Team:Worldshaper-XSHS/Results
Results
Standard curve of RR
We prepare reactive red x (RR) into different concentrations of solution, and test the absorbance at 530nm to get the data. After taking the average value of absorbance, we analyzed the correlation between the concentration of RR and it. After fitting the function, we can see that there is a good linear relationship between RR concentration and absorbance.
Figure 1. Relationship between RR concentration and absorbance
laccase decolorization ability
We tested the function of two laccase, ScLac and CotA. We divided the scLac into two groups: one group was induced by IPTG, which was labeled as ScLac +, while the other group without IPTG was labeled as ScLac -. Similarly, CotA was divided into two groups for control experiment. We detect the absorbance at regular intervals, and after statistical analysis, we get the results of Fig. 2 and Fig. 3.
Figure 2. Absorbance change of RR catalyzed by cotA
Figure 3. Absorbance change of RR under the catalysis of scLac
It can be seen from Figure 2 that under the catalysis of cotA, the dye has not been significantly degraded in the first 12 hours. However, after 12 hours of degradation, there were significant differences between the IPTG induced group and the control group, and the absorbance value decreased significantly, which proved that the degradation effect was significant after IPTG induced expression. After 48 hours, the absorbance value has been reduced to 0.7, which is better than that of the control group, indicating that cotA has a better decolorization effect under the induction of IPTG.
From the analysis in Figure 3, we can see that the degradation speed of ScLac group is faster than that of CotA group, the decolorization effect increases significantly after 12h, and the absorbance has decreased to 0.4 after 48h, which is significantly different from the control group.
At the same time, we also tested the control bacterial solution to exclude the influence of plasmid and bacteria on dye degradation. After analyzing the experimental results, we get Figure 4. Control + was induced by IPTG and control - was not induced by IPTG. It can be seen from the figure that the two groups of data have a high degree of agreement, and whether or not IPTG is added has little effect on the degradation of dyes. However, with the extension of time, the absorbance values of the two groups of dyes decreased to a certain extent. This trend is fluctuant, indicating that the bacteria may have weak degradation capacity for dyes, but it does not play a leading role in the decolorization of dyes.
In order to determine whether the degradation of dyes is caused by bacteria or there is a certain degradation of dyes themselves, we made a group of dye control, that is, only adding dyes without adding any bacteria. After analyzing the data, we get Figure 5. We found that the absorbance of the dye itself changed little with time.
Figure 4. Absorbance change of RR in control bacterial solution
Figure 5. Absorbance change of dye RR
At the same time of the absorbance detection of the supernatant, we also retained the bacteria and observed them. It can be seen from Fig. 6 that in IPTG induction group, the color of bacteria is purple, which is closer to the color of dye. However, in the control group without IPTG, the color of bacteria was yellow, which was more similar to the color of the mixture of bacteria and pigment, so it was speculated that its adsorption on dye was very weak. According to the color observation of the supernatant in Figure 7, after 48h, the dye has been degraded to a large extent, and its color is obviously different from the original color.
Figure 6. Cell color change under cotA catalysis
Figure 7. Color change of supernatant under the catalysis of cotA
Similar results were obtained in two groups of experiments catalyzed by scLac (Fig. 8, Fig. 9). The color of the bacteria was compared with that of the bacteria catalyzed by CotA. It was found that the color of the bacteria was darker when it was catalyzed by ScLac, which may be that when E. Perhaps because of this reason, scLac showed stronger degradation ability than cotA. After 48 hours, the color of the supernatant under the catalysis of scLac decreased obviously.
Figure 8. Cell color change under the catalysis of scLac
Figure 9. Color change of supernatant under the catalysis of scLac
Test results of Azor decolorization ability
We also tested the decolorization ability of AzoR in azoreductase. However, because the decolorization function needs to be carried out in the oxygen free condition, and the experimental conditions are strict, so we are not so smooth. In the end, it didn't get the expected effect.
In order to simulate the anaerobic environment, we put each group of bacterial solution into EP pipe and sealed it with sealing strip. The supernatant was detected by absorbance every certain time. However, no matter whether there is IPTG induction or not, we have not seen obvious degradation of dyes.
Then we looked up the literature and found that CuCl2 may work normally only when it exists. So we did two more experiments, Azor + added CuCl2, AzoR - did not add CuCl2. After two days of experiment, we got disappointed data again. After a simple statistic of this set of data, we see two very gentle curves (Figure 10). No matter whether CuCl2 is added or not, the absorbance of the dye decreases slightly, which is consistent with the degradation of the dye itself, but the decrease of CuCl2 is slightly larger, which indicates that the presence of CuCl2 can promote the catalytic effect of AzoR to some extent, but after 48 hours, the absorbance of the supernatant of the two groups still hovers around 1.5, which indicates that Azor is not in our experimental condition. Show excellent decolorization ability.
Figure 10. Decolorization ability of Azor to dye RR in different environments
Actual sewage test
The wastewater from the printing and dyeing factory was added with the same concentration of dye RR as the previous experiment for RR degradation test. In order to ensure the consistency of the experimental method, we have done the dye degradation test of the sewage group and the experimental group at the same time, and recorded the light absorption value of the dye in 24 hours, 48 hours and 72 hours. The results are as follows:
In the experimental group, we repeated the experiment according to the method of functional test. In this result, cotA and scLac showed good degradation effect on RR. It can be seen that the dye had been degraded by naked eyes at 48 hours, but the degradation effect of ScLac was significantly better than that of CotA, and the color removal was more obvious.
In the sewage group, our two laccase can work normally, but there are also unexpected findings: in the sewage, the degradation effect of the enzyme is faster. At the same time, the control group which was not induced by IPTG also had obvious decolorization phenomenon. In view of this situation, we suspect that the reason may be the presence of degradation substances in sewage.
In order to verify the specific cause of this phenomenon, we did the following negative control experiments: adding wastewater treatment solution WR to LB culture medium directly, and taking dye RR in LB culture medium + pure water as the control. The results are as follows:
It can be seen that the sewage group without bacteria has a certain degradation effect. However, we have also observed mixed bacteria in the culture medium, but the possible degradation factors of the sewage itself can not be determined whether it is bacteria or other unknown substances, and further experiments are still needed to determine.
SDS-PAGE
We also performed SDS-PAGE experiments, and obtained the following results. AzR-, AzoR-, CotA- and ScLac- represent no induction with IPTG, and AzR+, AzoR+, CotA+ and ScLac+ represent after induction by IPTG. By reviewing the literature, we found that AzR has a protein size of 19.3 kDa, AzoR of 23.1 kDa, CotA of 59.9 kDa, and ScLac of 70.9 kDa. After comparison with the images, we found that all proteins were successfully expressed under the induction of IPTG. However, in the previous decolorization experiments, only the CotA+ and ScLac+ groups showed good decolorization ability, while the AzR+ and AzoR+ decolorization ability was not good. We speculated that this may be related to the experimental environment. Decolorization of dyes by AzR+ and AzoR+ requires a strict anaerobic environment. We sealed the EP tube with a sealing strip and could not meet the anaerobic requirements of the enzyme.
