Decolorization of diazo dye Direct Red 81 by a novel bacterial ...

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Nishant Junnarkar; D. Srinivas Murty; Nikhil S. BhattEmail author; Datta Madamwar ... Azo dyes bacterial consortium Direct Red 81 dye decolorization starch.
Ó Springer 2005

World Journal of Microbiology & Biotechnology (2006) 22: 163–168 DOI 10.1007/s11274-005-9014-3

Decolorization of diazo dye Direct Red 81 by a novel bacterial consortium Nishant Junnarkar1, D. Srinivas Murty1, Nikhil S. Bhatt1,* and Datta Madamwar2 1 Biogas Research Centre and Post Graduate Department of Microbiology, Gujarat Vidyapith, Sadra 382 320, Dist. Gandhinagar, Gujarat, India 2 Post Graduate Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar, 388 120, Gujarat, India *Author for correspondence: Tel.: +91-79-23274274, Fax: +91-79-27542547, E-mail: [email protected] Received 20 September 2004; accepted 29 June 2005

Keywords: Azo dyes, bacterial consortium, Direct Red 81, dye decolorization, starch

Summary Samples collected from various effluent-contaminated soils in the vicinities of dyestuff manufacturing units of Ahmedabad, India, were studied for screening and isolation of organisms capable of decolorizing textile dyes. A novel bacterial consortium was selected on the basis of rapid decolorization of Direct Red 81 (DR 81), which was used as model dye. The bacterial consortium exhibited 90% decolorization ability within 35 h. Maximum rate of decolorization was observed when starch (0.6 g l)1) and casein (0.9 g l)1) were supplemented in the medium. Decolorization of DR 81 was monitored by high performance thin layer chromatography, which indicated that dye decolorization was due to its degradation into unidentified intermediates. The optimum dye-decolorizing activity of the culture was observed at pH 7.0 and incubation temperature of 37 °C. Maximum dye-decolorizing efficiency was observed at 200 mg l)1 concentration of DR 81. The bacterial consortium had an ability to decolorize nine other structurally different azo dyes.

Introduction Synthetic dyes are extensively used in textile dyeing, paper, printing, color photography, pharmaceutics, cosmetics and other industries (Marmion 1991). Amongst these, azo dyes represent the largest and most versatile class of synthetic dyes (Keharia et al. 2004). Approximately 10–15% of the dyes are released into the environment during manufacturing and usage (Spadary et al. 1994; He et al. 2004). Since some of the dyes are harmful, dye-containing wastes pose an important environmental problem (Verma & Madamwar 2003). These dyes are poorly biodegradable because of their structures and treatment of wastewater containing dyes usually involves physical and/or chemical methods (Kim & Shoda 1999) such as adsorption, coagulation-flocculation, oxidation, filtration and electrochemical methods (Calabro et al. 1991; Lin & Peng 1994, 1996). However, these methods are expensive and have operational problems. Furthermore, dyestuffs cannot be converted to CO2 by physical and chemical methods. Complete degradation of dyestuff can only be accomplished by chemical or biological oxidation (Kapdan et al. 2000). Over the past decades, biological decolorization has been investigated as a method to transform, degrade or mineralize azo dyes (Banat et al. 1996). Moreover, such

decolorization and degradation is an environmentally friendly and cost-competitive alternative to chemical decomposition processes (Verma & Madamwar 2003). Unfortunately, most azo dyes are recalcitrant to aerobic degradation by bacterial cells (Pagga & Brown 1986; Bras et al. 2001). However, there are few known microorganisms that have the ability to reductively cleave azo bonds under aerobic conditions (Horitsu et al. 1977; Ogawa et al. 1986; Wong & Yuen 1996; Coughlin et al. 2002). Efforts to isolate bacterial cultures capable of degrading azo dyes started in the 1970s with reports of Bacillus subtilis (Horitsu et al. 1977), then Aeromonas hydrophila (Idaka & Ogawa 1978) followed by Bacillus cereus (Wuhrmann et al. 1980). Numerous bacteria capable of dye decolorization, either in pure cultures or in consortia, have been reported (Yatome et al. 1981; Banat et al. 1996; Hu 1996; Rajaguru et al. 2000; Coughlin et al. 2002; Pearce et al. 2003; Verma & Madamwar 2003). The utilization of microbial consortia offers considerable advantages over the use of pure cultures in the degradation of synthetic dyes. The individual strains may attack the dye molecule at different positions or may use decomposition products produced by another strain for further decomposition (Forgacs et al. 2004). The benefits and drawbacks of the use of microbial

164 consortia for the decomposition and decolorization of various dyes have been reviewed by Banat et al. (1996). For sulfonated azo dyes, both aromatic sulfonic and azo groups contribute to their xenobiotic nature as these are rare among natural products. In this article, we report studies on the bacterial decolorization of Direct Red 81 (C.I. No. 28160, Figure 1), a sulfonated diazo dye that is soluble in water, acidic in nature and has a kmax of 509 nm. We also report the optimization of parameters required for the bacterial consortium NBNJ6 to decolorize the dyes efficiently in a short period.

Materials and methods Chemicals All chemicals used were of analytical grade. The common name of all the dyes have been used for convenience. Dyes were procured from Vim Dye Chem and Hue Lab, Vatva, Ahmedabad, India. Dyes used in our study were Direct Red 81 (DR 81), Reactive Black B, Reactive Blue 172, Reactive Violet 5R (C.I. No. 18097), Reactive Red 5B, Reactive Black RL, Ponceau 4R (C.I. No. 16255), Raspberry Red, Tartrazine (C.I. No. 19140), Sunset Yellow FCF (C.I. No. 15985). Direct Red 81 was used as a model dye for the optimization experiments. Medium The microbial consortium, NBNJ6 was routinely grown at 37 °C in the basal culture medium, Bushnell and Hass medium (BHM) containing the following in g l)1: MgSO4, 0.2; CaCl2, 0.02; KH2PO4, 1.0; K2HPO4, 1.0; (NH4)2NO3, 1.0; FeCl3, 0.05 supplemented with glucose (0.9 g l)1) and yeast extract (0.9 g l)1) and DR81 (100 mg l)1). Isolation of bacterial cultures

N. Junnarkar et al. conditions at 37 °C. Samples, which showed decolorization in liquid media, were repeatedly tested further by adding fresh dye-containing medium till stable dyedecolorizing cultures were obtained, showing consistent growth and decolorization in every successive transfer. The samples that showed consistent growth and decolorization in dye-containing medium were transferred on to BHM agar plates containing 100 mg DR 81 l)1 along with glucose and yeast extract. This resulted in isolation of 10 different bacterial cultures. Various permutations were made using these isolates to improve the decolorization of DR 81. Amongst these, NJ38, NJ1, NJLC1 and NJLC3, when combined, formed a bacterial consortium NBNJ6, having the ability to decolorize several different dyes. Gram staining of the bacterial isolates was carried out to determine the nature of the microorganisms. Study of physico–chemical parameters Decolorization was studied using various co-substrates and at different dye concentrations (50–400 mg l)1), inoculum size (5, 10, 15, 20, 25 and 30% (v/v)), pH (6– 10) and temperature (25, 30, 35, 37, 40, 45 and 50 °C). Decolorization was determined by measuring absorbance of culture supernatants at the absorbance maxima (kmax) of the respective dyes (kmax: Reactive Black B, 610 nm; Reactive Blue 172, 605 nm; Reactive Violet 5R, 558 nm; Reactive Red 5B, 500 nm; Reactive Black RL, 586 nm; Ponceau 4R, 506 nm; Raspberry Red, 510 nm; Tartrazine, 450 nm; Sunset Yellow FCF, 400 nm) using UV–Visible spectrophotometer (Shimadzu UV–VIS 16A). All the experiments were performed in quadruplicate and the mean as well as the standard deviation (vertical bars in Figures 2–5) of the data was considered. Metabolite isolation by HPTLC Degradation of dye, DR 81, was monitored by TLC using silica gel (silica gel 60 F254) plates supplied by

The soil samples collected from effluent-contaminated sites in the vicinities of dyestuff industries located in industrial estates of Ahmedabad, Gujarat, India, were used for isolation of dye-decolorizing bacteria using BHM amended with dye (DR 81, 100 mg l)1) as a sole source of carbon or along with glucose (0.9 g l)1) and yeast extract (0.9 g l)1). Dye-containing media (100 ml) in 250 ml Erlenmeyer flasks were inoculated with 10 ml of 1% (w/v) soil suspension and incubated on an orbital shaker (150 rev min)1), as well as, under stationary

Figure 1. Chemical structure of Direct Red 81 dye.

Figure 2. Decolorization of Direct Red 81 by bacterial consortium NBNJ6 under stationary and agitated conditions, at 37 °C.

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Decolorization of diazo dye Direct Red 81

15 ll of the sample was spotted on TLC plates in which micro syringe (Linomat V, Camag, Germany) was used. The solvent system used was propane-2-ol/liquor ammonia (strength of 25%, v/v) in the ratio of 7:3 (v/v). The dye chromatogram was analyzed by a Camag Scanner at 509 nm and was observed by exposing it to ultraviolet light (254 nm) and in visible light.

Results and discussion Isolation of dye-decolorizing bacteria

Figure 3. Decolorization rate of Direct Red 81 by bacterial consortium NBNJ6 at different initial pH of medium at 37 °C under stationary conditions.

Figure 4. Decolorization rate of Direct Red 81 by bacterial consortium NBNJ6 at different incubation temperatures under stationary conditions.

The soil samples collected from effluent contaminated sites in the vicinities of dyestuff industries located in industrial estate of Ahmedabad, Gujarat, India, were used for isolation of dye-decolorizing bacteria employing Direct Red 81 (DR 81) dye as a sole source of carbon and energy. Despite repeated attempts we were not successful in isolating bacteria capable of decolorizing and utilizing DR 81 dye as a sole source of carbon and energy. The obligate requirement of labile carbon source for functioning of dye-decolorizing bacteria has been reported, therefore, isolation was also attempted by employing glucose and yeast extract as co-substrates (Banat et al. 1996; Coughlin et al. 1997). Samples which showed the consistent growth and decolorization in dyecontaining media in every successive transfers, were transferred on BHM agar plates amended with DR 81 dye (100 mg l)1) and glucose and yeast extract. This resulted in isolation of 10 different dye-decolorizing bacteria. Various permutations and combinations were made using these isolates to improve the decolorization of DR 81. Amongst these combinations, a consortium named NBNJ6 consisting of bacterial cultures labeled as NJ38, NJ1, NJLC1 and NJLC3 was found to be more efficient in decolorizing DR 81 dye. NBNJ6 could also decolorize several different dyes with decolorization efficiency in the range of 84–95% (Table 2) and was thus chosen for further studies. Gram staining of bacterial isolates was carried out to determine the nature of microorganisms. Bacterial cultures NJ38, NJ1 and NJLC3 were found to be Gram-negative short rods, whereas, bacterial culture NJLC1 consisted of Gram-positive cocci. When pure cultures of these isolates were tested individually for their decolorizing ability in liquid medium, none of these cultures showed complete decolorization of DR 81, even on extended incubation. When all the four bacterial cultures were mixed and inoculated in the liquid medium, complete decolorization of DR 81 was observed. Thus, this clearly suggests a synergistic role of bacterial species in decolorization (Knapp & Newby 1995).

Figure 5. Decolorization rate of Direct Red 81 by bacterial consortium NBNJ6 at different initial dye concentration in medium at 37 °C under stationary conditions.

Effect of culture conditions

Merck, Germany. The culture supernatant at different periods during decolorization was used for analysis. A

The bacterial consortium NBNJ6, exhibited dye-decolorizing activity only when incubated under the stationary conditions, whereas, negligible decolorization (17%)

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was noticed under the agitating conditions. Stationary cultures exhibited apparently complete decolorization (90%) of DR 81 within 35 h of incubation (Figure 2) and further incubation upto 40 h did not improve decolorization. Under aerobic conditions azo dyes are generally resistant to attack by bacteria (Hu 1998). Azo dye decolorization by bacterial species if often initiated by enzymatic reduction of azo bonds, the presence of oxygen normally inhibits the azo bond reduction activity since aerobic respiration may dominate utilization of NADH; thus impeding the electron transfer from NADH to azo bonds (Chang & Lin 2001). Effect of co-substrate on dye decolorization Bacterial consortium NBNJ6 exhibited maximum decolorization of DR81 dye when starch and casein were supplemented in the medium (Table 1). In absence of co-substrate, the bacterial culture was unable to decolorize the dye, which indicates that availability of supplementary carbon source seems to be necessary for growth and decolorization of dyes (Nigam et al. 1996). The ability of our mixed culture to use starch and casein as co-substrates was encouraging from a commercial point of view. Other combinations of two carbon sources also seemed to be reasonably effective. In order to optimize the concentration of starch in the medium for maximum decolorization, the rate of decolorization was monitored at starch concentrations varying from 0.3 to 1.5 g l)1. Fastest decolorization rate (2.41 mg l)1 h)1) was observed at a starch concentration of 0.6 g l)1. Similarly, concentrations of casein were varied from 0.3 to 1.5 g l)1, in order to optimize the concentration of casein in the medium. Decolorization efficiency of the culture increased with increase in casein concentration from 0.3 to 0.9 g l)1 showing increase in decolorization rate from 2.01 to 2.51 mg l)1 h)1, respectively. Further increase in casein concentration did not improve the decolorization efficiency of our culture. Kapdan et al. (2000) reported lactose (5 g l)1) and yeast extract (50 mg l)1) to be the most effective carbon-nitrogen source in decolorization of Everzol Red RBN by bacterial consortium PDW as compared to the other nutrient sources used in their study.

Effect of pH and temperature on dye decolorization Bacterial cultures generally exhibit maximum decolorization at pH values near 7. The rate of decolorization for NBNJ6 was optimum in the narrow pH range from 7.0 (2.11 mg l)1 h)1) to 8.0 (1.87 mg l)1 h)1) with marked reduction in decolorizing activity at pH 6.5 (Figure 3). Both, E. coli and P. luteola, exhibited best decolorization rate at pH 7 with constant decolorization rates upto pH 9.5 (Chang & Lin 2001). K. pneumoniae RS-13 completely degraded methyl red in pH range from 6.0 to 8.0 (Wong & Yuen 1996). Mali et al. (2000) found that a pH value between 6 and 9 was optimum for decolorization of triphenylmethanes and azo dyes by Pseudomonas sp. Moreover, it has been reported that generally azo dye reduction by bacterial cultures to more basic aromatic amines leads to a rise in pH of the medium by about 0.8–1.0 values (Hu 1994; Knapp & Newby 1995). The dye decolorization activity of our culture was found to increase with increase in incubation temperature (Figure 4) from 25 to 37 °C with maximum activity attained at 37 °C (2.46 mg l)1 h)1). Further increase in temperature resulted in marginal reduction in decolorization activity of consortium NBNJ6. Decline in decolorization activity at higher temperature can be attributed to the loss of cell viability or to the denaturation of the azo-reductase enzyme (Pearce et al. 2003). Wong & Yuen (1996) reported that Klebsiella pneumoniae RS-13 exhibited decolorization of methyl red at temperatures varying from 23 to 37 °C, whereas, at 45 °C decolorization was completely inhibited. Maximum potential of Pseudomonas sp. to decolorize Malachite green, Fast green, Brilliant green, Congo red and Methylene blue was noticed at 37 °C (Mali et al. 2000). Effect of dye and inoculum concentration on decolorization Decolorizing activity of the bacterial consortium was studied using DR 81 at different initial concentrations varying from 50 to 400 mg l)1(Figure 5). Rate of decolorization increased with increase in initial dye concentration up to 200 ppm (2.29 mg l)1 h)1). Further

Table 1. Effect of various co-substrates on decolorization of Direct Red 81 (100 mg l)1) by mixed bacterial consortium NBNJ6, at 37 °C under stationary conditions (Initial concentration of each co-substrate was used as 1 g l)1) No.

Co-substrates

Incubation period (h)

% Decolorization

1 2 3 4 5 6 7 8 9 10 11

BHM BHM+Dextrose+Yeast extract BHM+Sucrose+Yeast extract BHM+Cellobiose+Yeast extract BHM+Starch+Yeast extract BHM+Starch+Peptone BHM+Starch+Casein BHM+Starch+Tryptone BHM+Starch+Meat extract BHM+Starch BHM+Casein

60 46 46 46 46 50 46 48 50 60 60

10±1.5 75±2.1 74±1.8 72±1.6 74±2.2 72±1.7 89±2.2 83±1.9 73±1.8 24±1.7 34±2.1

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Decolorization of diazo dye Direct Red 81 Table 2. Decolorization of different dyes by mixed bacterial consortium NBNJ6 at 37 °C under stationary conditions No.

Dye

Type of dye

Dye concentration (mg l)1)

Incubation period (h)

% Decolorization

1. 2 3 4 5 6 7 8 9

Reactive Black B Reactive Blue 172 Reactive Violet 5R Reactive Red 5B Reactive Black RL Ponceau 4R Raspberry Red Tartrazine Sunset Yellow FCF

Diazo Diazo Mono Mono Mono Mono Mono Mono Mono

250 250 250 250 250 500 1000 300 300

35 32 30 30 35 24 24 24 24

88±2.3 89±1.9 85±2.2 90±1.8 87±2.2 91±1.9 84±2.2 95±1.9 89±2.3

azo azo azo azo azo azo azo

increase in dye concentration resulted in reduction in decolorization rates. Lower decolorization efficiency is due to higher inhibition at high dyestuff concentration (Verma & Madamwar 2002). O’Neill et al. (1999) reported that the dye concentration in the reactive dye bath effluent was observed within narrow range of 0.1– 0.2 g l)1. Our consortium could decolorize the dye at concentrations much above those reported in wastewaters and thus it can be successfully employed for treatment of dye-bearing industrial wastewaters. In order to find out the optimum inoculum needed for faster and higher percentage decolorization by our consortium, decolorizing ability was tested at different inoculum concentrations starting from 5 to 30% (v/v). The decolorization rate increased with increase in the inoculum size, reaching maximum (2.53 mg l)1 h)1) at 20% (v/v) inoculum size. However, beyond 20% (v/v) inoculum size rate of decolorization did not vary significantly. There was no proportionate increase in the percentage of decolorization with increase in the inoculum size of Kurthia sp. when inoculated in textile effluent (Sani & Banerjee 1999). Spectrum of dyes decolorized by the bacterial consortium As industrial effluent consists of a mixture of various dyes, the ability of our culture to decolorize different azo dyes was studied (Table 2). The bacterial consortium NBNJ6 showed rapid decolorization of all dyes within 24–35 h at 37 °C. Culture could decolorize upto 250 mg l)1 of Reactive Black B, Reactive Blue 172, Reactive Violet 5R, Reactive Red 5B and Reactive Black RL, within 30–35 h at 37 °C in the range of 85– 90%. Food colors like Ponceau 4R (500 mg l)1), Raspberry Red (1000 mg l)1), Tartrazine (300 mg l)1) and Sunset Yellow FCF (300 mg l)1) were decolorized within 24 h in the range of 84–95%. The rate of decolorization of individual dyes varied. Decolorizing efficiency of our consortium is different for different dyes. Similar observations were also made by Paszcezynski et al. (1992). Metabolite isolation by HPTLC The dye decolorization study of our bacterial consortium was further supported by TLC analysis. The dye

chromatograms in visible light showed a decrease in intensity of dye bands in the lanes corresponding to the spots of decolorized medium (Rf value of DR 81=0.48). When the dye chromatogram was observed in UV light, fluorescent bands with Rf value (0.40, 0.51, 0.60 and 0.62) different from that of dye were detected in the lanes corresponding to the spots of decolorized medium, and no such bands were observed for spots of uninoculated medium, indicating that decolorization was due to dye degradation. Furthermore, the intensity of these bands was found to decrease upon increase in incubation period suggesting further transformation. The initial step in bacterial degradation of dye is due to the reduction of azo bonds leading to the formation of aromatic amines. These aromatic amines are likely to be formed during reductive cleavage of azo bonds through which the amines are linked in the dye. Different bands, which were formed in the initial period of incubation of inoculated medium, disappeared upon extended incubation supporting degradation. The present study confirms the ability of bacterial consortium NBNJ6 to decolorize 10 structurally different synthetic dyes with decolorization efficiency of above 80%, thus suggesting its application for decolorization of dye-bearing industrial wastewaters. The culture requires starch and casein as medium supplements for dye decolorization which seems encouraging from economic point of view. Normally, dye concentration in the effluent varies within a narrow range of 0.1–0.2 g l)1 (O’Neill et al. 1999). A consortium NBNJ6 could decolorize dyes much above the reported dye concentration in wastewaters. However, the potential of the culture needs to be demonstrated for its application in treatment of real dye-bearing wastewaters using appropriate bioreactors. References Banat, I.M., Nigam, P., Singh, D. & Marchant, R. 1996 Microbial decolorization of textile dye containing effluents: a review. Bioresource Technology 58, 217–227. Bras, R., Ferra, I.A., Pinheiro, H.M. & Goncalves, I.C. 2001 Batch tests for assessing decolorization of azo dyes by methanogenic and mixed cultures. Journal of Biotechnology 89, 155–162. Calabro, V., Drioli, E. & Matera, F. 1991 Membrane distillation in the textile wastewater treatment. Desalination 83, 209–224.

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