Abstract

Various textile industries are discharging toxic effluents containing azo dyes and they adversely affect the aquatic life, water resources, soil fertility and ecosystem integrity. With the goal of using microorganisms as the agent for the bioremediation in the waste water treatment containing dye has been done over the last two decades, to our best of knowledge considerable work has been not been done using actinomycetes in the treatment of dye containing waste water. In the present study, actinomycetes strain namely Micromonospora sp were isolated from textile effluent adopted soil of Tiruppur district, Tamil Nadu, India and screened for their ability to decolourize Reactive Red 35 at different parameters includes pH, agitation, nitrogen source and medium. Comparision of decolourising capacity data revealed that more effective decolorization occurs by using minimal media with peptone under static and moderately decolourised under shaking condition. The decolourising capability is found to be high at pH 8 followed by pH 6. FTIR spectrum was compared between the spectrum of RR35 and the products obtained after decolorization confirmed the biodegradation of dye. This decolorization potential suggests the applicability of this isolate for dye removal in effluent from textile industries.

1. Introduction

Major problems that human are facing is the water pollution mainly it is caused by the effluent generated by the textile industries which when released in the ponds, rivers and on the land reduces the esthetical value of the water. Azo dyes were the major group of the dyes used in the textile, leather industries, pharmaceutical, food and cosmetics industries ^{1, 19}. Dye contents in the range of 10–200 mg per litre were found in the textile processing wastewaters which were highly colored. The chemical structure of such dyes were characterized by highly substituted aromatic rings joined by one or more azo groups [–N=N]. Due the presence of such ring structures make these molecules recalcitrant since they were not degraded by conventional wastewater treatment processes ². Various techniques have been employed for the treatment of industrial effluent ^{3, 4}. It includes precipitation, adsorption, ion exchange membrane and electro chemical technologies. Literature study show that these technologies are highly expensive, and are not environmental friendly and usually dependent on one of concentration of waste ⁵. Their initial investment and operational costs are also so high that they can be widely used in developing countries ^{7, 8, 9}. Biological treatment was the cheapest way to treat the waste water containing textile effluent i.e. by using microorganism as compared to costly chemical, physical and physicochemical treatments. The biological decolourization treatment of textile waste water by purely biological process may be possible even without the inclusion of other carbon sources [eg] municipal waste water. The effectiveness of these treatment systems was based on the adaptability and survival of microorganisms during the treatment processes ^{10, 11}. It had been reported that the ability to decolourize azo dyes could be achieved by many microorganisms belonging to different taxonomic groups of bacteria ¹⁰, fungi ¹², actinomycetes ¹³ and algae ¹⁴. The potential in decolourization of textile dyes by actinomycetes was not exploited well when compared to the exploitation towards the production of the life saving drugs called antibiotics. Using reactive red 35 few studies had been attempted for the biodecolourization studies. Reactive red 35 [RR35], a vinyl sulfone based monoazo dye were proved hazardous to human as it was responsible for causing skin, eyes and respiratory irritation ¹⁵.

2. Materials and Methods

Sample was collected from textile effluent adopted soil from Tiruppur district, Tamil Nadu, India for isolation and identification of actinomycetes decolourising azo dyes. The soil sample was aseptically transferred to the sterile bottles and brought up to the laboratory.

The azo dye used in this study, Reactive red 35 was obtained from local dye manufacturing market of Tiruppur district, Tamil Nadu, India.

One gram of the soil sample was serially diluted up to 10^-6 dilution. One ml of diluted sample was permitted into the actinomycetes isolation agar medium amended with reactive red 100 ppm and pure strain of actinomycete was selected after 7-15 days at 28°C. The azo dye adopted actinomycete was identified from several aspects including visual determination by observing agar plates for colour of the colony, nature of the mycelium, morphology of colony, production of pigments, reverse side colour and biochemical tests as described in Bergey’s Manual of Determinative Bacteriology ¹⁶.

The Micromonospora sp isolate is analyzed for the decolourisation of Reactive Red 35 dye in broth cultures condition. The flask containing 100 mL of Minimal broth medium [Di Potassium hydrogen phosphate 7.0g, Sodium Citrate 0.5g, Ammonium Sulfate 1.0 g, Magnesium Sulphate 0.1g, Dextrose 1.0g, Distilled water 1000L] and 20 ppm dye was inoculated with 10% (v/v) of 3 days old actinomycetes suspension. The culture flasks were incubated at 28°C for 7 days. The flasks without inoculation of the strain were kept as control. OD values were measured spectrophotometrically in UV-Vis Spectrophotometer at 540 nm to estimate the decolorization process.

The rate of decolourization was calculated using the following formula. ¹⁷

100 mL of minimal media, minimal media with 0.1gm yeast extract and minimal media with 0.1gm peptone was added into 250 mL Erlenmeyer conical flask respectively and sterilized. Reactive Red 35 dye, at the concentration of 100 ppm was added to the broth independently. 10 mL of 3 days old cultures of Micromonospora spp was inoculated to the broth and incubated at 28°C for 7 days, under shake (100rpm, 200rpm) and static condition. Control were maintained without the inoculum. 5 mL of the incubated broth was drawn after doclourization and centrifuged at 7000 rpm for 15 min. Absorbance of the supernatant was recorded using UV vis spectrophotometer at 540 nm for reactive red 35 dye and compared with the control. The percent decolorization of the dye was calculated as mentioned earlier.

The actinomycetes isolate were analyzed for the decolourisation of Reactive Red 35 dye in ISP No.1 broth and ISP No.4 broth cultures respectively. The flask containing ISP No.1 broth and ISP No.4 broth was added into 250 mL Erlenmeyer conical flask respectively and 100 ppm dye was inoculated in the actinomycetes suspension. The culture flasks were incubated at 28°C for 7 days. The flasks without inoculation of the strain were kept as control. OD values were measured spectrophotometrically in UV-Vis Spectrophotometer at 540 nm to estimate the decolorization process. The rate of decolourization was calculated using above mentioned formula.

100 mL of ISP No.1 broth was added into 250 mL Erlenmeyer conical flask of six quantities respectively. Reactive Red 35 dye, at the concentration of 100 ppm was added to the broth independently. Various levels of pH were optimized for effective dye decolorization by the isolate Micromonospora spp. pH 2.0, 4.0, 6.0, 8.0 and 10.0 of the medium were adjusted using dilute acidic and alkaline solution of hydrochloric acid and sodium hydroxide respectively and sterilized. 1 mL of 3 days old cultures of Micromonospora spp was inoculated to the broth and incubated at 28°C for 7 days. Control was maintained without the inoculum. 5 mL of the incubated broth was drawn after doclourization and centrifuged at 7000 rpm for 15 min. Absorbance of the supernatant was recorded using UV vis spectrophotometer at 540 nm for reactive red 35 dye and compared with the control. The percent decolorization of the dye was calculated as mentioned earlier.

The degradation changes of azo dyes were characterized by FTIR spectroscopy. The analysis results were compared with control dye.

3. Results and Discussion

Actinomycete strains were isolated from effluent treated soil. By microscopic observation of mycelium, spore morphology and biochemical test revealed that the isolated strain was Micromonospora sp [Table 1]. Secondary screening was performed for dye decolourization and it is carried out in 250 ml flasks containing 100 ml of minimal medium with 20 ppm of Reactive red. Experiment was carried out for three days at 28°C. The results revealed that decolourization of RR were effective by Micromonospora sp.

The percent decolourization of reactive red dye by potential isolate Micromonospora spp at shaking and static conditions were as shown in the Table 2. The strain Micromonospora spp showed maximum decolourisation of 60% for reactive red dye in minimal media with peptone followed by 53% of decolorisation in minimal media with yeast extract under static condition. It was found that the isolate Micromonospora spp showed more percent decolorization of 50% using minimal media with yeast extract followed by 40% using minimal media with peptone under 200rpm shaking conditions. Under 100rpm shaking condition the isolate showed maximum decolourisation of 42% using minimal media with peptone followed by 30% using minimal media with yeast extract. These results showed that the isolate Micromonospora spp was more effective and potential in decolourising reactive red dye under still conditions than shaking conditions [Table 2].

No decolourization is found in actinomycetes inoculated ISP NO.4 broth when compared to the actinomycetes inoculated ISP NO.1 broth ¹⁸ due its impact on nitrogen source. ISP1 broth is taken for further studies to check the effect of different pH.

The pH is the important factor for the optimal physiological performance of microbial cultures and decolourization of dyes. It affects the cell growth and various biochemical and enzymatic mechanisms ²⁰. The actinomycetes culture was inoculated with the pH range at 2.0, 4.0, 6.0, 8.0 and 10.0 respectively. Studies have shown that the maximum decolourization by Micromonospora spp was observed at pH 8.0 of about 84% next to which at pH 6 the percentage of decolourization is found to be 74% [Table 3] when compared to other broth at different pH.

Fourier Transform Infrared Spectroscopy [FTIR] analysis was done for the control and the decolorized sample [Figure 2 and Figure 3] the results of which showed various peaks. Comparison between FTIR spectrum of RR35 and products obtained after decolorization confirmed the bioabsorption of dye. Spectra of control RR35 showed the presence of different peaks. The peak at 3374/cm was observed for N–H stretching, whereas the peak at 1017/cm represented N=N stretching, which indicates the presence of azo group of RR35, peak representing O–H stretching was observed at 3661/cm and 3678/cm, S=O stretching was observed at the peak of 1413/cm and 1346/cm indicating presence of sulphonated compound. C–O stretching was observed at 1113/cm. C-H stretching was observed at 2946/cm and 2841/cm, FTIR spectra of metabolites showed disappearance of sharp peak at 1017/cm, strongly supports predicted symmetric cleavage of azo bond in the molecule of reactive red dye carried out by induced activity of azoreductase, observed during dye decolorization.

The peak at 3500/cm represent N–H stretching of primary amines and peak at 1374/cm represent N-O stretching of nitro compound. The disappearance of some major peaks like 3374/cm, 2841/cm and 1017/cm from spectrum of parent dye and appearance of new peaks at 1758/cm and 1245/cm in spectrum support the biotransformation of RR35 into metabolites.

4. Conclusion

The current study concludes that the isolated, identified the Micromonospora sp have decolorization activity of textile azo dye, Reactive Red 35. optimized parameters for the maximum decolorization of Micromonospora sp showed maximum decolourisation of reactive red 35 at static condition of 60% using minimal media with peptone. Maximum decolourisation has been found at the pH 8. The optimum pH for decolorization of dyes is often at a neutral pH value or slightly acidic/alkaline pH. The rate of dye decolorization tends to decrease rapidly at strongly acid or strongly alkaline pH values. Thus Micromonospora spp can be used as bioagent for the decolourisation of textile azo dye, Reactive Red 35 effectively. However, there is a need for further investigation to understand the enzymes and other mechanisms involved in the decolorization of the azo blue dye by the isolate Micromonospora spp.

Acknowledgments

We thank the PG and Research Department of Microbiology, Dhanalakshmi Srinivasan College of Arts and Science for Women [Autonomous], Perambalur and MIET Arts and Science College, Trichy for supporting us to carry out this work.

References