Study On Isolation,Identification And Degradation Characteristics Of Azo Dye Degrading Strains

Azo dye is by far one category of synthetic dyes with the largest usage amount, and it is widely used in the textile, leather, food and cosmetics industries, because of its low cost and outstanding dyeing performance. Azo dyes have abilities of anti-light and anti-oxidation so that it can exist in the water stablely, affecting water chromaticity and the survival of aquatic organisms. In addition, most of the azo dyes and their metabolic intermediate are carcinogenic, teratogenic and mutagenic. Therefore, water pollution caused by azo dyes has been the focus of attention at home and abroad. At present, lots of ways can be used to treat azo dye wastewater. Relatively speaking, biological method is efficient, safe, low-energyconsuming and environmentally friendly. Therefore, screening microbes, which are capable of efficiently degrading azo dyes, has important practical significance. This study focuses on the isolation, identification of bacteria and exploring its growth and degradation characteristics to obtain its optimal growth conditions. A preliminary inquiry on enzymology characteristics is taken into action at the same time, in order to provide more adequate microbial resources for dye wastewater degradation, and provide strong support for the theoretical research and application on microbial degradation.In the study, two strains L-1 and L-4, which can efficiently degrade and decolor methyl red, were isolated from the environment contaminated with dyes for a long time. Based on morphology observation, physiological and biochemical characteristics, and 16S rRNA gene sequence analysis, L-1 and L-4 were identified as Dyadobacter sp.and Paracoccus sp respectively. They were also isolated from the species for the first time, which has the capablity of degrading methyl red.Stain L-1 has high similarities with Dyadobacter sp. D.fermentans DSM 18053^T, D. soil JCM 16232^T and D. beijingensis CGMCC 1.6375^T, reaching 99.2%, 98.9% and 99.2% respectively. The main cellular fatty acids of strain L-1 contain iso-C15 : 0?C16 : 1?5c?isoC17 : 0 3-OHand summed feature 3?C16: 1 ?7cand /or C16: 1 ?6c?. In addition, C16:0?iso-C15 : 0 3-OH and C16 : 0 3-OH are also an important part of the bacteria. Compared with model bacterium, distributionand and content are very similar. The DNA G+Cmol % content of the strain L- 1 is 51.3%. DNA-DNA hybrid homology results show that L-1 has low genomic relatedness with D.fermentans, D. soil and D. beijingensis, reaching 38.6±2.6%?41.2±1.8% and 35.0±2.1%, respectively. To summarize, we identified L-1 as a new species of the genus Dyadobacter, and named it as Dyadobacter jiangsuensis sp. nov.The study also explored the optimal growth conditions of the strain L-4. It's found that the strain is able to use a variety of carbon and nitrogen source in experiments. In LB liquid medium, OD600 nm can achieve 2.2. The optimum temperature and pH for growth of the strain were 30 ? and 7.0, respectively. Growth of the strain affected by ventilation volume generally presents a downward trend when fluid volume increases, but the impact is not obvious. Some metal ions have a strong inhibitory effect on the growth of the strain, and the inhibitory effect of Co²⁺ is the strongest.Experiments of degradation characteristics of strain L-4 show that the optimum temperature for degrading methyl red is 25³0?, and the suitable value of pH is 6.0⁷.0. Zn²⁺, Co²⁺, Cu²⁺, Ag+, Al³⁺, Fe³⁺, Fe²⁺ have a stronger inhibitory effect on the degradation of methyl red. But the effect of ventilation volume is less, and anoxic conditions can be helpful to degradation of methyl red. When the amount of liquid was3125m L at 12 h, the degradation rate of methyl red was still over 40%. The strain also has good decolorization degradation ability on high concentration of methyl red, when the initial concentration of the mass was?200 mg/L, methyl red decolorization rate remained above 90% at 24 h, and when the amount of liquid was 3300 mg/L, the decolorization rate also can reach 43.35%.The experiment results of enzymology characteristics of L-4 show that the enzyme is a kind of constitutive expression enzymes. Most is outside the cell, and a small amount exists in the periplasmic space. The optimum enzymatic reaction system is shown as follow. The temperature is 30?, and the pH is 7.0. The amount of crude enzyme, which is added into the reaction system of 5ml, is 100?L, and the reaction time is 15 min. The enzyme with the condition of 30?, pH6.0⁷.0 has higher stability, which can reach 95.98%. 1mmol/L of Fe²⁺ can promote the degradation of methyl red, and Ca²⁺didn't promote or inhibit the degradation of methyl red. And other metal ions have different degrees of inhibition. The enzyme is the most stable in acetone, methanol and ethanol, and relative energy is over 100%, and it can reach 116.88% in ethanol. Other organic solvents have inhibitory effect on the enzymatic activity. Among them, the inhibition effect of chloroform was the strongest, the relative enzymatic activity was only 9.09%.