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

• Applicant: Privatdozentin Dr. Eva Spieck
• Subject Area: Microbial Ecology and Applied Microbiology
• Term: from 2012 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 230977559

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.

• DFG Programme: Research Grants