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Biodegradation and Decolorization of Reactive Dye Red ME4BL by Bacillus subtilis

Velmurugan. S, Ravikumar. R*

Department of Botany, Jamal Mohamed College, Tiruchirappalli, Tamil Nadu

Abstract: A bacterial strain SVM1 with remarkable ability to decolorization textile dye Reactive Red ME4BL was isolated from the activated sludge soil collected from a common effluent treatment plant (CETP) in Perunduari, Erode district Tamil Nadu. Different parameters such as temperature and pH were optimized for decolorization of Red ME4BL by using isolated bacteria. The most promising bacterial isolated was used to further dye degradation studies. The 16 s RNA gene sequencing isolated organisms as Bacillus subtilis strain SVM1 the strain showed 93% decolorization of the selected dye Reactive Red ME4BL 200mg/1000ml within 42 hours in static and shaking condition. The optimum pH and temperature for the decolorization was 8 and 37°C respectively. The biodegradation was analysis by UV-Vis, FTIR and HPLC. The result suggested that the isolated organisms Bacillus subtilis strain SVM1 can be used useful tool to treat waste water containing textile reactive dye.

Keywords: Decoloriaztion, Bacillus subtilis, Red ME4BL dye

Received August 28, 2014; Revised September 11, 2014; Accepted December 21, 2014

Citation: Velmurugan. S, Ravikumar. R, Biodegradation and Decolorization of Reactive Dye Red ME4BL by Bacillus subtilis. International Journal of Environmental Bioremediation & Biodegradation, 2 (6). 250-255. January 2014. doi:10.12691/ijebb-2-6-1

1. Introduction

The synthetic dyes are often found in environment due to their wide use in various industries. The industries such as plastics, Textile, food, paper, and cosmetics are using these dyes in large quantities to color their products. However, the textile industry ranks first in the consumption of dyes. It is estimated that approximately 10-15% of total amount of dyes produced were released into the environmental mainly through waste water [1]. Dyes can be removed from waste water by chemical and physical methods including adsorption, coagulation- flocculation, oxidation and electro chemicals methods have many disadvantages in application, such as high energy costs, high – sludge production formation of by- products [2]. Biological methods are generally considered environmental friendly as they can lead to complete mineralization of organic pollutants at low cost [3]. Bioremediation may be the most effective methods of treating industrial dyes waste water [4]. Dyes include a broad spectrum of different chemical structures, primarily based on substituted aromatic and heterocyclic groups such as aromatic amine (C₆H₅-NH₂), which is a suspected carcinogen, phenyl (C₆H₅-CH₂) and naphthyl (NO₂-OH), the only thing in common is their ability to absorb light in the visible region [5].

At present a number of studies have focused on microorganisms, which have able to decolorize and biodegrade the dyes. Several combined anaerobic and aerobic microbial treatments have been suggested to enhance the degradation of azo dyes [6]. Decolorization experiment were run under various culture conditions was done by varying one at time at keeping others constants [7]. However biological processes provide an alternative to existing technologies since they are more cost effective, environment friendly, and do not produce large quantities of sludge [8]. Several scientific research have reported biodegradation of synthetic organic colorant, especially azo dyes [9,10]. Some bacterial strains such as Bacillus cereus, pseudomonas putida, pseudomonas fluorereus, streanotrophomonas acidaminiphila have been used in the biodegradation of azo dyes [11]. In contrast, remediation of dyeing industry effluent by using microorganisms has proved to be the best solution [12]. Reactive dyes are commonly used in textile industries because of their favorable characteristics of bright colour, water-fastness, and simple application techniques with low energy consumption [13]. The ubiquitous nature of microorganism makes them invaluable tools in the effluent bio treatment [14]. The advantages of mixed cultures are apparent as some microbial consortia can collectively carry out biodegradation tasks that no individual pure strain can undertake successfully [15].

The microbial decolorization and degradation of dyes and effluents have been of considerable interest since it is inexpensive, eco-friendly, and produces a less amount of sludge [16]. The effectiveness of microbial decolorization depends on the adaptability and the activity of selected microorganisms. Wide range of microorganisms including bacteria, fungi, yeasts, actinomycetes and algae capable of degrading azo dye have been reported [17,18]. Among the reactive dyes, three most common groups are azo, anthraquinone and phthalocyanine dyes [19]. The resulting products such as aromatic amines are further degraded by multiple-step bioconversion occurring aerobically or anaerobically [20,21]. Azo dyes, which are aromatic compounds with one or more (–N=N–) groups, are the most important and largest class of synthetic dyes used in commercial applications [22]. In the present study an attempt has been made for the degradation and decolorization of an reactive dye by using Bacillus subtilis.

2. Material and Methods

2.1. Chemical and Media

The dye Reactive Red ME4BL was kindly provided by the Jamara textile dyeing industry, Perunduai, Erode, Tamil Nadu. The structures of the dyes are shown in (Figure 1). All the analytical grade reagents were purchased from Hi Media and used without further purification. The nutrient agar medium at used all the batch experiments containing (g/l): NaCl 5.0, bacteriological peptone 10.0, yeast extract 2.0, beef extract 1.0 and agar agar 15.0, stored at 4°C until use [23]. The organisms from stock culture were used for the decolorization studies after pre-culturing in Nutrient Broth (g/l): peptone 10.0, NaCl 5.0, yeast extract 2.0 and beef extract 1.0 at 37±2°C for 16 hours under shaking condition (120 rpm) at neutral pH.

Figure 1. chemical structure of Reactive Red ME4BL dye

2.2. Isolation and Identification of Decolorizing Bacteria

Sludge soil sample collected from common effluent treatment plant (CETP), textile dyeing industry area in Perunduari, Erode, Tamil Nadu. Aliquots (10g) sludge soil samples were serially diluted by following the standard protocol and the dilution series of 10⁻² to 10⁻⁷ was plated in nutrient agar medium. Each dilution was maintained in triplicates. All the plates were incubated at 37°C for 24 hours [24]. Cultures were identified based on their morphologically different colonies were streaked and transferred aseptically into sterile agar slants for raising pure cultures to perform further study. The Decolorizing ability of the bacterial isolates were named by SVM1 to SVM6 was tested individually on the Reactive Red ME4BL dye. Decolorization experiments were performed in triplicates sets. A loopful of log phase pure culture was inoculated into 250 ml Erlenmeyer flask containing 100 ml nutrient broth with the dye concentration of 200mg/l000 [25]. The flask was incubated at 37°C for 24 hours in a shaker-incubator at 120 rpm. The decolrization ability bacteria was identified as SVM1. Identification of the isolated strain was performed by 16s rRNA sequence analysis. The taxonomical studies were carried out according to Bargeys manual [26]. The 16s rRNA gene in this strain was amplified by 30 cycle of PCR using genomic DNA from strain SVM1 as the template and two specific primes, 27F (5’AGAGTTTGATCMTGGCTCAG 3’) and 1492 R (5’TACGGYTACCTTGTTACGACTT 3’). Each cycle was carried out at 94°C for 45 sec, at 45°C for 60 sec, and at 72°C for 60 sec. DNA fragments were amplified about 1,400 bp in the case of bacteria. Sequencing reactions were performed using a ABI PRISM BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq DNA polymerase (FS enzyme) (Applied Biosystems) and Bacterial Genomic DNA was isolated by using the InstaGene TM Matrix Genomic DNA isolation kit Catalog # 732-6030.

2.3. Effect of the Culture Growth

pH ranges from 6 to 8 of the 100 ml nutrient broth medium inoculated with isolated identified bacterial culture incubated at 37°C in the static and shaking condition. The shaking incubator at 120 rpm bacteria growth of the culture was measured.

2.4. Decolorization Study

Aliquots 5ml of the culuture media were withdrawn at time interval of 1 hours over 42 hours and centrifuged 10,000 rpm for 15min. decolorization was quantitatively analyzed by measuring the absorbance of the supernatant using a UV- visible spectrophotmeter Jasco 550 at maximum wavelength max 540 nm for Red ME4BL. The decolorization rate was calculated using the equation [27].

2.5. Effect of Different Temperature

The experiments were conducted in 250ml Erlenmeyer flasks containing 100ml of reactive dye Red ME4BL and SVM1 bacteria. To evaluate the effects of operation and environmental factors on the efficiency of color removal the batch decolorization were carried out of concentration 200mg/1000ml, bacterial strain inoculated with 5 ml, temperature 32°C, 37°C, 40°C and 50°C and pH was fixed with 8. The batch decolorization experiment was prformed under a static and shaking incubation condition.

2.6. Biodegradation Analysis

FTIR analysis was carried out using Simadzu 8400s spectrophotometer in the mid – infrared of 400 – 4000cm- with 16 scan speed. HPLC analysis was performed in an isocratic water 2690 system equipped with dual absorbance detector using C18 column (4.6X 250nm) and HPLC grade method as mobile phase with flow rate 1ml min-1.

3. Result and Discussion

3.1. Identification of a Bacterial Strain SVM1

Isolated bacterial strain SVM1 was rod shaped and gram negative bacteria. Molecular studies revealed its characterization as DNA fragments are amplified about 1400bp nucleotide in length identified as Bacillus subtilis and 16s ribosomal RNA gene partial sequences show similarity 99%. The sequencing data was submitted to genbank. The phylogenetic tree showed the grouping of Bacillus subtilis (Figure 2).

Figure 2. Phylogenetic tree of the Bacillus subtilis on 16s RNA sequences

Figure 3. Growth rate of Bacillius subtilis in 5ml/100ml of nutrient broth

3.2. Effect of pH and Temperature on the Growth of Bacillus subtilis.

pH ranging from 6 to 8 of the medium, the cell growth was tested at 37°C for 23 hours (Figure 3). The bacterium showed slow growth rate at pH 6. We employed pH8 showed better growth rate of the bacterium. Previously reported that growth rate of this organism was slow and leg phase was long [28].

3.3. The Effect of Temperature and pH

The temperature effect on decolorization rate was significant (Figure 4). Different temperature the dye concentration and pH were fixed at 200mg/1000ml and pH8 respectively. The most optimum temperature for decolorization was 37°C shaking and static condition. This also demonstrates that decolorization of dye was through microbial which relies on optimal temperature and not by adsorption [29].

Figure 4. Effect on decolorization of Reactive Red ME4BL by Bacillus subtilis

3.4. Decolorization of the Reactive Red ME4BL under Static and Shaking Conditions

The decolorization potential of bacillus subtilis in nutrient medium at different time intervals after 2 hours in analysis to 42 hours was studied. The selected bacterial isolated was found to be potential enough to decolorize the reactive dye red ME4BL. The decolorzation rate of Bacillus subtilis in 42 hours was maximum 93%. The biodegradation of the Red ME4BL dye was monitored by UV-Vis analysis. For untreated Red ME4BL dye presented absorbance peaks at 540, 289 and 290 nm (Figure 5a). For treated dye, after biodegradation of the reactive dye in the static and shaking treated solution, the absorbance peaks in the visible region disappeared, indicating complete decolorization (Figure 5b, Figure 5c). Many bacteria capable of reducing azo dyes reported were isolated from textile effluent contaminated sites [18].

Figure 5a. UV-Vis spectra of the Red ME4BL before treatment; b. UV-Vis spectra of the Red ME4BL after shaking condition; c. UV-Vis spectra of the Red ME4BL after non shaking condition

3.5. Analysis of FT-IR

The FT-IR spectra obtained from the untreated dye samples showed several peaks in the region where C-S and S-O stretching is normally observed (2500- 3500 cm¹). After the shaking and non shaking treatment a significant reduction in absorption was observed in this region. The bonds located in the range from 2500- 3500 cm¹ disappeared in the shaking and non shaking stage after reductive treatment (Figure 6 a,b,c). The biodegradation of the azo dyes was monitored by UV-Vis and FTIR analysis [24].

3.6. HPLC Analysis

HPLC spectrum of Red ME4BL showed the peaks at retention time 3.662, 4.915,9.497,10.578 and 12.180 min (Figure 7a) and the metabolites obtained after shaking its degradation by: Bacillus subtilis. SVM 1 showed the peaks at retention time 3.091 and 4.927 min (Figure 7b). and the non shaking degradation by Bacillus subtilis showed peak at retention time 3.093 and 4.913 min (Figure 7c). Similarly, HPLC analysis of the reactive red dye was also reported that the retention times showed the disappearance of the dye implying its mineralization from the solution [30].

Figure 6. a. FT-IR Spectra of the Reactive Red ME4BL before treatment; b. FT-IR Spectra of the Reactive Red ME4BL after shaking condition; c. FT-IR Spectra of the Reactive Red ME4BL after non shaking condition

Figure 7a. HPLC analysis of the Reactive Red ME4BL before treatment; b. HPLC analysis of the Reactive Red ME4BL after shaking condition; c. HPLC analysis of the Reactive Red ME4BL after non shaking condition

4. Conclusion

In the present study Bacillus subtilis was isolated from sludge soil. Analytical studies of the supernatant of reactive dye red ME4BL have found that the decolorization of reactive dye red ME4BL dye into non toxicity. It was concluded that at optimum temperature was 37°C and pH8 this organism can grow even at higher concentration above 200mg/l and can remove 93% of Red ME4BL anaerobic and aerobically in flask studies. The Bacillus subtilis can be also used for the treatments of dye decolorization and degradation.

Acknowledgements

Authors are grateful to the University Grants Commission, New Delhi for providing financial support.

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