专一性PCR和变性梯度胶电泳协助从焦化废水处理装置中分离优势功能菌Thauera属菌株

Specific-PCR and denaturing gradient gel electrophoresis assistant isolation of Thauera spp. from a coking wastewater treatment plant

摘要:

【目的】在专一性PCR和变性梯度胶电泳(DGGE)的协助下，从废水处理装置的微生物群落中分离较难分离的功能菌Thauera。【方法和结果】本研究首先使用Thauera特异性PCR-DGGE的方法鉴定了焦化废水处理装置反硝化池生物膜中的Thauera在6种培养基、好氧/厌氧条件下的生长情况。挑选Thauera多样性较高的培养基1/10 NB与MMQ在好氧条件下进行分离培养。然后使用Thauera特异性PCR方法确定分离得到的菌落是否呈阳性，并使用DGGE的方法检验其是否为纯菌。使用不同培养基对含有Thauera的混合菌落进行进一步纯化，DGGE检测发现MMP培养基对混合细菌菌落Q20中的Thauera有明显的富集作用。经过Thauera特异性PCR结合DGGE检测对Thauera属细菌进行追踪，将混合菌落在MMP培养基上多次划线，最终分离得到纯菌。通过这种方法，从反硝化池样品中分离获得了3株在样品中最为主要的Thauera菌株。【结论】以特异性分子标记为导向分离培养细菌，不仅提高了分离效率及细菌筛选的灵敏度，还能协助分离常规方法难以分离的细菌。

Abstract:

[Objective] We used specific-PCR and denaturing gradient gel electrophoresis (DGGE) to isolate Thauera spp. from a coking wastewater treatment plant. [Methods and results] To isolate Thauera from the denitrifying bioreactor of a coking wastewater treatment, biofilm was inoculated to six different media and cultured them under both aerobic and anaerobic conditions. We then compared the composition of Thauera spp. using Thauera-specific PCR-DGGE method. The media 1/10 NB and MMQ which grew higher diverse Thauera spp. and fewer colonies, were used to isolate Thauera sp. under aerobic condition. The colonies were then screened by Thauera-specific PCR. The purity of the colonies that shown Thauera-specific PCR positive signal was then checked by DGGE. The colonies with multiple species were further streaked on different media. DGGE analysis showed that Thauera in colony Q20 was enriched in medium MMP. The colony was finally purified by streaking on MMP medium for several rounds. The composition of the colonies were tracked by Thauera-specific PCR and DGGE at each step. Finally, three strains were purified, which were identified as Thauera sp. according to their 16S rRNA gene sequences. [Conclusion] Guiding with specific biomarker, the efficiency and sensitivity of bacteria isolation can be largely improved.