International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 2(8), 37-43, August (2013) Int. Res. J. Environment Sci. International Science Congress Association 37 Effect of Process Parameters on Anaerobic Decolourization of Reactive Azo Dyes Using Bacillus licheniformis isolated from Textile effluent contaminated site in Perundurai, IndiaSudha D. and Balagurunathan R.Department of Microbiology, Periyar University, Periyar Pakalai Nagar, Salem-636011, TN, INDIAAvailable online at: www.isca.in Received 26th June 2013, revised 8 July 2013, accepted 11 August 2013 AbstractA bacterial strain Bacillus licheniformis with remarkable ability to decolourize the reactive azo dyessuch asreactive red, reactive yellow, reactive brilliant red and reactive brilliant blue was isolated from the textile effluent contaminated site.The effluent sample was collected from Infra Tex textile industry, Perundurai in Erode district situated at Tamil nadu state and country of India. Static conditions with 10 g/l glucose, pH 9, 37°C, 20% inoculum concentration, 50 mg/l of dye concentration, 3 g/l of NHNO, were considered to be the optimum decolourizing conditions. Bacillus licheniformis grew well in these optimum conditions, resulting in 82% decolourization extent 7 days of incubation. Phenotypic characterization and phylogenetic analysis of the 16S rRNA sequence indicated that the bacterial strain belonged to the genus Bacillus. UV (Ultra Violet) analysis of bacterial isolate suggested that it exhibited decolourizing activity, rather than inactive surface adsorption. This decolourization extent and facile conditions show the potential for this bacterial strain to be used in the biological treatment of textile effluent or dyes. Keywords: Azo dyes, Bacillus licheniformis, optimization, decolourization. Introduction The textile industry plays an important role in the world economy as well as in our daily life, but at the same time, it consumes large quantities of water and generates large amount of waste water. The chemical reagents used in textile sector are diverse in chemical composition ranging from inorganic to organic. Azo dyes, aromatic moieties together by azo (-N=N-) chromophores, represent the largest class of dyes used in textile processing and other industries such as cosmetic, food colorants, printing and pharmaceutical industries. As a characteristic of the textile-processing industry, a wide range of structurally diverse dyes are used within short periods in one and the same textile industry and therefore, effluents from textile industry are extremely variable in composition. Dyes bearing effluents are complex, most often non-biodegradable and exhibit toxicity to both aquatic and non aquatic biota. Around 10,000 different dyes with an annual production of more than 7 × 105 metric tons worldwide are commercially available. In India an average discharges about 1.5 million litres of dye-contaminated effluent per day, which leads to chronic and acute toxicity to the living things. The coloured wastewater treatment methods based on physical and chemical procedures are effective but suffer from shortcomings such as high expenditure, intensive energy requirements and formation of perilous by products1,5 whereas, biological degradation of these dyes does not face such problems. Microbial methods have recently received much attention owing to its ease of application, low cost and environmental benignity. In the present study, we focused on the isolation and identification of microorganisms from textile effluents having decolourizing ability for several different dyes. Various parameters have been optimized to achieve maximum dye decolourization for reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue. Material and Methods Sample and Dyes Collection: The effluent sample was collected from Infra Tex textile industry, Perundurai (Lat: 11°13’18.6”N, Long: 77°39’18.5”E) in Erode district situated at Tamil nadu state and country of India for decolourization studies shown in figure-1. The sample was transported to the laboratory without delay and preserved in the refrigerator at 4°C before and after the microbial analysis. The dyes used for this study (reactive red, reactive yellow, reactive brilliant blue and reactive brilliant red) were also procured from the same industry. Isolation of Microorganism: Microorganism was isolated from the effluent and soil sample by preparing aliquot (10 ml) dilutions. Nine millilitre of sterile water was placed in test tubes and labeled as 10-1 to 10-6, after 1ml of effluent sample was transferred into each tubes using sterile pipette. Then 1ml was taken from both soil and effluent aliquots and plated on dye fermentation agar medium containing reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 2(8), 37-43, August (2013) Int. Res. J. Environment Sci. International Science Congress Association 38 respectively and incubated at 37°C for 72 hrs. The culture capable of growth on this medium was isolated and purified by sub-culturing on dye fermentation agar medium. The well grown culture in the medium was taken for further studies. Molecular Characterization of bacterial isolate: The 16S rRNA sequencing of potential bacterium was carried out at Sri Ramachandra Medical University, Porur Chennai, as recommended by Robert Farrell. A portion of the bacterial 16S rRNA gene was amplified using the primers, 16SF (AGAGTTTGATCMTGGCTCAG) and 16SR (TACGGYTACCTTGTTACGACTT). The reaction mixture was set up on ice and it included: 1X TAE buffer (with Mg2+) (5 µL), dNTP (3.5 µL), 2 µL each for forward and reverse primer. Then, Taq DNA polymerase (0.5 µL), template DNA (3 µL) and an addition of double distilled HO up to 20 µL as the final volume. The PCR program was denaturized at 96°C for 10 seconds and at 94°C for 30 seconds, while it was annealed at 50°C for 5 seconds and extended at 60°C for 4 min, these steps cycled a total of 30, while the program was finally extended at 72°C for 10 min. Subsequently, the PCR product was separated by 2% agarose gel electrophoresis and the band of expected size was cut off and purified with a purification kit. The expected bands were sequenced by RUN 3730, Applied Bio-system 3.0 versions. Decolourization Assay: Decolourization studies were followed in static condition by bacterial isolate, such as Bacillus licheniformis by using various carbon sources, nitrogen sources, and combination of carbon + nitrogen sources at different pH were followed. Four reactive azo dyes were selected for decolourization purpose that is reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue. Carbon sources such as glucose, sucrose, lactose, maltose 10 g/l and nitrogen sources such as ammonium nitrate, ammonium sulphate were used. In addition to this effect of pH at 7, 8 and9, temperature at 28°C, 37°C and 45°C, effect of dye concentrations of 50, 100, 150 and 200 mg/l and effect of inoculum concentrations (5-20%) were carried out for decolourization studies. It was taken into separate conical flask and microbial inoculum was added to each and incubated for 7 days, by using mineral salt medium (K2 HPO4 - 1.6 g, KH2 PO4 - 0.2 g, (NH) SO4 - 1.0 g, MgSO.7HO - 0.2 g, FeSO.7HO - 0.01 g, NaCl - 0.1, COCl. 2HO - 0.002 g, glucose - 3 g, yeast extract - 1.0 g/l, pH.7)10. Then results were noted based on turbidity and Optical Density (OD) value by using following formula11. o – At Decolourization (%) =  × 100 o Where: A =Absorbance of the dye solution, At = Absorbance of the treated dyes solution at specific time, t. Results and Discussion Isolation and Identification of Microorganism: Among the 7 isolated bacterial strains, one strain was selected based on its ability to form high dye decolourization zone on fermentation agar medium. Biochemical test and physiological tests were performed to identify the isolated strain. These strain was gram positive rods, had spores, motile, occur single rod or in chains, yellowish colour colony, and showed positive to Voges-Proskauer test, catalase test and negative to methyl red test, indole test, urease activity and citrate utilization. Then, 1200 bp of 16S rRNA gene of the isolated strain (GenBank Accession No.KC866382) was determined. The nucleotide alignment of the strain showed most phylogenetic similarity to Bacillusgenus. The isolated strain was 99% similar to Bacilluslicheniformis. In the present study revealed the findings of Bacilluslicheniformis a bacterial isolate, isolated from textile effluent which was beneficial for the degradation of reactive azo dyes. The morphological and biochemical character of the isolated strain was performed12. The isolated strain was identified, belonging to the Bacillus sp by 16S rRNA sequence analysis. Microbial Decolourization: The development of dye decolourizer Bacillus licheniformis from the effluent sample collected from contaminated site of textile industry indicates the natural adaptation of this organism to survive in the presence of toxic dyes13. Among seven isolated bacteria, Bacillus licheniformis was screened for their ability to decolourize reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue. This isolate efficiently decolourize 82 % of reactive red, 71 % of reactive yellow, 69 % of reactive brilliant red and 69 % of reactive brilliant blue (50 mg/l) also showed good growth. With dyes reactive brilliant red, reactive brilliant blue however lesser decolourization and growth was noted even on complete incubation. The result inferred from our work involving screening of isolate to decolourize structurally different dyes prompted us to conclude that the bacterial isolate would be better option for decolourization work. In the presence of these four dyes in the mineral salt medium indicated that they were not toxic to the culture. The differential rates of decolourization will all the four dye/organism, providing the different reduction capacity of bacterium and different sensitivity of dyes. The ability of microbial isolates to achieve a high percentage of decolourization on single dyes has been reported in previous studies14. In the present study the bacterial isolate have the ability to achieve high percentage decolourization on four reactive azo dyes. The preponderance of biodegration reports are on anareobic treatment probably because earlier reports indicated that azo dyes were resistant to bacterial degradation under aerobic conditions while anaerobic decolourization was perceived to be easy to achieve15. In the present study decolourization was performed in static conditions, azo dye acts as artificial electron acceptor and activity is rather nonspecific with respect to the azo compounds involved thus, static condition was preferred in decolourization. Evaluation of Optimum Conditions: Effect of pH: The effect of pH (7, 8, and 9) on decolourization with incubation period of International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 2(8), 37-43, August (2013) Int. Res. J. Environment Sci. International Science Congress Association 39 four reactive dyes at 50 mg/l concentration of dye with 20% inoculums is shown in figure-2. The figure shows that the percentage removal of dye increased with increase in incubation period at pH 9. The maximum removal 80%, 73%, 63% and 63.5% dyes were found at pH 9 after 7 days of incubation period. In pH 7 and 8 resulted in decreased percentage removal of dye when compared to pH 9, the optimum pH was found to be 9 for maximum removal of dye. The pH plays great influence in decolourization of four reactive dyes. Bacillus licheniformisprefers alkaline range of higher decolourization extent. Chen et al. reported that the optimum pH was found to be 6-8 for maximum removal of dye. The pH has a major effect on the efficiency of dye decolourization and the optimal pH for colour removal is often between 6.0 and 10.0 for most of the dyes13. The pH tolerance of decolourizing bacteria is quite important because reactive azo dyes bind to cotton fibers by addition or substitution mechanisms under alkaline conditions and at high temperature16. Effect of Temperature: Figure 3 shows decolourization of dye with incubation period at three different temperatures (28°C, 37°C and 45°C) at 50 mg/l dye concentration and 20% inoculums. It is clear from the figure that percentage removal of dye increased at 37°C and there was no decolourization activity at 28°C, at 45°C there was no activity and cell death occurred slowly. Our results similar to that Ponraj et al. they reported that the range of activity on decolourization of orange 3R with 37°C was 78.57%, Bacillus spwas found to be the most effective decolourizer17. Ceten Domnoz reported that a suppressed decolourizing activity at 45°C, this might be due to the loss of cell viability or deactivation of the enzymes responsible for decolourization at higher temperature18. Effect of Various Carbon Sources: The effect of glucose, sucrose, lactose and maltose (10 g/l) on decolourization with time performed at 50 mg/l initial concentration of dyes, 20% of inoculum concentration and at 37°C in static condition. The figure 4 clearly shows that maximum removal of dye 82% for reactive red, 80% for reactive yellow, 79% for reactive brilliant red and 78% reactive brilliant blue was achieved after 7 days of incubation period using glucose as a carbon source. Whereas with co substrate sucrose shows that 71%, 68%, 63% and 62.5% of dye decolourization by reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue respectively. But it was less when compared to glucose used as a carbon source and also figure shows lower decolourization at lactose that was 67%, 62%, 65% and 69%, maltose that was 69%, 62%, 68% and 65% for reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue respectively. The reason for low decolourization at lactose and maltose might be that this carbon sources could not meet the good growth requirements for the bacterial isolate. In the present study it was found that glucose was optimum carbon source for decolourization of reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue dyes. Dyes being deficient in carbon sources the biodegradation of dyes without any extra carbon source is very difficult19 and therefore, in our study, glucose, sucrose, maltose and lactose were used as carbon source was supplemented in the mineral salt medium. Moosvi et al. reported maximum decolourization (93%) of reactive violet 5R by bacterial consortium JW-2, with glucose (1 g/l) 10. Saraswathi and Balakumar also reported that Bacillusfirmusand Bacillus laterosporus show maximum decolourization of azo dye pigment red 208 in the presence of glucose as a carbon source20. Effect of Inoculum Concentrations: Figure 5 shows the effect of inoculum concentration (5-20%) with incubation period on decolourization of dye at 50 mg/l dye concentration. It is clear from the figure that percentage removal of dye increased with an increase in incubation period at 20% inoculums concentration. At this concentration the dye removal was found to be 85.2%, 73%, 63% and 67% for reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue respectively when compared to 5%, 10% and 15% inoculums concentrations. This is the optimum inoculums concentration. Decolourization activity of Bacillus licheniformis has high in 20% of inoculum. In previous studies Ponraj et al. reported that decolourization activity of Bacillus sp has high (86.72%) in 4 % of inoculums17. Effect of Dye Concentrations: Figure 6 shows the effect of dye concentration ranging from 50-200 mg/l of dye at pH 9, 20% inoculum concentration and at 37°C. It is clear from the figure that percentage removal of dye increased with an increase in incubation period at 50 mg/l of dye concentration. Percentage removal of dye found to be 85%, 86%, 73% and 79% for reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue respectively. Uponincreasing the dye concentrations from 50-200 mg/l gradual decrease in both decolourization and growth observed probably due to the toxic effect of dyes or different structures our results similar with Sponza and Isik21. Sani and Banerjee also found that dyes were easily decolourized at concentration of 10 mM by Kurthia sp but colour removal was reduced when dye concentration was increased to 30 mM as the colour reduction capability of the cells was not enough to degrade all the transferred dye through the cell membrane22. Effect of Nitrogen Sources: Figure 7 shows the effect of nitrogen sources (NHNO and NHSO3 3 g/l) on decolourization with incubation period at 50 mg/l of dye concentration, 20% inoculum concentration and at 37°C at static conditions. The figure clearly shows that maximum removal of dye 67%, 57%, 66% and 50% for four reactive dyes respectively was achieved after 7 days of incubation period using NHNOas a nitrogen source and 56%, 65%, 60% and 51% of decolourization when NHSO used as a nitrogen sources. These results indicated that the nitrogen sources were less efficient than carbon source availed by microorganism. International Research Journal of Environment Vol. 2(8), 37-43, August (2013) International Science Congress Association Effect of Nitrogen + Carbon Sources Combination nitrogen + carbon sources (NH NHSO+g lucose) on decolourization with incubation of four reactive dyes at 50 mg/l dye concentrations with 20% inoculum is shown in figure- 8. The figure clearly shows that the percentage removal of dye increased with increase in incubation time. The maximum remov al 68%, 68%, 69% and 52% of dyes were achieved at 7 days of incubation period using NHNO +glucose and 61%, 59%, 50% and 49% of decolourization was achieved with NH combinations. The results from the figure showed that the decolourization perce ntage was higher than the medium contains only nitrogen source and lower than the medium Sampling area of textile effluent, Perundurai Effect of different pH on decolourization of reactive dyes by  7 3040Decolourization (%) reactive red reactive yellow Environment Sciences_______________ _________________________ International Science Congress Association Effect of Nitrogen + Carbon Sources Combination : Effect of nitrogen + carbon sources (NH NO+glucose, lucose) on decolourization with incubation of four reactive dyes at 50 mg/l dye concentrations with 20% inoculum 8. The figure clearly shows that the percentage removal of dye increased with increase in incubation al 68%, 68%, 69% and 52% of dyes were achieved at 7 days of incubation period using +glucose and 61%, 59%, 50% and 49% of decolourization was achieved with NH SO+glucose combinations. The results from the figure showed that the ntage was higher than the medium contains only nitrogen source and lower than the medium contains only carbon source. The growth of bacteria was not significantly different in the fermentation medium. cereus grew best in ammonium nitrate/glucose a best in ammonium nitrate/sucrose combination 81% textile dyes (reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue) is degradable under static conditions with a concerted effort of bacterium isolated from effluent. Nutrients (carbon and nitrogen sources) and physical parameters (pH, temperature and inoculums size, dye concentration) had significant effect on dye decolourization. Bacilluslicheniformis decolourizes four reactive azo dyes effectively during optimization throughout Figure-1 Sampling area of textile effluent, Perundurai Figure-2 Effect of different pH on decolourization of reactive dyes by Bacillus licheniformis (37°C, 50 mg/l, 20 % inoculum, 7 7 89728052732140631841.163.5pH reactive yellow reactive brilliant red reactive brilliant blue _________________________ ______ ISSN 2319–1414 Int. Res. J. Environment Sci. 40 contains only carbon source. The growth of bacteria was not significantly different in the fermentation medium. Bacillus grew best in ammonium nitrate/glucose a nd decolourizes best in ammonium nitrate/sucrose combination 81% . The textile dyes (reactive red, reactive yellow, reactive brilliant red and reactive brilliant blue) is degradable under static conditions with a concerted effort of bacterium isolated from textile dye effluent. Nutrients (carbon and nitrogen sources) and physical parameters (pH, temperature and inoculums size, dye concentration) had significant effect on dye decolourization. decolourizes four reactive azo dyes throughout the study. (37°C, 50 mg/l, 20 % inoculum, 7 th day of incubation) reactive brilliant blue International Research Journal of Environment Vol. 2(8), 37-43, August (2013) International Science Congress Association Effect of different temperature on decolourization of reactive dyes by (pH 9, 50 mg/l, 20 % inoculum, 7 Effect of different Carbon Sources on decolourization of reactive dyes by (37°C, pH 9, 50 mg/l, 20 % inoculum, 7 Effect of different inoculum concentration on decolourization of reactive dyes by (37°C, 50 mg/l, pH 9, 7  Decolourization (%) reactive red reactive yellow 444982 42 424580 41.4 305079424978 \n   357GlucoseDecolourization (%) reactive red reactive yellow \n 3575%203955 33 344849404547.7203940Decolourization (%) reactive red reactive yellow Environment Sciences_______________ _________________________ International Science Congress Association Figure-3 Effect of different temperature on decolourization of reactive dyes by Bacillus licheniformis (pH 9, 50 mg/l, 20 % inoculum, 7 th day of incubation) Figure-4 Effect of different Carbon Sources on decolourization of reactive dyes by Bacillus licheniformis (37°C, pH 9, 50 mg/l, 20 % inoculum, 7 th day of incubation) Figure5 Effect of different inoculum concentration on decolourization of reactive dyes by Baci llus licheniformis (37°C, 50 mg/l, pH 9, 7 th day of incubation) 28C37C4579736163.3Temperature reactive yellow reactive brilliant red reactive brilliant blue 42 48.27132396720 41.4 506834376222.23351.2634044.26520303562.5313269 22 3573573SucroseLactoseCarbon Sources g/l reactive yellow reactive brilliant red reactive brilliant blue 357357310%15% 33 364449426050424650545359.160313944556364524448.257.7485155 42 Inoculum Concentrations reactive yellow reactive brilliant red reactive brilliant blue _________________________ ______ ISSN 2319–1414 Int. Res. J. Environment Sci. 41 Bacillus licheniformis Bacillus licheniformis llus licheniformis reactive brilliant blue 2469256232.368 22 246557Maltose reactive brilliant blue 5720%7385697356.163 42 5067 reactive brilliant blue International Research Journal of Environment Vol. 2(8), 37-43, August (2013) International Science Congress Association Effect of different dye concentration on decolourization of reactive dyes by (37°C, 20 % inoculum, pH 9, 7 Effect of different nitrogen source on decolourization of reactive dyes by (37°C, 50 mg/l, 20 % inoculum, pH 9, 7 Effect of different nitrogen + carbon sources on decolourization of reactive dyes by (37°C, 50 mg/l, 20 % inoculum, pH 9, 7  \n   357 3 5040428544395286 44 504473 31.2 496079Decolourization(%) reactive red reactive yellow 323933.142404430.1  35Decolourization (%) NH reactive red reactive yellow  35303421 35 24 44 30Decolourization (%)NHNO3 + Glucose reactive red reactive yellow Environment Sciences_______________ _________________________ International Science Congress Association Figure6 Effect of different dye concentration on decolourization of reactive dyes by Bacillus licheniformis (37°C, 20 % inoculum, pH 9, 7 th day of incubation) Figure-7 Effect of different nitrogen source on decolourization of reactive dyes by Bacillus licheniformis (37°C, 50 mg/l, 20 % inoculum, pH 9, 7 th day of incubation) Figure8 Effect of different nitrogen + carbon sources on decolourization of reactive dyes by Bacillus (37°C, 50 mg/l, 20 % inoculum, pH 9, 7 th day of incubation) 3 573573100150507131345220 44 517434355018 31.2 4060314539 19.2 404162202547 13 Dye Concentrations (mg/l) reactive yellow reactive brilliant red reactive brilliant blue 672935572233663033.134502334735 NH NO3 NHSO 3 reactive yellow reactive brilliant red reactive brilliant blue 735423133502124 44 51252233.5363130 + Glucose NHSO3 + Glucose reactive yellow reactive brilliant red reactive brilliant blue _________________________ ______ ISSN 2319–1414 Int. Res. J. Environment Sci. 42 Bacillus licheniformis licheniformis Bacillus licheniformis 57200294820402039 13 19.129 reactive brilliant blue 56656051 reactive brilliant blue 40425439 + Glucose reactive brilliant blue International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 2(8), 37-43, August (2013) Int. Res. J. Environment Sci. International Science Congress Association 43 ConclusionThe present study concludes that dye-degrading microorganism; Bacillus licheniformis from an effluent contaminated site of textile dyeing industry have potential of decolourization. This observation has established that the bacteria are adaptive in nature and can degrade the dye contaminants. The ability of the strain Bacillus licheniformis to tolerate, decolorize and degrade reactive azo dyes at high concentration gave it an advantage for treatment of textile industry wastewaters. However, potential of culture needs to be demonstrated for its application in treatment of real dye bearing wastewaters using appropriate decolourization methodology and also anaerobic conditions favours the growth of Bacillus licheniformis in decolourization broth. This biological method can be promoted to degrade the variety of reactive dyes from the textile industries. The treated textile dyes when disposed to the land has several applications, includes, soil fertility improvement, easy transport of compost, humus rich, very slow release of nutrient and increases in water holding capacity of the soil. This potential strain may be used for the treatment of the dye industrial effluents, may also be applicable for the treatment of sewage and other polluted water. 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