5.1 Introduction - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/7234/13/13_chapter 5.pdf · metabolism of azo dyes requires specific enzymes (aerobic azoreductases), which

Chapter 5: Community genomics.........

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5.1 Introduction

Industrial estates, situated within the ‘Golden Corridor’ (a highly industrialized zone

from Vapi to Mehsana in Gujarat, India) manufacture dyes, paints and pigments,

pharmaceuticals, textiles and many other commercial products. These industries

release liquid wastes containing dyes, xenobiotic compounds and many other

unnatural products in the environment and thus are the major cause of ground and

surface water pollution in these areas. Dyestuff effluents are one of the major

pollutants that are released into the environment. Even very low concentrations of

dyes (less than 1 mg/l) can be highly visible in solutions. Synthetic dyes are very

soluble in water and are recalcitrant to microbial degradation because they contain

substituents such as azo, nitro or sulfo groups. They are frequently found in a

chemically unchanged form even after waste-water treatment and hence they are

regarded as recalcitrant pollutants (Pagga and Brown, 1986; Shaul et al., 1991). Azo

dyes are the largest class of dyes and are widely used in the textile, leather, food,

cosmetic and dyestuff manufacturing industries (Anliker, 1979; Reisch, 1996; Blumel

et al., 2002) because of their chemical stability, ease of synthesis and versatility

(Nakanishi et al., 2001). In year 2000, more than 7×105 tons of these dyes were

produced worldwide (Suzuki et al., 2001). Azo dyes, characterized by the presence of

one or more azo groups (-N=N-), have become a great concern in effluent treatment

due to their colour, bio recalcitrance and potential toxicity to animals and human.

The treatment system of effluents based on physico-chemical methods is effective but

suffers from shortcomings such as high cost, formation of hazardous by-products,

intensive energy requirements and production of a large amount of sludge. In contrast,

biological degradation of these dyes does not have similar problems. To establish

biological waste-water treatment of azo dyes, it is essential to discover the

microorganisms that encode azo dye-degrading enzymes (Sugiura et al., 2006).

Diverse set of enzymes like azoreductases and peroxidases have so far been reported

in dye degradation. Azoreductases of microorganisms are favourable for the

development of biodegradation systems for azo dyes, because these enzymes catalyze

reductive cleavage of azo groups (-N=N-) under mild conditions. In addition, bacterial

enzymes can be readily overproduced (Nakanishi et al., 2001). The most generally


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accepted hypothesis is that many bacterial strains possess cytoplasmic enzymes,

which act as ‘azoreductases’ when under the pressure of azo dyes.

Azo bonds in azo dyes are reduced, under anaerobic conditions, leading to formation

of corresponding amines. Intermediate aromatic amines are further mineralized under

aerobic conditions (Nakayama et al., 1983; Blumel at al., 2002; Chen et al., 2005).

Till now mainly combined anaerobic-aerobic microbial treatments of dye wastes have

been used, due to limited knowledge about microorganisms having oxygen tolerant

enzymes that are involved in decolourization of azo dyes. Aerobic treatment of

dyestuff containing wastewaters possess significant potential, however, the aerobic

metabolism of azo dyes requires specific enzymes (aerobic azoreductases), which

catalyze the NAD(P)H-dependent reduction of azo compounds to the corresponding

amines (Blumel et al., 2002; Chen et al., 2005). Very few aerobic bacteria which can

grow on/with azo compounds have been reported such as azoreductase from Bacillus

sp. OY1-2 (Suzuki et al., 2001) and Xenophilus azovorans (Blumel et al., 2002).

Moreover, Flavobacterium can aerobically degrade 4,4’-dicarboxyazobenzene ring

(Overney, 1979; Blumel et al., 2002) and Sphingomonas sp. azoreductases can cleave

several sulphonated naphthol and benzol rings (Stolz, 1999).

Any individual microorganism is incapable of processing all the metabolic reactions

to degrade any of the environmental pollutants, however a group of diverse organisms

form a community and collectively process all the metabolic reactions for

bioremediation. The individual strains may attack the dye molecule at different

positions or may use decomposition products produced by another strain for further

decomposition. To gain insights into these processes, it becomes inevitable to identify

and characterize the dye decolorizing genetic machinery.

One approach is metagenomics or community genomics, which aims to access the

genomic potential of an environmental sample either directly or after enrichment for

specific purpose, respectively. Soil metagenome can be enriched in various ways

keeping in mind specific gene targets that have potential applications in

bioremediation, industry, medicine or agriculture. Bacteria capable of xenobiotic

degradation are widely distributed in the environment. These bacteria have evolved to


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utilize a variety of compounds that are present in the environment. Another approach,

direct cloning of gene fragments from dye degrading bacterial consortia wherein

genes can be targeted by carrying out PCR using gene specific/degenerate primers,

may also give better insights. Moreover, transformation of azoreductase expressing

clones to an easily manipulative host can serve for large-scale enzyme production or

setting of a bioreactor with known parameters.

In order to search for azoreductases which can withstand the aerobic conditions and

possess broad range of substrate specificity, attempts were made to clone oxygen

tolerant azoreductases from dye decolourizing enriched mixed cultures.

5.2 Materials and methods

5.2.1 Bacterial strains, plasmids and growth conditions

E. coli strains DH5α, DH10B and BL21(DE3) were cultured at 37°C in Luria Broth

(Himedia, Mumbai, India). Recombinant strains were also cultured under similar

conditions with appropriate antibiotics (ampicillin-100 µg/ml and kanamycin-50

µg/ml). The pGEM-T kit (Promega, Madison, USA) was used for cloning of the

azoreductase gene. The plasmids pUC19 and pET28a+ were used for expression

studies of the azoreductase gene. All plasmid isolations were carried out using

plasmid preparation kit obtained from Qiagen (Hilden, Germany).

5.2.2 Chemicals

Co-factors (FAD, FMN, NAD, NADP.Na2, NADH.DPNH and NADPH.Na4) were

obtained from Sigma-Aldrich (Missouri, USA); restriction and modification enzymes

were obtained from NEB (Ipswich, USA); antibiotics were obtained from Himedia

(Mumbai, India); agarose and DNA molecular weight markers from Bangalore Genei

(Bangalore, India); primers from MWG Biotech (Edersburg, Germany) and dNTPs

from Bioron (Ludwigshafen, Germany). Reactive Violet 5 (RV5) was of commercial

grade. All other essential chemicals were of molecular biology grade.

5.2.3 Contaminated site

The soil samples were collected from contaminated banks of the Khari-cut canal (N

22° 57. 878′; E 072° 38. 478′), flowing through GIDC (Vatva, Ahmedabad, Gujarat,

India) and into the Khari river.


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5.2.4 Development of V9 consortium

Soil samples were inoculated (10%) in Bushnell Haas Minimal medium (BHM: 1.7

mM MgSO4, 0.2 mM CaCl2, 7.4 mM KH2PO4, 5.7 mM K2HPO4, 12.5 mM NH4NO3

and 0.3 mM FeCl3) supplemented with glucose and yeast extract and under selective

pressure of the azo dye (Reactive Violet 5). For dye degradation under anoxic (static)

condition, BHM with Reactive Violet 5 (100 ppm) was supplemented with glucose

(0.1% w/v) and yeast extract (0.1% w/v). For dye degradation under aerobic (120

rpm) condition, BHM with Reactive Violet 5 (100 ppm) was supplemented with

glucose (0.1% w/v) and yeast extract (0.5% w/v). Once the dye was decolourized,

parts of these cultures were used as inoculum (10%) in fresh medium. Fifty transfers

were carried out and each time dye decolourization was observed. The stabilized

consortia were used for further studies. Dye degradation was monitored visually and

also by determining the absorbance of the supernatant at 558 nm (λmax of RV5) using

an UV-visible spectrophotometer (Analytik Jena AG Specord®

210, Jena, Germany).

5.2.5 Characterization of V9 consortium

5.2.5.1 Determination of carbon source

BHM supplemented with yeast extract (0.1% w/v) along with different carbon sources

such as glucose, sucrose, lactose, pyruvate and starch (0.1% w/v) and Reactive Violet

5 (100 ppm) were inoculated with V9 consortium (10% v/v) and incubated at 37°C. A

control, without any carbon source, was kept under similar conditions. To find the

minimum amount required for dye decolourization, various concentrations (0.01,

0.05, 0.1, 0.2, 0.3 and 0.5% w/v) of the selected carbon source were studied.

5.2.5.2 Determination of nitrogen source

BHM supplemented with glucose (0.1% w/v) along with different nitrogen sources

such as yeast extract, peptone, ammonium nitrate, sodium nitrate, potassium nitrate

and urea (0.1% w/v) and Reactive Violet 5 (100 ppm) were inoculated with V9

consortium (10% v/v) and incubated at 37°C. A control, without any nitrogen source,

was kept under similar conditions. To find the minimum amount required for dye

decolourization, various concentrations (0.01, 0.05, 0.1, 0.2, 0.3 and 0.5% w/v) of the

selected nitrogen source were studied.


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5.2.5.3 Optimum temperature for dye decolourization

BHM supplemented with glucose (0.1% w/v), yeast extract (0.1% w/v) and Reactive

Violet 5 (100 ppm) was inoculated with V9 consortium (10% v/v) and incubated at

different temperatures such as 4°C, 20°C, 30°C, 37°C, 45°C and 55°C.

5.2.5.4 Effect of salinity on dye decolourization

BHM supplemented with glucose (0.1% w/v), yeast extract (0.1% w/v) and Reactive

Violet 5 (100 ppm) along with various NaCl concentrations (0, 1, 2, 3, 4, 5, 6, 7, 8, 9,

10, 20 and 30 g/l) were inoculated with V9 consortium (10% v/v) and incubated at

37°C.

5.2.5.5 Spectrum of dyes as substrates

BHM supplemented with glucose (0.1% w/v), yeast extract (0.1% w/v) and twenty

four kinds of dyes (100 ppm), respectively were inoculated with V9 consortium (10%

v/v) and incubated at 37°C. Decolourization was determined at respective absorbance

maxima of dyes.

5.2.6 Isolation and identification of cultivable bacteria constituting the V9

consortium

The appropriate dilutions of enriched consortium were spreaded on rich medium

(Luria agar) as well as minimal media (BHM agar and R2A) and incubated at various

temperatures (20°C, 28°C and 37°C) for various growth periods (1 day, 2 days, 5

days, 10 days and 20 days). Based on morphological characteristics different colonies

were picked and subsequently maintained on Luria agar plates.

The 16S rRNA genes were amplified from the bacterial cultures by colony PCR.

Amplification was carried out in a 30 l PCR reaction consisting of 1X buffer (10

mM Tris pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100), 0.33 mM each of

dNTPs, 0.66 þmoles each of custom synthesized universal primers 27F (5’-

GAGTTTGATCCTGGCTCA-3’) and 1385R (5’-CGGTGTGTRCAAGGCCC-3’),

and 1.5 U of Taq DNA polymerase. Amplification program was performed with

initial denaturation step at 94°C for 5 min; followed by 30 cycles of 1 min

denaturation step at 94°C, 1 min annealing step at 55°C, and 1.2 min elongation step

at 72°C and a final extension step at 72°C for 20 min using Biorad iCycler version


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4.006 (Biorad, CA, USA). The ~1.5 kb PCR product was sequenced by automated

DNA Analyzer 3730 using BigDyeTM

Terminator 3.1 sequencing chemistry (Applied

Biosystems, Foster City, CA). Full length 16S rRNA gene sequences were analyzed

using SEQMATCH tool of the Ribosomal Database Project (RDP-II) to identify the

bacteria. The 16S rRNA gene sequences have been submitted to GenBank and the

obtained accession numbers have been described in section 5.3.2.

5.2.7 Isolation of consortial DNA

The V9 consortium, after dye decolourization, was used for total community DNA

preparation using Zhou et al. (1996) protocol with some modifications. The

precipitation was done differently by adding polyethylene glycol (PEG) 10,000 at a

final concentration of 5% followed by incubation at 4°C overnight. DNA

concentration was measured using NanoDrop ND-1000 spectrophotometer

(NanoDrop Technologies Inc., Delaware, USA) and further analysed by gel

electrophoresis.

5.2.8 Amplification, cloning and sequencing of azoreductase gene

In order to clone the azoreductase gene, three sets of primers were designed from the

sequences available in NCBI database [(1) AZR1F 5’-

ATGAAACTAGTCGTTATTAAC-3’ and AZR1R 5’-

TCACTCCACTCCTAGTTGTTTTTT-3’; (2) AZR2F 5’-

TAGCAAAACTTGAAGTGG-3’ and AZR2R 5’-CGTATCATTTTGAACAGG-3’;

(3) AZR3F 5’-GGAATTCATATGAACATGTTAGTCATAAA-3’ and AZR3R 5’-

CGGGATCCAACAAATCCCGGCGTTYAGA-3’]. Amplification of the

azoreductase gene from consortial DNA was carried out as described in section 5.2.6.

The amplified products were purified by adding a solution (20% PEG 8000 in 2.5 M

NaCl) to the reaction mixture in a ratio of 1:0.66 and incubating at 37°C for 1 hour

followed by two 70% ethanol washes. All the centrifugations (Kubota 6500, Bunkyo-

Ku, Tokyo, Japan) were carried out at 20,000 x g for 20 minutes at 4°C. The pellet

was air-dried and resuspended in Milli-Q water (Millipore, Billerica, USA). The

electrocompetent cells were prepared as described in section 2.2.4.4.2 of chapter 2.

Purified amplified products were ligated into pGEM-T vector (according to manual of

pGEM-T vector) and transformed into E. coli DH5α competent cells by


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electroporation as described in section 2.2.4.5.2 of chapter 2. Recombinant plasmids

were isolated and subjected to sequencing using M13 primers by automated DNA

Analyser 3730 using ABI PRISM® BigDye

TM Terminator Cycle Sequencing 3.1

(Applied Biosystems, CA, USA). The sequence has been submitted to GenBank and

the accession number is described in section 5.3.4. DNA and protein sequences with

homology to the sequenced gene were searched using BLASTn and BLASTx in

NCBI databases, respectively.

5.2.9 Phylogenetic analysis

Three hundred sequences of each of azoreductase genes and proteins were

downloaded from NCBI database in February 2010. The sequenced gene and ORF of

its translated product were aligned with downloaded azoreductase genes and proteins

sequences, respectively using Clustal W 1.6 program at

(http://www.ebi.ac.uk/clustalw/). Phylogenetic analyses was performed (for both

DNA and protein sequences) using aligned sequences by the neighbour joining

algorithm (Saitou and Nei, 1987) using Kimura 2 parameter distance and

bootstrapping with 550 replicates (Felsenstein, 1985) in MEGA 4.0 software (Tamura

et al., 2007). Branches corresponding to partitions reproduced in less than 50%

bootstrap replicates were collapsed. The resulting tree was drawn to scale, with

branch lengths in the same units as those of the evolutionary distances used to infer

the phylogenetic tree. The evolutionary distances were computed using the Maximum

Composite Likelihood method (Tamura et al., 2004) and the Poisson correction

method (Zuckerlandl and Pauling, 1965) for DNA and protein sequences,

respectively. All positions containing alignment gaps and missing data were

eliminated only in pair wise sequence comparisons.

5.2.10 Expression of an azoreductase gene in E. coli

To facilitate cloning of azoreductase gene in expression vectors, BamHI and HindIII

restriction site sequences were added in set 1 primers (AZR1FB 5’-

GGCGGATCCATGAAACTAGTCGTTATTAAC-3’ and AZR1RH 5’-

GGCAAGCTTTCACTCCACTCCTAGTTGTTTTTT-3’). The underlined bases in

AZR1FB and AZR1RH represent sites for BamHI and HindIII, respectively.

Amplified azoreductase gene and vectors pUC19 and pET28a+ were digested with


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BamHI and HindIII enzymes. The ligations were performed in 10 μl reactions

containing 50 ng vector, 150-200 ng insert DNA and 3 units of T4 DNA ligase in T4

DNA ligase buffer at 16ºC overnight. Two μl of the reaction was used for

transformation of 100 μl E. coli DH10B competent cells by electroporation (Bio-Rad

MicroPulser, Hercules, California, USA) at a voltage of 1.8 kV and 2.5 kV for 0.1 cm

and 0.2 cm cuvettes, respectively. The preparation of competent cells and

electroporation are described in detail in section 2.2.4.4.2 and 2.2.4.5.2, respectively

of chapter 2. For facilitating expression of the azoreductase gene, a second

transformation of recombinant plasmid isolated from E. coli DH10B clone pET1 was

carried out in E. coli BL21(DE3) competent cells.

5.2.11 Whole cell extract preparation

Cells were harvested and washed with 20 mM sodium phosphate buffer (pH 7.0). All

the centrifugations were carried out at 20,000 x g for 20 minutes at 4°C. The cells

were resuspended in the same buffer and two freeze-thaw cycles at -20ºC and 4ºC

were carried out followed by disruption using an Ultrasonic probe (Sonics Vibra Cell

500, USA) at 35% amplitude and 9 second pulses with 5 second intervals for 15 times

or till complete cell lysis (clear lysate). The lysate was then centrifuged and the

obtained supernatant was collected and termed as crude extract.

5.2.12 Azoreductase activity assay

Enzyme assay was carried out in 2 ml 20 mM sodium phosphate buffer system

containing 2 mg protein (crude extract), 50 ppm Reactive Violet 5 dye and 1 mM co-

factor. The experimental reactions were incubated at 37°C till dye degradation and

control reactions under similar conditions for three hours. Dye degradation was

measured by the absorbance of the supernatant at 558 nm (λmax). The amount of

protein was calculated according to the method of Lowry et al. (1951).

5.2.13 Studies on suitable co-factor

To find out the co-factor required by the cloned azoreductase gene, enzyme assays

were carried out with different co-factors (FAD, FMN, NAD, NADP.Na2,

NADH.DPNH and NADPH.Na4) at a concentration of 1.5 mM. To determine the


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minimum amount of the selected co-factor required for expression, enzyme assays

were carried out with different concentrations (0 mM, 0.5 mM, 1 mM and 1.5 mM).

5.2.14 Azoreductase assay of whole cell crude extracts of E. coli strains, strains

with vectors and strains with recombinant vectors

Azoreductase assay were performed for cell extracts of E. coli DH10B clone pUC1,

E. coli DH10B clone pET1 and E. coli BL21(DE3) clone pET1. As controls, assays

were also performed for cell extracts of E. coli DH5α, E. coli DH10B, E. coli

BL21(DE3) and E. coli strains with vectors pUC19 and pET28a+. The assays were

carried out as described in section 5.2.12.

5.3 Results and discussion

5.3.1 Contaminated site

The persistent release of toxic pollutants from industrial units (situated in GIDC,

Vatva, Ahmedabad, Gujarat, India) manufacturing dyes, chemicals, solvents and other

xenobiotic compounds have contaminated the surrounding water and soil bodies. The

soil samples collected from contaminated banks of the Khari-cut canal, flowing

through Vatva and into the Khari river were used for developing consortium, enriched

with bacterial cultures capable of either degrading dye or growing in presence of dye

stress.

5.3.2 V9 consortium

Reactive Violet 5 was chosen as a model dye on account of its complexity. It contains

three aromatic rings, three sulphonated groups as well as copper as a metal ion. The

structure of Reactive Violet 5 is shown in Fig. 5.1.

Fig. 5.1: Chemical structure of Reactive Violet 5 (RV5) dye.


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b

a

The developed bacterial V9 consortium was able to decolourize 100 ppm of Reactive

Violet 5 (RV5) within 24 h at 37ºC. The degradation of dye was observed visibly

(Fig. 5.2a) as well as by the decrease in the absorbance at 558 nm (λmax of RV5)

(Fig. 5.2b).

Fig. 5.2: Dye decolourization profile depicted by (a) photograph and (b) UV-Visible

overlay spectra. ( Inoculated and Dye degraded)


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To achieve efficient dye decolourization, various biotic and abiotic parameters were

studied. V9 consortium was characterized for carbon source as well as nitrogen source

and their concentrations. Various carbon sources such as glucose, sucrose, lactose,

pyruvate and starch were screened for dye degradation studies and amongst them

glucose was found to be the best (Fig. 5.3a). As observed from Fig. 5.3a, 95% of dye

was decolourized within 24 h in presence of glucose. To find the minimum amount of

glucose required for dye decolourization, various concentrations (0.01, 0.05, 0.1, 0.2,

0.3 and 0.5% w/v) were studied (Fig. 5.3b). From Fig. 5.3b, it can be observed that

when glucose concentration was 0.1% w/v, 95% of dye was decolourized within 24 h.

Various nitrogen sources such as yeast extract, peptone, ammonium nitrate, sodium

nitrate, potassium nitrate and urea were screened for dye degradation studies and

amongst them yeast extract was found to be the best (Fig. 5.4a). As observed from

Fig. 5.4a, 95% of dye was decolourized within 24 h in presence of yeast extract. It

was observed that peptone leads to even faster rapid degradation of dye, however the

dye decolourization is temporary and after further 24 h, colour reappears.

Consequently, yeast extract was preferred over peptone. To find the minimum amount

of yeast extract required for dye decolourization, various concentrations (0.01, 0.05,

0.1, 0.2, 0.3 and 0.5% w/v) were studied (Fig. 5.4b, c). Amount of nitrogen source

requirement varied for anoxic and aerobic conditions. In anoxic conditions, 90% of

dye was decolourized within 24 h in presence of 0.1% yeast extract (Fig. 5.4b).

Conversely, in aerobic conditions 90% of dye was decolourized within 30 h in

presence of 0.5% yeast extract (Fig. 5.4c). Lesser concentrations of yeast extract did

not lead to optimum dye degradation in aerobic conditions. Thus, the minimum

essential amount of yeast extract required was higher in aerobic (0.5%) than in anoxic

(0.1%) conditions. Reasons can be attributed to the fact, that oxygen might be

competing for the electrons. Moreover, one peculiar phenomenon was observed that

for dye degradation, presence of yeast extract in medium is inevitable whereas,

absence of glucose did not hamper the metabolic process.


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Fig. 5.3: (a) Effect of various carbon sources on decolourization of RV5 by V9

consortium under anoxic condition at 37ºC; (b) Effect of different glucose

concentrations on decolourization of RV5 by V9 consortium under anoxic condition

at 37ºC.

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Fig. 5.4: (a) Effect of various nitrogen sources on decolourization of RV5 by V9

consortium under anoxic condition at 37ºC; (b) Effect of different yeast extract

concentrations on decolourization of RV5 by V9 consortium under anoxic condition

at 37ºC; (c) Effect of different yeast extract concentrations on decolourization of RV5

by V9 consortium under aerobic condition at 37ºC.

Moreover, optimum temperature required for efficient and faster dye decolourization

was characterized. Optimized BHM medium with Reactive Violet 5 (100 ppm) was

inoculated with V9 consortium (10% v/v) and incubated at different temperatures

such as 4°C, 20°C, 30°C, 37°C, 45°C and 55°C (Fig. 5.5). The decolourization ability

of V9 consortium was highest at 37°C showing 93% decolourization of RV5 within

24 h.

Fig. 5.5: Effect of different temperatures on decolourization of RV5 by V9

consortium under anoxic condition.

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c


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Fig. 5.6 shows overall decolourization profile of RV5 and growth pattern of V9

consortium under anoxic (Fig. 5.6a) and aerobic (Fig. 5.6b) conditions. Under anoxic

conditions V9 consortium was able to decolourize 95% of RV5 within 24 h, while in

aerobic conditions (120 rpm) 85-90% of RV5 was decolourized within 30-36 h at

37ºC.

Fig. 5.6: Dye decolourization and growth profile of V9 consortium under (a) anoxic

and (b) aerobic conditions at 37ºC.

0

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The V9 consortium was able to efficiently decolourize dye upto 500 ppm

concentration and tolerate dye up to 2000 ppm. Moreover, generally contaminated

sites are reported to have high salinity. Consequently, dye decolourization efficiency

of V9 consortium in presence of high salinity was determined. BHM supplemented

with glucose (0.1% w/v), yeast extract (0.1% w/v) and Reactive Violet 5 (100 ppm)

along with various NaCl concentrations (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 and 30 g/l)

were inoculated with V9 consortium (10% v/v) and incubated at 37°C (Fig. 5.7). The

consortium showed more than 90% dye decolourization even at higher concentrations

of NaCl, which demonstrated its ability of decolourizing dye RV5 over a wide range

of saline conditions.

Fig. 5.7: Effect of salinity on decolourization of RV5.

To determine whether developed V9 consortium can decolourize broad range of

substrates, various kinds of azo dyes (having different - groups, metal ions, ring

formations, etc.) were used as substrates in the study. BHM supplemented with

glucose (0.1% w/v), yeast extract (0.1% w/v) and twenty four kinds of dyes (100

ppm), respectively were inoculated with V9 consortium (10% v/v) and incubated at

37°C (Fig. 5.8). Decolourization was determined at respective absorbance maxima of

dyes. Decolourization in the range of 85-95% within 24 h was observed for 22 out of

24 dyes used in the study.


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Fig. 5.8: Spectrum of dyes decolourized by the V9 consortium.

Determination of azo-dye degrading organisms is essential for developing new

bioremediation strategies in waste-water treatment plants (Suzuki et al., 2001).

Consequently, cultivable organisms constituting the V9 consortium and playing a role

in dye degradation were identified. Based on morphological characteristics seven

different cultures were isolated. Subsequently their 16S rRNA genes were amplified

(Fig. 5.9) and sequenced.

Fig. 5.9: 16S rRNA genes amplified by colony PCR. Lanes 1 to 7: 16S rRNA genes

from seven different pure cultures; Lane 8: Supermix DNA ladder.

1.5 kb

1 2 3 4 5 6 7 8


154

These 16S rRNA gene sequences were analyzed using SEQMATCH tool of

Ribosomal Database Project (RDP-II). The organisms were identified as

Pseudomonas citronellolis V91DM (JN400328), Lysinibacillus fusiformis V92DM

(JN400329), Gordonia cholesterolivorans V93DM (JN400330), Ochrobactrum

pseudintermedium V94DM (JN400331), Stenotrophomonas sp. V95DM (JN400332),

Enterococcus casseliflavus V96DM (JN400333) and Citrobacter sp. V97DM

(JN400334). The genera Pseudomonas, Stenotrophomonas and Citrobacter belong to

class Gammaproteobacteria of phylum Proteobacteria. The genus Ochrobactrum

belongs to class Alphaproteobacteria of phylum Proteobacteria. The genera

Enterococcus and Lysinibacillus belong to class Bacilli of phylum Firmicutes. The

genus Gordonia belongs to class Actinobacteria (class) of phylum Actinobacteria.

Many reports are available describing the roles of strains, belonging to genera

identified in V9 consortium, in azo dye degradation. Azo reduction activity by strain

Pseudomonas putida MET94 (Mendes et al., 2011) and decolourization of adsorbed

textile dyes by mixed cultures of Pseudomonas sp. SUK1 and Aspergillus ochraceus

NCIM-1146 (Kadam et al., 2011) have been reported. Lysinibacillus sp. strain AK2 is

capable of decolourizing sulfonated azo dye Metanil Yellow (Anjaneya et al., 2011).

Ochrobactrum intermedium ANKI is able to decolourize azobenzene (Vakkerov-

Kouzova, 2007). Azoreductase (AzoA) from Enterococcus faecalis is a very active

enzyme with broad spectrum of substrate specificity (Chen et al., 2008). Macwana et

al. (2010) have isolated and identified an azoreductase from Enterococcus faecium

which is 67% similar to azoreductase from Enterococcus faecalis but uses different

co-factors NADH and NADPH instead of FMN. A bacterial strain Stentrophomonas

maltophilia AAP56 isolated from a polluted soil was able to decolorize recalcitrant

dyes of an industrial effluent: SITEX Black (Said et al., 2010). Petty et al. (2010)

sequenced complete genome of Citrobacter rodentium and they have identified an

azoR gene (FMN dependent NADH-azoreductase).

5.3.3 Consortial DNA

The stabilized V9 consortium (transferred 50 times and each time dye was

decolourized) was used for extraction of total consortial DNA. The DNA isolation

was carried out according to protocol described by Zhou et al. (1996) with some


155

modifications. The consortium besides bacterial cultures also contained aromatic

amines and other dye degradation products. Thus it was essential that these

contaminants were removed from the extracted DNA as they would have interfered in

further molecular experiments. Consequently, the precipitation was done using 5%

PEG 10,000 and incubated at 4°C overnight. The advantage of using PEG 10,000 was

that no additional purification step such as gel permeation chromatography was

required. The extracted DNA (Fig. 5.10) was free of impurities and amenable to PCR,

ligation and other molecular studies. The quantity and quality of extracted DNA is

described in Table 5.1.

Fig. 5.10: Consortial genomic DNA electrophoresed on 0.8% (w/v) agarose gel. Lane

1: Supermix DNA ladder; Lanes 2 and 3: Consortial genomic DNA.

Table 5.1: Details of consortial genomic DNA.

V9 consortium after dye degradation 200 ml

Extracted consortial genomic DNA 3.2 µg

A260 0.188

A280 0.098

A230 0.093

260/280 1.92

260/230 2.02

33.5/24.5 kb

1 2 3


156

5.3.4 Azoreductase gene

Three sets of primers were designed from the sequences of azoreductases available in

the NCBI database. Azoreductase gene was amplified (Fig. 5.11), by set 1 primers –

AZR1F and AZR1R, from consortial DNA as template. The other two sets of primers

could not amplify azoreductase gene from V9 consortium. The amplified

azoreductase gene was cloned into the pGEM-T vector and sequenced. Amplified

azoreductase gene (JN400335) had a length of 537 bp (Fig. 5.12) containing an ORF

of 178 amino acids. BLASTn analysis of the sequence showed 97% identity to

azoreductase gene of Rhodobacter sphaeroides, while BLASTx analysis showed 98%

identity to azoreductase of Bacillus cereus G9241.

Fig. 5.11: Azoreductase gene amplified from consortial genomic DNA. Lanes 1 and

2: Azoreductase gene; Lane 3: Supermix DNA ladder.

atgaaactagtcgttattaacggtacaccaagaaaatttggtagaactcgcgtggtggcaaaatatattgcggatcaatttgaa

ggggagttatatgatttagcatttgaggagttacctttatacaatagagaagagtcacaacgtgatttagaggcagtgaaaaa

attaaaaacgttagtgaaagctgcggatggggttgtattatgtacaccagaatatcataatgcgatgagcggtgcgctgaaa

gactctttagattacttaagtagtaatgaatttattcataaaccagttgctttgttagcggttgctggtggcggtaaaggtggaat

aaatgcattaaacagcatgcgaacggtcgctagaggtgtttatgcaaatgcaattccaaaacaagttgttcttgatggattaca

cgtgcaagatggtgaacttggggaagatgcaaaaccattaattcatgatgtagttaaagaattgaaagcatatatgagcgtat

ataaagaggtgaaaaaacaactaggagtggagtga

Fig. 5.12: Nucleotide sequence of the amplified azoreductase gene (JN400335).

1 2

3

0.5 kb

1 2 3


157

To find out the phylogenetic affiliation of the gene, 300 sequences each of

azoreductase gene and protein were downloaded from the NCBI database. However,

similar sequences from each cluster were removed to make the resulting trees less

complex. The phylogenetic tree for azoreductase genes (Fig. 5.13a) and proteins (Fig.

5.13b) showed that the sequenced gene was clustered with the azoreductases of

phylum Firmicutes. The gene is closely related to azoreductase of Bacillus cereus Q1.

Within the phylogenetic tree of azoreductase genes, genes from

Gammaproteobacteria class were not near to each other and were interspersed with

azoreductase genes from Firmicutes. Conversely, within the phylogenetic tree of

azoreductase proteins, those of Gammaproteobacteria and Firmicutes were not

interspersed and cluster clearly apart.

The above observation can be explained by the fact that several enzymes are endowed

with promiscuous activities, azoreductases being one of them. The term 'catalytic

promiscuity' describes the capability of an enzyme to catalyse different chemical

reactions, called secondary activities, at the responsible active site. Many of the

enzymes active in degradation pathways are linked from their protein phylogeny and

not strictly linked to the taxonomical affiliation of the bacteria (Perez-Pantoja et al.,

2009), indicating that the genes encoding those catabolic enzymes are involved in

very dynamic events. To characterize the catabolic potential for biodegradation it is

necessary to take into consideration the broad diversity of catabolic routes evolved by

microorganisms and also the diversity of enzymes of a given gene family or even

between gene families.


158

Escherichia coli CFT073 Escherichia coli str K-12 Klebsiella pneumoniae Citrobacter sp 30

Salmonella enterica serovar Enterobacter sp 638 Enterobacter sakazakii ATCC BAA-894 Cronobacter turicensis

Sodalis glossinidius Yersinia pestis biovar Yersinia enterocolitica

Serratia proteamaculans 568 Erwinia tasmaniensis Et1/99 Dickeya zeae Ech1591 Pectobacterium carotovorum Erwinia carotovora

Pectobacterium wasabiae WPP Photorhabdus asymbiotica Aggregatibacter actinomycete Haemophilus parasuis SH0165 Burkholderia cenocepacia J2 Klebsiella pneumoniae 342

Acinetobacter baumannii AB3 Vibrio harveyi ATCC BAA-1116 Vibrio parahaemolyticus RIM

Vibrio vulnificus YJ016 Tolumonas auensis DSM 9187

Photobacterium profundum SS Shewanella sediminis HAW-EB Shewanella loihica PV-4

Shewanella denitrificans OS2 Shewanella frigidimarina NC

Shewanella putrefaciens CN-3 Shewanella sp MR-7 Shewanella oneidensis MR-1

Rhodopirellula baltica SH 1 Aspergillus fumigatus Af293

Colwellia psychrerythraea 3 Marinomonas sp MWYL1

Pseudovibrio sp JE062 Teredinibacter turnerae T79 Rhizobium leguminosarum bv

Acinetobacter baumannii AB00 Escherichia coli UMN026

Acaryochloris marina MBIC110 Pseudovibrio sp JE062 Chitinophaga pinensis DSM 2 Teredinibacter turnerae T79

Rhodococcus erythropolis PR4 Burkholderia cenocepacia J23

78Herminiimonas arsenicoxydans Erythrobacter litoralis HTC

Rhodobacter sphaeroides KD1 Granulibacter bethesdensis

Sulfitobacter sp EE-36 Roseobacter sp GAI101

Burkholderia mallei ATCC 103 Burkholderia multivorans AT Bradyrhizobium sp BTAi1 Stenotrophomonas sp SKA14

Methylobacterium extorquens Burkholderia multivorans

Ricinus communis Pseudomonas aeruginosa PAO1 Pseudomonas mendocina

Pseudomonas fluorescens Pf-5 Pseudomonas entomophila L48 Pseudomonas putida W619 Pseudomonas putida GB-1

Rhodobacteraceae bacterium K Colwellia psychrerythraea 34

Mycoplasma pulmonis UAB CTI Mycoplasma penetrans HF-2 Mycoplasma mobile 163K Mycoplasma conjunctivae

Bacillus weihenstephanensis Bacillus cereus

Bacillus clausii KSM-K16 Bacillus anthracis strain Ames Bacillus weihenstephanensis Bacillus halodurans C-125 Listeria monocytogenes EGD-e

Listeria innocua Clip11262 Lysinibacillus sphaericus

Bacillus licheniformis Bacillus halodurans C-125 Brevibacillus brevis NBRC 10 Exiguobacterium sibiricum 2 Coprothermobacter proteolyticus Lactococcus lactis

Clostridium botulinum B Listeria monocytogenes HCC23

Paenibacillus sp JDR-2 Bacillus cereus

Exiguobacterium sibiricum 2 Staphylococcus haemolyticus Staphylococcus aureus Mu50

Geobacillus kaustophilus HTA Bacillus licheniformis Bacillus halodurans C-125 Bacillus subtilis Bacillus pumilus SAFR-032

Bacillus cereus G9241 Bacillus subtilis strain 168

Leptospira interrogans Staphylococcus epidermidis

Staphylococcus aureus MRSA2 Bacillus licheniformis

Bacillus cereus Q1 Cloned azoreductase gene

Bacillus cereus B4264 Bacillus cereus G9842 Bacillus weihenstephanensis

Bacillus cereus cytotoxis N Bacillus cereus E33L Bacillus cereus ATCC 10987 Bacillus thuringiensis

Gamma proteobacterium NOR5-3 Shewanella amazonensis SB2B

Shewanella loihica PV-4 Shewanella piezotolerans WP

Shewanella sp MR-7 Shewanella sp ANA-3

Shewanella oneidensis MR-1 Ricinus communis

Dickeya dadantii Ech586 Azoarcus sp BH72

Stenotrophomonas maltophilia

99

99

99

99

99

9599

99

8599

99

99

99

98

98

98

98

70

97

96

5896

87

95

95

95

89

62

53

95

94

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90

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89

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a


159

Escherichia coli SE11 Escherichia coli CFT073 Klebsiella pneumoniae Citrobacter sp 30 2 Salmonella enterica Citrobacter koseri ATCC BAA-895 Enterobacter sakazakii Cronobacter turicensis Enterobacter sp 638

Sodalis glossinidius Yersinia pestis CO92 Yersinia enterocolitica Serratia proteamaculans 568 Pectobacterium wasabiae WPP Dickeya zeae Ech1591

Photorhabdus asymbiotica Erwinia tasmaniensis Et1/99

Aggregatibacter actinomycete Photobacterium profundum SS Vibrio harveyi ATCC BAA-1116

Vibrio sp Ex25 Vibrio vulnificus YJ016

Shewanella frigidimarina NC Shewanella loihica PV-4 Shewanella sediminis HAW-EB Shewanella denitrificans OS2 Shewanella sp MR-7 Shewanella sp W3-18-1 Shewanella baltica OS155

Acinetobacter baumannii AB3 Burkholderia cenocepacia J2 Klebsiella pneumoniae 342

Haemophilus parasuis SH0165 Tolumonas auensis DSM 9187

Pseudovibrio sp JE062 Teredinibacter turnerae T79

Rhodobacteraceae bacterium K Burkholderia multivorans ATCC17616 Burkholderia mallei JHU Sulfitobacter sp EE-36 Roseobacter sp. GAI101

Colwellia psychrerythraea 34 Erythrobacter litoralis HTC Rhodobacter sphaeroides KD1

Herminiimonas arsenicoxydans Granulibacter bethesdensis

Chitinophaga pinensis DSM 2 Colwellia psychrerythraea 3

Marinomonas sp MWYL1 Pseudovibrio sp JE062 Rhizobium leguminosarum bv Burkholderia multivorans Acaryochloris marina MBIC110

Rhodococcus erythropolis PR4 Escherichia coli UMN026

Methylobacterium extorquens Stenotrophomonas sp SKA14 Bradyrhizobium sp BTAi1

Ricinus communis Acinetobacter baumannii AB0057 Pseudomonas aeruginosa PAO1 Pseudomonas aeruginosa PA7 Pseudomonas mendocina ymp

Pseudomonas syringae pv Pseudomonas fluorescens Pf-5

Pseudomonas entomophila L48 Pseudomonas putida F1 Pseudomonas putida W619

Bacillus cereus G9241 ORF of cloned azoreductase gene

Bacillus cereus Q1 Bacillus cereus 03BB102 Bacillus weihenstephanensis Bacillus cereus Bacillus licheniformis ATCC14580

Staphylococcus epidermidis Staphylococcus aureus

gamma proteobacterium NOR5-3 Leptospira interrogans serovar

Shewanella amazonensis SB2B Shewanella piezotolerans WP3

Shewanella loihica PV-4 Shewanella sp ANA-3 Shewanella sp MR-7

Shewanella oneidensis MR-1 Azoarcus sp BH72 Stenotrophomonas maltophilia

Rhodopirellula baltica SH 1 Aspergillus fumigatus Af293

Burkholderia cenocepacia J23 Mycoplasma penetrans HF-2 Mycoplasma pulmonis UAB CTIP Mycoplasma mobile 163K

Mycoplasma conjunctivae Bacillus licheniformis ATCC14580 Bacillus amyloliquefaciens

Bacillus pumilus SAFR-032 Geobacillus kaustophilus HTA426 Bacillus halodurans C-125

Staphylococcus aureus Staphylococcus haemolyticus Paenibacillus sp JDR-2 Exiguobacterium sibiricum 255-15 Exiguobacterium sp AT1b

Bacillus cereus Bacillus anthracis Bacillus cereus G9842 Listeria monocytogenes HCC23

Clostridium botulinum B Coprothermobacter proteolyticus

Lactococcus lactis Exiguobacterium sibiricum 255-15 Lysinibacillus sphaericus C3-41

Listeria monocytogenes EGD-e Listeria innocua Clip11262

Brevibacillus brevis NBRC 10 Bacillus halodurans C-125

Bacillus licheniformis ATCC14580 Bacillus subtilis Bacillus halodurans C-125 Bacillus anthracis strain Ames Bacillus cereus G9842

Bacillus clausii KSM-K16 Bacillus cereus

Bacillus thuringiensis Bacillus cereus ATCC 14579 Bacillus cereus B4264

Bacillus cereus AH187 Bacillus subtilis

Ricinus communis Dickeya dadantii Ech586

896799

100

8295

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Fig. 5.13: Phylogenetic relationship of the cloned gene sequence (a) and its deduced

amino acid sequence (b) with gene and protein sequences of azoreductases available

in database, respectively. The trees were constructed using neighbour joining

algorithm with Kimura 2 parameter distances in MEGA 4.0 software. Numbers at

nodes indicate percent bootstrap values above 50 supported by 550 replicates. The bar

indicates the Jukes-Cantor evolutionary distance. The names of the downloaded

sequences are as described in GenBank. The cloned gene is named as cloned

azoreductase gene in (a) and as ORF of cloned azoreductase gene in (b).

5.3.5 Expression of the azoreductase

In order to clone the azoreductase gene, BamHI and HindIII sites were added in

forward and reverse primers of set 1, respectively. The azoreductase gene, amplified

using modified set 1 primers, was digested with BamHI and HindIII enzymes. The

vectors pUC19 and pET28a+ were isolated and digested with Bam HI and Hind III

enzymes (Fig. 5.14). The digested products were ligated into digested vectors. The

constructs were transformed in E. coli DH10B. The presence of inserts in pUC19 was

verified by carrying out amplification of azoreductase gene using M13 primers as

shown in Fig. 5.15.

The resulting recombinant clones were named as E. coli DH10B clone pUC1 (pUC19

with azoreductase gene) and E. coli DH10B clone pET1 (pET28a+ with azoreductase

gene). Moreover, for higher expression of the azoreductase gene, a second

transformation of recombinant plasmid, isolated from E. coli DH10B clone pET1, was

carried out in E. coli BL21(DE3) cells and the recombinant clone was named as E.

coli BL21(DE3) clone pET1. The recombinant strains were checked for their in vivo

azo dye degradation capabilities. However, no degradation was observed and reason

can be attributed to the fact that recombinant E. coli strains were unable to uptake

sulphonated azo dyes. This result corroborates with earlier similar observations by

Blumel et al. (2002). Moreover, they had also suggested that besides uptake other

limitation factor could be availability of NADPH, essential for the reduction of azo

dyes. Consequently, for measuring in vitro enzyme activity, assays were carried out

with 2 mg crude cell extracts (enzyme) in presence of co-factor and 50 ppm substrate

(Reactive Violet 5 dye). The experimental reactions were incubated till dye

degradation and control reactions were incubated for an extended period of three

hours.


161

Fig. 5.14: Agarose gel (1%) analysis of vectors. Lane 1: Supermix DNA ladder; Lane

2: Isolated pUC19 vector; Lane 3: pUC19 vector digested with BamHI and HindIII

enzymes; Lane 4: Isolated pET28a+ vector; Lane 5: pET28a+ vector digested with

BamHI and HindIII enzymes.

Fig. 5.15: Amplification of azoreductase gene using M13 primers. Lane 1:

Azoreducase gene; Lane 2: 100 bp ladder.

5 kb

3 kb

600 bp

1 2 3 4 5

1 2


162

Among several reported azoreductases, the enzymes of X. azovorans KF46F (Blumel

et al., 2002), P. kullae K24 (Blumel and Stolz, 2003), E. coli (Nakanishi et al., 2001),

Ent. faecalis (Chen et al., 2004) and R. sphaeroides AS1.1737 (Bin et al., 2004) use

NADH as electron donors. However, the enzymes of E. coli (Nakanishi et al., 2001)

and Ent. faecalis (Chen et al., 2004) are also FMN-dependent. On the other hand,

enzymes of B. sp. OY1-2 (Suzuki et al., 2001) and S. aureus (Chen et al., 2005) use

NADPH as electron donor. Consequently, to find out the co-factor required by the

cloned gene product, enzyme assays were carried out with different co-factors as

shown in Fig. 5.16. Co-factors FAD (Fig. 5.16a), FMN (Fig. 5.16b), NAD (Fig.

5.16c) and NADP.Na2 (Fig. 5.16d) did not activate the enzyme as only 6%, 4.7%, 8%

and 8.5% dye degradation was observed, respectively. Co-factor NADH.DPNH (Fig.

5.16e) did facilitate dye degradation but only 56%. On the other hand, co-factor

NADPH.Na4 (Fig. 5.16f) facilitated 85-90% dye degradation. A similar kind of fact

has also been reported earlier (Zimmerman et al., 1982; Blumel et al., 2002). They

had found azoreductases, which preferably used NADPH as co-factor and NADH

only when present in significantly higher Km values. Consequently, co-factor

NADPH.Na4 was best amongst all and was selected for further studies. To determine

the minimum amount of NADPH.Na4 required by the gene product, enzyme assays

with different concentrations of co-factor were carried out (Fig. 5.17). As we can see,

6.8%, 46.7%, 83% and 85.3% dye degradation was observed with 0 mM (Fig. 5.17a),

0.5 mM (Fig. 5.17b), 1 mM (Fig. 5.17c) and 1.5 mM (Fig. 5.17d) NADPH.Na4

concentrations, respectively. Thus it was clear that the cloned gene product requires at

least 1 mM of NADPH.Na4 for the desired activity.


163

f e

d

b a

c

Fig. 5.16: Dye degradation by pET1 E. coli BL21(DE3) clone with different co-

factors: (a) FAD, (b) FMN, (c) NAD, (d) NADP.Na2, (e) NADH.DPNH and (f)

NADPH.Na4. ( 0 min, 10 min, 20 min and 30 min)


164

d c

b a

Fig. 5.17: Dye degradation by pET1 E. coli BL21(DE3) clone at different

concentrations of NADPH.Na4 (a) 0 mM, (b) 0.5 mM, (c) 1 mM and (d) 1.5 mM.

( 0 min, 10 min and 20 min)


165

Azoreductase assays were carried out for cell extracts of clones carrying azoreductase

gene - E. coli DH10B clone pUC1, E. coli DH10B clone pET1 and E. coli

BL21(DE3) clone pET1. As controls, assays were also carried out for cell extracts of

E. coli DH5α, E. coli DH10B, E. coli BL21(DE3) and E. coli strains with vectors

pUC19 and pET28a+. The comparison of dye degradation capabilities of the

recombinant strains with the control strains is shown in Fig. 5.18. Strains such as E.

coli BL21(DE3) (Fig. 5.18a), E. coli DH5α (Fig. 5.18b) and E. coli DH10B (Fig.

5.18c) could degrade only 26.1%, 32.6% and 22.2% dye, respectively after 3 hours.

Strains with vectors pUC19 (Fig. 5.18d) and pET28a+ (Fig. 5.18e) could also

degrade only 23.5% and 24.4% dye, respectively after 3 hours. Whereas, recombinant

pUC19 with azoreductase gene transformed in E. coli DH10B named as E. coli

DH10B clone pUC1 (Fig. 5.18f) and recombinant pET28a+ with azoreductase gene

transformed in E. coli DH10B named as E. coli DH10B clone pET1 (Fig. 5.18g)

could degrade 24.4% and 59% dye, respectively after 3 hours. On the other hand,

recombinant plasmid, isolated from E. coli DH10B clone pET1, transformed in E. coli

BL21(DE3) named as E. coli BL21(DE3) clone pET1 (Fig. 5.18h) was able to

degrade 90% of dye within 7 minutes.

Thus it can be observed that only 20-30% of total dye was degraded with cell extracts

of strains such as E. coli DH5α, E. coli DH10B, E. coli BL21(DE3) and E. coli strains

containing vectors pUC19 and pET28a+ after 3 hours. Even cell extracts of E. coli

DH10B clone pUC1 and E. coli DH10B clone pET1 could degrade 24.4% and 59%

dye, respectively after 3 hours. In comparison to these controls and recombinant

clones in E. coli DH10B, E. coli BL21(DE3) clone pET1 was able to degrade 90% of

dye and that too within 7 minutes only. Moreover, this also shows that the selection of

right host strain is very important. Because when recombinant pET28a+ was

transformed in E. coli DH10B it could degrade only 59% of total dye in 3 hours.

Whereas, when the same was transformed in E. coli BL21(DE3) it was able to 90% of

dye and that too within 7 minutes only.


166

e f

d c

b a


167

h g

Fig. 5.18: Comparison of dye degradation by cloned gene product with experimental

controls: (a) E. coli B21(DE3), (b) E. coli DH5α, (c) E. coli DH10B, (d) Strain with

vector pUC19, (e) Strain with vector pET28a+, (f) pUC1 E. coli DH10B clone, (g)

pET1 E. coli DH10B clone and (h) pET1 E. coli BL21(DE3) clone. [ 0 min and

3 h for (a-g) / 7 min for (h)]

5.4 Conclusion

The present study helps us to understand the importance of consortium (mixed

cultures) in bioremediation processes. The cloned azoreductase gene, having a length

of 537 bp and containing an ORF of 178 amino acids, on phylogenetic analysis

clustered with azoreductase of Bacillus cereus Q1 (phylum Firmicutes). The

recombinant strains possessed in vitro azoreductase activities. However, they did not

show in vivo activities and reason can be attributed to the fact that recombinant E. coli

strains were unable to uptake azo dyes. The cloned azoreductase requires at least 1

mM of co-factor NADPH.Na4. In comparison to control strains (20-30% of total dye

in 3 hours) and E. coli DH10B recombinant clones (25-60% of total dye in 3 hours),

E. coli BL21(DE3) clone pET1 was able to degrade 90% of dye within 7 minutes

only. The expression analysis of azoreductase gene will be of immense help in

developing novel recombinant strains for application in dye contaminated waste-water

treatment technologies.


168

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strains: Bacillus sp. strain AK1 and Lysinibacillus sp. strain AK2. J Hazard

Mater. 190: 351-358.

Anliker, R. (1979) Ecotoxicology of dyestuffs-a joint effort by industry. Ecotox.

Environ Safety. 3: 59-74.

Bin, Y., Jiti, Z., Jing, W., Cuihong, D., Hongman, H., Zhiyong, S., Yongming, B.

(2004) Expression and characteristics of the gene encoding azoreductase from

Rhodobacter sphaeroides AS1.1737. FEMS Microbiol Lett. 236: 129-136.

Blumel, S., Knackmuss, H.J., Stolz, A. (2002) Molecular cloning and characterization

of the gene coding for the aerobic azoreductase from Xenophilus azovorans

KF46F. Appl Environ Microbiol. 68: 3948-3955.

Blumel, S., Stolz, A. (2003) Cloning and characterization of the gene coding for the

aerobic azoreductase from Pigmentiphaga kullae K24. Appl Microbiol

Biotechnol. 62: 186-190.

Chen, H., Hopper, S.L., Cerniglia, C.E. (2005) Biochemical and molecular

characterization of an azoreductase from Staphylococcus aureus, a tetrameric

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Chen, H., Wang, R.F., Cerniglia, C.E. (2004) Molecular cloning, overexpression,

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Chen, H., Xu, H., Kweon, O., Chen, S., Cerniglia, C.E. (2008) Functional role of Trp-

105 of Enterococcus faecalis azoreductase (AzoA) as resolved by structural and

mutational analysis. Microbiology. 154: 2659-2667.

Felsenstein, J. (1985) Confidence limits on phylogenies: An approach using the

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Kadam, A.A., Telke, A.A., Jagtap, S.S., Govindwar, S.P. (2011) Decolorization of

adsorbed textile dyes by developed consortium of Pseudomonas sp. SUK1 and

Aspergillus ochraceus NCIM-1146 under solid state fermentation. J Hazard

Mater. 189: 486-494.

Lowry, H.O., Rosenbrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein

measurement with the Folin phenol reagent. J Biol Chem. 193: 265-75.

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Documents

5.1 Introduction - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/7234/13/13_chapter 5.pdf · metabolism of azo dyes requires specific enzymes (aerobic azoreductases), which