shah mp, patel ka, nair ss, darji am. molecular characterization … · 2013. 7. 12. · maulin p...
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Shah MP, Patel KA, Nair SS, Darji AM. Molecular characterization and optimization of Azo dye degrading Bacillus subtillis ETL-2013. OA Molecular & Cell Biology 2013 Apr 01;1(1):2.
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Molecular characterization and optimization of
Azo dye degrading Bacillus subtillis ETL-2013
Shah MP*, Patel KA, Nair SS, Darji AM
Industrial Waste Water Research Laboratory,
Division of Applied & Environmental Microbiology Lab
Enviro Technology Limited, Plot No: 2413/14,
GIDC, Ankleshwar-393002
Gujarat, India
Corresponding author. Maulin P Shah, Industrial Waste Water Research Laboratory, Applied &
Environmental Microbiology Lab, Enviro Technology Limited (CETP), Ankleshwar- 393 002, Gujarat, India, Tel:
+91-90 999 65504, Fax No: +91-2646-250707, E-mail: [email protected],
1) Abstract:
Triphenylmethane dyes belong to the most important group of synthetic colorants and are used
extensively in the textile industries for dying cotton, wool, silk, nylon, etc. They are generally
considered as the xenobiotic compounds, which are very recalcitrant to biodegradation. The
organism isolated from contaminated sites of textile industry soil sample that degrade bromo
phenol blue was subjected to 16s rRNA and the organism was characterized as Bacillus subtilis.
It was able to decolorize 88% of bromo phenol blue dye (5 mg/l) within 12hr at optimized
conditions pH 6, temperature 40ºC, and carbon source (glucose).
Keywords: Bromo phenol blue, decolorization, Bacillus subtilis ETL-1982
2) Introduction
Rapid industrialization has necessitated the manufacture and use of different chemicals in day to
day life (Shah et al., 2013). Reactive dyes, including many structurally different dyes, are
extensively used in the textile industry because of their wide variety of color shades, high wet
fastness profiles, ease of application, brilliant colors, and minimal energy consumption. The
three most common groups are azo, anthraquinone and phthalocyanine (Shah et al., 2013). A
major class of synthetic dyes includes the azo, anthroquinone dyes. Approximately 10,000
different dyes and pigments are used industrially and over 0.7 million tons of synthetic dyes are
produced annually worldwide. Azo dyes belong to the most important group of synthetic
colorants and are used extensively in the textile industries for dying cotton, wool, silk, nylon, etc.
Azo dyes, which are aromatic compounds with one or more(–N=N–) groups, accounts for the
majority of all textile dyestuffs produced and have been the most commonly used synthetic dyes
in the textile, food, paper making, printing, leather and cosmetic industries. They are generally
considered as the xenobiotic compounds, which are very recalcitrant to biodegradation (Zollinger,
1991; Stolz, 2001). During the dyeing processes about 10–90% of the dye stuff do not bind to the
fibers and therefore, released into the sewage treatment system or the environment (Zollinger
1991; Abdullah, et al., 2000). The inefficiency in dying process has resulted in 10-15% of
unused dye stuff entering the wastewater directly. Improper textile dye effluent disposal in
aqueous ecosystems leads to the reduction in sunlight penetration which in turn decreases
photosynthetic activity, dissolved oxygen concentration, water quality and depicts acute toxic
effects on aquatic flora and fauna, causing severe environmental problems worldwide. In
addition to their visual effect, triphenylmethane dyes and their metabolites are toxic,
carcinogenic, mutagenic, leading to potential health hazard to humankind Removal of hazardous
industrial effluents is one of the growing needs of the present time (Mittal et al. 2005). Dyes are
synthetic aromatic water-soluble dispersible organic Colorants, having potential application in
various industries (Mohan et al. 2002). The dye degradation of physical/chemical methods has
inherent drawbacks: economically unfeasible (more energy and chemicals), unable to remove the
recalcitrant triphenylmethane dyes and their organic metabolites completely from such effluents,
generate a significant amount of sludge may cause secondary pollution problems, substantially
increases the cost of these treatment methods and complicated procedures. Biological
degradation, being inexpensive and ecofriendly, is considered a valuable removal method for
many toxic pollutants. Several microorganisms, including a number of bacteria, yeast, and fungi,
have been investigated for their ability to biodecolorize triphenylmethane dyes (Wamik et al.
1988; Sani and Banerjee 1999; Cha et al. 2001; An et al. 2002; Liu et al. 2004; Jadhav and
Govindwar 2006; Ren et al. 2006b; Utkarsha et al. 2008). Biochemical studies of the
decolorization process indicate that laccase, peroxidase, and lignin peroxidase and
triphenylmethane reductase (TMR) are involved in the enzymatic decolorization of dyes (Tekere
et al. 2001; Shin and Kim 1998; Jang et al. 2005). This study aims to investigate the potential of
Bacillus subtillis isolated from textile soil for decolorizing a solution containing a bromo phenol
blue dye at various optimized conditions like temperature, pH, and carbon source and dye
concentration.
3) Experiment
3.1) Sample collection: Soil sample was collected from contaminated sites of Ankleshwar
textile industry, Ankleshwar, Gujarat, India. The collected soil sample was placed in a sterile
polythene bags and stored at 4ºC.
3.2) Isolation of bacteria: The soil sample collected from textile industry was subjected to
serial dilution (Madigan et al. 2000). From that 10-5 and 10-6 dilutions are plated on Nutrient agar
(peptone 5g/l, NaCl 5g/l, yeast extract 1.5g/l, beef extract 1.5g/l, agar20g/l temperature-25ºC,
pH7.4±0.2) by spread plate method. The single isolated colonies were sub cultured on LB (casein
enzymatic hydrolysate-10g/l, yeast extract-5g/l, sodium chloride-10g/l, agar-15g/l and ph(at
25)7.5±0.2) agar plates. And pure culture was maintained on LB (Luria Bertani) agar plates.
3.3) Dye Degradation assay: The purified two colonies from 10-5 dilution were streaked on the
screening media (Prepare 30ml LB agar medium and 10mg/lit bromophenolblue dye was added
to medium) and were kept for incubation for 2days for preliminary identification of dye
degrading bacteria. A control plate was also maintained for comparison.
3.4) Genomic DNA isolation: Method described by Krsek and Wellington (1999), with some
modifications. The enzymatic, chemical and mechanical lysis was performed. For the enzymatic
lysis, the overnight grown culture was suspended in 2 ml of a lysozyme solution (150mM NaCl,
100 mM EDTA, 5 mg/ml lysozyme). The tube was inverted several times to mix the contents
and placed in a 37°C water bath for 2hr. Subsequently, 500µl proteinase K (2.5mg/ml) was
added, and the contents of the tube gently mixed and placed in a 55°C water bath for 15min. For
the chemical lysis, 2ml SDS solution (500 mM NaCl, 500 mM Tris-HCl, 10% w/v SDS) was
added to the tube and the contents mixed by inversion several times for 5 min. Finally, for the
mechanical lysis process, the samples were subjected to three cycles of freezing in liquid
nitrogen and thawing at 100ºC. An equivalent volume (4.5ml) of phenol- chloroform-isoamyl
alcohol (25:24:1) was added and the mixture gently vortexed for 1 min. To precipitate DNA, 0.7
vol. cooled isopropanol and 1/10 vol. 3M sodium acetate were added to the supernatant. The
mixture was gently mixed (5-10 times) and kept at -20°C overnight. The sample was centrifuged
at 4100rpm for 10min, and the pellet was washed three times with cold 70% ethanol and then
resuspended in 100µlTE buffer 10/0.1 (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). For
visualizing the DNA extract, 10µl of each extract was electrophoresed on 1% agarose gel in 1X
TAE buffer, which were then stained with ethidium bromide and examined under UV light.
3.5) Strain characterization: The Genomic DNA was isolated from the overnight grown
bacterial culture was amplified with universal bacterial primers. The 25μl of reaction mixture
contains, 15µl of master mix (10X assay buffer, DNTP’s, Taq), 1µl of forward primer
(GGCGAACGGGTGAGTAA), 1µl of reverse primer (ACTGCTGCCTCCCGTAG), 2µl of
template DNA and 6µl of distilled water. PCR was carried out by the thermal cycler PCR under
the following conditions-- 30 cycles of 94ºC for 4min, 94ºC for 1min, 52ºC for 1min, 72ºC for
1.15min followed by final extension at 72ºC for 1min and holding temperature at 10ºC for 1min.
The amplified DNA fragments were observed by agarose gel electrophoresis in 2% agarose gel
and sequenced. The unknown bacterium was identified using GenBank database. Partial 16S
rRNA sequences obtained from isolates were assembled in a contig using the
phred/Phrap/CONSED program (Godon et al., 1997; Ewing et al., 1998). Identification was
achieved by comparing the contiguous 16S rRNA sequences obtained with the 16S rRNA
sequence data and type strains available in the public databases GenBank using the BLASTn
sequence match routines. The sequences were aligned using the CLUSTAL X program and
analyzed with the MEGA software 2001 (Thompson et al., 1994; Kumar et al., 2004).
Evolutionary distances were derived from sequence-pair dissimilarities, calculated as
implemented in MEGA, using Kimura’s DNA substitution model (Kimura, 1980). The
phylogenetic reconstruction was done using the neighbourjoining (NJ) algorithm, with bootstrap
values calculated from 1000 replicate runs, using the routines included in the MEGA software
(Saitou and Nei, 1987).
3.6) Decolorization experiments: Decolorization of dyes was determined as relative in decrease
in absorbance for each dye at their absorbance maximum at particular time interval. A loop full
of microbial culture (10µl) was inoculated in 250ml of Erlenmeyer flask containing 100ml LB
broth and incubated at 30ºC for 24hr. After 24hr of incubation, bromo phenol blue dye was
added at concentration of 5 mg/l and 3ml of the culture media was withdrawn at different time
intervals. Aliquot was centrifuged at 8000rpm for 10 minutes to separate the bacterial cell mass,
clear supernatant was used to measure the decolorization at the absorbance maxima of the
respective dyes. Abiotic controls (without microorganism) were always included. (Saratale, G.D,
Kalme, S.D and Govindwar, S.P, 2006). Controls were carried out in the same conditions but
without dyes or inoculum. Concentration of dyes were selected which obeys lamberts- beer law
The percentage decolorization was calculated as follows
% Degradation = Initial absorbance - Observed absorbance
_________________________________×100
Initial absorbance
3.7) Effect of initial dye concentration on decolorization: The decolorization of bromo phenol
blue blue was studied at different concentrations of the dye (5, 10, 20, 30, 40, 50 and 60 mg/l) as
described by (El-Naggar et al. 2004). The bromo phenol blue blue dye was added separately to
150 ml Erlenmeyer flasks containing 50 ml LB medium. The flask was inoculated with 1ml
Bcillus subtilis ETL-2013 (isolated from Industrial Textile soil). Dye absorbance was measured
by spectrophotometer at 620nm.
3.8) Effect of pH on decolorization: The effect of pH on bromophenolblue dye decolorization
was studied by performing the experiment at different pH values from pH 6-pH 10 using 1M
NAOH and 1M HCL and at 620nm absorbance was measured by spectrophotometer (Oranusi
and Ogugbue 2005).
3.9) Effect of Temperature on decolorization: For studying the effects of temperature on
decolorization of bromophenolblue dye, the bacterial culture was incubated at different
temperatures from 10ºC-40ºC. After 24hrs incubation the bromophenolblue dye was added
separately to cultures. Dye absorbance was measured by spectrophotometer at 620nm.
3.10) Effect of carbon source on decolorization: For enhancing the decolorization of
bromophenolblue was observed by addition of different carbon sources to culture media. The
rate of decolorization was decreased with increasing concentration of dye. Absorbance was
measured by spectrophotometer at 620nm (Oranusi, N.A. and Mbah, A.N. 2005).
4) Results and Discussion:
4.1) Dye Degradation assay:
In LB agar medium, after two days incubation the plates were observed for decolorization of the
dye at and around the culture position on comparison with control.
4.2) Genomic DNA isolation:
Result of isolated genomic DNA from dye industry. Agarose gel electrophoresis of genomic
DNA. Lanes 1 and 2 were isolated Genomic DNA from dye industry sample.
4.3) Effect of initial dye concentration on decolorization:
The decolorization of bromophenolblue was studied at various increasing concentration of dye
i.e. from 5, 10, 20, 30, 40, 50, 60 mg/L. The rate of decolorization was decreased with increasing
concentration of dye. 61% decolorization of bromophenolblue was observed at 5mg/lt,
concentration within 24 h respectively. Only 51%, 45%,36%,28%,23% and 22% decolorization
was observed at 10, 20, 30, 40, 50, 60 mg/L dye concentration respectively.
4.4) Effect of PH on decolorization: The effect of pH on dye decolorization was studied by
performing the experiment at pH6- pH10. 68% decolorization of BPB was observed at Ph8,
within 12h respectively. Only 64%. 63%, 60% and 39% decolorization was observed at ph
8,10,9 and 7 dye concentration respectively.
4.5) Effect of Temperature on decolorization: For studying the effects of temperature on
decolorization of Bromophenolblue the culture was incubated at 10-40ºC. 88% decolorization of
bromophenolblue was observed at 40ºC, concentration within 12h respectively. Only 83%, 52%,
27% and no decolorization was observed at temperature dye concentration respectively.
4.6) Effect of carbon source on decolorization: In LB medium, only 70% decolorization of
BPB was observed in 24 h. In an attempt to enhance decolorization performance with extra
supplements of carbon source in LB medium, found 92% decolorization was observed with
glucose in 12h. Only 58% and 46% decolorization observed at fructose and starch respectively.
Conclusions:
The present research indicates the potential aspects of Bacillus subtilis to 88% decolorize and
degrade bromophenol blue. The culture has ability to decolorize bromophenolblue in optimizing
the various parameters like pH, temperature and initial dye concentration within less time, which
is significant for its commercial and industrial application. The results showed that the
decolorization depend on dye concentration, pH and temperature. These results indicate that the
color removal by Bacillus subtilis may be largely attributed to biodegradation. Overall results
suggested the ability of Bacillus subtilis for the decolorization of azo dye and ensured the
ecofriendly degradation of bromophenolblue. It is proposed that Bacillus subtillis ETL-1982 has
a remarkable practical application potential in the biotransformation of various dye effluents
Acknowledgements: Authors are highly thankful to the management of Enviro Technology
Limited., Ankleshwar, Gujarat, India for allowing us to carry our such a noble work for the
sustainable environment.
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