Nitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes and plays a central role in the global nitrogen cycle. Our earlier investigations based on 16S rRNA and amoA amplicon analysis, amoA quantitative PCR and metagenomics of groundwater-fed biofilters indicated a consistently high abundance of comammox Nitrospira Here, we hypothesized that these nonclassical nitrifiers drive ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 16S rRNA amplicon sequencing to identify the active members in the biofilter community when subjected to a continuous supply of NH4 + or NO2 - in the presence of 13C-HCO3 - (labeled) or 12C-HCO3 - (unlabeled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira dominated ammonia oxidation in the biofilter community. A total of 78 (8 by RNA-SIP and 70 by DNA-SIP) and 96 (25 by RNA-SIP and 71 by DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We also detected significant HCO3 - uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobacter, and Acidovorax under conditions that favored ammonia oxidation. Canonical Nitrospira alone drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia-oxidizing taxa was not detected in the SIP fractions. This study provides the first in situ evidence of ammonia oxidation by comammox Nitrospira in an ecologically relevant complex microbiome.IMPORTANCE With this study we provide the first in situ evidence of ecologically relevant ammonia oxidation by comammox Nitrospira in a complex microbiome and document an unexpectedly high H13CO3 - uptake and growth of proteobacterial and acidobacterial taxa under ammonia selectivity. This finding raises the question of whether comammox Nitrospira is an equally important ammonia oxidizer in other environments.

中文翻译：

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DNA和RNA SIP揭示了硝化螺旋体 作为地下水供给的生物滤池中硝化的主要驱动力。

硝化作用是由微生物驱动的将氨从氨转化为亚硝酸盐和硝酸盐的氧化过程，在全球氮循环中起着核心作用。我们基于16S rRNA和amoA扩增子分析，amoA定量PCR以及由地下水喂养的生物滤池的宏基因组学的早期研究表明，comammox Nitrospira的含量一直很高。在这里，我们假设这些非经典硝化剂会驱动氨N氧化。因此，我们使用DNA和RNA稳定同位素探测（SIP）结合16S rRNA扩增子测序来确定在连续供入NH4 +或NO2-在13C-HCO3存在下，生物滤池中的活性成员-（标记为）或12C-HCO3-（未标记）。添加烯丙基硫脲（ATU）和氯酸钠分别抑制自养氨和亚硝酸盐氧化细菌。我们的结果证实，谱系II硝化螺旋藻在生物滤池群落中占主导地位。共有78种（RNA-SIP为8种，DNA-SIP为70种）和96种（RNA-SIP为25种，DNA-SIP为71种）硝化螺旋体系统型（99％16S rRNA序列相似性）被鉴定为完全氨和亚硝酸盐分别氧化。我们还检测到在有利于氨氧化的条件下，嗜酸菌亚组10，Pe微生物，根瘤菌和嗜酸菌明显吸收了HCO3。单独的规范硝化螺菌会推动生物滤池中亚硝酸盐的氧化，而在SIP馏分中未检测到古细菌氨氧化类群的活性。这项研究提供了在生态上相关的复杂微生物组中，comammox Nitrospira对氨氧化的第一个原位证据。重要事项通过这项研究，我们提供了在复杂的微生物组中comammox Nitrospira在生态学上与氨相关的氨氧化的第一个原位证据，并记录了在氨选择性下意外高的H13CO3-细菌和酸性细菌类群的吸收和生长。这一发现提出了一个问题，即在其他环境中，comammox Nitrospira是否是同等重要的氨氧化剂。