1. Introduction

The growth of the world population, the development of various industries, and the use of fertilizers and pesticides in modern agriculture have overloaded not only the water resources but also the atmosphere and the soil with pollutants ^[10]. In the last few decades the handling of wastewater appeared to be one of the most important. Textile industry which is one of the largest water consumers in the world produces the wastewater comprising of various recalcitrant agents such as dye, sizing agents and dying aid. Therefore it has to be really concerned in releasing these types of wastewater to the environment. In the disposal of textile wastewater, color is of very important due to the aesthetic deterioration as well as the obstruction of penetration of dissolved oxygen and sun light into natural water bodies ^[24]. The degradation of the environment due to the discharge of polluting wastewater from industrial sources is a real problem in several countries. This situation is even worse in developing countries like India where little or no treatment is carried out before the discharge ^{[3, 4, 25]}. In spite of the many steps taken to maintain and improve the quality of surface and groundwater, the quantities of wastewater generated by these industries continue to increase and municipalities and industries are confronted with an urgent need to develop safe and feasible alternative practices for wastewater management. Bioremediation is a pollution-control technology that uses natural biological species to catalyze the degradation or transformation of various toxic chemicals to less harmful forms. Xenobiotic compounds are not naturally available and hence the locally occurring microorganisms cannot readily degrade them. Hazardous materials may render harm to humans, livestock, wildlife, crops or native plants through handling, ingestion, application to land or other distributions of the contaminated materials into the environment. The textile industry leaves about 50 % of the textile azo dyes in free state to be discharged in the factory effluent and eventually to the surrounding environment. Azo compounds constitute the largest and the most diverse group of synthetic dyes and are widely used in a number of industries such as textile, food, cosmetics and paper printing ^[21]. The reactive azo dyes-containing effluents cause serious environmental pollution. Therefore, industrial effluents containing azo dyes must be treated before discharging into the environment to remove the dye toxicity from textile effluents ^{[11, 23, 27, 29]}. This study aims to investigate the potential of bacterial cultures isolated from industrial dye effluent for decolorization of a textile dye, Crystal violet. Dye decolorization by bacterial cultures was optimized with respect to various nutritional sources (carbon and nitrogen), environmental parameters (temperature, pH).

2. Materials & Methods

Ankleshwar is one of the most industrialized cities in India. It is known as the chemical hub and was chosen for effluent sample collection. The Effluent sample was collected from the middle point of the area. Standard procedures (Spot and Grab) were followed during sampling. The Temperature and pH were determined at the sampling site. The pH was determined by using pH meter (Cyber scan pH meter) and temperature with laboratory thermometer. The sample was transported to laboratory at 4ºC as in accordance with the standard methods ^[31]. The physicochemical parameters such as (Colour, Biological Oxidation Demand (BOD) Chemical Oxygen Demand (COD), Total Suspended Solids (TSS), and Total Dissolved Solids (TDS) were determined as soon as the sample was brought to the laboratory. Sample colour was analyzed by spectrophotometer (Shimadzu UV-1700). BOD was determined by employing evaporation method by DO meter while COD was measured by COD instrument directly.

The textile dye, Crystal violet (λmax 523 nm) was obtained from Ankleshwar Textile Industries, Ankleshwar. Nutrient broth. A stock solution of the dye (1000 mg L^–1) was prepared in de-ionized water and used for all studies.

The Textile Effluent was collected in sterile collection tubes from the sludge and wastewater of the ditches at industrial site located in Ankleshwar Textile Industries, Ankleshwar. The sample collected from the textile mill was screened for azo dye (Crystal violet) decolorizing bacterial strains by inoculating 10 ml of sludge solution into 250ml. Erlenmeyer flask containing 100ml nutrient broth (gL^–1 Peptone-5, Meat extract-1, Yeast extract-2, NaCl-5, pH-7). The flasks were incubated at 35°C under shaking conditions (140rpm). After 48h of incubation, 1.0ml. of the culture broth was appropriately diluted and plated on Nutrient Agar containing 20 mg L^–1 Crystal violet. The Morphologically distinct bacterial isolates showing clear zones around their colonies due to decolorization of dye were selected for further studies. The pure culture stocks of these isolates were stored at 4°C on Nutrient Agar slopes containing 1000 mg L^–1 of Crystal violet. These isolates were screened for their ability to decolorize Crystal violet in liquid culture. The Screening process in liquid media was carried out by inoculating a loop full of cultures exhibiting clear zones into Nutrient broth containing Crystal violet under static conditions. After 24h of incubation, 1ml. of cell suspension was transferred to fresh nutrient broth containing Crystal violet to screen the strains with color removing ability. The Screening procedure in liquid medium was continued until complete decolorization of broth. A small amount of decolorized broth was transferred to nutrient agar plates containing Crystal violet (50 mg L^–1). The bacterial isolate which tolerated higher concentration of the Azo dye was isolated by streaking plate method. The Azo dye decolorizing bacteria was identified from several aspects including morphology characters, biochemical tests as described in Bergey's manual of determinative bacteriology (Indole, Methyl Red, Voges-Proskauer test, Citrate, Catalase, Oxidase, Nitrate Reduction test, Hydrolysis of Casein, Starch, Urea and Gelatin). Assimilation of various sugars such as D-glucose, D-fructose, galactose, mannitol and D-maltose as sole carbon source were determined by inoculating the isolate into carbohydrate broth supplemented with respective carbon source. After inoculation the tubes were incubated at 37ºC for 24-48h.

The decolorizing activity was expressed in terms of the percentage decolorization by the modified method described previously ^[8]. The Decolorization process was carried out using shaking culture and static culture by inoculating 1ml. of precultured (O.D 0.85-1) Bacillus spp. ETL-79 into 100ml. of sterilized Nutrient broth in 250 ml. Erlenmeyer flask and incubated on rotary shaker (130 rpm) at 35ºC for 24h ^[15]. Filter sterilized (0.22 λm) Crystal violet (100 mgL^–1 ) was added to the culture and incubated in shaking conditions at 140rpm and in static conditions at room temperature for decolorization to occur. At regular intervals, 4 ml.sample was withdrawn aseptically and centrifuged at 8,500 rpm for 15min. The cell free supernatant was used to determine the percentage decolorization of Crystal violet. Decolorization of dye was determined by monitoring the decrease in absorbance at the maximum wavelength of Crystal violet (λmax 523 nm) by using a UV-Visible spectrophotometer (UV-1700 Pharmaspec, Shimadzu, Japan). The uninoculated dye Medium supplemented with respective dye was used as blank ^[13]. Decolorization activity (%) was calculated by the following formula and all assays were done in triplicate:

The decolorization efficiency of Bacillus spp. ETL-79 strain was compared over a wide range of pH (5-9) by adjusting the pH with hydrochloric acid or sodium hydroxide. Decolorization at different Temperatures (RT, 35°C, 37°C, 40°C, 45°C, 50°C) was carried out by adjusting the pH to 8. Varying Carbon sources 1% each (dulcitol, starch, maltose, sucrose, dextrose, mannitol, d-xylose, lactose, mannose) and Nitrogen sources 1% each (urea, potassium nitrate, sodium nitrate, malt extract, ammonium sulphate, ammonium nitrate, ammonium chloride, peptone) were used to check the decolorizing potential of the strain. All the flasks were incubated in static conditions at pH 8 and at 35°C.

3. Results & Discussion

The effluent sample collected from a small scale Textile Industries, Ankleshwar, Gujarat, India was dark black in color, with pungent smell and pH of slightly above neutral level and was within the permissible limits (Table 1). The temperature of the effluent was high. Total Suspended Solids (TSS) and Total Dissolved Solids (TDS) in the textile effluent were very high. The solids present in ground water, besides effecting the growth of the plants directly, also affect the soil structure, permeability and aeration, indirectly effecting the plant growth. The Chemical Oxygen Demand (COD) and Biological Oxygen Demand (BOD) values were within the permissible limits in the effluent sample. Different bacterial strains isolated from the textile effluent were screened for their ability to decolorize the textile Azo dye (Crystal violet) and the potential strains were characterized morphologically and biochemically.

Table 1. Physico-chemical characterization of the textile effluent collected from Textile Industries, Ankleshwar

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The study was started by screening for potential textile Azo dye decolorizing bacteria isolated from the textile industry effluent. Colonies surrounded by a nearly decolorized zone were isolated and then tested for dye removal capability using submerged culture. Strains isolated from the white colonies were inoculated in 100ml. of Nutrient broth in a 250ml. conical flask and incubated at 35°C under static conditions. ETL-79 exhibiting highest decolorizing activity was chosen for further studies. The gram staining test showed the isolate to be non-motile, gram positive, spore forming, and rod-shaped bacteria. The spore was terminally located and ellipsoidal in shape. Biochemical characterization of the isolate revealed it to be negative for Indole, Methyl Red test, Voges-Proskauer, Citrate, Catalase, oxidase test and Nitrate Reduction test. The isolate showed negative result for the hydrolysis of casein, gelatin, starch and urea. The strain utilized various sugars, D-Maltose, D-Glucose, D-Fructose, Mannitol and Galactose as sole carbon sources and was found to be positive.

The decolorization efficiency of Bacillus spp. ETL-79 was compared across a wide range of pH (5-9). The maximum decolorization (90%) was recorded at pH 8. At neutral pH the strain exhibited percentage decolorization value of 80%. Whereas it was 45% and 40% at pH 6 and 9. The percentage decolorization decreased markedly at pH 5 (10%) due to acidic conditions (Figure 2). The optimum pH for growth and decolorization was found to be 8. The dye decolorization activity of the strain was found to decrease with increasing incubation temperature. Highest decolorization was achieved at 35°C (95%) and least percentage decolorization was at Room Temperature (RT) (25%). At 37°C there was 85% decolorization noted followed by 70%, 55% and 25% at 40°C, 45°C and 50°C respectively at the end of 24h incubation (Figure 1). No specific decolorization was observed in shaking conditions (140 rpm).

Figure 1. Effect of temperature on Crystal violet decolorization

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Figure 2. Effect of pH on Crystal violet decolorization

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Figure 3. Effect of Carbon source on Crystal violet decolorization

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Results of Crystal violet decolorization by ETL-79 with different Carbon (Figure 3) and Nitrogen sources (Figure 4) are depicted. Dextrose resulted in higher decolorization efficiency to 92% followed by starch (80%) and mannose (65%) at the end of 24h incubation period. The decolorization efficiency decreased with dulcitol (52%), mannitol (45%), lactose (35%), d-xylose (30%) and sucrose (25%). Least decolorization was observed with maltose (10%). Maximum decolorization with nitrogen sources was achieved with Peptone (90%) and least was with Malt extract (15%). Urea and Ammonium sulphate exhibited good decolorization with 75% and 65%. The decolorization efficiency decreased markedly with Ammonium nitrate (50%), Sodium nitrate (25%), Potassium nitrate (22%) and Ammonium chloride (15%).

Figure 4. Effect of Nitrogen source on Crystal violet decolorization

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Throughout India, there is a grave concern and constant attention given to the treatment of industrial effluent from textile and dye manufacturing units. Several researchers have demonstrated the possibility of utilizing microorganisms for biotreatment of textile waste water. In India, most textiles units are scattered and/or operated from private homes. Therefore, it is necessary to collect and treat the waste in common effluent treatment plants. Microbiological methods are quite simple to use and cost of operation is low. Industrial effluent is not stable and it varies often in a wide range depending upon the process practiced. South Asian countries are experiencing severe environmental problems due to rapid industrialization. This phenomenon is very common where the polluting industries like textile dyeing, leather tanning, paper and pulp processing, sugar manufacturing, etc. thrive as clusters. Among these the Textile industries are large industrial consumers of waters as well as producers of wastewater. The effluent discharged by this industry leads to serious pollution of groundwater and soils and ultimately affects the livelihood of the poor ^[14]. The physico-chemical characterization of the collected textile effluent sample from Textile Industries, Ankleshwar showed a high load of pollution indicators. Colour is contributed to a water body by the dissolved compounds (dyes and pigments). The effluent color was dark black due to mixture of various dyes and chemicals used in the dyeing process. The pH of the study sample was slightly alkaline when compared to the acidic pH of the dyeing effluent in a previous study ^[26]. The pH of the effluent alters the physico-chemical properties of water which in turn adversely affects aquatic life, plant and humans. The soil permeability gets affected resulting in polluting underground resources of water ^[28]. The temperature of the effluent was high in comparison with the temperature of another effluent in one study ^[19]. High temperature decreases the solubility of gases in water which is ultimately expressed as high BOD/COD. The values of BOD and COD were within the permissible limits in the present sample in comparison to the very high values of BOD and COD. TDS and TSS values of effluent sample were higher than the permissible limits. Sediments rate is drastically increased because of High value of Total Dissolved Solids which reduces the light penetration into water and ultimately decrease the photosynthesis. The decrease in photosynthetic rate reduces the DO level of wastewater which results in decreased purification of wastewater by microorganisms ^[9]. The current sample exhibited high values of heavy metals which were of the same order of magnitude reported in another effluent sample ^[18]. The nutrients of the surrounding soils are depleted as a result of high value of heavy metals thereby affecting soil fertility. High chloride contents are harmful for agricultural crops if such wastes containing high chlorides are used for irrigation purposes ^[2]. Majority of the textile effluent samples have permissible limits of sulphate ions. The effluent showed phenolic contents greater than 0.1 ppm which is though permissible limit of the phenolic compounds that are very toxic to fish even at very low concentrations ^[5]. The bleaching and dyeing process are the main causes of pollutants which include caustic soda, hypochlorite and peroxides. The isolation of different microorganisms from the effluent sample collected from the Textile Industries, Ankleshwar indicates natural adaptation of microorganisms to survive in the presence of toxic chemicals and dyes. Interest in the bioremediation of pollutants using bacteria has intensified in recent years, as many researches demonstrated the efficacy of bacterial bioremediation over fungal and Actinomycetes. Many bacteria capable of reducing Azo dyes reported were isolated from Textile effluent contaminated sites^[7]. A strain of bacterium Bacillus spp. ETL-79 with strong decolorizing ability was isolated from Textile effluent to decolorize the textile Azo dye Crystal violet (100 mgL^–1) within 24h in aerobic and static conditions. The reason for the decreased decolorization under shaking conditions could be competition of oxygen and dye compounds for the reduced electron carriers under aerobic conditions. The percentage decolorization of Crystal violet by Bacillus spp. ETL-79 strain under static conditions was 90% within 24h of incubation which was equal to a similar study but with 35h of incubation period ^[17]. In another study conducted with Pseudomonas putida, P.fluorescence, Bacillus cereus and Stentrophomonas acidaminiphila to decolorize Acid Red 88 showed their efficiencies at 35%, 31%, 40% and 50% respectively ^[12]. Under aerobic conditions azo dyes are generally resistant to attack by bacteria ^[6]. The optimal pH for complete decolorization of Crystal violet was 8 which is slightly lower in accordance with Cosmarium spp. Decolorizing malachite green at pH 9 ^[30] and Klebsiella pneumonia RS-13 which completely degraded Methyl Red in pH range of 6 to 8 ^[20]. Optimal growth temperature was found to be 35°C which is consistent with the highest decolorization temperature in our study. Maximum potential of Pseudomonas sp. to decolorize Malachite green, fast green was noticed at 37°C ^[1]. Vibrio logei and Pseudomonas nitroreducens showed the highest Methyl Red degradation activity at 30-35°C ^[16]. Dextrose and Peptone were found to be the most effective carbon and nitrogen sources for decolorization of Crystal violet in the present study compared to Lactose and Yeast extract in another similar study for decolorization of Everzol Red RBN ^[22].

4. Conclusion

The study concluded that pH, temperature, various carbon & nitrogen source have a significant influence on dye removal efficiency by Bacillus spp. ETL-79. This shows that the isolated bacterium have enormous potential to degrade the textile dyes and resolve the problem of unnecessary dyes present in the effluents of textile industries. Further pilot scale studies are required with these strains for actual industrial applications, and detailed study is needed to explore the mechanism involved. Although decolorization is a challenging process to both the textile industry and the waste water treatment, the result of this findings and literature suggest a great potential for bacteria to be used to remove color from dye wastewaters. The bacterial strain Bacillus spp. HMM-87 showed decolorizing activity through a degradation mechanism rather than adsorption. This observation has established that the bacteria are adaptive in nature and can degrade contaminants. The ability of the strain to tolerate, decolorize azo dyes at high concentration gives it an advantage for treatment of textile industry waste waters. However, potential of the strain needs to be demonstrated for its application in treatment of real dye bearing waste waters using appropriate bioreactors. Application of traditional waste water treatment requires enormous cost and continuous input of chemicals which becomes uneconomical and causes further environmental damage. Hence, economical and eco-friendly techniques using bacteria can be applied for fine tuning of waste water treatment. Biotreatment offers easy, cheaper and effective alternative for colour removal of textile dyes.

Acknowledgement

Authors are highly grateful to the management of Enviro Technology Limited., Ankleshwar, Gujarat, India for allowing us to carry out such a noble work for the sustainable environment.

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