[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.
国家自然科学基金(20677041); 国家“863计划”重点项目(2007AA021301); 上海市国际合作项目(05SR07107); 上海市重点学科建设项目资助(B203)