Journal of Environmental Biology �January, 2009�

© Triveni Enterprises, Lucknow (India) J. Environ. Biol.

ISSN : 0254-8704 30(1), 89-92 (2009)

http : //www.jeb.co.in info@jeb.co.in

A simple method to screen for azo-dye-degrading bacteria

M.A. Syed, H.K. Sim, A. Khalid and M.Y. Shukor*

Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,

University Putra Malaysia, 43400 Serdang, Selangor, Malaysia

(Received: December 18, 2007; Revised received: June 10, 2008; Accepted: June 20, 2008)

Abstract: A stab-culture method was adapted to screen for azo dyes-decolorizing bacteria from soil and water samples. Decolorized azo

dye in the lower portion of the solid media indicates the presence of anaerobic azo dyes-decolorizing bacteria, while aerobic decolorizing

bacteria decolorizes the surface portion of the solid media. Of twenty soil samples tested, one soil sample shows positive results for the

decolourisation of two azo dyes; Biebrich scarlet (BS) and Direct blue 71 (DB) under anaerobic conditions. A gram negative and oxidase

negative bacterial isolate was found to be the principal azo dyes degrader. The isolate was identified by using the BiologTM identification

system as Serratia marcescens.

Key words: Azo dyes, Stab-culture, Screening method

PDF of full length paper is available with author (*yunus@biotech.upm.edu.my)

Introduction

Many of the known bacterial isolates that could decolorize

azo dyes can only do so under static or anaerobic conditions. In

addition, growth and decolorizing ability are often a two-stage

process. Here, we report on a simple method to screen for azo dye

decolorizing bacteria in soil and water samples. Since the first

commercial synthetic dye, Mauveine, was discovered in 1856, more

than 100 000 dyes have been generated worldwide with an annual

production in excess of 7 x 105 metric tonnes (Zollinger, 1987). Azo

dyes are the largest class of commercially available synthetic dyes

and have found wide applications in textiles, food, cosmetics, plastic,

laboratories, leather, paper printing, colour photography,

pharmaceutical and toy industries (Chung, 1983; Garrigós et al.,

2002; Mathur and Bhatnagar, 2007; Pant et al., 2008; Laowansiri et

al., 2008). Their widespread use, especially in textile and dyestuff

industries, coupled with the fact that azo dyes are not readily degraded

in conventional aerobic treatment systems makes this class of

xenobiotic a significant environmental problem. Until today there is

still no effective and economical treatment of azo dyes that contain

effluents. The employment of physico-chemical methods, although

effective, is cost prohibitive and often lead to extra solid wastes. As a

viable alternative, biological processes have received increasing

interest owing to their cost effectiveness (Banat et al., 1996). Over

the past decades, many microorganisms that are capable of

degrading azo dyes, including bacteria (Haug et al., 1991), fungi

(Demir et al., 2007; Singh et al., 2007), yeasts (Banat et al., 1996),

actinomycetes (Zhou and Zimmerman, 1993) and algae (Jinqi and

Houtian, 1992) have been documented.

The anaerobic reductive cleavage of azo bonds is often

always favoured over the aerobic condition as oxygen molecules

will compete with the azo group for electrons in the oxidation of

reduced electron carrier, i.e. NADH (Banat et al., 1996). The low

specificity of azoreductase often involves low molecular weight redox

mediators (e.g., flavins or quinones) and allows for unspecific

reduction, thus providing a wider range of dyes reduction (Keck et

al., 1997). Many bacteria have been reported to readily decolourise

azo dyes under anaerobic conditions. Bacteroides sp., Eubacterium

sp., Clostridium sp., Proteus vulgaris and Streptococcus faecalis

(Bragger et al., 1997; Rafii et al., 1990). However, the aerobic

decolourisation of azo dyes can also be carried out in the presence

of external carbon sources and, presumably, does not use azo dyes

as the sole carbon or energy sources (Padmavathy et al., 2003).

Zissi et al. (1997) had reported on the cometabolic reductive cleavage

of p-aminoazobenzene to aniline during aerobic growth on glucose.

The reductive decolourisation of sulfonated azo dyes by Bacillus

sp., Pseudomonas sp., Sphingomonas sp. and Xanthomonas sp.

under aerobic conditions in the presence of additional carbon sources

has also been reported (Dykes et al., 1994; Sugiura et al., 1999).

Most of the known azo dye decolourizer was isolated from sludge,

soil and water samples. In all of the studies, the initial screening was

carried out either in shake flasks or solid azo dye media. Since the

majority of azo dye-degrading bacteria occur under anaerobic

conditions, isolation of azo dye-degrading bacteria is rather slow. It

can require up to four days to observe the decolourization zone due

to the high oxygen tension surrounding the bacterial colonies. The

usage of stab culture, as far as microbes are concerned, is usually

restricted to bacterial maintenance (Wise et al., 2006). To our

knowledge, the use of stab culture to screen for anaerobic and

aerobic xenobiotics-degrader has never been reported. Here, we

report on a simple method to screen for azodye-decolorizing bacteria,

using the well-known stab-culture method often used for culture

storage. Using this method, results can be obtained within 24 hours.

Materials and Methods

Isolation of bacteria: Soil samples, each measuring approximately

10 grams were taken randomly to a depth of 5 cm from the topsoil

using sterile spatula and stored in sterile screw-capped polycarbonate

Journal of Environmental Biology �January, 2009�

Syed et al.

Table - 1: Growth of S. Marcescens on various carbon sources in

BIOLOG’S GN microplates

Carbon sourceGrowth

(After 24 hr incubation)

Water (control) -

α-Cyclodextrin -

Dextrin +

Glycogen +/-

Tween 40 +

Tween 80 +

N-Acetyl-D-Galactosamine +

N-Acetyl-D-Glucosamine +

Adonitol +

L-Arabinose -

D-Arabitol -

D-Cellobiose -

i-Erythritol +

D-Fructose +

L-Fructose +

D-Galactose +

Gentiobiose -

α-D-Glucose +

m-Inositol +

α-D-Lactose -

Lactulose -

Maltose +

D-Mannitol +

D-Mannose +

D-Melibiose -

β-Methyl-D-Glucoside +

D-Psicose +

D-Raffinose -

L-Rhamnose -

D-Sorbitol +/-

Sucrose +

D-Trehalose +

Turanose -

Xylitol +

Pyruvic Acid Methyl Ester +

Succinic Acid-Mono-Methyl-Ester +

Acetic Acid -

Cis-Aconitic Acid +

Citric Acid +

Formic Acid +

D-Galactonic Acid Lactone -

D-Galacturonic Acid +/-

D-Gluconic Acid +

D-Glucosaminic Acid -

D-Glucuronic Acid -

α-Hydroxy butyric Acid -

β-Hydroxy butyric Acid -

γ- Hydroxy butyric Acid +/-

p-Hydroxy Penylacetic Acid +

Itaconic Acid -

α-Keto Butyric Acid -

α-Keto Glutaric Acid +

α-Keto Valeric Acid -

D,L-LacticAcid +

Malonic Acid -

Propionic Acid -

Quinic Acid -

D-Saccharic Acid -

Sebacic Acid -

Succinic Acid +

Bromosuccinic Acid +

Succinamic Acid -

Glucuronamide +/-

L-Alaninamide -

D-Alanine +

L-Alanine +

L-Alanyl-Glycine +

L-AsparticAcid +

L-Glutamic Acid +

Glycyl-L-Aspartic Acid -

Glycyl-L-Glutamic Acid +/-

L-Histidine +

Hydroxyl-L-Proline +/-

L-Leucine -

L-Ornithine -

L-Phenylalanine -

L-Proline +

L-Pyroglutamic Acid -

D-Serine -

L-Serine +

L-Threonine +/-

D,L-Carnitine -

γ-Amino Butyric Acid -

Urocanic Acid -

Inosine +

Uridine +

Thymidine +

Phenylethyl-amine -

Putrecine +/-

2-Aminoethanol -

2,3-Butanediol -

Glycerol +

D,L-α-Glycerol Phosphate +

α-D-Glucose-1-Phosphate +

+ = positive reaction

- = negative reaction

-/+ = borderline

90

Journal of Environmental Biology �January, 2009�

A simple method to screen for azo-dye-degrading bacteria

tubes. The soil samples were taken from several locations in Malaysia

near textile or dye-based industries. The exact locations were; (1)

Taman Tanjung and Lengkok Burmah, Penang, (2) Taman Miharja,

Selangor and (3) Bintulu, Sarawak. The samples were immediately

placed on ice until returned to University Putra Malaysia for further

examinations. Approximately one gram of each soil sample was

serially diluted ten times in 0.85% saline. Bacterial samples from

diluted soils were inoculated via stab inoculation into loosely-capped

microbiological test tubes containing the screening medium, semi-

solidified with 5.0 g l-1 of agar. The medium used was a modified

version of Hayase et al. (2000) and contained 2.34 g K2HPO

4, 1.33

g KH2PO

4, 0.20 g of MgSO

4.7H

2O, 1.00 g of (NH

4)2SO

4, 0.50 g of

NaCl, 0.10 g of yeast extract, 1.00 g of glucose and 1.0 ml of trace

element solution per liter, adjusted to the final pH of 7.0 with 3 M

NaOH and HCl. The trace element solutions contained 11.90 mg l-1

of CoCl2.6H

2O, 11.80 mg l-1 of NiCl

2, 6.30 mg l-1 of CrCl

2, 15.70 mg l-1 of

CuSO4.5H

2O, 0.97 g l-1 of FeCl

3, 0.78 g l-1 of CaCl

2.2H

2O and 10.00

mg l-1 of MnCl2.4H

2O. Bacteria from soil samples were tested for their

abilities to decolourise four azo dyes; Orange G (OG), Ponceau 2R

(P2R), Biebrich Scarlet (BS) and Direct blue 71 (DB). The final dye

concentration was 0.10 g l-1. Colour changes were qualitatively

observed.

Identification of bacteria: Isolates exhibiting distinct colonial

morphologies were isolated by repeated sub culturing into basal salt

medium and solidified basal salt medium until purified strains were

obtained. Identification at species level was performed by using

Biolog GN microplate (Biolog, Hayward, CA, USA) according to the

manufacturer’s instructions. Briefly,a pure culture of the bacterium

was grown on a Biolog Universal Growth agar plate. The bacteria

were swabbed from the surface of the agar plate, and suspended to

a specified density in GN/GP inoculating fluid. A hundred fifty µl of a

bacterial suspension was pipetted into each well of the micro-plate.

The micro-plate was incubated at 30oC or 35oC depending upon the

nature of the organism for 4-24 hr according to manufacturer’s

specification. The micro-plate was read with the Biolog MicroStationTM

system and compared to database.

Results and Discussion

Figure 1 shows typical screening results using the stab culture

method after 24 hr of incubation at room temperature. Of the twenty

soil samples tested, one soil sample showed the ability to decolorize

two azo dyes- Biebrich scarlet (BS) and Direct blue 71 (DB). The

location of the decolourization zones in both dye tubes indicates the

requirement for anoxic conditions. We were unable to isolate aerobic

azo dyes-decolorizing bacteria. If we had done so, the decolourization

zone would occur near or on the surface of the agar. The selective

advantage of a low oxygen tension environment on the

decolourisation of azo dyes, as observed, was probably due to the

electron-withdrawing properties of the azo bond itself that produces

electron deficiency at the site of cleavage and could only thus be

cleaved in the presence of reducing agents, generated during

anaerobic metabolism on static incubation (Rieger et al., 2002). After

further screening, a gram and oxidase negative bacterial isolate

was found to be the principal azo dyes degrader. The isolate was

identified by using the 95 carbon Biolog GN Microplate biochemical

tests (Table 1) as Serratia marcescens with 99% probability (0.749

similarity; 3.80 distance).

Methyl red degradation occurs both under aerobic and

anaerobic conditions. Aerobic reduction has been reported to

occur in several bacteria such as Pseudomonas sp. (Kulla et al.,

1983), Bacillus sp. (Maier et al. (2004) and Klebsiella pneumoniae

(Wong and Yuen, 1996). On the other hand, anaerobic conditions

are required in S. cerevisiae (Jadhav et al., 2007). Acetobacter

liquefaciens is unique in that decolourisation of methyl red occurs

efficiently under both aerobic and anaerobic (static) conditions

(So et al., 1990). The aerobic azoreductases from the carboxy-

orange-degrading Pseudomonas strains K22 and KF46

reductively cleave several sulfonated azo dyes (Kulla et al.,

1983). Therefore, the presence of an aerobic azoreductase in

Isolate 13 was probably responsible for the aerobic degradation

of methyl red dye. Under aerobic conditions, Biebrich Scarlet is

not readily metabolized by the isolate. However, under anaerobic

conditions, many bacteria reduce the highly electrophilic azo

bond in the dye molecule, reportedly by the activity of low

specificity cytoplasmic azoreductases, to produce colourless

aromatic amines (Pearce et al., 2003). The clear zone observed

at the bottom of the test tube suggests that the degradation of

Biebrich Scarlet occurs under anaerobic conditions. The

decolourization of azo dyes can easily be seen from the side of

the tube. The overall design is simple, yet effective, in screening

for aerobic and anaerobic azo dye-degrading bacteria.

Acknowledgments

This project was supported by funds from The Ministry of

Science, Technology and Innovation, Malaysia (MOSTI) under

IRPA-EA grant no: 09-02-04-0854-EA001.

Fig. 1: Stab inoculation of soil isolate in agar deep tubes containing BS (left)

and DB (right). The control uninoculated dye tubes are at the left of each pair

of tubes

BIEBRICH SCARLET (BS) DIRECT BLUE (DB)

Control Innoculated Control Innoculated

91

Journal of Environmental Biology �January, 2009�

References

Banat, I.M., P. Nigam, D. Singh and R. Marchant: Microbial decolourisation

of textile-dye-containing effluents, a review. Bioresource Technol., 58,

217-227 (1996).

Bragger, J.L., A.W. Lloyd, S.H. Soozandehfar, S.F. Bloomfield, C. Marriot

and G.P. Martin: Investigations into the azo reducing activity of a

common colonic microorganism. Inter. J . Pharm ., 157, 61-71

(1997).

Chung, K.T.: The significance of azo-reduction in the mutagenesis and

carcinogenesis of azo dyes. Mutat. Res., 114, 269-281 (1983).

Demir, G., H.K. Ozcan, N. Tufekci and M. Borat: Decolorization of Remazol

Yellow RR Gran by white rot fungus Phanerochaete chrysosporium.

J. Environ. Biol., 28, 813-817 (2007).

Dykes, G.A., R.G. Timm and V.A. Holy: Azoreductase activity in bacteria

associated with the greening of instant chocolate puddings. Appl. Environ.

Microbiol., 60, 3027-3029 (1994).

Garrigos, M.C., F. Reche, M.L. Marín and A. Jimenez: Determination of

aromatic amines formed from azo colorants in toy products. J.

Chromatogr. A., 976, 309-317 (2002).

Haug, W., A. Schmidt, B. Nörtemann, D.C. Hempel, A. Stolz and H-J.

Knackmuss: Mineralization of the sulfonated azo dye Mordant Yellow

3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium.

Appl. Environ. Microbiol., 57, 3144-3149 (1991).

Hayase, N., K. Kouno and K.J. Ushio. Isolation and characterization of

Aeromonas sp. B-5 capable of decolorizing various dyes. J. Biosci.

Bioeng., 90, 570-573 (2000).

Jadhav, D.J.P., G.K. Parshett i, S.D. Kalme and S.P. Govindwar:

Decolourization of azo dye methyl red by Saccharomyces cerevisiae

MTCC 463. Chemosphere, 68, 394-400 (2007).

Jinqi, L. and L. Houtian: Degradation of azo dyes by algae. Environ. Pollut.,

75, 273-278 (1992).

Keck, A., J. Klein, M. Kudlich, A. Stolz, H.J. Knackmuss and R. Mattes:

Reduct ion of azo dyes by redox mediators originat ing in the

naphthalenesulfonic acid degradation pathway of Sphingomonas sp.

strain BN6. Appl. Environ. Microbiol., 63, 3684-3690 (1997).

Kulla, H.G., F. Klausener and U. Meyer: Interference of aromatic sulfo groups

in the microbial degradation of the azo dyes Orange I and Orange II.

Arch. Microbiol., 135, 1-7 (1983).

Laowansiri, Sunantha, Soydoa Vinitnantharat, Pawinee Chaiprasert and Sung

Ryong Ha: Anaerobic degradation kinetics of reactive dye with different

carbon sources. J. Environ. Biol., 29, 309-314 (2008).

Maier, J., A. Kandelbauer, A. Erlacher, A. Cavaco-P and M. Gubitz: A new

alkali-thermostable azoreductase from Bacillus sp. strain SF. Appl.

Environ. Microbiol., 70, 837-844 (2004).

Mathur, Nupur and Pradeep Bhatnagar: Mutagenicity assessment of textile

dyes from Sanganer (Rajasthan). J. Environ. Biol., 28, 123-126 (2007).

Padmavathy, S., S. Sandhya, K. Swaminathan, Y.V. Subrahmanyam, T.

Chakrabarti and S.N. Kaul: Aerobic decolorization of reactive azo

dyes in presence of various cosubstrates. Chem. Biochem. Eng., 17,

147-151 (2003).

Pant, Deepak, Anoop Singh, Yamini Satyawali and R.K. Gupta: Effect of

carbon and nitrogen source amendment on synthetic dyes decolourizing

efficiency of white-rot fungus, Phanerochaete chrysosporium. J.

Environ. Biol., 29, 79-84 (2008).

Pearce, C.I., J.R. Lloyd and J.T. Guthrie: The removal of colour from textile

wastewater using whole bacterial cells, a review. Dyes Pigm., 58,

179-196 (2003).

Rafii, F., W. Franklin and C.E. Cerniglia: Azoreductase activity of anaerobic

bacteria isolated from human intestinal microflora. Appl. Environ.

Microbiol., 56, 2146-2151 (1990).

Rieger, P.G., H.M. Meier, M. Gerle, U. Vogt, T. Groth and H.J. Knackmuss:

Xenobiotics in the environment, present and future strategies to obviate

the problem of biological persistence. J. Biotech., 94, 101-123 (2002).

Singh, K.D., S. Sharma, A. Dwivedi, P. Pandey, R.L . Thakur and V. Kumar:

Microbial decolorization and bioremediation of melanoidin containing

molasses spent wash. J. Environ. Biol., 28, 675-677 (2007).

So, K.O., P.K. Wong and K.Y. Chan: Decolorization and biodegradation of

methyl red by Acetobacter liquefaciens. Toxic. Assess., 5, 221-235 (1990).

Sugiura, W., T. Miyashita, T. Yokoyama and M. Arai: Isolation of azo-dye-

degrading microorganisms and their application to white discharge

printing of fabric. J. Biosci. Bioeng., 88, 577-581 (1999).

Wise, A.A., Z. Liu and A.N. Binns: Culture maintenance of Agrobacterium

strains. Methods Mol. Biol., 343, 3-13 (2006).

Wong, P.K. and P.Y. Yuen: Decolorization and biodegradation of methyl red

by Klebsiella pneumoniae rs-13. Water Res., 30, 1736-1744 (1996).

Zhou, W. and W. Zimmermann: Decolorization of industrial effluents containing

reactive dyes by actinomycetes. FEMS Microbiol. Lett., 107, 157-162

(1993).

Zissi, U., G. Lyberatos and S. Pavlou: Biodegradation of p-aminobenzene

by Bacillus subtilis under aerobic conditions. J. Ind. Microb. Biotech.,

19, 49-55 (1997).

Zollinger, H.: Color chemistry–syntheses, properties and applications of

organic dyes and pigments. VCH, New York. pp. 12-13 (1987).

Syed et al.92