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Decolorization of Reactive Black 5 by a newly isolated bacterium Bacillus sp. YZU1
International Biodeterioration & Biodegradation 76 (2013) 41e48
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Decolorization of Reactive Black 5 by a newly isolated bacterium Bacillus sp. YZU1 Z.W. Wang a, b, J.S. Liang b, *, Y. Liang a, c, * a
Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University, Kowloon, Hong Kong SAR, PR China College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, Jiangsu, PR China c Centre for Food Safety and Environmental Technology, Guangzhou Institutes of Advanced Technology, Chinese Academy of Sciences, Guangzhou 511458, China b
a b s t r a c t Keywords: Biodegradation Decolorization Reactive Black 5 Bacillus
A bacterial strain, YZU1, with remarkable ability to decolorize Reactive Black 5 (RB-5), was isolated from soil samples collected around a textile factory. Phenotypic and phylogenetic analyses of the 16S rDNA sequence indicated that YZU1 belonged to Bacillus sp. Bacillus sp. YZU1 showed great capability to decolorize various reactive textile dyes, including azo dye. Static conditions with pH 7.0 and 40 C were considered to be optimum for decolorizing RB-5. Bacillus sp. YZU1 grew well in medium containing high concentration of dye (100 mg/l), resulting in approximately 95% decolorization in 120 h, and could tolerate up to 500 mg/1 of RB-5. Enzyme assays demonstrated that Bacillus sp. YZU1 possessed azoreductase and played the most important role in decolorization, while a small percentage of decolorization occurred via passive surface adsorption. High biodegradation extent under a mild condition suggested that Bacillus sp. YZU1 had great potential to be applied in dye effluent treatment. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Synthetic dyes are essential for textile, paper, pharmaceutical, cosmetics and food industries. Over 800,000 tons/year of more than 100,000 dyes are produced worldwide (Husain, 2006). The production in China accounts for 40% of the world total, increasing at a rate of more than 30% annually (Fan et al., 2008). Azo dyes account for up to 70% of dyestuffs applied in textile processing, due to the ease and cost-effectiveness in their synthesis, stability and availability of variety of colors compared to natural dyes (Wang et al., 2008; Saratale et al., 2009). One major concern is that at least 10e15% of the used dyes are discharged with wastewater (Robinson et al., 2001), and dyes are generally resistant to fading on exposure to light, water and many chemicals due to their chemical structure. The recalcitrant dyes are not toxic themselves, but biodegradation of dyes particularly azo dyes may generate colorless but carcinogenic compounds such as aromatic amines, which may adversely influence human and environmental health (Husain, 2006). Recent studies also showed that chlorination of azo dyes generates mutagenic disinfection byproducts, which may contaminate drinking water (Oliveira et al., 2006). Additionally, dyes with trace amount (10e15 mg/L) are highly visible, affecting water recreational value, light penetration in water and as a consequence reduced photosynthesis and dissolved oxygen (Dafale et al., 2010). * Corresponding authors. Tel.: þ852 3411 7751; fax: þ852 3411 7743. E-mail addresses: [email protected] (J.S. Liang), [email protected] (Y. Liang). 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2012.06.023
Physicochemical methods, such as coagulation, adsorption, membrane filtration, electro- and photo-chemical removal, have been used for the treatment of dye containing wastewater, but large amounts of sludge are generated and very expensive (Husain, 2006). In contrast, biological methods using bacteria and fungi have been shown to be efficient and more cost-effective (Kuhad et al., 2004). Aerobic removal of azo dyes by bacteria and fungi, particularly white rot fungi, is mostly mediated by a variety of oxidative enzymes such as peroxidases, Mnþ peroxidases, laccases and lignin peroxidases, and complete removal can be achieved (Kuhad et al., 2004; Husain, 2006). Under anaerobic conditions, the color removal is effective mostly higher than 70% removal, and the processes generally are driven by bacterial azoreductases, leading to the cleavage of dyes’ azo linages and formation of aromatic amines, while the aromatic amines can be degraded in a consequent aerobic treatment (Van der Zee and Villaverde, 2005). Although substantial research has been conducted on the decolorization of dye by bacteria, isolation of new especially indigenous microorganisms capable of degrading various dyes with high effectiveness has always been the focus. Upon in-depth characterization of degradation biologically and chemically, mechanisms of the processes will be clarified. The information will help promote the relevant application. In this study, we attempted to isolate indigenous bacteria from local dye wastewater treatment plant. Following characterizing the bacteria, we hope that the bacteria would be utilized in wastewater treatment.
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2. Materials and methods
Decolorization percentage ð%Þ ¼
2.1. Azo dyes Reactive Black 5 (C.I., 55% dye, SigmaeAldrich, RB-5), a commonly used azo dye, was chosen for the screening of degrading bacteria. Other reactive dyes with different structures, including C.I. Methyl Red, C.I. Methyl Orange, C.I. Scarlet Red, C.I. Reactive Red M5B, C.I. Reactive Yellow 17, C.I Cibacron Brilliant Yellow 3G-P, C.I. Reactive Blue 171, C.I. Brilliant Blue G, C.I. Trypan Blue, C.I. Evans Blue, were chosen as structurally different dyes to examine decolorizing capability of the isolated bacterium. 2.2. Isolation and identification of dye degrading bacteria Nutrient broth containing dye RB-5 (100 mg/l) was inoculated with 10% (w/v) of a soil sample collected from a textile processing factory around Suzhou city in China. The flask was incubated at 30 C statically for 48 h. Then, 1.0 ml of the culture was diluted and 100 ml of the diluted culture were spread-plated on the nutrient agar plate containing 100 mg/l RB-5. Colonies on the agar showing clear zone around were isolated and acclimated to increasing concentrations of RB-5 in the broth (from 25, 50 to 100 mg/l). Eventually, one bacterial strain with the highest RB-5 decolorizing ability was preserved at 70 C in the broth (with 15% glycerol) before further identification. The isolated bacterial cells were recovered and cultured in nutrient broth, and harvested by centrifugation (8000 g, 10 min). The cells were then subjected to sequential digestion by lysozyme (2.5 mg/ml, 37 C, 1 h) and proteinase K (200 mg/ml in 1% SDS, 55 C for 1 h), followed by incubation in 1% CTAB and 0.7 M NaCl at 65 C for 15 min. DNA extracted by phenol/chloroform was recovered by ethanol precipitation and then dissolved in ddH2O. The 16S rDNA gene was amplified by PCR in a 25 ml reaction system using primers 27F and 1492R (DeLong, 1992). The conditions were: 1U Ex Taq Buffer, 0.2 mM of each dNTP, 0.2 mM of each primer and 1U Ex Taq polymerase (Takara). An initial denaturing period of 15 min was followed by 30 cycles at 94 C for 30 s, 55 C for 30 s, 72 C for 1.5 min. The final extension (72 C) time was 5 min. The PCR products were sequenced by Generay Biotech (Shanghai) Co. Ltd. (China). The sequence was uploaded in NCBI to be identified by BLAST search. Physiological studies were also conducted according to procedures outlined in Bergey’s Manual of Determinative Bacteriology (Staley et al., 2001). 2.3. Induction of mutagenesis and selection of mutants Ten ml of the bacterial cell suspension (107 CFU/ml) in saline water (0.9% NaCl) were transferred in an open petri-dish and exposed to a germicidal UV lamp. The distance between surface of the cell suspension and the lamp was 40 cm, while the time of exposure varied from 1 to 5 min. Then, 1 ml of the UV radiation exposed cell suspension was transferred to 9 ml saline water. After a series of dilution, the cells were plated on nutrient agar containing 50 mg/l RB-5 and incubated at 37 C for 24 h. Colonies formed were counted and each colony was inoculated into one tube containing nutrient broth respectively and incubated overnight. Using the wild strain as the control, a positive mutant (strain YZU1) with the highest decolorization percentage (%), upon incubation in nutrient broth containing 100 mg/l of RB-5, was selected and preserved for further dye degradation experiments. The decolorization percentage (%) was determined by measuring the absorbance of the culture supernatant at 597 nm using a UVeVis spectrophotometer (GE), calculated as:
OD1 ODt 100 OD1
(1)
where OD1 referred to the initial absorbance, ODt referred to the absorbance after incubation, and t referred to the incubation time. 2.4. Decolorization studies of RB-5 in nutrient broth For all of the decolorization studies, each experiment was performed in triplicate. A cell suspension of YZU1 (0.15 ml) was inoculated into an 8 ml nutrient broth containing 100 mg/l RB-5, while the same amount of autoclaved bacterial cell suspension (dead cells) was also transferred to another 8 ml nutrient broth as a control. The cultures were incubated at 37 C for 120 h, and samples from both cultures were scanned from 400e800 nm using a UVeVis spectrophotometer (GE). Variation in decolorization was then calculated and recorded. Effects of environmental factors, including temperature (varied between 20e45 C with 5 C interval), dye concentration (25, 50, 100, 150, 200, 300, 400 and 500 mg/l), initial pH (5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), dissolved oxygen (static and shaking culture) and salt concentration (0, 0.5, 1, 2, 4, 6, 8, 10 g/l), were investigated as well on the decolorization effectiveness. Briefly, the bacterium was firstly cultured in a nutrient broth at 200 rpm, 37 C overnight. The bacterial cells were then transferred into 8 ml nutrient broth. The cultures were incubated at 37 C for 24 h, and the extent of decolorization was calculated and recorded. Influence of initial substrate concentration on degradation rate was analyzed using Monod model:
V ¼
Vmax $S S þ S2 =Ki þ Ks
(2)
where Vmax was the maximum decolorization rate (mg/l h), S was the concentration of substrate(mg/l), Ks was the substrate saturation, Ki was the substrate inhibition constant. 2.5. Azoreductase assay of bacterial crude enzyme extract Bacterial cells were harvested (8000 rpm, 10 min), washed by 10 mM buffer (pH 7) and suspended in an equal volume of 50 mM phosphate buffer (pH 7). The cells were then disrupted by cold sonication (15 min, 70% amplitude). After cell debris and undisrupted cells were removed by centrifugation (15,000 g, 20 min, 4 C) (Maier et al., 2004), the supernatant was then freeze-dried and preserved in a desiccators as the bacterial crude enzyme extract. The crude extract was dissolved in 50 mM phosphate buffer before enzyme assays. The assay was carried out in a cuvette (path length ¼ 1 cm, 1 ml). Four hundred mL 50 mM phosphate buffer was mixed with varied concentrations of crude extract and 200 ml of RB-5 (500 mg/l, resulting in a final concentration of 100.8 mM dye). The reaction started by adding NADH, and the mixture was monitored photometrically at 597 nm. The slope of the initial linear decrease in absorption was for calculating the azoreductase activity based on the molar absorption coefficient of RB-5 (3 ¼ 21.329 mmol1 cm1). One unit of enzyme activity was defined as the amount of enzyme required to decolorize 1 nmol of dye per min under the assay conditions. Enzyme activities among different levels of protein in the crude extract (0.3, 0.6, 0.9, 1.2, 1.5 mg/ml; NADH 2 mM), as well as among varied NADH concentrations (0.5, 1.0, 1.5, 2.0, 2.5 mM), were determined. All the assays were conducted in triplicate.
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2.6. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with LSD multiple comparisons. 3. Results and discussion 3.1. Isolation and identification of decolorizing bacteria Upon initial screening and mutation induction, a strain of bacterium YZU1 having remarkable decolorizing ability on RB-5 was isolated. Significant decolorization (60%) was observed after 24 h incubation, and a maximum value (95%) was achieved after incubation for 120 h (Fig. 1). Colony of the bacterial isolate was circular or nearly circular, flat, smooth and white. The cells were gram-positive rod-shaped (short). Sequence analysis of 16S rDNA and comparison with other related bacteria in the GenBank database showed that the isolated bacterium had the highest similarity (99%) with the species of Bacillus cereus ZQN6 (accession number: GU384236.1). Based on the phenotypic and phylogenetic analyses, the bacterial strain YZU1 was identified as Bacillus sp. strain YZU1.
Fig. 2. Vis spectra of Reactive Black 5 decolorization at different bacterial treatment time.
3.2. Physical adsorption vs biodegradation In the cultures added with heat-killed bacterial cells, only 3.10% decolorization was observed after 120 h incubation (Fig. 1), probably due to the adsorption by heat-killed bacterial cells. Colored cell pellets at the bottom of the culture was also observed in this study. In contrast, 95.0% decolorization was achieved in 120 h, in the culture inoculated with live bacterial cells (Fig. 1), and the cells were not pigmented. Additionally, vis spectral scan (400e800 nm) data of the supernatants showed that the maximum absorbance wavelength was blue shifted in control cultures (Fig. 2). This indicated that molecular structure change in RB-5 occurred by the bacterial biodegradation. In fact, both ways of decolorization of dyes, adsorption and biodegradation, were expected in the process (Aravindhan et al., 2007; Kumar et al., 2007). Indeed extracellular polysaccharides of Bacillus subtilis could be utilized as biosorbents in removal of dye from industrial effluents (Binupriya et al., 2010). Obviously, in this study biodegradation played a more important
role, as the major peak decreased accompanied by a shift to shorter wavelengths (blue) (Yu and Wen, 2005). This suggested that azo bonds cleaved during the reaction leading to damage/break-up in the primary chromophore, most likely mediated by azoreductase (Oturkar et al., 2011). Enzyme assay result showed that bacterial crude extract contained NADH dependent azoreductase (7.50 U/mg) (Fig. 3a). With the presence of a same level of NADH, decolorization depended on the amount of crude extract used, the more crude extract, the faster of the reaction. Furthermore, it showed that decolorization of RB-5 by the crude extract was slow before 60 s (Fig. 3a), but more rapid right after. On the other hand, the azoreductase activity increased as NADH concentration increased, up to about 1.5 mM (Fig. 3b). Above this level, further addition of NADH did not accelerate the reaction. Characteristics of azoreductase in the crude extract in this study were in consistent with those reported by Maier et al. (2004) on NADH dependent azoreductase derived from Bacillus, that anaerobic condition was required to initiate azoreductase activity. 3.3. Effect of temperature Bacillus sp. strain YZU1 showed high decolorizing capability in the temperature range of 35 Ce45 C (Fig. 4-1). Although the percentage of decolorization after 24 h was found to be comparatively low at 20 C, it increased to a greater level at 40 C. Decolorizing activity was substantially inhibited with other temperatures, mostly likely because of deactivation of enzymes responsible for decolorization or loss of cell viablility (Panswad and Luangdilok, 2000; Cetin and Donmez, 2006). 3.4. Effect of pH
Fig. 1. Decolorization of Reactive Black 5 by live and dead cells (heat-killed) of Bacillus sp. YZU1.
The best decolorization was achieved at pH 7.0, with 60% decolorization after 24 h (Fig. 4-2). This could be due to the fact that the optimum pH for the growth of Bacillus sp. YZU1 was neutral. Decolorization was observed at pH 6.0e9.0 after 24 h, but was significantly lower at relative strongly acidic (pH 4.0 and 5.0) conditions. pH is an important parameter of microbial growth and azo dye degradation, and the optimal levels of pH for color removal were often between 6.0 and 10.0 (Chen et al., 2003; Guo et al., 2007; Kilic et al., 2007). In fact, studying pH tolerance of
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Fig.3. Decolorization of Reactive Black 5 with Bacillus sp. YZU1 cell extract. (a) Different amounts of cell extract and 2.0 mM NADH. (b) Different concentrations of NADH and 1.2 mg/ ml cell extract (100.8 mM Reactive Black 5, 50 mM phosphate buffer, pH 7.0).
decolorizing bacteria is crucial because alkaline conditions facilitated binding between azo dyes and fibers (Aksu, 2003), and pH of the dye wastewater discharged from textile factory usually ranged between 8 and 9. This demonstrated that this strain worked under a wide range of pH (6e9), making it as a promising strain for practical bio-treatment of dye wastewater.
anaerobe. The bacterial growth was promoted with the presence of O2 but the yield of dye decolorization/degradation related enzyme was suppressed. The result supported previous reports that aerobic condition inhibited bacterial biodegradation of dyes (Khehra et al., 2005; Moosvi et al., 2005). Instead of azo groups in the dyes, oxygen was more preferred as an electron acceptor, resulting in an inhibition of the dye reduction process (Pearce et al., 2003).
3.5. Effect of O2 3.6. Effect of salt concentration Decolorization of RB-5 was about 60% in static condition, and significantly decreased in aerobic condition when the speed of shaker increased from 100 rpm to 250 rpm. However, the growth of Bacillus sp. YZU1 was the greatest under aerobic condition (Fig. 43). This indicated that Bacillus sp. YZU1 was a facultative
Salt level greatly influenced decolorization (Fig. 4-4). Decolorization after 24 h reached >40% when salt concentration is less than 2 g/l, significantly (P < 0.05) higher than that in other salt concentrations (<30% decolorization). Moreover, decolorization started to
Fig.4. Effect of (1) temperature, (2) pH, (3) dissolved oxygen and (4) salt concentration on decolorization after 24 h.
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decrease when the salt concentration exceeded 0.5 g/l. This is due to the fact that high salt concentration affected osmotic pressure in Bacillus sp. YZU1, inhibited bacterial growth and even caused cell death. Similar salt effect on bacterial decolorization of azo dyes was also observed in other studies (Kolekar et al., 2008). Nevertheless, currently as high salt level in dye waste has been unavoidable, isolating and investigating salt-tolerant microbes have become one centre of studies (Gopinath et al., 2009; Anjaneya et al., 2011). Our future research will also be led towards this direction.
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Table 1 Parameter and equation of kinetics of decolorization of Reactive Black 5 by Bacillus sp. YZU1. Concentration (mg/l)
Equation of kinetic
K0 (h1)
Correlation coefficient (r2)
50 100 150 200 300 400 500
y ¼ 0.0357x þ 3.5845 y ¼ 0.0288x þ 4.4713 y ¼ 0.0294x þ 4.9698 y ¼ 0.0221x þ 5.1953 y ¼ 0.0245x þ 5.7298 y ¼ 0.0156x þ 6.0831 y ¼ 0.0147x þ 6.3383
0.0357 0.0288 0.0294 0.0221 0.0245 0.0156 0.0147
0.9863 0.9904 0.9686 0.9423 0.9300 0.9732 0.9767
3.7. Effect of initial dye concentration With the same culture time decolorization reduced with increase in initial dye concentration (Fig. 5). For example, in the dye culture of 50 mg/l, time required to reach a 90% decolorization was 60 h, whereas in that of 300 mg/l the time required doubled (120 h). In general, the decolorizaiton was well described by a 1st order kinetic model (Table 1). Furthermore, data also revealed that decolorizaiton was faster at the initial stage (e.g. as early as 6 h), and a calculation of decolorization rate was conducted. With the use of Origin 8.0 software, we fitted curve of kinetics Eq. (2) of decolorization for Haldane model. The Haldane model was:
V ¼
7:9855$S S þ S2=615:2196 þ 130:7367
(3)
Fitting situation of predicted and experiment value was showed in Fig. 6. The correlation coefficient (r2) was 0.9817, well described the dynamic characteristics of decolorization rate in the initial stage (0e6 h) with diverse dye concentrations. The pattern of decolorization rate at initial stage suggested that 300 mg/l was an optimum concentration, allowing highest decolorization occur. With the increase in dye concentration toxic effect of dye/dye metabolites became dominant, leading to inhibition in decolorization. Similar patterns were also observed in the past (Khehra et al., 2005; Kalme et al., 2007). Using the above mentioned model, an optimal concentration (Sopt) was calculated:
Sopt ¼
pffiffiffiffiffiffiffiffiffiffiffiffi Ki $Ks
(4)
In this study, the Sopt value for Bacillus sp. YZU1 was 283.6 mg/l, with a greatest rate of decolorization (Vopt) of 4.1549 mgl1h1:
Fig. 5. Effect of initial dye concentration on decolorization.
Vopt ¼
Vmax sffiffiffiffiffi Ks 1þ2 Ki
(5)
Compared with decolorization of RB-5 using Bacillus sp. reported in the literature, the strain YZU1 reached >84% decolorization in this study, higher than the maximum level (80%) reported by Modi et al. (2010) with their isolated Bacillus cereus. However, at 12 h, YZU1 only achieved 17% decolorization, less effective than a strain of Bacillus sp. AK1 (31%) isolated by Anjaneya et al. (2011). Nonetheless, maximum decolorization of Bacillus sp. YZU1 in the present study reached 95%, comparable to other methods such as physical adsorption (45%, Eren and Acar, 2006), photocatalytic degradation (98%, You et al., 2010), electrochemical oxidation (95%, CeroonRivera et al., 2004) and microbial degradation (90% using Enterobacter sp. EC3, Wang et al., 2009). 3.8. Decolorization of various textile dyes Azo dyes used in this study were listed in Table 2. Decolorization of various azo dyes after 120 h was listed in Table 3. Among the 10 structurally different azo dyes (100 mg/l), a maximum decolorization of 99.14% was recorded in Methyl Red after 10 h, and for Methyl Orange and Reactive Yellow, the values were respectively 96.08% and 97.88% after 72 h. Decolorization of the above three azo dyes by dead cells were 1.68%, 2.31% and 4.19%, respectively. This variation might be due to the structural difference in the dyes (Kalyani et al.,
Fig. 6. Fitted curve of kinetics equation of decolorization model: Bacillus sp. YZU1.
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Table 2 Characteristics of main azo dyes used in this study. Azo dye
Molecular structure
Wavelength (nm)
Methyl red
430
Methyl orange
464
Reactive yellow
435
Scarlet red
525
Reactive red M5B
513
Brilliant blue
595
Reactive blue
598
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Table 2 (continued ) Azo dye
Molecular structure
Wavelength (nm)
607
Trypan blue
611
Evans blue
428
Cibacron brilliant yellow 3G-P
Table 3 Decolorization of various textile dyes by Bacillus sp. YZU1. Dyes
a
Reactive red M5B Scarlet red Brilliant blue G Reactive blue 4 Trypan blue Cibacron brilliant yellow 3G-P a b c
Decolorization (%) 87.73% 87.62% 87.54% 49.89% 46.29% 16.71%
b
Acknowledgments Decolorization (%)
c
11.28% 8.48% 3.23% 5.73% 12.12% 8.66%
100 mg/l dye concentration. Decolorization after 120 h by live cells. Decolorization after 120 h by dead cells.
2008). Nevertheless, decolorization of Bacillus sp. YZU1 against the reactive dyes tested in this study (except for Cibacron Brilliant yellow 3G-P) was >45%, and more than 70% was achieved in treating Scarlet Red, Reactive Red M5B, Brilliant Blue and Evans Blue. This suggested that this strain showed great potential in decolorizing complex dye effluent containing various reactive dyes. However, it also appears that more research is needed, especially in optimizing treating conditions, in the future. 4. Conclusions In this study, an effective RB-5 decolorizing bacterial strain, Bacillus sp. YZU1, was isolated. Bacillus sp. YZU1 showed azoreductase activity in the degradation, not simply a physical surface adsorption. In degradation, Bacillus sp. YZU1 simply needs a mild condition, and it showed remarkable tolerance to high concentrations of Reactive Black 5 (500 mg/l). Bearing high decolorizing activities against various reactive dyes commonly used in the textile industries, it is proposed that Bacillus sp. YZU1 had a practical application potential in the biotransformation of various dye effluents.
Financial support from the Faculty Research Grant, Hong Kong Baptist University (No. FRG2/09-10/072) is gratefully acknowledged.
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Moosvi, S., Keharia, H., Madamwar, D., 2005. Decolourization of textile dye Reactive Violet 5 by a newly isolated bacterial consortium RVM 11.1. World Journal of Microbiology & Biotechnology 21, 667e672. Oliveira, D.P., Carneiro, P.A., Rech, C.M., Zanoni, M.V., Claxton, L.D., 2006. Mutagenic compounds generated from the chlorination of disperse azo-dyes and their presence in drinking water. Environmental Science & Technology 40, 6682e6689. Oturkar, C.C., Nemade, H.N., Mulik, P.M., Patole, M.S., Hawaldar, R.R., 2011. Mechanistic investigation of decolorization and degradation of Reactive Red 120 by Bacillus lentus BI377. Bioresource Technology 102, 758e764. Panswad, T., Luangdilok, W., 2000. Decolorization of reactive dyes with different molecular structures under different environmental conditions. Water Research 34, 4177e4184. Pearce, C.I., Lloyd, J.R., Guthrie, J.T., 2003. The removal of colour from textile wastewater using whole bacterial cells: a review. Dyes and Pigments 58, 179e196. Robinson, T., McMullan, G., Marchant, G., Nigam, P., 2001. Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresource Technology 77, 247e255. Saratale, R.G., Saratale, G.D., Kalyani, D.C., Chang, J.S., Govindwar, S.P., 2009. Enhanced decolorization and biodegradation of textile azo dye Scarlet R by using developed microbial consortium-GR. Bioresource Technology 100, 2493e2500. Staley, J.R., Boone, A.R., Brenner, D.J., Vos, P.D., Garrity, G.M., Goodfellow, M., Krieg, N.R., Rainey, F.A., Schleifer, K.H., 2001. Bergey’s Manual of Determinative Bacteriology, second ed. Springer. Van der Zee, F.P., Villaverde, S., 2005. Combined anaerobic-aerobic treatment of azo dyes e a short review of bioreactor studies. Water Research 39 (8), 1425e1440. Wang, Y., Zhao, D., Ma, W., Chen, C., Zhao, J., 2008. Enhanced sonocatalytic degradation of azo dyes by Au/TiO2. Environmental Science & Technology 42 (16), 6173e6178. Wang, H., Zheng, X.W., Sua, J.Q., Tian, Y., Xiong, X.J., Zheng, T.L., 2009. Biological decolorization of the reactive dyes Reactive Black 5 by a novel isolated bacterial strain Enterobacter sp. EC3. Journal of Hazardous Materials 171, 654e659. You, S.J., Damodar, A.R., Hou, S.C., 2010. Degradation of Reactive Black 5 dye using anaerobic/aerobic membrane bioreactor (MBR) and photochemical membrane reactor. Journal of Hazardous Materials 177, 1112e1118. Yu, Z.S., Wen, X.H., 2005. Screening and identification of yeasts for decolorizing synthetic dyes in industrial wastewater. International Biodeterioration & Biodegradation 56, 109e114.
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Decolorization of Reactive Black 5 by a newly isolated bacterium Bacillus sp. YZU1 Z.W. Wang a, b, J.S. Liang b, *, Y. Liang a, c, * a
Croucher Institute for Environmental Sciences, Department of Biology, Hong Kong Baptist University, Kowloon, Hong Kong SAR, PR China College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, Jiangsu, PR China c Centre for Food Safety and Environmental Technology, Guangzhou Institutes of Advanced Technology, Chinese Academy of Sciences, Guangzhou 511458, China b
a b s t r a c t Keywords: Biodegradation Decolorization Reactive Black 5 Bacillus
A bacterial strain, YZU1, with remarkable ability to decolorize Reactive Black 5 (RB-5), was isolated from soil samples collected around a textile factory. Phenotypic and phylogenetic analyses of the 16S rDNA sequence indicated that YZU1 belonged to Bacillus sp. Bacillus sp. YZU1 showed great capability to decolorize various reactive textile dyes, including azo dye. Static conditions with pH 7.0 and 40 C were considered to be optimum for decolorizing RB-5. Bacillus sp. YZU1 grew well in medium containing high concentration of dye (100 mg/l), resulting in approximately 95% decolorization in 120 h, and could tolerate up to 500 mg/1 of RB-5. Enzyme assays demonstrated that Bacillus sp. YZU1 possessed azoreductase and played the most important role in decolorization, while a small percentage of decolorization occurred via passive surface adsorption. High biodegradation extent under a mild condition suggested that Bacillus sp. YZU1 had great potential to be applied in dye effluent treatment. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Synthetic dyes are essential for textile, paper, pharmaceutical, cosmetics and food industries. Over 800,000 tons/year of more than 100,000 dyes are produced worldwide (Husain, 2006). The production in China accounts for 40% of the world total, increasing at a rate of more than 30% annually (Fan et al., 2008). Azo dyes account for up to 70% of dyestuffs applied in textile processing, due to the ease and cost-effectiveness in their synthesis, stability and availability of variety of colors compared to natural dyes (Wang et al., 2008; Saratale et al., 2009). One major concern is that at least 10e15% of the used dyes are discharged with wastewater (Robinson et al., 2001), and dyes are generally resistant to fading on exposure to light, water and many chemicals due to their chemical structure. The recalcitrant dyes are not toxic themselves, but biodegradation of dyes particularly azo dyes may generate colorless but carcinogenic compounds such as aromatic amines, which may adversely influence human and environmental health (Husain, 2006). Recent studies also showed that chlorination of azo dyes generates mutagenic disinfection byproducts, which may contaminate drinking water (Oliveira et al., 2006). Additionally, dyes with trace amount (10e15 mg/L) are highly visible, affecting water recreational value, light penetration in water and as a consequence reduced photosynthesis and dissolved oxygen (Dafale et al., 2010). * Corresponding authors. Tel.: þ852 3411 7751; fax: þ852 3411 7743. E-mail addresses: [email protected] (J.S. Liang), [email protected] (Y. Liang). 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2012.06.023
Physicochemical methods, such as coagulation, adsorption, membrane filtration, electro- and photo-chemical removal, have been used for the treatment of dye containing wastewater, but large amounts of sludge are generated and very expensive (Husain, 2006). In contrast, biological methods using bacteria and fungi have been shown to be efficient and more cost-effective (Kuhad et al., 2004). Aerobic removal of azo dyes by bacteria and fungi, particularly white rot fungi, is mostly mediated by a variety of oxidative enzymes such as peroxidases, Mnþ peroxidases, laccases and lignin peroxidases, and complete removal can be achieved (Kuhad et al., 2004; Husain, 2006). Under anaerobic conditions, the color removal is effective mostly higher than 70% removal, and the processes generally are driven by bacterial azoreductases, leading to the cleavage of dyes’ azo linages and formation of aromatic amines, while the aromatic amines can be degraded in a consequent aerobic treatment (Van der Zee and Villaverde, 2005). Although substantial research has been conducted on the decolorization of dye by bacteria, isolation of new especially indigenous microorganisms capable of degrading various dyes with high effectiveness has always been the focus. Upon in-depth characterization of degradation biologically and chemically, mechanisms of the processes will be clarified. The information will help promote the relevant application. In this study, we attempted to isolate indigenous bacteria from local dye wastewater treatment plant. Following characterizing the bacteria, we hope that the bacteria would be utilized in wastewater treatment.
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2. Materials and methods
Decolorization percentage ð%Þ ¼
2.1. Azo dyes Reactive Black 5 (C.I., 55% dye, SigmaeAldrich, RB-5), a commonly used azo dye, was chosen for the screening of degrading bacteria. Other reactive dyes with different structures, including C.I. Methyl Red, C.I. Methyl Orange, C.I. Scarlet Red, C.I. Reactive Red M5B, C.I. Reactive Yellow 17, C.I Cibacron Brilliant Yellow 3G-P, C.I. Reactive Blue 171, C.I. Brilliant Blue G, C.I. Trypan Blue, C.I. Evans Blue, were chosen as structurally different dyes to examine decolorizing capability of the isolated bacterium. 2.2. Isolation and identification of dye degrading bacteria Nutrient broth containing dye RB-5 (100 mg/l) was inoculated with 10% (w/v) of a soil sample collected from a textile processing factory around Suzhou city in China. The flask was incubated at 30 C statically for 48 h. Then, 1.0 ml of the culture was diluted and 100 ml of the diluted culture were spread-plated on the nutrient agar plate containing 100 mg/l RB-5. Colonies on the agar showing clear zone around were isolated and acclimated to increasing concentrations of RB-5 in the broth (from 25, 50 to 100 mg/l). Eventually, one bacterial strain with the highest RB-5 decolorizing ability was preserved at 70 C in the broth (with 15% glycerol) before further identification. The isolated bacterial cells were recovered and cultured in nutrient broth, and harvested by centrifugation (8000 g, 10 min). The cells were then subjected to sequential digestion by lysozyme (2.5 mg/ml, 37 C, 1 h) and proteinase K (200 mg/ml in 1% SDS, 55 C for 1 h), followed by incubation in 1% CTAB and 0.7 M NaCl at 65 C for 15 min. DNA extracted by phenol/chloroform was recovered by ethanol precipitation and then dissolved in ddH2O. The 16S rDNA gene was amplified by PCR in a 25 ml reaction system using primers 27F and 1492R (DeLong, 1992). The conditions were: 1U Ex Taq Buffer, 0.2 mM of each dNTP, 0.2 mM of each primer and 1U Ex Taq polymerase (Takara). An initial denaturing period of 15 min was followed by 30 cycles at 94 C for 30 s, 55 C for 30 s, 72 C for 1.5 min. The final extension (72 C) time was 5 min. The PCR products were sequenced by Generay Biotech (Shanghai) Co. Ltd. (China). The sequence was uploaded in NCBI to be identified by BLAST search. Physiological studies were also conducted according to procedures outlined in Bergey’s Manual of Determinative Bacteriology (Staley et al., 2001). 2.3. Induction of mutagenesis and selection of mutants Ten ml of the bacterial cell suspension (107 CFU/ml) in saline water (0.9% NaCl) were transferred in an open petri-dish and exposed to a germicidal UV lamp. The distance between surface of the cell suspension and the lamp was 40 cm, while the time of exposure varied from 1 to 5 min. Then, 1 ml of the UV radiation exposed cell suspension was transferred to 9 ml saline water. After a series of dilution, the cells were plated on nutrient agar containing 50 mg/l RB-5 and incubated at 37 C for 24 h. Colonies formed were counted and each colony was inoculated into one tube containing nutrient broth respectively and incubated overnight. Using the wild strain as the control, a positive mutant (strain YZU1) with the highest decolorization percentage (%), upon incubation in nutrient broth containing 100 mg/l of RB-5, was selected and preserved for further dye degradation experiments. The decolorization percentage (%) was determined by measuring the absorbance of the culture supernatant at 597 nm using a UVeVis spectrophotometer (GE), calculated as:
OD1 ODt 100 OD1
(1)
where OD1 referred to the initial absorbance, ODt referred to the absorbance after incubation, and t referred to the incubation time. 2.4. Decolorization studies of RB-5 in nutrient broth For all of the decolorization studies, each experiment was performed in triplicate. A cell suspension of YZU1 (0.15 ml) was inoculated into an 8 ml nutrient broth containing 100 mg/l RB-5, while the same amount of autoclaved bacterial cell suspension (dead cells) was also transferred to another 8 ml nutrient broth as a control. The cultures were incubated at 37 C for 120 h, and samples from both cultures were scanned from 400e800 nm using a UVeVis spectrophotometer (GE). Variation in decolorization was then calculated and recorded. Effects of environmental factors, including temperature (varied between 20e45 C with 5 C interval), dye concentration (25, 50, 100, 150, 200, 300, 400 and 500 mg/l), initial pH (5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), dissolved oxygen (static and shaking culture) and salt concentration (0, 0.5, 1, 2, 4, 6, 8, 10 g/l), were investigated as well on the decolorization effectiveness. Briefly, the bacterium was firstly cultured in a nutrient broth at 200 rpm, 37 C overnight. The bacterial cells were then transferred into 8 ml nutrient broth. The cultures were incubated at 37 C for 24 h, and the extent of decolorization was calculated and recorded. Influence of initial substrate concentration on degradation rate was analyzed using Monod model:
V ¼
Vmax $S S þ S2 =Ki þ Ks
(2)
where Vmax was the maximum decolorization rate (mg/l h), S was the concentration of substrate(mg/l), Ks was the substrate saturation, Ki was the substrate inhibition constant. 2.5. Azoreductase assay of bacterial crude enzyme extract Bacterial cells were harvested (8000 rpm, 10 min), washed by 10 mM buffer (pH 7) and suspended in an equal volume of 50 mM phosphate buffer (pH 7). The cells were then disrupted by cold sonication (15 min, 70% amplitude). After cell debris and undisrupted cells were removed by centrifugation (15,000 g, 20 min, 4 C) (Maier et al., 2004), the supernatant was then freeze-dried and preserved in a desiccators as the bacterial crude enzyme extract. The crude extract was dissolved in 50 mM phosphate buffer before enzyme assays. The assay was carried out in a cuvette (path length ¼ 1 cm, 1 ml). Four hundred mL 50 mM phosphate buffer was mixed with varied concentrations of crude extract and 200 ml of RB-5 (500 mg/l, resulting in a final concentration of 100.8 mM dye). The reaction started by adding NADH, and the mixture was monitored photometrically at 597 nm. The slope of the initial linear decrease in absorption was for calculating the azoreductase activity based on the molar absorption coefficient of RB-5 (3 ¼ 21.329 mmol1 cm1). One unit of enzyme activity was defined as the amount of enzyme required to decolorize 1 nmol of dye per min under the assay conditions. Enzyme activities among different levels of protein in the crude extract (0.3, 0.6, 0.9, 1.2, 1.5 mg/ml; NADH 2 mM), as well as among varied NADH concentrations (0.5, 1.0, 1.5, 2.0, 2.5 mM), were determined. All the assays were conducted in triplicate.
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2.6. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with LSD multiple comparisons. 3. Results and discussion 3.1. Isolation and identification of decolorizing bacteria Upon initial screening and mutation induction, a strain of bacterium YZU1 having remarkable decolorizing ability on RB-5 was isolated. Significant decolorization (60%) was observed after 24 h incubation, and a maximum value (95%) was achieved after incubation for 120 h (Fig. 1). Colony of the bacterial isolate was circular or nearly circular, flat, smooth and white. The cells were gram-positive rod-shaped (short). Sequence analysis of 16S rDNA and comparison with other related bacteria in the GenBank database showed that the isolated bacterium had the highest similarity (99%) with the species of Bacillus cereus ZQN6 (accession number: GU384236.1). Based on the phenotypic and phylogenetic analyses, the bacterial strain YZU1 was identified as Bacillus sp. strain YZU1.
Fig. 2. Vis spectra of Reactive Black 5 decolorization at different bacterial treatment time.
3.2. Physical adsorption vs biodegradation In the cultures added with heat-killed bacterial cells, only 3.10% decolorization was observed after 120 h incubation (Fig. 1), probably due to the adsorption by heat-killed bacterial cells. Colored cell pellets at the bottom of the culture was also observed in this study. In contrast, 95.0% decolorization was achieved in 120 h, in the culture inoculated with live bacterial cells (Fig. 1), and the cells were not pigmented. Additionally, vis spectral scan (400e800 nm) data of the supernatants showed that the maximum absorbance wavelength was blue shifted in control cultures (Fig. 2). This indicated that molecular structure change in RB-5 occurred by the bacterial biodegradation. In fact, both ways of decolorization of dyes, adsorption and biodegradation, were expected in the process (Aravindhan et al., 2007; Kumar et al., 2007). Indeed extracellular polysaccharides of Bacillus subtilis could be utilized as biosorbents in removal of dye from industrial effluents (Binupriya et al., 2010). Obviously, in this study biodegradation played a more important
role, as the major peak decreased accompanied by a shift to shorter wavelengths (blue) (Yu and Wen, 2005). This suggested that azo bonds cleaved during the reaction leading to damage/break-up in the primary chromophore, most likely mediated by azoreductase (Oturkar et al., 2011). Enzyme assay result showed that bacterial crude extract contained NADH dependent azoreductase (7.50 U/mg) (Fig. 3a). With the presence of a same level of NADH, decolorization depended on the amount of crude extract used, the more crude extract, the faster of the reaction. Furthermore, it showed that decolorization of RB-5 by the crude extract was slow before 60 s (Fig. 3a), but more rapid right after. On the other hand, the azoreductase activity increased as NADH concentration increased, up to about 1.5 mM (Fig. 3b). Above this level, further addition of NADH did not accelerate the reaction. Characteristics of azoreductase in the crude extract in this study were in consistent with those reported by Maier et al. (2004) on NADH dependent azoreductase derived from Bacillus, that anaerobic condition was required to initiate azoreductase activity. 3.3. Effect of temperature Bacillus sp. strain YZU1 showed high decolorizing capability in the temperature range of 35 Ce45 C (Fig. 4-1). Although the percentage of decolorization after 24 h was found to be comparatively low at 20 C, it increased to a greater level at 40 C. Decolorizing activity was substantially inhibited with other temperatures, mostly likely because of deactivation of enzymes responsible for decolorization or loss of cell viablility (Panswad and Luangdilok, 2000; Cetin and Donmez, 2006). 3.4. Effect of pH
Fig. 1. Decolorization of Reactive Black 5 by live and dead cells (heat-killed) of Bacillus sp. YZU1.
The best decolorization was achieved at pH 7.0, with 60% decolorization after 24 h (Fig. 4-2). This could be due to the fact that the optimum pH for the growth of Bacillus sp. YZU1 was neutral. Decolorization was observed at pH 6.0e9.0 after 24 h, but was significantly lower at relative strongly acidic (pH 4.0 and 5.0) conditions. pH is an important parameter of microbial growth and azo dye degradation, and the optimal levels of pH for color removal were often between 6.0 and 10.0 (Chen et al., 2003; Guo et al., 2007; Kilic et al., 2007). In fact, studying pH tolerance of
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Fig.3. Decolorization of Reactive Black 5 with Bacillus sp. YZU1 cell extract. (a) Different amounts of cell extract and 2.0 mM NADH. (b) Different concentrations of NADH and 1.2 mg/ ml cell extract (100.8 mM Reactive Black 5, 50 mM phosphate buffer, pH 7.0).
decolorizing bacteria is crucial because alkaline conditions facilitated binding between azo dyes and fibers (Aksu, 2003), and pH of the dye wastewater discharged from textile factory usually ranged between 8 and 9. This demonstrated that this strain worked under a wide range of pH (6e9), making it as a promising strain for practical bio-treatment of dye wastewater.
anaerobe. The bacterial growth was promoted with the presence of O2 but the yield of dye decolorization/degradation related enzyme was suppressed. The result supported previous reports that aerobic condition inhibited bacterial biodegradation of dyes (Khehra et al., 2005; Moosvi et al., 2005). Instead of azo groups in the dyes, oxygen was more preferred as an electron acceptor, resulting in an inhibition of the dye reduction process (Pearce et al., 2003).
3.5. Effect of O2 3.6. Effect of salt concentration Decolorization of RB-5 was about 60% in static condition, and significantly decreased in aerobic condition when the speed of shaker increased from 100 rpm to 250 rpm. However, the growth of Bacillus sp. YZU1 was the greatest under aerobic condition (Fig. 43). This indicated that Bacillus sp. YZU1 was a facultative
Salt level greatly influenced decolorization (Fig. 4-4). Decolorization after 24 h reached >40% when salt concentration is less than 2 g/l, significantly (P < 0.05) higher than that in other salt concentrations (<30% decolorization). Moreover, decolorization started to
Fig.4. Effect of (1) temperature, (2) pH, (3) dissolved oxygen and (4) salt concentration on decolorization after 24 h.
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decrease when the salt concentration exceeded 0.5 g/l. This is due to the fact that high salt concentration affected osmotic pressure in Bacillus sp. YZU1, inhibited bacterial growth and even caused cell death. Similar salt effect on bacterial decolorization of azo dyes was also observed in other studies (Kolekar et al., 2008). Nevertheless, currently as high salt level in dye waste has been unavoidable, isolating and investigating salt-tolerant microbes have become one centre of studies (Gopinath et al., 2009; Anjaneya et al., 2011). Our future research will also be led towards this direction.
45
Table 1 Parameter and equation of kinetics of decolorization of Reactive Black 5 by Bacillus sp. YZU1. Concentration (mg/l)
Equation of kinetic
K0 (h1)
Correlation coefficient (r2)
50 100 150 200 300 400 500
y ¼ 0.0357x þ 3.5845 y ¼ 0.0288x þ 4.4713 y ¼ 0.0294x þ 4.9698 y ¼ 0.0221x þ 5.1953 y ¼ 0.0245x þ 5.7298 y ¼ 0.0156x þ 6.0831 y ¼ 0.0147x þ 6.3383
0.0357 0.0288 0.0294 0.0221 0.0245 0.0156 0.0147
0.9863 0.9904 0.9686 0.9423 0.9300 0.9732 0.9767
3.7. Effect of initial dye concentration With the same culture time decolorization reduced with increase in initial dye concentration (Fig. 5). For example, in the dye culture of 50 mg/l, time required to reach a 90% decolorization was 60 h, whereas in that of 300 mg/l the time required doubled (120 h). In general, the decolorizaiton was well described by a 1st order kinetic model (Table 1). Furthermore, data also revealed that decolorizaiton was faster at the initial stage (e.g. as early as 6 h), and a calculation of decolorization rate was conducted. With the use of Origin 8.0 software, we fitted curve of kinetics Eq. (2) of decolorization for Haldane model. The Haldane model was:
V ¼
7:9855$S S þ S2=615:2196 þ 130:7367
(3)
Fitting situation of predicted and experiment value was showed in Fig. 6. The correlation coefficient (r2) was 0.9817, well described the dynamic characteristics of decolorization rate in the initial stage (0e6 h) with diverse dye concentrations. The pattern of decolorization rate at initial stage suggested that 300 mg/l was an optimum concentration, allowing highest decolorization occur. With the increase in dye concentration toxic effect of dye/dye metabolites became dominant, leading to inhibition in decolorization. Similar patterns were also observed in the past (Khehra et al., 2005; Kalme et al., 2007). Using the above mentioned model, an optimal concentration (Sopt) was calculated:
Sopt ¼
pffiffiffiffiffiffiffiffiffiffiffiffi Ki $Ks
(4)
In this study, the Sopt value for Bacillus sp. YZU1 was 283.6 mg/l, with a greatest rate of decolorization (Vopt) of 4.1549 mgl1h1:
Fig. 5. Effect of initial dye concentration on decolorization.
Vopt ¼
Vmax sffiffiffiffiffi Ks 1þ2 Ki
(5)
Compared with decolorization of RB-5 using Bacillus sp. reported in the literature, the strain YZU1 reached >84% decolorization in this study, higher than the maximum level (80%) reported by Modi et al. (2010) with their isolated Bacillus cereus. However, at 12 h, YZU1 only achieved 17% decolorization, less effective than a strain of Bacillus sp. AK1 (31%) isolated by Anjaneya et al. (2011). Nonetheless, maximum decolorization of Bacillus sp. YZU1 in the present study reached 95%, comparable to other methods such as physical adsorption (45%, Eren and Acar, 2006), photocatalytic degradation (98%, You et al., 2010), electrochemical oxidation (95%, CeroonRivera et al., 2004) and microbial degradation (90% using Enterobacter sp. EC3, Wang et al., 2009). 3.8. Decolorization of various textile dyes Azo dyes used in this study were listed in Table 2. Decolorization of various azo dyes after 120 h was listed in Table 3. Among the 10 structurally different azo dyes (100 mg/l), a maximum decolorization of 99.14% was recorded in Methyl Red after 10 h, and for Methyl Orange and Reactive Yellow, the values were respectively 96.08% and 97.88% after 72 h. Decolorization of the above three azo dyes by dead cells were 1.68%, 2.31% and 4.19%, respectively. This variation might be due to the structural difference in the dyes (Kalyani et al.,
Fig. 6. Fitted curve of kinetics equation of decolorization model: Bacillus sp. YZU1.
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Table 2 Characteristics of main azo dyes used in this study. Azo dye
Molecular structure
Wavelength (nm)
Methyl red
430
Methyl orange
464
Reactive yellow
435
Scarlet red
525
Reactive red M5B
513
Brilliant blue
595
Reactive blue
598
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47
Table 2 (continued ) Azo dye
Molecular structure
Wavelength (nm)
607
Trypan blue
611
Evans blue
428
Cibacron brilliant yellow 3G-P
Table 3 Decolorization of various textile dyes by Bacillus sp. YZU1. Dyes
a
Reactive red M5B Scarlet red Brilliant blue G Reactive blue 4 Trypan blue Cibacron brilliant yellow 3G-P a b c
Decolorization (%) 87.73% 87.62% 87.54% 49.89% 46.29% 16.71%
b
Acknowledgments Decolorization (%)
c
11.28% 8.48% 3.23% 5.73% 12.12% 8.66%
100 mg/l dye concentration. Decolorization after 120 h by live cells. Decolorization after 120 h by dead cells.
2008). Nevertheless, decolorization of Bacillus sp. YZU1 against the reactive dyes tested in this study (except for Cibacron Brilliant yellow 3G-P) was >45%, and more than 70% was achieved in treating Scarlet Red, Reactive Red M5B, Brilliant Blue and Evans Blue. This suggested that this strain showed great potential in decolorizing complex dye effluent containing various reactive dyes. However, it also appears that more research is needed, especially in optimizing treating conditions, in the future. 4. Conclusions In this study, an effective RB-5 decolorizing bacterial strain, Bacillus sp. YZU1, was isolated. Bacillus sp. YZU1 showed azoreductase activity in the degradation, not simply a physical surface adsorption. In degradation, Bacillus sp. YZU1 simply needs a mild condition, and it showed remarkable tolerance to high concentrations of Reactive Black 5 (500 mg/l). Bearing high decolorizing activities against various reactive dyes commonly used in the textile industries, it is proposed that Bacillus sp. YZU1 had a practical application potential in the biotransformation of various dye effluents.
Financial support from the Faculty Research Grant, Hong Kong Baptist University (No. FRG2/09-10/072) is gratefully acknowledged.
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