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Decolorization and biodegradation of triphenylmethane dyes by a novel Rhodococcus qingshengii JB301 isolated from sawdust

Annals of Microbiology volume 64, pages 1575–1586 (2014)Cite this article

Abstract

A sawdust sample collected from a wet environment was used to screen for crystal violet-decolorizing and methyl violet-decolorizing bacterial strains. A strain with degrading and decolorizing abilities was isolated and identified as Rhodococcus qingshengii JB301. The decolorization of crystal violet and methyl violet was improved by optimizing culture conditions. Based on liquid chromatography–mass spectrometry (LC-MS) analysis, similar degradation products of crystal violet and methyl violet were detected, while [N,N-dimethylaminophenyl] [aminophenyl] benzophenone or [N-methylaminophenyl] [N-methylaminophenyl] benzophenone was different from previous reports. A common degradation pathway was established to demonstrate the mechanism of crystal violet and methyl violet decolorization. It was found that lignin peroxidase activity was greatly enhanced after addition of crystal violet (by 514 %) and methyl violet (by 698 %) at 12 h, while NADH-DCIP (dichlorophenolindophenol) reductase activity was inhibited by the dyes. The growth of Triticum aestivum was inhibited by both dyes and the toxicity of the dyes was weakened after the degradation by R. qingshengii JB301.

Introduction

Triphenylmethane dyes, including crystal violet, methyl violet, malachite green and brilliant green, are widely applied in several industries such as textiles, cosmetics, paper and as biological stains (like gram stain) (Beveridge 2001; Parshetti et al. 2006; Nohynek et al. 2010; Kumar et al. 2011). Improper disposal of dye effluents in lakes and rivers leads to reduced sunlight penetration, which in turn decreases both photosynthetic activity and dissolved oxygen concentration. Due to hazardous products released during decomposition, dye pollutants from these industries had toxic effects on flora and fauna (Kumar et al. 2005; Soloman et al. 2010). In addition, human exposure to these dyes may cause a severe risk due to their toxicological and carcinogenic properties on the immune system and respiratory system (Fessard et al. 1999; Kumar et al. 2005; Culp et al. 2006).

Several physical and chemical methods, such as flocculation, coagulation, adsorption, membrane filtration, precipitation and irradiation/ozonization, are applied to treat wastewater containing colored effluents (Franciscon et al. 2010). However, the main drawbacks of physical and chemical methods, such as the high cost, inability to remove recalcitrant dyes and production of large amounts of sludge, limit their applications (Robinson et al. 2001; Mittal et al. 2005; Khataee et al. 2009). Much attention has been devoted to the microbial decolorization and degradation of dyes because of the advantages of being inexpensive and producing less amounts of toxic sludge. It has been reported that a variety of dyes can be biodegraded by a wide range of microorganisms, including fungi (Chander and Arora 2007), bacteria (Manal et al. 2005), yeasts (Jadhav and Govindwar 2006), actinomycetes (Yatome et al. 1993) and algae (Khataee et al. 2011).

Rhodococcus sp. bacteria are widely distributed in soil and possess the ability to degrade a range of compounds. Due to their metabolic properties, Rhodococcus species have been employed for the degradation of toxic compounds and chemicals (Behki et al. 1993; Bell et al. 1998; Dean-Ross et al. 2001), production of enzymes (Kamble et al. 2010; de las Heras et al. 2011) and the synthesis of bio-surfactant and flocculant (Marques et al. 2009; Chang et al. 2009). The degradation products and the pathway of dyes degradation have been analyzed in many studies (Yatome et al. 1993; Chen et al. 2008, 2010; Kumar et al. 2011; Parshetti et al. 2011). However, no intensive work has been carried out to investigate the possible mechanism of methyl violet and crystal violet degradation by Rhodococcus sp. In the present study, a dye decolorizing bacterial strain has been isolated from sawdust and identified as Rhodococcus qingshengii JB301. The decolorization ability of the isolated strain was studied under different culture conditions using crystal violet and methyl violet. The phytotoxic properties and the degradation products formed during the dye degradation were analyzed.

Materials and methods

Dyes and chemicals

Crystal violet and methyl violet used in this study were purchased from Sinopharm Chemical Reagent Co. (Beijing, China). Azure B (3-methylamino-7-dimethylaminophenothiazin-5-ium chloride) and ABTS [2,2'-azino-di-(3-ethyl-benzothiazolin-sulphonate)] were obtained from Shanghai Sangon Biotechnology (Shanghai, China).

Screening for microorganism and culture conditions

A sawdust sample collected from a wet environment (the riverside of Taihu Lake, Wuxi, China) was screened for dye-decolorizing bacterial strains. The sample was cultured with sterilized saline water and the culture broth was spread on an aniline blue agar plate. The culture broth was composed of (g/l): peptone 10, yeast extract 5, and NaCl 5 (pH 7.0). The aniline blue agar plates contained (g/l): peptone 10, beef extract 3, NaCl 5, aniline blue 1, and agar 20 (pH 7.0). Colonies producing transparent circles on the plate were isolated and inoculated into Luria-Bertani (LB) medium at 30 °C and 150 rpm. Twenty-five milligrams of crystal violet and methyl violet per liter were separately added to the culture broth after 24 h cultivation and the decolorizing ability of the isolated strain was measured. After the screening, one strain showing the greatest ability of decolorization was identified as R. qingshengii JB301 and preserved for further decolorization analysis.

16S rDNA sequencing

Fragments of the 16S rRNA gene were amplified by polymerase chain reaction (PCR) as described by Weisburg et al. (1991). The primers used for amplifications were:

  • 5′-AGAGTTTGATCCTGGCTCAG-3′ (primer F) and

  • 5′-CGGTTACCTTGTTACGACTT-3′ (primer R).

The amplified 16S rRNA gene was sequenced with a Dye Terminator Cycle-sequencing FS Ready Reaction kit and a model ABI 3130 automatic DNA sequencer (Applied Biosystems) by Shensushengwu Co., Ltd (Shanghai, China). A homology search was performed with the GenBank database to determine the strain species.

Decolorization assay

In order to evaluate the decolorization ability of the isolated strain, 25 mg/l of crystal violet and methyl violet were added to the culture medium after 24 h incubation. The repetitive decolorization capacity of the cells was determined by consecutive addition of 5 mg/l crystal violet and 5 mg/l methyl violet to the culture broth separately. The culture broth was withdrawn at different time intervals and treated as previously described to measure the decolorization of crystal violet and methyl violet (Manal et al. 2005; Jadhav et al. 2008; Yang et al. 2011a, b). The control experiment was the same condition of the two dyes decolorization without the inoculation. Each experiment was performed in triplicates. The results were shown as the averages of three replicates, with error bars indicating the standard deviation.

Effect of culture conditions on decolorization

The decolorization of the two dyes was evaluated under different conditions, including pH (4 - 10), various temperatures (20 - 45 °C), different inoculation sizes of 0.1 ∼ 10 % (volume per volume), and static and shaking (150 rpm) conditions. The concentrations of crystal violet and methyl violet were 15 mg/l each.

In order to check the effect of dye concentration on decolorization, 15, 30, 50, 100, 150 and 200 mg/l of crystal violet and methyl violet were separately added to the culture medium. The effect of dye (15 mg/l crystal violet and 15 mg/l methyl violet) addition time on decolorization was studied after incubation for 12, 24, 36, 48 and 60 h.

Enzyme activity assay

Rhodococcus qingshengii JB301 was grown in LB medium for 24 h and cells were collected by centrifugation at 8,000 rpm for 20 min. The cell precipitate was dissolved in 50 mM potassium phosphate buffer (pH 7.4) for sonication (sonics-vibracell ultrasonic processor). The sonifier output was kept at 60 A with eight strokes, 30 s each, at 2 min intervals. The temperature was maintained at 4 °C and the cell extract was used as an enzyme source without centrifugation (Parshetti et al. 2009).

The cell extract obtained by sonication was used to determine the activity of intracellular enzymes. The activity of lignin peroxidase (LiP) was measured using veratryl alcohol as the substrate at 300 nm (Tien and Kirk 1984). Laccase activity was determined spectrophotometrically at 420 nm using 10 % ABTS as the substrate in 0.1 M acetate buffer (pH 4.9) (Kalyani et al. 2008). Tyrosinase activity was determined at 495 nm in a mixture containing 0.01 % catechol with 0.1 M phosphate buffer (pH 7.4) (Kalyani et al. 2008). NADH-DCIP reductase activity was measured at 595 nm in the 5 ml reaction mixture contained 50 μM DCIP, 50 μM NADH, 50 mM potassium phosphate (pH 7.4) and 0.1 ml appropriately diluted crude culture broth with the molar extinction coefficient of 19 mM-1 cm-1 (Salokhe and Govindwar 1999). The protein content in the cell extract was measured as described by Bradford (1976).

Decolorization and biodegradation analysis

The culture broth from crystal violet and methyl violet decolorization was used to obtain degradation products. The supernatant was treated as previously described and the crystallized degradation products were dissolved for further determination by LC-MS (Jadhav et al. 2008). We used a ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC–Q-TOF MS) system equipped with a Waters Acquity SDS pump connected to a Waters Acquity PDA detector, Sunfire C18 column (150 mm × 2.1 mm, 5 μm), and time-of-flight mass spectrometer with electrospray ionization source. Solvent A was acetonitrile and Solvent B was 20 mM ammonium acetate. The proportion of A was increased linearly from 30 % to 70 % for 10 min, kept at equilibrium for 2 min, and then switched to 30 % A after 12.10 min. The flow rate was 0.3 ml/min. Chromatograms were analyzed at an integrated range between 200 nm and 500 nm. Mass spectra data were obtained in the positive ion mode by full scanning from 50 to 1, 200 m/z. A Masslynx software workstation was used for UPLC-MS instrument control, data acquisition and data processing (Liu et al. 2010).

Phytotoxicity assay

In order to assess the phytotoxicity of dyes and their degradation products, the length of plumule and radical of Triticum aestivum were measured (Shedbalkar et al. 2008). The measured T. aestivum seeds were cultured by 10 ml sterilized water (control) or a solution of degradation products. The degradation products were obtained from the ethyl acetate extraction of metabolites of dyes degradation and then dissolved in sterilized water.

Statistical analysis

All the experiments were independently performed in triplicate and differences among treatments were one-way analysis of variance (ANOVA) with a Tukey-kramer multiple comparisons test.

Results

Screening and identification

Twenty bacterial samples were isolated from sawdust to test for their potential ability to degrade aniline blue. Of these isolated strains, one strain possessed an excellent ability to decolorize crystal violet and methyl violet. This strain was identified by 16S rDNA sequencing and compared with sequences available at GenBank. The isolated strain showed a high similarity with species in the Rhodococcus genus and the phylogenetic tree is shown in Fig. 1. The isolated strain was deposited at the China Center for Type Culture Collection (CCTCC M 2012064), and the 16S rDNA sequence of this strain was deposited at GenBank (Accession No. JQ900306).

Fig. 1
figure 1

Bootstrap N-J tree of Rhodococcus qingshengii JB301 and the relationship of strain JB301 to other members of Rhodococcus genus based on similarity of a 16S rRNA fragment. The scale bar indicates 0.5 % sequence dissimilarity

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Effect of various culture conditions on dye decolorization

Effect of pH

The effect of pH on dye decolorization was investigated over a pH range from 4.0 to 10.0. The results showed that the decolorization of crystal violet and methyl violet was similar and the maximum decolorization was achieved between pH 5.0 - 9.0 and pH 6.0 - 9.0, respectively (Fig. 2a). The decolorization of crystal violet and methyl violet by R. qingshengii JB301 remained steady at a pH ranging from 6.0 to 9.0 and was inhibited at pH 4.0 and 10.0.

Fig. 2
figure 2

Decolorization of crystal violet (■) and methyl violet (●) under different pH (A) and temperatures (B) by Rhodococcus qingshengii JB301

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Effect of temperature

The effect of temperature on decolorization of the two dyes was determined at temperatures ranging from 20 to 50 °C. Figure 2b demonstrates that temperature greatly influenced the decolorization of methyl violet, while it had little effect on crystal violet decolorization. The maximum decolorization of crystal violet and methyl violet were both achieved between 28 and 37 °C.

Effect of inoculation ratio

The decolorization of crystal violet remained at approximately 85 % and was slightly enhanced by increasing the inoculation ratio of R. qingshengii JB301 (Fig. 3a). However, the decolorization of methyl violet was greatly enhanced when the inoculation size (volume per volume) increased to 2 %.

Fig. 3
figure 3

Effect of inoculum size and culture conditions on dye decolorization. A Decolorization of crystal violet (■) and methyl violet (●) using various inoculum sizes. B Decolorization of crystal violet (square) and methyl violet (circle) under shaking (filled symbols) and static (empty symbols) conditions

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Effect of static and shaking conditions

The 90 % decolorization of crystal violet and methyl violet at static conditions was observed after 24 h and 18 h, respectively. Compared with the static condition, the time of 90 % decolorization of crystal violet and methyl violet were reduced (Fig. 3b).

Effect of dye addition time and concentration on decolorization

The effects of dye addition time and dye concentration on crystal violet and methyl violet decolorization were similar (Fig. 4). High decolorization efficiency was obtained when both dyes were added after 24 h, and the decolorization time was decreased. Figure 5 shows that the decolorization of crystal violet and methyl violet under all tested concentrations remained constant after 24 h. When the concentration of dyes exceeded 50 mg/l, the decolorization decreased sharply.

Fig. 4
figure 4

Decolorization of crystal violet (A) and methyl violet (B) after different addition times: 12 h (■), 24 h (●), 36 h (▲), 48 h (▼), 60 h (◆)

Full size image
Fig. 5
figure 5

Effect of different concentrations of crystal violet (A) and methyl violet (B) on decolorization. 15 mg/l (■), 30 mg/l (●), 50 mg/l (▲), 100 mg/l (▼), 150 mg/l (◆), 200 mg/l (◄)

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Effect of different dyes on cell growth

The effect of the added dyes on cell growth of R. qingshengii JB301 was investigated, and the results indicated that cell growth was inhibited by 15 mg/l crystal violet and methyl violet, compared to the control without dyes (Fig. 6).

Fig. 6
figure 6

Effect of culture conditions on cell growth. (●) crystal violet, (▲) methyl violet and (■) control without dyes

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Decolorization after repeated additions of dyes

Consecutive dye decolorization cycles were studied by repeatedly adding crystal violet (5 mg/l) and methyl violet (5 mg/l) separately. Figure 7 shows that 79.67 % decolorization of crystal violet and 85.72 % decolorization of methyl violet were achieved by R. qingshengii JB301 within 8 h. Although the decolorization rate of both dyes was slow after five circles, a high decolorization efficiency was maintained by R. qingshengii JB301.

Fig. 7
figure 7

Repeated use of Rhodococcus qingshengii JB301 in nutrient broth for decolorization of crystal violet (A) and methyl violet (B)

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Degradation products analysis

The intermediate products of crystal violet and methyl violet during the decolorization were analyzed by LC-MS. The crystal violet intermediates were identified as leucocrystal violet with a molecular weight of 373, leucomethyl violet with a molecular weight of 359, N,N-dimethyl-N′, N′-dimethylpararosaniline (DDPR) or N,N-dimethyl-N′-methyl-N″-methylpararosaniline with a molecular weight of 345, Michler’s ketone with a molecular weight of 268, [N,N-dimethylaminophenyl] [N-methylaminophenyl] benzophenone with a molecular weight of 254, [N,N-dimethylaminophenyl] [aminophenyl] benzophenone or [N-methylaminophenyl] [N-methylaminophenyl] benzophenone with a molecular weight of 240 (Table 1). The intermediates of methyl violet were similar to those of crystal violet except leucocrystal violet.

Table 1 Retention times and accurate mass measurements obtained from LC-MS spectra of the crystal violet and methyl violet biodegradation products by Rhodococcus qingshengii JB301
Full size table

Based on these results, a degradation pathway of crystal violet and methyl violet was proposed (Fig. 8). Crystal violet was firstly demethylated to leucomethyl violet, and then formed N,N-dimethyl-N′, N′-dimethylpararosaniline or N,N-dimethyl-N′-methyl-, N″-methylpararosaniline by removal of one methyl group. These two components were split into Michler’s ketone and [N,N-dimethylaminophenyl] [aminophenyl] benzophenone, or [N,N-dimethylaminophenyl] [N-methylaminophenyl] benzophenone and [N-methylaminophenyl] [N-methylaminophenyl] benzophenone, respectively.

Fig. 8
figure 8

Degradation pathway of crystal violet and methyl violet by Rhodococcus qingshengii JB301

Full size image

Analysis of enzyme activities

The activities of LiP, laccase, tyrosinase and NADH-DCIP reductase both in cell extract and supernatant were monitored before and after decolorization. Table 2 shows that only LiP and NADH-DCIP reductase were detected in the cell extract. No laccase or tyrosinase activity was detected in either cell extract or supernatant. Compared to control samples, LiP activity was greatly enhanced after addition of crystal violet (514 %) and methyl violet (698 %) after 12 h. However, reduced activity of NADH-DCIP reductase was observed during the decolorization of both dyes.

Table 2 Enzyme activities of Rhodococcus qingshengii JB301 during decolorization
Full size table

Phytotoxicity study

Table 3 shows that the germination of T. aestivum seeds was greatly inhibited by crystal violet and methyl violet when compared to metabolites obtained after decolorization and control (sterilized water). However, the plumule and radical of T. aestivum seeds cultured with degradation products returned to normal growth and their lengths were slightly decreased compared with the control. Thus, the toxicity of dyes could be removed by degradation through R. qingshengii JB301.

Table 3 Phytotoxicity comparison of dyes and their extracted metabolites
Full size table

Discussion

A wide range of microorganisms have been reported to possess the ability to decolorize dyes. Due to their ability to produce thermostable enzymes, some fungi have been regarded as suitable candidates for decolorization (Murugesan et al. 2007). However, unlike lengthy fungal cultures, the stationary phase of bacteria can be achieved in a relatively short time under simple fermentation procedures (Maki et al. 2009). In this study, a strain highly capable of triphenylmethane dye decolorization was isolated from sawdust and was identified as R. qingshengii JB301. This is the first report discussing the mechanism of triphenylmethane dyes decolorization by R. qingshengii JB301.

In previous studies, some members of the genus Rhodococcus exhibited a wide range of catabolic activities in the degradation of hazardous materials including 2,4-dinitrophenol, 2,4,6-trichlorophenol, pyridine, etc. (Briglia et al. 1996; Yoon et al. 2000a, b). Xu et al. (2007) reported that a strain possessed the ability to degrade carbendazim was identified as R. qingshengii sp. nov. In this work, the isolated strain showed a high homology to R. qingshengii sp. nov. and exhibited the capacity to degrade the methyl violet and crystal violet.

Many reports have shown that the optimal pH changed greatly for decolorization of various dyes by different microorganisms. In some bacteria, the maximum rate of decolorization was observed at the range of pH 5.0 ∼ 7.0 (Zheng et al. 2009; Chen et al. 2011). In this study, the decolorization of crystal violet and methyl violet remained constant over an extensive range of pH (6.0 to 9.0), which would be favorable for bioprocessing dye-containing wastewater and useful for industrial applications (Chen et al. 2003).

In some studies, the decolorization ability of microorganisms, especially fungi, has been attributed to the secretion of efficient enzymes degrading a wide range of pollutants (Hilden et al. 2009; Maalej-Kammoun et al. 2009; Yang et al. 2011a, b). Therefore, temperature has a great influence on the decolorization ability of microorganisms by stimulating or deactivating the enzymes responsible for decolorization (Dantan-Gonzalez et al. 2008; Ayed et al. 2009). Decolorization in this study was slightly affected by temperatures ranging from 20 to 45 °C. This result indicated that the bioprocess of wastewater could be achieved at normal temperatures and, thus, decreased the costs for heating.

In this work, the biomass of R. qingshengii JB301 was monitored after different initial inoculum sizes, and the results indicated that the decolorization of crystal violet and methyl violet was not strongly enhanced by increasing the inoculum size above 2 %. Radha et al. (2005) reported that the removal of methyl violet increased significantly after an increase in the initial inoculum size, and a similar result was also obtained by Chen et al. (2011). However, it has been shown that cell concentrations of Kurthia sp. had different effects on the decolorization of effluents from textile production, and the phenomenon has been attributed to a different group of dyes (Sani and Banerjee 1999).

It has been reported that the decolorization under shaking conditions was partially or completely suppressed in comparison to static conditions (Kalme et al. 2007; Saratale et al. 2009). In a study by An et al. (2002), the removal of crystal violet by Citrobacter sp. under static conditions was comparatively slower than under shaking conditions, but eventually achieved the same decolorization at the end of culturing. In contrast to previous studies, our present work demonstrated that shaking conditions were beneficial to decolorization of both dyes.

The mechanisms of crystal violet and methyl violet decolorization differ for various microorganisms. Ren et al. (2006) reported that an isolated bacterium was capable of using crystal violet both as a carbon source and an energy source, and that the cell mass was greatly increased by adding crystal violet. However, the cell growth of R. qingshengii JB301 was inhibited at 15 mg/l of crystal violet and 15 mg/l methyl violet. Repeated use of microorganisms for dye decolorization is important for commercial application and has been reported by several studies (Kalyani et al. 2008; Parshetti et al. 2011). The decolorization rate after the second addition of a microorganism decreased in subsequent additions and the decrease was attributed to nutrient depletion (Moosvi et al. 2005; Jadhav et al. 2008). However, R. qingshengii JB301 exhibited a great ability for crystal violet and methyl violet decolorization after ten rounds, which highlights its applicability in wastewater treatment processes.

As revealed by the LC-MS analysis, the same intermediates were found for both dyes. This indicated that crystal violet and methyl violet had a similar degradation pathway, which was in contrast to previous studies (Parshetti et al. 2006, 2009; Ren et al. 2006) and may be attributed to the similar chemical structure of crystal violet and methyl violet. It has been reported that crystal violet and methyl violet were reduced to their leucoderivatives during decolorization (Chen et al. 2008, 2010). Both leucocrystal violet and leucomethyl violet were detected in our analysis, indicating that these leucoderivatives were important intermediates for further dye degradation steps. Chen et al. (2010) reported that two dyes were directly split into Michler’s ketone and N-dimethylamonophenol (N-methylamonophenol) by ring cleavage. However, demethylation has been found to occur before ring cleavage in methyl violet decolorization by Aspergillus sp. (Kumar et al. 2011). In our proposed degradation pathway, leucomethyl violet was demethylated and then degraded by ring cleavage. Crystal violet is composed of three N-dimethylaminophenol rings and the ring cleavage resulted in the same compound. Michler’s ketone is the major degradation product in many crystal violet decolorizations (Yatome et al. 1991, 1993; Chen et al. 2008). In our study, four intermediates, including Michler’s ketone, were detected and proposed to be produced by degradation of DDPR and N,N-dimethyl-N′-methyl-N″-methylpararosaniline. [N,N-dimethyl-aminophenyl] [N-methylaminophenyl] benzophenone has been detected in crystal violet decolorization by Shewanella sp., whereas [N,N-dimethyl-aminophenyl] [aminophenyl] benzophenone and [N-methyl-aminophenyl] [N-methylaminophenyl] benzophenone with a molecular weight of 240 has not been reported yet. Additionally, Chen et al. (2010) reported that small amounts of N-methylaminobenzaldehyde and N-methylaminophenol were detected during decolorization by Shewanella decolorationis. However, these two compounds were not detected in this study, which may be attributed to the rapid degradation.

The degradation of dyes in microbial systems is the result of enzymatic transformation (Raghukumar et al. 1996). Therefore, the enhancement of enzyme activity is beneficial to decolorizing dye effluents. Saratale et al. (2009) reported that an increased activity of NADH-DCIP reductase and azoreductase was observed during Navy blue HER decolorization. However, in this study NADH-DCIP reductase was repressed by crystal violet and methyl violet, which was similar to previously observed cotton blue decolorization by Penicillium ochrochloron (Shedbalkar et al. 2008). LiP is a member of Lignin-modifying enzymes (LMEs), which also contain laccase (EC 1.10.3.2), manganese peroxidase (MnP, EC 1.11.1.13) and versatile peroxidase (VP, EC 1.11.1.16) (Martínez 2002; Hammel and Cullen 2008). It has been reported that LiP possesses the ability to degrade the dyes, include Direct Blue-6 and azo dyes (Selvam et al. 2003; Kalme et al. 2007). Therefore, the increased decolorization of crystal violet and methyl violet may be caused by the enhancement of Lip activity during the fermentation. Furthermore, no enzyme activity was detected in the supernatant, indicating that the degradation of dyes was an intracellular process.

Conclusions

A newly isolated bacterium R. qingshengii JB301 possessed the ability to decolorize and degrade crystal violet and methyl violet. A potential degradation pathway for these two dyes was suggested to demonstrate their decolorization mechanism. [N,N-dimethylaminophenyl] [aminophenyl] benzophenone or [N-methylaminophenyl] [N-methylaminophenyl] benzophenone were detected during the decolorization, which differed from previous reports. LiP and NADH-DCIP reductase were detected in the cell extract and LiP activity was enhanced by addition of crystal violet and methyl violet. This might be the main reason for the high decolorization efficiency of R. qingshengii JB301.

References

  • An SY, Min SK, Cha IH, Choi YL, Cho YS, Kim CH, Lee YC (2002) Decolorization of triphenylmethane and azo dyes by Citrobacter sp. Biotechnol Lett 24:1037–1040

    Article  CAS  Google Scholar 

  • Ayed L, Cheriaa J, Laadhari N, Cheref A, Bakhrouf A (2009) Biodegradation of crystal violet by an isolated Bacillus sp. Ann Microbiol 59:267–272

    Article  CAS  Google Scholar 

  • Behki R, Topp E, Dick W, Germon P (1993) Metabolism of the herbicide atrazine by Rhodococcus strains. Appl Environ Microbiol 59:1955–1959

    CAS  PubMed Central  PubMed  Google Scholar 

  • Bell KS, Philp JC, Aw DWJ, Christofi N (1998) The genus Rhodococcus. J Appl Microbiol 85:195–210

    Article  CAS  PubMed  Google Scholar 

  • Beveridge TJ (2001) Use of the Gram stain in microbiology. Biotech Histochem 76:111–118

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Briglia M, Rainey FA, Stackebrandt E, Schraa G, Salkinoja-Salonen MS (1996) Rhodococcus percolatus sp. nov., a bacterium degrading 2,4,6-trichlorophenol. Int J Syst Evol Microbiol 46:23–30

    CAS  Google Scholar 

  • Chander M, Arora DS (2007) Evaluation of some white-rot fungi for their potential to decolourise industrial dyes. Dyes Pigments 72:192–198

    Article  CAS  Google Scholar 

  • Chang WN, Liu CW, Liu HS (2009) Hydrophobic cell surface and bioflocculation behavior of Rhodococcus erythropolis. Process Biochem 44:955–962

    Article  CAS  Google Scholar 

  • Chen KC, Wu JY, Liou DJ, Hwang SCJ (2003) Decolorization of the textile dyes by newly isolated bacterial strains. J Biotechnol 101:57–68

    Article  CAS  PubMed  Google Scholar 

  • Chen CH, Chang CF, Ho CH, Tsai TL, Liu SM (2008) Biodegradation of crystal violet by a Shewanella sp. NTOU1. Chemosphere 72:1712–1720

    Article  CAS  PubMed  Google Scholar 

  • Chen CH, Chang CF, Liu SM (2010) Partial degradation mechanisms of malachite green and methyl violet B by Shewanella decolorationis NTOU1 under anaerobic conditions. J Hazard Mater 177:281–289

    Article  CAS  PubMed  Google Scholar 

  • Chen CC, Chen CY, Cheng CY, Teng PY, Chung YC (2011) Decolorization characteristics and mechanism of Victoria Blue R removal by Acinetobacter calcoaceticus YC210. J Hazard Mater 196:166–172

    Article  CAS  PubMed  Google Scholar 

  • Culp SJ, Mellick PW, Trotter RW, Greenlees KJ, Kodell RL, Beland FA (2006) Carcinogenicity of malachite green chloride and leucomalachite green in B6C3F(1) mice and F344 rats. Food Chem Toxicol 44:1204–1212

    Article  CAS  PubMed  Google Scholar 

  • Dantan-Gonzalez E, Vite-Vallejo O, Martinez-Anaya C, Mendez-Sanchez M, Gonzalez MC, Palomares LA, Folch-Mallol J (2008) Production of two novel laccase isoforms by a thermotolerant strain of Pycnoporus sanguineus isolated from an oil-polluted tropical habitat. Int Microbiol 11:163–169

    CAS  PubMed  Google Scholar 

  • de las Heras LF, Mascaraque V, García Fernández EG, Navarro-Llorens JM, Perera J, Drzyzga O (2011) ChoG is the main inducible extracellular cholesterol oxidase of Rhodococcus sp. strain CECT3014. Microbiol Res 166:403–418

    Article  Google Scholar 

  • Dean-Ross D, Moody JD, Freeman JP, Doerge DR, Cerniglia CE (2001) Metabolism of anthracene by a Rhodococcus species. FEMS Microbiol Lett 204:205–211

    Article  CAS  PubMed  Google Scholar 

  • Fessard V, Godard T, Huet S, Mourot A, Poul JM (1999) Mutagenicity of malachite green and leucomalachite green in in vitro tests. J Appl Toxicol 19:421–430

    Article  CAS  PubMed  Google Scholar 

  • Franciscon E, Piubeli F, Fantinatti-Garboggini F, de Menezes CR, Silva IS, Cavaco-Paulo A, Grossman MJ, Durrant LR (2010) Polymerization study of the aromatic amines generated by the biodegradation of azo dyes using the laccase enzyme. Enzym Microb Technol 46:360–365

    Article  CAS  Google Scholar 

  • Hammel KE, Cullen D (2008) Role of fungal peroxidases in biological ligninolysis. Curr Opin Plant Biol 11:349–355

    Article  CAS  PubMed  Google Scholar 

  • Hilden K, Hakala TK, Lundell T (2009) Thermotolerant and thermostable laccases. Biotechnol Lett 31:1117–1128

    Article  CAS  PubMed  Google Scholar 

  • Jadhav JP, Govindwar SP (2006) Biotransformation of malachite green by Saccharomyces cerevisiae MTCC 463. Yeast 23:315–323

    Article  CAS  PubMed  Google Scholar 

  • Jadhav UU, Dawkar VV, Ghodake GS, Govindwar SP (2008) Biodegradation of Direct Red 5B, a textile dye by newly isolated Comamonas sp. UVS. J Hazard Mater 158:507–516

    Article  CAS  PubMed  Google Scholar 

  • Kalme SD, Parshetti GK, Jadhav SU, Govindwar SP (2007) Biodegradation of benzidine based dye Direct Blue-6 by Pseudomonas desmolyticum NCIM 2112. Bioresour Technol 98:1405–1410

    Article  CAS  PubMed  Google Scholar 

  • Kalyani DC, Patil PS, Jadhav JP, Govindwar SP (2008) Biodegradation of reactive textile dye Red BLI by an isolated bacterium Pseudomonas sp. SUK1. Bioresour Technol 99:4635–4641

    Article  CAS  PubMed  Google Scholar 

  • Kamble AL, Meena VS, Banerjee UC (2010) Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Lett Appl Microbiol 51:413–420

    Article  CAS  PubMed  Google Scholar 

  • Khataee AR, Pons MN, Zahraa O (2009) Photocatalytic degradation of three azo dyes using immobilized TiO(2) nanoparticles on glass plates activated by UV light irradiation: influence of dye molecular structure. J Hazard Mater 168:451–457

    Article  CAS  PubMed  Google Scholar 

  • Khataee AR, Dehghan G, Zarei M, Ebadi E, Pourhassan M (2011) Neural network modeling of biotreatment of triphenylmethane dye solution by a green macroalgae. Chem Eng Res Des 89:172–178

    Article  CAS  Google Scholar 

  • Kumar KV, Sivanesan S, Ramamurthi V (2005) Adsorption of malachite green onto Pithophora sp., a fresh water algae: Equilibrium and kinetic modelling. Process Biochem 40:2865–2872

    Article  CAS  Google Scholar 

  • Kumar CG, Mongolla P, Basha A, Joseph J, Sarma VUM, Kamal A (2011) Decolorization and Biotransformation of Triphenylmethane Dye, Methyl Violet, by Aspergillus sp. Isolated from Ladakh, India. J Microbiol Biotechnol 21:267–273

    CAS  PubMed  Google Scholar 

  • Liu RJ, Jin QZ, Tao GJ, Shan L, Liu YF, Wang XG (2010) LC-MS and UPLC-quadrupole time-of-flight MS for identification of photodegradation products of aflatoxin B(1). Chromatographia 71:107–112

    Article  CAS  Google Scholar 

  • Maalej-Kammoun M, Zouari-Mechichi H, Belbahri L, Woodward S, Mechichi T (2009) Malachite green decolourization and detoxification by the laccase from a newly isolated strain of Trametes sp. Int Biodeterior Biodegrad 63:600–606

    Article  CAS  Google Scholar 

  • Maki M, Leung KT, Qin WS (2009) The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass. Int J Biol Sci 5:500–516

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Manal MA, El-Naggar S, El-Aasar A, Khlood IB (2005) Bioremediation of crystal violet using air bubble bioreactor packed with Pseudomonas aeruginosa. Water Res 39:5045–5054

    Article  CAS  PubMed  Google Scholar 

  • Marques AM, Pinazo A, Farfan M, Aranda FJ, Teruel JA, Ortiz A, Manresa A, Espuny MJ (2009) The physicochemical properties and chemical composition of trehalose lipids produced by Rhodococcus erythropolis 51T7. Chem Phys Lipids 158:110–117

    Article  CAS  PubMed  Google Scholar 

  • Martínez AT (2002) Molecular biology and structure-function of lignin-degrading heme peroxidases. Enzym Microb Technol 30:425–444

    Article  Google Scholar 

  • Mittal A, Kurup L, Gupta VK (2005) Use of waste materials - Bottom Ash and De-Oiled Soya, as potential adsorbents for the removal of Amaranth from aqueous solutions. J Hazard Mater 117:171–178

    Article  CAS  PubMed  Google Scholar 

  • Moosvi S, Keharia H, Madamwar D (2005) Decolourization of textile dye Reactive Violet 5 by a newly isolated bacterial consortium RVM 11.1. World J Microbiol Biotechnol 21:667–672

    Article  CAS  Google Scholar 

  • Murugesan K, Nam IH, Kim YM, Chang YS (2007) Decolorization of reactive dyes by a thermostable laccase produced by Ganoderma lucidum in solid state culture. Enzym Microb Technol 40:1662–1672

    Article  CAS  Google Scholar 

  • Nohynek GJ, Antignac E, Re T, Toutain H (2010) Safety assessment of personal care products/cosmetics and their ingredients. Toxicol Appl Pharmacol 243:239–259

    Article  CAS  PubMed  Google Scholar 

  • Parshetti G, Kalme S, Saratale G, Govindwar S (2006) Biodegradation of malachite green by Kocuria rosea MTCC 1532. Acta Chim Slov 53:492–498

    CAS  Google Scholar 

  • Parshetti G, Saratale G, Telke A, Govindwar S (2009) Biodegradation of hazardous triphenylmethane dye methyl violet by Rhizobium radiobacter (MTCC 8161). J Basic Microbiol 49:36–42

    Article  Google Scholar 

  • Parshetti GK, Parshetti SG, Telke AA, Kalyani DC, Doong RA, Govindwar SP (2011) Biodegradation of crystal violet by Agrobacterium radiobacter. J Environ Sci-China 23:1384–1393

    Article  CAS  PubMed  Google Scholar 

  • Radha KV, Regupathi I, Arunagiri A, Murugesan T (2005) Decolorization studies of synthetic dyes using Phanerochaete chrysosporium and their kinetics. Process Biochem 40:3337–3345

    Article  CAS  Google Scholar 

  • Raghukumar C, Chandramohan D, Michel FC, Redd CA (1996) Degradation of lignin and decolorization of paper mill bleach plant effluent (BPE) by marine fungi. Biotechnol Lett 18:105–106

    Article  CAS  Google Scholar 

  • Ren SZ, Guo J, Zeng GQ, Sun GP (2006) Decolorization of triphenylmethane, azo, and anthraquinone dyes by a newly isolated Aeromonas hydrophila strain. Appl Microbiol Biotechnol 72:1316–1321

    Article  CAS  PubMed  Google Scholar 

  • Robinson T, McMullan G, Marchant R, Nigam P (2001) Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresour Technol 77:247–255

    Article  CAS  PubMed  Google Scholar 

  • Salokhe MD, Govindwar SP (1999) Effect of carbon source on the biotransformation enzymes in Serratia marcescens. World J Microbiol Biotechnol 15:259–263

    Article  Google Scholar 

  • Sani RK, Banerjee UC (1999) Decolorization of triphenylmethane dyes and textile and dye-stuff effluent by Kurthia sp. Enzym Microb Technol 24:433–437

    Article  CAS  Google Scholar 

  • Saratale RG, Saratale GD, Chang JS, Govindwar SP (2009) Decolorization and biodegradation of textile dye Navy blue HER by Trichosporon beigelii NCIM-3326. J Hazard Mater 166:1421–1428

    Article  CAS  PubMed  Google Scholar 

  • Selvam K, Swaminathan K, Chae KS (2003) Decolourization of azo dyes and a dye industry effluent by a white rot fungus Thelephora sp. Bioresour Technol 88:115–119

    Article  CAS  PubMed  Google Scholar 

  • Shedbalkar U, Dhanve R, Jadhav J (2008) Biodegradation of triphenylmethane dye cotton blue by Penicillium ochrochloron MTCC 517. J Hazard Mater 157:472–479

    Article  CAS  PubMed  Google Scholar 

  • Soloman PA, Basha CA, Velan M, Balasubramanian N (2010) Electro oxidation of malachite green and modeling using ANN. Chem Biochem Eng Q 24:445–452

    CAS  Google Scholar 

  • Tien M, Kirk TK (1984) Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization, and catalytic properties of a unique H2O2-requiring oxygenase. Proc Natl Acad Sci U S A 81:2280–2284

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Biotechnol 173:697–703

    CAS  Google Scholar 

  • Xu JL, He J, Wang ZC, Wang K, Li WJ, Tang SK, Li SP (2007) Rhodococcus qingshengii sp. nov., a carbendazim-degrading bacterium. Int J Syst Evol Microbiol 57:2754–2757

    Article  CAS  PubMed  Google Scholar 

  • Yang XQ, Wang JR, Zhao XX, Wang Q, Xue R (2011a) Increasing manganese peroxidase production and biodecolorization of triphenylmethane dyes by novel fungal consortium. Bioresour Technol 102:10535–10541

    Article  CAS  PubMed  Google Scholar 

  • Yang YY, Hu H, Wang G, Li ZL, Wang B, Jia XM, Zhao YH (2011b) Removal of malachite green from aqueous solution by immobilized Pseudomonas sp. DY1 with Aspergillus oryzae. Int Biodeterior Biodegrad 65:429–434

    Article  CAS  Google Scholar 

  • Yatome C, Ogawa T, Matsui M (1991) Degradation of crystal violet by Bacillus subtilis. J Environ Sci Health A 26:75–87

    Google Scholar 

  • Yatome C, Yamada S, Ogawa T, Matsui M (1993) Degradation of crystal violet by Nocardia corallina. Appl Microbiol Biotechnol 38:565–569

    CAS  Google Scholar 

  • Yoon JH, Cho YG, Kang SS, Kim SB, Lee ST, Park YH (2000a) Rhodococcus koreensis sp. nov., a 2,4-dinitrophenol-degrading bacterium. Int J Syst Evol Microbiol 50:1193–1201

    Article  CAS  Google Scholar 

  • Yoon JH, Kang SS, Cho YG, Lee ST, Kho YH, Kim CJ, Park YH (2000b) Rhodococcus pyridinivorans sp. nov., a pyridine-degrading bacterium. Int J Syst Evol Microbiol 50:2173–2180

    Article  CAS  Google Scholar 

  • Zheng TL, Wang H, Su JQ, Zheng XW, Tian Y, Xiong XJ (2009) Bacterial decolorization and degradation of the reactive dye Reactive Red 180 by Citrobacter sp CK3. Int Biodeterior Biodegrad 63:395–399

    Article  Google Scholar 

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Acknowledgements

This research was supported by a grant from the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, People’s Republic of China

    Guohui Li, Lin Peng, Zhongyang Ding, Liang Zhang & Guiyang Shi

  2. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Guohui Li, Lin Peng, Zhongyang Ding, Zhenghua Gu, Liang Zhang & Guiyang Shi

  3. School of Food Science and Technology, Jiangnan University, Wuxi, 214122, People’s Republic of China

    Yuanfa Liu

  4. Key Laboratory of Eco-Textiles, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, People’s Republic of China

    Guohui Li

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  1. Guohui Li
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  2. Lin Peng
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  3. Zhongyang Ding
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  4. Yuanfa Liu
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Correspondence to Zhongyang Ding.

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Li, G., Peng, L., Ding, Z. et al. Decolorization and biodegradation of triphenylmethane dyes by a novel Rhodococcus qingshengii JB301 isolated from sawdust. Ann Microbiol 64, 1575–1586 (2014). https://doi.org/10.1007/s13213-014-0801-7

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Annals of Microbiology

ISSN: 1869-2044

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