氨氧化是废水生物脱氮系统（BNR）的限速步骤。检测、比较废水BNR系统不同氨氧化微生物丰度可以探讨不同氨氧化途径/微生物对氨氧化的相对贡献，有助于控制、改进和优化废水BNR工艺，其关键是针对特定功能基因片段设计出特异性引物。关于硝化细菌（AOB）氨氧化功能基因（amo）和特异性引物的研究开展得最早，所设计的特异性引物数量也最多。受限于对废水BNR系统异养硝化菌（HNB）遗传多样性的认识不足，此前AOB引物研发时对HNB同源性干扰评价较少。近年来有研究发现采用AOB特异性引物成功扩增出了HNB类amoA基因，然而，目前尚缺乏系统研究准确界定这种HNB同源性扩增现象对AOB amo基因扩增效果、遗传发育多样性等研究结果的具体影响。本文首先全面调研了废水BNR系统HNB遗传发育多样性，据此筛选8株HNB作为废水BNR系统HNB代表种，重新评价了HNB同源性干扰下典型AOB amo功能基因引物的特异性。研究结果将有助于基于AOB amo基因的PCR扩增结果和AOB遗传发育多样性的准确解释，有助于准确评价AOB和HNB在废水BNR系统氨氧化过程中的相对贡献，具有非常重要的理论与实际意义。

本研究的主要研究结果如下：

（1）通过全面的文献调研整理了分离自废水BNR系统的134株HNB的分类学信息。废水BNR系统HNB属于4个菌门，其中变形菌门HNB隶属于3个菌纲15个菌目25个菌科35个菌属；厚壁菌门HNB隶属于1个菌纲1个菌目3个菌科4个菌属；放线菌门HNB隶属于1个菌纲2个菌目7个菌科7个菌属；只有一株HNB隶属于拟杆菌门。大部分废水BNR系统HNB隶属于变形菌门（78%），其中52%隶属于γ-proteobacteria纲。本文最终选取并购买了Pseudomonas stutzeri CGMCC 1.6516、Bacillus cereus CGMCC 1.229、Acinetobacter sp. CGMCC 1.9045、Alcaligenes faecalis CGMCC 1.924、Paracoccus pantotrophus CGMCC 1.8851、Ochrobactrum anthropi CGMCC 1.2501、Corynebacterium glutamicum CGMCC 1.1886和Arthrobacter aurescens CGMCC 1.1892等8株HNB作为AOB amo基因引物特异性再评价研究的HNB代表种。

（2）完成了基于废水BNR系统HNB多样性的引物对amoA 1F/amoA 2R特异性再评价。对于应用最广泛的AOB amoA基因引物amoA 1F/amoA 2R而言，DNA模板浓度/体积、引物浓度、循环数和退火温度等PCR反应条件对HNB类amoA基因扩增效果的影响较大，而体系总体积影响较小。在引物浓度为10 μM、退火温度为49-58℃、总循环数为35-40的多种组合条件下，通过降落PCR反应扩增出了8株HNB类amoA片段，在此基础上完成测序、BLAST比对和系统发育树构建。这8株HNB的类AMO均与N. europaea AMO系统发育相近，表明文献报道的基于amoA基因的AOB克隆文库结果中部分N. europaea基因可能来自于HNB。

（3）完成了基于废水BNR系统HNB多样性的其它AOB引物特异性再评价。对于其它AOB特异性引物对A189/A682、amoA 34f/amoA 2R、amoBMf/ amoBMr和amoA 3F/amoB 4R，通过优化PCR反应条件最终都能从HNB扩增出类amo基因片段。测序、BLAST比对和系统发育树构建结果表明这些引物的特异性均不高，与HNB基因组DNA均存在一个或多个结合位点。其中，A189/A682、amoBMf/amoBMr和amoA 3F/amoB 4R从HNB基因组DNA扩增得到的PCR产物与氨氧化基因均无明显关联，根据产物序列特征可以准确推算出原菌株的分类特征；amoA 34f/amoA 2R从Acinetobacter sp.基因组DNA中扩增出类amoA基因序列，与N. europaea AMO系统发育相近。

Ammonia oxidation is often recognized as the rate-limiting step in wastewater biological nitrogen removal (BNR) processes. The detection of gene abundance of five groups of ammonia-oxidizing microorganisms (including ammonia-oxidizing bacteria (AOB), heterotrophic nitrifying bacteria (HNB), etc.) allows a better understanding of the relative contribution of different ammonia-oxidizing pathways/microorganisms, which is often essential to control, improve, and optimize BNR processes. The accuracy of gene abundance detection is considerably determined by the specific primers that target the functional genes. Since the publication of the first primers to amplify amo genes from AOB in 1995, the number of available AOB amo-targeted primers has increased remarkably. However, the lack of the overall information on the HNB diversity in BNR systems has hampered the specificity evaluation on AOB amo-targeted primers in the presence of the homologous amo–like genes of HNB. In recent years, some researchers have successfully amplified amoA-like genes from the genomic DNA of some HNB isolates using some AOB amo-targeted primers. However, there is still no systemetic investigation to explain how the homologous amo genes of HNB affect the PCR results and AOB diversity analyses (ie., the cloning library) when these AOB amo-targeted primers are used. This thesis systematically investigated phylogenetic diversity of HNB isolates from wastewater BNR systems in previous publications, from which 8 HNB species was chosed as the representative HNB. Under the conditions, the specificity of typical AOB amo-targeted primers was revaluated in terms of HNB in wastewater BNR systems, which will help to accurately evaluate the results of AOB quantification and diversity analyses with AOB amo-targeted primers, and further to compare the contribution of AOB and HNB to ammonia oxidization in wastewater BNR systems.

The first part of this thesis summarized the taxonomical information of 134 HNB isolates from wastewater BNR systems in previous papers. These HNB isolates are taxonomically affiliated to four bacterial phyla. The HNB isolates in the phylum Proteobacteria are from 3 classes, 15 orders, 25 families, and 35 genera; the HNB isolates in the phylum Firmicutes are affiliated to 1 class, 1 order, 3 families, and 4 genera; the HNB isolates in the phylum Actinobacteria are affiliated to 1 class, 2 orders, 7 families, and 7 genera; only one HNB isolate is affiliated with the phylum Bacteroidetes. Finally, 8 HNB species, including Pseudomonas stutzeri CGMCC 1.6516, Bacillus cereus CGMCC 1.229, Acinetobacter sp. CGMCC 1.9045, Alcaligenes faecalis CGMCC 1.924, Paracoccus pantotrophus CGMCC 1.8851, Ochrobactrum anthropi CGMCC 1.2501, Corynebacterium glutamicum CGMCC 1.1886 and Arthrobacter aurescens CGMCC 1.1892, were chosed and purchased as representative HNB for specificity revaluation of AOB amo-targeted primers.

The primer pair amoA 1F and amoA 2R was the earliest, widely-used one to amplify AOB amoA in most environmental studies. When it was used in this study, it was found that the concentration and volume of template DNA, the primer concentration, cycle number and annealing temperature exhibited significant effect on PCR amplifications of the amoA-like gene from HNB, while the amplifications at differernt reaction volumes showed no signficant differences. When the touchdown PCR reaction was carried out at 10 μM each primer, 49-58℃ annealing temperature and 35-40 cycles, the amoA-like genes were successfully amplified from all 8 HNB species. After sequencing, alignment and phylogenetic tree construction, the amoA-like genes from 8 HNB species are found to be evolutionarily related with amoA from N. europaea. Under the conditions, the N. europaea amoA sequences in the clone library might be amplified from HNB in previous papers.