Triphenylmethanes, Malachite Green and Crystal Violet Dyes Decolourisation by Sphingomonas Paucimobilis

Annals of Microbiology, 59 (1) 57-61 (2009)

Triphenylmethanes, malachite green and crystal violet dyes decolourisation by Sphingomonas paucimobilis

Jihane CHERIAA, Amina BAKHROUF*

Laboratoire d’Analyse, Traitement et Valorisation des Polluants de l’Environnement et des Produits, Faculté de Pharmacie, Rue Avicenne, 5000 Monastir, Tunisie

Received 23 October 2007 / Accepted 30 January 2009

Abstract - In this study, Gram negative bacterium, Sphingomonas paucimobilis, was used to test its ability to decolourise two triphenylmethane dyes: malachite green (MG) and crystal violet (CV) in mineral salts medium (MSM). Decolourisation was examined with dye concentrations (2.5, 5, 15, 25, 30 and 50 mg/l), glucose (0, 1.4, 2.8, 4.2, 5.6 and 7 mM) and yeast extract concentrations (0, 0.05, 0.10, 0.15% w/v). Our results showed that Sphingomonas paucimobilis used at 1 OD (L 600 nm), equivalent to 14 x 107 CFU/ ml cell concentration, remove MG and CV colour with 35 and 55%, respectively, for 2.5 mg/l dye concentration in MSM. The best removal efficiencies for decolourisation were 93.43% for MG and 71.29% for CV at 50 mg/l dye concentration, obtained with 7 mM of glucose used as source of carbon. Whereas the optimum concentration of yeast extract which allowed high value on decolourisation was determined at 0.1% for MG and 0.05% for CV. We obtained the high percentage of decolourisation which reaches 100% within 10 h. Moreover, at the same conditions the source of nitrogen yeast extract enhanced more rapidly the decolourisation of dyes colour removal by a new natural isolates Sphingomonas paucimobilis which was significantly affected by adding nutrient sources.

Key words: decolourisation; crystal violet; glucose; malachite green; Sphingomonas paucimobilis; triphenylmethane dyes; yeast extract.

INTRODUCTION biodegraded (Aksu, 2005). Contact of malachite green with humans causes skin irritation and eye injury (Kumar et al., The triphenylmethane group of dyes is used extensively 2005). It has been reported that crystal violet is mutagenic in textile manufacturing (Kumar and Banerjee, 1999). It and toxic to the mammalian cells (Clemmensen et al., 1996; is reported that 10-20% of dyes are lost in wastewater as Fessard et al., 1999). The use of some bacteria’s potential for consequence of inefficiency in the dyeing process (Zollinger, dyes biodegradation seems to be promising. A wide range of 1987). For the treatment of wastewater containing dyes, the bacteria were studied for their ability to decolourise synthetic microbial decolourisation has been studied (Cooper, 1995; dyes. Some of these strains included Aeromonas hydrophila Parshetti et al., 2006). (Chen et al., 2003), Pseudomonas luteola (Chang et al.,

Synthetic dyes are used extensively in textile, printing 2001), Escherichia coli NO3 (Chang and Kuo, 2000) and and other industrial applications. Malachite green and crystal Pseudomonas mendocina MCM B-402 (Sarnaik and Kanekar, violet are two triphenylmethane dyes belonging to a basic 1999). Aerobic degradation of triphenylmethane dyes has dyes class, used extensively for dyeing silk, wool and cotton been demonstrated frequently; however these dyes resist to (Parshetti et al., 2006) and have wide range applications, e.g. the degradation in activated sludge system. It has been found in textile industries for both dyeing and printing, in the inks that four triphenylmethane dyes were biodegraded using manufacturing, and as colouring agents for papers, toys and Pseudomonas pseudomallei (Yatome et al., 1993). In addition, plastic varieties (Sarnaik and Kanekar, 1999; El-Naggar et al., Parshetti et al. (2006) demonstrated that malachite green 2004). The effluents of these industries are highly coloured was transformed into non-toxic compound by Kocuria rosea and cause serious environmental pollution. Removed colour strain. from wastewater by conventional treatment methods is very This study aims to investigate the capacity of Sphingomonas difficult (Kumar et al., 2006). Dyes usually have complex paucimobilis isolated from textile wastewater to decolourise aromatic molecular structures which make them difficult to be triphenylmethane dyes (malachite green and crystal violet). The correlation of the kinetic properties of dyes concentra- * Corresponding Author. Phone: +216 73 461-000; tion and the addition of other supplement (glucose and yeast Fax: +216 73 461-830; E-mail: [email protected] extract) in the medium were also studied. 58 J. CHERIAA and A. BAKHROUF

MATERIALS AND METHODS ZnSO4 0.2 mg/l, CuSO4 0.2 mg/l, FeSO4 0.14 mg/l in 1000 ml of distilled water; pH 7.0 (Moutaouakkil et al., 2002); The Screening of dye decolourizing microorganisms. Activated medium was sterilised at 120 °C for 15 min. Stock solutions of sludge samples were collected from a textile dyeing industry. yeast extract, glucose and dyes were sterilised through a 0.2 μm Firstly, one millilitre was spread on Petri dishes containing nutri- membrane filter and added to sterilised MSM. ent agar (Bio-Rad) medium. Secondly, all isolated microorgan- Experiments were performed in flasks containing 100 ml isms were screened onto MSM-agar-dye composed of (g/l): MSM medium added with 50 mg/l of dyes, initial inoculum

6 Na2HPO4, 0.5 NaCl, 3 K2HPO4, 0.1 MgSO4, 0.14 NH4Cl, 3.0 size of selected bacterium was 1 OD at L = 600 nm equivalent Bacto-Casamino Acids (Difco), 15 Agar-agar (Fluka), and 50 mg/l to 14 x 107 CFU/ml (El-Naggar et al., 2004). The flasks were of dyes. All plates were incubated at 30 °C for 48 h. incubated at 37 °C under shaking at 150 rpm during 24 h. The flask containing dyes and MSM without bacterium was Identification of bacterium and culture condition. used as control. Samples of supernatant (5 ml) were taken Microorganisms were selected according to their ability to and centrifuged at 5000 rpm for 15 min every 2 h (Demet decolourise dyes by forming clear zones around colonies. and Göonöul, 2006). The absorbance of supernatant was The most interesting bacterial isolate produd a clear spectrophotometrically determined using spectrophotometer zone of diameter ≈10 mm. The strain was identified with (Shimadzu UV-2401 PC model Kyoto, Japan). All essays microscopic observation of Gram stain and basing on were performed in duplicate and compared with the control other bacteriological tests such as: mobility, reduction of sample. Decolourisation percentage was calculated as nitrates, ONPG, oxidase, indole, gelatinase, urease (Pinon indicated in many studies (Dhanve et al., 2008; Daneshvar et al., 1987). The identification was completed by using et al., 2007; Parshetti et al., 2006). API 20NE Test System (bio-Mérieux, France), at 30 °C (I-F) within 48 h. Decolourisation % = x 100 I Dyes and chemicals. The triphenylmethane dyes: where I = initial absorbance and F = absorbance of malachite green (dimethylamino-4-alpha-phenyl- decolourised medium. benzylidene-4-cyclohexadiene-2,5-ylidene dimethyl ammonium) and crystal violet (N, N, N’, N’, N’’, N’’- Effect of concentration dyes. Various dyes concentrations hexamethylpararosaniline) were obtained from the Sigma were tested for MG and CV: 2.5, 5, 15, 25, 30, 40 and 50 mg/l Chemical Company, MO, USA. The dyes concentrations in MSM medium deprived of nutrient sources. The flasks were were measured with a spectrophotometer UV-Visible prepared, incubated at 37 °C under shaking condition (150 rpm) (Shimadzu UV-2401 PC model Kyoto, Japan) at maximum and the percent of colour removal was calculated as indicated absorption wavelengths Lmax= 618 nm and 592 nm for above. The decolourisation was observed during 24 h. malachite green (MG) and crystal violet (CV), respectively (Table 1). Optimisation of medium supplements. The effects of different initial concentrations of glucose and yeast extract Decolourisation assay. A decolourising activity was evaluated were performed as described by Khehra et al., 2005. The with decolourisation percentage as indicated by Khehra et al. MSM medium with 50 mg/l dye concentration, for both (2005) and determined by monitoring the decrease of absorb- dye tested was supplemented by different concentrations ance at maximum absorption wavelengths. All experiments were of glucose (0, 1.4, 2.8, 4.2, 5.8 and 7 mM) and by realised in mineral salts medium (MSM) and prepared by adding yeast extract (0, 0.05, 0.1 and 0.15% w/v). Flasks were the following components: MgSO4 0.1 g/l, (NH4)2SO4 0.6 g/l, incubated at 37 °C, under shaking (150 rpm) in aerobic NaCl 0.5 g/l, K2HPO4 1.36 g/l, CaCl2 0.02 g/l, MnSO4 1.1 mg/l, conditions.

TABLE 1 - Chemical structure of triphenylmethane dyes

Dyes Chemical structure Lmax

CH H3C 3 N Cl

Crystal violet 592 nm

H3C CH3 N N

H3C CH3

Malachite green 618 nm

Me2N NMe2 Ann. Microbiol., 59 (1), 57-61 (2009) 59

FIG. 1 - Effect of initial concentration of malachite green on FIG. 2 - Effect of initial concentration of crystal violet on decolourisation by Sphingomonas paucimobilis in MSM. decolourisation by Sphingomonas paucimobilis in MSM. £: 2.5, r: 5, €: 15, +: 25, ô: 30, Ø: 50 mg/l. £: 2.5, r: 5, €: 15, +: 25, ô: 30, Ø: 50 mg/l.

RESULTS AND DISCUSSION bonds and aromatic ring, second, a single pure bacterium was involved in the decolourisation assays. We could deduce that CV A Gram negative rod strain, identified as Sphingomonas pauci- was more toxic than MG and its lower rate might be attributed mobilis, was isolated from textile wastewater treatment plant. to intermediates metabolites. Moreover, Khehra et al. (2005) It was able to generate large clear zone on MSM-agar-dye and suggested that the decrease in decolourisation efficiency might was used for decolourisation assay. Many studies reported that be due to the toxic effect of dyes. However, Pérez-Estrada et isolation and screening of microorganisms collected from water al. (2007) reported that some products transformation gener- and sediments contaminated by wastewater textile industry, at ated during photolytic degradation of MG were more toxic to the high dyes concentrations, were adapted to removal colour (An et marine bacterium Vibrio fischeri than the parent compound. al., 2002; El-Rahim et al., 2003; Manjinder et al., 2005). The isolate strain showed capacity to decolourise triphenyl- The decolourisation by Sphingomonas paucimobilis of MG methane dyes in MSM without adding nutrient sources. MSM and CV was, respectively, more than 35 and 55%, at 2.5 mg/l dye was supplemented with different initial glucose concentrations, concentration within 24 h (Fig. 1 and 2). The increase of dyes con- in order to enhance the lower rates of dyes removal at 50 mg/l centration induced decrease on decolourisation efficiency, in fact, as shown in Fig. 1 and 2. The presence of glucose increased colour removal efficiency depended on various initial dye concen- the decolourisation efficiency of solutions containing MG and CV trations and Shingomonas paucimobilis was not able to decolourise (Fig. 3 and 4). The results indicated that glucose is essential for dyes at higher concentrations, from 40 mg/l (data not shown) for decolourisation of MG and CV by Sphingomonas paucimobilis MG and 25 mg/l for CV (Fig. 1 and 2). Similar observation was at 50 mg/l concentration dye. In the absence of glucose, only reported during decolourisation of MG by Kocuria rosea MTCC 1532 0.16% of MG colour, was removed. Though, with adding glucose, strain (Parshetti et al., 2006) and diazo dye CI acid black 24 by decolourisation efficiency increased proportionally and the higher zero-valent iron powder reactive violet (Chang et al., 2006). rate colour removal (93.43%) was achieved with 7 mM glucose Although, the decolourisation efficiency of MG was lower than in 24 h (Fig. 3). The same effect of glucose was observed on that of CV, we do not think that two different enzymatic activities decolourisation of CV, the rate of colour removal climbs from 4.82 were involved. First, the dyes were closely related, same dye to 71.29% with 7 mM glucose in 24 h (Fig. 4). Moreover, Dhanve class and high similarity of chemical structures (Table 1) with et al. (2008) showed that glucose improves colour removal.

FIG. 3 - Effect of glucose concentrations on decolourisation of FIG. 4 - Effect of glucose concentrations on decolourisation of malachite green (50 mg/l) in MSM by Sphingomonas crystal violet (50 mg/l) in MSM by Sphingomonas pau- paucimobilis. Ø: 0, £: 1.4, Õ: 2.8, r: 4.2, € : 5.6, ô: cimobilis. Ø: 0, £: 1.4, Õ: 2.8, r: 4.2, € : 5.6, ô: 7 mM. 7 mM. 60 J. CHERIAA and A. BAKHROUF

FIG. 5 - Effect of different yeast extract concentrations on FIG. 6 - Effect of various yeast extract concentrations on decolourisation of malachite green 50 mg/l in MSM by decolourisation of crystal violet 50 mg/l in MSM, by Sphingomonas paucimobilis. £: 0.05%, Ø: 0.1%, r: Sphingomonas paucimobilis. £: 0.05%, Ø: 0.1%, r: 0.15%. 0.15%.

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