耐高浓度苯酚菌株的筛选及其降解特性研究

Screening of high-tolerance-phenol strain and its degrading capacity

通过液体富集培养,平板培养分离法从焦化废水的污泥中分离出1株可耐受2 000mg/L苯酚的菌株,经16S rDNA序列分析,鉴定为施氏假单胞菌(Pseudomonas stutzeri)。该菌能以苯酚为唯一碳源和能源生长。通过摇瓶试验和高效液相色谱(HPLC)分析法可知,在pH=7.5,温度为30℃的条件下,苯酚质量浓度在50～400mg/L时,该菌细胞生长和对苯酚的降解转换快速同步进行。当苯酚质量浓度在800～900mg/L,菌细胞依次出现快速生长、延缓生长、次快速生长3个生长时期,在前两时期内苯酚降解率低于5%,在次快速生长期内苯酚降解率从低于5%快速增加到100%。气相色谱-质谱联用仪(GC-MS)测定结果表明,该菌可将苯酚转化成4-羟基-2-氧代戊酸、邻苯二酚、对苯二酚、3,4-二羟基苯甲酸和对羟基苯甲酸等中间产物。

The aim of this study is to isolate and characterize a strain capable of high-phenol-tolerance so as to determine the kinetics of biodegradation on the intermediates based on phenol degradation. To achieve our research goal, we have adopted the liquid culture enrichment and plate-spreading method for isolating the strain from the acti- vated sludge of coking plant sewage with it being identified by 16S rDNA gene-sequencing method as Pseudomonas stutzeri and nominated as Pseudomonas stutzeri N2. And, next, the strain cultured under the temperature of 37 ℃ was allowed to grow on a plate with 2 000 mg/L of phenol till it turns to be a sole carbon energy source. And, later, the cultivating process was conducted with the fast cell-growth and phenol transformation, with no need to retard the growth period, so as to ameliorate the phenol-removing efficiency to 100% in 30 hours. Such an efficiency could also be made possible by shaking the testing flask at 150 r/min in the aerobic condition and high-efficient liquid chromatography （HPLC） with pH = 7.5, under 30 ℃, at the initial concentrations of phenol from 50 mg/L to 400 mg/L. However, when the initial concentrations of phenol were within 800 - 900 mg/L, three cell growth periods would be needed, that is, the fast growth, retarding growth, and inferior fast growth, correspondingly. The transformation rate of phenol proves to be low （only less than 5 % ） in the fast growth period and retarding growth period, whereas the transformation rate within the inferior fast growth periods would likely to increase from 5 % to 100% quickly. The removal efficiency of phenol by the strain would also be made to reach 100% in the liquid medium with 800 mg/L phenol in 50 hours, and the phenol degradation rate would be made to reach 100% in the liquid medium with 900 mg/L phenol for 90 hours. And, finally, we have also investigated the metabolites with trimethylsilylation of the sample by N, O-Bis（trimethylsilyl） acetamide, with the gas chromatography/mass spectrometry （GC/MS） analysis, and five intermediates of phenol degradation detected by Pseudomonas stutzeri N2.