活性污泥中抗性细菌筛选及拉曼光谱检测

Screening of Antibiotic-Resistant Bacteria in Activated Sludge and Study of Their Raman Spectroscopy

抗生素制药废水是污水处理中比较难处理的废水。抗生素与微生物作用的机理研究对于解决抗生素废水处理问题具有重要意义。拉曼光谱作为一种对样品无损,非侵入型,并且对水不敏感的检测手段,已越来越多地用于生物、医学的研究。从普通污水处理反应器的活性污泥里通过涂布法和平板划线法分离得到了4株对头孢他啶具有抗性的菌株,研究了其对头孢他啶的降解效果,降解率最高达45.44%。采用16S rRNA基因测序进行菌种鉴定,得到这些菌种分别属于气单胞菌属,芽孢杆菌属和红球菌属。对筛选出的细菌进行不同条件下拉曼光谱测定,探讨了细菌拉曼光谱的测试条件,结果显示CaF_2是测定生物样品拉曼光谱时效果最好的基底。通过拉曼光谱技术和主成分分析法对菌种进行了差异分析,提取了三个主成分,累积荷载达到83.9%。其中,第一主成分主要包括748,1 003和1 126cm^(-1)等比较明显的峰位,第二主成分包括1 661和1 448cm^(-1),第三主成分只有855cm^(-1)。本研究利用拉曼光谱对活性污泥筛选的抗性菌株进行生物大分子的差异表征,通过拉曼光谱对抗性菌种进行了较好的区分,与基因测序的结果相吻合,证明了拉曼光谱用于抗生素废水生物处理作用机理研究的可行性及优越性。

Antibiotic pharmaceutical wastewater is difficult to be treated in wastewater treatment field.The study of interaction mechanism between antibiotic and microorganism is of great significance for solving antibiotic wastewater treatment problem.As Raman spectroscopy offers a nondestructive,noninvasive,and water-insensitive test tool,it has been more and more widely applied in the study of biology and medicine.This work isolated 4 strains of antibiotic-resistant bacteria from activated sludge taken from sewage treatment reactor by using streak plate method and spread plate method,and the drug degradation effect of these strains is assessed,which shows the highest degradation rate reached 45.44%.Besides,sequence of these strains’16S rRNA gene was analyzed to identify them,and the result shows that these strains belong to Aeromonas sp.,Bacillus sp.and Rhodococcus sp..Raman spectroscopies of these stains under different conditions were taken to investigate the best test condition of bacteria’s Raman spectroscopy,and the result shows that CaF 2 can be the best substrate.Then the Raman spectroscopies of these strains were obtained and the difference among them is analyzed by using principal component analysis,and three principal components were extracted,of which the accumulated load reached 83.9%.Among all the variables,748,1003,1 126 cm^-1 belong to the first principal component,1 661,1 448cm^-1 belong to the second,and 855 cm^-1 belongs to the third.Thisresearch characterized biological macromolecules of stains screened from activated sludge by Raman spectroscopy and well distinguished them,proving the reliability and superiority of Raman spectroscopy for applying to mechanism study of microorganism interaction in wastewater treatment.