Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill

Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill

Chemosphere xxx (2014) xxx–xxx Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Technica...

790KB Sizes 9 Downloads 208 Views

Chemosphere xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Technical Note

Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill Hilor Pathak a, Dhaval Soni a, Kishor Chauhan b,⇑ a

Department of Microbiology, P.D. Patel Institute of Applied Sciences, Charotar University of Science and Technology, Changa 388 421, Gujarat, India Department of Integrated Biotechnology, Ashok and Rita Patel Institute of Integrated Studies and Research in Biotechnology and Allied Sciences, Sardar Patel University, New VallabhVidyanagar 388 121, Gujarat, India b

h i g h l i g h t s  Morganella sp. HK-1 degraded Reactive Black B dye (20 g L

1

) within 24 h.

 This is the first report that describes the pathway for degradation of RB-B dye.  Degraded dye metabolites demonstrated non-phytotoxic and non-genotoxic behavior.

a r t i c l e

i n f o

Article history: Received 13 August 2013 Received in revised form 31 December 2013 Accepted 3 January 2014 Available online xxxx Keywords: Sulphonated reactive azo dye Morganella sp. Degradation pathway Phytotoxicity Genotoxicity

a b s t r a c t Reactive Black-B (RB-B) – one of the multi-sulphonated reactive azo dye – is being used extensively in textile as well as paper industries. Reactive azo dyes comprise of a significant group of synthetic compounds categorized as xenobiotics and its abatement from the environment still remains a challenge. In the present study, a newly isolated indigenous bacterial strain Morganella sp. HK-1 was exploited for its ability to decolorize and degrade RB-B dye. The isolate completely degraded RB-B (20 g L 1) within 24 h under static conditions. Furthermore, the visible and FTIR spectral analysis established the bio-degradation of RB-B. The degraded metabolites of RB-B by Morganella sp. HK-1 were identified by GC–MS analysis as disodium 3,4,6-triamino-5-hydroxynaphthalene-2,7-disulfonate, 4-aminophenylsulfonylethyl hydrogen sulfate, naphthalene-1-ol, aniline and benzene. Based on this information, a putative pathway of degradation of RB-B by Morganella sp. HK-1 has been proposed. This study is the first report on elucidation of mechanism of bacterial degradation of RB-B dye. Furthermore, phytotoxicity, genotoxicity and aquatic acute toxicity studies of the parent dye and the bio-degraded dye products revealed drastic reduction in the toxicity of metabolites as compared to the parent dye. This implies that the biotreatment of the dye is of non-toxic nature. This study thus indicates the effectiveness of Morganella sp. HK-1 for the treatment of textile effluents containing sulphonated azo dyes. Ó 2014 Published by Elsevier Ltd.

1. Introduction The annual global market of azo dyes is estimated to be around 1 Mt for more than about 10,000 structurally dissimilar azo dyes (Moosvi et al., 2007). About 0.3 Mt of different dyestuffs are used annually for textile dyeing operations. One of this is the sulphonated reactive azo dyes, which contain a chromophoric azo group whose nitrogen atoms are linked to sp2-hybridized carbon atoms of the aromatic ring and in addition carry sulphonic acid groups.

⇑ Corresponding author. Tel.: +91 2692 229189. E-mail addresses: [email protected] (H. Pathak), [email protected] aribas.edu.in (K. Chauhan).

Sulfo and azo groups are man-made compounds and are recalcitrant to degradation. The dyestuff, textile, paper, and leather industries release highly colored effluents that pose severe environmental hazards (Chang and Lin, 2001; Zhao and Hardin, 2007; Saratale et al., 2009; Pathak et al., 2011). It is estimated that 10–15% of the total dye be consumed in dyeing processes may found in effluents owing to the inefficiency of the dyeing process (Moosvi et al., 2007). Its removal from effluents is important due to their potential mutagenicity, carcinogenicity and intense coloration (Ghodake et al., 2009). Moreover, these dyes may significantly affect the photosynthetic activity of aquatic life due to reduced light penetration. Textile effluent treatment includes several physiochemical methods like filtration, specific coagulation, use of activated carbon,

0045-6535/$ - see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.chemosphere.2014.01.004

Please cite this article in press as: Pathak, H., et al. Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.004

2

H. Pathak et al. / Chemosphere xxx (2014) xxx–xxx

electron beam radiation and chemical flocculation (Pathak et al., 2011; Paul et al., 2011). However, these processes are cost as well as labor intensive and besides, less efficient (Forgacs et al., 2004; Zhang et al., 2004). Therefore, bioremediation provides a cheaper and eco-friendly option. The microorganisms have proven significant tools in the effluent treatment and a number of potential biotechnological approaches have been reported (Salokhe and Govindwar, 2003; Parikh and Madamwar, 2005; Saratale et al., 2007; Telke et al., 2008; Pathak et al., 2011; Paul et al., 2011). Although microbial systems have already been described for dye degradation, there is still a necessitation for research to discover the potential of various microbes for the amelioration of polluted ecosystems. Moreover, to enhance the dye degradation efficacy by micro-organisms, an understanding of the possible mechanisms underlying dye decolorization is essential. Further, the non-toxic behavior of the degraded metabolites also needs to be established. The present study emphasizes the decolorization and degradation of a sulphonated reactive azo dye – RB-B by indigenously isolated bacterial strain identified as Morganella sp. HK-1. Different parameters influencing dye degradation by Morganella sp. HK-1 were examined in vitro. The biodegradation products were analyzed using various analytical methods. Phytotoxicity as well as genotoxicity potential of the parent dye RB-B before and after degradation was also examined. The present study, therefore, holds a significant magnitude in understanding the existing in vitro biodegradation potential of Morganella sp. HK-1 for sulphonated reactive azo dye that can leave a foundation to explore its ability for in situ/ex situ bioremediation.

2. Materials and methods 2.1. Dyes Commercial grade RB-B (kmax 605 nm) was acquired from Bodal chemicals, Padra, Baroda, Gujarat, India. This dye has multi sulfonated complex structure comprising of naphthalene and benzene rings and one or more azo linkages.

2.2. Chemicals and culture medium All chemicals used in the study were of analytical grade. The culture media – Nutrient agar and Bushnell and Haas Mineral Broth (BHM) were procured from Hi Media (Mumbai, India). An indigenously isolated gram negative bacterium capable of degrading RB-B was isolated from dye contaminated industrial landfill at Baroda, Gujarat, India. The isolate HK-1 was identified, using 16S rRNA gene sequencing, as Morganella sp. The gene sequence has been submitted to NCBI with the Accession No. JX000462. For inoculum preparation, the strain HK-1 was routinely grown at 30 °C in BHM broth with glucose (0.35% w/v) and yeast extract (1.35% w/v) under static (anoxic) condition for 24 h. 10 mL of this was used as inoculum unless specified.

2.3. Azo reductase assay for RB-B decolorization by Morganella sp. HK-1 Azo reductase activity was measured according to the method described by Vijaykumar et al. (2007). The azo-reductase activity was determined spectrophotometrically at room temperature by monitoring the decrease in absorbance at 605 nm. One unit of enzyme activity was defined as the amount of enzyme that catalyses the conversion of 1 lmol of substrate min 1.

2.4. Effect of supplemental nitrogen sources, carbon sources and electron donors on RB-B decolorization by Morganella sp. HK-1 To study the effect of supplemental carbon (0.35% w/v) and nitrogen (1.35% w/v) sources on the decolorization ability of Morganella sp. HK-1, BHM broth was supplemented with seven different carbon sources (starch, fructose, lactose, maltose, mannitol, dextrose, sucrose) and eight different nitrogen sources (peptone, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium dihydrogen ortho phosphate, yeast extract, beef extract and casein hydrolysate) respectively. Various electron donors (succinic acid, sodium citrate, sodium acetate, sodium carbonate, sodium pyruvate and NADH) at concentration of 1 mM were tested for RB-B decolorization. Flasks were incubated under static conditions for 24 h at 30 °C and decolorization was measured from culture supernatants as described elsewhere (Pathak et al., 2011). 2.5. Continuous dye degradation of repeated RB-B inputs by Morganella sp. HK-1 Twenty-four hour old Morganella sp. HK-1 culture was inoculated (10%) in BHM broth spiked with 10 g L 1 RB-B as final concentration. Assessment of dye decolorization was performed after every 24 h (i.e. considered as one decolorization cycle) followed by addition of 10 g L 1 RB-B dye as final concentration in the same flask. Culture flasks were consecutively supplemented with 10 g L 1 RB-B dye for 12 such cycles. 2.6. Analysis of degradation products of RB-B by Morganella sp. HK-1 Following complete decolorization of RB-B by Morganella sp. HK-1, the culture broth was centrifuged at 6000g for 15 min to remove cells and the supernatant was extracted with equal volumes of ethyl acetate. The extract was dried over anhydrous Na2SO4 and was dissolved in methanol to be used for further analysis. The metabolic intermediates were investigated by a double beam UV– Visible spectrophotometer (Systronics 2203, India) and FTIR spectroscopy (NICOLET 6700, Thermo Scientific, USA). The metabolic intermediates were identified using Gas Chromatography coupled to Mass spectrometry (GC–MS) (Perkin–Elmer, USA). The mass spectra of the metabolic intermediate were analyzed using the NIST library. A pathway for degradation of RB-B by Morganella sp. HK-1 is proposed in the present study based on the information compiled from the above analysis as well as the azo reductase activity of the culture. 2.7. Toxicity studies of degraded metabolites 2.7.1. Phytotoxicity studies Phytotoxicity test was carried out as described by Telke et al. (2008) using Vigna radiata and Vigna aconitifolia seeds which are widely used in Indian agriculture. Germination (%), length of plumule (cm) and radical (cm) were documented after 7 d. 2.7.2. Genotoxicity studies The genotoxicity test was performed as described by Phugare et al. (2011). Briefly, the locally obtained onion bulbs of Allium cepa (diameter, 15–20 mm; weight, 5–10 g) were washed under running tap water. The loose outer scales were removed and the dry bases were scraped to expose the root primordials at a concentration of 1 g L 1 of RB-B dye and its degraded metabolites. The experiment was conducted with three bulbs per exposure at room temperature (30 ± 4 °C) under a 12 h light/dark cycle. The mean values for root length, mitotic index (MI – the ratio between the number of cells in mitosis and the total number of cells), cells in interphase and undergoing division, nuclear aberrations, such as

Please cite this article in press as: Pathak, H., et al. Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.004

3

H. Pathak et al. / Chemosphere xxx (2014) xxx–xxx

micronuclei, and multinucleated cells at each point were examined (Carita and Marin-Morales, 2008). 2.7.3. Aquatic acute toxicity test In order to evaluate the toxicity of parent dye RB-B as well as its degraded metabolites on invertebrates, a static bioassay was performed using the nematode, Melodogyne incognita. An acute toxicity test was performed according to the procedure of Donkin and Williams (1995). In order to estimate the toxicity on nematodes they were exposed to the parent dye and the degraded metabolites at a concentration of 5 mg L 1. After 24 h of exposure mortality rate was calculated. 3. Results and discussion

a 100 98

3.1. Azo reductase assay The major mechanism involved in the microbial biodegradation of reactive azo dyes is based on their bio-transformation by several enzymes (Saratale et al., 2009) with reductase as the key enzyme. The initial step involved in the biodegradation of azo dyes is the reductive cleavage of azo bond ([email protected]) with the azo reductase enzyme (Chang and Lin, 2001). The decolorization of azo dye by azo reductase activity was monitored over the time intervals of 4 h for 24 h. Fig. 1 depicts the decolorization of RB-B along with the respective azo reductase activity with time intervals for isolate Morganella sp. HK-1. The azo reductase activity was observed congruent to percentage dye decolorization. A similar inductive pattern of azo reductase was observed during decolorization of sulfonated azo dyes by Kerstersia sp. strain VKY1 (Vijaykumar et al., 2007) and Galactomyces geotrichum MTCC 1360 (Jadhav et al., 2010). From these results it is evident that the isolate Morganella sp. HK-1 decolorizes the RB-B dye by enzymatic reduction with azo reductase enzyme.

96 94 92 90 88 Peptone

Amm. Chloride

Yeast extract

Beef extract

Casein hydrolysate

b 99 98 97 96 95 94 93 92 91 sodium citrate

3.2. Effect of supplemental carbon sources, nitrogen sources and electron donors on RB-B decolorization by Morganella sp. HK-1

Fig. 1. Azo reductase assay for RB-B decolorization by Morganella sp. HK-1 at 30 °C and pH 7.0.

Ammonium Amm. Nitrate Phosphate

100

succinic acid

Azo dyes by themselves do not act as carbon and/or nitrogen source for the organism for their growth and consequently dye decolorization (Sani and Banerjee, 1999; Saratale et al., 2011). Thus, supplemental carbon and nitrogen sources are a pre-requisite to study dye decolorization efficacy of organisms. Among the carbon sources examined, dextrose was found to be the best allowing 98% decolorization of RB-B dye (Fig. 2a). The metabolism of glucose results in the production of reduced nucleotides (NADH, FADH) which lead to enhanced decolorization efficiency (Khehra

Amm. Sulfate

sodium sodium sodium acetate carbonate puruvate

NADH

control

c Fig. 2. Effect of (a) carbon sources, (b) nitrogen sources and (c) electron donors on dye decolorization by Morganella sp. HK-1 at 30 °C and pH 7.0.

Table 1 Repeated decolorization of Reactive Black B dye by Morganella sp. DH-1. Cycle no.

Decolorization (%)

1 2 3 4 5 6 7 8 9 10 11 12

96 98 98 97 97 97 96 96 95 94 92 89

et al., 2005; Jain et al., 2012). Moreover, reports are available on decolorization of certain textile dyes like Orange II, AO8 and AR88 by Sphingomonas sp. strain ICX exclusively when carbohydrate is present (Coughlin et al., 1997). The ability of the culture to decolorize the RB-B dye in the presence of different nitrogen sources was also examined. All the nitrogen sources tested showed >95% dye decolorization (Fig. 2b) except for ammonium nitrate and peptone where the efficiency

Please cite this article in press as: Pathak, H., et al. Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.004

4

H. Pathak et al. / Chemosphere xxx (2014) xxx–xxx

Fig. 3. FTIR analysis of parent dye (a) and degraded dye metabolites (b).

was lower (92% and 93% respectively). This indicates that Morganella sp. HK-1 can utilize not only the organic nitrogen sources but also the inorganic nitrogen sources equally efficiently for dye decolorization. The metabolism of organic and inorganic nitrogen sources is considered essential to the regeneration of NADH that acts as electron donor for the reduction of azo bonds (Pathak et al., 2011). To study the effect of electron donors on dye decolorization, several electron donors were employed at 1 mM concentrations. Sodium citrate, sodium acetate and sodium pyruvate improved dye decolorization by magnitude of 0.5–1% as compared to 98% in control (Fig. 2c). This improvement can be considered insignificant. Other electron donors did not improve dye decolorization than control. This can be explained as the NADH generated by glucose and yeast extract metabolism suffices the need of electron donor for reduction of azo bonds.

3.3. Repeated degradation of RB-B dye inputs by Morganella sp. HK-1 This study was conducted to investigate the efficacy of Morganella sp. HK-1 to combat repeated inputs of RB-B dye. Initial 10 g L 1 of RB-B dye was degraded near to 97% within 24 h at 30 °C under static condition. The organism sustained nearly 90% dye decolorization ability up to 12 repeated inputs of dye (Table 1). This ability of the organism proposes its potential application in continuous bioremediation of reactive dyes. 3.4. Analysis of RB-B dye degradation products The decolorized cell-free broth was processed and investigated for the degraded metabolites of RB-B dye by Morganella sp. HK-1 by UV–Visible scan, FTIR and GC–MS. The UV–Visible scan of RBB dye before and after biodegradation (Fig. SM-1 in Supplementary

Please cite this article in press as: Pathak, H., et al. Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.004

5

H. Pathak et al. / Chemosphere xxx (2014) xxx–xxx

O

O H S

O

N

N

OH

S

O

O

N

O

Reactive Black 5 M.W. 903

H NH 2 N

O

O S

S O

O

O O

O

S

O

O

OH

Azo reductase

O H S

O

O S

O H O

O

O

H

Reductive cleavage

NH 2

O 2N

H

O

O

S

H NH 2

n

Desulfonation O 2N

H O

S

O

Desulfonation

2N

OH

O

OH

O 2N

S

CH 3

S

H

O

NH2 H NH2

2, 8, 9 triamino naphthalene 1-ol M.W. 189, (m/z) 191

4(ethanesulfonyl) aniline M.W. 183, (m/z) 185

Deamination

Desulfonation

2N

OH

Naphthelene 1-ol M.W. 144, (m/z) 146

H

Aniline M.W. 93, (m/z) 93 Deamination

Benzene M.W. 78, m/z 78 Fig. 4. Proposed pathway for the degradation of RB-B dye by Morganella sp. HK-1.

material (SM)) depicted the disappearance of the peak at 605 nm implying that the RB-B dye has been degraded. To support this data, FTIR spectral scan of RB-B dye before and after biodegradation was performed (Fig. 3a and b). The FTIR spectrum of the parent dye (Fig. 3a) showed presence of peaks at 3445 cm 1 for ANH3 stretching of aromatic amine, 1593 cm 1 for [email protected] stretching of azo bond, 1494 cm 1 displayed aromatic [email protected] bending, 1132 cm 1 for ASO stretching of sulfide, 1046 cm 1 for ASO stretching of sulphonic acid. The FTIR spectrum of degraded metabolites (Fig. 3b) depicts presence of different peaks at 2929 cm 1 for ACH stretching of alkanes, 1717, 1699, 1668 cm 1 for [email protected] stretching of carboxylic acid, 1418 and 1384 cm 1 for ACH bending of alkanes, 1140 cm 1 for CAO stretching of carboxylic acid. Thus disappearance of different

characteristics of aromatic system and azo bond certify that Morganella sp. HK-1 degrades the dye efficiently. 3.5. Mechanism of RB-B dye degradation by Morganella sp. HK-1 To acquire further insights into the degradation mechanism of RB-B by Morganella sp. HK-1, GS–MS analysis of intermediate and degraded metabolites was performed. Most of the mechanisms for reactive azo dye degradation by microorganisms generally follow one of the two main routes for degradation i.e. either symmetrical or asymmetrical cleavage of azo bonds (Jadhav et al., 2010). Gas chromatogram of the degradation products showed peaks of four intermediates (data not shown). The structures of the detected compounds were assigned from the fragmentation pattern

Please cite this article in press as: Pathak, H., et al. Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.004

6

H. Pathak et al. / Chemosphere xxx (2014) xxx–xxx

Table 2 Toxicity studies of degraded metabolites of Reactive Black B dye by Morganella sp. DH-1. Parameter

Phytotoxicity studies Vigna radiata

Panel (A) Distilled water Parent dye Degraded metabolites

Parameters analyzed

Vigna aconitifolia

Germination (%)

Radical (cm)

Germination (%)

Radical (cm)

100 40 100

1.9 ± 0.3 0.7 ± 0.2 2.4 ± 0.4

100 30 100

1.7 ± 0.5 0.4 ± 0.2 2.0 ± 0.4

Genotoxicity studies of Allium cepa

Panel (B) RL MN MI TCA

Control (Distilled water)

Parent dye (1000 mg L

4.0 ± 0.5 0 2.20 2705

2.5 ± 0.4 9 22.90 2405

1

)

Extracted metabolites (1000 mg L

1

)

4.3 ± 0.3 2 2.24 2557

Aquatic acute toxicity test using Meladogyne incognita Nematode mortality (%) Concentration (mg L Panel (C) 1 3 5

1

)

Parent dye

Degraded metabolites

50 75 100

12.5 25 50

RL – root length (cm), MN – micronuclei, MI – mitotic index and TCA – total cells analyzed.

and m/z values (Table SM-1). The intermediates were identified as 2,8,9-triamino naphthalene 1-ol with molecular weight of 189 (peak at 191 m/z), 4-ethanesulfonyl aniline with molecular weight of 183 (peak at 183 m/z), naphthalene 1-ol with molecular weight of 144 (peak at 147 m/z), aniline with molecular weight of 93 (peak at 93 m/z) and benzene with molecular weight of 78 (peak at 78 m/ z). Based on the results of intermediate product analysis, we propose the degradation pathway of RB-B by Morganella sp. HK-1 (Fig. 4). The reductive cleavage by the azo reductase of Morganella sp. HK-1 attacks the azo bonds of RB-B dye and yields a naphthalene ring containing intermediate which upon desulfonation forms another intermediate (2,8,9 triamino naphthalene 1-ol) and a benzene ring intermediate (4-ethanesulfonyl aniline). Further deamination of these intermediates gives rise to benzene with mass peak (m/z 78) and naphthalene 1-ol with mass peak (m/z 145). Several studies depict that these products are further metabolized by gentisate pathway (Jain et al., 2012). Moreover, several reports on degradation mechanism of various reactive dyes like Reactive Red 141, Mentil Yellow, Reactive Red HE3B, Reactive Violet 5 are available (Telke et al., 2008; Phugare et al., 2011; Jain et al., 2012). However, till date there are sparse reports on aerobic degradation mechanism of RB-B dye by microorganisms. To the best of our knowledge, this is the first report on pathway elucidation for degradation of RB-B dye by Morganella sp. HK-1.

mination was obtained with its degradation metabolites. Pathak et al. (2011) showed the non-toxic behavior of degraded metabolites of a synthetic dye mixture comprising of four reactive azo dyes. Jadhav et al. (2010) also depicted the non-phytotoxic nature of the biodegraded metabolites of Reactive Orange 16 (RO16) on seeds of Triticum aestivum and Phaseolus mungo.

3.6. Toxicity studies

3.6.2. Genotoxicity studies A. cepa was used for in situ monitoring of the cytotoxicity and genotoxicity of the parent dye RB-B before and after treatment. The cytotoxicity level of a test compound can be determined based on the increase or decrease in the MI (Carita and Marin-Morales, 2008). The MI of cells exposed to undegraded RB-B dye was 22.90 whereas that of degraded metabolites was 2.24 which is almost equal to those exposed with distilled water (2.20) (Table 2part B). The decreased MI implies decreased proliferation rate of the cells exposed to RB-B dye thus indicating the cytotoxic behavior of the parent dye. The increased MI in case of degraded dye metabolites implies their non-cytotoxic behavior. A similar finding was observed by Phugare et al. (2011) with Reactive Red H3EB and its degraded metabolites. Furthermore, formation of micronuclei is another indicator of the genotoxic potential of the candidate under study (Carita and Marin-Morales, 2008; Phugare et al., 2011). A total of 9 micronuclei formation was observed with parent dye whereas a drastically decreased number of micronuclei formations was evident with the degraded metabolites (Table 2-part B). This again emphasizes the reduced genotoxicity of the degraded metabolites (Fig. SM-2).

3.6.1. Phytotoxicity studies Plant populations are of great importance as they possess commercial as well as agricultural significance and they may also be used as biosensors of genetic toxicity of the environmental pollutants. Table 2 (part A) depicts the phytotoxicity study using V. radiata and V. aconitifolia in presence of parent and degraded dye sample. The control set was performed using distilled water. RBB dye inhibited germination of the seeds by 60% whereas 100% ger-

3.6.3. Aquatic acute toxicity test using M. incognita Test was performed by monitoring the mortality rate of free living nematode M. incognita, exposed to different concentrations of parent and degraded RB-B dye by Morganella sp. HK-1 (Table 2-part C). The mortality rate of the nematodes decreased drastically (50%) with degraded metabolites of RB-B dye as compared to the parent dye indicating the degraded metabolites as non-toxic as compared to parent dye.

Please cite this article in press as: Pathak, H., et al. Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.004

H. Pathak et al. / Chemosphere xxx (2014) xxx–xxx

4. Conclusion The present bioremediation strategy demonstrates the potency of Morganella sp. HK-1 to effectively decolorize and degrade a multi sulphonated azo dye – RB-B. Morganella sp. HK-1 depicted the ability to retain 90% of its dye degradation ability even up to 12 consecutive fresh inputs of the dye. Based on the study of the intermediates of RB-B dye degradation by Morganella sp. HK-1, the degradation pathway of RB-B dye is proposed herein. The degraded metabolites were found to be non-phytotoxic and non-cytotoxic in nature. Appendix A. Supplementary material Supplementary material associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2014.01.004. References Carita, R., Marin-Morales, A., 2008. Induction of chromosome aberrations in the Allium cepa test system caused by the exposure of seeds to industrial effluents contaminated with azo dyes. Chemosphere 18, 311–316. Chang, J., Lin, C., 2001. Decolorization kinetics of a recombinant Escherichia coli strain harboring azo-dye-decolorizing determinants from Rhodococcus sp. Biotechnol. Lett. 23, 631–636. Coughlin, M., Kinkle, B., Tepper, A., Bishop, P., 1997. Characterization of azo dye degrading bacteria and their activity in biofilms. Water Sci. Technol. 36 (1), 215–220. Donkin, G., Williams, L., 1995. Influence of developmental stage, salts and food presence on various endpoints using Caenorhabditis elegans for aquatic toxicity testing. Environ. Toxicol. Chem. 14, 2139–2147. Forgacs, E., Cserhati, T., Oros, G., 2004. Removal of synthetic dyes from wastewaters: a review. Environ. Int. 30, 953–971. Ghodake, G., Telke, A., Jadhav, P., Govindwar, P., 2009. Potential of Brssica juncea in order to treat textile effluent contaminated sites. Int. J. Phytoremediat. 11, 297– 312. Jadhav, P., Kalyani, C., Telke, A., Phugare, S., Govindwar, P., 2010. Evaluation of the efficacy of a bacterial consortium for the removal of color, reduction of

7

heavy metals and toxicity from textile dye effluent. Bioresour. Technol. 101, 165–173. Jain, K., Shah, V., Chapla, D., Madamwar, D., 2012. Decolorization and degradation of azo dye – reactive violet 5R by an acclimatized indigenous bacterial mixed cultures-SB4 isolated from anthropogenic dye contaminated soil. J. Hazard. Mater. 213, 378–386. Khehra, S., Saini, S., Sharma, K., Chadha, S., Chimmi, S., 2005. Decolorization of various azo dyes by bacterial consortium. Dyes Pigm. 67, 55–61. Moosvi, S., Kher, X., Madamwar, D., 2007. Isolation, characterization and decolorization of textile dyes by a mixed bacterial consortium JW-2. Dyes Pigm. 74, 723–729. Parikh, A., Madamwar, D., 2005. Textile dye decolorization using cyanobacteria. Biotechnol. Lett. 27, 323–326. Pathak, H., Patel, S., Rathod, M., Chauhan, K., 2011. In vitro studies on degradation of synthetic dye mixture by Comamonas sp. VS-MH2 and evaluation of its efficacy using simulated microcosm. Bioresour. Technol. 102, 10391–10400. Paul, J., Rawat, P., Sarma, S., Sabharwal, S., 2011. Decoloration and degradation of Reactive Red-120 dye by electron beam irradiation in aqueous solution. Appl. Radiat. Isot. 69, 982–987. Phugare, S., Kalyani, D.C., Patil, A.V., Jadhav, J.P., 2011. Textile dye decolorization by bacterial consortium and subsequent toxicological analysis of dye and dye metabolites using cytotoxicity, genotoxicity and oxidative stress studies. J. Hazard. Mater. 186, 713–723. Salokhe, D., Govindwar, P., 2003. Effect of carbon source on the biotransformation enzymes in Serratia marcescens. World J. Microb. Biot. 15, 229–232. Sani, R., Banerjee, U., 1999. Decolorization of triphenylmethane dyes and textile and dyestuff effluent by Kurthia sp. Enzyme Microb. Technol. 24, 433–437. Saratale, D., Humnabadkar, P., Govindwar, P., 2007. Study of mixed function oxidase system in Aspergillus ochraceus (NCIM 1146). Indian J. Microbiol. 47, 304–309. Saratale, G., Saratale, D., Chang, S., Govindwar, P., 2009. Decolorization and biodegradation of textile dye Navy Blue HER by Trichosporon beigelii NCIM3326. J. Hazard. Mater. 166, 1421–1428. Saratale, R., Saratale, D., Chang, J., Govindwar, P., 2011. Bacterial decolorization and degradation of azo dyes: a review. J. Taiwan Inst. Chem. E 42, 138–157. Telke, A., Kalyani, D., Jadhav, J., Govindwar, S., 2008. Kinetics and mechanism of Reactive Red 141 degradation by a bacterial isolate Rhizobium radiobacter MTCC 8161. Acta Chim. Slov. 55, 320–329. Vijaykumar, H., Vaishampayan, A., Shouche, Y., Karegoudar, B., 2007. Decolorization of naphthalene-containing sulfonated azo dyes by Kerstersia sp. strain VKY1. Enzyme Microb. Technol. 40, 204–211. Zhang, F., Yediler, A., Liang, X., Kettrup, A., 2004. Effects of dye additives on the ozonation process and oxidation by-products: a comparative study using hydrolyzed CI Reactive Red 120. Dyes Pigm. 60, 1–7. Zhao, X., Hardin, R., 2007. HPLC and spectrophotometric analysis of biodegradation of azo dyes by Pleurotus ostreatus. Dyes Pigm. 73, 322–325.

Please cite this article in press as: Pathak, H., et al. Evaluation of in vitro efficacy for decolorization and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from dye contaminated industrial landfill. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.01.004