Bioresource Technology 98 (2007) 3168–3171

Short Communication

Decolorisation and detoxification of Direct Blue-15by a bacterial consortium

Koel Kumar, Sivanesan Saravana Devi, Kannan Krishnamurthi,Dipanwita Dutta, Tapan Chakrabarti *

Environmental Biotechnology Division, National Environmental Engineering Research Institute, Nagpur 440 020, India

Received 18 July 2006; received in revised form 16 October 2006; accepted 22 October 2006Available online 26 February 2007

Abstract

Studies were carried out on decolorisation and biotransformation of the dye Direct Blue-15 into 3,3 0-dimethoxybenzidine (O 0-dian-isidine) and a sulphonated derivative by a five-member bacterial consortium. Chromatographic studies revealed further complete biodeg-radation of 3,3 0-dimethoxybenzidine coupled with release of ammonia, but the recalcitrant sulphonated derivative persisted. Themicroorganisms identified in the mixed consortium by 16S rDNA sequence analysis were Alcaligenes faecalis, Sphingomonas sp. EBD,Bacillus subtilis, Bacillus thuringiensis and Enterobacter cancerogenus. The cytotoxicity data showed a significant reduction in the toxicity(P < 0.001) of the degraded dye as evidenced from the number of viable human polymorphonuclear leukocyte cells present.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Direct Blue-15; Benzidine; Decolorisation; Biotransformation; Cytotoxicity

1. Introduction

The major classes of synthetic dyes used for textile dye-ing and other industrial applications include azo, anthra-quinone and triaryl methane dyes (Padmavathy et al.,2003; Chang et al., 2001). Azo dyes are by far the mostwidely used (Manning et al., 1985; Chen et al., 2004) andcolor index lists more than 2000 azo compounds(SCCNFP, 2002). Around 15% of the total world produc-tion of dyes and intermediates are released into environ-ment during synthesis, processing and usage (Spadaryet al., 1994; Zollinger, 1991).

Benzidine-based azo dyes contain benzidine attached toother substituents by diazo linkages. Benzidine by itself istumorigenic (Haley, 1975) and a human urinary bladdercarcinogen (Brown, 1977; National Institute for Occupa-tional Safety and Health, 1980).

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2006.10.037

* Corresponding author. Tel.: +91 712 2249757; fax: +91 712 2249961.E-mail address: t_chakrabarti@neeri.res.in (T. Chakrabarti).

The NIOSH review also indicated benzidine-based dyesto be carcinogenic due to their biotransformation tobenzidine.

According to the EU (European regulations, SCCNFP,2002), Direct Blue-15 is one of the azo dyes, which splits intocarcinogenic amine. Although Direct Blue-15 has beenbanned in India since 1997 (Ministry of Environment andForest, 1997), it is still available in the world market(EEC, 2000) and is manufactured in India for export. Theaim of the present study was to carry out decolorisationand biodegradation studies on benzidine-based DirectBlue-15 using a mixed bacterial culture. Further, the toxicitypotential of the dye on human polymorphonuclear lympho-cytes was compared before and after microbial treatment.

2. Methods

2.1. Reagents

Commercial grade Direct Blue-15 was obtained from adye-manufacturing unit. A stock solution of the dye

K. Kumar et al. / Bioresource Technology 98 (2007) 3168–3171 3169

(1000 mg L�1) was prepared in de-ionized water and usedfor all studies. Diethyl ether, dichloromethane, methanoland DMSO used were of HPLC grade (Merck, India).All other chemicals used in the experiment were of analyt-ical grade (Merck, India).

2.2. Experimental setup

2.2.1. Screening and selection of microorganisms

Acclimated activated sludge was sourced from an aero-bically maintained bioreactor treating textile wastewater.Around 10 g of sludge was added in 100 mL of minimalbasal medium (g L�1, Na2HPO4 – 6, KH2PO4 – 3, NH4Cl– 1, NaCl – 0.5, and 1 M MgSO4 – 1 mL L�1) and kept inorbital shaker for 12 h at 150 rpm and 37 �C. Supernatantwas collected after allowing the sludge to settle down for2 h. The microorganisms capable of degrading dye werescreened by inoculating the Luria broth agar plates(g L�1, Casein enzymic hydrolysate – 10, yeast extract –5, NaCl – 5, agar-15; Hi-media laboratories, India) con-taining 10 mg L�1 of Direct Blue-15 with the supernatant.The bacterial growth was picked up on the basis of its abil-ity to form clear zones on these plates. These cultures weresubsequently transferred to Luria broth medium contain-ing different concentrations of the dye. For identification,these cultures were further isolated on Luria agar andmaintained on slants. Five colonies were picked up accord-ing to their different morphological appearance and identi-fied on the basis of 16S rDNA sequence analysis.

2.2.2. Decolorisation and biodegradation studies

Experiments were performed in flasks. Different concen-trations of Direct Blue-15 dye (50,100 and 250 mg L�1)were added in Luria broth medium inoculated with0.10 OD (600 nm) of culture. These flasks were incubatedat 37 �C at static condition till decolorisation and degrada-tion were completed. The samples were drawn at differenttime intervals, centrifuged at 12,000g and analysed fordecolorisation and biodegradation as described below.

2.3. Analyses

2.3.1. Decolorisation and ammonia analyses

The UV–Vis spectra of the samples were recorded from200 to 800 nm using a spectrophotometer (Perkin ElmerUV–Vis-NIR Lambda-900). Decolorisation was measuredat different time intervals at the wavelength in the visiblerange where maximum absorbance was obtained(604 nm). Ammonia was estimated by Nesslerizationmethod as described in Standard Methods (APHA,AWWS, WPCF, 1991).

2.3.2. Biomass study

The pellet from the centrifuged sample (Section 2.2.2)was suspended in 5 mL of distilled water. This was thenhomogenized using a vortex and filtered using pre-weighed

glass fibre circles [GFC (42.5 mm)]. The biomass on GFCwas determined gravimetrically.

2.3.3. Extraction and HPLC analysis

The dye degradation metabolites were monitored byHPLC (Waters, Model No.: 501) as the decolorisation con-tinued. Ten milliliter sample was taken at different daysinterval (day 4 and 10) centrifuged at 12,000g, and filteredthrough 0.45 lm membrane filter (Millipore). The filtratewas then extracted three times with diethyl ether and flashevaporated in rotary vacuum evaporator in temperaturecontrolled water bath (45–50 �C) and residue dissolved in2 mL methanol. This extracted sample was analysed byHPLC having the mobile phase of 50:49.6:0.4% metha-nol:water:disodium hydrogen phosphate, C-18 columnwith a flow rate of 0.8 mL min�1, chart speed of1 cm min�1 and UV detector at 280 nm.

2.3.4. Extraction and GC/MS analysis

Samples on the 4th day, and after conclusion of theexperiment, were centrifuged and then filtered through0.45 lm membrane filter. The filtrate was then extractedthrice with diethyl ether and flash evaporated in rotary vac-uum evaporator in temperature controlled water bath (45–50 �C) and residue was dissolved in methylene chloride forGC/MS analysis.

GC/MS conditions: The GC/MS analysis of metabo-lite(s) was carried out using Varian/Saturn 2200 GC/MS/MS equipped with gas chromatograph CP-3800 with capil-lary column CP-Sil-8CB. Helium was used as carrier with aflow rate of 1.1 mL min�1. The injector temperature wasmaintained at 300 �C and the analysis was carried out asper the protocol of EPA-8270. The compounds were iden-tified using NIST library on the basis of mass spectra andretention time.

2.3.5. Cytotoxicity study

The cytotoxicity studies were carried out using trypanblue exclusion assay (Kiang et al., 1998) on human poly-morphonuclear leukocyte cells. Two milliliter sample wastaken, extracted thrice by diethylether and flash evapo-rated. The left over residue was dissolved in 1 mL of0.1% dimethyl sulphoxide. This extract was used to chal-lenge the cells for cytotoxicity study. The average and per-centage of cell viability were calculated from the results ofat least five replicate experiments. Statistical analysis wasdone using student ‘‘t’’ test by Analyse-it software.

3. Results and discussion

The individual microorganisms identified in the mixedculture by 16S rDNA sequence analysis were Alcaligenes

faecalis, Sphingomonas sp. EBD, Bacillus subtilis, Bacillus

thuringiensis and Enterobacter cancerogenus. The 16SrDNA sequences of Alcaligenes faecalis, Sphingomonassp. EBD, Bacillus subtilis, Bacillus thuringiensis and Enter-

obacter cancerogenus have been deposited in the GenBank

3170 K. Kumar et al. / Bioresource Technology 98 (2007) 3168–3171

with accession numbers EF011115, DQ855091, DQ666275,DQ837529 and EF011116 respectively. Decolorisation wasinitially monitored every 4 h interval up to 24 h and thenevery 24 h up to the 10th day. Significant decolorisationof 92.14%, 94.46% and 95.63% was observed at 24th h atthe concentrations of 50, 100 and 250 mg L�1, respectively.Bioadsorption study was also carried out (data not shown)while monitoring decolorisation process of the dye as it hasbeen reported that many azo dyes get adsorbed initially onthe cell surface (Ambrosio and Campos-Takaki, 2004;Kumar et al., 2006). It was noticed that no such phenome-non took place with Direct Blue-15 azo dye during thedecolorisation process. The growth in biomass was moni-tored gravimetrically (Fig. 1), which showed a continuouslog phase up to the 6th day after which a gradual decreasewas observed.

As the decolorisation proceeded, the release of ammonia(NH3–N) was observed; maximum ammonia was releasedon the 3rd and 4th day (Fig. 2), which coincided with theformation of 3,3 0-dimethoxybenzidine (on the 4th day). Ini-tial release of ammonia up to 3rd or 4th day was due to themineralization of the Luria broth medium containing

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Fig. 1. Biomass growth.

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Fig. 2. Release of ammonia.

nitrogen sources. The ammonia production continued upto the 10th day, which might be because of the deaminationof 3,3 0-dimethoxybenzidine (resulting in degradation of thedye as revealed through HPLC analysis), though deamina-tion metabolites could not be identified. The HPLC analy-sis of the dye sample taken at the beginning of staticincubation (0 h incubation) showed a major peak of thedye, (2,2 0-{4,4 0-[3,3-bis(methoxy)]biphenylenebis(azo)}bis-[8-amino-1-hydroxynapthalene-3,6-disulfonic acid] tetraso-dium salt) with the retention time of 4.0 min. After fourdays, as the degradation proceeded, the HPLC chromato-gram showed an additional new peak with retention timeof 5.4 along with the peak at 4.0 min. The peak at5.4 min was identified as 3,3 0-dimethoxybenzidine (O 0-dianisidine) on comparing with 3,3 0-dimethoxybenzidinestandard. The peak at 4.0 min was identified as naphtha-lene disulphonic acid derivative on comparing with thestandard. On analyzing the 10th day sample, the peak at5.4 min was barely present, suggesting biodegradation ofthe intermediate 3,3 0-dimethoxybenzidine. However, thepeak at 4.0 min was still present showing the degradationof Direct Blue-15 as partial and the recalcitrant nature ofthe sulphonated naphthalene derivative. The 4th day sam-ple showed only one intermediate when injected into theGC/MS. The mass spectrum identified was that of 3,3 0-dimethoxybenzidine with retention time of 22.546 minand molecular weight of 244. It has been reported that3,3 0-dimethoxybenzidine-based dyes could be metabolizedto 3,3 0-dimethoxybenzidine, which is human carcinogen(NTP, 2002). On further analysis of the 10th day sample,3,3 0-dimethoxybenzidine was not found.

The toxicity of Direct Blue-15 dye was evaluated beforeand after degradation at concentrations of 50, 100 and250 mg L�1. The viability of the control cells was >90%(0.1% DMSO). Cytotoxicity studies revealed that biodegra-dation of the dye by the mixed cultures for a period of tendays resulted in detoxification of the dye as could be seenfrom the absence of significant total cell death at all inter-vals (0–3 h) and at all the concentrations (50, 100 and250 mg L�1) tested (Fig. 3).

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Fig. 3. Cytotoxicity study of Direct Blue-15 (50, 100, 250 mg L�1).

K. Kumar et al. / Bioresource Technology 98 (2007) 3168–3171 3171

4. Conclusions

From the results obtained in this study, it was concludedthat acclimated bacterial consortium was capable of decol-orisation, biotransformation and detoxification of the toxicbenzidine-based dyes. This bacterial consortium could beused to augment activated sludge biomass engaged indegrading and detoxifying benzidine-based dyes.

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