Title: Isolation and Process Optimization of textile dye degrading bacteria

Abstract:
Bacteria can remove dyes from wastewater and soil, leading to a decrease in their toxicity. The detoxification rate depends upon media and culture conditions. The decolourization/removal of textile dyes like bromophenol blue, methylene blue, methyl green, and malachite green, in addition to various other industrial dyes, using bacteria isolated from soil has been an important area of research for bioremediation and the present work is focused on this specific aspect. The effect of independent variables such as time, temperature, pH, and agitation on decolorization efficiency of bacteria has been optimized. Biodegradation of methylene blue and bromophenol blue was demonstrated by monitoring the effects of the different parameters and determining optimal conditions for degradation activity. Introduction
Several industrial activities, such as textile dyeing, olive oil extraction and the manufacture of pulp and paper are characterized by intensive water consumption rates. They release huge amounts of more or less coloured effluents into the environment. As far as synthetic dye release is concerned, textile dyeing facilities and the manufacture of dyestuffs are two major polluting sources. In addition, traditional textile dyeing processes generate a large amount of coloured effluents, because about 100 litres of water are required to process 1 kg of dyed fabrics. Moreover, up to 15% of applied dyestuffs are lost to the effluents due to dyeing process inefficiencies. Colour itself could be very pernicious to the receiving water courses not only for aesthetic reasons and toxicity towards many aquatic organisms, but also because coloured compounds reduce water transparency, which, in turn, affects photosynthetic activity, thus causing severe damage to the ecosystems. (Nermeen and El-Sersy, 2001)
Industrial textile dyes have been designed and synthesized to be highly resistant to washing, chemical agents, including solvents, and environmental factors, such as the actionof sunlight, water and microbial attack. On the other hand, heavy metal complexed dyes are of public health concern. There are currently more than 10,000 different textile dyes commercially available in the world market, which can be classified according to the application process and chemical class. The chemical structures of selected textile dyes illustrating the following chromophoric groups – azo, indigoid, anthraquinone, triphenylmethane and phthalocyanine – are presented below. Among them, azo, indigoid and anthraquinone are the major chromophores used in the textile industries, azo dyes being the largest class. (Mohee and Mudhoo, 2004)

Indigoid (A), anthraquinone (B), triphenylmethane (C), azo (D), phthalocyanine (E)

A large portion of dyes, that is lost during the dyeing process, could remain more or less intact, given the fact that both traditional physic-chemical and biological wastewater treatments are unable to perform an acceptable degradation and decolorization of the majority of available dyes. For example, Weber and Stickney (1999) have reported that the half-life of reactive blue 19 is 46 years at 25°C and pH 7.0. Combined with physico-chemical treatments, activated sludge is the process most widely used by the textile industry (Weber and Stickney, 1999). In this process, the effluent is mechanically agitated in the presence of air and microbial biomass. In spite of removing upt o 80% of dye content, most of it (40-80%) is only absorbed or adsorbed into the biomass, producing sludge with high dye concentration, which prevents its furthur utilization. Besides displaying high levels of sludge production, it is also very sensitive to effluent composition, particularly as far as the content of toxic substances is concerned. Moreover, activated sludge treatment is almost ineffective with reactive textile dyeing effluents. (Dos Santos et al., 2007; El Ahwany, 2008).
The presence of dyes in effluents is a major concern due to their adverse effect to many forms of life. Colored waters are one of the most important hazard in industrial effluents, which needs to be treated because the presence of dyes in water reduces light penetration, precluding the photosynthesis of aqueous flora. Besides that, some dyes may cause allergy, dermatitis, skin irritation and cancer to humans in addition to being mutagenic. Synthetic dyes are extensively employed in textile, paper, photo electrochemical cells, printing, leather, food, cosmetics, etc. industries, which employ these substances to color their final products. The treatment of aqueous water containing soluble dyes thus requires complete removal followed by secure disposal. The most commonly used techniques for color removal include chemical precipitation, ion exchange, reverse osmosis, ozonation and solvent extraction etc. However, these techniques have certain disadvantages such as high capital cost and operational costs or secondary sludge disposal problem. Microbial decolorization processes offer a complete cleanup of pollutants in a natural way and appear to be an attractive alternative.
Anaerobic treatment with the production of methane, carbon dioxide and water requires less energy effort and produced low sludge quantities. It has been shown that reductive decolourization of azo dyes could be achieved by the action of bacterial strains under anaerobic conditions. However, the production of potentially carcinogenic aromatic amines, which resist further degradation, has been reported. Furthermore, re-colorization of anaerobic-treated effluents may take place upon exposure to air. These findings triggered the screening of alternative biological systems as well as its performance evaluation. In recent years, a growing number of research papers have been putting in evidence for the feasibility of dye decolourization by fungi and their oxidative enzymatic systems (Mabrouk and Yousef, 2007). Another promising process for the treatment or final polishment of textile effluents is the phytoremediation technology with constructed wetlands.

Aim and Objective:
Aim:
The aim of this project is to isolate species of textile dye degrading bacteria and optimize the growth conditions required for maximum degradation of the selected dyes.

Objectives:
1) Different soil samples collected from different textile industries of Noida will be screened for Isolation of textile dye degrading bacteria by serial dilution method
2) Optimization of growth conditions and degrading capacity of isolated bacteria
3) Characterization of Isolated bacterial species.

Experimental Strategy:
The basic strategy followed in this experiment was:
1.) Preparation of Serial Dilutions of the collected samples.
2.) Growing cultures by preparing spread plates of different serial dilutions
3.) Further purification of bacterial cultures by streaking identified colonies on to fresh plates
4.) Subculturing of pure cultures
5.) Preparation and inoculation of agar plates and luria broth containing known concentrations of selected dyes.
6.) Identification of colonies of textile dye degrading bacteria.
7.) Optimization of growth conditions: Temperature, pH, Incubation Time and Agitation rate.
8.) Characterization of bacterial species by staining methods.

Review of Literature:
A dye is a coloured substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber. (Aksu, 2005)
Both dyes and pigments appear to be colored because they absorb some wavelengths of light more than others. In contrast with a dye, a pigment generally is insoluble, and has no affinity for the substrate. Some dyes can be precipitated with an inert salt to produce a lake pigment, and based on the salt used they could be aluminum lake, calcium lake or barium lake pigments. (Chander and Arora, 2007)
Dyed flax fibers have been found in the Republic of Georgia dated back in a prehistoric cave to 36,000 BP. Archaeological evidence shows that, particularly in India and Phoenicia, dyeing has been widely carried out for over 5,000 years. The dyes were obtained from animal, vegetable or mineral origin, with no or very little processing. By far the greatest source of dyes has been from the plant kingdom, notably roots, berries, bark, leaves and wood, but only a few have ever been used on a commercial scale. (Mabrouk and El-Ahwany, 2008)
Natural Dye:
The majority of natural dyes are from plant sources – roots, berries, bark, leaves, and wood, fungi, and lichens. Textile dyeing dates back to the Neolithic period. (Browne and Zerban, 1948) Throughout history, people have dyed their textiles using common, locally available materials. Scarce dyestuffs that produced brilliant and permanent colors such as the natural invertebrate dyes Tyrian purple and crimson kermes were highly prized luxury items in the ancient and medieval world. Plant-based dyes such as wood, indigo, saffron, and madder were raised commercially and were important trade goods in the economies of Asia and Europe. Across Asia and Africa, patterned fabrics were produced using resist techniques to control the absorption of color in piece-dyed cloth. Dyes from the New World such as cochineal and logwood were brought to Europe by the Spanish treasure fleets, and the dyestuffs of Europe were carried by colonists to America. (Acemioglu et al., 2010)
The discovery of man-made synthetic dyes late in the 19th century ended the large-scale market for natural dyes.
Synthetic dye:
The first human-made (synthetic) organic dye, mauveine, was discovered serendipitously by William Henry Perkin in 1856. Many thousands of synthetic dyes have since been prepared.
Synthetic dyes quickly replaced the traditional natural dyes. They cost less, they offered a vast range of new colors, and they imparted better properties to the dyed materials. Dyes are now classified according to how they are used in the dyeing process.
Dye Types:
Acid dyes are water-soluble anionic dyes that are applied to fibers such as silk, wool, nylon and modified acrylic fibers using neutral to acid dye baths. Attachment to the fiber is attributed, at least partly, to salt formation between anionic groups in the dyes and cationic groups in the fiber. Acid dyes are not substantive to cellulosic fibers. Most synthetic food colors fall in this category. (Sandhya et al., 2004)
Basic dyes are water-soluble cationic dyes that are mainly applied to acrylic fibers, but find some use for wool and silk. Usually acetic acid is added to the dyebath to help the uptake of the dye onto the fiber. Basic dyes are also used in the coloration of paper.
Direct or substantive dyeing is normally carried out in a neutral or slightly alkaline dyebath, at or near boiling point, with the addition of either sodium chloride (NaCl) or sodium sulfate (Na2SO4) or sodium carbonate (Na2CO3). Direct dyes are used on cotton, paper, leather, wool, silk and nylon. They are also used as pH indicators and as biological stains.
Mordant dyes require a mordant, which improves the fastness of the dye against water, light and perspiration. The choice of mordant is very important as different mordants can change the final color significantly. Most natural dyes are mordant dyes and there is therefore a large literature base describing dyeing techniques (Sandhya et al., 2004). The most important mordant dyes are the synthetic mordant dyes, or chrome dyes, used for wool; these comprise some 30% of dyes used for wool, and are especially useful for black and navy shades. The mordant,potassium dichromate, is applied as an after-treatment. It is important to note that many mordants, particularly those in the heavy metal category, can be hazardous to health and extreme care must be taken in using them. (Pandey et a., 2007; El Ahwany 2008)

Vat dyes are essentially insoluble in water and incapable of dyeing fibres directly. However, reduction in alkaline liquor produces the water soluble alkalimetal salt of the dye, which, in this leuco form, has an affinity for the textile fibre. Subsequent oxidation reforms the original insoluble dye. The color of denim is due to indigo, the original vat dye. (Pandey et al., 2007; El Ahwany 2008)

Reactive dyes utilize a chromophore attached to a substituent that is capable of directly reacting with the fibre substrate. The covalent bonds that attach reactive dye to natural fibers make them among the most permanent of dyes. "Cold" reactive dyes, such as Procion MX, Cibacron F, andDrimarene K, are very easy to use because the dye can be applied at room temperature. Reactive dyes are by far the best choice for dyeing cotton and other cellulose fibers at home or in the art studio. (Pandey et al., 2007; El Ahwany 2008)

Disperse dyes were originally developed for the dyeing of cellulose acetate, and are water insoluble. The dyes are finely ground in the presence of a dispersing agent and sold as a paste, or spray-dried and sold as a powder. Their main use is to dye polyester but they can also be used to dye nylon,cellulose triacetate, and acrylic fibres. In some cases, a dyeing temperature of 130 °C is required, and a pressurised dyebath is used. The very fine particle size gives a large surface area that aids dissolution to allow uptake by the fibre. The dyeing rate can be significantly influenced by the choice of dispersing agent used during the grinding. (Pandey et al., 2007; El Ahwany 2008)
Azoic dyeing is a technique in which an insoluble azo dye is produced directly onto or within the fibre. This is achieved by treating a fibre with both diazoic and coupling components. With suitable adjustment of dyebath conditions the two components react to produce the required insoluble azo dye. This technique of dyeing is unique, in that the final color is controlled by the choice of the diazoic and coupling components. This method of dyeing cotton is declining in importance due to the toxic nature of the chemicals used.
Sulfur dyes are two part "developed" dyes used to dye cotton with dark colors. The initial bath imparts a yellow or pale chartreuse color, This is aftertreated with a sulfur compound in place to produce the dark black we are familiar with in socks for instance. Sulfur Black 1 is the largest selling dye by volume.
Food dyes:
One other class that describes the role of dyes, rather than their mode of use, is the food dye. Because food dyes are classed as food additives, they are manufactured to a higher standard than some industrial dyes. Food dyes can be direct, mordant and vat dyes, and their use is strictly controlled by legislation. Many are azo dyes, although anthraquinone and triphenylmethanecompounds are used for colors such as green and blue. Some naturally-occurring dyes are also used. (Acemioglu et al., 2010)

Chemical classification:
By the nature of their chromophore, dyes are divided into:
 Category:Acridine dyes, derivates of acridine
 Category:Anthraquinone dyes, derivates of anthraquinone
 Arylmethane dyes
 Category:Diarylmethane dyes, based on diphenyl methane
 Category:Triarylmethane dyes, derivates of triphenyl methane
 Category: Azo dyes, based on -N=N- azo structure
 Diazonium dyes, based on diazonium salts
 Nitro dyes, based on a -NO2 nitro functional group
 Nitroso dyes, based on a -N=O nitroso functional group
 Phthalocyanine dyes, derivatives of phthalocyanine
 Quinone-imine dyes, derivativees of quinone
 Category:Azin dyes
 Category:Eurhodin dyes
 Category:Safranin dyes, derivates of safranin
 Indamins
 Category:Indophenol dyes, derivates of indophenol
 Category:Oxazin dyes, derivates of oxazin
 Oxazone dyes, derivates of oxazone
 Category:Thiazin dyes, derivatives of thiazin
 Category:Thiazole dyes, derivatives of thiazole
 Xanthene dyes, derived from xanthene
 Fluorene dyes, derivatives of fluorene
 Pyronin dyes (Acemioglu et al., 2010)

The dyes used in the present study are described below:
Bromophenol Blue: Bromophenol blue (3',3",5',5"-tetrabromophenolsulfonphthalein) is used as an acid-base indicator, a color marker and a dye.
Its appearance is tan to orange, light pink to purple or red crystals or powder. It is odorless. It is slightly soluble in water.
Its melting point is 273°C, with a density of 1gm/cc
As an acid-base indicator its useful range lies between pH 3.0 and 4.6. It changes from yellow at pH 3.0 to purple at pH 4.6; this reaction is reversible. Bromophenol blue is structurally related to phenolphthalein (a popular indicator).

C19H10Br4O5S

Bromophenol blue is also used as a color marker to monitor the process of agarose gel electrophoresis and polyacrylamide gel electrophoresis. Since bromophenol blue carries a slight negative charge at moderate pH, it will migrate in the same direction as DNA or protein in a gel; the rate at which it migrates varies according to gel density and buffer composition, but in a typical 1% agarose gel in TAE buffer or TBE buffer, bromophenol blue migrates at the same rate as a DNA fragment of approximately 500 base pairs, in 2% agarose as 150 bp. Xylene cyanol and Orange G may also be used for this purpose.
Bromophenol blue is also used as a dye. At neutral pH, the dye absorbs red light most strongly and transmits blue light. Solutions of the dye therefore are blue. At low pH, the dye absorbs ultraviolet and blue light most strongly and appears yellow in solution. In solution at pH 3.6 (in the middle of the transition range of this pH indicator) obtained by dissolution in water without any pH adjustment, bromophenol blue has a characteristic green red color.
Very toxic to aquatic organisms may cause long-term adverse effects in the aquatic environment. Cumulative effects may result following exposure Cumulative effects may result following exposure.
It is harmful if ingested or absorbed through the skin. It has also been linked to several chronic conditions.
Maximum Absorbance: 592 nm
Methylene Blue: Methylene blue (CI 52015) is a heterocyclic aromatic chemical compound with the molecular formula C16H18N3SCl. It has many uses in a range of different fields, such as biology and chemistry. At room temperature it appears as a solid, odorless, dark green powder, that yields a blue solution when dissolved in water. The hydrated form has 3 molecules of water per molecule of methylene blue.[1] Methylene blue should not be confused with methyl blue, another histology stain, new methylene blue, nor with the methyl violets often used as pH indicators.
The International Nonproprietary Name (INN) of methylene blue is methylthioninium chloride.

Methylene blue (MB) has wide applications, which include paper coloring, temporary hair colorant, dying cottons, and wools. Although not strongly hazardous, it can cause some harmful effects in humans such as heartbeat increase, vomiting, shock, cyanosis, jaundice, quadriplegia, and tissue necrosis. The effluents of the manufacturing and textile industries are discarded into rivers and lakes, changing their biological life (Ho and McKay, 1998; Walker et al., 2003; Stydini et al., 2004). The problems associated with dye pollution could be reduced or minimized by physical, chemical and biological processes; for example, by microbial degradation.
Methylene blue is a suspected carcinogen and hence must be removed from soil and wastewater. Methylene blue exposure is linked to several adverse effects on the central nervous system.

Malachite Green: Malachite green is an organic compound that is used as a dyestuff and has emerged as a controversial agent in aquaculture. Malachite green is traditionally used as a dye for materials such as silk, leather, and paper. Although called malachite green, the compound is not related to the mineral malachite — the name just comes from the similarity of color.
Malachite green is classified in the dyestuff industry as a triarylmethane dye. Formally, Malachite green refers to the chloride salt [C6H5C(C6H4N(CH3)2)2]Cl, although the term Malachite green is used loosely and often just refers to the colored cation. The oxalate salt is also marketed. The chloride and oxalate anions have no effect on the color. The intense green color of the cation results from a strong absorption band at 621 nm (extinction coefficient of 105 M−1cm−1). Malachite green is prepared by the condensation of benzaldehyde and dimethylaniline to give leuco malachite green (LMG):
C6H5CHO + 2 C6H5N(CH3)2 → C6H5CH(C6H4N(CH3)2)2 + H2O
Second, this colorless leuco compound, a relative of triphenylmethane, is oxidized to the cation that is MG:
C6H5CH(C6H4N(CH3)2)2 + HCl + 1/2 O2 → [C6H5C(C6H4N(CH3)2)2]Cl + H2O
A typical oxidizing agent is manganese dioxide. Malachite green is traditionally used as a dye. Millions of kilograms of MG and related triarylmethane dyes are produced annually for this purpose.[3]
MG is active against the oomycete Saprolegnia, which infects fish eggs in commercial aquaculture, and other fungi. Furthermore, MG is also used as a parasiticide and antibacterial. (Acemioglu et al., 2010) It is a very popular treatment against ichthyophthirius in freshwater aquaria. The principal metabolite, LMG, is found in fish treated with malachite green, and this finding is the basis of controversy and government regulation.
In 1992 Canadian authorities determined that eating fish contaminated with malachite green posed a significant health risk (El-Sersy, 2001; Mohana et al., 2008). Malachite green was classified a Class II Health Hazard. Due to its low manufacturing cost, malachite green is still used in certain countries with less restrictive laws for non-aquaculture purposes. In 2005, analysts in Hong Kong found traces of malachite green in eels and fish imported from China and Taiwan. In 2006 the United States Food and Drug Administration (FDA) detected malachite green in seafood imported from China, among others, where the substance is also banned for use in aquaculture. In June 2007, the FDA blocked the importation of several varieties of seafood due to continued malachite green contamination.[7] The substance has been banned in the United States since 1983 in food-related applications. It is banned in the UK also.

Methyl green: Methyl Green is most commonly used as a nuclear stain. The material can produce chemical burns within the oral cavity and gastrointestinal tract following ingestion. The material can produce chemical burns to the eye following direct contact. Vapors or mists may be extremely irritating.

Repeated or prolonged exposure to corrosives may result in the erosion of teeth, inflammatory and ulcerative changes in the mouth and necrosis (rarely) of the jaw. Bronchial irritation, with cough, and frequent attacks of bronchial pneumonia may ensue.

Maximum Absorbance: 630 nm

Materials and Methods:

Growth Media Composition:
Luria Bertani Agar Media (I000ml):
Agar -20gm
NaCl -5gm
Yeast Extract -5gm
Tryptone - 10gm

20ml per petri plate.

Minimal Growth Media (1000ml):
Agar – 20gm
K2HPO4 – 2.34gm
KH2PO4 – 1.33gm
MgSO4 – 0.2gm
(NH4)2SO4 – 1gm
NaCl – 0.5gm
Yeast Extract – 0.1gm
Glucose – 1gm
Trace Elements – 1ml pH – 7.0

Trace Element Solution (1000ml):
CoCl2.6H20 – 11.9mg
NiCl2 - 11.0mg
CrCl2 – 6.3mg
CaCl2.2H20 – 0.78gm
MnCl2.4H20 – 10mg
CuSO4.5H20 – 15.7mg
FeCl3 – 0.97gm

Dye Stock Solution Preparation:
For preparation of stock solution, 0.25gm of dye was added to 1ml of water in an eppendorf tube..
Prepare Stock Solution for each Dye:
1.) Bromophenol Blue
2.) Methylene Blue
3.) Malachite Green
4.) Methyl Green

Preparation of Luria Bertani Broth:
Tryptone – 10gm
Yeast Extract – 10gm
NaCl – 10gm pH – 7.0

Preparation of Luria Agar + Dye:
100µl of dye stock solution was added to 1000ml of Luria Agar Media.

Preparation of Minimal Media + Dye:
100µl of dye stock solution was added to 1000ml of Minimal media soln.

Preparation of Luria Broth + Dye:
100µl of dye stock solution was added to 1000ml of Luria Broth Media.

Experimental Methods:
1.) Collection of Soil Samples from three different sites:
The samples used in this project: Soil samples collected from effluent pipes in different locations, in and around Noida, Uttar Pradesh.
The Locations are:
Sample 1: Karan Textile Mills A-19, Sector 7, Noida - 201301 #0120-2579795

Sample 2: Indus Textile Mills Pvt. Ltd. 16A, Udyog vihar, Greater Noida – 201306 #0120-6523978

Sample 3: Roll Mill Industries Ltd. E-30, Sector 11, Noida – 201301 #0120-3082555

2.) Isolation of Textile Dye Degrading Bacteria from Soil
The collected soil samples were serially diluted 10 fold up to 10-9 dilution.

Procedure:
1.) 500ml of distilled water was autoclaved.
2.) 9 test tubes were labeled as 10-1 to 10-9
3.) In the first tube, take 1gm soil + 10ml water
4.) Mixed well, to uniform suspension, then using a pipette, pipetted out 1ml of the suspension.
5.) Filled each of the remaining test tubes with 9ml of water.
6.) Added the 1 ml pipetted out from the 10-1 test tube to the test tube marked 10-2.
7.) Mixed well, and then, using a fresh pipette tip, took out another 1ml from the second test tube, and add it to the third test tube (10-3)
8.) Repeated the above procedure for the remaining test tubes.
9.) Now, using a micropipette, took 100µl suspension from 10-7, 10-8, 10-9 dilutions from each of the three samples, and made two spread plate cultures for each dilution of each sample. (Total 18 petri plates)

3.) Purification of Cultures by Sub-culturing (or picking off) Technique
After incubation has been completed in spread plates, and appearance of the discreet, well separated colonies has been examined, the next step is to subculture some of the cells from one of the colonies to separate agar plates with a sterilized inoculation loop for further examination and use. Each of the new cultures represents a pure or stock culture.
Subculturing is the term used to describe the procedure of transferring of microorganisms from thei parent growth source to a fresh one or from one medium to another.
Requirements:
Luria Agar Plates from Step 2, with growing cultures, Inoculating loop, Bunsen Burner, Fresh agar plates, permanent marker.
Procedure:
1.) With a marker, labeled the fresh plates with the Sample No., Dilution Factor, and colony number and morphology of isolated colony.
2.) Sterilized the inoculating loop by holding it in the hottest portion of the bunsen burner flame.
3.) Flamed until the entire wire became red hot
4.) Allowed the loop to cool for a few seconds or cooled it by dipping it in the centre of the fresh agar plate.
5.) Touched the tip of the loop to the surface of a selected discrete colony of the chosed spread plate
6.) Lift the lid of the luria agar plate at 45° and inoculated the plate in the centre over the hardened agar surface and recapped the lid of the petri plate
7.) Sterilized the inoculating loop in the bunsen burner flame.
8.) After allowing the loop to cool, touched the inoculating loop to the fresh agar plate at the point of inoculation and spread the loop around in a zig zag fashion. Sterilized the loop once or twice before starting a fresh zig zag from the last point. This to further separate the cells in to single celled colonies.
9.) After inoculation, resterilized the inoculation loop to kill off existing organisms
10.) Incubated for 24 hrs at 37°C
11.) Carefully labeled the petri dishes by Sample Number and Dilution Factor.
12.) Observed growth after 24 hours, and single colonies were isolated

4.1) Identification of Dye Degrading Bacteria by growing on Minimal Media + Dye
The aim of this study was to isolate bacteria capable of degrading textile dyes, by growing on minimal media + dye. Therefore, in order to do this, the different isolated pure cultures were checked for their dye degrading capabilities.
Therefore, four test tubes containing each of the four dyes and minimal media were used for each isolated bacterial colony.
The procedure for this technique is given below:
Requirements:
Minimal Media+Dye (15ml+25µl)
Test Tubes
Inoculation loop
Spirit Lamp

Procedure:
1.) The working area of the laminar flow bench was cleaned with 70% ethanol.
2.) Labeled the test tubes carefully, with colony number and morphology.
3.) Sterilized the inoculation loop and allowed it to cool, then touched the loop to an isolated, pure bacterial colony, then dipped the loop into the test tube containing minimal media and dye.
4.) Once all the test tubes were inoculated, incubate them all in an incubator at 37°C for 24-48 hours.
5.) Observed each test tube for the appearance of turbidity.

4.2) Identification of Dye Degrading bacteria by growing on Luria Bertani Broth + Dye
The isolated and pure colonies were inoculated into luria broth that contained dye. Separate flasks were used for each dye.
The growth was observed at intervals of 24, 36 and 48 hours to see the extent of degradation.
Requirements:
Cotton plugs, several 250ml flasks containing 100ml each of LB broth + dye, Inoculating loop, Spirit lamp,
Procedure:
1.) Each isolated bacterial colony was inoculated onto the freshly prepared luria broth media containing dye.
2.) One flask of each of the four dyes was used as a control.
3.) The 250ml flasks were incubated at 37°C
4.) After incubation, the results were examined for the extent of degradation.
5.) The selected bacterial colonies which were capable of maximum degradation of dye in 24 hrs period were chosen and further characterized.

5.) Isolation of Bacteria on Luria Agar + Dye
Bacterial colonies were isolated from the flasks which were showing dye degradation on LA + dye containing plates. The bacterial cells were isolated using serial dilutions followed by spread plating.

Optimization of Growth Conditions:
The degradation capacity of the isolated bacterial colonies must be optimized and optimum conditions of growth i.e., temperature, inoculation time, pH and effect of agitation, was experimentally determined.
The procedures for the different optimization strategies used are described below:

Optimization of Time:
Requirements: Luria Broth + Dye, 500ml flasks, Inoculation loop, Spirit lamp
Procedure:
1.) Prepared several 250ml solutions of Luria Broth + Dye, making sure all the apparatus and media was properly autoclaved.
2.) Marked the four inoculation times on four flasks for each dye, and each bacterium.
3.) 4 flasks of each dye were inoculated with the same dye degrading bacterium
4.) 1 flask of each dye was used as control
5.) Incubated all the flasks at 37°C for durations of 24 hrs and 48 hrs.
6.) Observed the extent of degradation of dyes for every inoculation duration.

Optimization of Temperature:
Requirements: Luria Broth + Dye, 500ml flasks, Inoculation loop, Spirit lamp
Procedure:
1.) Prepared several 250ml solutions of Luria Broth + Dye, making sure all the apparatus and media was properly autoclaved.
2.) Marked the 4 different temperatures on the respective flasks for each dye and bacterium.
3.) 4 flasks of each dye was inoculated with the same bacterium
4.) 1 flask of each dye was used as control
5.) Incubated one flask of each dye and bacterium at each of the four different temperatures: 4°C, 28°C, 37°C, 50°C
6.) Observed the extent of degradation for each sample at the different temperatures after 24 hours.

Optimization of pH:
Requirements: Luria Broth + Dye, 500ml flasks, Inoculation loop, Spirit lamp
Procedure:
1.) Prepared several 250ml solutions of Luria Broth + Dye, calibrated the pH of each sample to the required level, making sure all the apparatus and media was properly autoclaved.
2.) Marked the 3 different pH values on the respective flasks, along with dye name and colony number.
3.) 3 flasks of each dye at the different pH values – 5, 7 and 9 were inoculated with dye degrading bacteria
4.) One flask of each dye was used as a control
5.) Incubated the samples at 37°C, for 24 hours.
6.) Observed the extent of degradation at the different pH values.

Effect of Agitation:
The effect of agitation on the growth and activity of the bacteria was determined
To compare the efficiency of static and shaking conditions (100 rpm) for activity of the bacterial strain used in decolourization of the dye, we kept one inoculated sample in a shaking incubator for 24 hours, and another in a normal incubator for 24 hours, and one sample was used as a control.(without inoculation)

Characterization of Isolated Strains:
The different isolated dye-degrading strains were characterized by gram staining and endospore staining techniques:
Gram Staining:
To perform Gram staining of isolated bacterial culture

Requirements:
1) 24 hr old bacterial culture
2) Gram Staining Reagents: Crystal Violet, Gram's iodine solution,95% Ethyl alcohol and safranin.
3) Staining tray
4) Wash bottle of Distilled water
5) Dropper
6) Inoculating loop
7) Glass Slides
8) Blotting Paper
9) Bunsen burner/spirit lamp
10) Microscope

Procedure:
• Thin smear of bacterial culture on glass slide was prepared
• Air dried the smear
• Heat fixed the smear
• Covered the smear with crystal violet for 30 sec
• Washed the slide with distilled water for few seconds using wash bottle
• Covered the smear with gram's iodine solution for 60 sec(1min)
• Washed off the iodine solution with 95% ethyl alcohol by adding drop-wise, until no more colour flowed from the smear
• washed the slide with DW and drain
• applied safranin on the smear for 30 sec
• washed with DW and blot dried with blotting paper/absorbent paper
• observed under oil-immersion microscope.

Endospore Staining:

Requirements:
• Bacterial culture(48 hrs old)
• Malachite Green(5% aqueous)
• Safranin(0.5% aqueous)
• Glass slides
• Inoculating loop
• Blotting paper
• Spirit lamp
• Microscope
Procedure:
• Made smear of bacterial culture on a clean glass slide
• Air dried and heat fix the smear
• Flooded the smear with malachite green
• Heated the slides to streaming and steam for 5 min,adding more stain to the smear from time to time
• washed the slide under slow running tap water
• Counterstained with safranin for 30 sec
• Washed smear with DW
• Blot dried slide with blotting paper /absorbent
• Observed under oil immersion microscope

Results and Discussion:

1.) Bacteria is isolated from soil by the process of serial dilution, followed by Spread Plate Method for isolation of single colonies.
Results:
Primary Isolated Cultures: Colony D (Smooth) (Methyl Green) Colony A (Rough) (Methylene Blue)

Colony II (Bromophenol Blue) Colony B (Rough) (Methylene Blue)

2.1) Identification of dye degrading bacteria: By growing on minimal media + dye
There was no growth of bacterial cells on minimal media containing dye.
Results:

Methyl Green + Minimal Media (After 24 hours) Bromophenol Blue + Minimal Media (after 24 hours)

2.2) Identification of dye degrading bacteria: By growing in Luria Bertani Broth + dye
Results: Bromophenol blue and methylene blue containig flasks were showing degradation after 24 hours of incubation Luria Broth + Methylene Blue + Inoculum (after 24 hours)

Luria Broth + Bromophenol Blue (after 24 hours)
Malachite green and methyl green flasks did not show any degradation after 72 hours of incubation.
Isolation of Bacteria on Luria Agar + Dye:
Two types of colony morphologies were observed:
Rough – colony A , colony C (methylene blue)
Smooth – colony B (methylene blue), colony D (methyl green)
Colony II of Bromophenol Blue

Optimization of Growth Conditions:
Optimization of Incubation Time:
Results: In these flasks shown from left to right the flask on the left hand side showed no degradation. It was used as control. The flask kept next to that flask showed partial degradation. It was kept for 24 hours. The flask on the extreme right showed ful degradation of dye by bacteria. It was kept for two complete days, i.e. 48 hours. It denotes that the degradation of bacteria is totally dependent on time. The same case was seen with Bromophenol blue, where the flask on the extreme right showed maximum degradation. Degradation of Methylene Blue at time intervals: (from left) control, 24 hours, 48 hours

Degradation of Bromophenol Blue at time intervals: (from left) control, 24 hours, 48 hours

Optimization of pH:
Results:

Optimization of pH: (from left) : pH – 5.0, pH – 7.0, pH – 9.0
In this case, the flask with the pH of 7.0 showed maximum degradation, whereas the flask with pH – 5.0 showed a little degradation.

Optimization of Temperature:
Results:

Optimization of Temperature: (from left) - 4°C, 28°C, 37°C

Effect of Agitation:
Results: The culture on the left, i.e., the culture that was grown in a shaking incubator, showed maximum degradation. This proves that agitation enhances the degradation rate. Left: With Shaking Right: Static Culture

Characterization of Isolated Strains: Gram-stain Results

Result:
The cultures observed are gram(+) ve because they stained purple/violet which indicates (+) ve gram's stain
Colonies:
• Colony-II of bromophenol blue
• Colony -"d" and "h"(smooth) both of methylene blue
• Colony-"b"(rough) of methylene blue.

Observation: Arrangement culture "h":Gram (+) ve "rod"(small) Single culture "d": Gram (+) ve "rod"(large) Single culture "b": Gram(+) ve "rod" (larger) Single culture "II":Gram(+) ve "rod"(small)

Endospore Staining Results
Result:
colony "b":Endospore present colony "h:Endospore present colont II: Endospore present

Discussion:
Textile industries have been using synthetic dyes intensively because of their ease and cost effectiveness. The textile dyes are highly reactive and therefore, during processing, difficult to treat. During the past decade the use of microbial degradation methods have been under active development in textile and dyestuff industry. As can be seen from the results of this project, the isolated bacterium, is a choice organism reported to be a potent decolourizer of the effluent.
In the present work, an attempt has been made to study the common soil inhabiting bacteria isolated from the effluent pipes of textile mills. It can be assumed that the chosen source will provide samples containing the bacteria with the desired activity, i.e., degradation of textile dyes.
The decolourization of the various dyes used in this experiment shows that the decolourization of the dye is not due to biosorption but is due to the metabolism of the dye by the Bacteria because a high rate of decolourization of the dye is possible through metabolism of the dye rather than biosorption. Furthurmore, the decolourization is faster when agitation is employed rather than in static reaction conditions. For this reason, all the incubation in this experiment was carried out in a shaking incubator.
Physio-chemical parameters such as temperature, pH and bioavailability of the substrate determine the bioremediation process.

The chosen dyes are representative of the range of textile dyes available in the market, and which cause soil and wastewater pollution.
Methylene Blue is an azo dye and resembles commonly used textiles dyes in the textile industry. Bromophenol blue is a triphenylmethane dye used as a laboratory reagent. The decolorization of these dyes by isolated bacterial strains is discussed and the effect of independent variables such as pH, Temperature, Incubation Time, and agitation on the decolorization efficiency was observed to study the factors for the optimization of bacterial growth conditions for dye decolorization. Decrease in absorbance is considered as an indicator of dye removal and decolorization.
The isolated bacterial strain has the potential to decolourize dye effluents. Isolated bacterial strains showed maximum decolorization of dye at temperatures 28°C and 37°C., pH – 7, and under agitated conditions. The colour reduction increased linearly with increase in incubation time.
Some other physical parameters in pH, temperature, concentration of xenobiotic dyes in the wastewater thus might have implications in the dye removal efficiency of most bacteria. The results may be extrapolated and upscaled for the potential application in industrial effluents and wastewater treatment.

References:
• Sayadda R. Ghuznavi, Rangeen – Natural dyes of Bangladesh, Vegetable Dye Research and Development Society, Bangladesh, 1987.
• 22, Development Technology Unit, University of Warwick, Coventry CV4 7AL, UK. 1988 Dalby, Gill and Dean, Jenny, Natural Dyes in Luapula Province (Zambia): Evaluation of Potential for Production, Use and Export. Working Paper
• Hans-Samuel, Josef Stawitz and Kluas Wnderlich (2005): “Anthraquinone Dyes and Intermediate” in Ullmann’s, Encyclopedia of industrial chemistry Wiley-VCH, Weinheim: 2000.
• Harley, J.P. and L.M. Prescott. Basic laboratory and culture techniques. In: Laboratory excercises inMicrobiology. 2nd Ed. W.C. Brown Publishers, Dubuque, 1993, 14-46
• Coughlin, M.F., B.K.Kinkle, A. Tepper and P.L.Bishop, Characterization of aerobic azo dye-degrading bacteria and their activity in biofilms. Water Sci.Technol., 1997,36: 215-220.
• Feingold, BF. Hyperkinesis and learning disabilities linked to artificial food flavors and colors. Am J Nurs. 1975;75: 797–803.
• Ali, N., Hameed, A, and Ahmed, SW. J. Microbiol. Biotechnol.2008, 24 : 1067–1072
• Tan NGG. Prenafeta-Boldu FX, Opsteeg JL, Lettinga G, Field JA (1999) Appl. Microbiol. Biotechnol. 51:865-871
• Pandey, A., Singh, P., Iyengar, L., 2007. Bacterial decolorization and degradation of azo dyes. International Biodeterioration and Biodegradation 59, 73–84.
• Olukanni, O.D., Osuntoki, A.A., Gbenle, G.O., 2006. Textile effluent biodegradation potentials of textile effluent-adapted and non-adapted bacteria. African Journal of Biotechnology 5, 1980–1984
• Text book of microbiology K.R.Aneja
• Hunger, K., ed. (2003). Industrial Dyes. Chemistry, Properties, Applications. Weinheim: Wiley-VCH
• Bioremediation of Methylene Blue by Bacillus thuringiensis 4 G1: Application of Statistical Designs and Surface Plots for Optimization: Nermeen A. El-Sersy Biotechnology 01/2007;
• Bioremediation and Sustainability: Research and Applications By Romeela Mohee, Ackmez Mudhoo

• Biodegradation of Textile Dyes, Bromophenol Blue and Congored by Fungus Aspergillus Flavus Lokendra Singh* and Ved Pal Singh Applied Microbiology and Biotechnology Laboratory, Department of Botany, University of Delhi, Delhi- 110 007, India.

ANNEXURES
A. Equipment
• Test tubes
• Pippets & tips
• Petri plates
• Cotton plugs
• Laminar air flow
• Spirit and Spirit lamp
• Inoculation loop
• Flask
• Orbital Shaker
• Eppendorf

B. Reagents
• Malachite green Dye
• Methyl green Dye
• Bromophenol blue Dye
• Methylene blue Dye
• Distilled water
• Tryptone
• Yeast Extract
• Nacl
• K2HPO4
• MgSO4
• Glucose
• Trace Elements
• KH2PO4
• (NH4)2SO4