Co-metabolic degradation of diazo dye—Reactive blue 160 by enriched mixed cultures BDN

Journal of Hazardous Materials 279 (2014) 85–95

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Co-metabolic degradation of diazo dye—Reactive blue 160 by enriched mixed cultures BDN Kshama H. Balapure a , Kunal Jain b , Sananda Chattaraj b , Nikhil S. Bhatt a , Datta Madamwar b,∗ a

Post Graduate Department of Microbiology, Biogas Research and Extension Centre, Gujarat Vidyapeeth, Sadra 382320, Gujarat, India Environmental Genomics and Proteomics Lab, BRD School of Biosciences, Satellite Campus, Sardar Patel University, Vadtal Road, Post Box No. 39, Vallabh Vidyanagar 388120, Gujarat, India b

h i g h l i g h t s • • • •

Decolourization of RB160 requires co-substrate. BDN efficiently decolourized 1500 mg/L RB160 exhibiting broad substrate specificity. Complete degradation led to detoxification of RB160 assessed by phytotoxicity. BDN successfully colonized soil environment and decolourized RB160.

a r t i c l e

i n f o

Article history: Received 12 March 2014 Received in revised form 30 May 2014 Accepted 19 June 2014 Available online 2 July 2014 Keywords: Bioremediation Decolourization Co-substrate Microaerophilic Microcosms

a b s t r a c t Mixed cultures BDN (BDN) proficient in decolourizing diazo dye—reactive blue 160 (RB160) consist of eight bacterial strains, was developed through culture enrichment method from soil samples contaminated with anthropogenic activities. The synthrophic interactions of BDN have led to complete decolourization and degradation of RB160 (100 mg/L) within 4 h along with co-metabolism of yeast extract (0.5%) in minimal medium. BDN microaerophilicaly decolourized even 1500 mg/L of RB160 under high saline conditions (20 g/L NaCl) at 37 ◦ C and pH 7.0. BDN exhibited broad substrate specificity and decolourized 27 structurally different dyes. The reductase enzymes symmetrically cleaved RB160 and oxidative enzymes further metabolised the degraded products and five different intermediates were identified using FTIR, 1 HNMR and GC–MS. The phytotoxicity assay confirmed that intact RB160 was more toxic than dye degraded intermediates. The BDN was able to colonize and decolourized RB160 in soil model system in presence of indigenous miocroflora as well as in sterile soil without any amendment of additional nutrients, which signifies it useful and potential application in bioremediation. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Azo dyes (R1 N N R2 ) are known to be one of the oldest industrially synthesized organic chemicals [1]. These dyes are designed such that the vast numbers of substitution makes them highly versatile and environmentally stable. Also, substitution like sulphonic acid, imparts water solubility to the dye molecule and because of their persistent character they proved to be toxic. Dye manufacturing units, textile sectors and other dye consuming

∗ Corresponding author. Tel.: +91 2692 229380; fax: +91 2692 236475. E-mail addresses: [email protected] (K.H. Balapure), [email protected] (N.S. Bhatt), datta [email protected] (D. Madamwar). http://dx.doi.org/10.1016/j.jhazmat.2014.06.057 0304-3894/© 2014 Elsevier B.V. All rights reserved.

industries are on the major sources which pollutes environment [2]. However, some of the parent dyes are not directly toxic, but their precursors and/or biotransformed products (e.g. aromatic amines) have shown toxic, mutagenic or carcinogenic properties [1,3]. Thus the technology employed must be viable enough not only that decolourize dye but simultaneously it must completely degrade even intermediate compounds. The limited success and inherent disadvantages of physicochemical technologies have led to the development of selfsustained, environmentally acceptable bioremediation methods mainly through microbes. It offers a great advantage of astonishing catabolic diversity of innate microbial population inhabiting the polluted environment. Due to selective pressure of pollutants, microbial capacity for degrading recalcitrant xenobiotics is

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constantly evolving that can be harnessed for the removal of pollutants. The efficacy of bioremediation is highly dependent on establishing an effective acclimatized community and providing suitable environmental conditions that support the growth and activity of the enriched community. The competent community required to be characterized for their optimized need on environmental factors to enhance the bioremediation potential of the community. Generally, azo dye degradation is a two step process. The first step is reductive cleavage of the azo bonds under anaerobic or microaerophilic environment, followed by aerobic break down of corresponding aromatic amines (these amines are reported to be toxic and carcinogenic) [2,4–6] by a pure culture or mixed bacterial population leading to their complete mineralization [7]. Thus, the bacterial population employed in a dye effluent treatment system must be able to work under both anaerobic and aerobic conditions so as to achieve mineralization of azo compounds. In this work, we have demonstrated the efficiency of mixed cultures BDN (BDN) for its inherent ability to degrade diazo dye reactive blue 160 (RB160) and characterized the nutritional requirements and environmental parameters to enhance its degradation potential. The comprehensive study was carried out to understand the degradation profile of RB160 using ultraviolet–visible (UV–vis) and Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (1 HNMR) spectrometry and gas chromatography–mass spectrometry (GC–MS). The efficacy of dye decolourization by BDN and its potential use in bioremediation was assessed through soil microcosm and phytotoxicity studies.

2. Materials and methods 2.1. Dyes, chemicals and microbiological media Diazo dye reactive blue 160 (RB160) (C.I. No. 137160) (Fig. S1) is widely used in textile and accessories industries, thus selected as model azo compound in this study. RB160 is polyaromatic, multisulphonated, dichloro-triazene high molecular weight dye. RB160 and other dyes used in this study were procured from Ganesh Dye Industry, Vatva, Gujarat, India. All other chemicals and reagents used in the study were of analytical grade. Bushnell Haas medium (BHM) along with varying concentration of yeast extract (0.1–0.5%) amended with RB160 (100 mg/L) was used for development of bacterial mixed cultures.

2.2. Sampling, development and characterization of RB160 degrading mixed cultures BDN by culture enrichment method

Serially diluted enriched mixed cultures were spreaded on Luria Agar, R2A (Reasoner’s 2A), R3A and Bushnell Haas agar and incubated at 25 and 37 ◦ C for 1 to 8 days. Distinct colonies with unique morphology were further screened to pure culture to enumerate culturable bacteria present in the mixed cultures. Genomic DNA of all individual bacteria comprising RB160 degrading mixed cultures BDN (henceforth BND) was extracted following Ausubel et al. [8]. 16S rRNA gene was amplified using universal eubacterial primers 8F and 1492R as described by Desai and Madamwar [9]. The purified PCR product of each strain were differentiated using ARDRA and on the basis different and unique banding pattern, 1.5 kb product was sequenced by automated DNA Analyzer 3730 using ABI PRISMTM Terminator Cycle Sequencing v3.1 chemistry (Life Technologies, USA). 2.3. Inoculum development BDN was grown in 100 mL BHM amended with yeast extract (0.5%) and RB160 100 mg/L under microaerophilic conditions for 24 h. Four millilitre of grown BDN was harvested at 8000 × g for 10 min at 4 ◦ C. The obtained cell mass was resuspended in sterile 1 mL distilled water and used as an inoculum for optimization of nutritional and environmental parameters. 2.4. Decolourization assay and cell growth Aliquots of 2.0 mL were withdrawn from experimental and control medium at regular intervals of 2 h till 48 h (or until complete decolourization was observed) and cell mass was harvested at 8000 × g for 10 min at 4 ◦ C to obtain clear supernatant. RB160 decolourization was measured at max (610 nm) by UV–vis spectrophotometer (Elico, India) against media blank. Cell growth was monitored by resuspending the cell pellet in distilled water and measuring absorbance at 600 nm. Uninoculated media containing 100 mg/L of RB160 served as abiotic control. All experiments were performed in triplicates. 2.5. Study of nutritional and environmental parameters The influence of different co-substrates (carbon and nitrogen sources) on RB160 decolourization and degradation was studied to enhance dye decolourization ability of BDN. The effects of various environmental parameters on decolourization were optimized (pH (4–12), temperature (20–55 ◦ C), NaCl concentration (0–40 g/L) and RB160 concentration (100–1500 mg/L)). Potential of BDN to decolourize structurally different dyes (100 mg/L) was also studied and their decolourization was determined at respective absorbance maxima. 2.6. Enzymatic studies

Naroda Industrial Area, GIDC, Naroda near Ahmedabad, Gujarat, India harbours several dye and dye intermediate industries since last couple of decades. Thus it is a pertinent site to obtain well acclimatized bacterial community adapted for azo dye degradation. Sub-surface (8 cm below) soil samples were collected from polluted zones to isolate and to develop bacterial mixed cultures capable of degrading RB160. One gram of soil samples were added in BHM, supplemented with yeast extract (0.5%) and RB160 (100 mg/L) and incubated at 37 ◦ C under shaking (150 rpm) and microaerophilic (static) conditions. On demonstrating complete decolourization mixed cultures was repeatedly transferred into fresh medium [amended with RB160 (100 mg/L) and yeast extract (0.5%)] to acclimatize the mixed cultures in dye mineralization. With each consecutive transfer consistent decolourization and stable growth was observed.

2.6.1. Preparation of cell free extract BDN and its constituent pure cultures were grown in BHM amended with RB160 (100 mg/L) along with yeast extract (0.5%). Cell mass was harvested (regular time interval of 2 h) at 10,000 × g, 4 ◦ C, for 20 min and resuspended in 3 mL potassium phosphate buffer (50 mM, pH 6.8). Cells were lysed using ultrasonic probe (Sonics Vibracell, USA), while maintained at 4 ◦ C in ice bath, keeping sonicator output at 30% amplitude and 7 strokes each of 25 s with 1 min rest. Obtained homogenate was centrifuged at 10,000 × g, 4 ◦ C, for 20 min and supernatant was used as source of crude enzymes. 2.6.2. Enzyme assays Azo-reductase activity was measured by monitoring the decrease in concentration of methyl red at 440 nm as described

K.H. Balapure et al. / Journal of Hazardous Materials 279 (2014) 85–95

by Telke et al. [10] with few modifications. NADH–DCIP reductase and lignin peroxidase activity was determined as mentioned earlier [11,12]. Laccase activity was estimated using the method of Hatvani et al. [13]. Tyrosinase activity was measured using the method described by Zhang and Flurkey [14]. One unit of enzymatic activity was defined as change in absorbance U/min/mg of protein of the enzyme under ambient conditions [15]. However, procedures for each enzyme are described in details in supplementary information. Proteins were estimated by Lowry et al. [16] with bovine serum albumin as standard. All enzyme activities were assayed under ambient conditions and performed in triplicates.

Table 1 Experimental set up for microcosms study to access the competence of BDN for its ability to degrade RB160 in soil environment. No.

Experimental sets

Experimental parameter (RB160 100 mg/g)

1.

Set A

2.

Set B

3.

Set C

4.

Set D

5.

Set E

Sterile soil amended with RB160; to determine abiotic loss of dye Sterile soil amended with RB160 and inoculated with BDN, to determine dye degradation ability of BDN in absence of indigenous microflora Non sterile soil amended with RB160, to evaluate the intrinsic ability of soil to biodegrade the dye Non sterile soil amended with RB160 and inoculated with BDN, to check whether the strain accelerated dye degradation while competing with indigenous microorganisms Sterile soil (without dye) inoculated with BDN, to determine the ability of BDN to grow on the soil nutrients and colonize there in

2.7. Biodegradation studies BDN was gown in presence of RB160 (100 mg/L) in BHM along with yeast extract (0.5%) under microaerophilic condition at 37 ◦ C. It was harvested at 10,000 × g for 20 min after 0, 4 and 12 h to obtained cell free supernatant. RB160 degraded intermediates were extracted using equal volumes of ethyl acetate and dried in a SpeedVac (Thermo Electron Corporation, Waltman, MA). These extracted metabolites were used for HPTLC, FTIR, 1 HNMR and GC–MS analysis. High performance thin layer chromatography (HPTLC) was performed using 2-butanol:1-propanol:ethyl acetate:water (2:8:4:6, v/v) as a mobile phase. Spots were observed under 254 nm. FTIR analysis was carried out using Perkin Elmer, Spectrum GX spectrophotometer in the mid infrared region of 400–4000 cm−1 with 16-scan speed. Further 1 HNMR studied using 13 C NMR-400 MHz (Bruker, USA). GC–MS analysis of metabolites was performed using Auto-system XL (Perkin Elmer, USA) as mentioned in previous study [17]. However, procedures for 1 HNMR and GC–MS are described in details in supplementary information.

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conditions. Percentage dye decolorization and CFU per gram of dry soil were monitored after 12 h of incubation. 2.9.2. Dye extraction, analysis and quantification Fifty milliliters of distilled water was added into soil and kept on shaker (150 rpm) for 30 min. The supernatant was centrifuged at 12,000 × g for 15 min. For quantification of the residual dye, the absorbance of the supernatant was measured at 610 nm using UV–vis spectrophotometer (Elico, India) and the number of colony forming units (CFU) was counted per gram of dry soil. 2.10. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) with Tukey–Kramer multiple comparisons test.

2.8. Phytotoxicity studies

3. Results and discussion

Phytotoxicity experiments were performed as described by Phugare et al. [18] using Phaseolus mungo and Triticum aestivum. The seeds of both the plants was irrigated separately by providing 10 mL of RB160 (100 mg/L) and its degraded products daily for 8 days. Simultaneously a separate set of experiment was carried out where 10 mL of distilled water was sprinkled on both the seeds. Both the sets were incubated under ambient conditions and percent germination, length of the plumule and radical was recorded for 8 days.

The omnipresence of bacteria has established their versatility and ability to colonize under diverse and extreme environment. The evolutionary process through selective pressure have given bacteria an enormous catabolic potentiality, which now enable them to utilized and degrade recalcitrant xenobiotic compounds through synergism. Thus, this study has been designed to exploit the catabolic diversity of attenuated bacteria for dye degradation. 3.1. Development and characterization of BDN

2.9. Bioremediation of RB160 using simulated microcosm The soil model system was prepared from agricultural soil near Sadra, Gandhinagar, Gujarat, India. The soil pH was near neutral. Overnight grown BDN was inoculated in five different sets of soil amended with RB160 (100 mg/g) and dye decolorization was observed at different intervals. Cell growth was monitored by standard plate count and expressed as CFU/g of soil. 2.9.1. Simulated microcosm studies Simulated microcosms to assess the capability of BDN to decolourize dyes in soil system were studied as mentioned by Pathak et al. [19]. Simulated microcosms, each containing 50 g of soil were established in 200 mL glass beakers. Five distinct experiments were set up in triplicates as mentioned in Table 1. Soil was sterilized (for Set A and Set B) at 121 ◦ C for 60 min. Water was sprinkled up to the 40% moisture level. After inoculation (4%, w/v) the soil was thoroughly homogenized and incubated under ambient

BDN was developed through enrichment culture method from dye contaminated soil samples demonstrating consistent growth and dye decolourization at each successive transfer in fresh medium. BDN comprised of eight distinct bacterial strains namely Alcaligenes sp. BDN1 (NCBI Accession No. KF500593), Bacillus sp. BDN2 (KF500594), Escherichia sp. BDN3 (KF500595), Pseudomonas sp. BDN4 (KF500596), Provedencia sp. BDN5 (KF500597), Acinetobacter sp. BDN6 (KF500598), Bacillus sp. BDN7 (KF500599) and Bacillus sp. BDN8 (KF500600) representing three bacterial classes ␤-proteobacteria, Bacilli and ␥-proteobacteria respectively. Fig. S2 describes their taxonomic relationship. These groups of bacteria were previously described to possess dye degradation ability in several studies [20,21]. 3.2. Nutritional parameters The main rationale of dye bioremediation employing microorganisms is to use dye as a carbon (even sole carbon source) and

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No Carbon Rahmnose Lactose

a

Glucose Fructose Xylose

Sucrose Cellulose Galactose

Starch Maltose

100 90

Decolourization (%)

80 70 60 50 40 30 20 10 0 0

2

4

6

8

10

12

14

16

18

20

22

24

Time (h) No Nitrogen Yeast Extract Malt Extract Potass ium Nitrate

b 100

Tryptone Beef Extract Urea Ammonium Molybdate

Peptone Gelatin Ammonium Chloride

90

Decolourization (%)

80 70 60 50 40 30 20 10 0 0

2

4

6

8

10

12 14 Time (h)

16

18

20

22

24

Fig. 1. Effect of (a) carbon sources and (b) nitrogen sources (co-substrates) on decolourization of RB160 (100 mg/L) by BDN in minimal medium under microaerophilic condition at 37 ◦ C, pH 7.0.

nitrogen source. However azo dyes are considered as carbon and electron deficient compound and functional groups like SO3 generates electron deficiency due to electron-withdrawing capacity, which makes dye less susceptible to degradation [22]. Therefore, additional source of co-substrate which provide carbon as well as energy source can sustain the growth to support decolourization. Moreover, by replenishing the nitrogen it always found to enhance the decolourization potential. In this study, the similar phenomena was observed, where decolourization of RB160 by BDN was negligible in minimal medium and only 17% dye was decolourized within 24 h. (Fig. 1a). But on providing nitrogenous compound (yeast extract) complete decolourization was observed within 4 h, i.e. there was 20 fold

increase in decolourization efficiency of BDN (Fig. 1b). Thus, initial study revealed that decolourization of RB160 was dependent on cosubstrate. Although previous studies by Seesuriyachan et al. [23] and Silveira et al. [24] observed that carbon sources did enhance the degradation potential of bacteria; however, they does not influence decolourization in our study. However obligatory requirement of nitrogenous compounds in dye metabolism was well reported [5]. On providing sucrose, glucose, starch and fructose 71, 38, 31 and 26% RB160 was decolorized within 4 h, respectively, but with increase in incubation time (to 24 h) above 78% decolourization was observed (Fig. 1a). With other carbon sources, above 50% decolourization was obtained but time required was higher as

K.H. Balapure et al. / Journal of Hazardous Materials 279 (2014) 85–95

Decolorization (Microaerophilic)

Decolorization (Shaking)

Growth (Microaerophilic)

Growth (Shaking) 1

90

0.9

80

0.8

70

0.7

60

0.6

50

0.5

40

0.4

30

0.3

20

0.2

10

0.1

0

A600

Decolourization (%)

100

0 0

2

4

6

10

12

14

16

18

20

22

24

Time (h)

Fig. 2. Effect of aeration and microaerophilic conditions on decolourization of RB160 (100 mg/L) by BDN (and its growth) in minimal medium at 37 ◦ C, pH 7.0.

compared to yeast extract. Similarly, other sources of organic nitrogenous compounds were assessed and it was observed that tryptone and peptone supported 22 and 38% dye decolourization within 4 h, respectively (Fig. 1b). As observed with carbon sources, with increase in time, decolourization above 50% was obtained except ammonium salts of chloride and molybdate. Thus inorganic nitrogen compounds were found to be poor source to support decolourization. Kapdan et al. [25] during their study on mixed bacterial consortium PDW reported that yeast extract can provide carbon and nitrogen sources simultaneously, required for dye decolourization. 3.3. Environmental parameters The efficiency of bioremediation is greatly influenced by operational parameters. The level of oxygen, temperature and pH must be optimized for better dye reduction. Besides physical factors, composition of textile wastewater varies in terms of salts, dye concentration as well as range of different dyes [26]. Thus effect of each factor must be studied before BDN can be used to treat industrial effluent. 3.3.1. Effect of oxygen, pH and temperature Molecular oxygen has been found to inhibit dye decolourization. This perception is been well supported in present study, results in Fig. 2, clearly demonstrate that oxygen has inhibitory effect on RB160 decolourization. Under oxygen rich condition 40% decolourization was observed within 24 h (24% within 4 h), whereas more than 90% decolourization was achieved within 4 h under microaerophilic condition. Thus, decolourization rate increases nearly 4.1 fold under microaerophilic environment. Furthermore, as expected, growth rate of BDN was relatively faster under aeration as compared to oxygen limiting (microaerophilic) environment. In presence of oxygen, electrons required for dye reduction are preferentially being consumed by oxygen because of its high redox potential impeding dye decolorization. Yeh and Chang [27] during their study on Escherichia coli NO3 and E. coli CY1 (harbouring decolourizing gene from Rhodococcus sp.) observed that decolourization of reactive black B was inhibited at DO level higher than 0.35 mg/dm3 , i.e. complete decolourization of dye was observed under microaerophilic conditions within 60 h.

89

The maximum decolourization of RB160 was observed at pH 7.0 and 37 ◦ C under microaerophilic condition and nearly complete decolourization was obtained within 4 h. Results in Fig. 3a indicated that decolourization rate increased by 10.3 fold as the pH was raised from 5.0 to 7.0 and nearly 1.4 fold when temperature was increased from 25 to 37 ◦ C. With increase in pH it was observed that nearly 86 and 76% of RB160 was decolourized at pH 8.0 and 9.0, respectively. Moreover, at higher pH (11.0) decolourization efficiency of BDN decreased and 61% dye was decolourized within 24 h. Chang et al. [28] observed the similar increase in decolourization efficiency (2.5 fold) of Pseudomonas luteola by raising pH from 5.0 to 7.0. It was observed that reactive red 3B-A was optimally decolourized by Clostridium bifermentans SL186 at alkaline pH (10.0) [29]. Similarly, at lower temperature of 20 ◦ C, 64% dye was decolourized and at higher temperature of 50 ◦ C, above 57% RB160 was decolourized (Fig. 3b). Thus, it was inferred that, though maximum dye decolourization often corresponds with the optimum cell growth conditions, BDN were well adapted to wide range of environmental conditions for efficient dye decolourization. 3.3.2. Effect of salt (NaCl), dye concentration and spectrum of dyes Different salts are essentially required in both dye manufacturing and consuming industries and generally industrial effluent with reactive dyes contains salt concentration as high as 60–100 g/L [30]. Likewise, it was observed that when sodium hydroxide are used to increase the dyeing efficiency, pH of the dye bath can be as high as 10, which results in elevated level of sodium in wastewater [31]. Therefore, bacterial system employed for dye remediation must be able to tolerate such high salt concentration and they should be metabolically active. Results from Fig. 4a indicated that RB160 was proficiently been decolourized by BDN in presence of 2 g/L NaCl, within 6 h. Even at 8 g/L salt concentration above 90% decolourization was observed within 12 h (Fig. 4a). Generally it was observed that above 3 g/L sodium concentration bacterial cell activities decrease, which may affect dye decolourization [32]. But, these results evidently indicates that with further increase in incubation, 90 and 50% of RB160 was decolourized even at 20 g/L NaCl within 18 and 24 h, respectively (Fig. 4a). Thus, BDN were able to tolerate and have acclimatized for efficient dye decolourization under high saline environment. Dye concentration has inversely influenced the decolourization rate. The study showed that 50 mg/L of diazo dye RB160 was effortlessly been decolourized in less than 2 h whereas 200 mg/L required not more than 7 h. Conversely, after an initially rapid decolourization, rate of colour removal decreases at higher concentrations. As observed from Fig. 4b, when dye concentration was increased to 800 mg/L, 90% dye was decolourized within 16 h and that for 1500 mg/L, 87% of RB160 was decolourized within 24 h. The decrease in the efficiency of colour removal may be attributed to the toxicity of dye and dye metabolites that were formed during dye metabolism at higher concentrations [26]. The study further demonstrated that BDN efficiently decolourize repeated addition of 100 mg/L RB160 up to 6 cycles without supplementing fresh nutrient (Fig. S3). Decolourization efficiency was unaltered for the first three successive cycles, but with accumulation of degraded intermediates, depletion of nutrients and cell mass, decolourization efficacy decreases and nearly 70% dye was decolourized at 6th cycle and reduced sharply by 7th cycle. Industrial effluents of both textile and dye processing units are complex mixture of various structurally distinct dyes of synthetic origin. BDN was therefore assessed for its inherent potential to decolourize different dyes other than RB160. Fig. S4 revealed

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a 4

5

6

7

8

9

10

11

12

100

Decolourization (%)

90 80 70 60 50 40 30 20 10 0 0

2

4

6

8

10

12

14

16

18

20

22

24

Time (h)

b 20°C

25°C

30°C

37°C

45°C

50°C

55°C

100

Decolourization (%)

90 80 70 60 50 40 30 20 10 0 0

2

4

6

8

10

12

14

16

18

20

22

24

Time (h) Fig. 3. Effect of initial (a) pH and (b) temperature on RB160 decolourization by BDN in minimal medium under microaerophlic condition.

that more than 80% decolourization was observed in twenty three different dyes used in the study. Amongst them reactive red M8B, reactive red BS, reactive blue 160, reactive blue 222, reactive violet 5R, reactive golden yellow RNL and food colour red was decolourized ≥90%. The variation in efficiency of decolourization may be influenced by chemical nature and type and position of substitutions present on dye compound [33]. 3.4. Enzymatic study Degradation of recalcitrant compounds through microbial systems is greatly influenced by oxidoreductive enzymes [34,35]. In particular, azo dyes are mainly reduced via azoreductases generating corresponding aromatic amines. Subsequently, these amines generate highly reactive free radicals via action of oxidative enzymes which undergo complex series of spontaneous/enzyme

induced cleavage to form lower molecular weight compounds that may enter central metabolism pathways. As it can be observed from Table 2, higher activities of azoreductases and NADP-DCIP reductases was measured up to complete decolourization of RB160 from BDN. But, oxidative enzymes like ligning peroxidase, laccase and tyrosinase exhibited comparatively lower enzymatic activities. The observed results indicated that reduction of azo bonds were predominantly carried out by strains BDN4, BDN7, BDN5 and BDN6 since they exhibited higher azoreductase activity compared to remaining five strains. Strains BDN6, BDN8, BDN5 and BDN1 demonstrated higher lignin peroxidase activity, while BDN6, BDN5 and BDN1 exhibited higher laccase activity. Thus reduced dye products were further detoxified by the collective action of oxidative enzymes where each bacterium of BDN playing their significant role in dye mineralization.

K.H. Balapure et al. / Journal of Hazardous Materials 279 (2014) 85–95

a

No Salt 8 g/L 30 g/L

1 g/L 10g/L 35 g/L

2 g/L 15 g/L 40g/L

91

4 g/L 20 g/L

6 g/L 25 g/L

100 90

Decolourization (%)

80 70 60 50 40 30 20 10 0 0

2

4

6

8

10

12

14

16

18

20

22

24

Time (h)

b

50 mg/L 400 mg/L 1000 mg/L

100 mg/L 600 mg/L 1200 mg/L

200 mg/L 800 mg/L 1500 mg/L

100 90

Decolourization (%)

80 70 60 50 40 30 20 10 0 2

0

4

6

8

10

12

14

16

18

20

22

24

Time (h) Fig. 4. Effect of initial (a) NaCl and (b) RB160 concentrations on dye decolourization by BDN in minimal medium under microaerophlic condition at 37 ◦ C, pH 7.0.

Table 2 Oxido-reductive enzyme profile of consortium BDN and its constituent strains after 12 h incubation of 100 mg/L RB160. Enzymes

BDN1

BDN2

Lignin peroxidasea Laccasea Tyrosinasea Azoreductaseb NADH–DCIPc

0.11** 0.07* ND 0.4*** 2.6***

± 0.02 0.05** ± 0.05 0.02* 0.01* ± 0.1 0.3*** ± 2.1 1.5***

BDN3 ± ± ± ± ±

BDN4

0.04 0.06** ± 0.08 0.03** 0.1 0.001* ± 0.1 0.03* 0.002 0.02* ± 0.001 0.02* 0.4 0.07*** ± 0.18 5.2*** 3.2 0.9*** ± 2.8 12.7***

BDN5 ± ± ± ± ±

0.03 0.09 0.004 0.1 4.2

0.12** 0.08* 0.01* 2.2*** 8.1***

Values are mean of three experiments ± SEM. Significantly different from control cells at comparison test. a Enzyme U/min mg protein. b ␮M Methyl red reduced/min/mg protein. c ␮g DCIP reduced/min/mg protein, ND = not detected.

*

BDN6 ± ± ± ± ±

0.26** 0.09* ND 1.2*** 2.8***

0.09 0.01 0.002 0.08 3.8

P < 0.05,

**

BDN7 ± 0.05 ± 0.02 ± 0.31 ± 2.7

P < 0.01 and

***

0.07** 0.01* ND 3.7*** 5.4***

BDN8 ± 0.08 ± 0.16 ± 0.5 ± 4.4

0.15** 0.02* 0.04* 0.1*** 1.2***

Consortium BDN ± ± ± ± ±

0.03 0.85** 0.11 0.06* 0.005 0.1** 0.6 15.7*** 3.8 34.5***

± ± ± ± ±

0.02 0.03 0.02 0.4 2.9

P < 0.001 by one way ANOVA with Tukey–Kramer

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Fig. 5. UV–visible overlay spectra of RB160 and its ethyl extracted degraded products.

3.5. Biodegradation analysis Decolourization and degradation profile of RB160 was studied using UV–vis and FTIR spectroscopy, HPTLC, 1 HNMR and GC–MS. UV–vis spectral analysis (400–800 nm) of pure dye produce broad and intense peak at 610 nm (max ) (Fig. 5). During decolourization, intensity of peak at 610 nm gradually decreased and finally disappeared up on complete decolourization within 4 h without any shift in max . Dye degradation pattern was analysed by FTIR spectroscopy at mid-IR fingerprinting region (400–4000 cm−1 ). The intact RB160 exhibited characteristic peaks of various functional substitutions. Azo groups of RB160 showed absorption of stretching vibrations of azo bonds ( N N ) near 1625 cm−1 . Peaks at 1186 and 1396 cm−1 indicated the presence of stretching and asymmetric vibrations of S O, respectively (Fig. S5). NH stretching vibrations exhibited peak at 3434 cm−1 , while C N stretching at 1082 cm−1 and C H stretching vibrations in CH3 and CH2 groups at 2955 cm−1 . The aromatic nature of RB160 was confirmed by multiple peaks between 900 and 675 cm−1 . The cleavage of azo bonds is essential and foremost step that mark the beginning of dye degradation. Peak at 1625 cm−1 gradually disappeared (after 4 h) to indicate that RB160 decolourization was due to azo bond breakdown. At the same time number of peaks at aromatic region decreases indicating the opening of aromatic rings (Fig. S6). While, peaks at 3417 and 1104 cm−1 showed NH stretching of aromatic amides suggested the formation of primary and secondary amines. The absolute loss of aromatic nature was confirmed by the absence of corresponding peaks between 900 and 675 cm−1 after 12 h (Fig S7). Further peak at 2925 cm−1 represented the asymmetric stretching of alkenes, indicating the complete breakdown of RB160. Moreover, disappearance of peaks at 1330 and 1186 cm−1 signified the cleavage of S O bonds from SO3 groups. These results evidently suggested that reductases catalyzed the reductive cleavage of azo bonds of RB160 and further degradation was facilitated by oxidative enzymes. Above results were further supported by HPTLC analysis of degraded products at different time intervals (i.e. 4 and 12 h). HPTLC chromatogram of 4 h extracted samples showed six distinct bands on TLC plates with Rf values corresponds to 0.06, 0.16, 0.42, 0.47, 0.55 and 0.65 (data not shown), whereas number of bands from 12 h samples decreased to two (Rf 0.12 and 0.28). Thus, RB160 decolourization, formation of intermediates and their breakdown were clearly observed by IR spectroscopy and HPTLC analysis. RB160 and its degraded products were also analyzed using 1 HNMR spectroscopy. Intact dye molecule showed downshift

signals of aromatic protons between ı 6.8 and 7.4 of high intensity and signals between ı 3.0 and 4.0 corresponds to C Cl confirming the presence of halide moiety on RB160 (Fig. 6a). During dye degradation several intermediatory products have formed along with aromatic nucleus showing signals between ı 6.8 and 8.5, but their intensities were much lower. Signals of lower intensity in the range of ı 5.3 to 6.2 correspond to protons from alkenes formed during breakdown of benzene rings (Fig. 6b). The absence of any detectable signals in aromatic region of NMR spectrum (of 12 h) evidently proved that all benzene rings were metabolized by BDN (Fig. 6c). During breakdown formation of lower molecular weight aliphatic hydrocarbons were detected at multiple signals of lower intensities in the region between ı 1.0 and 3.0. The above analysis of dye and its degraded products indicated that RB160 was symmetrically cleaved into lower molecular weight intermediates. The GC–MS behaviour of the same samples evidently supported the symmetric cleavage of azo bonds leading to the formation of 1-phenylmethanediamine having mass peak (m/z) of 121 (data not shown). The catalytic breakdown of RB160 (after 2 h) was resulted in the formation of two more intermediates, benzene sulfonate (m/z, 157) and benzene-1,4-disulfonate (m/z, 283). With the progress of time (i.e. 4 h), transformed products were further converted into lower molecular weight compounds, benzene (m/z, 78) and aniline (m/z, 93). The oxidation of aniline to catechol leading to TCA cycle was anticipated (though it was not detected in the study), due to detectable activity of tyrosinase (Table 2). Thus, all the above dye intermediates would have entered the central metabolic pathway leading to the mineralization of RB160. As observed in FTIR and NMR analyses peaks corresponding to high molecular weight compounds were not detected in GC–MS analysis after 12 h, which clearly indicated the complete cleavage of RB160 into lower molecular weight (aliphatic) compounds. Patil and Jadhav during their study on phytoremediation of RB160 degradation by Tagetes patula L. [36], observed that dye was degraded into six intermediates, amongst them three intermediates (1-phenylmethanediamine, benzene sulfonate and benzene-1,4-disulfonate) were also observed in our study. 3.6. Microcosms studies Imitating natural ecosystem as simulated microcosms would provide good connection between standard laboratory results and field experiments. Because results obtained from experimental conditions and augmented bacteria under laboratory conditions seldom represent the open environment and are not site specific. Therefore, to substantiate the proficiency of BDN in soil ecosystem for colonizing and dye metabolising ability, simulated microcosm study was performed. As observed from Fig. 7, BDN was able to colonize in sterile soil (set B) even in presence of autochthonous microbial population (set D). The proliferation ability was directly correlated with RB160 decolourizing efficiency of BDN, where 90% decolourization was observed in sterile soil. While autochthonous microbial population has improved the decolourization proficiency of BDN and nearly 100% decolourization was observed in non sterile soil (set D). 3.7. Phytotoxicity studies Seed germination and plant growth studies using two agriculturally important plants P. mungo and T. aestivum were performed to determine the degree of toxicity of RB160 and its degraded products. The observed results suggested that RB160 in its native form inhibits seed germinations, as 58 and 52% of P. mungo and T. aestivum, seeds failed to geminates under test conditions, respectively (Table 3). Whereas, in the presence of RB160 degraded metabolites

K.H. Balapure et al. / Journal of Hazardous Materials 279 (2014) 85–95

Fig. 6.

1

93

HNMR spectra of (a) RB160, (b) ethyl extracted degraded products of RB160 after 4 h (c) after 12 h.

92 and 96% of P. mungo and T. aestivum seeds geminated, respectively. After germination, in presence of RB160, young plants of P. mungo and T. aestivum demonstrated retarded growth as shoot and

root height was decreased by 74 and 85% and 58 and 73%, respectively. But when seeds were cultivated along with RB160 degraded products, young plants produced shoots and roots of much higher length than observed in the previous case. Thus, results indicated

Table 3 Phytotoxicity of RB160 (100 mg/L) and its degraded products extracted after 12 h incubation for Phaseolus mungo and Triticum aestivum. Observation

Germination (%) Plumule (cm) Radical (cm)

Phaseolus mungo

Triticum aestivum

Distilled water

RB160

Extracted metabolites

Distilled water

RB160

Extracted metabolites

100 8.2 ± 1.2 6.1 ± 1.3

42 2.1** ± 1.4 0.9** ± 1.1

92 6.6* ± 1.6 5.5* ± 1.8

100 14.11 ± 1.3 12.80 ± 1.0

48 5.8** ± 1.4 3.2** ± 1.3

96 13.7* ± 1.5 12.1* ± 1.2

Values are mean of three experiments, SEM (±), significantly different from the control (seeds germinated in distilled water) at * P < 0.05, ** P <0.001, by the one way analysis of variance (ANOVA) with Tukey–Kramer multiple comparison test.

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Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat. 2014.06.057.

References

Fig. 7. Simulated microcosm study accessing competence of BDN for its ability to colonize and degrade RB160 in soil environment. (A) Sterile soil amended with RB160, (B) sterile soil amended with RB160 and inoculated with BDN, (C) non sterile soil amended with RB160, (D) non sterile soil amended with RB160 and inoculated with BDN, (E) sterile soil (without dye) inoculated with BDN.

that dye toxicity was essentially due to original compound rather than its biotransformed products. The BDN simultaneously detoxified RB160 intermediate that would have generated during dye metabolism.

3.8. Decolorization profile of pure culture in compared to BDN Individual bacterial strains of consortium BDN examined for their potential to decolorize RB160 under microaerophilic condition. Results indicated that Pseudomonas sp. BDN4, Providencia sp., BDN5 and Bacillus sp. BDN7 were able to decolorize 86, 84, and 80% at RB160 (100 mg/L) within 12 h. While, Acinetobacter sp. BDN6, Alcaligenes sp. BDN1 and Bacillus sp. BDN8 were able to decolorize, respectively, 74, 79 and 75% RB160 within 18 h. Although Bacillus sp. BDN2 (65%) and Escherichia sp. BDN3 (61%) were not efficient dye degrading strains, but their presence might be playing an important role in resistance toward dye compounds. As mentioned above, these bacterial species were previously been described to possess dye decolourization ability either in pure form or as consortia/mixed cultures [22,37].

4. Conclusions Bacterial mediated remediation of recalcitrant dyes of anthropogenic origin offers great opportunity for the restoration of dye contaminated environments in an ecologically acceptable manner. As noted above bacteria in community rather than as individual pure cultures are well efficient for complete mineralization of any pollutants. Therefore, we have developed the potent bacterial mixed cultures BDN that not only cleaved substituted sulphonated aromatic dye but simultaneously detoxify the degraded intermediates at a faster rate under ambient conditions. The consistent growth of BDN at flask conditions and ability to colonize in soil system with minimal nutrient requirement and concurrent degradation of toxic dye compounds signifies its potential application for in situ bioremediation.

Acknowledgements Authors are thankful to Sophisticated Instrumentation Centre of Applied Research and Training (SICART), Vallabh Vidyanagar, Gujarat India for providing facilities for FTIR and GC–MS.

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