Beach Natalie K, Noguera Daniel R
Department of Civil and Environmental Engineering, University of Wisconsin-Madison, Madison, WI, United States.
Front Microbiol. 2019 Jan 31;10:36. doi: 10.3389/fmicb.2019.00036. eCollection 2019.
Published PCR primers targeting the ammonia monooxygenase gene () were applied to samples from activated sludge systems operated with low dissolved oxygen (DO) to quantify total and clade-level that perform complete ammonium oxidation (comammox); however, we found these existing primers resulted in significant artifact-associated non-target amplification. This not only overestimated comammox copies but also resulted in numerous false positive detections in the environmental samples tested, as confirmed by gel electrophoresis. Therefore, instead of attempting to quantify comammox diversity, we focused on accurately quantifying the candidate comammox species. We designed specific and sensitive primers targeting 3 candidate species: () Nitrospira nitrosa, N. inopinata, and N. nitrificans. The primers were tested with templates of these candidate species and used to quantify comammox at the species level in low DO activated sludge systems. We found that comammox related to N. nitrosa were present and abundant in the majority of samples from low DO bioreactors and were not detected in samples from a high DO system. In addition, the greatest abundance of N. nitrosa was found in bioreactors operated with a long solids retention time. N. inopinata and N. nitrificans were only detected sporadically in these samples, indicating a minor role of these comammox in nitrification under low DO conditions.
已发表的靶向氨单加氧酶基因（）的聚合酶链式反应（PCR）引物被应用于低溶解氧（DO）运行的活性污泥系统的样本中，以量化进行完全铵氧化（全程氨氧化）的总细菌和进化枝水平的细菌；然而，我们发现这些现有的引物导致了与假象相关的显著非靶标扩增。这不仅高估了全程氨氧化细菌的拷贝数，还导致在所测试的环境样本中出现大量假阳性检测结果，凝胶电泳证实了这一点。因此，我们没有尝试量化全程氨氧化细菌的多样性，而是专注于准确量化候选全程氨氧化细菌物种。我们设计了针对3种候选物种的特异性和灵敏性引物：（）亚硝化螺菌、意外亚硝化螺菌和硝化亚硝化螺菌。这些引物用这些候选物种的模板进行了测试，并用于在低溶解氧活性污泥系统中对全程氨氧化细菌进行物种水平的量化。我们发现，与亚硝化螺菌相关的全程氨氧化细菌存在于大多数低溶解氧生物反应器的样本中且数量丰富，而在高溶解氧系统的样本中未检测到。此外，在长固体停留时间运行的生物反应器中发现亚硝化螺菌的丰度最高。意外亚硝化螺菌和硝化亚硝化螺菌仅在这些样本中偶尔被检测到，表明这些全程氨氧化细菌在低溶解氧条件下的硝化作用中起次要作用。
