Optimization of Process Conditions for Effective Degradation of Azo Blue Dye by Streptomyces DJP15.
MATERIALS AND METHODS
The previously isolated potential strain of Streptomyces DJP15 (20) was grown and maintained on enrichment media (21) amended with 50 mg/L of azo blue dye at a temperature of 37[degrees]C under agitation at 180 rpm. Decolurisation experiment were carried out in 50 mL of starch casein broth the medium (soluble starch 10.0g, [K.sub.2]HP[O.sub.4] 2.0 g, KN[O.sub.3] 2.0g, NaCl 2.0 g, Casein 0.3 g, MgS[O.sub.4] 0.05 g, CaC[O.sub.3] 0.02 g, FeS[O.sub.4] 0.01g, Distilled [H.sub.2]O 1000 mL, pH 7.0) amended with 50 mg/L of the test dye. The efficiency of degradation percentage of the azo blue dye by Streptomyces DJP15 was studied with respect to the varying effects of shake condition, static condition, dye concentration, inoculum size, pH and temperature for optimization of the degradation process. All experiments were done in triplicates.
Analytical methods for dye decolourisation studies
Aliquots (5 mL) of the culture media were withdrawn at time intervals of 6 h over 54 h and centrifuged at 7000 rpm for 15 min. Decolourisation was quantitatively analyzed by measuring the absorbance of the supernatant using a UV-visible spectrophotometer (Spectronic[R] GENESYS TM 2 PC; at maximum wavelength, [lambda]max, of 620 nm for azo blue dye. The decolourisation rate was calculated using the equation (22).
Dye Decolourisation percentage = Initial absorbance-Final absorbance/Initial absorbance X 100
Optimization of process conditions for effective dye degradation
Optimization of important conditions such as shake condition, static condition, dye concentration, inoculums size, pH and temperature for effective dye degradation by the potential isolates of Streptomyces was carried out (23). The potential Streptomyces DJP15 strain was examined for the degradation of azo blue dye. Effect of one parameter at a time, keeping others constant was followed. Influence of shake condition and static conditions on the maximum degradation of dye, at empirical conditions was carried out primarily before the examination of other mentioned conditions.
Influence of shake and static conditions
50 mL of starch casein broth was added into 100 mL Erlenmeyer conical flask and sterilized. 1 mL of azo blue dye, at the concentration of 50 mg/L was added to the broth independently. 1 mL of 3 days old cultures of test isolate Streptomyces DJP15 was inoculated to the broth and incubated at 35[degrees]C for 3 days, under shake condition (on shakers at 180 rpm) as well as static conditions. 5 mL of the incubated broth was drawn at every 6 h and centrifuged at 7000 rpm for 15 min. Absorbance of the supernatant was recorded using UV vis spectrophotometer at 620 nm for azo blue dye. The percent degradation of the dye was calculated as mentioned earlier.
Optimization of dye concentration
The maximum dye degradation under static state by the potential isolate Streptomyces DJP15at different concentrations of dye was assessed following broth culture method as mentioned earlier. Degradation of azo blue dye was examined at the concentrations of 50, 100, 150, 200, 250 and 300 mg/L. The percent dye degradation was calculated as mentioned earlier. Optimization of inoculum size
Inoculum size was optimized for effective dye degradation by the test isolate Streptomyces DJP15, following broth culture method as mentioned above. Inoculum size of 3 days old test isolate at 1, 2, 3, 4 and 5% (v/v) were assessed for maximum dye degradation.
Optimization of pH
Various levels of pH were optimized for effective dye degradation by the test isolate Streptomyces DJP15, following broth culture method as mentioned above. pH 6.0, 6.5, 7.0, 7.5 and 8.0 of the medium were adjusted using dilute acidic and alkaline solution of hydrochloric acid and sodium hydroxide respectively.
Optimization of temperature
Various ranges of temperatures were optimized for effective dye degradation by the test isolate Streptomyces DJP15, following broth culture method as mentioned above. The effect of temperature on the maximum dye degradation was examined by keeping the inoculated broth at 25, 30, 35, 40 and 45[degrees]C respectively. The percent dye degradation by the test isolate at different ranges of temperatures was calculated as mentioned earlier.
Optimization of process conditions for effective dye degradation
In the present study, an attempt was made to optimize the degradation of azo blue dye by the potential Streptomyces strain DJP15. Effect of various process parameters like shake condition, static condition, dye concentrations, inoculum size, pH and temperature were studied. The efficiency of Streptomyces DJP15 isolate was evaluated for the degradation of azo blue dye. The effect of shake condition, still condition, dye concentration, inoculum size, pH and temperature was studied with an aim to determine the optimal conditions required for degradation of the azo blue dye in starch casein broth.
Influence of static and shake conditions
The percent degradation of azo blue dye by potential isolate Streptomyces DJP15 at shaking and static conditions were as shown in the Figure 1. The strain Streptomyces DJP15 showed maximum degradation of 65.26% for azo blue dye under continuous shaking conditions at 48 h of incubation time. Under still condition, a sudden increase in the percent degradation of 12.63% by Streptomyces DJP15 for blue dye was observed. The isolate Streptomyces DJP15 exhibited 77.89 % of maximum degradation at a incubation time of 48 h under static conditions. It was found that the isolate Streptomyces DJP15 showed more percent degradation under static conditions than shaking conditions. These results showed that the isolate Streptomyces DJP15 was more effective and potential in degrading azo blue dye under still conditions than shaking conditions.
To the best of our knowledge, it is the first report on degradation of sulfonated reactive di azo textile dyes (azo blue) by Streptomyces strains. Optimization of dye concentration
Figure 2 shows the effect of initial concentration of dye ranging from 50-300 mg/L. The percent degradation of azo blue dye after 48 h of incubation by Streptomyces DJP15 was found to be 76.66, 71.25, 68.42, 64.78, 61.53 and 45% at initial dye concentrations of 50, 100, 150, 200, 250 and 300 mg/L respectively. It was further noted that the degradation of the dye was concentration dependent. It was clear from the observation that, percent degradation of dye increased with an increase in time, irrespective of initial dye concentration. Further, percent degradation of dye decreased with an increase in dye concentration. i. e. lower the concentration higher the degradation efficiency and vice-versa. In our study, the diazo dye, reactive blue 222 (azo blue) degraded up to 76.66% at 48 h of incubation with an initial dye concentration of 50 mg/L by the isolate Streptomyces DJP15. Optimization of inoculum size
Effect of inoculum size (1-5% v/v) with time on degradation of azo blue dye was represented in the Figure 3. The result in Figure 3 depicts that, at every dose of inoculum, dye degradation increased with time during 6 to 48 h incubation. After 48 h, the percent degradation of azo blue dye was found to be 76.03, 77.68, 80.99, 81.81 and 81.81% at inoculum sizes of 1, 2, 3, 4, and 5% v/v respectively. When the inoculum size was increased up to 3.0% (v/v), the extent of degradation increased to 80.99% at 48 h of incubation. No drastic or considerable increase or decrease in the percent degradation was observed when the inoculum size was increased to 4.0 and 5.0% (v/v). The maximum dye degradation (80.99 %) was attained at 3.0% (v/v) inoculum at 48 h. Therefore, 3.0% (v/v) dose of Streptomyces DJP15 inoculum was selected as optimum for the degradation of azo blue dye.
Optimization of pH
Effect of pH (6.0-8.0) on the degradation of azo blue dye by Streptomyces DJP15 was shown in Figure 4. In our study, it was noticed that, an increase in pH from 6 to 7 enhanced the rate of degradation significantly. However, degradation rate was the highest between 7-7.5 pH. Highest degree of degradation occurred at optimum pH 7.0 at 48 h of incubation. The results further revealed that, any deviation in the pH from optimum, decreased the extent of dye degradation. From the Figure 4 it was clearly noted that, the percent degradation of azo blue dye increased with increase in time irrespective of pH. The maximum percent degradation (76.31%) of dye was found at pH 7 after 48 h of incubation period. Good percent degradation (72.10%) was observed at pH 7.5. Further, increase in pH from 7.5 to 8.0, decreased the percent degradation of azo blue to 67.89%. Least percent degradation (57.36%) was recorded at pH 6.0. 62.63% degradation was noticed at pH 6.5. It was clearly understood that, degradation was lower in acidic pH than alkaline pH.
Optimization of temperature
Figure 5 shows degradation of azo blue dye by Streptomyces DJP15 with time at different temperatures (25, 30, 35, 40 and 45[degrees]C). The percentage degradation of azo blue dye at 25, 30, 35 40 and 45[degrees]C was found to be 57.89, 74.21, 79.47, 68.94 and 64.73% respectively. It was clear that, percent degradation of dye increased with an increase in temperature from 25 to 35[degrees]C. The percentage removal of dye was decreased with further increase in temperature up to 45[degrees]C. Degradation activity was significantly suppressed at 25[degrees]C than other temperatures, which might be due to the loss of cell viability or deactivation of the enzymes responsible for degradation at 25[degrees]C (Cetin 2006).Further, increase in the temperature resulted in the decrease in the percent degradation. This may be due to the that at higher temperatures, thermal deactivation to the enzyme responsible for degradation may occur.
Extended period of incubation further to 6 h (54 h), decreased the percent degradation may be due to the decline phase of the isolate growth curve in all the conditions subjected for the study.
Microbes posses more than one mechanism for dye degradation (24). Decolorisation of dye was enhanced by static condition as previously reported by the researchers (25). Other researchers also reported that, static conditions were suitable for dye degradation process (26, 27, 28). Generally stationary culture condition dominates over shake culture condition (29, 30, 31). The present study supports that still / static condition is suitable for dye degradation process as other researchers reported (32, 33). The more efficient decolorisation of similar structurally complex dyes under shaking condition was reported (34). Azo dye degradation of 20% in shake culture and more than 95% in still culture by Proteus mirabilis was reported (35). The initial biodegradation of azo dye occurs under anoxic condition leading to reductive cleavage of azo bond which causes decolorization of the dye. During shaking condition, presence of oxygen leads to deprive the azoreductase required for azo bond cleavage. In the present study degradation was noticed both in shaking and static conditions by both the isolates but effective degradation was recorded only under static condition than shaking condition. The competition between azo dyes and oxygen for reduced electron carriers under aerobic condition was the reason for decreased decolorization at shaking condition (32). This reveals that the enzyme azoreductase involved in the initial step of azo bond reduction must be an oxygen insensitive (9, 36). The dye concentration can influence the efficiency of microbial decolourization through a combination of factors including the toxicity imposed by dye at higher concentration 16 37 and the dye degradation efficiency depends on the initial dye concentration 38. The degradation of 80% of the synthetic dyes by Pseudomonas sp at 50 mg/L concentration in more than 7 days was reported (39). The 80% decolorisation of navy blue 3G at 50mg/L by Brevibacillus laterosporus MTCC2298 within 48 h under static condition was also reported (40). It was noted that beyond certain size of inoculum there was no proportionate increase in degradation with further increase in size of inoculum (41). Rate of terasil black effluent decolorization enhanced with increase in inoculum size of B. cereus from 2.5 to 10%; however, further increase of inoculum up to 20% did not cause any change in the intensity of color (42). The pH has a major effect on the efficiency of dye decolorization, and the optimal pH for color removal is often between 6.0 and 10.0 for most of the dyes (35). The effect of pH in degradation of the dye may be due to the transport of dye molecules across the cell membrane, which is considered as a rate limiting step for dye decolorization (43). The high decolourization of Reactive Black 5 by Enterobacter EC3 at pH 7 was reported (29). The maximum decolourization of Reactive Red 195 by Georgenia sp at pH 7 was also observed (44). E. coli and P. luteola both exhibited best decolorization rate at pH 7.045. Our findings also in accordance with these reports, where maximum dye degradation was noticed at pH 7.0. Inhibition of Klebsiella pneumoniae RS-1, and Alcaligens liquefaciens S-1 biodecolourisation activity at 45[degrees]C was reported (45). Optimum temperature of 37[degrees]C was observed for the decolorization of acid orange 10 and disperse blue 79 by Bacillus fusiformis kmk 546. The optimum temperature of 30-40[degrees]C as for decolorization of crystal violet by Shewanella Sp NT0Vl was observed (47). The decrease in the decolorization activity at higher temperature can be attributed to the loss of cell viability or to denaturation of the azoreductase enzyme (48). The pH and temperature exert major effect on the efficiency of dye decolorization and that optimal conditions vary between pH 7.0-10.0 and 30-40 [degrees]C, respectively18, 26, 35.
The isolate Streptomyces DJP15 found to be very effective and potential in degrading the textile dye azo blue. The significant and striking observation of the resistance to higher levels of azo blue dye toxicity by the strain Streptomyces DJP15 enables their use for in situ bioremediation because it indicates the ability of strain to withstand shock loads of dye during the bioremediation process. The results of incubation temperature showed no deactivation of the degradation ability of the isolates up to 45[degrees]C which indicates the thermo tolerance ability of the isolates. Therefore, the isolate Streptomyces DJP15 could be useful for on-field process in a country like India, where temperatures reaches to above 40[degrees]C in some parts of the country during summer season. However, there is a need for further investigation to understand the enzymes and other mechanisms involved in the degradation of the azo blue dye by the isolate Streptomyces DJP15 in order to harness its property for bioremediating the dye contaminated habitats for clean environment and clean nature for all life.
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H.P. Jai Shanker Pillai
Faculty of Public Health, St. Theresa International College, 1 Moo 6, Rangsit-Nakhonnayok Road (Klong 14), Bungsan, Ongkharak - 26210, Thailand.
(Received: 20 October 2017; accepted: 30 November 2017)
* To whom all correspondence should be addressed.
Caption: Fig. 1. Degradation of azo blue by potential isolate Streptomyces DJP15 at shake and static conditions
Caption: Fig. 2. Degradation of azo blue by Streptomyces DJP15 at different dye concentrations
Caption: Fig. 3. Degradation of azo blueby Streptomyces DJP15 at different inoculum size
Caption: Fig. 4. Degradation of azo blue by Streptomyces DJP15 at different pH
Caption: Fig. 5. Degradation of azo blue by Streptomyces DJP15 at different temperature
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|Author:||Pillai, H.P. Jai Shanker|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Dec 1, 2017|
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