A Simple Method To Screen For Azo-dye Degrading Bacteria

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J. Environ. Biol. 30(1), 89-92 (2009) [email protected]

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 (*[email protected])

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.

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.

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

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


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Table - 1: Growth of S. Marcescens on various carbon sources in BIOLOG’S GN microplates Carbon source

Growth (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

+ +/+ + + + + + + + + + + + + + + + +/+ + + + + + + + +/+ -

+ = positive reaction - = negative reaction -/+ = borderline

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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

+/+ + + + + +/+ + + + + +/+ +/+ + +/+ + + +/+ + +

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, semisolidified 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 K2HPO4, 1.33 g KH2PO4, 0.20 g of MgSO4.7H2O, 1.00 g of (NH4)2SO4, 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 CoCl2.6H2O, 11.80 mg l-1 of NiCl2, 6.30 mg l-1 of CrCl2, 15.70 mg l-1 of CuSO4.5H2O, 0.97 g l-1 of FeCl3, 0.78 g l-1 of CaCl2.2H2O and 10.00 mg l-1 of MnCl2.4H2O. 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

91 BIEBRICH SCARLET (BS)

Control

Innoculated

DIRECT BLUE (DB)

Control

Innoculated

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

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 carboxyorange-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. Journal of Environmental Biology


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