Try phenyl methane dyes have been found in soil and river sediments as consequences of improper chemical waste disposal. 10000 dyes and pigments are produced annually world wide amounting to 7*107tones which are hazardous and pose serious environmental problems. It is estimated that 10-15% of the dye is lost in the effluent during the dying process. Try phenyl methane dye decolorizing bacteria have been isolated; there are few reports of specific enzymes that decolorize these dyes. Isolate bacterial strains which had the capability to decolorize textile dye like bromophenol blue, crystal violet and malacate green. We estimate the decolorization percentage for all the three tri phenyl methane dyes and quantify the activity of the TMR enzyme that degrades try phenyl methane dye and characterize the dye degrading organism as pseudomonas species.
MICROBIAL DEGRADATION OF TRIPHENYL METHANE DYES
Abstract
Try phenyl methane dyes have been found in soil and river sediments as consequences of improper chemical waste disposal. 10000 dyes and pigments are produced annually world wide amounting to 7*107tones which are hazardous and pose serious environmental problems. It is estimated that 10-15% of the dye is lost in the effluent during the dying process. Try phenyl methane dye decolorizing bacteria have been isolated; there are few reports of specific enzymes that decolorize these dyes. Isolate bacterial strains which had the capability to decolorize textile dye like bromophenol blue, crystal violet and malacate green. We estimate the decolorization percentage for all the three tri phenyl methane dyes and quantify the activity of the TMR enzyme that degrades try phenyl methane dye and characterize the dye degrading organism as pseudomonas species .
Keywords: Try phenyl methane, TMR enzyme, Dyes and pseudomonas species.
Introduction:
Dyes are coloring pigments that impart color to the substrate when they are in solution form. Technically dyes are distinguished from the intermediates based on the presence of auxochrome, the group that allows the basic unit to attach and impart color to the substrate. Dyes are derived synthetically from raw materials like hydrocarbons, benzene, toluene, naphthalene and anthracene using coal tar obtained from distillation of coal (Alinsafi A, et al., 2006). Both organic and inorganic materials are needed to make dyes and intermediates. The raw material sequence for making dyes is petroleum hydrocarbons intermediates dyes (Bell J, et al., 2000). N Dyes are retained in substrates by physical absorption, metal complex formation or by the formulation of covalent chemical bonds and they obtain their color due to electronic transitions between various molecular orbital where intensity of the color is determined by the probability of transitions (Cariell, C.M, et al., 1996). Dye is a substance (generally an organic compound), which is used for imparting permanent color to textiles - silk, wool and other substances. There are two types of dyes. Natural dyes occur in nature e.g. Indigo (a blue dye), alizarin (a red dye).Synthetic dyes are man-made dyes e.g. Crystal violet (a bluish green dye), azo dye, aniline yellow, orange etc. A colored substance can act as a dye only if it can be fixed to the material being dyed (Daneshvar N, et al., 2007). At the same time it should be resistant to the action of light, water and soap. The important condition for a colored compound to act as a dye is the Presence of chromophore. These are the groups, which are responsible for producing a color to a dye because they are capable of absorbing light in the ultra violet region. Some important chromophores are: -N=O, -N=N-, -C=N, (CH=CH). A chromogen without auxochrome can never act as a dye. 10,000 dyes and pigments are produced annually worldwide amounting to 7x105 tones which are hazardous and pose serious environmental problems. It is estimated that 10-15% of the dye is lost in the effluent during the dying process (Lacalle M, et al., 2007).
The recent high profile of color pollution is mainly the result of increasing public awareness and expectations of the environment; coinciding with rising levels of color discharges (Oxspring DA, et al., 1996). One of the more pressing environmental problems that have been facing the textile industry is the removal of the color from dye bath effluent prior to discharge to local sewerage treatment facilities or adjoining watercourses. Considerable efforts have been made on developing suitable treatment systems for these effluents. Wastewaters originating from reactive dye processes have created a particular problem because the dyes can exhibit low levels of fixation with the fiber. The brightly colored unfixed dyes are highly water-soluble and are not removed by conventional treatment systems (Robinson T, et al., 2001). This is particularly noticeable as the human eye can detect reactive dyes at a concentration as low as 0.005 mg/l in clear waters. Discoloration of textile dye effluent does not occur when treated aerobically by municipal sewerage systems. Though the formation in 1974 of the Ecological and Toxicological Association of the Dye stuff Manufacturing Industry (ETAD), aims were established to minimize environmental damage, protect users and consumers and to cooperate fully with Govt. and public concerns over the toxicological impact of their products. Over 90% of some 4000 dyes tested in an ETAD survey had LD50 values greater than 2x103 mg /kg. The highest rates of toxicity were found amongst basic and diazo direct dyes .
Materials and Methods
Isolation of the dye degrading organism
Soil is collected from the textile industries in and around Hyderabad. This soil is then serially diluted for isolation of bacterial colonies. The 10-5 diluted soil sample is and spread over nutrient agar media along with a dye (bromophenol blue, or malachite green)with concentrations of 20 µM, 40 µM, 60 µM,80 µM. Whereas for crystal violet the concentrations ranged from 20 µM,30 µM ,40 µM ,50 µM The petri plates are incubated for 48 hrs at 370C. The colony which has shown maximum clearing zone (≈10mm) was selected and maintained as pure culture in nutrient broth.
Decolorization assay
For degradation experiments, the pure cultures were inoculated into flasks at 4% (v/v) level. All experiments were conducted in the same conditions consisting of a 500-mL flask containing 250 mL nutrient broth (pH 7.5). Culture medium was supplemented with 50 mg/L of the dye to be tested in the presence of yeast extract (0.1%) and glucose (7 mM). Flasks were incubated at 37°C, under shaking (120 rpm) in a rotary shaker. Samples were collected at different time points (2, 4, 6, 8, and 24 h) to determine the dyes decolorization.
Decolorization Assay
For determination of CV, BB and MG color removal, 5 ml aliquots of the culture were sampled at different culture periods (2, 4, 6, 8 and 24 h), centrifuged at 6,000 rpm for 10 min to eliminate the bacterial cells, and the supernatant was examined by spectrophotometer at the (λmax) of 590 nm, 600 nm and 618 nm, respectively, for CV, BB and MG. The percentage of decolonization was calculated as following:
Decolonization (%) = [(Absorbance at t0) − (Absorbance at t1)]/ (Absorbance at t0) × 100]. Dye elimination was investigated with each selected bacterial isolate of that particular dye.
100 ml nutrient broth was prepared and poured into four conical flasks (25 ml each). Different concentrations of bromophenol blue (20µM, 40 μM, 60 μM, 80 μM) were added to each flask. Inoculate a 1 ml of culture from the nutrient broth and incubate the flasks at 37⁰c. Observe the OD values after 24 hrs of incubation at 600 nm. Similarly for each concentration of the dye, control is set up without the culture.
Depolarization % =O.D (control – test)/ O.D of control*100
TMR Enzyme assay
Since the dyes are triphenyl methane dyes and the organism is able to reduce the dye, therefore triphenyl methane reductase enzyme assay was done. The standard assay system for TMR comprises 20mM sodium phosphate buffer (pH 7), 20µM dye (bromophenol blue), 0.1mM NADH and a suitable amount of culture broth in a total volume of about 1ml. This is incubated at room temperature for about 2 mins and then OD is observed at 600nm.
The enzyme activity is calculated as follows
In general terms the enzyme activity (μmol /min/ml) = ΔAx 1000/ extinction coefficient of bromophenol blue x vol in cuvette x 1.0/vol used for assay.
Extinction coefficient of bromophenol blue = 60,500
Extinction coefficient of crystal violet =111000
Extinction coefficient of malachite green=137000
The final activity is obtained by multiplying by any dilution factor for micromoles per min per ml and dividing by the protein concentration in mg per ml for micromoles per min per mg.
Characterization of the bacterial isolates
Take a clean glass slide and place a drop of water over it. Now, sterilize the inoculating loop and pick a part of colony and spread over the water drop, make thin smears of it. Let the smears air dry and then heat fix. Stain the smear with crystal violet for 1-2 minutes, then wash it with distilled water, now cover the smear with grams iodine for about 1 min and wash the slide with distilled water, then add acetone (alcohol) drop by drop for about 30secs and immediately wash with distilled water. Lastly add saffranin to the smear for 2 minutes and wash with distilled water. Blots dry the slide using a blotting paper. Let the slide dry completely and then observe the fluid under 10x, then observe morphology under 45x and finally report the gram morphology and arrangement under oil immersion lens (100x).
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