Biochemical and molecular characterization of two novel

azodye- degrading microorganisms from contaminated ground

water and soil

In the present investigation, bacterial isolates were inoculated in to the media containing

various azodyes to study their biodegradability potential. The decolorization of various dyes was

measured spectrophotometrically after incubation of 24, 48 and 72 hours. Among all the isolates,

S1 and S2 exhibited strong decolorizing potential for five azodyes within 72 hours. These

isolates were later characterised as Achromobacter and Bacillus sp. through 16s rDNA gene

sequence analysis. The sequences were submitted to gene bank with accession number

AB936782 and AB936782. The bacterial isolates were found to contain plasmid DNA which

was effectively isolated by hot alkaline method. The presence of resistance traits on plasmids

was also evident from curing experiments. Among the different curing agent used, it was

observed that SDS and rifampicin at 9 mg/ml is effective in plasmid curing for these isolates.

Biochemical characterisation reveals that Achromobacter sp. is positive for tryptophanase, indole

and citrate permease and also able to ferment glucose with the production of gas. Bacillus sp.

showed presence of enzyme tryptophanase but negative for urease. The isolates were able to

sustain the salt concentration between 4%-10%, thus regarded as moderately halotolerant. In

order to check the heavy metal resistance, agar well diffusion method was used. The Bacillus sp.

was tolerant to heavy metal salts zinc and arsenic up to 8M; copper, cobalt and nickel to an

extent of 500mM. The efficient decolorizing activity in normal environmental condition

indicates the selected strain can be utilized in the treatment of effluents of dye industries.
Introduction

The rapid development of dye industry has accelerated dye production along with a series

of environmental hazards. The discharge of toxic effluents from various industries adversely

affects water resources, soil fertility, aquatic organisms and ecosystem integrity. Among various

industries, the textile dyeing industries discharge large volume of wastewater after the dyeing

process. Azo dyes are the largest and most diverse group of synthetic dyes and more than 50% of

annually produced dyes are belong to azo compounds (Szygula et al., 2008). Azo compounds

are solids of varying colour from yellow to red and violet to blue. The N=N group is called an

azo group and parent compound, HNNH, is called diimide. Azo dyes, being the largest group of

synthetic dyes, constitute up to 70% of all the known commercial dyes (Carliell et al., 1998). The

chemical structure of coloured dyes are characterized by highly substituted aromatic rings joined

by one or more azo groups (–N=N–). These substituted ring structures make these molecules

recalcitrant and, thus they are not degraded by conventional wastewater treatment processes.

These dyes released into the environment causes acute toxic effects on the flora and fauna of the

ecosystem. Moreover, many azo dyes and their metabolites are mutagenic and carcinogenic

(Chung et al., 1992). Recent studies have reported that azo dyes contribute to mutagenic activity

(Rajaguru et al., 2002). Thus, the colour removal from textile wastewater remains a major

environmental concern.

Several physicochemical and biological methods such as adsorption, flocculation-

coagulation and precipitation, photolysis, chemical oxidation and reduction have been used for

the removal of dyes from effluents. However the above mentioned ways for clean-up are

expensive, coupled with the formation of large amount of sludge and the emission of toxic

substances (Banat et al., 1996, Moreira et al., 2000, Mohanty et al., 2006). In contrast, biological
treatment provides a better alternative. Though many researches has been focused on selection of

microorganisms in degradation of azo dyes, still there is demand in finding an efficient microorganisms

which can be helpful in bioremediation process. Therefore the present study is focused on

isolation and identification of potent species capable of degrading of azo dyes, study their

potential in azo dye degradation, detection of plasmid associated with biodegradation and heavy

metal tolerance.

Materials and methods

Collection of sample and bacterial isolation

Water and soil sample was collected from the contaminated site of textile mills and used

for the isolation of bacteria capable of degrading the dyes.

Samples were enriched by making ten-fold serial dilution. 100µl of diluted sample was

spreaded on to Screening Media (SM), which was prepared by (g/l) Potassium dihydrogen

phosphate 3.0g, Disodium hydrogen phosphate (6.0g), Sodium chloride (5.0g), Ammonium

chloride (2.0g), Glucose (8.0g), Magnesium sulphate (0.1g). The plates were incubated at

37ºC for 48 hours. The colony with distinct morphology was picked and streaked to get single

isolated pure cultures.

Screening of micro-organisms capable of degrading dyes

Three different textile dyes; Navy blue, yellow, dark red and two laboratory stains; malachite

green and congo red were used for the microbial decolourization study. Different dyes, with final

concentration of 100mg/l, were added to the conical flasks containing SM broth and were

autoclaved at 121ºC and 1 atm for 40 minutes. A loop full of morphologically distinct bacterial

isolate was aseptically inoculated in to the flasks prepared as above. The flasks were incubated in

a mechanical shaker at 37±1ºC for 72 hours. Appropriate amount of the culture media was
withdrawn at 24 hours time intervals to study the biodegradation. Aliquots were centrifuged at

6000rpm for 20 minutes to separate bacterial cell mass. The clear supernatant was used to

measure the decolourization at the absorbance maxima (λmax) of the respective dyes (Malachite

green-500, Congo red-650, Navy blue-450, Yellow-590, Dark red-700). Abiotic control (without

bacterial inoculation) was always included. The percentage decolourization was calculated using

the following equation:

Ac − As
% 𝐷𝑒𝑔𝑟𝑎𝑑𝑎𝑡𝑖𝑜𝑛 = X 100
Ac

Where, Ac- absorbance of control, As- absorbance of sample

Morphological and biochemical characterisation

The colony morphology was studied with respect to colour, shape, size and pigmentation. The gram

staining was performed according to (Prescott, 2002). The biochemical test such as indole,

methyl red, Voges Proskauer, Simmons citrate test, urease test and carbohydrate fermentation

test (glucose, sucrose and lactose) were performed according to standard procedure ( Prescott,

2002).

PCR amplification and 16SrDNA sequence analysis

Bacterial genomic DNA was isolated by using DNA purification kit (Genie DNA Extraction

kit). The 16SrDNA was amplified by PCR using forward primer 5’ AGA GTT TGA TCC TGG

CTC AG 3’ and reverse primer 5’ AAG GAG GTG ATC CAG CC 3’. The reaction was

repeated for 30 cycles and the amplified product was sent for sequencing. The sequence data

were anlalysed using NCBI BLAST programme (www.ncbi.nlm.nih.gov/BLAST/) to identify

the bacteria and its closest neighbour based on the percentage of similarity.
Two organisms were sent to Bharat Biotech., Bangalore, for 16S rRNA sequencing. The

sequencing data were analysed using BLAST tool to identify the organism.

Plasmid isolation and curing

Plasmid isolation was carried out by hot alkaline lysis method (Ref.) . Isolated plasmid was

electrophoresed on 0.8 % Agarose gel to check its purity.

The 24 hours culture of S1 and S2 organism was used as inoculums for fresh SM broth (10 ml)

containing sodium dodecyl sulphate (SDS), and antibiotics like tetracycline, ampicillin and

rifampicin with varying concentrations (3mg/ml, 6mg/ml and 9mg/ml) and was incubated at

37°C for at least 72 hours. After incubation, the culture containing the highest concentration of

curing agent which still allowed visible growth was identified and was centrifuged for the

isolation of plasmid. A control, without any curing agent, was also run for cross check.

Heavy metal tolerance

S1 and S2 organisms were tested for heavy metal tolerance using various concentration

of CuSO4, ZnSO4, NaOAs3, NiSO4, CoCl2 by well diffusion method (Tarun Agarwal and

Rachna Singh, 2012). The heavy metal sensitivity at each concentration was expressed in terms

of mean of diameter of Zone of Inhibition (in mm).

The minimal inhibitory concentration (MIC) was determined by spreading the bacterial

culture uniformly onto the nutrient agar plates containing different heavy metals at different

concentrations based on their sensitivity.

Halotolerance
In order to test the capability to tolerate high salt concentration sodium chloride with different

concentration (4%, 7%, 10%, 13%, 16%, 18% and 20%) was added to the media (Tarun et al.,

2012). The tubes were inoculated with the 24 hour culture of S1 and S2 and incubated at 37◦C

overnight. The growth of organism was determined spectrophotometrically (600nm).

Result and Discussion

The percentages of the dye degradation by isolated organisms were calculated. The

isolated strains of Achromobacter and Bacillus, were able to degrade the azo dyes malachite

green, congo red, yellow SG H/c, navy blue 3G and dark red 28 (100mg/l concentration) to an

extent of 98%, 99.89%, 88%, 71% and 87.15% respectively Malachite green and congo red were

found to be degraded more efficiently with 99.89% and 98% degradation. S2 organism was

observed as more efficient in degrading all the dyes tested in the experiment.

The percentage of degradation of different dyes by both the isolates is represented in the

Figure No.1(a,b) and figure no.2(a-e).

 120

100

% of degradation
80

60 24 hours

40 48 hours
72 hours
20

Different dye types

Fig. 1(a) Graph showing % degradation by S1 organism at different time intervals

 120
100
% of degradation

80
60
40
20 24 hours
48 hours
0
72 hours

Different dyes type

Fig. 1(b) Graph showing % degradation by S2 organism at different time intervals

Morphological and biochemical characterization

Gram staining of S1 and S2 reveals as gram-negative and gram-positive rods. The result of

biochemical tests for both the isolates shows that isolate S1 can produce tryptophanase, indole

and citrate permease (Table 2). Isolate S2 showed presence of enzyme tryptophanase but both

the organism were negative for urease.

Table no.2 Biochemical characterization of S1 and S2 isolates

RESULT

BIOCHEMICAL TESTS S1 S2

Citrate Positive Positive

Indole Positive Negative

MR Positive Positive

VP Positive Positive

Triple Sugar Iron Positive; gas formation Negative

Urease Negative Negative

Molecular characterization

The sequence obtained from of 16s rRNA gene sequencing was used for homology search using
basic local alignment search tool (BLASTn). The result showed that 16s rRNA sequence of
isolate S1 is belongs to Achromobacter species (92% similarity) while isolate S2 having 98%
similarity to Bacillus species. The sequence was submitted to gene bank with accession No.
AB936782 and AB936783.

Plasmid curing

The bacterial resistance to antibiotics is a common phenotypic character that is often plasmid-

encoded. Among the different curing agent tested, it was observed that cells did not lose their

resistance towards rifampicin and SDS after treatment with the highest sublethal doses. However

no growth was observed at concentration above 9mg/ml. Concentration of 9mg/ml was therefore

chosen as the highest sublethal concentrations for rifampicin and SDS. The plasmid bands in
both the strains were detected clearly. From this experiment it was evident that the strainS1 and

S2 contain genes which are resistant to rifampicin and SDS.

Halophilic nature of microbes

The halophiles are the extremophiles that can tolerate heavy salt concentrations and moderately
halophilic organisms are those that can tolerate salt concentration from 0.5M to 2.5M. Being an
extremophile they possess huge genomic and metabolic diversity thus can be a useful for
detoxification and bioremediation of toxic compounds. Halophiles have been reported to be
involved in the dye decolourization (Ref).

The isolates (S1 and S2) were able to sustain the salt concentration between 4%-

10%, thus they were regarded as moderately halotolerant. From the growth analysis of isolated

strains in different salt concentration, it is observed that the growth rate of microbes is reduced

with increase in the percentage of salt in the media. The organism were found to be intolerant to

salt concentration above 10% ( Fig no.4a and 4b)

Test for halotolerance

1.8
1.6
Absorbance at 600nm

1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 5 10 15 20 25
Conc of NaCl ( %)

Fig. 4(b) Graph showing the growth of S1 organism at different concentrations

 Test for halotolerance
1.6

1.4

1.2
Absorbance at 600nm

0.8

0.6

0.4

0.2

0
0 5 10 15 20 25
Conc.NaCl ( %)

Fig. 4(b) Graph showing the growth of S1 organism at different concentrations

Heavy metal tolerance

The moderately halotolerant strain of Bacillus was tolerant to heavy metal salts such as

zinc and arsenic to an extent of 8M; copper, cobalt and nickel to an extent of 500mM.

In order to check the heavy metal resistance of the bacterial strain, agar well diffusion

method was used.

Table no.4 and Fig 6(a), displays the zone of inhibitions bacterial isolate against heavy metal

aqueous solutions used at various concentrations. The zone of inhibitions of S1 and S2 isolate

against various heavy metals at various concentrations are represented in table no .

Plasmid profiles of wild-type strains and their cured derivatives indicates that the loss of the
ability to decolorize azo dyes correlated to loss of a 3 kb plasmid, suggesting that the genes required for textile azo dye
degradation were located on this plasmid.
Figure. Evolutionary relationships of taxa

The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree
with the sum of branch length = 0.15786394 is shown. The evolutionary distances were
computed using the Maximum Composite Likelihood method [2] and are in the units of the
number of base substitutions per site. The analysis involved 15 nucleotide sequences. All
positions containing gaps and missing data were eliminated. There were a total of 1043 positions
in the final dataset. Evolutionary analyses were conducted in MEGA6 [3].

1. Saitou N. and Nei M. (1987). The neighbor-joining method: A new method for reconstructing
phylogenetic trees. Molecular Biology and Evolution 4:406-425.

2. Tamura K., Nei M., and Kumar S. (2004). Prospects for inferring very large phylogenies by
using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA)
101:11030-11035.
3. Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. (2013). MEGA6: Molecular
Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution30: 2725-2729.

Disclaimer: Although utmost care has been taken to ensure the correctness of the caption, the
caption text is provided "as is" without any warranty of any kind. Authors advise the user to
carefully check the caption prior to its use for any purpose and report any errors or problems to
the authors immediately (www.megasoftware.net). In no event shall the authors and their
employers be liable for any damages, including but not limited to special, consequential, or other
damages. Authors specifically disclaim all other warranties expressed or implied, including but
not limited to the determination of suitability of this caption text for a specific purpose, use, or
application.