Degradation and decolourization potential of an ligninolytic enzyme producing Aeromonas hydrophila for crystal violet dye and its phytotoxicity evaluation

Ecotoxicology and Environmental Safety 156 (2018) 166–175

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Degradation and decolourization potential of an ligninolytic enzyme producing Aeromonas hydrophila for crystal violet dye and its phytotoxicity evaluation

T

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Ram Naresh Bharagavaa, , Sujata Mania, Sikandar I. Mullab, Ganesh Dattatraya Saratalec a

Laboratory of Bioremediation and Metagenomics Research (LBMR), Department of Environmental Microbiology (DEM), Babasaheb Bhimrao Ambedkar University (A Central University), Vidya Vihar, Raebareli Road, Lucknow 226 025, U.P., India b Laboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China c Department of Food Science and Biotechnology, Dongguk University-Seoul, Ilsandong-gu, Goyang-si, Gyeonggi-do 10326, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Crystal violet Aeromonas hydrophila Biodegradation Ligninolytic enzyme GC-MS analysis Phytotoxicity

This study deals the biodegradation of crystal violet dye by a ligninolytic enzyme producing bacterium isolated from textile wastewater that was characterized and identified as Aeromonas hydrophila based on the 16 S rRNA gene sequence analysis. The degradation of crystal violet dye was studied under different environmental and nutritional conditions, and results showed that the isolated bacterium was effective to decolourize 99% crystal violet dye at pH 7 and temperature 35 °C in presence of sucrose and yeast extract as C and N source, respectively. This bacterium also produced lignin peroxidase and laccase enzyme, which were characterized by the SDS-PAGE analysis and found to have the molecular weight of ~ 40 and ~ 60 kDa, respectively. Further, the GC-MS analysis showed that CV dye was biotransformed into phenol, 2, 6-bis (1,1-dimethylethyl), 2′,6′-dihydroxyacetophenone and benzene by the isolated bacterium and the toxicity of CV dye was reduced upto a significant level as it showed 60%, 56.67% and 46.67% inhibition in seed germination. But, after the bacterial degradation/ decolourization, it showed only 43.33%, 36.67% and 16.67% inhibition in seed germination after 24, 48 and 72 h, respectively. Thus, this study concluded that the isolated bacterium has high potential for the degradation/ decolourization of CV dye as well to reduce its toxicity upto a significant level.

1. Introduction

becomes essential for the industrial wastewaters to be treated adequately before final discharge into the environment. For the treatment of textile wastewater, physical, chemical and biological methods are being used, but biological methods are regarded as environment friendly as physicochemical methods are very costly and also generate huge amount of sludge as secondary pollutant (Mani and Bharagava, 2017, 2016; Pandey et al., 2007). Intensive research is going on the microbial degradation and detoxification of dyes to search the effective microorganisms or enzymes to replace or supplement the present treatment processes. Ligninolytic enzymes namely laccase and LiP are highly specific in nature and extremely efficient catalysts. These enzymes are well reported to catalyze the degradation and detoxification of a variety of organic pollutants present in industrial wastewaters (Mugdha and Usha, 2012; Bharagava et al., 2009; Pandey et al., 2007). Many researchers have also reported that the microbial treatment of industrial wastewaters containing a mixture of recalcitrant dyes with different chemical structure as well as other pollutants by enzymes may be an ingenious approach (Chandra

Crystal violet (CV) is a triphenylmethane dye that has been used as a human and veterinary medicine, staining agent to classify microorganisms and an artificial coloring agent to color fabrics in textile industries since ancient time (Ahmad, 2009; Shah et al., 2013a). CV provides a deep purple color to paints and printing ink. In medical solutions, CV is used as a mutagenic and bacteriostatic agent and since, it acts as an antimicrobial agent, it had been also used in poultry feeds to avoid the fungal growth (Mani and Bharagava, 2016; Littlefield et al., 1985). In spite of its various uses, CV has been also reported as a recalcitrant dye molecule due to its longtime persistence in the environment (Mani and Bharagava, 2016; Azmi et al., 1998). CV, if introduced or absorbed by living organisms, it prevents or affects the cell division process, acts as a powerful carcinogenic, and clastogenic agent and also promotes tumor growth in fishes (Mani and Bharagava, 2016; Fan et al., 2009). Thus, CV dye is regarded as a hazardous chemical and therefore, it

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Corresponding author. E-mail addresses: [email protected], [email protected] (R.N. Bharagava).

https://doi.org/10.1016/j.ecoenv.2018.03.012 Received 6 November 2017; Received in revised form 4 March 2018; Accepted 6 March 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.

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2.3.2. Genomic DNA preparation and 16S rRNA gene sequence analysis The alkaline lyses method was used to prepare the genomic DNA from overnight grown bacterial culture (Kapley et al., 2007). The 16 S rRNA gene was amplified using 5 µl genomic DNA (50 ng) as template DNA and universal primers (27 F) 5 ´ -AGAGTTTGATCMTGGCTCAG3 ´ and (1492 R) 5 ´ -CGGTTACCTTGTTACGACTT-3 ´ . The thermocycling reactions were carried out using Veriti® 96-Well Thermal Cycler (Applied Biosystems, USA) as initial denaturation at 95 °C for 2 min followed by 30 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 52 °C, extension for 2 min at 72 °C and final extension for 15 min at 72 °C. The PCR amplified products (1289 bp) were gel purified using QIA gel extraction kit, Qiagen, Germany, and sequenced using 27 F primer. The partial sequences obtained were subjected to BLAST analysis using the online option available at www.ncbi.nlm.nih.gov/BLAST to get the closest homolog of the isolated bacterium (Altschul et al., 1997). In addition, the partial sequences were also submitted to GeneBank database to obtain the accession number for the isolated bacterium.

and Chowdhary, 2015; Mugdha and Usha, 2012; Chandra et al., 2008). Deng et al. (2008), reported a new strain of Bacillus cereus, DC11, which was capable to decolorize a broad spectrum of dyes. Under optimized parameters, this bacterium was found effective to decolorize 95–98% anthraquinone, malachite green, and Basic Blue X-GRRL dye under anaerobic conditions. Similarly, Ren et al. (2006) also isolated an Aeromonas hydrophila strain from activated sludge of a textile printing wastewater treatment plant, which was capable to decolorize more than 90% of triphenylmethane dyes such as crystal violet, basic fuchsin, brilliant green and malachite green at the concentration of 50 mg/l within 10 h. But, here we are looking for a bacterial strain capable to decolorize the higher concentration of crystal violet dye within the lesser time as reported in previous studies. Thus, the present study aimed to isolate and characterize a bacterium capable for the effective degradation and detoxification of crystal violet dye, to optimize various environmental as well as nutritional parameters to enhance its CV degrading potential, to characterize the ligninolytic enzymes Laccase and LiP produced by the isolated bacterium during the degradation of CV dye. In addition, the metabolic products produced during the bacterial degradation process were characterized by FT-IR and GC-MS analysis and the toxicity of CV dye before and after bacterial degradation and detoxification was evaluated in terms of germination percentage as well as seedling growth of seeds of Phaseolus mungo L.

2.4. CV degradation/decolourization studies The CV degradation/decolourization activity of isolated bacterium (SJ4) was studied in triplicate in 250 ml Erlenmeyer flasks containing 100 ml of sterilized modified MSM broth amended with 100 mg/l filter sterilized (0.22 µm) CV dye. The flasks were inoculated with 1% (v/v) overnight grown culture of isolated bacterium and incubated at 35 °C under shaking flask condition (110 rpm, LSI-3016R, Labtech, India) for 8 h. The degradation/ decolourization of CV dye was monitored spectrophotometrically (Evolution 201, Australia) in terms of bacterial growth and decrease in color intensity (absorbance) at 620 and 590 nm, respectively and was expressed in terms of percent decolourization (Deepak et al., 2004). At a regular interval of 1 h, 4 ml of sample was withdrawn from flasks, out of which, 2 ml was used to measure the bacterial growth at 620 nm and remaining 2 ml was centrifuged at 5000 ×g for 20 min at 4 °C and the supernatant obtained was used to measure the absorbance at 590 nm to calculate the percentage decolourization of CV as per the following formula:

2. Materials and methods 2.1. Crystal violet dye and media composition The crystal violet dye used in this study was purchased from Spectrochem, India (C.I. 42555) and used for the preparation of standard stock solution. The medium used throughout this study was CV dye amended Minimal-salt-medium (MSM) containing K2HPO4, 5.22; KH2PO4, 4.08; MgSO4·7H2O, 0.2; CaCl2, 0.55; NH4Cl, 0.4 and agar, 15 g/l. The seeds of Phaseolus mungo L. were purchased from the local market of Lucknow, Uttar Pradesh, India and used in phytotoxicity evaluation experiments.

% Decolourization = 100 × (A o−At)/ Ao 2.2. Collection of textile wastewater sample, isolation, and screening of dye decolorizing bacteria

where Ao is the absorbance value of initial dye concentration and At is the absorbance value of final dye concentration in samples after complete decolourization. In addition, at the end of experiment, the media containing bacterial culture was autoclaved in order to kill the bacteria and/or deactivate the enzyme activity. Now, the bacterial culture was centrifuged at 5000 ×g for 15 min at 4 °C followed by twice washing with sodium phosphate buffer (pH 6.5–7). The supernatant obtained was discarded and 5% (w/v) pellet was inoculated in 250 ml Erlenmeyer flasks containing 100 ml of sterilized modified MSM broth amended with 100 mg/l filter sterilized CV dye under the previously mentioned conditions and was used as control. The color of pellet was visually scrutinized to check that whether the dye was adsorbed onto the cell surface or being degraded by the bacterial cells. The uninoculated MSM broth amended with CV dye was used as blank. In addition, another control inoculated with E. coli ATCC 35218 was also used as a negative control.

The textile wastewater sample was collected from a Handloom Textile Company located at Panki Industrial Area, Kanpur, which is the most industrialized area of Kanpur city, Uttar Pradesh, India located at 26.4670° north, 80.3500° east. For the isolation of potential bacterial strains capable for CV dye decolourization, 10 ml of wastewater was inoculated into a flask containing 100 ml of MSM broth amended with CV dye (100 mg/l) followed by incubation at 35 °C under shaking conditions (110 rpm). After 48 h of incubation period, 1 ml of culture broth was appropriately diluted and plated on MSM-CV dye amended agar plates. Initially, ten (SJ1-SJ10) distinct bacterial colonies showing clear zones around their colonies indicating decolourization of CV dye were selected and further screened based on their ability to decolourize and tolerate the highest CV dye concentration on growing in MSM-CV dye amended agar plates. Finally, a bacterial strain (SJ4) was selected based on its growth performance and ligninolytic enzyme activity (i.e. production of Laccase and LiP enzymes) as well as potential for decolourization of CV dye and used in further experiments.

2.5. Optimization of different parameters for maximum degradation/ decolourization of CV dye The optimization of CV dye degradation/decolourization under different parameters was done to achieve its maximum degradation/ decolourization by the isolated bacterium.

2.3. Characterization and identification of isolated CV degrading bacterium 2.3.1. Biochemical characterization The isolated CV decolourizing bacterium SJ4 was characterized morphologically and biochemically as per the standard protocols of Bergey's Manual of Determinative Bacteriology (Whitman et al., 2012).

2.5.1. Effect of static and shaking condition The effect of static and shaking conditions on the degradation/decolourization of CV Dye was studied in an Erlenmeyer flask containing 167

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100 ml of MSM broth amended with filter sterilized 100 mg/l CV dye, inoculated with 1% (v/v) overnight grown culture of isolated bacterium and incubated under static and shaking conditions (110 rpm) at 35 °C for 8 h with parallel abiotic controls and the decolourization was monitored spectrophotometrically at a regular interval of 2 h and expressed in percentage as mentioned earlier (Shah et al., 2013b).

30 s, with 1 min of interval at 4 °C. The homogenate was centrifuged at 10,000 ×g for 20 min and supernatant obtained was used as crude enzyme. 2.6.2. Enzyme assays The laccase and lignin peroxidase activity was assayed spectrophotometrically at room temperature. E. coli ATCC 35218 was used as negative control and Pseudomonas aeruginosa MTCC 424 was used as a positive control in the experiments. The activity of laccase enzyme was measured by monitoring the absorbance of reaction mixture containing 100 µl of 0.1 M guaiacol dissolved in 100 mM Na-Acetate buffer at pH 5 and 10 µl of crude enzyme at 450 nm, which was proportional to laccase activity (Parshetti et al., 2011). The test tube showing brown color was considered as positive control. To determine the lignin peroxidase enzyme activity, the conversion of propanaldehyde was monitored at 300 nm for 30 min in a 100 µl of reaction mixture containing 100 mM of n-propanol, 250 mM of tartaric acid, 10 mM of H2O2 and 10 µl of enzyme extract (Azmi et al., 1998). An activity producing 1 µM product per min or consuming 1 µM substrate per min under the assay conditions has been defined as one enzyme unit (Mugdha and Usha, 2012).

2.5.2. Effect of physicochemical parameters The effect of various physicochemical parameters such as pH and temperature on the degradation/decolourization of CV dye was studied in an Erlenmeyer flask containing 100 ml of MSM broth amended with 100 mg/l filter sterilized CV dye, inoculated with 1% (v/v) overnight grown culture of isolated bacterium and incubated under shaking conditions (110 rpm) at different pH (5−9) and temperature (25–45 °C) for 8 h with parallel abiotic controls and the decolourization was monitored spectrophotometrically at a regular interval of 2 h and expressed in percentage as mentioned earlier (Shah et al., 2013b; Bharagava and Chandra, 2010b). 2.5.3. Effect of carbon and nitrogen sources To study the effect of different carbon and nitrogen sources on the degradation/decolourization of CV dye, the MSM broth amended with 100 mg/l filter sterilized CV dye was supplemented with different carbon sources like glucose, sucrose, starch, maltose, and fructose individually at a concentration of 0.1% and various organic and inorganic nitrogen sources such as yeast extract, peptone, urea, sodium nitrate and ammonium sulphate in another set was also added at a concentration of 0.5% each to MSM broth followed by inoculation with 1% (v/v) overnight grown culture of isolated bacterium and incubated at 35 °C under shaking conditions (110 rpm) for 8 h with parallel abiotic controls and the decolourization was monitored spectrophotometrically at a regular interval of 2 h and expressed in percentage as mentioned earlier (Shah et al., 2013b; Bharagava and Chandra, 2010b).

2.6.3. SDS-PAGE analysis The denaturing sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by using the Western Blotting Electrophoresis Unit (GX-SCZ2, Genetix Biotech Asia Pvt. Ltd) and 10% polyacrylamide in gels (Chandra et al., 2009; Bharagava et al., 2009). The samples were loaded in duplicate and concentration of enzyme was calculated by comparing with the standard curve at 595 nm absorbance. On completion of electrophoresis, the gel was subjected to coomassie brilliant blue R-250 staining followed by destaining of the gel. The protein bands obtained were correlated with enzymatic activity and the molecular weight of protein bands was estimated by comparing with standard protein marker (Molecular Standard Mixture Recombinant, 15–150 kDa, Sigma).

2.5.4. Effect of initial dye (CV) concentrations The effect of initial dye concentrations (200–1000 mg/l) on the degradation/decolourization of CV dye was also studied by amending the MSM broth with the increasing concentrations (200–1000 mg/l) of CV dye followed by inoculation with 1% (v/v) overnight grown culture of isolated bacterium and incubated at 35 °C under shaking flask conditions (110 rpm) for 8 h with parallel abiotic controls and the decolourization was monitored spectrophotometrically at a regular interval of 2 h and expressed in percentage as mentioned earlier (Shah et al., 2013b; Bharagava and Chandra, 2010b).

2.7. Metabolites characterization by FT-IR and GC-MS analysis 2.7.1. FT-IR analysis The degradation of CV dye was studied using FT-IR spectroscopy. The undegraded and degraded dye samples were mixed with pure KBr in 5–95 ratio to form a uniform pellet and then fixed in the sample holder to carry out the FT-IR analysis in the mid IR region of 400–4000 cm−1 (Bharagava et al., 2017). 2.7.2. Extraction of metabolites To extract the metabolites from bacteria treated samples, a portion of 200 ml of bacteria degraded samples was centrifuged at 5000 ×g for 10 min at 4 °C to separate bacterial biomass and other suspended particles. The supernatant obtained was vacuum evaporated at 40 °C to concentrate metabolites and reduce the volume upto 50%. Now, 100 ml aliquot was extracted thrice with the equal volume of ethyl acetate (Parshetti et al., 2011). The ethyl acetate extract was dried over anhydrous Na2SO4 and evaporated to dryness in a rotary evaporator. The dry residue obtained was dissolved in 1 ml of HPLC grade methanol and used for metabolites analysis (Parshetti et al., 2011).

2.5.5. Effect of repeated addition of (CV) dye aliquots Besides the initial dye concentrations, the effect of repeated addition of CV dye (100 mg/l) at each cycle was also studied and expressed in percentage as mentioned earlier (Shah et al., 2013b; Bharagava and Chandra, 2010b). 2.6. Enzyme assay for laccase and lignin peroxides activity and their characterization 2.6.1. Preparation of cell free extract To prepare cell free extract, the bacterial cells were inoculated in an 250 ml Erlenmeyer flasks containing 100 ml of sterilized MSM broth amended with 100 mg/l filter sterilized CV dye and pH 7. The flasks were incubated at 35 °C under shaking flask condition (110 rpm, LSI3016R, Labtech, India) for 8 h. After the incubation period, a sample of 2 ml was withdrawn from flasks and centrifuged at 10,000 ×g at 4 °C for 20 min. The culture supernatant obtained was directly used as extracellular enzymes in order to determine the enzymatic activity. In addition, the harvested cells were resuspended in potassium phosphate buffer at pH 7.4 and sonicated (Sonics-Vibracell Ultrasonic Processor, USA), keeping sonifier output at 40 (amps) and giving 7 strokes each of

2.7.3. GC-MS analysis The Gas Chromatography-Mass Spectrometry analysis (GC-MS/MS) of metabolites was carried out by using PerkinElmer (UK) equipped with a PE auto system XL gas chromatograph and a PE-5MS capillary column (20 m × 0.18 mm internal diameter, 0.18 mm film thickness). Helium gas was used as a carrier with a flow rate of 1 ml min−1 using split less injector (injector temperature was 280 °C). The column temperature was programmed as 50 °C (5 min); 50–300 °C (10 °C min−1, hold time: 5 min). The MS transfer line and ion source temperatures were kept at 200 and 250 °C, respectively. The MS was operated in full 168

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Fig. 1. Effect of different environmental (a: pH and b: temperature) and nutritional (c: carbon & nitrogen and d: sucrose concentration) parameters on degradation/decolorization of crystal violet dye.

toxicity effects were recorded in terms of percentage germination, radical length and phytotoxicity percentage after 3 days (Bharagava et al., 2017; Bharagava and Chandra, 2010a).

SCAN mode with the solvent delay of 3.0 min. In full-scan mode, the electron ionization (EI) mass spectra were recorded in a range of 30–550 (m/z units) at 70 eV (Parshetti et al., 2011; Chandra et al., 2011). The metabolites were identified by using the National Institute of Standards and Technology (NIST) library available with the instrument and by comparing the retention time (RT) and fragmentation pattern.

2.9. Statistical analysis All the experiments were performed in triplicates. The results obtained from each set of experiment were expressed in terms of mean and standard deviation.

2.8. Phytotoxicity study

3. Results and discussion

In order to determine the toxicity of untreated and bacteria treated CV dye, the dried ethyl acetate extracted metabolites of degraded CV dye was dissolved in sterile distilled water to make the final concentration of 500 mg/l. Since, pulses are the important crop of Indian agriculture and thus, the study was carried out by using the seeds of Phaseolus mungo at room temperature. Seedlings were raised in glass Petri plates (10 cm diameter). Surpass to sowing, the seeds were surface sterilized in 0.1% HgCl2 for 2 min and thoroughly washed with distilled water (Bharagava et al., 2008). Now, ten seeds of Phaseolus mungo were placed in glass Petri plates in between the filter papers and moistened with the equal volume (5 ml) of distilled water as control, CV dye (500 mg/l) and its degraded metabolites (500 mg/l) for 3 days. The

3.1. Isolated CV dye degrading bacterium In this study, initially ten bacterial strains (SJ1-SJ10) were isolated and screened based on their CV degradation and decolourization potential and finally, one bacterium (SJ4) showing the maximum degradation/decolourization of CV dye was selected for further studies. Further, based on the biochemical reactions, this bacterium was characterized as gram-negative, motile, rod-shaped with rounded ends having normal texture, even margin and flat elevation. This bacterium also showed positive reactions for catalase, oxidase, indole, casein 169

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Fig. 2. SDS-PAGE analysis of crude enzyme produced by SJ4 (Aeromonas hydrophila), PC (Positive control: Pseudomonas aeruginosa MTCC 424); NC (Negative control: E. coli ATCC 35218) (a) and Laccase and LiP production by Aeromonas hydrophila during the degradation and decolorization of crystal violet dye (b).

has taken place. The flasks having negative control strain showed no degradation/decolourization of crystal violet dye.

hydrolysis, glucose fermentation, starch hydrolysis and negative reactions for methyl red, voges-proskauer, citrate utilization, sucrose fermentation, lactose fermentation and hydrogen sulfide. In addition, the BLAST analysis of 16 S rRNA gene sequence of isolated bacterium has shown the closest relationship with that of Aeromonas hydrophila. The phylogenetic relationship of isolated bacterium with the other closely related bacterial strains found in GeneBank database as well as homology has also indicated that the most efficient bacterial strains capable of CV dye decolourization belongs to the phylogenetic branch of genus Aeromonas. Hence, based on the sequence similarity, the isolated bacterium SJ4 was identified as Aeromonas hydrophila with accession no. KU720586. Our findings are well corroborated with that of various studies, who reported that the genus Aeromonas is well known to have a strong ability to degrade/ decolourize CV dye (Kale and Thorat, 2014; Pan et al., 2013; Ren et al., 2006; Chen et al., 2003).

3.3. Effects of different optimized conditions on the degradation/ decolourization of CV dye 3.3.1. Effect of static and shaking conditions The decolourization efficiency of isolated bacterium i.e. Aeromonas hydrophila was evaluated under the static and shaking flasks (110 rpm) conditions at 35 °C and results showed that shaking/agitation favored the decolourization of CV dye resulting in about 95% decolourization within the 8 h of incubation period. But, under static conditions, only 45% decolourization was achieved within the same incubation period indicating that shaking has positive effect on the decolourization of CV dye. This higher decolourization of CV dye under shaking condition might be due to the proper mixing of oxygen in medium, increase in biomass and oxygen transfer between the cells and medium. Parshetti et al. (2011) have achieved 99% decolourization of CV dye (10 mg/l) under static conditions and Kurade et al. (2012) have recorded 98% decolourization of single dye Scarlet RR within 18 h under static conditions. Bouraie and El Din (2016) have observed only 76% decolourization of Reactive Black 5 dye under static condition and 56% decolourization under shaking condition.

3.2. Degradation and decolourization of CV dye by the isolated bacterium During the degradation/decolourization of CV dye, a marked increase in optical density (OD) for bacterial growth at 620 nm was observed indicating the fast growth of bacterium and results showed 93% color reduction without any externally provided conditions. Initially, the color reduction was only 0.76% thereafter, at 2, 4, 6 and finally 8 h, a continuous increase in color reduction was observed. Many authors like Pan et al. (2013), reported that Aeromonas hydrophila DN322p was capable for 99% (w/w) decolourization of crystal violet dye within 2.5 h under shaking condition at 30 °C for 50 mg/l of CV. Similarly, Ren et al. (2006) also observed more than 90% decolourization of triphenylmethane dyes such as crystal violet, basic fuchsin, brilliant green and malachite green at a concentration of 50 mg/l within 10 h by an Aeromonas hydrophila strain isolated from activated sludge of a textile printing wastewater treatment plant. But, our isolated bacterium was capable to decolourize 99% crystal violet dye at the concentration of 100 mg/l within 8 h of incubation time under optimized conditions and thus, we can say that this study is novel and important in compression to the previous studies made by various researchers. In addition, after decolourization process, the heat killed bacterial pellets collected were appeared to be non-colored indicating that biosorption has not been taken place, but bacterial degradation of CV dye

3.3.2. Effect of pH and temperature The pH of culture medium plays an important role in the growth, metabolic activity, production and activity of ligninolytic enzymes as well as degradation/detoxification of pollutants by the microorganisms. Hence, to study the effect of pH on CV dye decolourization by A. hydrophila, the initial pH of medium was adjusted within a range of 5–9. The results obtained indicated that A. hydrophila has the capability to decolorize CV dye efficiently within this range of pH 6–9. But, maximum decolourization (97%) was obtained at pH 7 (Fig. 1a), indicating that neutral conditions favor the bacterial activity for CV dye decolourization, whereas at pH 6, 8 and 9, the decolourization percentage was 85%, 95% and 90%, respectively. But, the percentage decolourization decreased sharply upto 9% at pH 5 due to acidic conditions. Mali et al. (1999) and Chang et al. (2000), also reported similar results on decolourization of dyes. Bouraie and El Din (2016) and Shah et al. (2013b) have recorded the maximum decolourization of Reactive Black 170

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Fig. 3. FT-IR analysis of undegraded (a) and degraded crystal violet dye sample (b).

at pH 8% and 95% decolourization at 35 °C and these findings are well supported by Wang et al. (2009).

dye at pH 7. Besides pH, the incubation temperature is also an important parameter that varies from microbe to microbe and a slight variation in it may affect the growth and enzyme activities of microbes. Therefore, the effect of incubation temperatures on decolourization of CV by A. hydrophila was also studied and it was found that the dye decolourizing activity of isolated bacterium decreased with the increase in incubation temperature above 35 °C (Fig. 1b). In this study, the maximum decolourization (97%) of CV dye was achieved at 35 °C and least decolourization (75%) at 25 °C. In addition, at 40 °C, the decolourization was 85% followed by 80% and 83% at 45 °C and 30 °C, respectively. Similarly, Shah et al. (2013a) have also recorded 90% decolourization

3.3.3. Effect of carbon and nitrogen sources In order to increase the decolourization efficiency of isolated bacterium, the modified MSM-CV amended medium was supplemented with different carbon sources such as glucose, sucrose, starch, maltose, and fructose at a concentration of 0.1% each. The maximum decolourization of CV dye obtained was 97.08% in presence of sucrose as an additional carbon source followed by glucose (90.56%), lactose (82.43%), maltose (74.12%) and starch (66.34%) as shown in Fig. 1c. The decolourization efficiency of A. hydrophila decreased in the absence 171

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Table 1 FT-IR analysis data of undegraded and degraded crystal violet dye by Aeromonas hydrophila. Wavelength (cm−1)

Expected group

1. 2. 3. 4.

1584.4 1360.6 1293.2 1168.5

5. 6. 7.

940.1 908.1 832.8

8. 9. 10.

755.7 721.9 616.8

11.

565.7

Pyridine derivatives, NH2 in amino acids, COO- in Carboxylic acid salts, NH2 primary alkyl amides COO- in Carboxylic acid salts, SO2 in sulfonyl chlorides, isopropyl group, NO2 in aromatic nitro compounds N+-O- in pyridine N-oxides, P = O in phosphorus oxyacids and phosphates, C-F in aliphatic fluoro compounds C-O-C in esters, lactones, SO3H in sulfonic acids, C-O-C in ethers, C-C-N in amines, SO2Cl in sulfonyl chlorides, C-OH in alcohols, SO2NH2 in sulfonamides, SO2 in sulfones P-O-C in organophosphorus compounds, CH=CH2 in vinyl compounds CH=CH2 in vinyl compounds 1,2,4-trisubst benzenes, R-NH2 primary amines, Si-C in organosilicon compounds, 1,2,5-trisubst benzenes, Si-CH3 in silanes, CH=CRR’ in trisubst alkenes, C-Cl in Chloro compounds 1,2,3-trisubst benzenes, C-S in sulfonyl chlorides, monosubst benzenes, o-disubst benzenes C-Cl alkyl chlorides -(CH2)n- in hydrocarbons, CH=CH in cis disubst alkenes Ar-OH in phenols, C-S in sulfides, O-C≡O in carboxylic acids, C˭C-H in alkynes, S-C≡N in thiocyanates, NO2 in aliphatic nitro compounds, C-Br in bromo compounds, napthalenes, O-C-O in esters, pyridines, N-C˭O in amides C˭O in amides, SO2 in sulfonyl chlorides, SO2 in sulfones, C-I in iodo compounds, C-C-CN in nitriles, NO2 in aromatic nitro compounds, ring in cycloalkanes, ring in benzene derivatives, SO2 in sulfonyl chlorides, C-CO-C in ketones C-O-C in ethers, NO2 in nitro compounds

S. No. Undegraded

12.

513.9 Degraded sample

1. 2. 3. 4.

3410.8 2959.1 2927.0 1651.2

5. 6. 7. 8.

1544.1 1452.7 1404.1 1238.3

9. 10.

1075.7 657.4

-NH2 in aromatic amines, primary amines and amides, -OH in alcohols and phenols -CH3 and –CH2- in aliphatic compounds -CH3 and –CH2- in aliphatic compounds C˭N in oximes, C˭O and NH2 in primary amides, C˭O in ureas, C˭C in alkenes,etc, C˭O in secondary amides, C˭O in benzophenones, C˭O in primary amides, C˭O in tertiary amides, C˭O in ß-ketone esters NH in secondary amides, triazine compounds, NO2 in aromatic nitro compounds N = N-O in azoxy compounds, CH2 in aliphatic compounds, CH3 in aliphatic compounds CH3 in aliphatic compounds, OH in carboxylic acids, C-N in primary amides N-O in pyridine N-Oxides, P = O in phosphorus oxyacids and phosphates, C-F in aliphatic fluoro compounds, Ar-O in alkyl aryl ethers, Si-CH3 in silanes, C-O-C in epoxides, C-N in aromatic amines, C-O-C in esters, lactones, t- butyl in hydrocarbons, SO3H in sulfonic acids, C-O-C in ethers C-O-C in aliphatic ethers, C-NH2 in primary aliphatic amines, Si-O-Si in siloxanes, SO3H in sulfonic acid C-Cl in Chloro compounds, C-S in sulfonyl chlorides, C-Cl alkyl chlorides, Ar-OH in phenols, C-S in sulfides, O-C˭O in carboxylic acids, C-C-CHO in aldehydes, C-OH in alcohols, C≡C-H in alkynes

textile industry wastewater varies from 60 to 250 mg/l. Therefore, in order to investigate the dye removal efficiency of isolated bacterium at optimized conditions, the degradation experiment was performed with the increasing concentrations of CV dye ranging from 200 to 1000 mg/l and it was observed that the isolated bacterium degraded/decolourized all the tested concentrations. However, at lower concentration (200 mg/l), the decolourization was 98%, but at higher concentrations (> 600 mg/l), the decolourization of CV dye were reduced significantly upto 35% after 24 h of incubation period. It revealed that the dye decolourization rate was maximum at 200 mg/l, but at higher concentration, CV greatly suppressed the decolourization ability of the isolated bacterium. It might be due to the toxicity of dye molecules on bacterial cells or the saturation of bacterial cells with dye products or blockage of active sites of azo reductase enzymes by the dye molecules (Sumathi and Manju, 2000; Sponza and Isik, 2004) Further, this study was also aimed to evaluate the degradation/decolourization efficiency of A. hydrophila under the repeated addition of 100 mg/l dye solution at optimized conditions. Results showed that A. hydrophila was capable to decolorize CV dye consecutively upto 10 cycles with the reduction in decolourization efficiency ranging from 99% to 35% for 1–10 cycles, respectively. In first cycle, 99% decolourization was observed within 8 h and then the successive addition of dye resulted in a faster rate of decolourization process till 6th cycle, but the decolourization efficiency of organism decreased and required more time to decolourize CV dye from 7th cycle to the last cycle. This might be due to the nutrient depletion in flasks that may lead to the less production of enzyme and reduction in growth of bacterium. Vijaya and Sandhya (2003) showed decolourization of azo dye methyl red only up to three cycles.

of carbon source suggesting the efficacy of external co-substrate in enhancing the degradation/ decolourization of CV dye. Gahlout et al. (2013) have observed 91–93% dye decolourization by using 2 g/l of mannose concentration. Parshetti et al. (2011), also reported similar results. On the other hand, in case of nitrogen sources, the presence of yeast extract has shown the maximum 95.30% decolourization of CV dye within the 8 h of incubation period followed by ammonium sulphate (87.26%), peptone (80.37%), sodium nitrate (65.34%) and urea (53.77%), respectively (Fig. 1c). Shah et al. (2013a) have also reported 90% decolourization of CV dye in presence of peptone as nitrogen source and Gahlout et al. (2013) has observed 96% dye decolourization in presence of yeast extract as nitrogen source. Mahmood et al. (2011) reported 75–100% decolourization of Remazol Black-B azo dye in presence of 4% yeast extract and Guo et al. (2008) showed similar results for K-2BP dye decolourization in presence of yeast extract or peptone as a nitrogen source. Further, the effect of different concentration of sucrose (1–5 g/ 100 ml) on dye decolourization was also studied and maximum decolourization 97.08% was obtained at 1 g/100 ml of sucrose and further increase in sucrose concentration resulted a decrease in dye decolourization (Fig. 1d). This decrease in % decolourization might be due to the acidic conditions or catabolite repression mechanism induced at high sucrose concentration, which leads to a negative effect on dye decolourization. These findings have indicated that the isolated bacterium was capable of 99% decolourization of CV dye at the optimized nutritional conditions i.e. 0.1% sucrose and 0.5% yeast extract and environmental conditions i.e. 35 °C at pH 7.

3.4. Effect of different concentrations of CV dye and repeated dye addition on decolourization at optimized conditions

3.5. CV degrading/decolourizing enzymes

According to Bhatt et al. (2005), the actual concentration of dyes in

The catalytic enzymes such as peroxidases and laccases present in 172

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microorganisms are only the agents to be responsible for the biodegradation of chemical pollutants (Ayed et al., 2010). The possible role of these enzymes in biodegradation of chemical pollutants has been well documented by many researchers (Jadhav et al., 2009; Telke et al., 2009). However, the enzymes extracted from Aeromonas hydrophila, Pseudomonas aeruginosa MTCC 424 (positive control) and E. coli ATCC 35218 (negative control) were separated by SDS-PAGE were treated for enzyme renaturation. During the enzyme profiling, Aeromonas hydrophila and Pseudomonas aeruginosa MTCC 424 have shown two protein bands corresponding to ~ 40 kDa and ~ 60 kDa with clear zone of dye decolourization suggesting the presence of positive lignin peroxidase and laccase enzyme activity, respectively whereas E. coli ATCC 35218 did not showed any kind of band. The molecular weight of lignin peroxidase and laccase enzyme was determined by comparing with that of standard protein marker (Molecular Standard Mixture Recombinant, 15–150 kDa; Sigma) (Fig. 2a). During the degradation of CV dye by isolated bacterium, enzyme lignin peroxidase was detected as major enzyme as compared to laccase at 8 h of incubation period at optimized conditions whereas Pseudomonas aeruginosa MTCC 424 used as positive control showed less lignin peroxidase and laccase enzyme activity in comparison to A. hydrophila. The enzyme activities shown by A. hydrophila were found to be associated with the degradation and decolourization of CV dye (Fig. 2b). Parshetti et al. (2011) have reported the similar results after 8 h of decolourization process. Yan et al. (2009) have reported the biodegradation mechanism of CV dye by laccase enzyme with a low molecular mass fraction (LMMF) extracted from white rot fungus Pleurotus ostreatus. Bumpus and Brock (1988) also observed the substantial degradation of CV dye by the non-ligninolytic cultures of Phanerochaete chrysosporium suggesting the existence of another mechanism for the degradation of CV dye. 3.6. Spectrophotometric analysis of CV dye degradation and decolourization The UV–visible spectrophotometric analysis of CV dye (control and degraded samples) was made at 590 nm to confirm the bacterial degradation/decolourization of CV dye. According to Chen et al. (2003), the dye removal can be associated with the biodegradation process, only if a major visible light absorbance peak completely disappears or a major peak appears. In this study, it was depicted that decolourization of dye is not only a visible decolourization, it may be substantially due to biodegradation. The FT-IR analysis of control/undegraded CV dye is compared with the degraded dye products and shown in Fig. 3 and also interpreted in Table 1. In case of control dye samples, the peak 1584.4 cm−1 obtained may be for -NH2 group in amino acids, -COO- in carboxylic acid salts, -NH2 in primary alkyl amides, peak 940.1 cm−1 can be for organophosphorus compounds, peak 832.8 cm−1 may be for 1,2,4-trisubst benzenes, R-NH2 primary amines, 1,2,5-trisubst benzenes, peak 755.7 cm−1 may be for 1,2,3-trisubst benzenes, monosubst benzenes, odisubst benzenes C-Cl alkyl chlorides and peak 616.8 cm−1 may be for Ar-OH in phenols, C-Br in bromo compounds, naphthalenes. The FT-IR spectra of degraded metabolites display a peak at 3410.8 cm−1, which may be for -NH2 group in aromatic amines, primary amines, and amides, -OH in alcohols and phenols, 1651.2 cm−1 peak can be for C˭O and NH2 in primary amides, C˭O in benzophenone, 1404.1 cm−1 peak may be for CH3 in aliphatic compounds, -OH group in carboxylic acids, C-N in primary amides and 657.4 cm−1 peak is interpreted to be C-OH in alcohols, C≡C-H in alkynes. But, the peaks at 832.8, 755.7 and 565.7 cm−1 become disappear indicating the loss of benzene derivatives after the degradation/decolourization of CV dye. Similar work has also been reported by Hemapriya and Vijayanand (2014) and Bouraie and El Din (2016). The identified intermediates such as Phenol, 2, 6-bis (1,1-dimethylethyl) (a), 2′,6′-Dihydroxyacetophenone (b) and Benzene (c) were

Fig. 4. Proposed degradation pathway of CV dye by the isolated bacterium Aeromonas hydrophila (KU720586).

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Table 2 Phytotoxicity of undegraded and degraded crystal violet dye metabolites. Experiment

Germination % 24 h

Control (tap water) Undegraded crystal violet dye Degraded crystal violet dye metabolites

76.67 ± 5.774 40.00 ± 10.00 56.67 ± 5.774

48 h 94.44 ± 5.774 43.33 ± 5.774 63.33 ± 5.774

Radicle length (avg. in cm)

Phytotoxicity %

4.122 0.744 2.378

– 81.941 ± 2.470 42.317 ± 0.467

72 h 94.44 ± 5.774 53.33 ± 11.547 83.33 ± 10.00

4. Conclusion

formed with the molecular weight 264.478 g/mol, 206.3239 g/mol, 152.149 g/mol with the corresponding mass spectrum at retention times (RT) 17.97, 10.36 and 7.72, respectively in comparison to control (i.e. crystal violet; 407.99 g/mol). Thus, based on the results obtained in this study, we proposed a hypothetical degradation pathway of crystal violet dye by A. hydrophila as shown in Fig. 4. Further, it has been also assumed that crystal violet dye may be first biotransformed into Phenol 2,6-bis (1,1-dimethylethyl) (product a), which might be transformed into 2′,6′-Dihydroxyacetophenone (product b) and finally the product (b) might have transformed into Benzene (i.e. product c). Many authors have also reported the conversion of crystal violet dye into colorless leuco-derivatives though various microorganisms (Yatome et al., 1991, 1993; Chen et al., 2008), but the literature on degradation of crystal violet dye by Aeromonas hydrophila has been less found. Yatome et al., (1991, 1993) and Chen et al. (2008) have reported Michler's Ketone as a major degradation product of crystal violet dye by Bacillus subtilis IFO 13719, Nocardia coralline and Shewanella decolorationis NTOU1, respectively. Parshetti et al. (2011) has reported phenol as the final degraded product of crystal violet, but we have found the derivative of phenol i.e. Phenol, 2, 6-bis (1,1-dimethylethyl). It is a colorless solid alkylated phenol, which along with its derivatives are used in industries as UV stabilizers and anti-oxidants for hydrocarbonbased products to prevent clogging in aviation fuels and most importantly, it has very low toxicity (LD50 9200 mg/kg). Besides it, 2′,6′dihydroxyacetophenone was also found along with other degradation products. It is an organic compound belongs to acetophenone family, and used in analysis of fragile peptides, disulphide bonding and small proteins (Gorman et al., 1996). However, benzene is the last product and is a clear, colorless and highly flammable liquid, which is used as a solvent in many commercials, industrials and research operations.

In the present study, the isolated CV degrading bacterium was found to be a gram-negative, motile, rod-shaped with rounded ends and identified as Aeromonas hydrophila based on the 16 S rRNA gene sequence analysis. The bacterium showed 93% decolourization of CV dye in absence of carbon and nitrogen sources, but at optimized conditions i.e. in presence of sucrose and yeast extract as C and N source, it decolourized 99% CV dye at pH 7 and temperature 35 °C within 8 h. During CV dye degradation, this bacterium produced ligninolytic enzymes i.e. lignin peroxidase and laccase that were characterized by the SDS-PAGE analysis and found to have ~ 40 and ~ 60 kDa molecular weight, respectively. Furthermore, the GC-MS analysis showed that during the degradation of CV dye, it was metabolized into Phenol, 2, 6bis (1,1-dimethylethyl), 2′,6′-dihydroxyacetophenone and benzene by the isolated bacterium. In addition, the toxicity of CV dye was also reduced upto a significant level as results showed that undegraded CV dye inhibited 60%, 56.67% and 46.67% seed germination, but after bacterial degradation/decolourization, it showed only 43.33%, 36.67% and 16.67% of inhibition in seed germination after 24, 48 and 72 h of incubation period, respectively. Thus, this study concluded that the isolated bacterium has good potential for the degradation/decolourization of CV dye as well to reduce its toxicity upto a significant level and can be applied at industry level for the effective treatment of textile wastewater for environmental safety. Acknowledgements The authors are highly thankful to the University Grants Commission (UGC), Government of India (GOI), New Delhi, India for providing RGNF Fellowship (F1–17.1/2014-15/RGNF-2014-15-SCUTT-67555) to Ms. Sujata for this work. Authors are also thankful to Dr. Gaurav Kaithwas, Associate Professor, Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow 226 025, India for providing MTCC and ATCC cultures.

3.7. Phytotoxicity of CV dye and its metabolites In this study, the toxicity of CV dye and its metabolites obtained after bacterial degradation has been studied and results are described in Table 2. It is well reported that CV dye has toxic effects on plants as it inhibits the seed germination and seedling growth and in animals, it causes cytotoxic and genotoxic effects (Mani and Bharagava, 2016). Results indicated that the undegraded CV dye inhibited 60%, 56.67% and 46.67% seed germination after 24, 48 and 72 h of incubation period, respectively. But, after the bacterial degradation/decolourization, the toxicity of CV dye was reduced significantly and showed only 43.33%, 36.67% and 16.67% inhibition in seed germination after 24, 48 and 72 h of incubation period, respectively. In addition to the inhibition in seed germination, both undegraded as well as degraded dye molecules also affected the seedling growth, but undegraded CV dye molecules affected the seedling growth more severely in comparison to the degraded dye molecules. Similarly, the percentage of phytotoxicity (i.e. 42.317%) was also found to be much reduced in plants treated with degraded dye molecules as compared to the undegraded CV dye (i.e. 81.941%). Thus, this study has shown that the isolated bacterium i.e. Aeromonas hydrophila has potential for the degradation/decolourization of CV dye as well to reduce its toxicity upto a significant level. Our findings are well supported by the earlier findings of Parshetti et al. (2011) and Littlefield et al. (1985).

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