(1) Enviro Technology Limited, Ankleshwar, India
* Corresponding author Email:
Azo dyes represent a major group of dyes causing environmental concern because of their colour, biorecalcitrance nature and potential toxicity to living beings. Various physico-chemical methods have been used to eliminate the coloured effluent from wastewaters. These methods have disadvantages of being highly expansive, coupled with the formation of huge amount of sludge and emission of toxic waste. Today is the era of bioremediation as biological methods are eco-friendly. The aim of this study was to observe Pseudomonas stutzeri ETL-4 in microbial degradation of the Congo red dye.
Materials and methods
In the present investigation, the bacterial isolate Pseudomonas stutzeri ETL-4 (isolated from Common Effluent Treatment Plant) has been reported to decolourize the dye (congo red) at a concentration of 100 mg L-1 upto 94% within 24 h in static conditions.
The temperature and pH for optimum growth and activity of the isolate were found as 37 oC and 7.0, respectively.
The isolate (ETL-4) may be a potential strain for biological treatment of effluents of tannery industries in future.
The use of synthetic chemical dyes in various industrial processes, including paper and pulpmanufacturing, plastics, dyeing of cloth, leather treatment and printing has increased considerably overthe last few years, resulting in the release of dye-containing industrial effluents into the soil andaquatic ecosystems. Since most of these dyes are toxic in nature, their presence in industrialeffluents is of major environmental concern because they are usually very recalcitrant to microbialdegradation. In some cases, the dye solution can also undergo anaerobic degradation to formpotentially carcinogenic compounds that can end up in the food chain. Moreover, highly colouredwastewaters can block the penetration of sunlight and oxygen, essential for the survival of variousaquatic forms.Textile industries utilize substantial volumes of water and chemicals for wet-processing of textiles.These chemicals, ranging from inorganic compounds and elements to polymers and organic productsare used for desiring, scouring, bleaching, dyeing, printing, and finishing. There are more than8,000 chemical products associated with the dyeing process listed in the Colour Index, includingseveral structural varieties of dyes, such as acidic, reactive, basic, disperse, azo, diazo, anthraquinonebasedand metal-complex dyes. The removal of colour from wastewaters is often more importantthan the removal of the soluble colourless organic substances, which usually contribute to the majorfraction of the biochemical oxygen demand (BOD). Methods for the removal of BOD from mosteffluents are fairly well established; dyes, however, are more difficult to treat because their syntheticorigin are mainly complex aromatic molecular structures, often synthesized to resist fading onexposure to sweat, soap, water, light or oxidizing agents[1,11]. This renders them more stable and lessamenable to biodegradation[10,27].Many approaches, including physical and/or chemical processes, have been used in the treatment ofindustrial wastewater containing dye but such methods are often very costly and not environmentallysafe[15,25]. Methods utilizing powdered activated carbon and activated bentonites have beencommonly used[19,33]. However, the large amount of sludge generated and the low efficiency oftreatment with respect to some dyes have limited their use. Colour removal using ozone is alsousually effective and fairly rapid, but not all the methods employed give satisfactory results especiallyfor some dispersed dyes. Another widely used treatment method for coloured effluents is thephysical-chemical flocculation with metal hydroxides assisted by polymer flocculants, while theapplication of pre-mixed polyelectrolyte complexes made by the interaction of aqueous solutions ofpolycation and polyanionis are accepted as a more practical method. Such complex particles are ableto bind disperse dyes effectively over large distances due to their size and structure via hydrophobic aswell as electrostatic interaction forces. However, because dye molecules or their aggregates areincomparably smaller than such inorganic particles, and in some cases also uncharged, it is necessaryto apply other flocculation principles.Interest in the pollution potential of textile dyes has been primarily prompted by concern over theirpossible toxicity and carcinogenicity. This is mainly because many dyes are made from knowncarcinogens, such as benzidine and other aromatic compounds, all of which might be transformedbecause of microbial metabolism. It has also been shown that azo- and nitro- compounds arereduced in sediments and in the intestinal environment, resulting in the regeneration of theparent toxic amines. Some disperse dyes have also been shown to bio-accumulate, whileheavy-metal ions from textile effluents have also been reported at high concentrations in both algaeand higher plants exposed to such effluents. In recent times, industries have been faced with morestringent effluent treatment regulations and are required to lower the colour content in their wastewaterbefore discharge into the surface water. This means that for most textile industries, developingon-site or in-plant facilities to treat their own effluents before discharge is a fast approaching actuality.New flocculation mechanisms are therefore attracting more attention.The removal of dyes from wastewater presents a formidable challenge, as most dyes are completelysoluble in aqueous solutions. Although dyes constitute only a small portion of the total volume ofwaste discharge in textile processing, these compounds are not readily removed by typical microbialbasedwaste-treatment processes. Furthermore, dyes can be detrimental to the microbialpopulation present in such treatment works and may lead to decreased efficiency or treatment failure insuch plants. Biological methods, being relatively cheap andsimple to use, have been the focus of recent studies on dye degradation and decolourization.Therefore, the objective of this study was to observe the azo dye degradation (congo red) using novel bacterial isolate from common effluent treatment plants under optimized cultural conditions.
The protocol of this study has been approved by the relevant ethical committee related to our institution in which it was performed.
Congo red, a commonly used azo dye, was chosen for the screening of dye degradative bacteria. All the chemicals used were of highest purity and of analytical grade.
Effluent and soil samples were collected from the common effluent treatment plant present at Ankleshwar GIDC, Gujarat, India. All the samples were collected in sterile containers and bags.
Isolation of Microorganism
Bacteria were isolated from thecommon effluent treatment plant and soil was irrigated with these effluents using the serial dilution technique. Approximately 0.1 ml aliquots of appropriate dilutions were poured on nutrient agar plates and incubated at 37 oC for 24 to 48 h. Individual bacterial colonies which varied in shape and colour were picked and purified by repeated sub culturing on the respective medium.
Screening of Microorganism
Inoculum was prepared aerobically by growing the cells at 37 oC for 24 h in Luria Bertani (LB) media at pH 7.0. For screening of isolates, LB media containing dye (100 mg L-1) was inoculated with 24 h old precultured cells 1.0% (v/v). The decolourization of dye was monitored after every 24 h intervals. Primary screening was done only on visibility basis i.e. change in colour of media containing respective dye.
In secondary screening, decolourization (%) was measured as a decrease in optical density using the spectrophotometer (UV-1800, Shimadzu, Japan). Decolourization (%) was calculated by the following formula:20.
Decolourization (%) = [(Ao-A1) / Ao] x 100
where, Ao: Initial absorbance; A1: Final absorbance
The effect of temperature on the decolourization was studied by incubating the LB media containing dye (100 mg L-1) under a range of temperatures (5 oC to 55 oC) at pH 7.0. (Figure 3).
The effect of pH on decolourization was studied by inoculating the LB media containing dye (100 mg L-1) at different pH values (5.0 to 10.0) keeping temperature constant (37 oC).
Total genomic DNA of bacteria was isolated using the Charles &Nester method5 with slight modifications. Pure bacterium culture was grown in 10 ml nutrient broth for 18 to 24 h. The bacterial pellet was washed in 1.5 ml of 0.85% NaCl, centrifuged for 2 min at 10,000 rpm and was resuspended in 0.4 ml Tris-EDTA buffer. Cell lysis was done by adding 20 μl of 25% SDS, 50 μl of 1% lysozyme and 50 μl of 5M NaCl followed by incubation at 68 oC for 30 min in a circulatory water bath. For protein precipitation, 260 μl of 7.5M ammonium acetate solution was added to the micro centrifuge tubes and kept in ice for 20 min followed by centrifugation at 10,000 rpm for 15 min at 20 oC. Supernatant was carefully pipetted out in another fresh, sterile microcentrifuge tube in which 1 μlRNase (4 mg ml-1) was added followed by incubation at 37 oC for 20 min. Equal volumes of chloroform was added in the tubes and RNA was precipitated by centrifuging at 10,000 rpm for 1 min. The top layer containing total cell DNA was pipetted out in a fresh microfuge tube and used for the next step. DNA was precipitated by adding 0.8 volume of isopropanol followed by incubation on ice for 30 min and pellet down by centrifuging at 10,000 rpm for 15 min. DNA was further washed with 0.5 ml of 70% ethanol and spun down at 10,000 rpm for 1 min. Pure DNA sample was then suspended in 20 μlTris-EDTA buffer or deionized water and stored at 4 oC for further use.
Agarose gel electrophoresis
The genomic DNA sample of bacteria was quantified though agarose gel electrophoresis by analysing the migration on 0.8% agarose gel prepared in 0.5 M Tris-borate-EDTA (TBE) buffer and run in an electrophoresis tank filled with the same concentration of TBE buffer. The genomic DNA was diluted with Tris-EDTA buffer so as to achieve a concentration of 50 ng in 10 μL to be used as a template DNA in a PCR amplification reaction.
16S rRNA PCR-Amplification
The universal forward and reverse primers were custom synthesized from “Bangalore Genei”, India. The sequences of the oligonucleotide primers used for amplification of 16S rRNA genes were:
16SR (5'- ACGGCTACCTTGTTACGACTT-3 ')
The stock solution (100 ng ml-1) of primers was prepared by reconstituting lyophilized primers in sterilized deionized (milliQ) water and stored at 20 oC.
Phylogenetic identity of bacteria was determined by theBLASTn result and sequences were aligned using alignment software i.e. ClustalW. Phylogeny calculations and dendrogram was constructed by the Mega 5.05 software package using the UPGMA method. Bootstrap analysis was conducted using 1000 replicate samplings of data.
From the samples collected, a total of 75 different bacterial isolates were obtained. About 40 isolates were obtained from tannery effluent and the remaining 35 from soils and fields irrigated with this effluent.
Out of 75 isolated bacteria, only 4 bacteria (Figure 1) possessed the capability of showing visible decolourization of congo red (100 mg L-1) within 5 days. Then these 4 selected bacteria were used in secondary screening.
% Decolourization of congo red
Four isolates selected from primary screening were further used in secondary screening. The rate of decolourization was calculated by a decolourization assay. Among 4 selected isolates, the isolate ETL-4 (Figure 2) showed significant decolourization (88.99%, 5 days). This potent isolate was further selected for optimization of cultural conditions.
Visual Decolourization of congo red
The isolate ETL-4 showed maximum decolourization at 37 oC (93.31% ± 0.32). It was observed that the rate of decolourization increased from 20 oC to 40 oC and afterwards it has been significantly affected with temperature change (Figure 1). Wong et al. reported methyl red (MR) degradation by KlebsiellapneumoniaeRS-13 and AcetobacterliquefaciensS-1. Their study indicated that both K. pneumoniaeRS-13 and A. liquefaciensS-1 decolourized MR by cleaving it into 2-aminobenzoic acid (ABA) and N, N-dimethyl-p-phenylenediamine (DMPD). Maximum degradation of DMPD was found in temperature ranges of 30 to 37 oC whereas no decolourization was observed at 45 oC. Also, Bacillus subtilis(RA-29) showed significant (95.67%) congo red decolourization at 37 oC and an increase in temperature beyond 37 oC led to a decline in decolourization activity of the strain (Figure 3).
Temperature effect on Decolourization
The effect of pH was studied by inoculating the 1% culture (v/v) into LB media containing dye (100 mg L-1) at different pH (5.0 to 10.0) and decolourization was measured at 560 nm. The optimum pH for maximum decolourization (94% ± 0.69) was found to be 7.0 (Figure 4). Decolourization of congo red increased with an increase of pH and has been drastically affected after pH 8.0. The gradual increase in congo red decolourization was observed from pH 5.0 to 8.0 by Bacillus subtilisalso and maximum decolourization was found at pH 8.0. Mali et al. found that pH between 6.0 and 8.0 was optimum for decolourization of triphenylmethane and azo dyes by Pseudomonas sp. Also, Pseudomonas putidahas been reported as the best decolourizer (Acid Orange 10; 90%).
pH effect of Decolourization
Molecular Characterization16S rRNA sequence analysis
PCR amplification of the 16S rRNA gene for isolate ETL-4 produced an amplification product of approximately 1489 bp. The alignment of the retrieved sequences from the NCBI database with 16S rRNA gene of ETL-4 showed sequence homology to Pseudomonas stutzeri (Figure 5).
Phylogenetic tree showing interrelationship of ETL-4 with other closely related species
In the present study, consortia of alkalophilic microorganisms from common effluent treatment plant are used to decolorize congo red in a continuous anaerobic/aerobic reactor system. In the system 100 percent colour removal was obtained at the end of the treatment. As the dye was decolourized, the azo bond of congo serves as a final electron acceptor and cleaved into aromatic amines. Although colour removal is greater at the anaerobic stage, the presence of the anaerobic stage alone is not effective for complete decolourization. If dye reduction is not taking place at the anaerobic stage, it will leave the aerobic stage intact. Therefore, for the removal of colour as well as for removal of the products that are produced as a result of dye decolourization at the anaerobic stage, a continuous anaerobic/aerobic system is crucial. In the study, greater colour removal efficiency was achieved at the anaerobic stage than the aerobic stage. The colour removal efficiency after the anaerobic stage was found to be 96.0%. On the other hand, the colour removal efficiency of the overall was found to be 100%. The colour removal efficiency of the aerobic reactor was only 4%. Thus, for complete colour removal, a continuous anaerobic/aerobic system was found to be essential. Qualitatively, dye decolourization products were identified by High Performance Chromatography (HPLC). The chromatograms of HPLC of the anaerobic/aerobic treatment process showed the formation of new products at the anaerobic stage and oxidation of the product at the aerobic stage. At the anaerobic treatment process a third new peak and another two peaks with a lower retention time and greater peak area were identified. This is due to the presence of one azo bond in congo red dye. According to, azo dye reduction is taking place primarily by anaerobic reduction of the azo bond of the dye followed by aerobic oxidation of biodegradation products. The peak areas of the three metabolites decrease to a greater extent. This suggests that continuous anaerobic/aerobic treatment of dye containing effluents is a promising technology for dye decolourization as well as oxidation of decolourization products. Based on the structure of most of the reactive azo dyes, the prediction is that under anaerobic condition, the products of dye reduction would result in the formation of aromatic compounds. And the chromatogram of the Thin Layer, showed the presence of two fragmented dyes. This is because the congo red under study was a monoazo dye and cleavage of the azo linkage would result in two structurally distinct aromatic amines. According to the Literature, biodegradation of dyes by bacteria could be due to adsorption or biodegradation. After biodegradation, half of the decolourized media was autoclaved and inoculated into another media containing congo red. The remaining non-autoclaved decolourized media was inoculated into another media containing congo red. Then the biodegradation was monitored daily both visually and in a UV-Visible Spectrophotometer. In effect it is the non-autoclaved decolourized media showed biodegradation. This is because biodegradation of dyes proceeds primarily by degradation than physical adsorption. The continuous anaerobic/aerobic reactor system was evaluated for Chemical Oxygen Demand (COD), total nitrogen and ammonia evolution. In the system, COD removal efficiency was 66.6% at the anaerobic stage and the overall removal efficiency was 94.26%. This shows that most of the chemicals and the dye are used by the consortia. Similarly, the removal efficiency of total nitrogen increases from 50.2 at the anaerobic stage to 77.9 in the overall. This is because the nitrogen sources in the original dye media were used as a nitrogen source for growth by the consortia. On the other hand, ammonia evolution increases after aerobic treatment than after anaerobic treatment in the continuous reactor system. During decolourization of congo red by consortia in a continuous anaerobic/aerobic system, the pH of the synthetic dye containing wastewater, when it was treated anaerobically and increases slightly. According to Sponza the lowering of pH at the anaerobic stage is due to the formation of acids. This shows that decolourization products can be further degraded into simpler molecules.
The congo red decolourizing isolate ETL-4 (isolated from Common Effluent Treatment Plant) has been identified as Pseudomonas stutzeri. The azo dye was maximally (94%) decolourized at optimal conditions (pH 7.0; Temp. 37 oC) under static conditions in 24 h. Thus ETL-4 could be exploited for its bioremediation ability to treat theazo dye contaminated aqueous ecosystem. Moreover, further studies on this isolate could explore new tools and techniques to evolve commercially viable and ecofriendly microbial solutions for treatment of dye industry effluents.
8. Dos Santos AB, Cervantes FJ, van Lier JB. Review paper on current technologies for decolourization of textile wastewaters: Perspectives for anaerobic biotechnology. Bioresour.Technol. 2007, 98, 2369–2385.
11. Khan AA, Husain Q. Decolorization and removal of textile and non-textile dyes from polluted wastewater and dyeing effluent by using potato (Solanumtuberosum) soluble and immobilized polyphenol oxidase. Bioresour. Technol. 2007, 98, 1012–1019.
21. Petzold G, Mende M, Lunkwitz K, Schwarz S,Buchhammer HM. Higher efficiency in the flocculation of clay suspensions by using combinations of oppositely charged polyelectrolytes. Colloid. Surf. 2003, A218, 47–77.
22. Petzold G, Nebel A, Buchhammer HM, Lunkwitz K. Preparation and characterization of different polyelectrolyte complexes and their application as flocculants. Colloid Polym. Sci. 1998, 276, 125–130.
23. Pinheiro HM, Touranud E, Thomas O. Aromatic amines from azo dye reduction: status review with emphasis on direct UV spectrophotometric detection in textile industry wastewaters. Dyes Pigm. 2004, 61, 121–139.
25. Sanayei Y, Ismail N, Teng TT, Morad N. Studies on flocculating activity of bioflocculant from closed drainage system (CDS) and its application in reactive dye removal. Int. J. Chem. 2010, 2, 168–173.
36. Carvalho MC, Pereira C, Gonc-alves IC, Pinheiro HM, Santos AR, Lopes A, and Ferra MI. Assessment of the biodegradability of a monosulfonated azo dye and aromatic amines. Int. Biodeterioration and biodegradation. 2008, 62: 96-103.