Real-Time PCR Quantification of Nitrifying Bacteria in a Municipal Wastewater Treatment Plant

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• 作者：Gerda Harms ; Alice C. Layton ; Hebe M. Dionisi ; Igrid R. Gregory ; Victoria M. Garrett ; Shawn A. Hawkins ; Kevin G. Robinson ; and Gary S. Sayler
• 刊名：Environmental Science & Technology
• 出版年：2003
• 出版时间：January 15, 2003
• 年：2003
• 卷：37
• 期：2
• 页码：343 - 351
• 全文大小：189K
• 年卷期：v.37,no.2(January 15, 2003)
• ISSN：1520-5851

文摘

Real-time PCR assays using TaqMan or MolecularBeacon probes were developed and optimized for thequantification of total bacteria, the nitrite-oxidizing bacteriaNitrospira, and Nitrosomonas oligotropha-like ammoniaoxidizing bacteria (AOB) in mixed liquor suspended solids(MLSS) from a municipal wastewater treatment plant(WWTP) using a single-sludge nitrification process. Thetargets for the real-time PCR assays were the 16S rRNAgenes (16S rDNA) for bacteria and Nitrospira spp. andthe amoA gene for N. oligotropha. A previously reportedassay for AOB 16S rDNA was also tested for its applicationto activated sludge. The Nitrospira 16S rDNA, AOB 16SrDNA, and N. oligotropha-like amoA assays were log-linear over 6 orders of magnitude and the bacterial 16SrDNA real-time PCR assay was log-linear over 4 ordersof magnitude with DNA standards. When these real-timePCR assays were applied to DNA extracted from MLSS,dilution of the DNA extracts was necessary to preventPCR inhibition. The optimal DNA dilution range wasbroad for the bacterial 16S rDNA (1000-fold) andNitrospira 16S rDNA assays (2500-fold) but narrow forthe AOB 16S rDNA assay (10-fold) and N. oligotropha-like amoA real-time PCR assay (5-fold). In twelve MLSSsamples collected over one year, mean cell per Lvalues were 4.3 ± 2.0 × 10¹¹ for bacteria, 3.7 ± 3.2 ×10¹⁰ for Nitrospira, 1.2 ± 0.9 × 10¹⁰ for all AOB, and7.5 ± 6.0 × 10⁹ for N. oligotropha-like AOB. The percentof the nitrifying population was 1.7% N. oligotropha-likeAOB based on the N. oligotropha amoA assay, 2.9% totalAOB based on the AOB 16S rDNA assay, and 8.6% nitrite-oxidizing bacteria based on the Nitrospira 16S rDNA assay.Ammonia-oxidizing bacteria in the wastewater treatmentplant were estimated to oxidize 7.7 ± 6.8 fmol/hr/cell basedon the AOB 16S rDNA assay and 12.4 ± 7.3 fmol/hr/cellbased on the N. oligotropha amoA assay.