Quantification of Gordona amarae Strains in Foaming Activated Sludge and Anaerobic Digester Systems with Oligonucleotide Hybridization Probes

Previous studies have shown the predominance of mycolic acid-containing filamentous actinomycetes (mycolata) in foam layers in activated sludge systems. Gordona (formerly Nocardia) amarae often is considered the major representative of this group in activated sludge foam. In this study, small-subunit rRNA genes of four G. amarae strains were sequenced, and the resulting sequences were compared to the sequence of G. amarae type strain SE-6. Comparative sequence analysis showed that the five strains used represent two lines of evolutionary descent; group 1 consists of strains NM23 and ASAC1, and group 2 contains strains SE-6, SE-102, and ASF3. The following three oligonucleotide probes were designed: a species-specific probe for G. amarae, a probe specific for group 1, and a probe targeting group 2. The probes were characterized by dissociation temperature and specificity studies, and the species-specific probe was evaluated for use in fluorescent in situ hybridizations. By using the group-specific probes, it was possible to place additional G. amarae isolates in their respective groups. The probes were used along with previously designed probes in membrane hybridizations to determine the abundance of G. amarae, group 1, group 2, bacterial, mycolata, and Gordona rRNAs in samples obtained from foaming activated sludge systems in California, Illinois, and Wisconsin. The target groups were present in significantly greater concentrations in activated sludge foam than in mixed liquor and persisted in anaerobic digesters. Hybridization results indicated that the presence of certain G. amarae strains may be regional or treatment plant specific and that previously uncharacterized G. amarae strains may be present in some systems.

已有研究证实，含分枝菌酸的丝状放线菌（mycolata）在活性污泥系统的泡沫层中占据优势群落地位。戈登氏菌（原诺卡氏菌属，Gordona amarae）通常被视为活性污泥泡沫中该类群的主要代表菌株。本研究对4株G. amarae的小亚基核糖体RNA基因进行了测序，并将所得序列与G. amarae模式菌株SE-6的序列进行了比对。比较序列分析结果显示，本次研究所用的5株菌株可划分为两个进化谱系：第1组包含菌株NM23与ASAC1，第2组则涵盖菌株SE-6、SE-102及ASF3。本研究设计了3种寡核苷酸探针：分别为针对G. amarae的种特异性探针、针对第1组的组特异性探针，以及针对第2组的靶向探针。通过解链温度测定与特异性验证实验对上述探针进行了性能表征，并对种特异性探针在荧光原位杂交（fluorescent in situ hybridizations）中的应用效能进行了评估。借助组特异性探针，可将额外分离得到的G. amarae分离株归入对应的进化谱系组别中。将本次设计的探针与已报道的探针联合应用于膜杂交实验，以测定采自美国加利福尼亚州、伊利诺伊州及威斯康星州泡沫型活性污泥系统的样本中，G. amarae、第1组、第2组、细菌、mycolata及戈登氏菌的核糖体RNA丰度。相较于活性污泥混合液，目标类群在泡沫中的浓度显著更高，且在厌氧消化池中仍可稳定存续。杂交实验结果表明，部分G. amarae菌株的分布可能具有区域性或污水处理厂特异性，且部分系统中可能存在此前未被鉴定的G. amarae菌株。