Microbial Degradation and Decolorization of Reactive Orange Dye by Strain of Pseudomonas Spp

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3.2. Effect of Physicochemical Conditions on the Decolorization Performance

The effect of various physicochemical conditions such as pH, temperature, dye concentration, effect of carbon and nitrogen sources on decolorization of Reactive orange by the isolate was studied in detail. All parameters were studied at 37°C under static condition. 10% inoculums with O.D₆₀₀ 1.0 were used at 50 mg/L dye concentration.

3.2.1. Effect of pH

Bacterial cultures generally exhibit maximum decolorization at pH values near 7-8. In the present study the culture exhibited decolorization activity in the range of pH 5-8. At pH 3 and 4 decolorization observed was 15 and 38% respectively. The isolate showed more or less constant decolorization from pH 5 to 8. Maximum (98%) being at pH 8 (Figure 2). The isolate exhibited decolorization ability in the range of pH 5-8. This is in accordance with previous reports; Pseudomons aeruginosa BCH exhibited best decolorization at pH 8 ^[29].

Figure 2. Effect of pH on decolorization

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3.2.2. Effect of Temperature

Temperature is another very important parameter for treatment of wastewater. Selected isolate is mesophilic bacteria because it showed better decolorization in the temperature range of 25 to 37 °C. Similar results were also reported by Guo et al. (2008). The mesophilic range is traditionally used ^[33] since it is generally thought that maintaining high temperature would be uneconomical, while degradation within the psychrophilic range is too slow. Pseudomonas spp. showed strong decolorizing activity from 27°C to 37°C (Figure 3). Although a lag phase was observed and the decolorization rate was comparatively low at 22°C, the decolorization extent increased to a similar level from 27°C to 37°C. Decolorizing activity was significantly suppressed at 42°C, which might be due to the loss of cell viability or deactivation of the enzymes responsible for decolorization at 42°C ^{[7, 21]}. The isolate showed complete decolorization at 30, & 37°C but rapid decolorization was observed at 37°C (Figure 3). At 42 & 45°C there was no decolorization. Hence temperature optimum for Reactive orange was found to be 37°C. This may be owing to a greater production of enzymes and optimal growth conditions of the isolate for its dye decolorizing ability. The decolorization at this optimum temperature may be owing to higher respiration and substrate metabolism. This also demonstrate that decolorization of the dye was through microbial reaction which relies on optimal temperature and not by adsorption. At 42 & 45°C there was no decolorization.

Figure 3. Effect of temperature on decolorization

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3.2.3. Effect of Initial Dye Concentration

Decolorizing ability of the culture in the present study increased with increase in dye concentration from 50-250 mg/L (Figure 4). The isolate could decolorize 95% of 50 mg/L of the dye in 5 h whereas it took 48 h to decolorize 80% of 250 mg /L. the culture shows decolorizing ability up to 200mg/L at a faster rate after which the rate began decreasing. It has been proposed that efficiency of microbial decolorization through a combination of factors including the toxicity imposed by dye at higher concentration ^[23]. Thus, the isolate which could decolorize dye up to the reported dye concentration in wastewater, can be successfully employed for treatment of dye bearing industrial wastewater.

Figure 4. Effect of initial dye concentration

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3.2.4. Effect of carbon and Nitrogen Sources

While trying to enhance decolorization performance of reactive orange, extra carbon and nitrogen sources was supplied in semi synthetic medium. There was no decolorization in the presence in the synthetic media. Percentage decolorization was maximum with purified substrate yeast 95% while 92 and 78% decolorization was showed when supplied with malt and meat extract respectively while less decolorization with other supplements of carbon and nitrogen source within 24h (Figure 5). Similar result was also reported for bacterium consortium RVM for decolorization of reactive violet 5 ^[22]. The culture showed moderate decolorization in the presence of sucrose (50%) lactose (45%) and starch (55%) and decolorization in the presence of glucose was reported (62%). In addition supplying urea as a nitrogen source exhibited less decolorizing ability (38%). In contrast, addition of carbon sources seem to less effective to promote the decolorization probably due to the preference of the cells in assimilating the added carbon source seemed to be less effective to promote the decolorization probably due to the preference of the cells in assimilating the added carbon sources over using the dye compound as the carbon source.

Figure 5. Effect of carbon & Nitrogen sources

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3.3. FTIR Analyses

Figure 6. FTIR Spectrum of control dye

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The FTIR spectrum of a control dye and extracted metabolites (24 hours) by Pseudomonas spp. was compared. The spectrum of the control dye (Figure 6) displayed a peak 3418.11 cm^-1 for –NH stretching. The stretching between C N was reported at 2271.30 cm^-1 and amide, 5-membered ring peak at 1706.86 cm^-1. The peak at 1637.21 cm^-1 showed carbonyl stretching vibration. Peak at 1374.15 cm^-1 showed unsaturated nitrogen compounds. Peak at 1229.06 cm^-1 showed S=O stretching vibrations. The peak at 1106.62 cm^-1 indicates the aromatic nature. The peak at 616.95 cm^-1 showed hydrocarbon chromophore-C-H bending. The FTIR spectrum of 24 hours extracted metabolites of Pseudomonas spp. (Figure 7) showed a significant change in positions of peak, when compared to the control dye spectrum. A new peak at 1636.59 cm^-1 represented –N=N- stretching vibration. The C-H deformation showed at 1398.13 cm^-1. The peak at 3408.37 cm^-1 showed N-H stretching vibration.

Figure 7. FTIR Spectrum of degraded dye

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