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

人们早就知道硝化是废水生物处理的关键过程，但最近才发现了一些令人惊讶的特征。其中大部分与第二步硝化有关，涉及亚硝酸盐氧化菌（NOB）。1)与教科书上关于两种不同微生物行业的知识不同，我们检测到氨完全氧化为硝酸盐（comammox），这是由众所周知的硝化螺旋菌完成的。(2)在欧洲一些气候温和的污水处理厂中，亚硝酸盐氧化菌最多的似乎是适应寒冷的硝化菌，而非硝化螺旋菌。(3)硝酸菌（Nitrolancea）是氯氟菌门的第一硝化菌，是废水中亚硝酸盐氧化的新候选菌。由此可见，仍有可能发现具有未知生理特征的新型氨和亚硝酸盐氧化微生物。我们的研究结果验证了环境变量决定生态位分离，反之亦然，利用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，我们成功地富集和部分分离了硝基螺旋藻和硝基托加的新种，以扩展表型和基因组研究的参考生物集。为了了解NOB多样性形成的驱动因素，对部分生理特性进行了比较。例如，42°C的高温孵育（同时降低氧含量）对于从活性污泥中获得的一种新型硝化螺旋菌的增殖至关重要。除了温度和底物浓度外，我们现在可以证明，在冷淡水养殖植物的生物过滤器中，与硝基螺旋藻相比，微酸性ph值对硝基螺旋藻的上调很重要。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，硝化螺旋菌和硝化菌的亚硝酸盐Km值明显低于硝化杆菌，从而显示出最大活性Vmax。qPCR结果显示，在低温（17°C）条件下，硝化菌对硝化螺旋菌具有优势。然而，两种混合NOB中的一种的优势明显受到pH （6.5 vs 7.4）的影响，并与样品来源有关。这种复杂的调控将在下一个项目阶段对更多的硝基螺旋藻和硝基藻进行更详细的分析。除了改变矿物培养基和培养条件外，我们还将关注与异养菌的共培养，在这种情况下，辅助细菌的帮助可能会促进挑剔的NOB的增殖。此外，在系统发育上与荷兰硝化菌（Nitrolancea hollandica）有亲缘关系的新型NOB的培养将拓宽我们对这一功能群体特定生长偏好的认识。

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.