Isolation and Characterization of Dye Degrading Micro-Organisms from Textile Waste Water

Index

1. ABSTRACT

2. INTRODUCTION

3. REVIEW OF LITERATURE
3.1. HISTORY OF DYES
3.2. DYES AND ENVIRONMENT
3.3. DYES AS ENVIRONMENTAL POLLUTANTS
3.4. BIOLOGICAL DEGRADATION OF TEXTILE DYES
3.5. ADVANTAGES OF AEROBIC BACTERIAL DEGRADATION OVER ANAEROBIC
3.6. BACTERIAL BIODEGRADATION OF TEXTILE DYES

4. OBJECTIVES OF THE STUDY:

5. MATERIALS AND METHODS
5.1. MATERIALS
5.2. METHOD

6. RESULTS AND DISCUSSION
6.1. PRELIMINARY ANALYSIS RESULTS
6.2. COLLECTION OF THE EFFLUENT SAMPLE AND BIOLOGICAL TREATMENT OF SAMPLE WATER
6.3. ENRICHMENT FLASK
6.4. ISOLATION OF DYE DEGRADING MICRO-ORGANISM ON MODIFIED NUTRIENT AGAR PLATE
6.5. ISOLATION OF MICROORGANISMS ON NUTRIENT AGAR PLATE
6.6. IDENTIFICATION OF BACTERIAL ISOLATES MORPHOLOGICALLY AND BY BIOCHEMICAL TEST

7. CALCULATIONS:

8. CONCLUSION:

FUTURE PROSPECTS:

REFRENCES:

ACKNOWLEDGEMENT

There are many people to whom I am greatly indebted for their support throughout my academic career. First and foremost, I must thank my research advisor, POOJA MISHRA and Dr. B. R. Throat, Coordinator, Department of Biotechnology to encourage me for the innovative work. Both truly epitomizes the qualities of an effective advisor, guiding their students through the process of developing strong research goals and strategies, while nurturing their sense of ownership of the project. I must specifically acknowledge the Dr. Swati Wavhal, Principal, Government of Maharashtra, Ismail Yusuf Arts, Science and Commerce College, Jogeshwari (East), Mumbai 60, India for their valuable guidance and encouragement. She is good guardian and supports me to complete my work. Enduring the struggles of total my research work, I spend number of hours in the laboratory for which requires a tremendous amount of support and sacrifice from family. I must thank my Family members and my friends Qureshi Mehak Naaz for their valuable support directly or indirectly to improve my research. I must thank to our Laboratory Non-Teaching staff NAEEM, ARCHANA and SAI for their direct and indirect help for the chemicals and glass wears required for the research work.

SAYYED ZARA ABDIN

1. ABSTRACT

The experiment was carried out to degrade the dyes by using bacterial isolates extracted from textile dye effluents. Five different bacterial isolates were screened and they have capability to degrade and decolorize the textile dye effluents. In the present study, an attempt was made to examine the potential of isolated bacterium for decolonization of lacto phenol cotton blue, Acetocarmine red & Eriochrome black T in batch reactors. Bacteria from textile waste water were subjected to acclimatization with dyes, in the basal nutrient media. The most promising bacterial isolate was used for further dye degradation studies. Biochemical characteristics revealed the isolated organism as Pseudomonas spp.The optimum pH and temperature for the decolonization was 6.0 and 37°C, respectively. This decolonization potential increased the applicability of this microorganism for the dye removal. The results suggest that the Pseudomonas spp. Can be used as a useful tool to treat waste water containing reactive dyes.

Key words: Textile dye, Effluent (waste water) Decolonization, Bioremediation and Bacteria.

2. INTRODUCTION

In modern life, rapid industrialization and urbanization resulted in the discharge of large amount of waste in to the environment, which in turn create lots of pollution. Water is essential for survival and existence of life on planet earth. The waste water and sewage released from the industries entering into the water bodies polluting whole water stream, it is one of major source of environment toxicity, it also affects the soil micro flora and aquatic ecosystem. The most environmental problem we face due to the textile dyeing industry. Industry produces large volumes of high strength of aqueous waste effluents. The discharge of dye effluents containing recalcitrant residue into rivers and lakes. The residual dyes from different source such as textile industries, cosmetics, paper mills, pulp industries, dyeing and dye intermediates and bleaching industries, more than 80,000 tons of dyes and pigments are produced in these industries. Especially in textile industries produced more than 70% of the total quantity of waste in India. India is the second largest exporter of dyestuffs and intermediates after China. The textile industry accounts for the largest consumption of dyes stuffs, at nearly 80%. Industrialization is vital to a nation's economy because it serves as a vehicle for development. However, there are associated problems resulting from the introduction of industrial waste products into the environment. Many of these products are problematic because of persistence (low biodegradability) and toxicity.

The effluent which is untreated is one of the major sources of consumed metal dyes, phenol, aromatic amines; several aromatic amines are known mutagens and carcinogens to human beings. Dyes also affect internal organ like kidney, liver. The recycling of these effluents using several techniques such as chemical degradation and some physical methods, Physico-chemical methods such as adsorption, irradiation, ion exchange, oxidative process, ozonation, coagulation on have been used to decolorize textile effluent but these methods are costly, inefficient and sometimes produce hazardous by-products, it also affects the environment during the degradation process. Extensive research in the field of biological dye effluent decolonization and degradation has shown promising results, but much of this work has been done with single model compounds. However, industrial textile wastewater presents the additional complexity of dealing with unknown quantities and varieties of many kinds of dyes, as well as low BOD/COD ratios, and present the high amount of auxiliary chemicals and heavy metals. Therefore, the effluents produced are markedly variable in chemical composition, including organics, nutrients, Sulphur compounds, salt sand different toxic substances. In biological treatment processes, various physicochemical operational parameters, such as the level of agitation, oxygen, temperature, pH, dye structure, dye concentration, supplementation of different carbon and nitrogen sources, electron donor and redox mediator, directly influence the bacterial decolonization performance of dye effluents.

Thus, to make the process more efficient, faster and practically applicable, prior determination of the effect of each factor on the bacterial decolonization of dye effluents is essential. This may affect the efficiency of the biological decolourization. Over the past decades, biological degradation has been investigated as method to transform, degrade or mineralization dye effluents. Moreover, such decolonization and degradation is an eco-friendly method and cost comparative alternative to chemical degradation process. Isolated bacteria are able to degrade dye either single (or) consortia methods. The objective of the present study is to analyze the physico-chemical characterization of textile dye effluents and isolate and characterization of dye degrading bacteria from dye effluents. In addition to the environmental problem, the textile industry consumes large amounts of potable water. In many countries where potable water is scarce, this large water consumption has become intolerable and wastewater recycling has been recommended in order to decrease the water requirements and also recycling of dyes to be used again. Without adequate treatment, these dyes are stable and can remain in the environment for an extended period of time. Therefore, this effluent must be treated before discharge into natural water stream. The most often used methods for decolonization and degradation of dyes are chemical and physical treatments whereas most of these methods have limitations such as high running cost and disposal of large amount of sludge produced during these processes. Consequently, most investigations have focused on using the most economical and the environmental friendly approaches such as radiation technology and biological processes. Industrial biological wastewater treatment systems are designed to remove pollutants from the environment using microorganisms. The microorganisms used are responsible for the degradation of the organic matter. Biological treatments have several advantages such as cheap, simple, produce smaller volumes of excess sludge and high flexibility, since it can be applied to very different types of effluents. The textile industries are considered one of the most important industries all over the world and considered as the 5th largest source of foreign currency, but it also considered as the main sources of water pollution because the textile companies in Egypt discharge their wastewater into soak way and in few cases to stream of potable water. Prior to displaying the effect of textile wastewater on the environment, the textile manufacturing process and the kind of toxic substances generated from this process must be known. There are main three stages involved; they are spinning, knitting or weaving and wet processing the later involves many steps like sizing, desizing, scouring, bleaching, mercerizing, dyeing, printing and finishing. Each of these operations generates huge amounts of wastewater and pollution from wet processing steps desizing is one of the largest sources of wastewater pollutants and often contributes up to 50% of the Biological Oxygen Demand (BOD) load in wastewater. The scouring process also has a high BOD and also uses the highest volumes of water in the preparatory stages.

The major pollution issues in the bleaching process are chemical handling, water conservation and high pH values. Also, using pentachlorophenol (PCP) during scouring, bleaching, dyeing and printing which is removed from the fabric and discharged into the wastewater. It is toxic due to its relative stability against natural degradation processes and it is also bio accumulative (EEAA, 2003). But the majority of wastewater containing residual dyes is generated after dyeing and printing. Colored wastes reportedly contribute about 10-30% of the total BOD and in many cases reach 90%. Dyes also contribute about 2-5% of then addition to the high BOD and Chemical Oxygen Demand (COD), while dye bath chemicals contribute about 25-35%.COD values. From the previous explanation of textile manufacturing process it is apparent that textile wastewater must be treated, although the concentration of dyes in wastewater is usually lower than the other chemicals present (less than 1 ppm for some dyes), they often receive the largest attention due to their strong color that render them highly visible even at very low concentration, thus causing serious aesthetic and pollution problems in wastewater disposal and water transparency and gas solubility in lakes, rivers and other water bodies (Kritikos et al., 2007). The removal of color from wastewater is often more important than the removal of soluble colorless organic substance, which usually contribute the major fraction of BOD (Banat et al., 1996) of dyes, toxicity to aquatic organisms and fish toxicity have also been reported. It is estimated that 280.000 tons of textile dyes are discharged every year in such industrial effluents worldwide (Maas and Chaudhary, 2005). Direct discharge of these effluents causes formation of toxic aromatic amines under anaerobic conditions in receiving media. In addition to their visual effect and their adverse impact in terms of COD, many synthetic dyes are toxic, mutagenic and carcinogenic (Jinn et al., 2007), therefore, water pollution control is currently one of the major areas of scientific activity.

3. REVIEW OF LITERATURE

3.1. HISTORY OF DYES

The history of dyes begins in 2600 BC, according to the earliest written record, with the use of dye stuffs in China. Dyes were originally obtained from animal and vegetable sources. For example, in the 15th century BC, Phoenicians already used Tyrian purple, which was produced from certain varieties of crushed sea snails, and the well-known plant dye indigo has been used since 3,000 BC. Also, Egyptian mummies were discovered to wrapped with dyed clothes made of madder plants. In South America, the Incas elaborated fine textures with different colors before being conquered by Spain. The chemical industry started in 1856 when the Englishman William Henry Perkin accidentally synthesized the first dye, “Mauve (aniline), a brilliant fuchsia color, while searching for a cure for malaria. In the following decades a considerable number of new dyes were synthesized (Welham, 2000). Azo dyes are the largest and the most important group of dyes, mainly due to their simple synthesis. The production of azo dyes began in 1858 when the German scientist P. Gries discovered the reaction mechanism diazotization for the production of azo compounds. Dyes are classified according to their application and chemical structure. They are composed of a group of atoms responsible for the dye color, called chromophores, as well as an electron withdrawing or donating substituent that cause or intensify the color of the chromophores, called auxochromes (Christie, 2001). The most important chromophores are azo (N=N-), carbonyl (-=0), methane (-CH=), nitro (-NO2) and quinoid groups. The most important auxochromes are amine (-NH3), carboxyl (-COOH), sulfonate (-SO3H) and hydroxyl (-OH). The auxochromes belong to the classes of reactive, acid, direct, basic, mordant, disperse, pigment, vat, anionic and ingrain, Sulphur, solvent and disperse dye (Welham,2000). It is estimated that almost 109 kg of dyes are produced annually in the world, of which azo dyes represent about 70% by weight. This group of dyes is characterized by reactive groups that form covalent bonds with OH-, NH-, or SH- groups in fibers (cotton, wool, silk, nylon). Azo dyes are mostly used for yellow, orange and red colors (Christie, 2001). To obtain the target color, normally a mixture of red, yellow and blue dyes is applied in the dye baths. These three dyes do not necessarily have the same chemical structure. They might contain in many different chromophores, in which azo, anthra-quinone and phtalocyanine dyes are the most important groups (Hao et al., 2000). Anthraquinone dyes constitute the second most important class of textile dyes, after azo dyes. Anthraquinone dyes have a wide range of colors in almost the whole visible spectrum, but they are most commonly used for violet, blue and green colors (Fontenot et al., 2003). In alkaline conditions, that is, pH 9 - 12 and salt concentration from 40-100 g/l, and at high temperatures (30-70°C), reactive dyes form a reactive vinyl sulfone (-S04-CH=CH2) group, which forms a bond with the fibers. However, the vinyl sulfone group undergoes hydrolysis, that is, a spontaneous reaction that occurs in the presence of water, and because the products do not have any affinity with the fibers, they do not form a covalent bond. Therefore, a high amount of dye constituents is discharged in the wastewater (Hao et al.,2000). The fixation efficiency varies with the class of azo dye used, which is around 98% for basic dyes and 50% for reactive dyes. Large amount of salts such as sodium nitrate, sodium sulphate and sodium chloride are used in the dye bath, as well as sodium hydroxide is widely applied to increase the pH to the alkaline range. It is estimated that during the mercerizing process, the weight of these salts can make up 20% of the fiber weight (EPA, 1997).

3.2. DYES AND ENVIRONMENT

The loss of dyes to effluent can be estimated to be 10% for deep shades, 2% for medium shades and minimal for light shades. Dyes are present in the effluent at concentrations of 10 to 50 mg/l with 1 mg/l being visible to the naked eye. They are complex organic compounds which are refractory in aerobic treatment systems. Some contain metals such as Cr, Cu and Zn. Only 50% (m/m) is dye, the remainder is non-hazardous filler and surfactant. There are 2 main factors involved in determining the risk assessment of chemicals, namely, hazard and exposure. Hazard describes the potential biological effects (for example, toxicity and carcinogenicity) that have a dose-response curve. Exposure is a measure of the expected environmental concentration of a chemical over time and distance. The data obtained from hazard and exposure studies will indicate what effects are possible, whereas risk assessment involves determining what is probable.

In the aquatic environment, dyes can undergo bio-concentration, ionization, abiotic oxidation, abiotic and microbial reduction, precipitation and ligand exchange. The ionic dyes such as acid, direct, basic and metal complex dyes will not volatilize whereas, in principle, solvent, disperse, vat and Sulphur dyes have the potential to be volatile. Sorption should also play a major role as dyeing is a sorption process. Hydrolytic reactions are not important because if the dyes survive the rigors of biological treatment processes, it is unlikely to degrade rapidly in the environment. Photochemical reactions may be important as dyes are good absorbers of solar energy.

However, little information is available on this. It is expected that anionic dyes would react with ions such as calcium and magnesium to form insoluble salts and thereby reduce the concentration available for other biological reactions. Similarly, for basic dyes, due to their interaction with humid material and hydrous oxides, redox reactions should also be considered; as in early vat dyeing processes, the dyes were reduced microbial before chemical replacements were introduced. Reduction in the environment would most likely occur under anaerobic conditions, however, the difficulties of working with anaerobic systems has limited research in this area. In general, there is very little literature available on the environmental behavior of dyes. This is probably due to the lack of suitable analytical techniques.

3.3. DYES AS ENVIRONMENTAL POLLUTANTS

Dyes contain chromophores, decolorized electron system with conjugated double bonds and auxochromes, electron-withdrawing or electron-donating substituents that cause or intensify the color of the chromophore by altering the overall energy of the electron system. Usual chromophores are - C = C. - C = N, - C = 0, -N = N, - NO2 and quinoa ring and auxochromes are - NH3, -COOH, - SO3H and OH (Van der Zee, 2002). Azo dyes are the largest group of dyes. More than 3000 different varieties of azo dyes are extensively used in the textile, paper, food, cosmetics and pharmaceutical industries (Maximo et al., 2003). Azo dyes are characterized by the presence of one or more azo groups -N=N-, which are responsible for their coloration and when such a bond is broken the compound loses its color. They are the largest and most versatile class of dye, but have structural properties that are not easily degradable under natural conditions and are not typically removed from water by conventional waste water system. Azo dyes are designed to resist chemical and microbial attacks and to be stable in light and during washing. Many are carcinoma-genic and may trigger allergic reactions in man. 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. Some of these compounds cause serious threat because of their carcinogenic potential or cytotoxicity (Adebayo et al., 2004).

Dyeing of textile requires water and generates a substantial quality of effluents containing mineral salts and dyes at high concentration. An estimated 700000 tons of dyes are produced annually worldwide of which 60-70% are azo dyes (Soars et al., 2004). Chronic effects of dyestuffs, especially of azo dyes, have been studied for several decades. Azo dyes in purified form are mutagenic or carcinogenic, except for some azo dyes, leads to for-motion of 7aromatic amines and several aromatic amines are known mutagens and carcinogens to human beings (Praveen et al., 2009). In mammals, metabolic reduction of azo dye is mainly due to bacterial activity in the anaerobic parts of the lower gastrointestinal tract. Various organs, especially the live and the kidney also reduce azo dyes. After azo dye reduction in the intestinal tract, the released aromatic amines are absorbed by the intestine and are excreted in the urine. The acute toxic hazard of aromatic amines is carcinogenesis, especially bladder cancer. International Agency for Research on Cancer (IARC) summarized the literature on suspected azo dyes, mainly amino substituted azo dyes, fat soluble dyes and benzidineazo dyes, as well as a few sulphonatedazo.

3.4. BIOLOGICAL DEGRADATION OF TEXTILE DYES

Dyes are stable against breakdown by many micro-organisms and most dyes do not biodegrade under the aerobic biological treatments in a municipal sewage plant. Many dyes, including the azo dyes, degrade under anaerobic conditions and the aromatic amines thus formed have been found to degrade further aerobically. Out of several methods that are used in the treatment of textile effluents to achieve decolourization, including physio-chemical methods like filtration, specific coagulation, use of activated carbon and chemical flocculation, some of the methods are effective but quite expensive. Bio treatment offers a cheaper and environmentally friendlier alternative for color removal in textile effluent (Dubrow et al., 1996). Bioremediation is a pollution control technology that uses biological systems to catalyze the degradation or transformation of various toxic chemicals to less harmful forms. This natural process of bioremediation, which includes bioengineering the capabilities of intrinsic microorganisms, to clean up the environment is an effective alternative to conventional remediation methods (Vidali, 2009).

3.5. ADVANTAGES OF AEROBIC BACTERIAL DEGRADATION OVER ANAEROBIC

A number of reports discourage the azo dye decolourization by microorganisms under aerobic conditions as it leads to the formation of corresponding aromatic amines. Even though their reductive cleavage is responsible for color removal, the formation of aromatic amines is highly undesired as they are reported to be carcinogenic. In the presence of oxygen, aromatic amines can be degraded. Dyes house effluents usually contain azo dyes which are highly resistant to biological treatment and these dyes are considered to be recalcitrant xenobiotic compounds because of the presence of N=N bond and other sulphonyl group which are together to be degraded. It was reported that some anaerobic bacteria can biodegrade dye stuffs by azoreductase activity. However, the effluent from biodegradation of dye stuffs could be toxic. Reverse colorization may take place when the degradation process is exposed to oxygen. Due to the following mentioned problems, full scale application of bacterial biodegradation is limited and research works have been reported on anaerobic degradation of azo dyes.

The decolourization of azo dyes has been found to be effective under anaerobic conditions. However, the anaerobic degradation yields aromatic amines which are mutagenic and toxic to humans and cannot be metabolized further under the conditions which generated them.

Due to these above mentioned problems, the full scale application of bacterial degradation is limited and research works have been reported on aerobic degradation of azo dyes.

3.6. BACTERIAL BIODEGRADATION OF TEXTILE DYES

Investigations to bacterial dye biotransformation have so far mainly been focused to the azo dyes. The electron withdrawing nature of the azo linkages obstructs the susceptibity of azo dye molecules to oxidative reaction. Therefore, azo dyes generally resist aerobic bacterial biodegradation. Only bacteria with specialized azo dye reducing enzymes (azoreductase)were found to degrade azo dyes under fully aerobic conditions. This anaerobic reduction implies decolourizations of the dyes to potentially harmful aromatic amines. Aromatic amines are generally not further degraded under anaerobic conditions. Anaerobic treatment must therefore be considered merely as the first stage of the complete degradation of azo dyes. The second stage involves conversion of the produced aromatic amines. For several aromatic amines, this can be achieved by biodegradation under aerobic conditions. Walker (1970) used facultative anaerobic bacteria where it was suggested that reduced Flavin’s generated by cytosolic Flavin dependent reductase were responsible for the unspecific reduction of azo dyes. Dubin and Wright (1975) investigated the decolourization of azo food dyes by Proteus vulgaris. It was shown that independently of the intracellular location of the azo reductase, theoretically, a redox mediator could facilitate the transfer of reducing equivalents from intracellular NADPH to the substrate dye. Meyer (1981) said that the azo dyes may be microbial degraded under anaerobic or aerobic conditions or in aerobic and anaerobic two-stage systems. Zimmermann et al. (1982) showed the reduction of azo compounds to occur under aerobic conditions. The aerobic azo reductases from the carboxyl orange degrading Pseudomonas strains had monomeric Flavin free enzymes that use NADPH and NADH as cofactors and reductively cleave several sulfonate azo dyes. Rafi et al. (1991) developed a plate assay for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, strains of anaerobic bacteria capable of reducing azo dyes were isolated from human faces and identified Eubacteriumhadrum, Clostridium clostridiiforme, Butyrivibriosp., Bacteroidessp., Clostridium paraputrificumand Clostridium nexile. Kudlich et al. (1997) suggested a different model for the non-specific reduction of azo dyes by bacteria which does not require transport of the azo dyes or reduced flavins through the cell membranes for Sphingomonasxenophaga. It was proposed that in this system, Quinone act as redox mediators which are reduced by quinonereducatse located in the cell membrane of S. xenophagaand that the formed reduce the azo dyes in the culture supernatant in a purely chemical redox reaction. Hu (1998) reported decolourization of dyes by facultative bacteria. COD/BOD reduction showed that decolourization occurs during the logarithmic growth phase and COD/BOD reduction during the maximum stationary growth phase. Kapdan et al. (2000) reported that decolourization of azo dye would not take place at a dissolved oxygen concentration higher than 0.45 mg/l and a slight increase in bacterial cell mass at the initial stage.

The low color removal at a temperature beyond 35°C may be attributed to the thermal deactivation enzymes and the low biomass. Yu et al. (2001) isolated different species of Pseudomonas sp. and showed a significant improvement on decolourization of recalcitrant non-azo dyes. The optimum decolourization activity was observed in a narrow pH range (7-8), a narrow temperature range (35-40°C), and at the presence of organic and ammonium nitrogen. Chen et al. (2002) isolated and selected. Aeromonashydrophilafor color removal from various dyes. More than 90% of red RBN was reduced in color within 8 days at a dye concentration of 3000 mg/l. This strain could also 10 decolorize the media containing the mixture of dyes within 2 days of incubation. Nitrogen sources such as yeast extractor peptone could enhance strongly the decolourization efficiency.

4. OBJECTIVES OF THE STUDY:

1. To study the physicochemical properties of textile waste water.
2. To isolate dye degrading Microorganisms from textile waste water.
3. To characterize isolated microorganisms on the basis of morphological and biochemical characteristics.
4. To determine the decolorization potential of isolated microorganisms.

5. MATERIALS AND METHODS

5.1. MATERIALS

a)Sample Waste water samples generated by the dying industry.

b) Dyes Acetocarmine Red, Lactophenol cotton Blue and ErichromeBlack T.

5.2. METHOD

a ) Collection of the effluent sample:

Aseptic techniques were followed during effluent collection. 200 ml samples were collected and put in the sterile beaker (300 ml capacity). The samples were subjected to immediate preliminary analysis. This sample served as the source for the isolation of dye degrading micro-organisms.

b) Preliminary analysis of effluent:

Absorbance, pH, COD, BOD, Temperature, Odor value of the effluent were measured.

c) Enrichment of the sample collected:

The collected dye waste water samples were subjected with modified Nutrient broth in 200ml flask for providing nutrition's to the microorganisms.

d) Isolation of dye degrading bacterial isolates from dye effluents:

The bacterial isolates present in the textile dye waste water were isolated by Streak plate technique. In this technique 1 loop full of culture from enrichment flask were streaked on three different plates of nutrient agar each containing three different dyes lacto phenol cotton blue, acetocarmine red, and eriochromeblack T. The Nutrient agar plates were incubated at 37°C for 24 hrs. After incubation, the bacterial colonies were isolated and purified from the plates. The well grown bacterial cultures used for further screening technique and stored at 4°C.

e) Screening of dye degrading bacterial isolates from effluents:

Three morphologically distinct bacterial isolates were tested for their ability to degrade the dye. The isolated bacteria were screened out by incubating them on 100 ml of nutrient agar medium with 10 ml dye effluents. The nutrient agar medium incubated at 37°C for 24 hours. After the incubation, plates were observed for clear zones. The screened culture was transfer to agar slant and store at 4°C for further study. Three morphologically distinct bacterial isolates showing more than 70% degradation of the added dye effluent. These efficient bacterial strains were selected for further studies.

f) Identification of selected isolates:

The three selected dye degrading bacterial strains based on their dye degrading ability were used and they were identified using morphological and biochemical properties of the standard protocol.

g) Dye decolonization experiments:

Dye decolonization experiments were carried out in three 250 ml Erlenmeyer flasks for three different effluent samples. Each flask containing 100 ml of Nutrient Broth with 15 ml of dye effluents. The pH was adjusted to 7±0.2. Then, the flasks were autoclaved at 121°C at 15 lbs. pressure for 15 minutes. The autoclaved flasks were inoculated with 5ml of bacterial inoculum of each isolates and bacterial consortium. The flasks were kept in mechanical rotary shaker and incubated at 37°C for 6 days. Samples were drawn at every 24 hours’ intervals for observation. About 10 ml of the dye solution was filtered and centrifuged at 5000 rpm for 20 minutes. Decolourization was assessed by measuring absorbance at 510 nm of the supernatant with the help of spectrophotometer at wavelength maxima of respective dye.

h) Decolourization assay

Decolourization assay was measured in the terms of percentage decolourization using spectrophotometer. The percentage decolorization was calculated from the following formula.

Total Decolorization(%) = Initial OD - Final OD X100

Final OD

6. RESULTS AND DISCUSSION

6.1. PRELIMINARY ANALYSIS RESULTS

Table-1: Physic-chemical characterization of textile dye effluent sample water

Abbildung in dieser Leseprobe nicht enthalten

6.2. COLLECTION OF THE EFFLUENT SAMPLE AND BIOLOGICAL TREATMENT OF SAMPLE WATER

The dye sample was collected from textile dye industry situated at Vasai Road, for the study of dye decolourization and bioremediation treatment of industrial effluents.

6.3. ENRICHMENT FLASK

Abbildung in dieser Leseprobe nicht enthalten

Fig1- Control(a) and Inoculated(b) flask containing dye degrading microorganism.

6.4. ISOLATION OF DYE DEGRADING MICRO-ORGANISM ON MODIFIED NUTRIENT AGAR PLATE

Abbildung in dieser Leseprobe nicht enthalten

Fig2- Dye degrading bacterial colonies in NA plate containing Lacto phenol cotton blue and Aceto carmine red dye

The textile dye effluent samples were inoculated with modified nutrient broth and soil as an enrichment medium for bacteria for inducing their growth and increasing their population and increasing their population and efficiency for dye decolorization process. The enriched flask was incubated at 37°C for their optimum growth for at least 15 days.

Control was kept to analyses the dye degrading activity by bacteria throughout the enrichment process.

6.5. ISOLATION OF MICROORGANISMS ON NUTRIENT AGAR PLATE

Abbildung in dieser Leseprobe nicht enthalten

Fig3- Isolated microorganism on Nutrient agar Plate

Isolated colonies were identified morphologically and biochemically test and are preserved at nutrient agar slants for future processes.

6.6. IDENTIFICATION OF BACTERIAL ISOLATES MORPHOLOGICALLY AND BY BIOCHEMICAL TEST

1) Biochemical Test

Abbildung in dieser Leseprobe nicht enthalten

2) Morphological Test Gram Staining of Isolate: -

Abbildung in dieser Leseprobe nicht enthalten

Fig4- Isolated micro-organism under 100x microscope

Colony characteristics.

Shape - Circular.

Margin - Entire.

Elevation - Raised.

Size - Punctiform, small.

Texture (Surface) - Smooth.

Appearance - Shiny.

Pigmentation - Non pigmented (Colorless).

Decolourization(%) of dyes by bacterial isolates in 7 days of incubation:

Abbildung in dieser Leseprobe nicht enthalten

7. CALCULATIONS:

% Decolorization = Initial OD-Final OD X 100

Initial OD

1. Lacto phenol cotton blue = 0.51-0.45 x 100 = 11% 0.51

0.45-0.42 x 100 = 6.6%

0.45

0.42-0.39 x 100 = 7.14%

0.42

2. Acetocarmine red = 0.45-0.37 x 100 = 17.7% 0.45

0.37-0.36 x 100 = 2.77%

0.37

0.36-0.30 x 100 = 16.6%

0.36

3. Eriochrome Black T = 1-0.90 x 100 = 10% 1

0.90- 0.85 x 100 = 5.55%

0.90

0.85-0.82 x 100 = 3.53%

0.85

8. CONCLUSION:

Dye decolourization experiment was carried out to isolate and characterize the dye degrading bacteria from textile waste water. The degradation of dye effluents by using several biological methods was successfully done. As the physico-chemical methods are economically limited. The biological methods are more effective and low expense of treatment by using bacteria isolated from textile dye effluents. The present study was focused on biodegradation of textile dye effluents and decolorization assay. Bacteria from textile waste water were subjected to acclimatization with dyes in the basal nutrient media. The most promising bacterial isolate was used for further dye degradation studies. Biochemical characteristics revealed the isolated organism as Pseudomonas spp which is to be confirmed by further tests. They have shown the maximum dye degrading activity, thus they can be used as bioremediating agents to overcome the removal problem of carcinogenic and hazardous dyes from the discharge of various textile dye industries. The physical and chemical analysis was also done of textile effluent water before carrying out the biological treatments of textile dye waters. The available evidence suggests that dye structure affects decolourization rates but the basis for establishing a corelation between these two parameter remains, so far, unknown.

FUTURE PROSPECTS:

The biodegradation of dyes is a process relevant to the treatment of waste waters from textile industries which are frequently heavily colored and resistant to conventional waste water treatment processes. This would allow the development of a pilot treatment plant to apply in a textile industry. Another possibility of this work would be to go on exploring the capability of other microorganisms as bioremediation agents of textile dyes, particularly the more dye degrading bacteria ‘bacteria can be isolated from contaminated sites of different textile industry. Also those with oxidative capabilities can be eventually useful for the degradation of other types of dyes. We can also explore some thermophilic strains for the treatment at higher temperatures and strains with a wider operational ph. In addition, it has to be emphasized that azo dyes are toxic and harmful. Microbes need to produce enzymes degrading compounds that can be tested extensively inside. Subsequently, there would be a possibility of development of bacterial consortium capable of completely degrading, with limited envoi mental maintenance and substrate input. Further research is therefore being carried to develop and test the consortium for enhancing their dye degrading activity. Biological processes are being designed to take advantage of the biochemical reaction that can be carried out in living cells of different dye degrading bacteria. Such processes can make use of this natural process to overcome this pollution problem.

REFRENCES:

1 Reminder Kaur, Siddhartha Vats, Summit Mekhi, AnkitBhardwaj, JharnaGoel, Ranjeet S. Tanwar and Komal. K. Gaur, Procedia Environmental Sciences, 2010, 2, 595–599.

2 Thoker Farook Ahmed, Manderia Sushill and Manderia Krishna, International Research Journal of Environment Sciences, 2012, 1(2), 41-45.

3 N. Manikandan, S. SurumbarKuzhali and R. Kumuthakalavalli, J. Microbiol. Biotech.Res., 2012, 2 (1), 57-62. 4K. Rajeswari, R. Subashkumar and K. Vijayaraman, J. Microbiol.Biotech. Res., 2013, 3 (5), 37-41.

5 K. Varunprasath and A.N. Daniel, Iranica. J. Energy Environ. 2010, 1, 315-320.

4 K. Rajeswari, R. Subashkumar and K. Vijayaraman, J. Microbiol. Biotech. Res., 2013, 3(5), 37- 41.

6 D. Suteu, C. Zaharia, D. Bibla, A. Muresan. R. Muresan and A. Popescu, Industria Textila, 2009, 5, 254-263.

7 Praveen Sharma, G.R. Chaudry and Thomes Edison, Applied Environmental Microbiology, 2009, 42(4): 641-648.

8 Sriram, N., D. Reetha and P. Saranraj, Middle-East Journal of Scientific Research, 2013,17 (12), 1695-1700.

9 N. Ramamurthy, S. Balasaraswathy and P. Sivasakthivelan. Romanian J. Biophys., 2011,21 (2), 113–123.

10 F. J. Cervantes, F.P.Van der Zee, G. Lettinga. Water Science and Technology, 2001, 44,123- 128.

11 J.L. Bragger, A.W. Lloyd, S.H. Soozandehfar, International Journal of Pharmacy, 1997,157: 61-71.

12 O. Khadijah, K. K. Lee. and Mohd Faiz F. Abdullah. Malaysian Journal of Microbiology, 2009, 5(1), 25-32.

13 Mir Tariq Ahmad, Manderia Sushil and Manderia Krishna, Internation Research Journal of Environment Science, 2012, 1(1), 50-53.

14 Sofia Nosheen, Haq Nawaz and Khalil-UR-Rehman, International Journal of Agriculture and Biology, 2000, 2(3), 232-233.

15 P. Saranraj, V. Sumathi, D. Reetha and D. Stella. Journal of Ecobiotechnology, 2010, 2(7): 12 - 16.

16 M.M. Hassan, M.Z. Alam and M.N. Anwar., International Research Journal of Biological Sciences, 2013, 2(8):27-31. 17 A. Karthikeyan and N. Anbusaravanan. IOSR Journal of Environmental Science, Toxicology and Food Technology, 2013, 7 (2): 51-57.22

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