无苯酚培养基分离到的降酚菌多样性的分子分析

用3种不含苯酚的培养基(YPG、10%YPG和LB)从上海焦化厂废水处理系统(A2/O法)好氧池的悬浮污泥分离到24株降酚菌株.通过16SrDNAPCR扩增产物的限制性酶切分析(amplifiedribosomalDNArestrictionanalysis,ARDRA)、ERICPCR指纹图谱分析、多组分苯酚羟化酶大亚基(thelargestsubunitofthemulticomponentphenolhydroxylase,LmPH)基因的PCR扩增及16SrRNA基因测序的方法对这些降酚菌株进行表征.通过ARDRA分型,将这24株降酚菌株分为8个类型;利用ERICPCR可以将这24个菌株分为17种类型,说明同一ARDRA类型内菌株具有多样性.对17个ERIC类型代表菌株的16SrDNA扩增产物进行克隆并测序,测序结果在GenBank和RDP中进行比对.结果表明,与这17个代表菌株同源性最高的菌中,有6株是未见报道具有降酚功能的菌株.在这24株降酚菌中,有19株在苯酚含量为200mg/L的MP培养基中培养5d,苯酚降解率为20%左右,其余5株苯酚降解率达到100%,且均属于Rhodococcus属.本研究在降酚菌生物多样性上作了有益的探索.图2表1参20

Molecular Analysis of Phenol-degrading Bacteria Isolated from Media Without Phenol

24 phenol-degrading isolates were isolated with three media without phenol from the suspended sludge of the aeration tank of Shanghai coking wastewater treatment. These isolates were analyzed using series of molecular methods: amplified ribosomal DNA restriction analysis (ARDRA), ERIC (Enterobacterial repetitive intergenic consensus) PCR fingerprinting analysis, LmPH gene amplification and 16S rRNA gene sequencing. They were classified into 8 types by ARDRA analysis and 17 types by ERIC-PCR profiling. The full-length 16S rRNA genes of the representative strains of 17 ERIC-PCR types were amplified and cloned. The partial nucleotide sequences of 16S rRNA genes were determined and analyzed in GenBank database. The results demonstrated that 6 strains were reported as phenol-degrading bacteria. The phenol degradation rates of these 24 strains were determined by the 4-aminoantipyrine method cultured in MP medium with phenol (200 mg/L) as a sole carbon source for 5 days. The rate of 5 strains belonging to Rhodococcus was up to 100% and that of the other 19 strains was only 20%. Fig 2, Tab 1, Ref 20