Crystal violet (CV), a recalcitrant molecule, used commonly as biological stain and commercial dye, was subjected to biodegradation in this study by Pseudomonas fluorescens NCIM 2100 and its biodegraded metabolic products were identified by UV, 1HNMR and IR Spectrophotometry. During degradationprocess CV dye was first broken down into an intermediate compound which was further converted into either 4-benzhydrylidene-cyclohexa-2,5-dienylamine hydrochloride or 4-benzhydrylidene-cyclohexa-2,5-dienol or 4-benzhydryl-cyclohexa-2,5- diennol in broth medium and were non-toxic in nature. Hence, P. fluorescens can be used for bioremediation of textile effluents containing CV dye in sequential cycles.Key words: Crystal violet, bioremediation, Pseudomonas fluorescens, metabolic products, spectrophotometry

Pandey, BV

Pandey, RR

Upadhyay, RS

English

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African Journal of Biotechnology Vol. 10 (4), pp. 624-631, 24 January, 2011     Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.1616 ISSN 1684–5315 © 2011 Academic Journals      Full Length Research Paper  Characterization and identification of Pseudomonas fluorescens NCIM 2100 degraded metabolic products of Crystal Violet  R. R. Pandey1*, B. V. Pandey2 and R. S. Upadhyay3  1Department of Life Sciences, Manipur University, Canchipur, Imphal-795 003, India. 2Department of Botany, Sri Agrasen Kanya Autonomous P.G. College, Varanasi-221 001, India. 3Laboratory of Plant Pathology and Microbial Technology, Department of Botany, Banaras Hindu University,  Varanasi-221 005, India.  Accepted 23 December, 2010  Crystal violet (CV), a recalcitrant molecule, used commonly as biological stain and commercial dye, was subjected to biodegradation in this study by Pseudomonas fluorescens NCIM 2100 and its biodegraded metabolic products were identified by UV, 1HNMR and IR Spectrophotometry.  During degradation process CV dye was first broken down into an intermediate compound which was further converted into either 4-benzhydrylidene-cyclohexa-2,5-dienylamine hydrochloride or 4-benzhydrylidene-cyclohexa-2,5-dienol or 4-benzhydryl-cyclohexa-2,5- diennol  in  broth medium and were non-toxic in nature. Hence, P. fluorescens can be used for bioremediation of textile effluents containing CV dye in sequential cycles.   Key words: Crystal violet, bioremediation, Pseudomonas fluorescens, metabolic products, spectrophotometry.    INTRODUCTION  Crystal violet (N, N, N', N', N", N"- hexamethylpara-rosalinine), a triphenylmethane dye, has been used extensively in textile dyeing and dye-stuff manufacturing industries, in human and veterinary medicines as a biological strain and in paper, leather, food and cosmetic processing industries (Bangert et al., 1977; Bumpus and Brock, 1988; Gregory, 1993). This dye has been cate-gorized as a recalcitrant molecule indicating that, it is poorly metabolized by microbes and therefore, is per-sistent in environment for longer period when discharged (Chen et al., 2007). Increasing concerns about the color and toxic ingredients in the dyes effluents are leading towards the worldwide efforts for developing their effective removal processes. Physical and chemical facili-ties available for wastewater treatments are often unable to remove completely the commercial dyes from contami-nated environments, thus, contributing to the  pollution  in     *Corresponding author. E-mail: pandey.rr@rediffmail.com. Tel: +919436025986. Fax: +91-385-2435327. aquatic habitat (Reife, 1993). Some triphenylmethane dyes were also proved to be mutagenic and carcinogenic to biota (Black et al., 1980). However, the processes used for the chemical treatment of dyes effluents are much costly, less efficient and produce large amount of sludge, hence, biological degradation processes are getting more attention since it is cost effective, eco-friendly and produce less quantity of sludge as compared to physical-chemical treatments (Azmi et al., 1998; Ayed et al., 2009).  Various microbes such as bacteria, fungi, actinomy-cetes and algae have been reported to degrade different pollutants (Bumpus and Brock, 1988; Leahy and Colwell, 1990; Spain, 1995; Azmi et al., 1998). Interest is now focused on the bacteria which can perform high rate of discoloration under anoxic conditions and provide detoxi-fication of aromatic products under aerobic environment (Stolz, 2001; Upadhyay, 2002; Chen et al., 2007). Seve-ral isolates of Pseudomonas, Alcaligenes, Acinetobacter and Bacillus are a few important bacteria useful in bio-remediation of halogenated organic compounds (Chaudhary and Chapalamadugu,  1991;  Yatome  et  al.,      1991). However, few reports are available on the biode-gradation products or intermediates of different dyes. Therefore, the objective of the present study was to characterize and identify spetrophotometrically, the bio-degradation metabolic products of crystal violet by a CV-degrading bacterium, Pseudomonas fluorescens NCIM 2100.   MATERIALS AND METHODS  Microbial strain and culture medium  The bacterial culture of P. fluorescens NCIM 2100, used in the pre-sent study for biodegradation of crystal violet, was obtained from National Collection of Industrial Microorganisms, National Chemical Laboratory, Pune, India, that was well adopted to grow on this dye. The test bacterium was grown on King’s B medium. The pH of broth medium was adjusted to 7.2 with phosphate buffer before sterilization.   CV-degradation by P. fluorescens NCIM 2100  The experiments were performed as described by Kar et al. (1997) into 250 ml conical flasks (in triplicate) containing 50 ml King's B broth (pH 7.2) supplemented with CV dye (ca. 200 mg/l in H2O). Cell suspension of the test bacterium containing 106 cfu/ml was used as inoculum. Cultures were established by inoculating the CV supplemented broth with 0.1 ml bacterial cells and the flasks were transferred into a temperature controlled incubator shaker. Ambient temperature of the cultures was maintained at 37°C. The cultures were then flushed with oxygen, resealed and further incubated for 4 days on a rotary shaker (200 rev/min).   Preparation of cell extract  The flasks were removed after 4 days of incubation period and were centrifuged at 4°C temperature for 5 min by high speed cooling centrifuge (Remi C-24) at 10000 rev/min. The test bacterial cells were harvested after centrifugation, washed with phosphate buffer (10 mM, pH 7.2) and then cells were disrupted by adding 1 mm glass beads into the suspension. The resulting suspension was again centrifuged and the supernatant was stored on ice until use. The bioremediation experiments with cell-free extract before and after degradation of crystal violet dye by P. fluorescens NCIM 2100 were performed spectrophotometrically.   UV spectrophotometry  The UV and visible spectra of the samples were measured in ethanol with a Perkin Elmer double beam UV-Vis Spectrophotometer 117. Quartz cells (1 cm sq) having 1.0 cm path length was used for the determination. Hydrogen discharge tungsten filament lamp was applied as a source of light and maximum absorbance was recorded.   1HNMR spectroscopy  The samples were subjected to 1HNMR spectroscopy. Proton nuclear magnetic resonance spectra of the test samples were run at Brukar AC 200 M Hz in D2O using TMS as an interval standard and 0.5 ml aliquot of the samples along with 0.5 ml of  solvent  were  Pandey et al.        625    taken in 5 ml O.D glass tube for the determination.   IR spectroscopy  The infra red (IR) spectra of the test samples were obtained by placing solid compound in Nujol between salt plates (NaCl, 0.1 mm cells), without a spacer, in one of the beams of a Perkin Elmer-783 spectrophotometer Nernst glower, molded rod containing a mixture of zirconium oxide, yttraium oxide and erbium oxide and heated to 1500°C by electrical means, which was used as a light sources of IR irradiation. Grating was employed to obtain monochromatic light. The stretching and bending position of bands were observed and compared with reference compounds. Band position was presented in wave number unit (cm-1 reciprocal) and band intensities were expressed as transmittance (T). The functional groups present were counted according to band positions (Dyer, 1991).   RESULTS AND DISCUSSION  Ultraviolet (UV) spectrum of crystal violet before degra-dation by P. fluorescens NCIM 2100 showed maximum absorbance in methanol at 578 nm (Figure 1). After degradation, maximum absorbances were obtained at 238 and 276.7 nm. The peaks were completely different which reveal that, Crystal Violet changed into another compound (Figure 2). Before degradation by the test bacterium, 1HNMR spectrum of crystal violet (CV) showed three singlets at 4.25, 6.00 and 7.25 ppm (Figure 3). After degradation, it also showed three singlets in aromatic region at 7.25, 6.1 and 4.32 ppm. It can be concluded that, there is the formation of a new compound, which possessed benzene rings attached by benzylic carbon that contained one proton (Figure 4). IR spectrum before degradation, revealed a medium peak at 1350 cm-1, a strong peak at 1450 cm-1, a medium peak at 1550 cm-1 and a broad peak at 3350 cm-1 (Figure 5). After degradation, IR spectrum showed a medium peak at 1350 cm-1, a strong peak at 1450 cm-1, two medium peaks at 1530 and 1620 cm-1 and two broad peaks at 3200 cm-1 and 3450 cm-1. This indicate the presence of C-O, C=O, C-H, OH or NH group in the compounds (Figure 6). Our findings on UV, 1HNMR and IR spectrum after bio-treatment of crystal violet by P. fluorescens NCIM 2100 revealed that, CV was degraded into an intermediate compound which further converted into either 4-benzhydrylidene-cyclohexa-2,5- dienylamine hydro-chloride or 4-Benzhydrylidene-cyclohexa-2,5- dienol or 4-Benzhydryl-cyclohexa-2,5- diennol in broth medium that were non-toxic in nature (Figure 7). Buckingham and Macdonald (1996) have also reported that, the end products obtained as a result of textile dyes biode-gradation by P. fluorescens were non-toxic. A similar result has been observed by Pandey and Upadhayay (2006) in case of Acid Yellow-9 dye which after degra-dation by P. fluorescens changed into three non-toxic intermediate compounds such as 2,4 dihydroxy-benzene sulphonic acid sodium salt, 2 amino-4 dihydroxy-benzene  626         Afr. J. Biotechnol.       Figure 1. UV spectrum of crystal violet before degradation by P. fluorescens NCIM 2100.       Figure 2. UV spectrum of crystal violet after degradation by P. fluorescens NCIM 2100. Pandey et al.        627      Figure 3. 1HNMR spectrum of crystal violet before degradation by P. fluorescens NCIM 2100.    sulphonic acid sodium salt or 4-amino- 2 hydroxybenzene sulphonic acid sodium salt. Bacterial strains have the initial degradative dehalogenation steps yielding hydroxy benzoic acid, which is subsequently metabolized through meta-ring cleavage (Mark et al., 1984;  Kobayashi  et  al., 1997). The degradation mechanism is mainly associated with the presence of NAD (P) which acts as electron donor. Bumpus and Brock (1988) reported the biodegradation and mineralization of CV by a white rot fungus,   Phanerochaete   chrysosporium   and  observed  628         Afr. J. Biotechnol.      Figure 4. 1HNMR spectrum of Crystal violet after degradation by P. fluorescens NCIM 2100.    that, the initial degradation starts with sequential deme-thylation of the tested dye which resulted into appearance of metabolic products that were identified as N, N, N', N', N"-penta-, N, N, N', N"-tetra- and N, N', N"-trimethylpara-rosalinine. Chen et al. (2007) found that Pseudomonas putida is also capable of degrading the industrial CV dye after determining their demethylation degradation path-way in which a sequential identification of primary and secondary metabolites  that is, a series of N-demethy-lates intermediates, were observed under visible spectral region. Several studies on degradation of crystal violet by the microorganisms have reported that, the dye is relatively resistant to decolorization in the environment (Michaels and  Lewis,  1986;  Yatome  et  al.,  1981)  and  Pandey et al.        629         (%)   Figure 5. Infra red spectrum of crystal violet before degradation by P. fluorescens NCIM 2100.      (%)   Figure 6. Infra red spectrum of crystal violet after degradation by P. fluorescens NCIM 2100.    attributed to the fact that CV is toxic to several bacterial strains and inhibits their growth and activity during treatment process.  Conclusion   Our findings suggest that, crystal violet is non-toxic to the  630         Afr. J. Biotechnol.      Figure 7. Possible pathway of degradation of crystal violet.    test bacterium strain, P. fluorescens NCIM 2100, which has the capability to successfully degrade crystal violet in sequential cycles. Further studies on enzymatic system responsible for CV degradation would enhance our understanding in determining the potential of this bacte-rial strain for effective bioremediation of the test dye effluents.        REFERENCES  Ayed L, Cheriaa J, Laadhari N, Cheree A, Bakhrouf A (2009). Biodegradation of crystal violet by an isolated Bacillus sp. Ann. 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Characterization and identification of Pseudomonas fluorescens NCIM 2100 degraded metabolic products of Crystal Violet

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Characterization and identification of Pseudomonas fluorescens NCIM 2100 degraded metabolic products of Crystal Violet

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