Influence of salinity on novel (moderately) thermophilic nitrite-oxidizing bacteria

硝化作用是指将氨生物氧化为亚硝酸盐并进一步氧化为硝酸盐，它对避免有害氨和亚硝酸盐的积累具有重要的环境意义，而有害氨和亚硝酸盐的积累可能导致人类和水生动物的健康风险。在污水处理厂中，即使在温度或盐度升高等不利条件下，也必须保持其功能。特别是对执行第二步硝化的挑剔亚硝酸盐氧化细菌（NOB）的研究还不够充分。从地热系统和温水污水中，已经培养出了硝基螺旋藻和不同的氯藻NOB，但宏基因组数据表明，根据关键酶亚硝酸盐氧化还原酶（NXR）的特定序列，推测NOB存在高度多样化的群落。在该项目的第一阶段，定向培养方法导致描述了新的（中等）嗜热的硝化螺和硝化菌。此外，来自不同门（Ca. dadabobacteria, Actinobacteria, Chloroflexi, Acidobacteria, (Delta)-Proteobacteria, Nitrospirae）的几种在高温（37-50°C）下生长的新型NOB候选菌选择性富集，并与特定的nxrA/B和16S rRNA基因序列以及不同的形态相关，但它们与下一个分类描述的细菌的16S rRNA基因相似性相当低（80-94%）。宏基因组组装基因组（MAGs）的分子表征首次揭示了它们的碳、氮和氧代谢，包括在一些绿杆菌和放线菌中发现rubisco基因，支持它们的化能自养生活方式，并将它们与反硝化菌区分出来。在以往的研究中，NOB菌株的分离主要是通过亚硝酸盐浓度、温度、DO、pH等多种选择因素进行的，而在第二部分的研究中，盐度胁迫（尤其是硝酸盐）与高温的结合将成为生态位分离的主要驱动因素。初步结果表明，dehalococoides -like NOB和新型放线菌具有较高的NaCl耐受性，并降低了硝化螺旋菌的丰度。目的是通过细胞分选高度富集/分离新的耐盐和耐热NOB，对其进行生理表征，并筛选新的代谢途径的宏基因组。在不同生长条件下获得的基于RNA的基因表达和亚转录组数据将验证新型nxr基因的活性，并为耐受性机制提供见解。大多数有希望的候选者将测试它们在盐和温度胁迫下在生物反应器系统中生长的能力，以用于生物技术应用。关于一个变暖的世界，人们对热条件下的硝化作用越来越感兴趣，必须确保农业硝化抑制剂仍然会影响不断变化的硝化微生物群落。此外，嗜热细菌作为早期地球的幸存者，在进化方面是有趣的。

Nitrification, the biological oxidation of ammonia to nitrite and further to nitrate, is of environmental importance to avoid accumulation of harmful ammonia and nitrite, which can result in human and aquatic animal health risk. In WWTPs its functioning has to be maintained even under adverse conditions caused for example by elevated temperature or salinity. Especially the fastidious nitrite-oxidizing bacteria (NOB) that perform the second step of nitrification are not sufficiently investigated. From geothermal systems and warm sewage, Nitrospira and different Chloroflexi NOB were already cultivated, but metagenome data indicated the presence of a highly diverse community of putative NOB by specific sequences of the key enzyme, the nitrite oxidoreductase (NXR). In the first phase of this project, directed cultivation approaches led to the description of new (moderately) thermophilic species of Nitrospira and Nitrolancea. Additionally, several novel NOB candidates growing at elevated temperature (37-50°C) from diverse phyla (Ca. Dadabacteria, Actinobacteria, Chloroflexi, Acidobacteria, (Delta)-Proteobacteria, Nitrospirae) were selectively enriched and correlated with specific nxrA/B and 16S rRNA gene sequences as well as distinct morphotypes, but their 16S rRNA gene similarities to the next taxonomically described bacteria are rather low (80-94%). Molecular characterization of metagenome assembled genomes (MAGs) gave first insights into their carbon, nitrogen and oxygen metabolism including the finding of rubisco genes for some Chloroflexi and Actinobacteria, supporting their chemolithoautotrophic lifestyle and differentiating them from denitrifiers. In the past, new NOB isolates were obtained by various selecting factors (nitrite concentration, temperature, DO, pH), whereas in the second part of the project, salinity stress (especially nitrate) will be the main driver for niche separation in combination with high temperature. Preliminary results indicated high NaCl tolerances of Dehalococcoides-like NOB and novel Actinobacteria and reduced abundance of Nitrospira. It is the goal to highly enrich/isolate novel salt- and heat-tolerant NOB by cell sorting, to characterize them physiologically and to screen metagenomes for new metabolic pathways. RNA based gene expression and metatranscriptome data obtained under different growth conditions will verify activity of novel nxr genes and give insights into tolerance mechanisms. Most promising candidates will be tested for their ability to grow in bioreactor systems under salt and temperature stress for biotechnological applications. With regard to a warming world, there is a growing interest in nitrification under thermal conditions and it has to be ensured that agricultural nitrification inhibitors will still affect a shifting community of nitrifying microorganisms. Furthermore, thermophilic bacteria as survivors of early Earth are interesting with regard to evolutionary aspects.