A renewed view on the complex community of nitrite oxidizing bacteria derived from sewage

人们很早就知道硝化作用是废水生物处理的关键过程，但最近仍发现了一些令人惊讶的特征。其中大部分与第二步硝化作用有关，并与亚硝酸盐氧化细菌(NOB)有关。I)与教科书上关于两个不同微生物公会的知识不同，检测到氨完全氧化为硝酸盐(Comammox)，这是由众所周知的硝酸螺旋菌执行的。Ii)在欧洲一些气候温和的污水处理厂中，耐寒的硝化细菌似乎是最丰富的亚硝酸盐氧化细菌，而不是硝酸螺旋菌。Nitroancea是氯软门的第一个硝化菌，是废水中亚硝酸盐氧化的新候选者。因此，很明显，仍然可以发现生理特征未知的新的氨氧化和亚硝酸盐氧化微生物。我们的发现证实，环境变量决定生态位分离，反之亦然，通过竞争条件的定向培养方法可以获得新的NOB。在项目的第一部分，我们成功地丰富和部分分离了硝化螺旋菌和硝化细菌的新物种，以扩大表型和基因组研究的参考生物集。为了了解形成NOB多样性的驱动力，对所选择的生理特性进行了比较。例如，在42°C的高温下(同时在氧含量较低的情况下)培养对从活性污泥中获得的一种新的硝化螺旋菌的增殖至关重要。除了温度和底物浓度，我们现在可以证明，在冷淡水水产养殖植物的生物滤池中，与硝化螺旋菌相比，微酸性的pH值对于Nitrotoga的上调是重要的。在动力学实验中，对5属13种NOB的底物亲和力进行了分析，结果表明，Nitrospira和Nitrotoga对亚硝酸盐的Km值明显低于Nitrobacter，而Nitrobacter的最大活性Vmax最高。首次在生物反应器中进行的竞争实验证实了Nitrotoga相对于Nitrospira在低温(17℃)下的优势，正如qPCR所揭示的那样。然而，两种混合NOB中的一种的优势度明显受pH(6.5vs7.4)的影响，这与Nitrotoga的样品来源相关。在下一个项目阶段，将更详细地分析这种复杂的规则，并对更多种类的硝化螺旋菌和硝化细菌进行分析。除了矿物培养基和培养条件的改进外，我们还将重点研究与异养细菌的共培养，其中辅助菌的帮助可能会促进挑剔的Nob的增殖。此外，培育与Chloroflei(荷兰硝化菌的近缘)有亲缘关系的新Nob将拓宽我们对这一功能协会特定生长偏好的了解。

It has been known since a long time that nitrification is a key process of biological wastewater treatment, but still surprising features have been discovered very recently. Most of them are related to the second step of nitrification and concern nitrite oxidizing bacteria (NOB). I) In contrast to textbook knowledge about two distinct microbial guilds, complete oxidation of ammonia to nitrate (comammox) was detected, performed by the well-known Nitrospira. II) In some WWTPs in Europe of moderate climate, the cold-adapted Nitrotoga and not Nitrospira seems to be the most abundant nitrite oxidizing bacterium. III) Nitrolancea, the first nitrifier belonging to the phylum Chloroflexi, is a new candidate for nitrite oxidation in wastewaters. Therefore, it became obvious that still novel ammonia and nitrite oxidizing microorganisms with unknown physiological features can be discovered.Our findings verify that environmental variables decide in terms of niche separation and vice versa, novel NOB can be obtained by a directed cultivation approach using competitive conditions. In the first part of the project, we successfully enriched and partly isolated novel species of Nitrospira and Nitrotoga to extend the set of reference organisms for phenotypic and genomic investigations. Selected physiological characteristics were compared in order to understand the driving forces for shaping diversity of NOB. For example, incubation at elevated temperature of 42°C (and simultaneously at reduced oxygen content) was critical for proliferation of a new species of Nitrospira obtained from activated sludge. Apart from temperature and substrate concentration, we now could demonstrate that a slightly acidic pH-value is of importance for upregulation of Nitrotoga in comparison to Nitrospira in a biofilter of a cold freshwater aquaculture plant. In kinetic experiments, the substrate affinity was analyzed for 13 species of NOB belonging to five genera and it was shown that the Km value for nitrite of Nitrospira and Nitrotoga is clearly lower than that of Nitrobacter, which otherwise revealed the highest maximum activity Vmax. First competition experiments in bioreactors could confirm the advantage of Nitrotoga versus Nitrospira at low temperature (17°C), as revealed by qPCR. However, the dominance of one of two mixed NOB was clearly influenced by pH (6.5 vs 7.4) in correlation with the sample origin of Nitrotoga. Such complex regulation will be analyzed in more detail with further species of Nitrospira and Nitrotoga in the next project phase. Besides modification of mineral media and incubation conditions, we will also focus on co-cultures with heterotrophs, where the assistance of helper bacteria might facilitate proliferation of fastidious NOB. Moreover, cultivation of novel NOB phylogenetically affiliated to the Chloroflexi (relatives of Nitrolancea hollandica) will broaden our knowledge about specific growth preferences of this functional guild.