International Journal of Environment 1: 34-39 (2011)

ORIGINAL ARTICLE

Decolorization of Methyl Red by Staphylococcus arlettae PF4 Isolated from Garden Soil Pijush Sarkar, ANM Fakhruddin, Md. Kamruzzaman Pramanik and Abdullah-Al-Mahin
Abstract
Azo dyes are toxicant to biological system and cause serious damage to environment. In the present study, an attempt was made to isolate methyl red, an azo dye, decolorizing bacteria from soil. Among six isolates with methyl red decolorizing ability, the potential bacterial isolates, PF4 was selected for identification and characterization. The organism was found to belong to the genus Staphylococcus and species arlettae. The isolate S. arlettae PF4 completely decolorized 600 and 800 mg/l methyl red within 24 and 48 h respectively. S. arlettae PF4 partially decolorized 1000 and 1200 mg/l methyl red within 72 h. The maximum decolorization rate (mg/l/h) for 600, 800, 1000 and 1200 mg/l methyl red concentrations were 25, 22.38, 17.08 and 16.29 mg/l/h respectively. Thus, due to high potential for methyl red decolorization the soil isolate S. arlettae PF4 can be used in the biological treatment plant of industrial effluent containing azo dyes. Key words: Methyl Red, Staphylococcus arlettae, Decolorization, Garden soil.

ISSN: 2186-0009 http://www.BENJapan.org/IJE

ARTICLE HISTORY Received: 09 April 2011 Revised: 13 August 20111 Accepted: 22 September 2011 Published online: 24 October 2011

2011, International Journal of Environment. All rights reserved. I. INTRODUCTION The industrial effluents contain toxic and hazardous pollutants. One particular class of synthetic chemicals which is of major concern is synthetic dyes and dye intermediates [1, 2]. More than 100000 commercially available dyes are known and close to one million tons of these dyes are produced annually worldwide [3]. Azo dyes are the largest and the most diverse group of synthetic dyes, which are essential for satisfying the ever growing demand in terms of quality, variety, and speed of coloration of large number of substances.
AUTHORS INFO Pijush Sarkar e-mail: pijushrobi@gmail.com ANM Fakhruddin e-mail: a.fakhruddin2@mail.dcu.ie Department of Environmental Sciences, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh. Md. Kamruzzaman Pramanik e-mail: kpramanik2003@yahoo.com Abdullah-Al-Mahin* e-mail: mahinmicro@yahoo.com Institute of Food and Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka-1344, Bangladesh. *Corresponding author e-mail: mahinmicro@yahoo.com Tel: +880-01738326000, Fax: +880-2-7789620 34

Azo dyes are the major group of dyes used in the textile industry and contribute between 50-56% of the colors in the textile dyes [4, 5]. Azo dyes are characterized by the presence of one or more azo groups -N=N- [6], which are responsible for their coloration and when such a bond is broken (degraded) the compound loses its color [7]. It is estimated that over 10% of the dye used in textile processing does not bind to the fibers and is therefore released to the environment [8, 9]. Some of these compounds pose a serious threat because of their carcinogenic potential or cytotoxicity [1014]. Dyes with striking visibility in recipients may significantly affect photosynthetic activity in aquatic environment due to the reduced light penetration and may also be toxic to some aquatic lives due to the effluents containing several types of chemicals such as dispersants, leveling agents, acids, alkalis, carriers and various dyes [15]. Methyl red is an indicator dye that turns red in acidic solutions. It is an azo dye and is a dark red crystalline powder. Its molecular formula is C15H15N3O2, molar mass is 269.3 g/mol, density is 0.791 g/cc and melting point is 179-182 C. Methyl red is a pH indicator; it is red in pH under 4.4, yellow in pH over 6.2, and orange in between, with a pKa of 5.1 [16]. Currently the major methods of textile waste water treatment involve physical and chemical process. However, such technologies usually involve complicated procedures or economically unfeasible. Oxidation, a physical treatment used in wastewater, requires high energy and

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produces hazardous by-products. Chemical methods also produce high concentration of sludge during treatment [17]. Microbial decolorization and degradation is an alternative approaches to physical and chemical degradation processes of color removal, which is environment friendly and cost effective [18]. Most studies on azo dye biodegradation have focused on bacteria and fungi, which are able to biodegrade and bioadsorb the dyes in textile industry effluents [2, 19, 20]. The organisms used in most of the studies were Staphylococcus sp, Escherichia coli, Bacillus sp, Clostridium sp, and Pseudomonas sp. in bacteria [21]. Mechanism for decolorization of azo dyes by microrganims has been proposed by Keck et al. [22]. Anaerobic and microaerophilic microorganisms reduce azo bonds non-specifically in anaerobic conditions leading to dye decolorization. Methyl red being the simplest azo dye was used in this study to understand the microbial decolorization of azo dyes. Objectives of present work are as follows Isolation, screening and identification of highly Potential azo dye (methyl red) decolorizing bacteria. Determination of color removal efficiency of methyl red by isolated bacteria. II. MATERIALS AND METHODS A. Sources of samples Soil samples (garden soil) were collected from different locations of Jahangirnagar University campus area. On the basis of their ability to decolorize methyl red, a total of six methyl red decolorizing bacteria were isolated from soil and then purified by repeated subculture. Screening was performed on the basis of stab culture method in the semi-liquid media for isolation of potential isolates [23]. Among the six isolates, three methyl red decolorizing bacteria were selected on the basis of their decolorization capacity (i.e. decolorization of maximum concentration within shortest time) of methyl red. B. Preparation of methyl red solution Methyl red (BDH, England) dissolved in 50 per cent ethanol. Stock of 5000 mg/l methyl red solution was prepared by dissolving 5.0 g of methyl red in 1000 ml 50 per cent ethanol. The methyl red solutions for the experiments were prepared by diluting the stock solution to give desire concentrations. C. Culture medium Basal salt media [24] together with methyl red was used for decolorization studies. The ingredients of basal salt medium were mixed

with distilled water in conical flask and the pH was adjusted to 7.0 with NaOH (3M) and HCl (3M). The trace salts solution was prepared separately in distilled water and was stored in a dark bottle for 6-8 weeks and used in experiments. Methyl red was added to the basal salt medium after sterilization. The ingredients of the per liter basal salt medium are as follows: (K2HPO4, 2.34g; KH2PO4, 1.33g; MgSO4.7H2O, 0.20g; (NH4)2SO4, 1.00g; NaCl, 0.50g; Yeast extract, 0.10g; Glucose, 1.00g; pH, 7.0). Trace salts solution was added in the basal salt medium at a concentration of 1 ml/l. The composition of the per 100 ml trace salts solution was as follows: (CaCl2.2H2O, 4.77g; FeSO4.7H2O, 0.37 g; CoCl2.6H2O, 0.37 g; MnCl2.4H2O, 0.10 g; Na2MoO4.2H2O, 0.02 g). D. Culture conditions The organisms were grown overnight in nutrient broth, centrifuged at 5000 rpm for 10 minutes and washed twice with 0.01M sodium phosphate buffer. Five ml of bacterial suspension (107 cells/ml) was used to inoculate in 95 ml sterile basal salt containing appropriate amount of methyl red concentration in 250 ml conical flasks. After inoculation, flasks were incubated in an orbital shaker at 120 rpm at 37oC. Control flasks were run in parallel. Samples were aseptically removed at regular intervals for analyzing the methyl red decolorization. The study period for methyl red decolorization was 0-72 h. E. Measurement of growth of the organisms An attempt was made to determine the cell growth by noting optical density measurement at 660 nm. However, cell flocculation in presence of methyl red did not allow monitoring cell growth spectrophotometrically. Again, flocculation size was too small to determine dry cell weight either. Therefore, growth associated methyl red decolorization was monitored by observing the increase of flocculation size with simple eye estimation. F. Assay of methyl red decolorization Decolorization of the methyl red was determined at its respective maximum absorption wavelength (430 nm) in the culture supernatants using UV spectrophotometer. Samples were aseptically taken into the centrifuge tube at different times during incubation. Bacterial cells were separated from culture supernatant by centrifugation at 5000 rpm for 10 minutes and optical density of cell free supernatants were then measured. The optical densities (OD) measured were converted to the methyl red concentration using the standard curve constructed from

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known concentrations (0 to 500 mg/l) of methyl red. The efficiency of color removal was expressed as the percentage ratio of the decolorized dye concentration to that of initial one based on the following equation [25]. Color removal (%) = [{Dye (i) Dye (r)} / Dye (i)] 100% Where, Dye (i) = initial dye concentration (mg/l), Dye (r) = residual dye concentration (mg/l). G. Identification of the bacterial isolate Bacterial staining, cultural, morphological and biochemical procedures were studied for the identification of bacterial isolate. Isolated bacteria were identified according to methods described in Bergey's Manual of Determinative Bacteriology [26]. H. Use of software and data analysis Data of study were analyzed using Sigma plot (2001). Statistical analysis was also performed for determining the methyl red concentration by using Sigma plot (2001) and Microsoft office excel (2007). ABIS6 online software was used for identifying the bacterial isolate. III. RESULTS AND DISCUSSION A. Isolation and screening of methyl red decolorizing bacteria Methyl red decolorizing bacteria were isolated from soil. On the basis of their decolorizing capacity, a total of six bacteria were isolated and purified by repeated subculturing. These six bacteria have different potentials to grow on basal salt media containing 400 to 800 mg/l of methyl red. Screening was performed on the basis of stab culture method in the semi-liquid medi a for isolation of potential isolates. Same screening method was used by Syed et al. [23]. Among the six isolates, three methyl red decolorizing bacteria were selected on the basis of their decolorization capacity of methyl red. These three isolates were designated as PF1, PF4 and PS3.

Fig. 1. Decolorization of 800 mg/l methyl red by isolates PF1, PF4 and PS3.

B. Methyl red decolorization studies in liquid culture Decolorization of 800 mg/l methyl red by these three isolates shown in Fig 1. All these isolates showed significant removal efficiency within 48 h incubation time. All isolates showed little differences in their removal rate. However, isolate PF4 showed a little better decolorizing capacity than the other two isolates. Therefore, the isolate was selected for identification and further studies. All of the three isolates grew well in liquid culture containing 800 mg/l methyl red in the presence of yeast extract and glucose and decolorize the azo dye. Decolorization of azo dye Direct Black 22 in presence of glucose and yeast extract has already been reported earlier [27]. Oforka and Oranusi [28] stated the presence of easily metabolizable carbon source as an obligatory for microbial decolorization of synthetic dye. C. Identification of bacterial isolate Preliminary identification test was performed for isolate PF4. Other identification techniques including morphological characteristics, biochemical test were also performed. Morphological characteristics, colony characteristics and other biochemical characteristics of the isolate PF4 are outlined in Table 1.

Table 1. Microscopic observation of cell, colony characteristics and other biochemical characteristics of the isolate PF4. VogesProskauer Elevation Bacterial Isolate Catalase Gram reaction Oxidase

Opacity

Gelatin -

Methyl Red

Shape

PF4

Positive Circular Small Coccus Cream Opaque Convex

Indole -

Cell shape

Color

Size

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Table 2. Carbohydrate utilization by bacterial isolate PF4 Bacterial Glucose Fructose Sucrose isolate PF4 + + + Xylose + Sorbitol Arabinose Mannitol Rhamnose Lactose Maltose + + + + +

+ = Positive reaction,

- = Negative reaction

A wide range of carbohydrate (1%, w/v) utilization tests by isolate PF4 was also performed. It was observed that the isolate PF4 was able to utilize most of the carbohydrates except arabinose and the results are shown in the Table 2. According to Bergeys manual of determinative bacteriology [26] and ABIS 6 software, the results showed the similar characteristics of Staphylococcus arlettae. So the identified isolate was named as Staphylococcus arlettae PF4.

The percentage of decolorization efficiency by S. arlettae PF4 for 600, 800, 1000 and 1200 mg/l methyl red concentrations were 100%, 100%, 89.5% and 68%, respectively (Fig. 4).

Fig. 3. The decolorization rate (mg/l/h) of methyl red at different concentrations by Staphylococcus arlettae PF4.

Fig. 2. Decolorization of methyl red at different concentrations by Staphylococcus arlettae PF4.

D. Decolorization studies of various concentrations of methyl red by the isolate Staphylococcus arlettae PF4 The results of the decolorization of various concentrations of methyl red by S. arlettae PF4 are shown in Fig. 2. S. arlettae PF4 could completely decolorize 600 and 800 mg/l methyl red within 24 h and 48 h respectively. But the isolate could partially decolorize 1000 and 1200 mg/l of methyl red within 72 h. The decolorization rates (mg/l/h) of 600, 800, 1000 and 1200 mg/l methyl red by the isolate S. arlettae PF4 were 25, 22.38, 8.88 and 7.71 mg/l/h respectively for the first 24 h (Fig. 3). The decolorization rates of 800, 1000 and 1200 mg/l/h methyl red were 10.96, 11.33 and 10 mg/l/h within 24-48 h. The decolorization rates of 1000 and 1200 mg/l methyl red were 17.08 and 16.29 mg/l/h respectively within 48-72 h. The maximum decolorization rates by the isolate for 600, 800, 1000 and 1200 mg/l methyl red concentrations were 25, 22.38, 17.08 and 16.29 mg/l/h, respectively (Fig. 3).

Fig. 4. Percentage of decolorization efficiency of methyl red within 72 h by Staphylococcus arlettae PF4.

So et al. [29] isolated Acinetobacter liquefaciens S-1 which could decolorize and degrade methyl red into two colorless compounds namely 2-aminobenzoic acid (ABA) and N-Ndimethyl--phenylene diamine (DMPD), within 7 days and metabolized a maximum of 400 mg/l of methyl red in the presence of 5.0 g/l of glucose. Wong and Yuen [30] isolated a bacterium Klebsiella pneumoniae RS-13, which could degrade methyl red 100 mg/l in presence of 0.55.0 g/l of glucose. Whereas in the present study, S. arlettae PF4 could

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38 [6] Manu B and Chaudhari S. 2002. Anaerobic decolorisation of simulated textile wastewater containing azo dyes. Bioresource Technology, 82: 225231. [7] ONeill C, Lopez A, Esteves S, Hawkes FR, Hawkes DL and Wilcox S. 2000. Azo-dye degradation in an anaerobic-aerobic treatment system operating on simulated textile effluent. Applied Microbiology and Biotechnology , 53: 249254. [8] Weber EJ and Stickney VC. 1993. Hydrolysis kinetics of reactive blue 19-vinyl sulfone. Water Research, 27: 6367. [9] Reisch MS. 1996. Asian textile dye makers are a growing power in changing market. Chemical Engineering News, 15: 1012. [10] Chung KT, Jr. Stevens SE and Cernigliar CR. 1992. The reduction of azo dyes by the intestinal microflora. CRC Critical Reviews in Microbiology, 18: 175190. [11] Waldmann I and Vakilzadeh F. 1997. Delayed hypersensitivity to red azo dyes in tattoos. Der Hautarzt., 48: 666670. [12] Hildenbrand S, Schmahl FW, Wodarz R, Kimmel R and Dartsch PC. 1999. Azo dyes and carcinogenic aromatic amines in cell cultures. International Archieves of Occupational and Environmental Health 72 (suppl.), pp. 5256. [13] Van der Zee, Frank P, Lettinga G and Field JA, 2001. Azo dye decolourisation by anaerobic granular sludge. Chemosphere, 44: 11691176. [14] Martins MAM, Queiroz MJ, Silvestre AJD and Lima N. 2002. Relationship of chemical structure of textile dye on the preadaptation medium and the potentialities of their biodegradation by Phanerochaete chrysosporium. Research in Microbiology, 153: 361368. [15] Fu Y and Virarahavan T. 2001. Fungal decolourization of dye wastewater: A Review. Bioresource Technology, 79: 251-262. [16] Clarke HT and Kirner WR. 1941. Methyl Red, Org. Synth., 1: 374. [17] Saraswathi K and Balakumar S. 2009. biodecolourization of azodye (pigmented red 208) using Bacillus firmus AND Bacillus laterosporus. J Biosci Tech., 1(1): 1-7. [18] Wesenberg D, Kyriakides I and Agathos SN. 2003. A Review: White rot fungi and their enzymes for the treatment of industrial dye effluents. 21: 194-202. [19] Pearce CI, Lloyd JR and Guthrie JT. 2003. The removal of color from textile waste water using whole bacterial cells: A review. Dyes and Pigments, 58: 179-196. [20] Eichlerova I, Homolka L and Nerud F. 2006. Synthetic dye decolorization capacity of white rot fungus, Dichomitus squalens. Bioresource Technology, 97: 21532159. [21] McMullan G, Meehan C, Connely A, Kirby N, Robinson T, Marchant R and Smyth WF. 2001. Microbial decolourization and degradation of textile dyes. Appl. Microbiology and Biotechnology, 56: 81-87. [22] Keck A, Klein J, Kundlich M, Stolz A, Knackmuss HJ and Mattes R. 1997. Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. strain BN6. Appl

completely decolorize 600 and 800 mg/l methyl red within 24 h and 48 h respectively in presence of only 1.0 g/l of glucose. In another study, Moutaouakkil et al. [31] observed that under optimal conditions, Enterobacter agglomerans decolorized 92% of 100 mg/l of methyl red within 6 h of incubation in synthetic medium at 37C. Whereas in the present study, S. arlettae PF4 could decolorize 100% of 600 mg/l of methyl red within 24 h of incubation at 37C. The high methyl red decolorization rate and decolorization efficiency of S. arlettae PF4 enable this bacterium to be used in the biological treatment of industrial effluent containing azo dyes. IV. CONCLUSION Following conclusions can be drawn from the present study Methyl red decolorizing bacteria were isolated and screened from soil and one of the potential bacterium was identified as Staphylococcus arlettae PF4. Complete decolorization of 600 and 800 mg/l methyl red by S. arlettae PF4 was found within 24 h and 48 h respectively. The decolorization efficiency by S. arlettae PF4 for 600 and 800 mg/l methyl red was 100%, Partial decolorization of 1000 and 1200 mg/l methyl red by S. arlettae PF4 was found within 72 h, which was 89.5% and 68% respectively. The maximum decolorization rates by the isolate S. arlettae PF4 for 600, 800, 1000 and 1200 mg/l methyl red concentrations were 25.00, 22.38, 17.08 and 16.29 mg/l/h respectively.

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2011, International Journal of Environment, 1:34-39