exploring the strength of pseudomonas aeruginosa … · exploring the strength of pseudomonas...

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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. Com

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Exploring the strength of Pseudomonas aeruginosa ETL-1942 in decolourisation and degradation of acid

orange dye to combat textile effluent: applied aspectsMP Shah*, KA Patel, SS Nair, AM DarjiBi

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AbstractIntroduction In this study, an attempt was made to examine the potential of two bac-terial 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, and it was identified as Pseudomonas aerugi-nosa and Bacillus cereus. The effect of pH, temperature and initial con-centration 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 decol-ouriser, being selected for further studies. Results The selected bacterium shows higher decolourisation in the static condi-tion compared to the shaking con-dition. The optimum pH was 7.0. It shows good decolourisation effi-ciency 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 ini-tial dye concentration of 250 mg/l.

Biodegradation and decolourisation was confirmed using UV-VIS spectro-photometry, thin layer chromatogra-phy and fourier transform infrared spectroscopy analysis. Conclusion The study confirmed the potential of P. aeruginosa ETL-1942 in the biore-mediation of Acid Orange 10.

IntroductionEffluent discharged from the textile industries has variable character-istics in terms of pH, dissolved oxy-gen, organic and inorganic chemical content, etc. Together with indus-trialisation, 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 wastewater1. Existing effluent treatment procedures uti-lise pH neutralisation, coagulation followed by biological treatment, but they are unable to remove recal-citrant dyes completely from efflu-ents. This is because of the colour fastness, stability and resistance of dyes to degradation2. Dyes are dif-ficult to biodegrade because of its synthetic origin and complex aro-matic molecular structures which make them stable3. 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 degrada-tion of complex organic substances can be brought about by bacterial enzymes4–9. Different dyes used in the textile industry usually have a

synthetic origin and complex aro-matic molecular structures, which make them more stable and diffi-cult 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 100 000 commercially available dyes with a production of over 7 × 105 tons per year3. Azo dyes, contain-ing one or more azo bonds (-N=N-), account for 60%–70% of all textile dyestuffs used10. It is estimated that about 10%–15% of the total produc-tion of colourants is lost during their synthesis and dyeing processes11,12. Whereas, in the case of reactive dyes almost 50% of the initial dye load is found in the dye bath effluents. Col-oured industrial effluent is the most obvious indicator of water pollution and the discharge of highly coloured synthetic dye effluents is aestheti-cally displeasing and causes consid-erable damage to the aquatic life. Although several physical–chemical methods have been used to eliminate the coloured effluents in wastewa-ter, 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. Inter-est is therefore now focused on the microbial biodegradation of dyes as a better alternative13. Some micro-organisms, including bacteria, fungi and algae, can degrade or absorb a wide range of dyes14. The biological mode of treatment of dye bath efflu-ents offers distinct advantages over the conventional modes of treat-ment. This method is more economi-cal and leads to less accumulation

*Corresponding authorEmail: [email protected]

Applied & Environmental Microbiology Lab, Enviro Technology Limited (CETP), GIDC, Ankleshwar, Gujarat, India

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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.

of relatively harmless sludge. Most importantly, biological treatment of dye bath effluents is eco-friendly. It causes mineralisation of dyes to sim-pler inorganic compounds which are not lethal to life forms. The basic step in the decolourisation and degrada-tion of azo dyes is the breakdown of azo bonds, leading to removal of col-our. Azo dyes are known to undergo reductive cleavage whereas the resultant aromatic amines are metab-olised under aerobic conditions15. So for complete mineralisation of azo dyes the microbial population form-ing 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 decolouri-sation of Acid Orange 10 (azo dye), being selected. The aim of this study was to explore the strength of Pseu-domonas aeruginosa ETL-1942 in the decolourisation and degradation of acid orange dye to combat textile effluent.

Material and methodsThe 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 effluentAnkleshwar Textile Industries in Gujarat, one of the most industrial-ised cities in India, was chosen for effluent sample collection. The efflu-ent sample was collected from the middle point of the area. Standard procedures (Spot and Grab) were followed during sampling. The tem-perature and pH were determined at the sampling site. The pH was deter-mined using a pH meter (Hanna digi-tal pH meter) and temperature with a laboratory thermometer. The sample was transported to the laboratory at 4°C as in accordance with the stand-ard methods16. The physicochemical parameters such as colour, biological oxygen demand (BOD), chemical oxy-gen demand (COD), total suspended

solids and total dissolved solids (TDS) were determined as soon as the sample was brought to the labo-ratory. 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 chemicalsThe textile dye, Acid Orange 10 was a generous gift from the local textile industry in Ankleshwar, Gujarat, India and used for this study with-out any further purification. All the other chemicals used were of the highest purity available and of analytical grade.

Experimental methodsThe bacterial cultures were trans-ferred to fresh nutrient medium con-taining 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 10 000 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 experi-ments were repeated in triplicates. Absorbance of the supernatant with-drawn at different time intervals were measured at the absorbance maximum wavelength 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 decolouri-sation was calculated from the differ-ence between initial and final values using the following formula:

% Decolourisation =

Initial absorbance value

final absorba

−nnce value

Initial absorbance value× 100

The bacterial strain giving maximum decolourisation values was selected and used for further decolourisation

experiments. Changes in COD and BOD were also studied using Stand-ard Methods for Examination of Water and Wastewater APHA, 1995.

Conical flask assayConical flask assay was performed for the detection of decolourising activ-ity of bacteria. The nutrient broth containing Acid Orange 10 was auto-claved at 121°C for 15 minutes. Five percentage inoculums of the selected culture showing maximum decolour-ising activity were added to nutrient broth flasks containing Acid Orange 10 (250 mg/l). The flasks were cov-ered with Aluminum foils and were incubated at 37°C for three days. The flasks were observed for decolouri-sation of the azo dye present in the medium.

Optimisation of parametersIn an attempt to study the effect of static and shaking (130 rpm) con-ditions, the selected most potent decolourising bacterial culture was cultivated for 24 hours in a nutrient broth and amended separately with 250 mg/l of Acid Orange 10. To deter-mine the effect of pH on decolouri-sation the fully grown culture was inoculated in conical flasks contain-ing 100 ml of nutrient broth of vary-ing 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 similar fashion, the optimum temperature of dye decol-ourisation by selected bacterium was determined by evaluating the dye decolourisation at 20°, 30°, 37°, 40°, 45° and 50°C. After different time intervals, an aliquot (5 ml) of the cul-ture media was withdrawn and super-natants obtained after centrifugation was used for analysis of decolourisa-tion by Shimadzu UV-Visible spectro-photometer (UV 1800), according to the methods explained earlier.

Concentration studiesThe selected culture was culti-vated for 24 hours in a conical

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flask containing 100 ml of nutrient broth. After 24 hours the media was amended with Acid Orange 10 at a concentration 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 analysisThe analysis was done using UV-VIS spectrometry, thin layer chroma-tography (TLC) and fourier trans-form infrared spectroscopy (FTIR). The supernatants obtained after decolourisation were 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.

Analytical methodsAbsorbance of the supernatant with-drawn at different time intervals was measured at the maximum absorp-tion wavelength for Acid orange 10 (λ max = 480 nm) in the visible region on a Shimadzu UV-Visible spectropho-tometer (UV 1800). The percentage of decolourisation was calculated from the difference between initial and final absorbance values.

Phylogenetic analysis Almost the full length of 16S rRNA genes of bacteria was amplified by PCR with the following sets of prim-ers 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 coli17. PCR products were sequenced directly using ABI PRISM Big Dye Terminator Cycle Sequencing Kit on an ABI 3100 DNA sequencer following the manufactur-er’s instruction. Multiple alignments of the sequences were performed, and a neighbour-joining phyloge-netic tree18,19 was constructed using the latest version (ver. 1.8) of the CLUSTAL W program20. Similarity

values of the sequences were calcu-lated by using the GENETYX com-puter program.

Extraction of biotransformed productsThe supernatants after decolourisa-tion, which might contain biotrans-formation products of the dyes, were extracted with dichloromethane and dried over anhydrous sodium sul-phate. The solvent was evaporated and the residue was first examined by TLC. Fractions were collected and subjected to IR spectroscopy.

ResultsIdentification of strains ETL-1942 and ETL-1949 The results of 16S rDNA sequence alignment and phylogenetic tree anal-ysis revealed that 16S rDNA sequence of strain ETL-1942 was 100% iden-tical to that of Figure 1. The DNA–DNA hybridisation bet ween strain ETL-1942 and a reference strain Pseudomonas aeruginosa JCM 5962T was 96%. The taxonomic character-istics of strain ETL-1942 were mostly

the same as those of P. aeruginosa JCM 5962T, that is, tests for produc-tion of catalase and oxidase, reduc-tion 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 hydrolyse the 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 char-acterised 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 nature21. Species of the genus Bacillus are rods, which sporulate in aerobic condi-tions. The endospores are resistant to heat, dehydration or other physi-cal and chemical stresses. According to The Bergey’s manual of system-atic bacteriology and considering the

Figure 1: Phylogram (neighbour-joining method) showing genetic relationship between strain ETL-1942 and other related reference microorganisms based on the 16S rRNA gene sequence analysis.

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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. Den-drogram (Figure 2) showed phyloge-netic 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-joining22. Among the des-cribed sub-species, the closest relative of isolate ETL-1949 was B. cereus. The strain ETL-1949 was a spore-forming, Gram-positive, rod-shaped, faculta-tive 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).

Evaluation of optimum conditionsThe dye decolourisation of azo dye Acid Orange 10 was studied under static condition with an ini-tial 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 decolourisa-tion value of 48% and P. aeruginosa ETL-1942 shows 94% decolourisa-tion (Figure 3). Thus, P. aeruginosa ETL-1942 was selected for further decolourisation experiments, to see its decolourising potential. It was

observed that under static anoxic conditions, the dye decolourisa-tion 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 bac-terial dye decolourisation in fur-ther 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 elec-trons needed for cleavage of azo dyes. Whereas under static condi-tions, these electrons are available to azoreductase from NADH to decol-ourise azo dyes23,24. The optimum pH for Pseudomonas spp. ETL-1942 was pH 7.0. However, Pseudomonas spp. ETL-1942 was also capable of decol-ourising the dye over a pH range of 7–9 with good efficiency (Figure 5). Majority of the azo dye reducing bacterial species were reported25–27 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

Figure 2: Phylogram (neighbour-joining method) showing genetic relationship between strain ETL-1949 and other related reference microorganisms based on the 16S rRNA gene sequence analysis.

Figure 3: Percentage decolourisa-tion of Acid Orange 10 (250 mg/l) by Pseudomonas spp. ETL-1942 and Bacillus spp. ETL-1949 at 37°C.

Figure 4: Effect of static and shak-ing condition on % decolourisation of Acid Orange 10 by P. aeruginosa ETL-1942.

Figure 5: Effect of pH on Acid Orange 10 decolourisation by P. aeruginosa ETL-1942.

Figure 6: Effect of temperature on Acid Orange 10 decolourisation by P. aeruginosa ETL-1942.

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in COD and BOD was also observed. The values for reduction in COD were 82% and BOD was 70%, respectively (Figure 7).

Effect of different concentration of dye on decolourisationPercentage 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, respec-tively 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 decolouri-sation of 62% and for dye concen-tration of 1000 mg/l, only 50% of decolourisation was achieved (Figure 8). This is because of the

toxic nature of azo dyes. The per-centage decolourisation is found to be decreasing with an increase in dye concentration as evident from 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 concentra-tion much higher than other bacterial strains28–30. 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 mM31. 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.

Identification of metabolic intermediatesInoculation of P. aeruginosa ETL-1942 to media containing azo dyes resulted in the decolourisation of Acid Orange 10. The biodecolouri-sation 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 concentra-tion 250 mg/l, which indicates that the decolourisation is due to dye deg-radation (Figure 9). The dye degrada-tion 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

Figure 7: Percentage Reduction in COD and BOD values of dye caused by P. aeruginosa ETL-1942.

Figure 8: Effect of increasing dye con-centration (static condition) on the percentage decolourisation of Acid Orange 10 by P. aeruginosa ETL-1942.

Figure 10: TLC experiments shows different Rf values of control dye solution and decolourised samples.

Figure 9: UV-Vis spectrum of Acid Orange 10, before and after decol-ourisation. Lowering of peak at the wavelength maximum (480 nm) of clear supernatant indicates azo cleavage by P. aeruginosa ETL-1942.

by different Rf values. This clearly indicates that decolourisation was due to degradation of dyes into inter-mediate products.

Spectroscopic analysisIncubation of the azo dyes with P. aeruginosa ETL-1942 resulted in the decolourisation of Acid Orange 10 and the biotransformed metabolites were character-ised by FTIR. The results of FTIR analysis of Acid Orange 10 and the sample obtained after decol-ourisation showed various peaks (Figure 11). The FTIR spectra of

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Acid Orange dye (Figure 11) dis-plays peaks at 3483, 2929, 1660 and 1440 cm–1 for -OH stretching vibration, aromatic -CH stretch-ing vibration, -C=C- stretching and -N=N- stretching vibration, respec-tively. While peak near 1065 cm–1 is for -S=O, indicates the sulphox-ide nature of the dye. The IR spec-tra of degradation product displays peak at 3263 cm–1 for -OH stretch-ing. During the degradation of aro-matic amines of Acid Orange there is a formation of aromatic alde-hyde as an intermediate, which was confirmed by the spot test using 2,4-dinitrophenyl hydrazine reagent which indicated colour tests due to presence of alde-hyde. Besides the signal in IR at 1660 and 2929 cm–1 which corre-sponds 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 naphtha-lene part of the dye was further biodegraded with opening of one ring, the formation of aldehyde as one of the intermediate is con-firmed from the IR data.

DiscussionIndustrial effluent is not stable and it varies often in a wide range depend-ing upon the process practiced. South-Asian countries are experienc-ing severe environmental problems due to rapid industrialisation. This

phenomenon is very common where the polluting industries like textile dyeing, leather tanning, paper and pulp processing, sugar manufactur-ing, etc. thrive as clusters. Among these, the textile industries are large industrial consumers of water as well as producers of waste water. The efflu-ent discharged by this industry leads to serious pollution of groundwater, soils and ultimately affects the liveli-hood of the poor32. The physicochem-ical characterisation of 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 study33. 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 water34. The temperature of the effluent was high in comparison with the tem-perature of another effluent in one study35. 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 permis-sible limits. Sediment rate is drasti-cally increased because of high value of TDS reducing the light penetration into water and ultimately decreas-ing photosynthesis. The decrease in photosynthetic rate reduces the DO level of waste water, which results in decreased purification of waste water by microorganisms36. The current sample exhibited high values of heavy metals which were of the same order

of magnitude reported in another effluent sample37. 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 purposes38. The major-ity of the textile effluent samples have permissible limits of sulphate ions. The effluent showed phenolic con-tents 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 concentrations39. The bleaching and dyeing process are the main causes of pollutants which include caustic soda, hypochlorite and peroxides.

ConclusionThis study confirms the ability of the newly isolated bacterial culture Pseudomonas aeruginosa ETL-1942 to decolourise Acid Orange 10 with decolourisation efficiency of 94%, thus suggesting its application for decolourisation of dye-bearing indus-trial wastewaters. Although decol-ourisation is a challenging process for the textile industry and for wastewa-ter treatment, the result of this find-ing 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 decolourisa-tion of Acid Orange 10 in this study was isolated from the textile dye industry waste effluent. The ability of the strain to tolerate and decol-ourise azo dyes at high concentration gives it an advantage for treatment of textile industry wastewaters. How-ever, potential of the strain needs to be demonstrated for its application in treatment of real dye-bearing waste-waters using appropriate bioreactors.

References1. Jadhav JP, Parshetti GK, Kalme SD, Govindwar SP. Decolourization of azo dye methyl red by Saccharomyces

Figure 11: FTIR spectrum of Acid Orange 10 and its degradation product.

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cerevisiae MTCC 463. Chemosphere. 2007;68:394–400. 2. Anjaneyulu Y, Sreedhara Chary N, Raj DSS. Decolourization of indus-trial effluents – available methods and emerging technologies – a review. Envi-ron Sci Biotechnol. 2005;4:245–73.3. Fu Y, Viraraghavan T. Fungal decol-orization of dye wastewaters: a review. Bioresour Technol. 2001;79:251–62.4. Meyer U. Biodegradation of syn-thetic organic colorants. In: Leisinger T, Cook AM, Hunter R, Nuesch J, editors. FEMS Symposium 12, Academic Press, London; 1981. pp. 371–85.5. Zollinger H. Colour chemistry- synthesis, properties of organic dyes and pigments. New York: VCH Publishers; 1987. pp. 92–100.6. Banat IM, Nigam P, Singh D, Marchant R. Microbial decolorization of textile dye containing effluents, a review. Biores Technol. 1996;15:507–9.7. Weber EJ, Adams RL. Chemical- and sediment-mediated reduction of the azo dye disperse blue 79. Environ Sci Tech-nol. 1995;29:1163–70.8. Clarke EA, Anliker R. Organic dyes and pigments. Handbook of environ-mental chemistry. Springer, USA Verlag; 1980.9. Chudgar RJ. Azo dyes. In: Kirk-Othmer Encyclopedia of Chemical Technology, Kroschwitz L.I. 4th ed. Vol. 3. New York: Wiley; 1985. pp. 821–75.10. Carliell CM, Barclay SJ, Naidoo N, Buckley CA, Mulholland DA, Senior E. Microbial decolourisation of a reactive azo dye under anaerobic conditions. Water SA. 1995;21(1):61–9.11. Easton J. The dye maker’s view. In: Cooper P, editor. Colour in dyehouse effluent. Bradford, UK: Society of Dyers and Colourists; 1995. p. 11.12. Maguire RJ. Occurrence and persis-tence of dyes in a Canadian river. Water Sci Technol. 1992;25:265–70.13. An SY, Min SK, Cha IH, Choi YL, Cho YS, Kim CH, et al. Decolorization of triphenylmethane and azo dyes by Citrobacter sp. Biotechnol Lett. 2002;24:1037–40.14. Robinson T, McMullan G, Marchant R, Nigam P. Remediation of dyes in textile effluent: a critical review on current

treatment technologies with a proposed alternative. Bioresour Technol. 2001; 77:247–55.15. Kapdan IK, Tekol M, Sengul F. Decolorization of simulated textile wastewater in an anaerobic-aerobic sequential treatment system. Proc Bio-chem. 2003;38:1031–7.16. Yatome C, Ogawa T, Hishida H, Taguchi T. Degradation of azo dyes by cell-free extract from Pseudomonas stutzeri. J Soc Dyers Colourists. 1990;106:280–3. 17. Brosius J, Palmer JL, Kennedy JP, Noller HF. Complete nucleotide sequence of a 16S ribosomal gene from Escherichia coli. Proc Natl Acad Sci USA. 1978;75:4801–5.18. Kimura M. A simple method for estimating evolutionary rates base sub-stitution through comparative stud-ies of nucleotide sequences. J Mol Evol. 1980;16:111–20.19. Saitou N, Nei M. The neighbor- joining method: a new method for recon-structing phylogenetic tree. Mol Biol Evol. 1987;4:406–25.20. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence weigh-ing, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–80.21. Drobniewski FA. Bacillus cereus and related species. Clin Microbiol Rev. 1993;6(4):324–38.22. Felsenstein J. PHYLIP (phylogenetic inference package). Version 3.5c. Seattle, WA, USA: Department of Genetics, University of Washington; 1993.23. Chang JS, Kuo TS. Kinetics of bac-terial decolorization of azo dyes with Escherichia coli NO3. Bioresour Technol. 2000;75:107–11.24. Stolz A. Basic and applied aspects in the microbial degradation of azo dyes. Appl Microbiol Biotechnol. 2001;56:69–80.25. Kalme S, Ghodake G, Gowindwar S. Red HE7B degradation using desul-fonation by Pseudomonas desmolyti-cum NCIM 2112. Int Biodeter Biodegr. 2007;60:327–33.26. Chang JS, Lin CY. Decolorization kinetics of a recombinant Escheria coli strain harboring azo dye decolorizing determinants from Rhodococcus sp. Bio-technol Lett. 2001;23:631–6.

27. Suzuki T, Timofei S, Kurunczi L, Dietze U, Schuurmann. Correlation of aerobic biodegradability of sulfonated azo dyes with the chemical structure. Chemosphere. 2001;45:1–9.28. Coughlin MF, Kinkle BK, Bishop PL. Degradation of azo dyes containing aminonapthol by Sphignomonas sp strain 1CX. J Inds Microbio Biotechnol. 1999;23:341–6.29. Asad S, Ammozegar MA, Sarbolouki MN, Dastgheib SMM. Biores Technol. Decolorization of textile azo dyes by newly isolated halophilic and halotoler-ant bacteria. 2007;98:2082–8.30. Nachiyaar CV, Rajkumar GS. Deg-radation of a tannery and textile dye, Navitan Fast Blue S5R by Pseudomonas aeruginosa. WJ Microbiol Biotechnol. 2003;19:609–14.31. Sheshadri S, Bishop PL, Agha AM. Anaerobic/aerobic treatment of selected azo dyes in wastewater. Wastes Manage. 1994;14:127–37.32. Jiunkins R. Pretreatment of textile waste water. Proceedings 37th Industrial waste Conference; 1982; Purdue Univer-sity, Lafayette, Ind. pp. 37–139.33. Tyagi OD, Mehra M. A textbook of environmental chemistry. New Delhi, India: Anmol Publications; 1990.34. Vandevivre PC, Bianchi R, Verstraete W. Treatment and reuse of wastewater from the textile wet-processing industry: review of emerging technologies. J Chem Technol Biotechnol. 1998;72:289–302.35. Kumar A. Environmental chemistry. New Delhi, India: Wiley Eastern Limited; 1989.36. Delee W, Niel CO, Hawkes FR, Pinheiro HM. Anaerobic treatment of tex-tile effluents: a review. J Chem Technol Biotechnol. 1998;73:323–5.37. Kim HT. Soil reaction. In: Environ-mental soil science. U.S.A: Marcel Dekker Inc.; 1994. p. 149.38. Agarwal SK. Industrial environment: assessment and strategy. New Delhi, India: APH Publishing Corporation; 1996.39. Coughlin MF, Kinkle BK, Tepper A, Bishop PL. Characterization of aerobic azo dye degrading bacteria and their activity in biofilms. Water Sci Technol. 1997;36:215–20.

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