For citation purposes:
Shah MP, Patel KA, Nair SS, Darji AM. Exploring the strength of Pseudomonas aeruginosa ETL-1942 in decolourisation and degradation of acid orange dye to combat textile effluent: applied aspects. OA Biotechnology 2013 Mar 01;2(2):12.
Biochemical Engineering & Bioprocess Engineering
Exploring the strength of Pseudomonas aeruginosa ETL-1942 in decolourisation and degradation of acid orange dye to combat textile effluent: applied aspects
In this study, an attempt was made to examine the potential of two bacterial strains for decolourisation of Acid Orange 10. The strain, isolated from textile effluent treatment plant was characterised on the basis of morphological, biochemical and genotypic characteristics,andit was identified as Pseudomonas aeruginosa and Bacillus cereus. The effect of pH, temperature and initial concentration of dye was studied with an aim to determine the optimal conditions.
Materials and methods
The bacterial strains used in this study were P. aeruginosa ETL-1942 and B. cereus ETL-1949. Out of these two, P. aeruginosa ETL-1942 emerged as the most potent decolouriser, being selected for further studies.
The selected bacterium shows higher decolourisation in the static condition compared to the shaking condition. The optimum pH was 7.0. It shows good decolourisation efficiency even in the alkaline region. The optimum temperature was 37°C. The strain could decolourise Acid Orange 10 (250 mg/l) by 94% within 24 hours under static conditions, pH 7.0, temperature of 37°C and initial dye concentration of 250 mg/l. Biodegradation and decolourisation was confirmed using UV-VISspectrophotometry, thin layer chromatography and fourier transform infrared spectroscopy analysis.
The study confirmed the potential of P. aeruginosa ETL-1942 in the bioremediation of Acid Orange 10.
Effluent discharged from the textile industries has variable characteristics in terms of pH, dissolved oxygen, organic and inorganic chemical content, etc. Together with industrialisation, awareness towards the environmental problems arising due to effluent discharge is of critical importance. Pollution caused by dye effluent is mainly due to durability of the dyes in wastewater. Existing effluent treatment procedures utilise pH neutralisation, coagulation followed by biological treatment, but they are unable to remove recalcitrant dyes completely from effluents. This is because of the colour fastness, stability and resistance of dyes to degradation. Dyes are difficult to biodegrade because of its synthetic origin and complex aromatic molecular structures which make them stable. Bioremediation is the microbial clean-up approach, microbes can acclimatise themselves to toxic wastes and new resistant strains develop naturally, which can transform various toxic chemicals to less harmful forms. Several reports have suggested that the degradation of complex organic substances can be brought about by bacterial enzymes[4,5,6,7,8,9]. Different dyes used in the textile industry usually have a synthetic origin and complex aromatic molecular structures, which make them more stable and difficult to be biodegraded. Due to their ease of manufacturing methodology, azo dye accounts for almost 80% of annual production of commercial dyes all over the world. There are over 100000 commercially available dyes with a production of over 7×105 tons per year3. Azo dyes, containing one or more azo bonds (-N=N-), account for 60%–70% of all textile dyestuffs used. It is estimated that about 10%–15% of the total production of colourants is lost during their synthesis and dyeing processes[11,12]. Whereas, in the case of reactive dyes almost 50% of the initial dye load is found in the dye bath effluents. Coloured industrial effluent is the most obvious indicator of water pollution and the discharge of highly coloured synthetic dye effluents is aesthetically displeasing and causes considerable damage to the aquatic life. Although several physical-chemical methods have been used to eliminate the coloured effluents in wastewater, they are generally expensive and produce large amounts of sludge.Usually these conventional modes of treatment lead to the formation of some harmful side products. Interest is therefore now focused on the microbial biodegradation of dyes as a better alternative. Some microorganisms, including bacteria, fungi and algae, can degrade or absorb a wide range of dyes.The biological mode of treatment of dye bath effluents offers distinct advantages over the conventional modes of treatment. This method is more economical and leads to less accumulation of relatively harmless sludge. Most importantly, biological treatment of dye bath effluents is eco-friendly. It causes mineralisation of dyes to simpler inorganic compounds which are not lethal to life forms. The basic step in the decolourisation and degradation of azo dyes is the breakdown of azo bonds, leading to removal of colour. Azo dyes are known to undergo reductive cleavage whereas the resultant aromatic amines are metabolised under aerobic conditions.So for complete mineralisation of azo dyes the microbial population forming a part of the treatment system should be able to work efficiently. In view of these problems, the most potent bacterial culture was selected in this study for maximum decolourisation of Acid Orange 10 (azo dye), being selected. The aim of this study was to explore the strength of P. aeruginosa ETL-1942 in the decolourisation and degradation of acid orange dye to combat textile effluent.
The protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed.
Sampling and analysis of effluent
Ankleshwar Textile Industries in Gujarat, one of the most industrialised statesin India,was chosen for effluent sample collection. The effluent sample was collected from the middle point of the area. Standard procedures (Spot and Grab) were followed during sampling. The temperature and pH were determined at the sampling site. The pH was determined using a pH meter (Hanna digital pH meter) and temperature with a laboratory thermometer. The sample was transported to the laboratory at 4°C as in accordance with the standard methods. The physicochemical parameters such as colour, biological oxygendemand (BOD),chemical oxygen demand (COD), total suspended solids and total dissolved solids (TDS) were determined as soon as the sample was brought to the laboratory. Sample colour was analysed by spectrophotometer (Shimadzu UV 1800). BOD was determined by employing evaporation method by DO meter while COD was measured by COD instrument directly.
Dyestuff and chemicals
The textile dye, Acid Orange 10 was a generous gift from the local textile industry in Ankleshwar, Gujarat, India and used for this study without any further purification. All the other chemicals used were of the highest purity available and of analytical grade.
The bacterial cultures were transferred to fresh nutrientmedium containing Acid Orange 10 (250 mg/l) and were incubated at 37°C, under static conditions for three days. After day 3, aliquots (5 ml) of the culture media were withdrawn,centrifuged at 10000 rpm for 10 minutes in a centrifuge at room temperature to separate the bacterial cell mass. The supernatant was used for analysis of decolourisation and all the experiments were repeated in triplicates. Absorbance of the supernatant withdrawn at different time intervals weremeasured at the absorbance maximumwavelength for the dye Acid Orange 10 (λ max =480 nm) in the visible region on a Shimadzu UV-Visible spectrophotometer (UV 1800).The percentage of decolourisation was calculated from the difference between initial and final values using the following formula:
The bacterial strain giving maximum decolourisationvalues was selected and used for further decolourisation experiments. Changes in CODand BOD were also studied using Standard Methods for Examination of Water and Wastewater APHA, 1995.
Conical flask assay
Conical flask assay was performed for the detection of decolourising activity of bacteria. The nutrient brothcontaining Acid Orange 10 was autoclaved at 121°C for 15 minutes. Five percentage inoculums of the selected culture showing maximum decolourising activity were added to nutrient brothflasks containing Acid Orange 10 (250 mg/l). The flaskswere covered with Aluminum foils and were incubated at 37°C for three days. The flasks were observed for decolourisation of the azo dye present in the medium.
Optimisation of parameters
In an attempt to study the effect of static and shaking (130 rpm) conditions, the selected most potent decolourisingbacterial culture was cultivated for 24 hours in a nutrient brothand amended separately with 250 mg/l of Acid Orange 10.To determine the effect of pH on decolourisation the fully grown culture was inoculated in conical flasks containing100 ml of nutrient broth of varying pH (5–10) and was amended with 250 mg/l of Acid Orange 10. The pH values were adjusted using 1N NaOH and 1N HCl. In a similarfashion, the optimum temperature of dye decolourisation by selected bacterium was determined by evaluating the dye decolourisation at 20°, 30°, 37°, 40°, 45° and 50°C. After differenttime intervals, an aliquot (5 ml) of the culture media was withdrawn and supernatants obtained after centrifugationwas used for analysis of decolourisation by Shimadzu UV-Visible spectrophotometer (UV 1800), according to themethods explained earlier.
The selected culture was cultivated for 24 hours in a conical flask containing 100 ml of nutrient broth. After 24 hours the media was amended with Acid Orange 10 at aconcentration of 100, 250, 500, 750 and 1000 mg/l separately to study the effect of increasing dye concentration on percentage dye decolourisation.
Biodecolourisation and biodegradation analysis
The analysis was done using UV-VIS spectrometry, thin layer chromatography (TLC)and fourier transform infrared spectroscopy (FTIR).The supernatants obtained after decolourisationwere extracted with dichloromethane and dried over anhydrous Na2SO4 and evaporated to dryness. The residue obtained was first examined by TLC. It is further subjected to FTIR spectroscopy.
Absorbance of the supernatant withdrawn at different time intervals was measured at the maximum absorption wavelength for Acid orange 10 (λ max = 480 nm) in the visible region on a Shimadzu UV-Visible spectrophotometer (UV 1800). The percentage of decolourisation was calculated from the difference between initial and final absorbance values.
Almost the full length of 16S rRNA genes of bacteria was amplified by PCR with the following sets of primers 5¢-GAGTTTGATCCTGGCTCAG-3¢ and 5¢-AAGGAGGTGATCCA GCC-3¢ corresponding to the positions 9–27 and 1525–1541, respectively in the 16S rRNA gene sequence of Escherichia coli. PCR products were sequenced directly using ABI PRISM Big Dye Terminator Cycle Sequencing Kit on an ABI 3100 DNA sequencer following the manufacturer’s instruction. Multiple alignments of the sequences were performed, and a neighbour-joining phylogenetic tree[18,19] was constructed using the latest version (ver. 1.8) of the CLUSTAL W program. Similarity values of the sequences were calculated by using the GENETYX computer program.
Extraction of biotransformed products
The supernatants after decolourisation, which might contain biotransformation products of the dyes, were extracted with dichloromethane and dried over anhydrous sodium sulphate. The solvent was evaporated and the residue was first examined by TLC. Fractions were collected and subjected to IR spectroscopy.
The results of 16S rDNA sequence alignment and phylogenetic tree analysis revealed that 16S rDNA sequence of strain ETL-1942 was 100% identical to that of Figure 1. The DNA-DNA hybridisation between strain ETL-1942 and a reference strain P. aeruginosa JCM 5962T was 96%. The taxonomic characteristics of strain ETL-1942 were mostly the same as those of P. aeruginosa JCM 5962T, that is, tests for production of catalase and oxidase, reduction of NO3 to NO2 and hydrolysis of casein and gelatin are positive, but o-nitrophenyl-b-D-galactopyranoside test and hydrolysis of starch were negative for both strains. However, ETL-1942 was not able to hydrolysethe lipids (supplied as tributyrin), maltose or D-mannose, all of which were hydrolysed by P. aeruginosa JCM 5962T. To verify identification of ETL-1949 as Bacillus cereus or Bacillus thuringiensis, this strain was characterised with phenotypic analysis. Strains ETL-1949 were positive for catalase, oxidase, positive for urease and the Voges-Proskauer reaction, and did not hydrolyse starch and Tween 80. B. cereus and Bacillus spp. are widely distributed in nature. Species of the genus Bacillus are rods, which sporulate in aerobic conditions. The endospores are resistant to heat, dehydration or other physical and chemical stresses.According to The Bergey’s manual of systematic bacteriology and considering the physiological and biochemical tests performed, the strain was tentatively named as Bacillus sp. strain ETL-1949. To confirm the identity of the isolate, PCR amplification and sequencing of the 16S rRNA gene were done. Dendrogram (Figure 2) showed phylogenetic relationships derived from 16S rRNA gene sequence analysis of strain ETL-1949 with respect to Bacillus species with validly published names. The tree was constructed using the neighbour-joining. Among the described sub-species, the closest relative of isolate ETL-1949 was Bacillus cereus. The strain ETL-1949 was a spore-forming, Gram-positive, rod-shaped, facultative anaerobic bacterium, which was motile by the means of one or two subpolar flagella. This strain grew well at various concentrations of NaCl ranging from 0% to 9% (w/v).
Phylogram (neighbour-joining method) showing genetic relationship between strain ETL-1942 and other related reference microorganisms based on the 16S rRNA gene sequence analysis.
Phylogram (neighbour-joining method) showing genetic relationship between strain ETL-1949 and other related reference microorganisms based on the 16S rRNA gene sequence analysis.
Evaluation of optimum conditions
The dye decolourisation of azo dye Acid Orange 10 was studied under static condition with an initial dye concentration of 250 mg/l using isolated bacterial culture of P. aeruginosa ETL-1942 and B. cereus ETL-1949. B. cereus ETL-1949 shows a percentage decolourisation value of 48% and P. aeruginosa ETL-1942 shows 94% decolourisation (Figure 3). Thus, P. aeruginosa ETL-1942 was selected for further decolourisation experiments, to see its decolourizing potential. It was observed that under static anoxic conditions, the dye decolourisation of Acid Orange 10 was 94% within 24 hours as compared to 44% under agitation, respectively (Figure 4). Hence, static conditions were preferred to investigate bacterial dye decolourisation in further experiments. The result bears similarity with those of studies on Pseudomonas desmolyticum and Pseudomonas luteola. It was found that under agitation conditions, presence of oxygen deprives the azoreductase from obtaining electrons needed for cleavage of azo dyes. Whereas under static conditions, these electrons are available to azoreductase from NADH to decolourise azo dyes[23,24]. The optimum pH for Pseudomonas spp. ETL-1942 was pH 7.0. However, Pseudomonas spp. ETL-1942 was also capable of decolourising the dye over a pH range of 7–9 with good efficiency (Figure 5). Majority of the azo dye reducing bacterial species were reported[25,26,27] were able to reduce the dye at a pH near 7. The decolourisation of the dye, Acid Orange 10 was studied with a temperature range of 20° to 50°C. The optimum temperature for P. aeruginosa ETL-1942 was 37°C (Figure 6). Considerable decrease in COD and BOD was also observed. The values for reduction in COD were 82% and BOD was 70%, respectively (Figure 7).
Percentage decolourization of Acid Orange 10 (250 mg/l) by Pseudomonas spp. ETL- 1942 and Bacillus spp. ETL-1949 at 37°C.
Effect of static and shaking condition on % decolourisation of Acid Orange 10 by P. aeruginosa ETL-1942.
Effect of pH on Acid Orange 10 decolourisation by P. aeruginosa ETL-1942.
Effect of temperature on Acid Orange 10 decolourisation by P. aeruginosa ETL-1942.
Percentage Reduction in COD and BOD values of dye caused by P. aeruginosa ETL-1942.
Effect of different concentration of dye on decolourisation
Percentage decolourisation of Acid Orange 10 by P. aeruginosa ETL-1942 was found to vary with initial concentrations (100–1000 mg/l) when studied up to 48 hours. The 94% decolourisation of Acid Orange 10 was observed within 12, 16 and 24 hours for the dye concentration of 100, 250 and 500 mg/l, respectively at optimum conditions of pH 7.0, temperature 37°C and under static batch study. However, for the dye Acid Orange 10 concentration of 750 mg/l, maximum decolourisation of 62% and for dye concentration of 1000 mg/l, only 50% of decolourisation was achieved (Figure 8).This is because of the toxic nature of azo dyes. The percentage decolourisation is found to be decreasing with an increase in dye concentration as evidentfrom Figure 6. The dye decolourising potential of P. aeruginosa ETL-1942 was quite high and it decolourises the dye with better efficiency even at high concentrations of Acid Orange 10. P. aeruginosa ETL-1942 was able to decolourise the dye at a concentration much higher than other bacterial strains[28,29,30]. P. aeruginosa ETL-1942 could tolerate Acid Orange 10 up to 1 g/l, which is in contrast to the toxic effect reported for the azo dye Acid Orange 10 in concentrations within 0.037–0.051 mM. This observation is of significance for bioremediation since it indicates the potential of P. aeruginosa ETL-1942 to withstand high concentration of the azo dye. Due to its high degrading potential, P. aeruginosa ETL-1942 could be used successfully in the treatment of textile wastewaters as they contain high concentration of azo dyes.
Effect of increasing dye concentration (static condition) on the percentage decolourisation of Acid Orange 10 by P. aeruginosa ETL-1942.
Identification of metabolic intermediates
Inoculation of P. aeruginosa ETL-1942 to media containing azo dyes resulted in the decolourisation of Acid Orange 10. The biodecolourisation was confirmed by UV-VIS spectrum. The absorbance was analysed from 300 to 800 nm. The initial dye solution showed a high peak at the wavelength of 480 nm. The decolourised sample showed lowering of the peak to a minimal absorbance value for dye concentration 250 mg/l, which indicates that the decolourisation is due to dye degradation (Figure 9). The dye degradation by P. aeruginosa ETL-1942 was further supported by TLC analysis. The spots observed in the initial dye solution were different from the spot observed in the supernatant obtained after decolourisation (Figure 10).The supernatant obtained after dye decolourisation was different from the original dye, which was suggested by different Rf values. This clearly indicates that decolourisation was due to degradation of dyes into intermediate products.
UV-Vis spectrum of Acid Orange 10, before and after decolourisation. Lowering of peak at the wavelength maximum (480 nm) of clear supernatant indicates azo cleavage by P. aeruginosaETL-1942.
TLC experiments shows different Rf values of control dye solution and decolourised samples.
Incubation of the azo dyes with P. aeruginosa ETL-1942 resulted in the decolourisation of Acid Orange 10 and the biotransformed metabolites were characterised by FTIR. The results of FTIR analysis of Acid Orange 10 and the sample obtained after decolourisation showed various peaks (Figure 11).The FTIR spectra of Acid Orange dye (Figure 11) displays peaks at 3483, 2929, 1660 and 1440 cm[-1] for -OH stretching vibration, aromatic -CH stretching vibration, -C=C- stretching and -N=N- stretching vibration, respectively. While peak near 1065 cm?1 is for -S=O, indicates the sulphoxide nature of the dye. The IR spectra of degradation product displays peak at 3263 cm[-1] for -OH stretching. During the degradation of aromatic amines of Acid Orange there is a formation of aromatic aldehyde as an intermediate, which was confirmed by the spot test using 2,4-dinitrophenyl hydrazine reagent which indicated colour tests due to presence of aldehyde. Besides the signal in IR at 1660 and 2929 cm[-1] which corresponds to aldehyde and a signal at 2869 cm[-1] for -CH stretching is similar to that of vanillin. Thus aldehyde, one of the intermediates, formed during degradation of Acid Orange is confirmed. The naphthalene part of the dye was further biodegraded with opening of one ring, the formation of aldehyde as one of the intermediate is confirmed from the IR data.
FTIR spectrum of Acid Orange 10 and its degradation product.
Industrial effluent is not stable and it varies often in a wide range depending upon the process practiced. South-Asian countries are experiencing severe environmental problems due to rapid industrialisation. This phenomenonis very common where the polluting industries like textiledyeing, leather tanning, paper and pulp processing, sugar manufacturing, etc.thrive as clusters. Among these, the textile industries are large industrial consumers of water as well as producers of waste water. The effluent dischargedby this industry leads to serious pollution of groundwater, soils and ultimately affects the livelihood of the poor. The physicochemical characterisationof the collected textile effluent sample from textile industries in Ankleshwar showed a high load of pollution indicators. Colour is contributed to a water body by the dissolved compounds (dyes and pigments). The effluent colour was black due to a mixture of various dyes and the sample was slightly alkaline when compared to the acidic pH of the dyeing effluent in a previous study. The pH of the effluent alters the physicochemical properties of water which, in turn, adversely affects aquaticlife, plants and humans. The soil permeability gets affected resulting in polluting underground resources of water. The temperature of the effluent was high in comparison with the temperature of another effluent in one study. High temperature decreases the solubility of gases in water, which is ultimately expressed as high BOD/COD. The values of BOD and COD were within the permissible limits in the present sample in comparison to the very high values of BOD and COD inone effluent study. TDS and total suspended solids values of effluent sample were higher than the permissible limits. Sediment rate is drastically increased because of high value of TDS reducing the light penetration intowater and ultimately decreasing photosynthesis. The decrease in photosynthetic rate reduces the DO level of waste water, which results in decreased purification of waste water by microorganisms. Thecurrent sample exhibited high values of heavy metals which were of the same order of magnitude reported in another effluent sample. The nutrients of the surrounding soils are depleted as a result of the high value of heavy metals thereby affecting soil fertility. High chloride contents are harmful for agricultural crops if such wastes containing high chlorides are used for irrigation purposes. The majority of the textile effluent samples have permissible limits of sulphate ions. The effluent showed phenolic contents greater than 0.1 ppm which is the permissible limit of the phenolic compounds but these compounds are very toxic to fish even at very low concentrations. The bleaching anddyeing process are the main causes of pollutants which include caustic soda, hypochlorite and peroxides.
This study confirms the ability of the newly isolated bacterial culture P. aeruginosa ETL-1942 to decolourise Acid Orange 10 with decolourisation efficiency of 94%, thus suggesting its application for decolourisation of dye-bearing industrial wastewaters.Although decolourisationis a challenging process for the textile industry and for wastewater treatment, the result of this finding and literature suggests a great potential for bacteria to be used to remove colour from dye wastewaters. Interestingly, the bacterial species used in carrying out the decolourisation of Acid Orange 10 in this study was isolated from the textile dye industry waste effluent. The ability of the strain to tolerate and decolourise azo dyes at high concentration gives it an advantage for treatment of textile industry wastewaters. However, potential of the strain needs to be demonstrated for its application in treatment of real dye-bearing wastewaters using appropriate bioreactors.
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