Decolorization of Remazol Black-B by Three Bacterial Isolates

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2.2. Isolation of Bacterial Strain by Enrichment Method

Bacterial strains were isolated from water and sludge samples. Isolates from each inoculum source were first enriched using MSM medium amended with an azo dye Remazol Black-B as the sole source of C and N (20). Dye was added at a concentration of 150 mg L-1. The cultures containing 200 mL of MSM broth with dye in 500 mL Erlenmeyer flasks were inoculated with 10 mL volume of wastewater or sludge suspensions. The flasks were incubated at 32°C for 7 days under static conditions. After incubation, cell suspensions from each flask were plated onto MSM agar medium and incubated at 32°C for 24 h. Microbial colonies that appeared on the agar medium were washed gently with sterile water and resuspended into the flasks containing fresh MSM broth spiked with the Remazol Black-B dye. About 50 actively growing colonies were selected for purification.

2.3. Purification of Bacterial of Isolates

Selected isolates were purified by streaking on MSM medium containing agar at the concentration of 20 g L-1. Streaking was done thrice in zig zag manner. The purified cultures were preserved in a refrigerator for subsequent study.

2.4. Screening Efficient Azo Dye Decolorizing Bacterial Isolates

Screening was done to find out the efficient bacterial strains capable of decolorizing the Remazol Black-B azo dye using modified MSM. For this purpose, 48 isolates having the ability to decolorize Remazol Black-B from all samples were selected. After that decolorization, ability of each isolate was tested in the liquid medium. Media inoculated with the respective inocula were incubated at 35°C for 24 h. After 24 h, the respective cells were harvested by medium centrifugation at 10,000 rpm (REMI R-23, ) for 10 minutes. Then decolorization was determined with the help of spectrophotometer (SHIMADZU, ) at 597 nm. Uninoculated blanks were run to determine abiotic decolorization. The three most effective bacterial isolates (ETL-1, ETL-2 & ETL-3) from the final screening were further examined for their decolorization potentials in test tubes at different time periods. Ten milliliters of the sterilized MSM broth containing Remazol Black-B at the concentration of 100 mg L-1 was added to autoclaved test tubes supplemented with 0.5% yeast extract as a co-substrate. The medium was inoculated with the respective bacterial strains by adding inocula of uniform cell density (OD: 0.6) at 597 nm. The test tubes were tightly sealed and incubated at 35°C under static conditions. Uninoculated test tubes with MSM containing azo dye plus yeast extract were incubated under similar conditions to check for abiotic decolorization of dye. Decolorization was measured after 6, 12, 18 and 24 h at 597 nm by spectrophotometer as described by Khalid et al. (2008).

2.5. Optimization of Environmental Factors for Efficient Decolorization

Factors like substrate concentration, temperature and pH were optimized during the experimentation for Different carbon sources (glucose, yeast extract, Mannitol and maltose) at the concentration of 4 g L-1 were also tested as co- substrate in the decolorization process. Optimization studies included various concentration of dye (50,75,100,125,150,200 and 250 mg L-1), pH values (5,6,7,8,9) and temperatures (25,30,35,40,45^oC). All the bacterial isolates ETL-1, ETL-2 & ETL-3 were tested to optimize their decolorization efficiency. While culture conditions were the same as used in decolorization experiment i.e., minimal salt medium was used along with the 100 mg L-1 of Remazol Black-B azo dye. Uninoculated blanks were run to check the abiotic decolorization during the experimentation.

2.6. Statistical Analysis

Data were entered in a Microsoft® Excel 2007 spreadsheet.

3. Results

Efficiency of the bacterial isolates to decolorize Remazol Black-B was examined by measuring color intensity in liquid medium. Based upon the relative decolorization efficiency of different isolates, five the most efficient isolates (ETL-1,ETL-2 & ETL-3) with more than 80% decolorizing efficiency were selected for further experiments (Data not shown).

3.1. Biodecolorization of Remazol Black-B by Selected Bacterial Isolates

Biodecolorization of Remazol Black-B by the selective bacterial isolates (ETL-1, ETL-2 & ETL-3) was confirmed by conducting another experiment in liquid medium at different time periods (Figure 1). It was found that different bacterial isolates had variable potential to remove Remazol Black-B in the growing cultures. The most efficient bacterial isolate to decolorize the Remazol black-B was ETL-1 with 98% color removal efficiency in 18 h incubation period while remaining isolates displayed maximum decolorization in 24 h. Isolate ETL-2 was the second most efficient bacterial isolate and it decolorized the Remazol Black-B up to 94% in 24 h. Similarly, ETL-3 isolates had decolorization potential of 80.

Figure 1. Biodecolorization of Remazol Black –B

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3.2. Factors Affecting Biodecolorization of Remazol Black-B in Liquid Medium

Potential of selected isolates (ETL-1, ETL-2, and ETL-3) was further investigated for the optimization of various incubation/ environmental conditions for decolorizing the azo dye in liquid medium. It was evident (Figure 2) that Remazol Black-B azo dye decolorization sharply increased up to 100 mg L-1 of substrate concentration and maximum decolorization was observed at 100 mg L-1 of substrate concentration. Then, there was a gradual decrease in the azo dye decolorization. Isolate ETL-1 was the most efficient azo dye decolorizing strain with more or less complete removal of the color i.e., 100% decolorization at 100 mg L-1 and minimum decolorization was recorded at 50 mg L-1 while after 100 mg L-1 substrate concentration, again ETL-1 showed a decreasing trend. Isolate ETL-2 was the second at the rank with 90% decolorization at 100 mg L-1. But, ETL-3 showed different trend from the other isolates, it indicated enhanced decolorization up to 200 mg L-1 (82%).

Figure. 2 Effect of Substrate Concentration

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Figure 3. Effect of different Sources of carbon on decolorization of Ramazol Black-B

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3.3. Types of Carbon Sources

Effects of different carbon sources such as maltose, mannitol, glucose and yeast extract were evaluated on Remazol Black-B decolorization by bacterial isolates (Figure 3). It was found that maximum decolorization occurred with 4% yeast extract in all selected strains (85 to 95%) that was followed by glucose in which decolorization occurred in the range of 20 to 25%. However, least decolorization was observed in the case of mannitol (10 to 15%). Similarly, maltose application also showed decolorization in the lower range (up to 18%).

3.4. Effect of pH

For studying effect of pH value, different levels of pH ranging from 5 to 9 were used and incubation of all selected isolates was done at these levels (Figure 4). Initially with the increase in pH value from 5 to 7, decolorization increased and maximum occurred at 7 pH. Similarly, further increase in pH from 7 to 9 had negative effect on decolorization capacity of various isolates. The maximum decolorization was observed with the isolate ETL-1 (98%) at pH 7 while minimum decolorization occurred at pH 9. Similar trends in remaining isolates ETL-2, and ETL-3 were observed at pH 7. Overall, it was noted that all the bacterial isolates showed optimum decolorization from pH 5 to 7.

Figure 4. Effect of pH on decolorization of Ramazol Black-B

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3.5. Effect of Incubation Temperature

Five levels (25,30,35,40, and 45°C) of temperature were used for assessing optimal biodecolorization of Remazol Black-B by selected bacterial isolates. It is evident (Figure 5) that when the temperature raised from 25 to 35°Cthere was inconsistent trend in decolorization by different isolates. The ETL-1 and ETL-2 isolates showed gradual increase in decolorization, while one isolate ETL-3 displayed maximum decolorization at 25°C. Remaining two bacterial isolates (ETL-1 and ETL-2) with a gradual rise from 25 to 35°C showed maximum decolorization at 35°C. As the temperature increased further from 35°C to 45°C, there was sharp decline in decolorization capacity in all the isolates. It was also observed that with rise in temperature, abiotic decolorization also increased. Maximum decolorization was observed with the isolate ETL-1 (98%) at 35°Cand it is followed by ETL-2 (94%) at the same temperature. Least decolorization was observed at 45°Cin all the selected isolates.

Figure 5. Effect of incubation temperature

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