4.7 /5.0) had a significantly negative effect on the growth of bacteria, so the decolorization of azo groups was inhibited. These results are also in good agreement with those found at lower pH as aforementioned (Fig. 2). Moreover, while the pH of the medium was adjusted to 7.0 by adding aseptic NaOH after 3 days cultivation, it is worthy of note that color removal of RED RBN in this culture was increased from 25 to 90% within 2 days (data not shown), and the color of cell surface changed visually from red to white (original color of the cell). According to above results, it is inferred that as consumption of glucose concentration increased, the rate of accumulation of organic acids

Fig. 7. Effect of glucose concentration on decolorization of RED RBN by A. hydrophila in SM supplemented with or without buffer at 30 8C under ANO culture after 2 days cultivation (initial pH 7.0). (j) Supplemented with phosphate buffer; (I) without buffer.

in the medium was also increased. The growth and decolorization of A. hydrophila were inhibited at lower pH in the medium. To confirm that glucose inhibited the decolorization activity and the cell growth was due mainly to the lower pH that was, in part, caused by the consumed glucose or converted organic acids, phosphate buffer was added into the medium to provide pH control during growth and dye decolorization (Fig. 7). The pH of the culture supplemented with phosphate buffer dropped much less than that without buffer because the phosphate buffer was proved to provide a good pH control as well as high decolorization activity and cell growth of A. hydrophila . The results clearly show that the inhibition of cell growth and bacterial decolorization of azo dye by glucose was attributed to the reduced pH in the surrounding medium through a

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biological conversion to organic acids. This demonstrates the importance of pH control to decolorization if some very biodegradable carbon sources were present in dye wastewater. Many aspects of the mechanisms involved in the inhibition of decolorization by glucose are still scarcely known.

4. Conclusions The results indicate that utilization of A. hydrophila was suitable for the decolorization of dyes (RED RBN and Remazol Black B) in the presence of a nitrogen source such as yeast extract. Certainly, the use of yeast extract as a nitrogen source for cell growth would be of low economic efficiency in the application of industrial treatment plant. In order to enhance process efficiency, the search for cheaper supplementary nitrogen sources would be essential in future works. In contrast to nitrogen sources, glucose showed inhibitory effects on the cell growth and the decolorization activity. Additionally, to ensure an effective azo dye decolorization with A. hydrophila required a rigorous control of the DO concentration ( B/ 0.45 mg l(1) in the biological process. High dye concentrations ( /3000 mg l(1) might have a toxic effect on the isolate. This strain could also decolorize synthetic effluent containing a mixture of different dyes. That is applicable to a wide variety of individual dyes and mixture of dyes.

Acknowledgements The authors acknowledge the nancial support of National Science Council of Republic of China under Grant No. NSC-89-2211-E-007-005.

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