Indian Journal of Experimental Biology Vol. 40, October 2002, pp. 1131-1136

Biodegradation of benzidine based azodyes Dir('ct red and Direct blue by the immobilized cells of Pseudomonas fluorescens D41

Puvaneswari N, ] Muthukrishnan & P Gunasekaran*

Centre for Advanced Studies in Functional Genomics, School of Biologic.!1 Sciences, Madurai Kamaraj Univers ity , Madurai 625 021. Indi a

Received 21 March 2002; revised 9 August 2002

Benzidine based azodyes are proven carcinogens, mutagens and have been linked to bladder cancer of human beings and laboratory animals. The textile and dyestuff manufacturing industry are ri]e two major sources that re leased azodyes in their effl uents. The dye, Direct blue contains two carcinogenic compou nds namely benzidine (BZ), 4-amino biphenyl (4-ABP), while the dye Direct red has benzidine (BZ) . Among 40 isolates o. Pseudomollas jluorescells screened, one isol ate designated as D~ 1 was found to be capable of p ~ tensive l y degrading the dyes D· . I:!ct blue and Direct red. Immobil ized cell s of P. jI!lorescens D41 efficient ly degraded Dir;:ct red (82%) and Direct blue (71 %) in the presence of glucose.

Benzidine (BZ) based azodyes are widely used in dye manufacturing, textile dyeing, color paper printing and leather industries I. The potential toxicity, mutagenicity and carcinogenicity of azodyes are well documented2. In 1980, the National Institute of Occu­pational Safety and Health (NIOSH) published the studies of carcinogenicity of BZ based dyes in ex­perimental animals and epidemiological studies of workers exposed to dyes3

. BZ dyes have long been reco~nized as a carcinogen in human urinary blad­der4. and tumorigenic in a variety of laboratory ani­mals6

. A higher incidence of hemorrhagic cystitis, recurrent papillomas and cancer of the urinary bladder in workers employed in the manufacturers benzidine based dyes with an average of 12 years of employ­ment. The effect appears to be due to the nitro (-N02) and amino (-NH2) groups of the intermediates. The hydrogen substitution in 1 and 4 position of benzene ring have a high affinity for binding carcinogenic sub­stances and the route of entry of benzidine into diffe­rent target organs of mts? The NIOSH declared BZ based dyes to be carcinogenic due to their biotrans­formation to BZ. Since BZ is used as a reactant in dye synthesis workers could be directly exposed to the carcinogen. Also exposure could occur via generation of BZ during the bleaching of dyed fabrics and as a result of r'je metabolisms. Experimental studies with rats , dogs, and hamsters have shown that benzidine and BZ congener based dyes wh ich excrete poten-

···Correspondent author: Phone: 91-452-458478,458209,459873 Fax No: 9 1-452-458478; Email: pguna@eth .net

tially carcinogenic aromatic amines and their N­acety lated derivatives in their urine 9-11. Bioremedia­tion approaches provide method of detoxification or degradation of carcinogenic compounds and wide spectrum of aromatic compounds from the environ­ment l2 . Here, we report the use of immobilized cells of Pseudomonas fluorescens D41 for the degradation of benzidine-based dyes Direct blue and Direct red.

Materials and Methods Benzidine based azodyes namely Direct blue (Di ­

azotised I-amino 2-naphthol-4-sulphonicacid with B­naphthol) and Direct red (Tetra azotised Benzedrine with napthionic ac id) were purchased from Texti Ie Committee, Government of India, Ministry of Tex­tiles, Quality & Ecolab, Yladurai. The r.ledia compo­nents were obtained fro'n Hi-Media Bombay, India.

Microorganisms Rhizosphere soi l samples were collected from vari ­

ous sources, and screened for organisms capable of degrading dyes in minimal agar plates containing (gil): (NH4hS04, 0.5; KH2P04, 2.66; Na2HP04,32; Agar, 20 and dyes (50 ppm). Colonies were selected on the basis of their ability to form clear zone on agar plate. They were subsequentl y grown in the same liq­uid medium contai ning the selected dye.

Immobilization of cells The seed cultures of P. fluorescens D41 was grown

in nutrient broth and the cells from the late log phase cu lture were harvested by centrifuging at 10,000 rpm for 10 minutes, and the cells were washed and sus­pended in 0. 1 % (w/v) NaCI. To 75 ml 0.1 % (w/v)

1132 INDIAN J EXP BIOL, OCTOBER 2002

NaCI solution, 3.5% (w/v) of sodium alginate was added, dissolved by constant agitation and then steril­ized . To the sterile sodium alginate slurry, cell sus­pension of P. fluorescens D41 , 25 ml of 0.1 % (w/v) NaCI were added and mixed thoroughly without forming any air bubble in the slurry. The slurry con­taining the cells was extended as drops through a tube of (2mm diameter) into 4% CaCI] sol ution 13 The drops formed into spherical beads of 2 mm size. The gel beads were kept in 4% CaCh sclution at 5°C for about an hour for complete gelation. Then the beads were washed with sterile distilled water and used.

Decolourization study The decolourization (%) was also calculated by the

modified method of Yatome et al 14. Measuring the decrease in absorbance at 610 and 525 nm respec­tively monitored as decolourization. The uninoculated medium was maintained as control. Decolourization activity was calculated and expressed in terms of per­centage[ Decolouation (%) = Initial absorbance - Ob­served absorbance I Initial absorbance x 100 ].

Experimental design Immobilized cells of P . . fluorescens D41 and the

dye Direct blue (50 ppm) or Direct red were added to

A

B

4 - amino biphenyl

[3cnzidinc

1

c

the 500 ml Erlenmeyer tlasks incubated at 30°C for 24 hr with mild agitation. At interva ls samples with­drawn, 2 III injection were analyzed by GC-MS .

The capillary gas chromatograph was equipped with a standard Schimadzu GC-17 A injector operated in the splitless. The injection port temperature was 270°e. The oven was initially maintained at 60°C for I min and heated at 6°C/min to 250°C, where it re­mained for 30 min, separation was achieved on DB5 -60 mrts 0-32~lg 60-m capillary column operated with 7 Ib/m2 of helium carrier gas. The capillary column was installed directly in to the Schimadzu GC-17 A Mass Selective Detector Mass Spectrophotometer via the standard GC-MS interface oven. The interface oven temperature was maintained at 280°e. The mass spectrometer was operated in the 70-V electron im­pact mode. Source temperature was 270°e. The ana­lyzer was scanned from 50 - 300 M / Z.

Results and Discussion

Screening of bacterial strains degrading dyes Soil samples were diluted and plated on the nutri­

ent agar containing different benzidine dyes (50 ppm). Colonies exhibiting clearing zone around them were scored. One of the bacterial strain exhibited clear

[3cnzidinc

, l

D

Benzidine

J .. Fig. I-GC-MS analysis of azodyes and thei r degradation products. GC-MS profiles of Direct blue (A) and Direct red (C) Immobi lized P. flllorescens 041 cells were inoculated into the minimal medium supplemented with 50 ppm of Direct blue and Direct red and 2 gil glucose and incubated at 37°C for 24 hr. The degradation products of Direct blue (8) and Direct red (D) were analysed by GC-MS.

PUVANESWARI et at. : BIODEGRADATION OF BENZIDINE BASED AZODYES 1133

zone in the medium containing benzidine dyes namely Direct blue and Direct red. Later, we have identified this Pseudomonas jl.uorescens strain and designated as Pseudomonas jl.uorescens D41.The efficiency of this strain for the decolorization of dyes were meas­ured. This strain efficiently decolorized the dye Direct red (82%) and the dye Direct blue (71 %). The dye Direct blue contains two carcinogenic aromatic com­pounds namely Benzidine (BZ), 4-amino biphenyl (4-ABP) and Direct red has single carcinogenic com­pound Benzidine (BZ) .The GC-MS profile of Direct blue and Direct red is shown in Fig. 1. The compound with the retention time of 35 mts and the mass spec­tral fragmentation pattern M+mlz 184 was identical to the authentic BZ. After the growth of the P. jl.uores­cens D41 in the medium containing dye, the metabo­lites were extracted from the spent medium by the following method. The treated sample is reduced with sodium dithionate, extracted once in diethyl ether , after drying the content is reconstituted in methonol. The metabolites identified in the extract were 4-amino biphenyl (4-ABP) with the retention time of 26.6min and mass spectral fragmentation pattern M+m/z 169 and identical to authentic compounds Benzidine with the retention time of 35min and the Mass spectral fragmentation pattern M+m/z 57 was identified. The structures of Direct blue and Direct red and the identi­fied metabolites was shown in (Fig. 2). BZ was the product of the reduction of two azo (-N=N-) bonds of Direct blue, whereas 4-ABP was ostensibly formed upon monodeamination of BZ.

A Direct blue OH NH2 H2N OH

MN=N-D-O-N=N-+

N ao:;s~ NaO:lS~SO:1'l

(Dlazotlsed 1- amino- 2- napthol- 4 - sulphonlc acid with B· napthol)

B Direct red

NH2 H~

~~N-D-O-N=N~ ~ ~

SO;ti SO;ti

(Tetraazotised benzklne with Naphthionic acid)

c NH2--O-O-NH2 0-0-

Benzidine (BZ) 4· Amlnobl phenyl · ( 4· ABP)

Fig. 2-Chemical structures of the dye Direct blue and Direct red and their possible reactive benzidine group. A. Direct blue B. Direct red C. Possible metabolites of benzidine group.

Dye deg radation by viable cells of P. jluorescens D41 The cells grown in the dye containing medium was

separated by centrifugation and the spent medium and pellet were independently inoculated into the fresh medium containing the dye and the decolourization efficiency was examined. Decolourization of dyes was observed in the medium inoculated with the cell pellet while there was no decolourization noticed in the medium inoculated with the spent medium. These results suggested the direct involvement of bacterial cells in the decolourization of the azodyes.

Dye decolourization may take place through dye removal by simple adsorption of the dye at the cell surface or degradation of the dyes by the cells. Cells would become deeply colored if the dyes were re­moved by adsorption but cells remain colorless if they degrade the dyes. There was no change in the color of the pellet of the culture of P. jl.uorescens D41 after their growth in dye containing medium. To find out optimum cell mass concentration for the decolouriza­tion of the azodyes, the cell mass concentration from 2.4 to 24 gil was used. There was no significant de­colourization of the dye was observed with the cell mass of 2.4 gil. About 40-50% decolourization was obtained with the cell mass of 4.8 to 7.2 gil. Greater than 60% decolourization of both dyes were obtained with the cell mass of 12 to 24 gil in 4hr (Fig. 3). There was considerable delay in decolourization in the pellet with low cell density. Similarly, when the heat-killed cells were incubated overnight in the medium contain­ing dyes, there was no significant color reduction. Colour removal was achieved Direct red (65 %) and Direct blue (55 %) with in 4 hr only by the use of ac­tively growing bacterial cells (Fig. 4). Therefore, the decolourization of the dye was through bacterial deg­radation of the dyes.

Dye degradation by immobilized cells of P. jl.uores­cens D41

Several bacterial strains are known to degrade a wide range of compounds and thereby detoxify them I5

-17

.

Application of whole cell immobilization technology is a promising method of exploiting this ability of the bacteria in degradation of the dyes. Immobiliza­tion prevents washout of cells and allows a high cell density to be maintained in a continuous reactor. The catalytic stability is often improved upon immobiliza­tion, microorganisms may tolerate and degrade higher concentrations of toxic compounds than do their non­immobilised counterparts 14. To investigate the degra­dation and decolourization percentage, free cells and

1134 INDI AN J EXP BIOL, OCTOBER 2002

immobilized cell s of P, jluorescens 0 4 1 with various inoculum sizes, (5 to 15% v/v) were used, Maximum

100 A

80

60

40

20

o

100 B

80

60

40

20

o 2 3 4 5

Incubation Time (hr)

Fig, 3 - Effect o f cell mass concentrat ions of P. jluorescens 041 on decolouri zation of the dye Direct blue and Direct red . The

different cell mass (. ) 2.4 gil ; (X) 4.8 gil ; (. ) 7.2 gil ; (6) 12 gil ; (_) 24 gil of P. jluorescells 04 1 was inoculated in to the mini mal med ium supplemented with 50 ppm of Direct blue (A) and Direct red (B) and 2 gil glucose incubated at 37°C fo r 4 hr. The residual dye concentration was calcula ted by measuring absorbance of cu lture supernatant at 610 nm (Direc t blue) and 525 (Direc t red).

deco lourization efficiency was obtained by the (15 % v/v) (Fig. 4) immobili zed cell s, and shown that im­mobi lised systems have an advantage over a free cell syste m (Figs 5, 6) resulting in sustained degradation abi lity. A number of reports suggested that the certain bacteria cou ld uti lize specific azodye and certain sul­fonated azo compounds as sole carbon and energy

100 A

80 C

60

40

n n t;f. 20 n n c 0 .S ~ N

·C 80

U D 0 '0 u 60 ~

Q 40

20

2 3 4

Incubation time (hr)

Fig. 4-Effect of free and immob ilized cell s of P. jluorescells 04 1 on decolourization of the dyes Direct red and Direct blue. P. jlllorescens 041 free cel ls (A , C) and of immobilized ce lls (B, D) we re inocu lated into the m inimal mediuIll supplemellled with 50 ppm o f Direct blue (C,D) and Direct red (A ,B) incubated at 3r C fo r 4 hr. The res idual dye concentration was calculated by meas­urin g absorbance of culture supernatant at 6 10 nm (Di rect blue) and 525 (Direct red). A - Free cells on degradat ion of Direct red; B - Immobili zed cel ls; C - Free cell s on degradation of Direct blue 0 - Immobili zed cel ls.

Fig. 5 - Biodegradation of dye-D irec t blue by P. .f7uorescells 04 1. A- COlllro l B- Free ce ll s C- Immobi lized cell s.

PUVANESWAR I et al.: BIODEGRADATION OF BENZIDINE BASED AZODYES 1135

Fig. 6-Biodegradation o f dye-D irect red by P. jluorescells 041. A- Control B- Free cells C- Immobili zed ce ll s

source. The enzyme azo benzene reductase were puri­fied and charecterised from Pseudomonas strain K24 18

. Several authors employed different microor­gani sms for degradation of vari ous chemicals and toxic compounds such as pentachlorophenol , 3,4-dichloroaniline, 2-chlorophenol 19-22 . From the present study, it can be concluded that the complete transfor­mation of carcinogenic compounds, benzidine (BZ), and 4- amino biphenyl (4-ABP) to non-toxic co m­pounds can be achieved with immobili zed cells of P. fluorescens D4l.

Acknowledgement One of the author (N.P) thanks UGC, New Delhi

for the financial support and A.P.A.College, Palani for deputing her for higher studi es . Centre for Excel­lence in Genomic Sciences, Madurai Kamaraj Univer­sity is gratefull y acknowledged for the support.

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