The present disclosure described herein, in general, relates to methods to degrade nitrogen containing dyes by using microorganism consortium isolated from textile effluents and soil, subculture on mineral salt medium.
BACKGROUND
[002] Disposal of industrial dyes waste directly into streams, rivers etc.
without proper management system are one of the major challenges and has negative effect on water quality of surface water bodies.
[003] Bioremediation is the process that allows the possibility to degrade
or render less toxic by-products using natural microorganisms.
[004] US patent number, US5543324A is granted for method to degrade
nitrogen containing phenol compounds by using consortium containing microorganisms from the genera Arthrobacter, Avrobacterium and Pseudomonas. The said method completely degrade picric acid to the level where no aromatic degradation products can be detected.
[005] US patent number, US5877014A illustrates a new strain of
penicillium species used for bioremediation to degrade azo, anthraquinone, or triarylmethane dyes and other aromatic pollutants.
[006] European patent number, EP1210407B1 related to a invention
regarding a novel bacterial consortium EBC1000 and a method for remedying biologically a halogenated compound and recalcitrant toxic chemicals containing in industrial waste water, waste material and soil by using this consortium.

[007] The present invention is related to develop novel microbial
consortium containing Bacillus, Paenibacillus, Brevibacillus and Paenochrobactrum species.
[008] The novel consortium of microbes having ability to use Disperse
Red 167.1 (Rubine BL) as a sole carbon source and degrade this.
[009] The invention provides novel bacterial consortium that can be used
for simple and cost-effective bioremediation and removal of toxic dyes from soil and textile effluents.
SUMMARY
[0010] The present invention describes a methods to degrade nitrogen
containing dyes by using microorganism consortium isolated from textile effluents and soil, subculture on mineral salt medium.
[0011] In one embodiment microbes are isolated from a contaminated
environment on mineral salt medium.
[0012] In another embodiment a microbial consortium is prepared with the
six microbes isolated from the contaminated environment on mineral salt medium.
[0013] In yet another embodiment the contaminated environment is
contacted and maintaining with the microbial consortium for a time that is effective for the microbial consortium to degrade textile dye or related compounds.
[0014] In yet another embodiment the produced treated effluent is less
toxic as compared to contaminated environment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The foregoing detailed description of embodiments is better
understood when read in conjunction with the appended drawing. For the purpose of illustrating the disclosure, there is shown in the present document example

constructions of the disclosure; however, the disclosure is not limited to the specific methods and apparatus disclosed in the document and the drawings.
[0016] Figure 1 illustrates six potent dye decolorizing purified bacterial
isolates. (A) Isolate 1, (B) Isolate 2, (C) Isolate 3, (D) Isolate 6, (E) Isolate 11 and (F) Isolate 15.
[0017] Figure 2 illustrates phylogenetic tree corresponding to Isolate 1,
Isolate 2 and Isolate 3.
[0018] Figure 3 illustrates results of biocompatibility assay between six
potent identified dye decolorizing strains.
[0019] Figure 4 illustrates GC-MS spectrum of the extract obtained after
biodegradation of the dye.
[0020] Figure 5 illustrates plausible biodegradation pathway of the dye.
[0021] Figure 6 illustrates FTIR spectrum of control dye and extract
obtained after biodegradation of the dye.
DETAILED DESCRIPTION
[0022] Hereinafter, specific embodiments in conjunction with the
accompanying drawings specific embodiments of the present invention is described in detail. It should be noted that the technical features or combinations of technical features described in the following examples should not be considered in isolation, they may be combined with each other so as to achieve better technical effect.
[0023] In one implementation, a process for treating textile dyes in textile
effluent given.
[0024] In accordance with an embodiment of the present invention
bacterial strains are isolated from textile effluent and soil.
[0025] In another embodiment, isolated strains Bacillus cereus, Bacillus
sphaericus, Paenibacillus, Paenochrobactrumglaciei, Bacillus subtilis and

Brevibacilluspanacihumi collected on Mineral salt medium (MSM) and purify after serial dilution and streaking.
[0026] In one of the embodiment the purified strains preserved in the
refrigerator at 4 °C.
Determination of absorption maximum of dye
[0027] The textile dye, Rubine BL was studied for absorption spectrum by
UV-Vis spectroscopy. The UV-Vis absorption spectrum was recorded in a range of 200 nm to 900 nm. Absorption maximum wavelength ()wx) was found to be 469 nm. The absorption maximum wavelength found in the above experiment was used to determine the decolorization ability of bacterial isolates as well as the consortium.
Isolation and purification of dye decolorizing bacteria
[0028] Bacterial strains were isolated from the textile effluent samples and
soil samples using the protocol of Khalid et al. 2008. The purification of isolated bacterial strains was done by streaking it on Mineral salt medium (MSM) in pentagonal manner. After purification the isolated cultures were preserved in the refrigerator at 4 °C for successive decolorization and degradation study of selected textile dyes under investigation.
[0029] Referring now to Figure 1, six bacterial isolates which were
isolated and purified from the effluent samples and soil samples were named as Isolate 1, Isolate 2, Isolate 3, Isolate 6, Isolate 11 and Isolate 15.
Biochemical, morphological and physiological characterization of bacterial isolates
[0030] The six isolated and purified bacterial isolates were characterized
based upon their biochemical, morphological and physiological properties. Characteristics including form, surface of colonies on petri plate, colony color, gram reaction and enzymatic activity (catalase, indole, urease, oxidase test, Voges-proskauer and methyl red tests) were evaluated. The bacterial isolates obtained were then recognized by observing the characteristic features and

comparative analysis of the standard description of bacterial strains in Bergey's Manual of Determinative Bacteriology.
[0031] Referring now to Table 1, the biochemical, morphological and
physiological characterization of bacterial isolates identified Isolate 1, Isolate 2, Isolate 3, Isolate 6, Isolate 11 and Isolate 15 as Bacillus cereus AU 50 (KX034566), Bacillus sphaericus (KX034564), Paenibacillus pocheonensis (KX034565), Paenochrobactrum glacei (KX034561), Bacillus subtilis (KX034562) andBrevibacilluspanacihumi (KX034559), respectively.
Table 1. Biochemical, morphological and physiological characterization data of
six bacterial isolates

Bacterial Isolates Isolate 1 Isolate 2 Isolate 3 Isolate 6 Isolate 11 Isolate 15
Morphology Irregular Round Irregular Irregular Irregular Circular
Surface Smooth Smooth; Flat Undulate; Concentric Smooth Dry; Flat Ellipsoidal
Colony color White Off-
White Off-White White White Cream-White
Gram reaction Positive Positive Positive Positive Positive Positive
Catalase Test Positive Positive Positive Positive Positive Positive
Indole Test Negative Negative Negative Negative Negative Negative
Urease Test Positive Positive Negative Negative Negative Negative
Oxidase Test Positive Positive Positive Positive Positive Negative
Voges Proskauer Test Negative Negative Negative — Negative Negative
Methyl Red Test Negative Negative Negative — Negative Negative
Molecular Identification Bacillus cereus AU50 Bacillus
sphaericu
s Paenibacil
lus pocheonen
sis Paenoch robactru
m glaciei Bacillus subtilis Brevibacill
us panacihumi
NCBI
Accession No. KX0345 66 KX03456 4 KX034565 KX0345 61 KX03456
2 KX034559
GenBank
Accession
Number EF03268 2.1 AJ31189 3.1 JN006265. 1 - - -
Molecular characterization of bacterial isolates
[0032] The molecular characterization of six isolated and purified
bacterial isolates was done by PCR amplification of 16S rRNA gene. The

BLAST analysis was done on the obtained sequences by comparing it with the nucleotide database of GenBank database. On the basis of the highest identity score, the sequences were chosen and aligned with ClustalW.
[0033] Referring now to Figure 2A, the optimal tree with the sum of
branch length = 0.00631033 is shown. The analysis involved 11 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1192 positions in the final dataset. Referring now to Table 2, the culture, which was labeled as Sample-AB1 (Isolate 1) was found to be Bacillus cereus strain AU50 16S ribosomal RNA gene (GenBank Accession Number: EF032682.1) based on nucleotide homology and phylogenetic analysis.
[0034] Referring now to Figure 2B, the optimal tree with the sum of
branch length = 0.00565091 is shown. The analysis involved 11 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1244 positions in the final dataset. Referring now to Table 3, the culture, which was labeled as Sample-AB2 (Isolate 2) was found to be Bacillus sphaericus partial 16S rRNA gene (GenBank Accession Number: AJ311893.1) based on nucleotide homology and phylogenetic analysis.
[0035] Referring now to Figure 2C, the optimal tree with the sum of
branch length = 0.00596575 is shown. The analysis involved 11 nucleotide sequences. Codon positions included were lst+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 947 positions in the final dataset. Referring now to Table 4, the culture, which was labeled as Sample-AB_3 (Isolate 3) was found to be Paenibacillus sp. C-2 16S ribosomal RNA gene (GenBank Accession Number: JN006265.1) based on nucleotide homology and phylogenetic analysis.
[0036] Isolate 6, 12 and 15 were identified as Paenochrobactrum glaciei,
Bacillus subtilis and Brevibacillus panacihumi, respectively based on nucleotide homology and phylogenetic analysis.

[0037] In another embodiment, bacterial consortium is prepared by
inoculating all strains in the previously sterilized nutrient broth media and enriched for 24 h at 35 °C to 45 °C.
Table 2. List of sequence producing significant alignments (Isolate 1).

Accession Description Max
score Total score Query coverage Max ident
EF032682.1 Bacillus cereus strain AU50 16S ribosomal RNAgene 2180 2180 97% 99%
KM212993.1 Bacillus cereus strain GS5 16S ribosomal RNAgene 2174 2174 97% 99%
HM104648.1 Bacillus cereus strain 43-4 16S ribosomal RNAgene 2172 2172 97% 99%
AB795549.1 Bacillus cereus gene for 16S ribosomal RNA 2169 2169 97% 99%
KP772339.1 Bacillus cereus strain
PALB6 16S ribosomal RNA
gene 2169 2169 97% 99%
LN829562.1 Bacillus sp. AR141 partial 16SrRNAgene 2169 2169 97% 99%
KP411923.1 Bacillus cereus strain f24 16S ribosomal RNAgene 2169 2169 97% 99%
KP986945.1 Bacillus subtilis strain
PVR-YHB-1-1 16S
ribosomal RNA gene 2169 2169 97% 99%
KP165011.1 Bacillus cereus strain
NXUGDS005 16S ribosomal
RNA gene 2169 2169 97% 99%
KM596856.1 Bacillus sp. ADD 16S ribosomal RNA gene 2169 2169 97% 99%
Biocompatibility assay of efficient dye degrading bacteria for dyes
[0038] Synergistic activity among isolated bacterial species for the
development of consortium was analyzed through biocompatibility assay by following the method described by Nikam et al. 2007. Sterilized discs of whatmann paper no 1 impregnated with bacterial suspension of individual isolates were placed on petri plates already having growth of cultures inoculated in different pattern to check the antagonistic/syneristic effect of bacterial cultures on each other. Zone of inhibitions were visualized after 24 h.

[0039] Referring now to Figure 3, after 24 h of the growth in the
biocompatibility study no zone of inhibition was observed in any of the bacterial culture; hence all the bacterial cultures are compatible to each other and can be combined as a good consortium.
Table 3. List of sequence producing significant alignments (Isolate 2).

Accession Description Max
score Total score Query coverage Max ident
AJ311893.1 Bacillus sphaericus partial 16SrRNAgene 2272 2272 95% 99%
KP 192023.1 Arthrobacter siccitolerans strain BGSLP45 16S ribosomal RNA gene 2270 2270 95% 99%
KP192021.1 Lysinibacillus fusiformis strain BGSLP43 16S ribosomal RNA gene 2270 2270 95% 99%
KP 192020.1 Lysinibacillus fusiformis strain BGSLP42 16S ribosomal RNA gene 2270 2270 95% 99%
KP192018.1 Lysinibacillus fusiformis strain BGSLP40 16S ribosomal RNA gene 2270 2270 95% 99%
KP192017.1 Lysinibacillus fusiformis strain BGSLP39 16S ribosomal RNA gene 2270 2270 95% 99%
KP 192008.1 Lysinibacillus fusiformis strain BGSLP30 16S ribosomal RNA gene 2270 2270 95% 99%
KP191979.1 Lysinibacillus fusiformis
strain BGSLP1 16S
ribosomal RNA gene 2270 2270 95% 99%
KJ127184.1 Lysinibacillus sp. T2-4 16S ribosomal RNA gene, 2270 2270 95% 99%
HG798878.1 Uncultured bacterium partial 16S rRNAgene 2270 2270 95% 99%
Development of consortium
[0040] Consortium of bacterial isolates was made by using the protocol of
Lade et al. 2015 and Das et al. 2017'. The consortium thus obtained was used as the source of inoculum for further studies.
Preparation of cell free extract

[0041] Enzymatic activity of the individual bacterial isolate and the
consortium was assessed using the protocol of Telke et al. 2010 and More et al. 2011. To conduct these enzymatic activity assays a cell free extract was prepared. The cell free extract was prepared by first rupturing the cells by sonication and then centrifuging the mixture to obtain a clear solution containing the enzymes.
Table 4. List of sequence producing significant alignments (Isolate 3).

Accession Description Max
score Total score Query coverage Max ident
JN006265.1 Paenibacillus sp. C-2 16S ribosomal RNA gene 1784 1784 93% 99%
AB089250.1 Paenibacillus sp. M-2b gene for 16S ribosomal RNA 1759 1759 93% 99%
JQ738845.1 Uncultured bacterium clone S2_42 16S ribosomal RNA gene 1724 1724 93% 99%
HM027926.1 Paenibacillus sp. KUDC4121 16S ribosomal RNA gene 1718 1718 93% 99%
EU182895.1 Paenibacillus sp. MH67 16S ribosomal RNA gene 1701 1701 93% 98%
AM162318.1 Paenibacillus sp. HI0-02 partial 16S Rrna 1696 1696 93% 98%
JF119428.1 Uncultured bacterium clone ncdl360g09cl 16S ribosomal RNA gene 1696 1696 93% 98%
HQ424943.1 Paenibacillus sp. Upl 16S ribosomal RNA gene 1696 1696 93% 98%
AB576894.1 Paenibacillus sp. DSL09-3 gene for 16S rRNA 1696 1696 93% 98%
JX566546.1 Paenibacillus sp. 1105 16S ribosomal RNA gene 1690 1690 93% 98%
Table 5. Results of azo reductase activity assay.

Sr. No. Organism Activity
1 B. cereus 3.81±0.05
2 B. sphaericus 3.16 ±0.05
3 Paenibacillus 4.76 ±0.04
4 P. glacei 6.12±0.10
5 B. subtilis 0.91 ±0.04
6 B. panacihumi 4.92 ±0.03
7 Consortium 6.85 ±0.04

Table 6. Results of laccase activity assay.

Sr. No. Organism Activity
1 B. cereus 0.96 ±0.07
2 B. sphaericus 1.16 ±0.03
3 Paenibacillus 1.95 ±0.02
4 P. glacei 2.03 ±0.05
5 B. subtilis 0.79 ±0.03
6 B. panacihumi 1.83 ±0.70
7 Consortium 2.86 ±0.03
Azo reductase and Laccase activity assay
[0042] Azoreductase is the key enzyme which takes part in the reduction
of azo bond present in the synthetic dyestuffs. Many bacteria initiate the cleavage of the azo bonds by using NADH/NADPH as an electron donor. Laccases are industrially important enzymes which show potential to degrade azo bonded, polymeric and heterocyclic organic pollutants and used for bioremediation process. Laccases belongs to the multi copper oxidase family, biocatalyzes the oxidation reaction of electron rich synthetic chemical based organic substrates and degrades them into harmless species without releasing toxic metabolites. Therefore, to get further insight about the decolorization and degradation mechanism of selected dyes, screening of azo-reductase and laccase enzyme activities were checked.
[0043] Referring now to Table 5, the azo reductase activity was found to
be maximum i.e. 6.85 ± 0.04 in bacterial consortium when compared with the azo reductase activity in individual bacterial isolates.
[0044] Referring now to Table 6, the laccase activity was found to be
maximum i.e. 2.86 ± 0.03 in bacterial consortium when compared with the laccase activity in individual bacterial isolates.
[0045] In one of the embodiment the method is suitable to treat effluent
from textile industries containing azo dyes.
[0046] In accordance with an embodiment of the present invention, the
bacterial consortium can be used as an adsorbent in the form of columns, membranes, granules, beads and filters.

[0047] In another embodiment, the bacterial consortium has a specified
composition which enhances the dye degradation.
[0048] In another embodiment the bacterial consortium works on a culture
mineral salt culture medium.
[0049] The Table 7 represents comparision of decolorization rate of
different bacterial isolates with consortium for selected dye at 50 mg/L in 24 h. All the experiments were performed in triplicates and the average was calculated to represent the decolorization rate in percentage (%).
[0050] The Table 8 represents the phytotoxicity assay of selected azo-
disperse dyes at three different concentrations and the degraded metabolites obtained after its decolorization assay.
Dye decolorization assay
[0051] Dye decolorization assay was conducted to assess the potential of
the consortium to degrade the dye. Referring now to Table 7, percent decolorization was found to be maximum by the consortium when compared with the decolorization caused by individual bacterial isolates. The consortium showed decolorization percentage up to 82%. Therefore, consortium was found to be efficient in dye decolorization in comparison to individual isolates.
Table 7. Results of dye decolorization assay.

Sr. No. Organism Decolorization percent
1 B. cereus 73.51±0.46
2 B. sphaericus 73.15±0.44
3 Paenibacillus 72.80±0.32
4 P. glacei 72.16±0.28
5 B. subtilis 71.13±0.32
6 B. panacihumi 78.13±0.37
7 Consortium 82.76± 0.25

Table 8. Phytotoxicity assay of selected azo-disperse dyes at three different concentrations and the degraded metabolites obtained after its decolorization assay

Group Treatment Concentration (mg/mL) Germination
(%) Shoot length (cm) Root length (cm)
1st Tl 50 80 3.00±0.10** 4.73±0.20"*

150 70 2.53±0.05*" 3.03±0.20***

250 60 1.83±0.05*** 2.40±0.10***
2nd T2 - 90 3.20±0.20$ 4.03±0.15$$
Control T3 - 100 5.46±0.45 6.73±0.20
Phytotoxicity Assay
[0052] The phytotoxicity assay was performed to assess the toxicity of
selected dye and the degraded metabolites with respect to wheat variety PBW 550 (Triticum aestivum) collected from Punjab Agricultural University, Ludhiana, commonly used in Indian agriculture. Toxicity study was conducted by following the method of Parshetti etal. 2006. The studied was conducted to assess the effects of dye metabolites on seed germination, shoot and root length of Triticum aestivum seeds.
[0053] Referring now to Table 8, the results of the toxicity study showed
that there was a reduction of rate of germination of seeds treated with different concentration of dye. Less germination was observed on highest concentration of dye i.e. 250 mg/L. However, relatively more germination was observed in the seeds treated with dye degradation metabolites. The seeds of Triticum aestivum moistened with 50, 150 and 250 mg/L disperse red 167.1 solutions exhibited 80%, 70% and 60% germination. Disperse Red 167.1 affected the length of root and shoot compared to its metabolites obtained after decolorization and degradation. This effect was noticed on increasing dye concentration from 50 mg/L to 250 mg/L. The shoot length was 3.00±0.10, 2.53±0.05 and 1.83±0.05 cm at 50, 150 and 250 mg/L, respectively. In comparison, the extracted metabolite showed the growth of shoot to length

3.20±0.20 cm, which was significantly different in contrast to that of parent dye. Likewise, in case of root the length was maximum at 50 mg/L (4.73 ±0.20 cm) which decreased up to 2.40±0.10 at 250 mg/L. In comparision, the root length was 4.73±0.15 cm in case of extracted metabolite.
Analysis of degradation products by GC-MS
[0054] Degradation products present in the extract were identified through
GC-MS analysis. Referring now to Figure 4, peaks at m/z 167, 172, 292 and
250 were identified which were attributed to N,N-diethyl-benzene-1,4-
diamine, 2-chloro-4-nitro-phenylamine, {[4-amino-3-
(formylamino)phenyl]imino}diethane-2,l-diyl diformate and N4,N4-bis(2-methoxyethyl)-N2-methylbenzene-l,2,4-triamine using the standard NIST library. Referring now to Figure 5, a plausible degradation pathway was made on the basis of identified degradation products.
Analysis of biodegradation by FTIR
[0055] The FTIR spectra of disperse red 167.1 control dye was compared
with the spectra of consortium of bacteria (free cells) and 24 h extracted metabolites obtained after decolorization.
[0056] Referring now to Figure 6A, the FTIR spectrum of extracted
metabolites indicated the biodegradation of the parent dye. The peak in the FTIR spectrum of control dye in the range of 1595-1600 cnT1 for the presence of azo bond (-N=N-), 831 cnT1 for C-H deformation in alkanes and N-0 stretching in nitrites, 734-750 cnT1 for the presence of benzene ring, 619.17 cnT1 for C-N stretching in acyclic compounds, C-N vibrations at 1120.68 cnT1, peak at 3500-3400 cnT1 for -OH stretching vibration, peak at 1500-1400 cnT1 for aromatic C=C stretching frequency were identified. Similar functional groups were found on Rubine GFL by Waghmode et al. (2012) and Pan etal. (2017).
[0057] Referring now to Figure 6B, the peaks in FTIR spectra of bacteria
consortium at 968.3 cm1, 1057.03 cm1, 1220.98 cm1, 1452.45 cm1, and 1541.18 cm"1 corresponds to -C=0 stretching vibration, -C-H bending,

-COOH and -P=0 vibrations, -C=C- stretching vibrations, -C-H stretching vibrations respectively (Lin et al. 1998; Jiang et al. 2004). The metabolites found after degradation of disperse red 167.1 by consortium of bacteria showed peak at 1516.1 cnT1 for the presence of nitro (aromatic) compound -N=0- stretch, peak at 2,857.42-2,926.39 cm-1 for alkanes, 1141.9 cm-1 for C-H deformation (Jadhav et al. 2009). The disappearance of peak at (1595-1600 cm"1) showed azo bond reduction by azo-reductase enzyme during decolorization of disperse red 167.1 by consortium (Waghmode et al. 2012). Absence of major peaks from FTIR spectra of control dye in contrast to the metabolite obtained after decolorization of disperse red 167.1 displayed the action of enzymatic system of individual isolates in the form of consortium responsible for the biotransformation of disperse red 167.1 (Waghmode et al. 2012).
[0058] The following nonlimiting examples illustrate further aspects of the
invention.
EXAMPLE 1
[0059] The textile dye, Rubine BL was first studied for absorption
spectrum by UV-Vis double beam spectrophotometer (UV-1800, Shimadzu scientific works) in a range of 200 nm to 900 nm. Absorption maximum wavelength ()wx) was found to be 469 nm.
EXAMPLE 2
[0060] Bacterial strains were isolated from the textile effluent samples and
soil samples which were collected from affected area on Mineral salt medium (MSM) with serial dilutions from 10_1-10"3 in physiological saline using protocol of Khalid et al. 2008.
EXAMPLE 3
[0061] Bacterial isolates isolated in example 1 were purified by streaking
it again on MSM in pentagonal manner. The purification step was repeated thrice. After purification the isolated cultures were preserved in the

refrigerator at 4 °C for successive decolorization and degradation study of selected dyes under investigation. Results are shown in Figure 1.
EXAMPLE 4
[0062] The six selected bacterial isolates were further examined for their
biochemical and colony characteristics including form, surface of colonies on petri plate, colony color and gram reaction. The biochemical and physiological properties of the selected bacterial isolates were examined by catalase, indole, urease, oxidase test (Cheesbrough, 2006), Voges- proskauer and methyl red tests (Olutiola et al. 2000). The bacterial isolates obtained were then recognized by observing the characteristic features and comparative analysis of the standard description of bacterial strains in Bergey's Manual of Determinative Bacteriology. Results are shown in Table 1.
EXAMPLE 5
[0063] The 16S rRNA investigation was done to identify the bacterial
strain which is efficient in dye decolorizing. The isolate 1, 2 and 3 were sent to Samved Biotech Pvt. Ltd., Ahmedabad (Gujrat), India for identification of these bacterial strain. The PCR was performed on the isolated DNAto amplify the Fragment of 16S rDNA gene. The amplified DNA of 1500 bp size was resolved on the agarose gel and further analyzed for undue contaminants. For the amplification, the primers 27F (Forward) and 1492R (Reverse) were used with BDT v3.1 Cycle sequencing kit on ABI 3730x1 Genetic Analyzer. The BLAST analysis was done on the obtained sequences by comparing it with the nucleotide database of GenBank database. On the basis of the highest identity score, the sequences were chosen and aligned with ClustalW. RDP database was employed for generating Distance matrix whereas MEGA 4 was employed for constructing the phylogenetic tree. For assessing the evolutionary history, the NJ method was employed (Saitou and Nei, 1987). The evolutionary distances were calculated with the help of the Kimura 2-parameter method (Kimura, 1980) and units of the number of base substitutions per site.

EXAMPLE 6
[0064] The isolate 6, 11 and 15 were sent to Yaazh Xenomics, East
Chennai, Tamilnadu for molecular characterization. Genomic DNA of the Bacteria was extracted using the InstaGeneTM Matrix Genomic DNA isolation kit. The protocol involves the following steps: firstly, colony of the isolated bacterial was collected and suspended in 1ml of sterile water in a microfuge tube. After that the sample is centrifuged for 1 minute at 10,000 rpm, the supernatant obtained is then removed. 200 ul of Insta Gene matrix was added to pellet and the microfuge was incubated for 15 minutes at 56 °C. Sample is then vortexed at high speed for 10 seconds and after that it is placed for 8 minutes at 100 °C in water bath in order to give the heat shock. Again, the content obtained was vortexed at high speed for 10 seconds and centrifuged for 2 minutes at 10,000rpm. For the PCR reaction, 20ul of the supernatant was used to prepare the 50 ul PCR reaction mixture. Then, 16S rRNA region was amplified with the help of MJ Research Peltier Thermal Cycler. The Primer 518F/800R were used for the sequencing of the PCR product. Sequencing was done with ABI PRISM® BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq® DNA polymerase (FS enzyme) (Applied Biosystems). The 16s rRNA sequence obtained was analyzed with the help of blast tool by assessing the similarity score of the nucleotide which is already present in the NCBI Database. The phylogeny analysis of the closely related sequence obtained on performing blast was evaluated with multiple sequence alignment tool, MUSCLE ver. 3.7 was used (Edgar, 2004). The result obtained by aligning sequences were amended using the program Gblocks ver. 0.91b. Finally, for the phylogeny analysis, PhyML ver. 3.0 aLRT program was used and HKY85 was used as the Substitution model. For rendering the phylogenetic tree, program Tree Dyn ver. 198.3 was employed (Dereeper et al. 2008).
EXAMPLE 7

[0065] The assay was performed by following the method described by
Nikam et al. 2007. The selected bacterial isolates were spreaded on the growth media and allowed to grow for 24 h at 37 °C. Sterilized discs (whatmann paper no 1) of 5 mm size impregnated with bacterial suspension of individual isolates were placed at the distance of 5 mm from the periphery of petri plates already having growth of cultures inoculated in different pattern to check the antagonistic/syneristic effect of bacterial cultures on each other. Zone of inhibitions were visualized after 24 h. The results of the study are shown in Figure 3.
EXAMPLE 8
[0066] Consortium of bacterial was made by using the protocol of Lade et
al. 2015 and Das et al. 2017'. Initially a loop full of individual bacterial pure cultures were inoculated separately in minimal media and incubated for 24 h at 30 °C. For the development of bacterial consortium, 6 h old cultures were then transferred aseptically into nutrient medium followed by 24 h incubation at 30 °C.
EXAMPLE 9
[0067] Cell free extracts were prepared for enzyme activity assyas. The
bacterial cells were first subjected to centrifugation at 10,000 rpm for 15-20 min at 4 °C) and the resulted supernatant was used for screening of enzyme activities. The separated biomass of bacterial cells were suspended in 50 mM potassium phosphate buffer of pH 7.4, mildly homogenized and further sonicated by giving 12 strokes each of 30 sec with one min interval based on 60 amplitude keeping sonifier output below 4 °C. The subsequent extracts were then used as enzyme source (Telke et al 2010; More et al. 2011).
EXAMPLE 10
[0068] Azo reductase activity was assayed by modifying the protocol of
Telke et al. 2010. The 2 ml reaction mixture contained 4.45 uM of azo-disperse dye, 50 uM NADH and 1.2 ml of potassium phosphate buffer (20

mM, pH 7.5). The addition of NADH was done after pre-incubating the mixture for 4 min at 37 °C. The reaction mixture was monitored for the decrease in absorbance at 430 nm in UV-VIS spectrophotometer. The reaction was initiated by addition of 0.2 ml of the enzyme solution. Azo-disperse dye reduction was calculated by using its molar extinction coefficient of 0.023 uJVT1 cnT1. One unit of azoreductase enzyme activity was defined as quantity of enzyme required to catalyze 1 uM of substrate min-1 mg of protein1. All the enzyme assays were run in triplicates. The results of the study are shown in Table 5.
EXAMPLE 11
[0069] Laccase activity was monitored by the oxidation of ABTS using
the protocol of More et al. 2011 with some modifications. The assay mixture contained 2.8 mL of 0.5 mM ABTS in 200 mM sodium acetate buffer of pH 5.5 and 0.2 mL of enzyme and incubated for 5 min at 35 °C. The oxidation of ABTS was determined by observing the increase in spectrophotometer readings (420 nm, 3.6x104 M^cm-1) due to an intense bluish-green color read at 420 nm against a suitable blank. One unit of laccase enzyme activity was defined as the amount of enzyme required to oxidize 1 umol of ABTS substrate min"1 and expressed as units per gram of dry substrate (U gds1). All the enzyme assays were run in triplicates. The results of the study are shown in Table 6.
EXAMPLE 12
[0070] Individual bacterial isolates and their consortium were cultured for
24 h in 250 ml conical flasks having 100 ml MSM broth and was modified separately by different concentration of dye (50mg/L, 150 mg/L and 250 mg/L). The dye degradation competency of each isolates was examined under shaking conditions at 150 rpm with temperature of 37 °C in orbital shaking incubator (Khalid et al. 2008). After different interval of time, the 5 ml of culture media was taken and the sample was centrifuged for 10 min at 10,000 rpm for the separation of bacterial cell mass and supernatant. Thus,

supernatant obtained was used for the analysis of decolorization ability. The test was conducted in triplicates. Abiotic controls were also included. The absorbance of supernatants withdrawn at different intervals of time were determined at their maximum absorption wavelength for particular dye which is present in the visible region by UV-Vis double beam spectrophotometer (UV-1800, Shimadzu scientific works). All experiments were run in triplicates. The results of the study are shown in Table 7. The decolorization percentage was assessed by the following formula.
Decolorization Percent (%) = (l.A) - (OB) x 100
(LA)
Where, I.A= Initial Absorbance and O.B = Observed Absorbance
EXAMPLE 13
[0071] The supernatant obtained after dye decolorization assay contains
the degraded or biotransformed products of dye. These degraded or biotransformed products were extracted by centrifugation at 10,000 rpm for 20min at 4 °C. The resultant supernatant was further added to equal volume of ethyl acetate and mixed strongly to dissolve metabolites and dried over anhydrous Na2S04. Further solvent layer was air-evaporated in rotary vacuum evaporator. The remaining fractions were scrapped, collected and dissolved in 2-3 mL of HPLC grade methanol. Finally, the samples were filtered through 0.22 urn syringe filter (Millipore Millex with PES membrane) and then subjected to GC-MS analysis to confirm biodegradation of dyes according to the procedure of Lade et al. 2015. The GCMS analysis of the fractions was performed by using a Perkin Elmer MS Engine, equipped with integrated gas chromatograph with a HP1 column (30 m long, 0.25 mm id, nonpolar). The flow rate of carrier gas (Helium gas) was kept 1 ml per minute. The injector temperature was maintained at 280 °C with alterations in oven conditions as follows: Initially maintained at 80 °C kept constant for 2 min and increased up to 200 °C with 10 °C min-1 and finally raised up to 280 °C with 20 °C min"1

rate (Jadhav et al 2009; Kurade et al. 2012). The compounds obtained as a result were then identified on the basis of mass spectra and using the standard NIST library.
EXAMPLE 14
[0072] The sample was prepared by following the method as explained in
example 13. Further, the samples for FTIR analysis were prepared by mixing it with spectroscopically pure KBr in the form of pellets and analyzed by the methods described by Lade et al. 2012. To check the presence of different functional groups, FTIR analysis of selected dye was done in the mid IR region of 400-4000 cm"1 with 16 scan speed. FTIR spectrum is shown in Figure 6.
EXAMPLE 15
[0073] The effects of selected azo-disperse dyes and decolorized dye
metabolites on environmental ecology was monitored by evaluating the effects of dye metabolites on seed germination, shoot and root length of Triticum aestivum seeds. In the first treatment group the seeds of Triticum aestivum were moistened with 50, 150 and 250 mg/L disperse red 167.1 solutions. For the second treatment group the seeds of Triticum aestivum were moistened with the solution of degraded metabolites obtained after its decolorization assay. For control group the seeds of Triticum aestivum were moistened with distilled water. The toxicity was assessed by comparing the length of root and shoot in two treatment groups. Toxicity was also assessed by comparing the reduction in germination rate of seeds with respect to the control group. The results of the study are shown in Table 8.

WE claim:

1.A microbial consortium consisting two or more microbes selected from a group consisting of Bacillus cereus AU 50, Bacillus sphaericus, Paenibacillus pocheonensis, Paenochrobactrum glacei, Bacillus subtilis, and Brevibacillus panacihumi, said microbial consortium being capable of biodegrading textile dyes preferably Rubine BL dye.
2. The microbial consortium of claim 1 wherein said microbes are isolated from a contaminated environment on mineral salt medium.
3. The microbial consortium of claim 2 wherein said contaminated environment is a member of the group consisting of a water and an aqueous slurry of soil or other particulate matter contaminated by textile effluent.
4. A method of treating the contaminated environment, said method comprising contacting the contaminated environment with the microbial consortium and maintaining the microbial consortium in contact with the contaminated environment for a time that is effective for the microbial consortium to degrade the textile dyes identified by decolorization of the dye.
5. The method of claim 4 further comprising the step of sequestering the contaminated environment in a vessel to produce a treated effluent.
6. The method of claim 5 wherein the treated effluent is less toxic as compared to the contaminated environment.
7. The method of claim 4 further comprising the step of adding to the contaminated environment a member selected from the group consisting of a nutritional source of nitrogen and a nutritional source of phosphorous for the microbial consortium.

8. The method of claim 4 wherein the contaminated environment is maintained at a temperature within a range of 30 °C to about 45 °C.
9. The method of claim 4 wherein the contaminated environment is maintained at a pH within a range of 6 to about 8.
10. The method of claim 4 wherein the bacterial consortium can be used as an adsorbent in the form of columns, membranes, granules, beads or filters.