| Molasses wastewater is a recalcitrant pollutant. At present, biological treatment is useful to reduce the color of molasses. Our group screened a strain, named Aspergillus flavus A5pl, has an ability to decolour the wastewater. In order to find out the mechanism of decolorization by A5p1, we chose melonoidin, a main pigment of molasses, as the object to study its mechanism, providing a theoretical basis for the bio-decolorization. At first, we determined conditions of culture of Aspergillus flavus A5P1, including the carbon source, concentration of carbon source, nitrogen source, concentration of nitrogen source and the initial pH. The results showed the rate of decolorization of melanoidin (concentration of20%) by Aspergillus flavus A5P1reached the highest value of65%after8days of cultivation in the following conditions:2.5%(w/v) of sucrose as the carbon source,0.2%(w/v) of sodium nitrate as the nitrogen source and initial pH of6.0. In the previous stage of the process of decolorization, the rates of decolorization of melanoidin and cells dry weight were positively related, and the pH was in the range of5.0to6.0. In order to study the mechanism, we investigated the effect of biological adsorption during process of decolorization by using inactivated cells of Aspergillus flavus A5p1. We used extracted crude intracellular and extracellular enzymes to study which kind of enzyme is the key during biological degradation. And then we measured the activities of the extracellular enzymes including laccase (Lac), Manganese peroxidase (MnP) and manganese-independent peroxidase (MIP). The results showed that, there was24.5percent rate of decolorization during the process of decolorization of melanoidin by inactivated cells of Aspergillus flavus A5p1, and this rate was accounted for1/3of the total rate of decolorization by activated cells. Through the results we also found that the intracellular enzymes play a more important role in biological degradation than the extracellular ones. Three kinds of extracellular, Lac, MnP and MIP, existed in the decolorization process, although there was no clear relationship in the trend with the rate of decolorization, but their function on degradation can not be excluded. Moreover, H2O2-producing activities during the process had related trend with decolorization, which suggested that peroxidation of hydrogen peroxide may be the one of the main mechanisms.Finally, we studied the effect of glucose oxidase (GOD) in the process of decolorization by Aspergillus flavus A5p1based on the above results, as glucose oxidase is one of the enzymes which produce hydrogen peroxide. By adding the sodium (activator), sodium bisulfate (inhibitor) and gluconic acid (catalytic products), we verified the effect of glucose oxidase. The results show that the trend of enzymatic activities of glucose oxidase, H2O2-producing activities and decoloring rate were positively related. That means glucose oxidase is one of enzymes which produce hydrogen peroxide to decolor melanoidin. However, the degree of change among these three parameters were different, we speculated that there may be other H2O2-produced enzymes during the decoloring process. And as Aspergillus flavus A5p1grew in Martin culture was not detected activity of glucose oxidase, we analysed that melanoidin might be the inducer of producing of glucose oxidase. According to the conclusion of glucose oxidase in system of melanoidin, we expored whether there is glucose oxidase in molasses wastewater, and how the glucose oxidase works in the decolorization of molasses wastewater. The results showed that glucose oxidase also effected on the decolorization of molasses wastewater, and the trend of glucose oxidase activities were also as the same as the ones of H2O2-producing activities and decoloring rate. But there might be other enzymes that effect on the decolorization, as glucose oxidase and hydrogen peroxide had less impacted on decolorization of molasses wastewater comparing with melanoidin. |
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