ew

tisthinaced T

ith rundZU1texformgf RB

eductase and played the most important role in decolorization, while a small percentage of decolor-

1. Introduction

extile,r 800,orldwiof the

y (Fanplied intheir

2006). Recent studies also showed that chlorination of azo dyesgenerates mutagenic disinfection byproducts, which may contam-inate drinking water (Oliveira et al., 2006). Additionally, dyes withtrace amount (10e15 mg/L) are highly visible, affecting waterrecreational value, light penetration in water and as a consequencereduced photosynthesis and dissolved oxygen (Dafale et al., 2010).

decolorization of dye by bacteria, isolation of new especiallyindigenous microorganisms capable of degrading various dyes withhigh effectiveness has always been the focus. Upon in-depthcharacterization of degradation biologically and chemically,mechanisms of the processes will be claried. The information willhelp promote the relevant application. In this study, we attemptedto isolate indigenous bacteria from local dye wastewater treatmentplant. Following characterizing the bacteria, we hope that thebacteria would be utilized in wastewater treatment.

* Corresponding authors. Tel.: 852 3411 7751; fax: 852 3411 7743.

Contents lists available at

r

.e

International Biodeterioration & Biodegradation 76 (2013) 41e48E-mail addresses: jsliang@yzu.edu.cn (J.S. Liang), yliang@hkbu.edu.hk (Y. Liang).availability of variety of colors compared to natural dyes (Wanget al., 2008; Saratale et al., 2009). One major concern is that atleast 10e15% of the used dyes are discharged with wastewater(Robinson et al., 2001), and dyes are generally resistant to fading onexposure to light, water and many chemicals due to their chemicalstructure. The recalcitrant dyes are not toxic themselves, butbiodegradation of dyes particularly azo dyes may generate colorlessbut carcinogenic compounds such as aromatic amines, which mayadversely inuence human and environmental health (Husain,

a variety of oxidative enzymes such as peroxidases, Mndases, laccases and lignin peroxidases, and complete removal canbe achieved (Kuhad et al., 2004; Husain, 2006). Under anaerobicconditions, the color removal is effective mostly higher than 70%removal, and the processes generally are driven by bacterialazoreductases, leading to the cleavage of dyes azo linages andformation of aromatic amines, while the aromatic amines can bedegraded in a consequent aerobic treatment (Van der Zee andVillaverde, 2005).

Although substantial research has been conducted on theat a rate of more than 30% annuallaccount for up to 70% of dyestuffs apSynthetic dyes are essential for tcosmetics and food industries. Ovethan 100,000 dyes are produced wproduction in China accounts for 40%

to the ease and cost-effectiveness in0964-8305/$ e see front matter 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2012.06.023paper, pharmaceutical,000 tons/year of morede (Husain, 2006). Theworld total, increasinget al., 2008). Azo dyestextile processing, duesynthesis, stability and

Physicochemical methods, such as coagulation, adsorption,membrane ltration, electro- and photo-chemical removal, havebeen used for the treatment of dye containing wastewater, butlarge amounts of sludge are generated and very expensive(Husain, 2006). In contrast, biological methods using bacteria andfungi have been shown to be efcient and more cost-effective(Kuhad et al., 2004). Aerobic removal of azo dyes by bacteriaand fungi, particularly white rot fungi, is mostly mediated by

peroxi-suggested that Bacillus sp. YZU1 had great potential to be applied in dye efuent treatment. 2012 Elsevier Ltd. All rights reserved.ization occurred via passive surface adsorption. High biodegradation extent under a mild conditionDecolorization of Reactive Black 5 by a n

Z.W. Wang a,b, J.S. Liang b,*, Y. Liang a,c,*aCroucher Institute for Environmental Sciences, Department of Biology, Hong Kong BapbCollege of Bioscience and Biotechnology, Yangzhou University, Yangzhou, Jiangsu, PR CcCentre for Food Safety and Environmental Technology, Guangzhou Institutes of Advan

Keywords:BiodegradationDecolorizationReactive Black 5Bacillus

a b s t r a c t

A bacterial strain, YZU1, wsoil samples collected arosequence indicated that Ydecolorize various reactiveconsidered to be optimumconcentration of dye (100tolerate up to 500 mg/1 o

International Biodeterio

journal homepage: wwwAll rights reserved.ly isolated bacterium Bacillus sp. YZU1

University, Kowloon, Hong Kong SAR, PR China

echnology, Chinese Academy of Sciences, Guangzhou 511458, China

emarkable ability to decolorize Reactive Black 5 (RB-5), was isolated froma textile factory. Phenotypic and phylogenetic analyses of the 16S rDNAbelonged to Bacillus sp. Bacillus sp. YZU1 showed great capability to

tile dyes, including azo dye. Static conditions with pH 7.0 and 40 C weredecolorizing RB-5. Bacillus sp. YZU1 grew well in medium containing high/l), resulting in approximately 95% decolorization in 120 h, and could-5. Enzyme assays demonstrated that Bacillus sp. YZU1 possessed azor-

SciVerse ScienceDirect

ation & Biodegradation

lsevier .com/locate/ ibiod

factory around Suzhou city in China. The ask was incubated at

exposed to a germicidal UV lamp. The distance between surface of

bacterial cells were then transferred into 8 ml nutrient broth. The

activity based on the molar absorption coefcient of RB-5

concentrations (0.5, 1.0, 1.5, 2.0, 2.5 mM), were determined. All

iorathe cell suspension and the lamp was 40 cm, while the time ofexposure varied from 1 to 5 min. Then, 1 ml of the UV radiationexposed cell suspension was transferred to 9 ml saline water. Aftera series of dilution, the cells were plated on nutrient agar con-taining 50 mg/l RB-5 and incubated at 37 C for 24 h. Coloniesformedwere counted and each colony was inoculated into one tubecontaining 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 innutrient broth containing 100 mg/l of RB-5, was selected andpreserved for further dye degradation experiments. The decolor-ization percentage (%) was determined by measuring the absor-bance of the culture supernatant at 597 nm using a UVeVis30 C statically for 48 h. Then, 1.0 ml of the culture was diluted and100 ml of the diluted culture were spread-plated on the nutrientagar plate containing 100 mg/l RB-5. Colonies on the agar showingclear zone around were isolated and acclimated to increasingconcentrations of RB-5 in the broth (from 25, 50 to 100 mg/l).Eventually, one bacterial strain with the highest RB-5 decolorizingability was preserved at 70 C in the broth (with 15% glycerol)before further identication.

The isolated bacterial cells were recovered and cultured innutrient 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 at65 C for 15 min. DNA extracted by phenol/chloroform was recov-ered by ethanol precipitation and then dissolved in ddH2O. The 16SrDNA gene was amplied by PCR in a 25 ml reaction system usingprimers 27F and 1492R (DeLong, 1992). The conditions were: 1U ExTaq Buffer, 0.2 mM of each dNTP, 0.2 mM of each primer and 1U ExTaq polymerase (Takara). An initial denaturing period of 15 minwasfollowed by 30 cycles at 94 C for 30 s, 55 C for 30 s, 72 C for1.5 min. The nal extension (72 C) time was 5 min. The PCRproducts were sequenced by Generay Biotech (Shanghai) Co. Ltd.(China). The sequence was uploaded in NCBI to be identied byBLAST search. Physiological studies were also conducted accordingto procedures outlined in Bergeys Manual of DeterminativeBacteriology (Staley et al., 2001).

2.3. Induction of mutagenesis and selection of mutants

Ten ml of the bacterial cell suspension (107 CFU/ml) in salinewater (0.9% NaCl) were transferred in an open petri-dish and2. Materials and methods

2.1. Azo dyes

Reactive Black 5 (C.I., 55% dye, SigmaeAldrich, RB-5),a commonly used azo dye, was chosen for the screening ofdegrading 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 BrilliantYellow 3G-P, C.I. Reactive Blue 171, C.I. Brilliant Blue G, C.I. TrypanBlue, C.I. Evans Blue, were chosen as structurally different dyes toexamine decolorizing capability of the isolated bacterium.

2.2. Isolation and identication of dye degrading bacteria

Nutrient broth containing dye RB-5 (100 mg/l) was inoculatedwith 10% (w/v) of a soil sample collected from a textile processing

Z.W. Wang et al. / International Biodeter42spectrophotometer (GE), calculated as:( 3 21.329 mmol1 cm1). One unit of enzyme activity wasdened as the amount of enzyme required to decolorize 1 nmolof dye per min under the assay conditions. Enzyme activitiesamong 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 NADHcultures were incubated at 37 C for 24 h, and the extent ofdecolorization was calculated and recorded. Inuence of initialsubstrate concentration on degradation rate was analyzed usingMonod model:

V Vmax$SS S2=Ki Ks

(2)

where Vmax was the maximum decolorization rate (mg/l h), S wasthe concentration of substrate(mg/l), Ks was the substrate satura-tion, Ki was the substrate inhibition constant.

2.5. Azoreductase assay of bacterial crude enzyme extract

Bacterial cells were harvested (8000 rpm, 10 min), washed by10 mM buffer (pH 7) and suspended in an equal volume of 50 mMphosphate buffer (pH 7). The cells were then disrupted by coldsonication (15 min, 70% amplitude). After cell debris and undis-rupted cells were removed by centrifugation (15,000 g, 20 min,4 C) (Maier et al., 2004), the supernatant was then freeze-driedand preserved in a desiccators as the bacterial crude enzymeextract. The crude extract was dissolved in 50 mMphosphate bufferbefore enzyme assays.

The assay was carried out in a cuvette (path length 1 cm,1 ml). Four hundred mL 50 mM phosphate buffer was mixed withvaried concentrations of crude extract and 200 ml of RB-5(500 mg/l, resulting in a nal concentration of 100.8 mM dye).The reaction started by adding NADH, and the mixture wasmonitored photometrically at 597 nm. The slope of the initiallinear decrease in absorption was for calculating the azoreductaseDecolorization percentage % OD1 ODtOD1

100 (1)

where OD1 referred to the initial absorbance, ODt referred to theabsorbance 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 per-formed in triplicate. A cell suspension of YZU1 (0.15 ml) was inoc-ulated into an 8 ml nutrient broth containing 100 mg/l RB-5, whilethe same amount of autoclaved bacterial cell suspension (deadcells) was also transferred to another 8 ml nutrient broth asa control. The cultures were incubated at 37 C for 120 h, andsamples from both cultures were scanned from 400e800 nm usinga UVeVis spectrophotometer (GE). Variation in decolorization wasthen calculated and recorded.

Effects of environmental factors, including temperature (variedbetween 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 saltconcentration (0, 0.5, 1, 2, 4, 6, 8, 10 g/l), were investigated as wellon the decolorization effectiveness. Briey, the bacterium wasrstly cultured in a nutrient broth at 200 rpm, 37 C overnight. The

tion & Biodegradation 76 (2013) 41e48the assays were conducted in triplicate.

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 identication of decolorizing bacteria

Upon initial screening and mutation induction, a strain ofbacterium YZU1 having remarkable decolorizing ability on RB-5was isolated. Signicant decolorization (60%) was observed after24 h incubation, and a maximum value (95%) was achieved afterincubation for 120 h (Fig. 1). Colony of the bacterial isolate wascircular or nearly circular, at, smooth and white. The cells weregram-positive rod-shaped (short). Sequence analysis of 16S rDNAand comparison with other related bacteria in the GenBank data-base 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 identied as Bacillus sp. strain YZU1.

on NADH dependent azoreductase derived from Bacillus, thatanaerobic condition was required to initiate azoreductase activity.

3.3. Effect of temperature

Bacillus sp. strain YZU1 showed high decolorizing capability inthe temperature range of 35 Ce45 C (Fig. 4-1). Although thepercentage of decolorization after 24 h was found to be compara-tively low at 20 C, it increased to a greater level at 40 C. Decol-orizing activity was substantially inhibited with othertemperatures, mostly likely because of deactivation of enzymesresponsible for decolorization or loss of cell viablility (Panswad andLuangdilok, 2000; Cetin and Donmez, 2006).

3.4. Effect of pH

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 thatthe optimum pH for the growth of Bacillus sp. YZU1 was neutral.Decolorization was observed at pH 6.0e9.0 after 24 h, but wassignicantly lower at relative strongly acidic (pH 4.0 and 5.0)conditions. pH is an important parameter of microbial growth andazo dye degradation, and the optimal levels of pH for color removalwere often between 6.0 and 10.0 (Chen et al., 2003; Guo et al.,

Z.W. Wang et al. / International Biodeteriora3.2. 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), prob-ably due to the adsorption by heat-killed bacterial cells. Colored cellpellets at the bottom of the culture was also observed in this study.In contrast, 95.0% decolorization was achieved in 120 h, in theculture inoculated with live bacterial cells (Fig. 1), and the cellswere not pigmented. Additionally, vis spectral scan (400e800 nm)data of the supernatants showed that the maximum absorbancewavelength was blue shifted in control cultures (Fig. 2). This indi-cated that molecular structure change in RB-5 occurred by thebacterial biodegradation. In fact, both ways of decolorization ofdyes, adsorption and biodegradation, were expected in the process(Aravindhan et al., 2007; Kumar et al., 2007). Indeed extracellularpolysaccharides of Bacillus subtilis could be utilized as biosorbentsin removal of dye from industrial efuents (Binupriya et al., 2010).Obviously, in this study biodegradation played a more importantFig. 1. Decolorization of Reactive Black 5 by live and dead cells (heat-killed) of Bacillussp. YZU1.role, as the major peak decreased accompanied by a shift to shorterwavelengths (blue) (Yu and Wen, 2005). This suggested that azobonds cleaved during the reaction leading to damage/break-up inthe primary chromophore, most likely mediated by azoreductase(Oturkar et al., 2011).

Enzyme assay result showed that bacterial crude extract con-tained NADH dependent azoreductase (7.50 U/mg) (Fig. 3a). Withthe presence of a same level of NADH, decolorization depended onthe amount of crude extract used, the more crude extract, the fasterof the reaction. Furthermore, it showed that decolorization of RB-5by the crude extract was slow before 60 s (Fig. 3a), but more rapidright after. On the other hand, the azoreductase activity increasedas NADH concentration increased, up to about 1.5 mM (Fig. 3b).Above this level, further addition of NADH did not accelerate thereaction. Characteristics of azoreductase in the crude extract in thisstudy were in consistent with those reported by Maier et al. (2004)

Fig. 2. Vis spectra of Reactive Black 5 decolorization at different bacterial treatmenttime.

tion & Biodegradation 76 (2013) 41e48 432007; Kilic et al., 2007). In fact, studying pH tolerance of

decolorizing bacteria is crucial because alkaline conditions facili-tated binding between azo dyes and bers (Aksu, 2003), and pH ofthe dye wastewater discharged from textile factory usually rangedbetween 8 and 9. This demonstrated that this strain worked undera wide range of pH (6e9), making it as a promising strain forpractical bio-treatment of dye wastewater.

3.5. Effect of O2

Decolorization of RB-5 was about 60% in static condition, andsignicantly decreased in aerobic condition when the speed ofshaker increased from 100 rpm to 250 rpm. However, the growth of

anaerobe. The bacterial growth was promoted with the presence ofO2 but the yield of dye decolorization/degradation related enzymewas suppressed. The result supported previous reports that aerobiccondition inhibited bacterial biodegradation of dyes (Khehra et al.,2005;Moosvi et al., 2005). Instead of azo groups in the dyes, oxygenwas more preferred as an electron acceptor, resulting in an inhi-bition of the dye reduction process (Pearce et al., 2003).

3.6. Effect of salt concentration

Salt level greatly inuenced decolorization (Fig. 4-4). Decolor-ization after 24 h reached>40%when salt concentration is less than

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).

Z.W. Wang et al. / International Biodeterioration & Biodegradation 76 (2013) 41e4844Bacillus sp. YZU1 was the greatest under aerobic condition (Fig. 4-3). This indicated that Bacillus sp. YZU1 was a facultativeFig.4. Effect of (1) temperature, (2) pH, (3) dissolved oxygen2 g/l, signicantly (P< 0.05) higher than that in other salt concen-trations (

decreasewhen the salt concentration exceeded 0.5 g/l. This is due tothe fact that high salt concentration affected osmotic pressure inBacillus sp. YZU1, inhibited bacterial growth and even caused celldeath. Similar salt effect on bacterial decolorization of azo dyes wasalso 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 onecentre of studies (Gopinath et al., 2009; Anjaneya et al., 2011). Ourfuture research will also be led towards this direction.

3.7. Effect of initial dye concentration

With the same culture time decolorization reduced withincrease in initial dye concentration (Fig. 5). For example, in the dyeculture of 50 mg/l, time required to reach a 90% decolorization was60 h, whereas in that of 300 mg/l the time required doubled(120 h). In general, the decolorizaiton was well described by a 1storder kinetic model (Table 1).

Furthermore, data also revealed that decolorizaitonwas faster at

Vopt Vmax

1 2KsKi

s (5)

Compared with decolorization of RB-5 using Bacillus sp. reported inthe literature, the strain YZU1 reached >84% decolorization in thisstudy, higher than themaximum level (80%) reported byModi et al.

3.8. Decolorization of various textile dyes

Table 1Parameter and equation of kinetics of decolorization of Reactive Black 5 by Bacillussp. YZU1.

Concentration(mg/l)

Equationof kinetic

K0 (h1) Correlationcoefcient (r2)

50 y0.0357x 3.5845 0.0357 0.9863100 y0.0288x 4.4713 0.0288 0.9904150 y0.0294x 4.9698 0.0294 0.9686200 y0.0221x 5.1953 0.0221 0.9423300 y0.0245x 5.7298 0.0245 0.9300400 y0.0156x 6.0831 0.0156 0.9732500 y0.0147x 6.3383 0.0147 0.9767

Z.W. Wang et al. / International Biodeterioration & Biodegradation 76 (2013) 41e48 45the initial stage (e.g. as early as 6 h), and a calculation of decolor-ization rate was conducted. With the use of Origin 8.0 software, wetted curve of kinetics Eq. (2) of decolorization for Haldane model.The Haldane model was:

V 7:9855$SS S2=615:2196 130:7367 (3)

Fitting situation of predicted and experiment value was showedin Fig. 6. The correlation coefcient (r2) was 0.9817, well describedthe dynamic characteristics of decolorization rate in the initialstage (0e6 h) with diverse dye concentrations. The pattern ofdecolorization rate at initial stage suggested that 300 mg/l was anoptimum concentration, allowing highest decolorization occur.With the increase in dye concentration toxic effect of dye/dyemetabolites became dominant, leading to inhibition in decolor-ization. Similar patterns were also observed in the past (Khehraet al., 2005; Kalme et al., 2007). Using the above mentionedmodel, an optimal concentration (Sopt) was calculated:

Sopt Ki$Ks

p(4)

In this study, the Sopt value for Bacillus sp. YZU1 was 283.6 mg/l,with a greatest rate of decolorization (Vopt) of 4.1549mgl1h1:Fig. 5. Effect of initial dye concentration on decolorization.Azo dyes used in this study were listed in Table 2. Decolorizationof various azo dyes after 120 h was listed in Table 3. Among the 10structurally different azo dyes (100 mg/l), a maximum decoloriza-tion of 99.14%was recorded inMethyl Red after 10 h, and for MethylOrange and Reactive Yellow, the values were respectively 96.08%and 97.88% after 72 h. Decolorization of the above three azo dyes bydead cells were 1.68%, 2.31% and 4.19%, respectively. This variationmight be due to the structural difference in the dyes (Kalyani et al.,(2010) with their isolated Bacillus cereus. However, at 12 h, YZU1only achieved 17% decolorization, less effective than a strain ofBacillus sp. AK1 (31%) isolated by Anjaneya et al. (2011). Nonethe-less, maximum decolorization of Bacillus sp. YZU1 in the presentstudy reached 95%, comparable to other methods such as physicaladsorption (45%, Eren and Acar, 2006), photocatalytic degradation(98%, You et al., 2010), electrochemical oxidation (95%, Ceroon-Rivera et al., 2004) and microbial degradation (90% using Enter-obacter sp. EC3, Wang et al., 2009).Fig. 6. Fitted curve of kinetics equation of decolorization model: Bacillus sp. YZU1.

Table 2Characteristics 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

Z.W. Wang et al. / International Biodeterioration & Biodegradation 76 (2013) 41e4846

ioraTable 2 (continued )

Azo dye Molecular structure

Trypan blue

Evans blue

Z.W. Wang et al. / International Biodeter2008). Nevertheless, decolorization of Bacillus sp. YZU1 against thereactive dyes tested in this study (except for Cibacron Brilliantyellow 3G-P) was >45%, and more than 70% was achieved intreating Scarlet Red, Reactive Red M5B, Brilliant Blue and EvansBlue. This suggested that this strain showed great potential indecolorizing complex dye efuent containing various reactive dyes.However, it also appears that more research is needed, especially inoptimizing treating conditions, in the future.

4. Conclusions

In this study, an effective RB-5 decolorizing bacterial strain,Bacillus sp.YZU1,was isolated.Bacillus sp.YZU1showedazoreductaseactivity in the degradation, not simply a physical surface adsorption.Indegradation,Bacillus sp. YZU1simplyneedsamild condition, and itshowed remarkable tolerance to high concentrations of ReactiveBlack 5 (500 mg/l). Bearing high decolorizing activities againstvarious reactive dyes commonly used in the textile industries, it isproposed that Bacillus sp. YZU1 had a practical application potentialin the biotransformation of various dye efuents.

Cibacron brilliant yellow 3G-P

Table 3Decolorization of various textile dyes by Bacillus sp. YZU1.

Dyesa Decolorization (%)b Decolorization (%)c

Reactive red M5B 87.73% 11.28%Scarlet red 87.62% 8.48%Brilliant blue G 87.54% 3.23%Reactive blue 4 49.89% 5.73%Trypan blue 46.29% 12.12%Cibacron brilliant yellow 3G-P 16.71% 8.66%

a 100 mg/l dye concentration.b Decolorization after 120 h by live cells.c Decolorization after 120 h by dead cells.Wavelength (nm)

607

611

428

tion & Biodegradation 76 (2013) 41e48 47Acknowledgments

Financial support from the Faculty Research Grant, Hong KongBaptist University (No. FRG2/09-10/072) is gratefully acknowledged.

References

Aksu, Z., 2003. Reactive dye bioaccumulation by Saccharomyces cerevisiae. ProcessBiochemistry 38, 1437e1444.

Anjaneya, O., Soucheb, S.Y., Santoshkumara, M., Karegoudara, T.B., 2011. Decolor-ization of sulfonated azo dye Metanil Yellow by newly isolated bacterial strains:Bacillus sp. strain AK1 and Lysinibacillus sp. strain AK2. Journal of HazardousMaterials 190, 351e358.

Aravindhan, R., Rao, J.R., Nair, B.U., 2007. Removal of basic yellow dye from aqueoussolution by sorption on green alga Caulerpa scalpelliformis. Journal of HazardousMaterials 142, 68e76.

Binupriya, R., Sathishkumar, M., Ku, C.S., Yun, S., 2010. Sequestration of ReactiveBlue 4 by free and immobilized Bacillus subtilis cells and its extracellularpolysaccharides. Colloids and Surfaces B: Biointerfaces 76, 179e185.

Ceroon-Rivera, M., Daavila-Jimeenez, M.M., Elizalde-Gonzaalez, M.P., 2004. Degra-dation of the textile dyes Basic yellow 28 and Reactive black 5 using diamondand metal alloys electrodes. Chemosphere 55, 1e10.

Cetin, D., Donmez, G., 2006. Decolorization of reactive dyes by mixed culturesisolated from textile efuent under anaerobic conditions. Enzyme and Micro-bial Technology 38, 926e930.

Chen, K.-C., Wu, J.-Y., Liou, D.-J., Hwang, S.-C.J., 2003. Decolorization of thetextile dyes by newly isolated bacterial strains. Journal of Biotechnology 101,57e68.

Dafale, N., Agrawal, L., Kapley, A., Meshram, S., Purohit, H., Wate, S., 2010. Selectionof indicator bacteria based on screening of 16S rDNA metagenomic library froma two-stage anoxiceoxic bioreactor system degrading azo dyes. BioresourceTechnology 101 (2), 476e484.

DeLong, E.F., 1992. Archaea in coastal marine environments. Proceedings of theNational Academy of Sciences of the United States of America 89 (12),5685e5689.

Eren, Z., Acar, F.N., 2006. Adsorption of Reactive Black 5 from an aqueous solution:equilibrium and kinetic studies. Desalination 194, 1e10.

Fan, L., Zhu, S.N., Liu, D.Q., Ni, J.R., 2008. Decolorization of 1-amino-4-bromoanthraquinone-2-sulfonic acid by a newly isolated strain of

Sphingomonas herbicidovorans. International Biodeterioration & Biodegrada-tion, 1e5 (online).

Gopinath, K.P., Murugesan, S., Abrahamb, J., Muthukumar, K., 2009. Bacillus sp.mutant for improved biodegradation of Congo red: random mutagenesisapproach. Bioresource Technology 100, 6295e6300.

Guo, J.B., Zhou, J.T., Wang, D., Tian, C.P., Wang, P., Uddin, M.S., Yu, H., 2007. Bio-catalyst effects of immobilized anthraquinone on the anaerobic reduction of azodyes by the salt-tolerant bacteria. Water Research 41, 426e432.

Husain, Q., 2006. Potential applications of the oxidoreductive enzymes in thedecolorization and detoxication of textile and other synthetic dyes frompolluted water: a review. Critical Reviews in Biotechnology 26 (4), 201e221.

Kalme, S., Parshetti, G., Jadhav, S., Govindwar, S., 2007. Biodegradation of benzidinebased dye Direct Blue-6 by Pseudomonas desmolyticum NCIM 2112. BioresourceTechnology 98, 1405e1410.

Kalyani, D.C., Patil, P.S., Jadhav, J.P., Govindwar, S.P., 2008. Biodegradation of reactivetextile dye Red BLI by an isolated bacterium Pseudomonas sp. SUK1. BioresourceTechnology 99, 4635e4641.

Khehra, M.S., Saini, H.S., Sharma, D.K., Chadha, B.S., Chimni, S.S., 2005. Comparativestudies on potential of consortium and constituent pure bacterial isolates todecolorize azo dyes. Water Research 39, 5135e5141.

Kilic, N.K., Nielsen, J.L., Yuce, M., Donmez, G., 2007. Characterization of a simplebacterial consortium for effective treatment of wastewaters with reactive dyesand Cr(VI). Chemosphere 67, 826e831.

Kolekar, Y.M., Pawar, S.P., Gawai, K.R., Lokhande, P.D., Shouche, Y.S., Kodam, K.M.,2008. Decolorization and degradation of Disperse Blue 79 and Acid Orange 10,by Bacillus fusiformis KMK5 isolated from the textile dye contaminated soil.Bioresource Technology 99, 8999e9003.

Kuhad, R.C., Sood, N., Tripathi, K.K., Singh, A., Ward, O.P., 2004. Developments inmicrobial methods for the treatment of dye efuents. Advances in AppliedMicrobiology 56, 185e213.

Kumar, K., Devi, S.S., Krishnamurthi, K., Dutta, D., Chakrabarti, T., 2007. Decolor-isation and detoxication of Direct Blue-15 by a bacterial consortium. Bio-resource Technology 98, 3168e3171.

Maier, J., Kandelbauer, A., Erlacher, A., Paulo, A.C., Georg, M., 2004. A new alkali-thermostable azoreductase from Bacillus sp. Strain SF. Applied and Environ-mental Microbiology 2, 837e844.

Modi, H.A., Rajput, G., Ambasana, C., 2010. Decolorization of water soluble azo dyesby bacterial cultures, isolated from dye house efuent. Bioresource Technology101, 6580e6583.

Moosvi, S., Keharia, H., Madamwar, D., 2005. Decolourization of textile dye ReactiveViolet 5 by a newly isolated bacterial consortium RVM 11.1. World Journal ofMicrobiology & Biotechnology 21, 667e672.

Oliveira, D.P., Carneiro, P.A., Rech, C.M., Zanoni, M.V., Claxton, L.D., 2006. Mutageniccompounds generated from the chlorination of disperse azo-dyes and theirpresence indrinkingwater. Environmental Science&Technology40, 6682e6689.

Oturkar, C.C., Nemade, H.N., Mulik, P.M., Patole, M.S., Hawaldar, R.R., 2011. Mecha-nistic investigation of decolorization and degradation of Reactive Red 120 byBacillus lentus BI377. Bioresource Technology 102, 758e764.

Panswad, T., Luangdilok, W., 2000. Decolorization of reactive dyes with differentmolecular structures under different environmental conditions. Water Research34, 4177e4184.

Pearce, C.I., Lloyd, J.R., Guthrie, J.T., 2003. The removal of colour from textilewastewater using whole bacterial cells: a review. Dyes and Pigments 58,179e196.

Robinson, T., McMullan, G., Marchant, G., Nigam, P., 2001. Remediation of dyes intextile efuent: a critical review on current treatment technologies witha proposed alternative. Bioresource Technology 77, 247e255.

Saratale, R.G., Saratale, G.D., Kalyani, D.C., Chang, J.S., Govindwar, S.P., 2009. Enhanceddecolorization and biodegradation of textile azo dyeScarlet R by using developedmicrobial 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. Bergeys Manual of DeterminativeBacteriology, second ed. Springer.

Van der Zee, F.P., Villaverde, S., 2005. Combined anaerobic-aerobic treatment ofazo 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 degra-dation 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. Biologicaldecolorization of the reactive dyes Reactive Black 5 by a novel isolated bacterialstrain 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 usinganaerobic/aerobic membrane bioreactor (MBR) and photochemical membranereactor. Journal of Hazardous Materials 177, 1112e1118.

Yu, Z.S., Wen, X.H., 2005. Screening and identication of yeasts for decolorizingsynthetic dyes in industrial wastewater. International Biodeterioration &Biodegradation 56, 109e114.

Z.W. Wang et al. / International Biodeterioration & Biodegradation 76 (2013) 41e4848

Decolorization of Reactive Black 5 by a newly isolated bacterium Bacillus sp. YZU11. Introduction2. Materials and methods2.1. Azo dyes2.2. Isolation and identification of dye degrading bacteria2.3. Induction of mutagenesis and selection of mutants2.4. Decolorization studies of RB-5 in nutrient broth2.5. Azoreductase assay of bacterial crude enzyme extract2.6. Statistical analysis

3. Results and discussion3.1. Isolation and identification of decolorizing bacteria3.2. Physical adsorption vs biodegradation3.3. Effect of temperature3.4. Effect of pH3.5. Effect of O23.6. Effect of salt concentration3.7. Effect of initial dye concentration3.8. Decolorization of various textile dyes

4. ConclusionsAcknowledgmentsReferences