对甲苯磺酸高效降解菌的筛

发布时间：2018-04-20 17:54

本文选题：p-TSA + 施氏假单胞菌Y-2　；参考：《南京农业大学》2015年硕士论文

【摘要】：对甲苯磺酸(p-TSA)是一种广泛应用于工业中的重要的化工原料,含p-TSA的废水大量排放到环境中,会对人类健康以及生态环境造成严重威胁。本论文从江苏丰山制药厂废水处理池活性污泥中,通过富集、分离,筛选出一株能以p-TSA为唯一碳源生长的菌株,命名为Y-2。该菌株能够在16h内将200mg/L的p-TSA完全降解。经鉴定,菌株Y-2为革兰氏阴性菌,根据其生理生化特征以及16s rRNA基因序列同源性比较,菌株Y-2被鉴定为施氏假单胞菌(Pseudomonasstutzeri)。菌株Y-2在LB培养基中生长良好,0-5h菌株处于延滞期,5h开始,菌株进入对数生长期,16h-25h菌株为稳定期,25h后菌株逐渐衰亡。菌株Y-2生长的最适温度为30°C,最适pH为7.0-8.0;菌株Y-2为严格好氧菌,通气量越多,菌株生长越好;NaCl浓度也对菌株的生长有影响,菌株Y-2在NaCl浓度为5-15g/L时长势较好,菌株生长最适的NaCl浓度为10g/L。降解菌Y-2可利用葡萄糖、果糖和麦芽糖等碳源,但是不能利用蔗糖、甘露醇、木糖等碳源;能利用蛋白胨、硫酸铵和氯化铵等氮源,也可以利用硝态氮,但不能利用尿素。菌株Y-2可以在16h之内完全降解浓度为200mg/L的p-TSA。菌株降解p-TSA的最适pH为7.0-8.0,最适温度为300°C;通气量越大越有利于p-TSA的降解。接种量对菌株Y-2对p-TSA的降解也有影响,当接种量在0.1%-2%的范围内时,加大接种量可以明显提高降解速率,接种量达到4%后,再增大接种量p-TSA降解速率基本不变。随着p-TSA初始浓度的增加,菌株Y-2的延滞期越来越长,但菌株能够在40h之内完全降解500mg/L的p-TSA,降解效率较高。添加200mg/L的葡萄糖作为额外碳源会促进p-TSA的降解,大大缩短降解时间。通过液质联用检测p-TSA降解过程中的代谢产物,发现主要的代谢产物有对羧基苯磺酸、对羟基苯磺酸、对苯二酚、丁烯二酸和乙二酸。根据检测结果推测可能的降解途径为:首先是甲基侧链氧化成羧基,紧接着羧基进一步脱碳生成羟基,生成对羟基苯磺酸,然后磺酸基脱落变成羟基,生成对苯二酚,然后对苯二酚开环,变成丁烯二酸和乙二酸,最后基本将其完全矿化。用劳麦方程对菌株Y-2降解p-TSA的降解动力学进行拟合,得出其动力学参数分别为 Vmax =46.95 mg/L/h,Ks=59.09 mg/L,当底物 p-TSA 浓度远远大于 46.95mg/L时,菌株Y-2对p-TSA的降解符合零级动力学模型,即p-TSA以最大速率降解,而与底物浓度无关。当底物p-TSA浓度远远小于46.95mg/L时,此时对甲苯磺酸降解速率与底物浓度呈一级反应关系,即底物浓度越高,降解速率越快。而在中间浓度时,菌株Y-2对p-TSA的降解处于混合级反应区。
[Abstract]:P-toluenesulfonic acid (p-TSA) is an important chemical raw material widely used in industry. Wastewater containing p-TSA is discharged into the environment in large quantities, which will pose a serious threat to human health and ecological environment. In this paper, a strain named Y-2 was selected from activated sludge of waste water treatment pond of Fengshan Pharmaceutical Factory in Jiangsu Province, which can grow with p-TSA as the sole carbon source by enrichment and separation. The strain could completely degrade the p-TSA of 200mg/L within 16 hours. The strain Y-2 was identified as a gram-negative strain. According to its physiological and biochemical characteristics and the homology of 16s rRNA gene sequence, strain Y-2 was identified as Pseudomonas Scheridoides. The strain Y-2 grew well in LB medium for 0-5h, and the strain began to decline after entering logarithmic growth stage (16h-25h) for 25 h. The optimum growth temperature and pH of strain Y-2 were 30 掳C and 7.0-8.0, respectively. Strain Y-2 was a strict aerobic bacterium, and the more aeration, the better the growth of strain Y-2, and the better the concentration of NaCl was, the better the growth of strain Y-2 was when the concentration of NaCl was 5-15g/L. The optimal concentration of NaCl for the growth of the strain was 10 g / L. The degradation bacteria Y-2 can use carbon sources such as glucose, fructose and maltose, but not sucrose, mannitol, xylose, nitrogen sources such as peptone, ammonium sulfate and ammonium chloride, but not urea. Strain Y-2 could completely degrade p-TSA with 200mg/L concentration within 16 hours. The optimum pH and temperature for p-TSA degradation were 7.0-8.0 and 300 掳C, respectively. The higher the aeration rate was, the more favorable the degradation of p-TSA was. The amount of inoculation also affected the degradation of p-TSA of strain Y-2. When the inoculation amount was in the range of 0.1-2%, the degradation rate of p-TSA could be increased obviously when the inoculation amount reached 4%, and the degradation rate of p-TSA was almost unchanged when the inoculation amount reached 4%. With the increase of initial concentration of p-TSA, the delay period of strain Y-2 was longer and longer, but the strain could completely degrade p-TSA of 500mg/L within 40 h, and the degradation efficiency was higher. The addition of 200mg/L as an additional carbon source could promote the degradation of p-TSA and shorten the degradation time. The main metabolites were p-carboxybenzenesulfonic acid, p-hydroxy benzenesulfonic acid, hydroquinone, butylenedioic acid and glycolic acid. According to the detection results, the possible degradation pathway is as follows: first, methyl side chain is oxidized to carboxyl group, then carboxyl group further decarbonizes to form p-hydroxy benzenesulfonic acid, then sulfonic group shedding to hydroxyl group to form hydroquinone. Then hydroquinone is ring-opening and becomes succinic acid and glycolic acid, and finally it is completely mineralized. The degradation kinetics of strain Y-2 degrading p-TSA was fitted by Laumai equation, and the kinetic parameters were obtained as follows: Vmax = 46.95 mg / L / L / L = 59.09 mg / L, respectively. When the p-TSA concentration of the substrate was much higher than that of 46.95mg/L, the degradation of p-TSA by strain Y-2 was in accordance with the zero-order kinetic model. That is, p-TSA degrades at maximum rate, independent of substrate concentration. When the concentration of substrate p-TSA is much lower than that of 46.95mg/L, the degradation rate of p-toluenesulfonic acid is first order reaction with the concentration of substrate, that is, the higher the substrate concentration, the faster the degradation rate of p-toluenesulfonic acid. At the intermediate concentration, the degradation of p-TSA by strain Y-2 was in the mixed reaction zone.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172

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