research study microbial degradation and decolourisation of reactive black by an application of

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For citation purposes: Shah MP, Patel KA, Nair SS, Darji AM. Microbial degradation and decolourisation of Reactive Black by an application of Pseudomonas stutzeri ETL-79. OA Biotechnology 2013 Mar 01;2(2):13.

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AbstractIntroduction The textile industry in India is one of the oldest industries. It provides direct employment to nearly thirty million people. Wastewater from the textile industries are a complex mix-ture of many polluting substances such as organo chlorine-based pes-ticides, heavy metals, pigments and dyes. The dye that has been cho-sen for this study is Reactive Black. This study focused on microbial degradation and decolourisation of Reactive Black by an application of Pseudomonas stutzeri ETL-79.Materials and methods The decolourising bacterium was identified as P. stutzeri ETL-79 based on physiological and biochemical characteristics as well as the 16S rRNA sequence. In this study, the effects of P. stutzeri ETL-79’s decol-ourising ability, for example, glucose concentration, temperature, pH and dye concentration, were investigated. Results The results show that under 40°C, pH 7.0, glucose concentration 0.2% and concentration of Reactive Black of 30 mg/L the decolourisation rate in 24 and 48 hours was 86.3% and 93.2%, respectively. Conclusion The study concluded that pH, tem-perature and various carbon sources have a significant influence on the dye removal efficiency by P. stutzeri ETL-79. This shows that the isolated

bacterium has enormous potential to degrade the textile dyes and resolve the problem of contamination by unnecessary dyes present in the efflu-ents of textile industries. Further pilot scale studies need to be conducted with such strains for actual industrial applications, and detailed studies are needed to explore the mechanisms involved. Although decolourisation is a challenging process for both textile industry and wastewater treatment, the result of these findings and lit-erature suggests a great potential for such bacteria to be used to decol-ourise dye wastewaters. P. stutzeri ETL-79 showed a decolourising activity through a degradation mech-anism, rather than adsorption. This observation has established that bac-teria are adaptive in nature and that they can degrade contaminants. The ability of the strain to tolerate and decolourise azo dyes at a high con-centration gives it an advantage for treatment of textile industry waste-waters. However, potential of the strains needs to be demonstrated for their application in treatment of real dye-containing wastewaters using appropriate bioreactors. Applica-tion of traditional wastewater treat-ment involves enormous cost and continuous input of chemicals, which becomes uneconomical and causes further environmental damage. Hence, economical and eco-friendly techniques using bacteria can be used for fine-tuning of wastewater treatment. Bio-treatment offers an easy, cheap and effective alternative for colour removal of textile dyes.

IntroductionThe textile industry is one of the old-est industries in India with over 1000 industries. Taking into account the

volume and composition of effluent, the textile wastewater is rated as the most polluting among all in the indus-trial sectors1,2. In general, the waste-water from a typical textile industry is characterised by high values of BOD, COD, colour and pH3,4. It is a complex and highly variable mixture of many polluting substances ranging from inorganic compounds and elements to polymers and organic products5. Incomplete use and washing opera-tions give the textile wastewater a considerable amount of dyes6. The untreated textile wastewater can cause rapid depletion of dissolved oxygen if it is directly discharged into the surface water sources due to its high BOD value. The effluents with high levels of BOD and COD values are highly toxic to biological life. The high alkalinity and traces of chromium, which is employed in dyes adversely affect the aquatic life and also interfere with the biological treatment processes7. It induces per-sistent colour coupled with organic load leading to disruption of the total ecological/symbiotic balance of the receiving water stream8. Dyes with striking visibility in recipients may lead to reduced light penetration in aquatic environment which will sig-nificantly affect the photosynthetic activity. The high concentration of nitrogen in the textile industrial effluents can cause the eutrophica-tion of closed water bodies. In addi-tion, coloured water is objectionable as it can spoil the beauty of water environments9,10. In view of the ear-lier mentioned adverse effects, the textile industry effluent should be discharged after proper treatment. The dyes are stable to light, heat and oxidizing agents, and it is difficult to remove the dyes from effluents.

Microbial degradation and decolourisation of Reactive Black by an application of Pseudomonas stutzeri ETL-79

MP Shah*, KA Patel, SS Nair, AM Darji

*Corresponding authorEmail: [email protected]

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

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This makes the effective and economic treatment of the effluents containing various dyes an important environ-mental problem. Traditionally, both physical and chemical methods such as coagulation, ozonation11, precipi-tation, adsorption by activated char-coal, ultrafiltration, nanofiltration12, electrochemical oxidation, electro-coagulation13,14, etc. were used in the treatment of the textile industrial effluents2. But both the methods have many shortcomings9,15,16. Chem-ical methods like coagulation often produce excess amount of chemi-cal sludge, which create problems in its disposal. Physical methods like adsorption by activated char-coal often need high capital invest-ment. Hence, most of the physical and chemical methods of effluent treatment are not accepted by the industries due to their high cost, low efficiency and inapplicability to a wide variety of dyes. Currently, much research has been focused on the biodegradation of the industrial effluents9,17,18. It mainly shows inter-est towards the pollution control using bacteria, fungi in combination with physicochemical methods19,20. The biomass can absorb the chromo-phores and also these chromophores can be reduced in low redox poten-tial environments. The attractive features of biological treatment are low cost, renewable and regenera-tive activity, and little or no second-ary hazard21–23. The conventional biological processes are not effec-tive because the dye content in the textile effluent is toxic to the micro-organisms used24,25. In situ degrada-tion of the effluent is a novel method under the biodegradation process. In this method, the microorganisms are isolated from the site of pollu-tion and the same microorganisms can be used for the treatment of the effluent26,8. The current study has evaluated the potential of isolated bacterial strain from textile effluent for their decolourisation efficiency of the textile dye, Reactive Black under

in vitro conditions and optimisation of the factors influencing the process.

Materials and methodsThe protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed.

Dyes and samplesThe Reactive Black dye used for this biodecolourisation study was obtained from the local textile indus-try in Ankleshwar, Gujarat. Heavily dye-contaminated soil and waste-water samples were obtained from surrounding areas of the textile industries and wastewater treatment factory in Ankleshwar, respectively. All samples were collected in sterile plastic containers.

Isolation of Reactive Black dye resistant and decolourising bacteriaSoil samples were used as inocu-lum and 1 g of soil was added into 50 ml of sterile modified base min-eral medium (BMM) in flasks and Reactive Black was added as the sole carbon source to give a final concentration of 30 mg/L. One litre of BMM contained 5.17 g K2HPO4, 1.70 g KH2PO4, 1.63 g NH4Cl and 10 ml of a salt solution. One litre of the salt solution contained 8.5 g MgSO4, 5 g MnSO4, 5 g FeSO4, 0.3 g CaCl2. The initial pH value of media was 7.227. The inoculated flasks were agitated on an orbital shaker at 130 rpm at 32°C for 72 hours. Then 2 ml of the culture medium was transferred to another 50 ml of fresh culture medium and cultivation was carried out under the same condi-tions for two to three times. The obtained suspensions were streaked on the nutrient agar plates. The medium composition is BMM, 30 mg/L of Reactive Black and 1.5% of agar. All the plates were incubated at 32°C for 48 hours. The morphologi-cally distinct bacterial isolates show-ing a clear decolourisation zone were

selected and preserved on Reactive Black dye-containing nutrient agar slant at 4°C.

Identification of an organismThe isolate with the largest decol-ourisation zone for the dye was selected and streaked on Reactive Black- containing nutrient agar for further purification and study. The purified isolate was used to inocu-late on several media for biochemi-cal testing. The physiological and biochemical characteristics of the Reactive Black decolourisation organisms were examined using standard procedures28. The utilisa-tion of some substrates as carbon sources and nitrogen sources by the isolate were performed based on Bergey’s Manual of Determina-tive Bacteriology29. Genomic DNA was isolated from the pure culture pellet using consensus primers and partial 16S rRNA genes were ampli-fied by PCR using forward primer (5′-GAGCGGATAACAATTTCACA-CAGG-3′), reverse primer (5′-CGC-CAGGGTTTTCCCAGTCACGAC-3′) and internal primer (5′-CAGCAGC-CGCGGTAATAC-3′). The amplified 16S rRNA gene was sequenced by Bangalore Genei (Bangalore, India). The obtained sequence data were aligned and analysed for identi-fication and finding the closest homology for the isolate. The next closest homology was found with Pseudomonas stutzeri and it was designated as P. stutzeri ETL-79.

Reactive Black decolourisation quantificationThe dye decolourising quantification studies were performed in a culti-vated medium. The cultivation was centrifuged at 8000 g for 20 minutes; the supernatant was scanned for absorbance maxim on a UV–VIS spec-trophotometer (Shimadzu, Japan). Decolourisation study in aqueous medium was performed by inoculat-ing 15% of (v/v) actively growing strain ETL-79 to another fresh 100 ml

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of medium containing Reactive Black dye in a 250 ml flask. All the tests were done in triplicates. One set of the flasks without inoculation was kept as control. After incubation, 10 ml of the inoculated and uninocu-lated samples were centrifuged at 8000 g for 20 minutes and the super-natant was analysed at 540 nm wave-length and decolourisation rate was calculated. Viable cell counts during Reactive Black decolourisation was followed from previous reports30.

Decolourisation experimentsThe Reactive Black stock solution of 3000 mg/L was prepared and using the required concentrations of dye, the solution was diluted. To study the effect of temperature (15°–50°C), pH (3.0–10.0), dye concentration (15–500 mg/L), glucose concentra-tion (0%–0.5%) in each experiment, one factor was changed, with the other factors remaining constant. The effects of these factors were evalu-ated by measuring the Reactive Black decolourisation rate and in triplicate.

ResultsIsolation of Reactive Black decolourisation bacteriaThere were 18 morphologically dis-tinct isolates showing decolouri-sation zones from 4 to 10 mm on nutrient agar containing 30 ppm Reactive Black. The isolated one, des-ignated as ETL-79, was with a larger decolourised zone (10 mm) for a col-ony size of 4–5 mm, and was selected for further studies. The other six isolates showed decolourisation zones between 4 and 7 mm and were resistant to 150 mg/L of Reactive Black, which may be the results of heavy contamination with dyes due to the consequence of natural adap-tation of the organism as the sam-ples from which the bacterial isolate was obtained were contaminated heavily with dyes. The strain ETL-79 had higher efficient decolourisation activity and could degrade the syn-thetic dye Reactive Black.

Identification of the ETL-79 isolateThe 16S rRNA gene sequences were compared by using BLAST similar-ity searches, and the closely related sequences were obtained from GenBank. On the basis of morpholog-ical and biochemical analysis in com-bination with phylogenetics analysis, the strain ETL-79 was identified as P. stutzeri and designated as P. stutzeri ETL-79 (Figure 1).

Effect of temperature on Reactive Black decolourisation by P. stutzeri ETL-79Temperature plays an important role in microbial growth and enzyme

activity and is one of the most impor-tant parameters taken into con-sideration in the development of biodecolourisation processes. The temperature range was 15°–50°C. The results (Figure 2) reveal a high decolourisation percentage at 35°C and 40°C. At 40°C and after 48-hour cultivation, the decolourisation per-centage was 93.2%, while it was only 81.2% and 58.3% at 35°C and 30°C, respectively. The viable cell count was evaluated during Reactive Black decolourisation. Cell density was about 106 cfu/mL at the beginning of the tests. At 40°C cell density increased rapidly and the num-ber of alive bacteria in the medium

Figure 1: Phylogram (neighbour-joining method) showing genetic relation-ship between P. stutzeri ETL-79 and other related reference microorganisms based on the 16S rRNA gene sequence analysis.

Figure 2: Effect of temperature on decolourisation of Reactive Black.

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could reach 108 cfu/mL, more than that under the condition at 35°C (1.3 × 107 cfu/mL) and 45°C (1.1 × 107 cfu/mL). ETL-79 strain could not stay alive when temperature exceeded 50°C, and the Reactive Black was not decolourised because of that. There-fore, the strain Reactive Black was a facultative heat-resistant bacteria and the optimum decolourisation temper-ature could be determined as 40°C.

Effect of initial pH on Reactive Black decolourisation P. stutzeri ETL-79To test the effect of pH on Reactive Black decolourisation by P. stutzeri ETL-79, the temperature was main-tained constant (40°C) for 48 hours and pH was varied from 3 to 10. Figure 3 shows that decolourisation percentage of Reactive Black was higher at pH 6–8. The highest decol-ourisation rate could be obtained at pH 7 for 48 hours, and the decolouri-sation rate also reached over 65% at pH 6 and 8, while the decolourisa-tion rate was 32.5% at pH 5 and was almost zero at lower (3 and 4) or higher (9 and 10) pH values. The vari-ation of the amount of viable bacteria under different pH was also tested and the results show (Figure 2) that there was no bacterial growth at pH values under 6 and above 9, and the high-est densities of viable bacteria were obtained between the pH range 6–8.5.

Effect of the glucose concentration on Reactive Black decolourisation P. stutzeri ETL-79To test the effect of glucose concen-tration on Reactive Black decolouri-sation by ETL-79 isolate, temperature and pH were kept constant at 40°C and pH 7, for 48 hours, and glucose concentration varied from 0% to 0.5%. Even though P. stutzeri ETL-79 had the ability to use Reactive Black as the sole carbon source, it grew slowly. But when glucose was added, the growth rate increased as well as the decolourisation rate. Figure 4 shows that decolourisation rate of Reactive Black was higher at

the range 0.2%–0.3%. The highest decolourisation rate is 92.5% when the glucose concentration is 0.2% while the rate is 59.8% and 82.6% when the glucose concentration is 0.1% and 0.3%, respectively. When the glucose concentration was over 0.3%, the decolourisation of Reactive Black rapidly decreased. Bacteria densities for a determined glucose concentration (Figure 3) varied greatly in the range 0%–0.5% with the highest value reached at 0.5%

(109 cfu/mL). P. stutzeri ETL-79 can use glucose as a carbon and energy source. Thus, it provided sufficient electrons for reductive conditions through the cleavage of the azo bond of Reactive Black. The increase of glucose concentration decreases the decolourisation rate of Reactive Black and it could be assumed that when there is enough glucose in the medium P. stutzeri ETL-79 prefers to use glucose instead of the dye Reac-tive Black.

Figure 3: Effect of pH on decolourisation of Reactive Black.

Figure 4: Effect of glucose concentration on Reactive Black decolourisation.

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Effect of Reactive Black concentration on decolourisation P. stutzeri ETL-79For the study of the effect of Reac-tive Black concentration on decol-ourisation, temperature, pH and glucose concentration were constant at 40°C, 7 and 0.2%, respectively for 48 hours, while Reactive Black varied from 15 to 500 mg/L. Figure 5 shows that decolourisation rate decreases with the increasing Reactive Black concentration.

UV–Vis absorption spectraFigure 6 shows that main vis-ible absorption peaks of Reactive Black decreased after 24 hours and almost disappeared at 48 hours, which indicated that the azo groups were broken. Moreover, it was also concluded that the ring was opened during the decolourisation of Reac-tive Black because its absorbance value at 330 nm was decreased at 24 hours and almost disappeared at 48 hours. It has been reported that the oxidation of azo bonds led to decolourisation of dye solutions with the formation of primary aro-matic amines and the formation of aromatic amines has also been reported in the natural anaerobic degradation of azo dyes31. The azo bond of Reactive Black was broken and the ring was opened during

decolourisation of Reactive Black by P. stutzeri ETL-79 in this study. This would lead to formation of aromatic amines in the medium. This is in accordance with previous reports.

Discussion The aim of this work was to biologi-cally degrade the dyes, which is by using bacteria that can survive in the conditions imposed by the efflu-ent. The bacterium that was isolated from the effluent was identified to be P. stutzeri ETL-79. Industrial efflu-ent is not stable and it varies often in a wide range depending upon the

process practiced. South-Asian coun-tries are experiencing severe envi-ronmental problems due to rapid industrialisation. This phenomenon is very common where the polluting industries like textile dyeing, leather tanning, paper and pulp process-ing, sugar manufacturing, etc. thrive as clusters. Among these, the tex-tile industries are large industrial consumers of water as well as pro-ducers of wastewater. The effluent discharged by this industry leads to serious pollution of groundwater and soils and ultimately affects the liveli-hood of the poor32. The physicochem-ical characterisation of the collected textile effluent sample from Ankle-shwar Textile Industries, Anklesh-war, Gujarat, India showed a high load of pollution indicators. Colour is contributed to a water body by the dissolved compounds (dyes and pig-ments). The effluent colour was black due to a mixture of various dyes and chemicals used in the dyeing pro-cess. The pH of the tested sample was slightly alkaline when compared to the acidic pH of the dyeing efflu-ent in a previous study33. The pH of the effluent alters the physicochemi-cal properties of water which in turn adversely affects aquatic life, plants and humans. The soil permeability gets affected resulting in polluting

Figure 5: Effect of Reactive Black concentration on decolourisation of Reactive Black.

Figure 6: UV-Vis absorption spectrum.

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underground resources of water. The temperature of the effluent was high in comparison with the tem-perature of another effluent in one study34. 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 in one effluent study. Sediment rate is drastically increased because of high value of TDS which reduces the light penetration into water and ultimately decreases photosynthe-sis. The decrease in photosynthetic rate reduces the DO level of waste-water which results in decreased purification of wastewater by microorganisms35. The current sam-ple exhibited high values of heavy metals which were of the same order of magnitude reported in another effluent sample36. The nutrients of the surrounding soils are depleted as a result of high value of heavy metals thereby affecting soil fertility. High chloride contents are harmful for agricultural crops if such wastes con-taining high chlorides are used for irrigation purposes37. The majority of the textile effluent samples have per-missible limits of sulphate ions. The effluent showed phenolic contents greater than 0.1 ppm which, though is the permissible limit of the phenolic compounds, are still very toxic to fish even at very low concentrations38. The bleaching and dyeing process are the main causes of pollutants, which include caustic soda, hypochlorite and peroxides. The isolation of different microorganisms from the effluent sample collected from the Ankle-shwar Textile Industries, Gujarat, India indicates natural adaptation of microorganisms to survive in the presence of toxic chemicals and dyes. Interest in the bioremediation of pol-lutants using bacteria has intensified in recent years, as many researches demonstrated the efficacy of bacte-rial bioremediation over fungal and

actinomycetes. Many bacteria capa-ble of reducing azo dyes reported were isolated from textile effluent-contaminated sites39. The effluent sample collected from Ankleshwar Textile Industries, Ankleshwar, Gujarat, India was black in colour with a pungent smell and pH of slightly above neutral level and was within the permissible limits. The tempera-ture of the effluent was high. TSS and TDS in the textile effluent were very high. The solids present in ground water, besides affecting the growth of the plants directly, also affect the soil structure, permeability and aeration, indirectly affecting plant growth. The BOD values were within the permis-sible limits in the effluent sample. Dif-ferent bacterial strains isolated from the textile effluent were screened for their ability to decolourise the tex-tile azo dye and the potential strains were characterised morphologically and biochemically for identifica-tion. The main benefit of employ-ing this technique is that the culture has an optimal temperature of 32°C and optimum pH of 7. In addition to this, the inherent advantages of the microorganism, like rapid growth, less space requirement, etc. makes this an efficient method for treat-ment of the textile industrial effluent. The massive mobilisation of organic pollutants in natural resources or the introduction of xenobiotics into the biosphere leads to the persis-tence of a number of chemicals in the biosphere and thus constitutes a source of contamination. Often these organic pollutants comprise a poten-tial group of chemicals, which can be dreadfully hazardous to human health and are resistant to degrada-tion. As they persist in the environ-ment, they are capable of long-range transportation, bioaccumulation in human and animal tissue and biomagnifications in food chains. Biodegradation is used to describe the complete mineralisation of the starting compound to simpler ones like CO2, H2O, NO3 and other inorganic

compounds. Microorganisms play an important role in the field of envi-ronmental science by degrading and transforming toxic compounds into non-toxic or less toxic forms. Microbes found in natural water and soil have a broad ability to utilise virtually all naturally and some syn-thetically occurring compounds as their sole carbon and energy sources thus, recycling the fixed organic car-bon back into harmless biomass and carbon dioxide resulting in a clean-up of the environment. Azoreductase can catalyse the reductive cleavage of azo bonds, which is the key enzyme expressed in azo dye-degrading bac-teria. In the last decade, enzymes with azoreductase activity have been identified and character-ised from many bacteria, such as P. chrysosporium, Xenophilus azovorans KF46F, Pigmentiphaga kullae K24, Enterococcus faecalis, Staphylococcus aureus40–44. Azoreductase has low substrate specificity and can break the dye molecules in the high-affin-ity electron azo bond, producing colourless aromatic amines utilis-ing NAD(P)H as an electron donor in vitro45,46. However, the involvement of intracellular azoreductases in bacterial decolourisation has been doubted in recent years due to their high polarities and complex struc-tures, many azo dyes are difficult to diffuse through cell membranes. In this study, the azo dye Reactive Black was degraded by P. stutzeri ETL-79. The other 18 isolates showed decol-ourisation zones of 4–7 mm and they were resistant to 150 ppm Reactive Black concentration. The tempera-ture, pH, concentration of Reactive Black, glucose content could change the strain ETL-79 biodecolourisa-tion rate and its rate reached 93.2% in 48 hours, under the condition of 40°C, pH 7.0, glucose concentration 0.2% and anaerobic culture, which is one of the fastest reported biode-colourisation organisms for Reactive Black. All these results showed that P. stutzeri ETL-79 had higher activity

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of the azo dye decolourisation than previous reports. Therefore, P. stutzeri ETL-79 is a potential can-didate for degrading and bioreme-diating azo dye wastewater and polluted soil. The next step, we will purify its azoreductase and test its physicochemical properties of the purified azoreductase, which may open new possibilities for its bio-technological applications and allow the use of P. stutzeri ETL-79 in the treatment of azo dyes in industrial effluents. Furthermore, we will also construct gene engineering bacte-rium to be used in a continuous pro-cess for the degradation of azo dyes in industrial effluents.

ConclusionThe study concluded that pH, tem-perature and various carbon sources have a significant influence on the dye removal efficiency by P. stutzeri ETL-79. This shows that the isolated bacterium has enormous potential to degrade the textile dyes and resolve the problem of contamination by unnecessary dyes present in the efflu-ents of textile industries. Further pilot scale studies need to be conducted with such strains for actual industrial applications, and detailed studies are needed to explore the mechanisms involved. Although decolourisation is a challenging process for both textile industry and wastewater treatment, the result of these findings and lit-erature suggests a great potential for such bacteria to be used to decolour-ise dye wastewaters. P. stutzeri ETL-79 showed a decolourising activity through a degradation mechanism, rather than adsorption. This obser-vation has established that bacte-ria are adaptive in nature and that they can degrade contaminants. The ability of the strain to tolerate and decolouriseazo dyes at a high con-centration gives it an advantage for treatment of textile industry waste-waters. However, potential of the strains needs to be demonstrated for their application in treatment of real

dye-containing wastewaters using appropriate bioreactors. Applica-tion of traditional wastewater treat-ment involves enormous cost and continuous input of chemicals, which becomes uneconomical and causes further environmental damage. Hence, economical and eco-friendly techniques using bacteria can be used for fine-tuning of wastewater treatment. Bio-treatment offers an easy, cheap and effective alternative for colour removal of textile dyes.

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