Research Proposal
MSc Part 2
Sem 3

CERTIFICATE

This is to certify that Mr. Akshay L. Shettigar has satisfactorily completed his proposal entitled “Biodegradation study of Triphenylmethane dye Crystal Violet by bacterium isolated from textile effluent.” for the year 2014-2015.

Signature of the Project guide Signature of the Examiner
(Prof. Mrs Radhika Birmole)

Date and college Stamp Signature of the H.O.D.
Proposal
(i) Project Title:
Biodegradation study of Triphenylmethane dye Crystal Violet by bacterium isolated from textile effluent.

(ii) Introduction:
Synthetic dyes are extensively used in textile dyeing, paper printing, colour photography, pharmaceutical, cosmetic and other industries [1]. Over ten thousand different dyes with an annual production of over 7x105 metric tons worldwide are commercially available [2]. 2% of dyes that are produced are discharged directly in aqueous effluent and 10% are subsequently lost during the textile coloration process [3]. Major classes of synthetic dyes include azo, triphenylmethane and anthraquinone dyes, some of them are known to be very toxic and mutagenic to living organism. With the increasing use of wide variety of dyes pollution by dye-waste water is becoming increasingly alarming. Colour removal, in particular, has recently become a major scientific interest. Although several physicochemical methods have been used to eliminate the coloured effluents in waste water, these methods are rather costly and sometimes produce hazardous byproducts and therefore other alternatives such as microbial biodegradation have attracted interest. Microbial decolorization and degradation is an environmentally friendly and cost-competitive alternative to chemical decomposition processes [4]. To develop and efficient biotechnology, the key step is to obtain broad-spectrum and highly efficient dye-decolorizing bacteria. Although many dye-decolorizing microorganisms have been reported [5-7], with exception of the decolorization of dyes by Pseudomonas pseudomallei 13NA and Citrobacter sp which decolorize both triphenylmethane and azo dyes by a single species of bacterium [8], there are no reports on decolorization of triphenylmethane, azo and anthraquinone dyes by a single strain of bacterium and no bacterium that has been reported was able to use crystal violet as sole carbon source and energy source for growth upto now.

* Origin of Research Problem
Triphenylmethane dyes are one of the major groups of dye stuffs used for textile, printing, paper making, leather, food and the cosmetics industry [6, 9]. These dyes are re-calcitrant molecules and can be anti-microbial, toxic to mammalian cells and mutagenic [5, 10, 11]. Therefore the decolorization of waste waters containing these dyes prior to discharge is mandatory by environmental regulations in most countries.
Although several physicochemical methods e.g. adsorption, chemical precipitation and flocculation, oxidation by chlorine, hydrogen peroxide and ozone, electrolysis, reduction, electrochemical treatment and ion-pair extraction have been used to eliminate the colour from these waste waters in the effluent [6, 12], they are generally expensive or produce large amount of sludge. Therefore these methods have limited applicability. As a result, the interest is focused on microbial biodegradation as a better alternative. * Interdisciplinary Relevance
The outcome of this project would interdisciplinary in nature. Physicists and chemists will be benefited with the results of proposed work. This is due to the fact that the dye molecules treated primarily by physicochemical method can be efficiently degraded using bacteria. The application of dye degrading bacteria would help environmental scientist to eliminate dyes and their toxic intermediates from the environment. This work of isolation of novel dye degrading bacterium can act as a guideline to environmental biotechnologist and environmental engineers to device a small scale model treatment plant for degradation of effluent containing dyes.

* Justification
Currently, various chemical and physical treatment methods including adsorption, chemical precipitation and flocculation and oxidation of chlorine, hydrogen peroxide and ozone, electrolysis, reduction, electrochemical treatment and ion-pair extraction were used to remove the dye [6, 13-15]. Because of the high cost, disposal problems and generation of toxic products most the chemical and physical methods for treating dye waste are not widely applied in the textile industries. Physicochemical methods of dye removal are effective only if the effluent volume is small and sometimes degradation products are toxic [16].
As a better alternative, therefore, the development of biological processes using microorganisms for treatment of dye containing waste water has become increasingly important [6, 16]. Microbial decolorization processes have the advantage of being environmentally friendly and low in cost compared to conventional treatments.

* Review of research and development of the subject
A number of scientific publications on dye degradation by bacterial cultures have increased exponentially. A number of research articles and papers are published in national and international journals.
International status:
Many researchers have employed pure cultures of bacteria Pseudomonas aeruginosa [17], Pseudomonas otitidis [18], Agrobacteium radiobacter [19] and Shewanella sp. NTOU1 [20] for biodegradation of triphenylmethane dyes such crystal violet ,malachite green and brilliant green. P. aeruginosa and P. otitidis carried out degradation of triphenyl methane dyes under shaker conditions whereas A. radiobacter and Shewanella sp.NTOU1 under static conditions. Various parameters were optimized and studied for their impact on the decolorization ability of the respective isolates. Effects of various metal ions, dye concentrations etc were also studied.
National status:
Nine white-rot fungal strains were screened for biodecolourization of brilliant green, cresol red, crystal violet, congo red and orange II. Dichomitus squalens, Phlebia fascicularia and P. floridensis decolourized all of the dyes on solid agar medium and possessed better decolourization ability than Phanerochaete chrysosporium when tested in nitrogen-limited broth medium.[21]. Malachite green (50 mg/L) was completely decolorized under static anoxic condition within 5 h by bacteria Kocuria rosea MTCC 1532; however decolorization was not observed at shaking condition. K. rosea have also shown decolorization of azo, triphenylmethane and industrial dyes (cotton blue, methyl orange, reactive blue 25, direct blue-6, reactive yellow 81, and red HE4B). Semi-synthetic media containing molasses, urea and sucrose have shown 100, 91, 81% decolorization respectively. Induction in the activities of malachite green reductase and DCIP reductase was observed during MG decolorization suggesting their involvement in the decolorization process. UV-Visible absorption spectrum, HPLC and FTIR analysis showed degradation of MG. Toxicity study revealed the degradation of MG into non-toxic products by K. rosea. [22]

Triphenylmethane dye CRYSTAL VIOLET

IUPAC name and General properties IUPAC name | Tris(4-(dimethylamino)phenyl)methylium chloride | Molecular formula | C25N3H30Cl | Molar mass | 407.979 g mol-1 | Melting point | 205 °C (401 °F; 478 K) |

Literature Survey
Triphenylmethane dyes are used extensively in textile industries for dying nylon, polyacrylonitrile modified nylon, wool, silk, and cotton. Some of the triphenylmethane dyes are used as medicine and biological stains. Paper and leather industries are also major consumers of triphenylmethane dyes. This group of dyes are also used for coloring plastics, gasoline, varnish, fats, oil, and waxes. Food and cosmetic industries also use different types of triphenylmethane dyes. Dyes are recalcitrant molecules difficult to degrade biologically. From the treatment point of view, the degradation of dyes has received considerable attention. Conventional wastewater treatement processes are suitable for stabilization of nonxenobiotic compounds whereas these processes do not work well with the xenobiotic compounds. Environmental regulations in most of the countries now have made it mandatory to decolorize the dye wastewater prior to discharge. Increasing concerns about color in the effluents are leading to the worldwide efforts to develop more effective color removal processes. A large number of methods are available to treat the wastewater containing dyes . Presently, most of the processes used for the treatment of dye wastewater are chemical processes which are costly, produce large amount of sludge, and are less efficient. Nowadays, biological processes are getting more and more attention since it is cost effective, environmently friendly, and does not produce large quantities of sludge. The decolorization may be defined as the removal of color only possibly by changing the chromophoric group to a nonchromophoric one while biodegradation is the breakdown of the substrate (dye) molecule by the biological process[9]. In 1980 reported the deposition of Crystal Violet and Malachite Green in the sediment and water of Buffalo river in New York (U.S.) They found aniline dyes in the aquatic environment[23]. From a study in 1980, it was shown that aniline dyes could be mutagenic and carcinogenic to biota[24]. These chemical have been suggested to be responsible for the promotion of tumor growth in several bottom-feeding species of fish.[25] Triphenylmethane dyes are some of the most widely used dermatological agents. Gentian Violet has been used in oral consumption for the treatment of pinworms and in topical applications in humans and domestic animals; it has been shown to be effective in controlling fungal growth under varying conditions[26]. Gentian Violet has also been added to poultry feed to control fungus, thus exposing the human population directly or indirectly to Gentian Violet through its extensive medicinal and commercial use. The cytogenic toxicity of Gentian Violet in Chinese hamster CHO cells in vitro has been studied. It was stated that this compound is a mitotic poison as well as a clastogen in vitro. Its clastogenic properties were confirmed in five other different mammalian cell types. Unless in viva studies prove otherwise. Gentian Violet and Crystal Violet may be regarded as biohazardous substances. * Treatment of triphenylmethane dyes
Chemical processes are extensively used for the decolorization of dyes by different dye manufacturing and dying industries. The ozonization of Malachite Green, a model compound of triphenylmethane dye was reported[27]. Reactions of dyes with ozone were studied from the treatment point of view. Such reactions are looked at in terms of reduction in the chemical oxygen demand (COD) and the decolorization; however, the ozonization mechanism has scarcely been touched. In 1970 the photofading of triphenylmethane dyes was reported[28]. The photoreaction of Malachite Green in water was reported to give 4-dimethylarnino benzophenone, the carbinol base of Malachite Green and p-dimethylamino phenol. The photoreaction of Crystal Violet has been reported to give pdimethylamino phenol, 4-4’-bis dimethylamino benzophenone, the leuco and demethylated derivative of Crystal Violet [29,30]. Textile and dye - stuff industrial wastes having different dyes are generally treated by physicochemical methods. These methods include adsorption, chemical precipitation and flocculation, oxidation by chlorine, hydrogen peroxide and ozone, electrolysis, reduction, electrochemical treatment, and ion-pair extraction, but none have been found to be very suitable as they produce a large quantity of sludge. All these methods possess significant differences in color removal results, volume capability, operating speeds, and capital costs [31]. For example, activated charcoal is extremely effective for removing color, but is capable of treating small effluent volume, operates at slow speeds, and has high capital costs. Membrane technology, ozone treatment, and coagulation and flocculation permit good color removal in large effluent volumes. Membrane technology is fast, but the capital cost for implementing this technology is high. Ozone treatment operates at moderate speeds and also requires high capital investment. Coagulation and flocculation techniques operate moderate to fast speeds and require a somewhat lower capital investment, but the biological methods have many advantages such as the possibility of degradation of dye molecules to carbon dioxide and water and the formation of less sludge. Also it is environment friendly. In 1981 the use of an activated sludge system for the treatment of dye wastes was reported [32]. Dye industries use the activated sludge processes to treat effluent; however, some dyes are toxic to the microbes and lessen their purifying function. The acclimatized microbes showed negative inhibition at a low dye concentration. Negative inhibition was tested by inoculating the triphenylmethane dyes (Methyl Violet) with the acclimatized microbes in the medium. They have also reported the elimination of Methyl Violet and New Fuchsin up to 90-100% by continuous culture of activated sludge. In 1985 the fate of Congo Red, Orange II, and Crystal Violet in activated sludges which were previously acclimatized with the medium and dyes was examined [33]. The effect of dye concentration on acclimatized sludges was also studied. It was observed that with the increase in dye concentration, the color loss was decreased. The inhibition of growth and respiration of activated sludges of various dyes was studied as well as examined for their inhibitory characteristic [34]. These tests were performed with unacclimatized sludges. Sludges were acclimatized to different triphenylmethane dyes which had adaptability to different concentrations of dyes [32]. With other dyes, Methyl Violet and Crystal Violet were used and were observed for the respiratory inhibition by these dyes. It was observed that the inhibition level of the unacclimatized microbes by Methyl Violet increased with the dye concentration. In comparison to the other dyes, Methyl Violet showed greater inhibitive action. The acclimatized microbes showed negative inhibition at a low dye concentration and a positive inhibition at a high concentration. The inhibition of microbial growth by certain basic dyes was reported [35].They used Bacillus subtilis as the test organism and observed that both the mean growth rate of the cell population at the logarithmic phase and the cell concentration at the stationary phase decreased with the addition of dyes. It is also reported that triphenylmethane dyes (Crystal Violet and Methyl Violet) strongly inhibited cell growth. The elucidation of inhibition mechanism by the dyes on chemical composition of cells was needed. For this purpose, they determined the nucleic acid content of the cells harvested both at the logarithmic and stationary phases. They found that the RNA content decreased with increasing concentration of Methyl Violet; this tendency was more remarkable in the logarithmic than stationary phase. The nucleic acids content ratio, i.e., (RNA)/ (DNA), decreased with increasing concentration of Methyl Violet, so they concluded that the dyes act more preferentially to lower the protein synthesis than inhibit cell division. Due to the inhibitive action, cell shape also varied. A report published [36] on the toxicity of wastewater to acclimatized sludge was examined. These reports described the mixtures of various kinds of organic and inorganic compounds in the sludge.
Crystal Violet has an antibacterial action [37] against Escherichia coli, Staphylococcus aureus, Streptococcus faecalis. and Bacillus subtilis. The Crystal Violet at a concentration of l-6 x lO-6 M was used. The effect of dye, measured as minimum inhibitory concentration in terms of retardation of growth, increased as the pH rose from 6 to 8. Out of the four species, E. coli was the most resistant to Crystal Violet. The mode of action of Crystal Violet is the formation of an unionized complex of bacteria with dye. Gram-negative organisms such as E. coli have high isoelectric points and contain less acidic components than Gram-positive bacteria which usually have lower isoelectric points, so the former combine with Crystal Violet less readily and are more resistant to dye. * Degradation by bacteria
There are few reports on the biodegradation of triphenylmethane dyes by bacteria. In 1981,[8] reported the biodegradation of triphenylmethane dyes by Pseudomonas pseudomallei 13 NA. At this time, the information related to the biodegradation of dyes other than azo dyes was insignificant. Four triphenylmethane dyes such as Basic Fuchsin, Methyl Violet, Crystal Violet, and Victoria Blue were used for biodegradation studies. They demonstrated that the decolorization of triphenylmethane dyes was inferior to that of azo dyes. They also showed that Methyl Violet and Crystal Violet were appreciably decolorized while Basic Fuchsin and Victoria Blue were not decolorized under experimental conditions. To determine whether decolorization was dependent on the chemical structure of the dyes, they attempted to correlate the decolorization of dyes with molecular weight and the octanol-water partition coefficient. The octanol-water partition coefficient of an organic compound gives an indication of the permeability of the organic compound through the cell membrane. The half-decolorization time of triphenylmethane dyes had no appreciable correlation with the molecular weight. In general, the decolorization of the dyes by P. pseudomallei is not related to molecular weight and the octanol-water partition coefficient of the dyes. It was also observed that the half-decolorization time for both Methyl Violet and Crystal Violet at a dye concentration of 1 X l0-5M was 54 h.
The viable cell count from the incubation mixture with Crystal Violet was found to be a function of incubation time. Under the experimental conditions, the viable cell count was constant up to l00-140 h. Viable cell count did not decrease during the incubation period. The ultraviolet (UV) spectrum of the reaction products of Crystal Violet with intact cells extracted in n-butanol showed the degradation of Crystal Violet. The shift of the peak and increase in absorbance were indicative that the reaction products are different from the original substrate.
In 1991, [38] reported the degradation of Crystal Violet by B. subtilis IF0 13719. Besides Crystal Violet. two other triphenylmethane dyes (Prarosaniline and Victoria Blue) were degraded by growing cells of B. subtilis IF0 13719. With the low cell growth, Crystal Violet was remarkably decolorized by B. subrilis after 8 h. The compound was totally decolorized in 24 h when the cell growth was higher. They observed that at a very low level of dye concentration (below 7 X 10-6 mol L-1, the decolorization of Crystal Violet occurred. The growth of the bacterium was completely inhibited at a dye concentration of 1.5-2.0 X l0-5 mol L-1. Other dyes like Basic Auramine 0, Basic Fuchsin, and Victoria Blue were decolorized in cultures of B. subhlis while cell growth was poor. They also tried other bacteria for decolorization studies. E. coli, although growing remarkably, did not decolorize Crystal Violet. Two other cultures, Pseudomonas cepacia and Pseudomonas cruciviae, also showed similar results. Twenty-one hydrophobic oleophilic bacterial strains which showed decolorization activity were isolated; the most active strain was Mycobacterium[39]. Crystal Violet and Malachite Green dyes were attacked by all active strains. Cells immobilized on the glass beads showed similar activity. Cell growth was inhibited when dyes were present but resumed when the dyes were transformed. A German patent [40] states a new process by which the xenobiotic dyes of triphenylmethane series are degraded using Covnebacterium and Mycobacterium sp. The process is rapid and simple and may be performed over a wide range of temperature ( 15-40°C) over l-12 h. No additional substrate supplements are needed. Both Crystal Violet and Malachite Green are removed from wastewater or soil extracts. For decolorization, Mycobacterium sp. was precultured at 32°C in a mineral salt medium containing 1% methanol as the sole energy and carbon source. After 72 h, cells were harvested and suspended in tap water at a cell concentration of 1010 ml-1. An aqueous solution ( 10.5 ml) of Malachite Green (20 mg mL-1) was inoculated with 1.5 ml cell suspension at 24°C. The dye concentration was reduced to 53% of the initial value within 2 h. At 32”C, the solution was completely decolorized after 22 h. the pH remained constant at 6.8. and the number of cells did not increase.

* Degradation by actinomycetes
The first report of biodegradation of triphenylmethane dyes by actinomycetes was published by [38]. With two actinomycetes, Nocardia corallina and Nocardia globerula, the decolorization of Crystal Violet was observed. They also showed that decolorization activity of actinomycetes is intracellular since there was no activity in the culture filtrate. The dyes were completely decolorized in 24 h. They also detected a degradation product of the Crystal Violet digestion as Michler’s Ketone (MK) by N. globerula.
In 1993, another report was published [41] regarding the degradation of triphenylmethane dyes by actinomycetes. They have shown that Crystal Violet was degraded to Michler’s Ketone by N. corallina; however, N, corallina could not decolorize Auramine 0, a typical dimethylmethane dye. N. corallina decolorized Methyl Violet, Ethyl Violet, Basic Fuchsin, and Victoria Blue to a much greater extent but Crystal Violet was the compound most remarkably decolorized. The decolorization rate was dependent upon the initial concentration of N. corallina in the medium. The decolorization rate was also dependent upon the growth phase of the precultured cells. The rates at early log phase (l0-20 h incubation), mid-logphase (20-30 h incubation), and late log phase (30-40 h incubation) were 10.6, 4.4, and 2.2 nmol mg-1 min-1, respectively. The decolorization of Crystal Violet was observed only at a low concentration (5 micro mol L-1) whereas the cell growth was completely inhibited at 7 micro mol L-1. The decolorization rates of Crystal Violet in LB, Bennett, and minimal medium were 10.6,6.9, and 2.8 nmol mg-1 min-1, respectively. With cell homogenates and extracellular extracts of N. corallina, the decolorization of Crystal Violet was not observed even after prolonged incubation. The decolorization activity was not observed in washed cells of N. corallina when the cells were incubated in buffer but the activity was regained when the cells were incubated in LB medium. The product of biodegradation was Michler’s Ketone.

* Degradation by yeast
Biodegradation of Crystal Violet by oxidative red yeasts is reported [42]. He described the role of yeasts or yeast-like fungi in the removal of environmental contaminants. He showed that oxidative yeasts such as Rhodotorula sp. and Rhodotorula rubra were capable of degrading Crystal Violet in the liquid broth. The growth media contains (g L-1) glucose, 10; Peptone, 10; Ox-bile, 15; and Crystal Violet, 0.01. No antibiotics were added in the media. The final concentration of test Crystal Violet in the growth media was at the level of 10 ppm. Crystal Violet degradation was measured in terms of primary degradation by following the disappearance of Crystal Violet from the flask broth. After four days of incubation, the absorbance of the supernatant at 600 nm became non measurable. This indicated the complete biodegradation of Crystal Violet by both the oxidative yeasts. In order to test the absorbance of the dye by the cells, the cells after biodegradation were sonicated in 70% (v/v) ethanol. The extract did not show any absorbance at 600 nm. It was also observed that the fermentative yeast S. cerevisiae did not degrade Crystal Violet in the liquid medium even after a prolonged incubation of 30 days. There may be little decrease in optical density due to the adsorption of the dye to the cells. The inability of the fermentative yeast S. cerevisiae to biodegrade Crystal Violet was apparently not due to any toxic effect from the dye. The yeast culture was found to grow equally well in both the control and test flasks in terms of cell dry weight and cell morphology as seen in the phase contrast microscope. There was a linear degradation of Crystal Violet by the two oxidative red yeasts between the 2nd and 4th days of incubation. This indicated the presence of an enzyme system for the degradation of Crystal Violet.

* Degradation of fungi
In 1988, [43] it was reported that the biodegradation of Crystal Violet in ligninolytic (nitrogen-limited) culture of the white rot fungus Phanerochaete chrysosporium. In the culture broth, the Crystal Violet disappeared with the appearance of its three products by the sequential N-demethylation of the parent compound. Demethylation of Crystal Violet was also performed with a H2O2 generating system in the extracellular fluid. The purified ligninase also catalyzed the N-demethylation of Crystal Violet. It proved that the lignin-degrading system is responsible for the biodegradation of Crystal Violet. Besides Crystal Violet, other triphenylmethane dyes are also degraded by this fungus. They also reported that the nonligninolytic culture of P. chrysosporium could also degrade the triphenylmethane dyes. This clearly suggested that in addition to the lignin-degrading system, another mechanism exists in this fungus which also degrades the dye. Adsorption to fungal mycelium may account for decolorization, but it is only 22% of the total decolorization observed. Six different triphenylmethane dyes were degraded by ligninolytic culture of P. chrysosporium. Involvement of the lignin-degrading system was confirmed by results showing the purified lignin peroxidase decolorizing these dyes. Brilliant Green, Malachite Green, and Ethyl Violet all contain N-alkyl groups, therefore, the initial oxidation of all these dyes proceeds via N-demethylation in a manner similar to that in Crystal Violet. Pararosaniline, Cresol Red, and Bromophenol Blue contain no alkyl groups; thus, oxidation of these dyes occurs by a mechanism different from that observed for Crystal Violet. These clearly indicate that the lignin degrading system of P. chrysosporium is relatively nonspecific. This nonspecific nature is due to the presence of lignin peroxidase in the organism. The initial oxidation of a wide variety of organic compounds is catalyzed by lignin peroxidases [44-46]. These enzymes have been shown to be able to catalyze a wide variety of reactions including benzylic oxidation, phenol dimerization, carbon-carbon bond cleavage, hydroxylation, and o-demethylation [46,47] .Decolorization of three triphenylmethane dyes (Crystal Violet. Bromophenol Blue, and Malachite Green) by three birds’ nest fungi, Cyathus bulleri, Cyathus stercoreus. and Cyathus striatus, was reported by [48]. Among the three organisms, C. bulleri was found to be most efficient in decolorization. The authors also reported the decolorization activity in extracellular fluid of the culture filtrate. The rate of Bromophenol Blue decolorization was very fast compared to the other dyes. This may be due to the less complex structure of Bromophenol Blue compared to the other two dyes tested. They obtained lactase and ligninase activity in C. bulleri and also observed lactase activity to be on the higher side than that of ligninase during the dye decolorization period. Under nitrogen-sufficient conditions and without addition of H2O2 the culture filtrate of C. bufkri was able to decolorize three triphenylmethane dyes (Crystal Violet, Malachite Green, and Bromophenol Blue) in a relatively simple medium. They reported the decolorization of Crystal Violet by ultrafiltered and dialyzed extracellular culture filtrate from C. bulleri. This could be due to the presence of active lactase in ultrafiltered and dialyzed extracellular fluid. The faster dye decolorization by whole cultures of fungus (96-100% in 4 days), compared with extracellular fluid (90% in 10 days), could be the result of the combined activity of some cell associated and extracellular ligninolytic enzyme. C. bulleri has been found to be capable of decolorizing the Crystal Violet up to 90 pM wheras P. chrysosporium has been shown to decolorize the dyes to a much lesser extent (12.3 micro molar) [43].
In 1995, [49] the decolorization of Crystal Violet by different fungi was reported. Three white-rot fungi such as Coriolus versicolor, Fun&a trogii, and Phanerochaete chrysosporium and one brown-rot fungus, Laetiporus sulphureus was used. The oxidation of Crystal Violet by commercial horseradish peroxidase was reported. A significant rate of oxidation was observed only when H2O2 was present. Without H2O2 the enzyme had no effect on the decolorization of dye, thus suggesting that a H2O2 dependent enzyme is probably involved in the oxidation of the dye.
The decolorization of several dyes by a group of wood-rotting fungi was reported [50]. Fruiting bodies of basidiomycetes fungi growing on the rotting wood were used for the decolorization experiment. Brilliant Green and Crystal Violet were completely decolorized by white-rot fungi. The ability of white-rot fungi to degrade a diverse array of xenobiotic compound [51] is often attributed to the use in the wide range of dye waste treatment. The Crystal Violet decolorization in a column bioreactor using P. chrysosporium was studied [52]. The decolorization was performed in a glass column bioreactor (31 cm X 5 cm) with an eight tier stainless steel inoculum holder through which the dye containing medium was recirculated by a peristaltic pump. Crystal Violet was passed through the column at a concentration of 0.002% with a recycling rate of 20 ml min-1 at 30°C. The data revealed that almost 92% decolorization is there in 82.4 h in recycled medium as compared to 64% in shake flasks in 17 days. With glucose, the peak decolorization is reached in about 3 days as compared to 4-6 days with sucrose. Various isoenzymes of lignin peroxidase (LIP) were purified from P. chrysosporium and were studied for their decolorization efficiencies on several dyes including two triphenylmethane dyes (Bromophenol Blue and Methyl Green) by crude lignin peroxidase and by three purified isoenzymes. The three isoenzymes are LIP 4.65, LIP 4.15. and LIP 3.85; various isoenzymes of lignin peroxidase LIP 4.65 (H2). LIP 4.15 (H7). and LIP 3.85 (H8) were reported to be different from each other with respect to their N-terminal amino acid sequence and the degree of glycosylation [53]. The specific activities of LIP 4.65, LIP 4.15. and LIP 3.85 were 26, 39, and 31 U mg-1, respectively. For decolorization of dyes, isoenzymes were purified by preparative isoelectric focusing.
In the presence of 2 mM veratryl alcohol, the crude lignin peroxidase was able to partially decolorize the dyes. The best decolorization was obtained for Bromophenol Blue (93%). The dyes were then studied with isolated isoenzymes, LIP 4.65, LIP 4.15, and LIP 3.85. of lignin peroxidase for decolorization. The dye decolorization in the presence of 2 mrvt veratryl alcohol with the isoenzymes was similar to that with the crude lignin peroxidase. The decolorization ability of the purified isoenzymes was greatly decreased when veratryl alcohol was not present in the reaction mixture. This suggests that veratryl alcohol acts as a mediator in the reaction but the omission of veratryl alcohol from the reaction mixture had almost no effect on the ability of the crude lignin peroxidase to decolorize the dyes. This may be due to the fact that the fungus itself synthesizes veratryl alcohol as a product of secondary metabolism. With the increase in the veratryl alcohol concentration the rate of decolorization of Methyl Green increased. In the presence of 1 mM veratryl alcohol, the crude lignin peroxidase was able to decolorize Methyl Green almost completely (initial concentration, 29 micro molar). The effect of other enzymes present in the crude enzyme on decolorization can not be excluded. but the effect of manganese peroxidase should be minor because the assay conditions for lignin peroxidase do not favour the enzymatic activity of manganese peroxidase.

Degradative pathway of Crystal Violet by N. corallina. [41] * Biodegradation products
Since the researchers had given main emphasis on the decolorization studies, very few reports are available on the biodegradation products or intermediates of triphenylmethane dyes. The degradation of Crystal Violet with Pseudomonas pseudomallei was studied and the reaction products of Crystal Violet and Methyl Violet by thin layer chromatography (TLC) were demonstrated [8]. The dichloromethane extract of the products of Crystal Violet digestion using methanol and dichloromethane (3:97 v/v) as a developing solvent produced a spot not for Crystal Violet (Rf 0.l), but for some unknown product (Rf 0.6). The results of the above experiments indicated the probability of biodegradation of triphenylmethane dyes. The biodegradation products or intermediates of the Crystal Violet digestion were not identified [39]. They concluded that the decolorization of Crystal Violet and Malachite Green by Mycobacterium sp. was irreversible probably due to the degradation and formation of leuco-derivatives and then to unidentified fluorescent- and UV-absorbing intermediates. It was reported that the major degradation product of Crystal Violet by growing cells of B. subtilis IF0 13719 was 4,4’ bis dimethylamino benzophenone (Michler’s Ketone MK) [38]. The same product was again obtained [41] with Nocardia corallina. The biodegradation product was identified by TLC and gas chromatography-mass spectrometry (GC-MS). The product was stable since it was not entirely degraded within 24-48 h by N. corallina (Figure above).
The biodegradation and mineralization of Crystal Violet by white-rot fungi was reported [43]. The disappearance of Crystal Violet as well as metabolite formation was monitored by high-Performance Liquid Chromatography (HPLC). They used methylene chloride extract for HPLC analysis. It was observed that the initial degradation starts with the sequential demethylation of Crystal Violet. Three degradation products were identified as N,N,N’,N’,N”-penta-, N,N,N’,N”-tetra-, and N,N’,N”-trimethylpararosaniline. Biodegradation appears to continue beyond that as they obtained two additional unidentified colored Crystal Violet metabolites during HPLC analysis, but finally, Crystal Violet degraded to a colorless product.

(iii) Aims & Objectives
Aim
Isolation and identification of a bacterial strain that decolorizes the triphenylmethane dye: crystal violet with an efficient rate.
Objectives
The main objectives of the study are : * Collection of natural samples. * Screening of the samples for dye degrading isolates. * Selection of dye degrading isolates. * Optimization of the physicochemical parameters. * Identification of the bacterium by 16s rRNA. * Analysis of degradation byproducts by FTIR. * Toxicity assessment of the degraded dye products.

(iv) Materials & Methods

1. Natural samples such as mangrove soil and water, sewage samples will be collected in sterile containers

2. The samples will be enriched in nutrient broth containing definite concentration of crystal violet and incubated at room temperature at shaker and static condition till decolorization is observed. Enriched culture from flask showing decolorization will be isolated on dye containing nutrient agar plates. On incubation at room temperature, morphologically distinct colonies will be studied for their dye decolorization characteristic using liquid culture method.

3. Individual isolates will be inoculated into the flasks containing nutrient broth with definite dye concentration. Dye decolorization will be monitored using UV-Vis spectrophotometer.

4. The selected dye degrading isolate will be identified by 16S rRNA technique.

5. Physicochemical parameters for dye degradation by selected isolate will be optimized. Factors such as media formulations (synthetic, complex), pH, temperature, inoculum density will be optimized.

6. The ability to degrade repetitively batches of dye by pre-grown culture of isolate will be checked

7. Confirmation of dye degradation by FTIR analysis

8. A range of dyes (representative dyes from different chemical classes and other triphenylmethane dyes) will be checked for their degradation by the isolate.

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Budget

HEADING | DETAILS | EXPENDITURE | CONSUMABLES | Media, chemical.Commercial dye samples.Glasswares. | Rs 30,000 | EQUIPTMENT | Minor euiptments like pH meter, digital weighing machine, colorimeter | Rs 50,000 | OTHER EXPENDITURE | Stationary, photocopy, printing, books and journals. | Rs 5,000 | MISCELLANEOUS | Out sourcing for identification of culture by 16s r-RNA analysis and for confirmation of dye degradation by HPLC | Rs 10,000 | TOTAL | | Rs 95,000 |

Infrastructure facilities extended by the institute * Water and electricity * AC room for equipment * Library * Laboratory space and furniture * Computer and internet facilities

Available equipment and accessories to be utilized for the project

* Available with the department

Instruments | Company name | Analytical balance | Contech | Autoclave | Equitrone | Incubator | Proto-tech | Shaker | Delux rotary shaker | UV-vis spectrophotometer | Systronics | FTIR | Agilent technologies Cary 630 |