A renewed view on the complex community of nitrite oxidizing bacteria derived from sewage
Final Report Abstract
Nitrification, the microbial oxidation of ammonia to nitrite and nitrate, is a key process in the biogeochemical nitrogen cycle. Recent studies demonstrated a high metabolic versatility of NOB in and outside the N-cycle and uncovered surprising features like ammonia oxidation in Nitrospira. Nevertheless, NOB are still understudied and knowledge of the diversity of NOB is still scarce. Since a stable nitrification is supported by biodiversity, we characterized several novel NOB to learn more about their niche partitioning. Our cultivation-dependent approaches revealed that various NOB can be isolated from a complex community as found in the WWTP Dradenau, Hamburg. The use of modified media and incubation conditions resulted in the enrichment of novel species or even genera. Especially temperature and substrate concentrations are main factors for niche differentiation and selective growth of NOB, but also the pH and DO are driving forces for shaping the NOB community structure. Whereas Nitrospira benefited from a lowered substrate concentration in contrast to Nitrobacter, novel species of Nitrospira and Nitrotoga were obtained by a low incubation temperature. On the other hand, a moderate thermophilic Nitrospira (Ns. elata) was enriched by increasing the growth temperature to 46°C (= low oxygen). In contrast to Nitrospira and Nitrotoga, Nitrolancea grew only in the presence of high nitrite concentrations (3 mM) at a temperature of 37°C. Because Nitrolancea hollandica lacks a nitrite reductase for N-assimilation, it was necessary to supply ammonium as described for this NOB. Using the same modified NOB medium, we achieved to isolate a new Nitrolancea species from a geothermal spring. Further on, different nxrA genes of so far unknown NOB in WWTPs were detected in cultures incubated at temperatures between 37-55°C. These findings indicate that further NOB exist, which is in line with the huge diversity of nxrA/B genes within uncultivated phyla detected recently by others. In the frame of this project, several of our NOB cultures (Nitrospira, Nitrolancea) were subject to genome annotation in cooperation with the groups of S. Lücker (Radboud University) and H. Daims (University of Vienna). A high flexibility in generating energy explains the ecological success and ubiquitous distribution of Nitrospira, but also revealed species-dependent adaptations. Nitrifying biofilms from aquaculture plants were subject of physiological studies with regard to salt adaptation. Using next generation sequencing, the bacterial core communities of marine, brackish and freshwater biofilters were compared and those fish-breeding conditions identified, which supported growth of comammox-Nitrospira. Competition is highly dependent on the substrate affinity and NOB are either r strategists with a cytoplasmic location of nitrite oxidoreductase (NXR) or K strategists with periplasmic NXR. Nitrotoga is rather a K strategist and the Nitrospira-like periplasmic location as well as a 46 KDa NxrB could be detected with monoclonal antibodies targeting the beta-subunit of the key enzyme. Half saturation constants Km for nitrite of marine NOB were relatively low in accordance with low nitrite concentrations in marine environments. All NOB had an inverse isotope effect and the value reflects location of the NXR. For the investigation of population dynamics and competition under changing environmental conditions, pure and enrichment cultures of NOB were mixed in lab-scale bioreactors under different conditions. Nitrotoga BS clearly had an advantage over Nitrospira defluvii (both from activated sludge) when its optimal growth parameters were used (pH 7.4 and 17°C), whereas a pH of 6.4 was more selective for Ns. defluvii. Our synthetic communities revealed that niche differentiation is influenced by complex interactions of environmental parameters and has to be evaluated for single species, before reliable nitrification models can be generated. Therefore, cultured representatives are needed to confirm the potential for nitrite oxidation for novel candidate NOB and to gain a deeper understanding of their physiological and genomic potential. Isolation of nitrifiers is a challenging task and the reason for failure might be the dependency on (facultatively) heterotrophic bacteria and complex interactions in the nitrifying consortia are hypothesized. For certain heterotrophs it is known that they enhance nitrification rates and it was surprising that accompanying bacteria belong to only a few bacterial phyla. In summary, we gained interesting and important new insights into the second step of nitrification and the responsible microorganisms.
Publications
- (2015) Improved isolation strategies allowed the phenotypic differentiation of two Nitrospira strains from widespread phylogenetic lineages. FEMS Microbiol Ecol 91(3)
Nowka B, Off S, Daims H, Spieck E
(See online at https://doi.org/10.1093/femsec/fiu031) - (2015). Comparison of oxidation kinetics of nitriteoxidizing bacteria: nitrite availability as a key factor in niche differentiation. Appl Environ Microbiol 81: 745-753
Nowka B, Daims H, Spieck E
(See online at https://doi.org/10.1128/AEM.02734-14) - (2015). Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira. Proc Natl Acad Sci USA. Sep 8;112(36):11371-6
Koch H, Lücker S, Albertsen M, Kitzinger K, Herbold C, Spieck E, Nielsen PH, Wagner M, Daims H
(See online at https://doi.org/10.1073/pnas.1506533112) - (2016). Relative abundance of Nitrotoga spp in a biofilter of a cold freshwater aquaculture plant appears to be stimulated by slightly acidic pH-value. Appl Environ Microbiol 82: 1838-45
Hüpeden, J, Wegen, S, Off, S, Lücker, S, Bedarf, Y, Daims, H, Kühn, C, Spieck, E
(See online at https://doi.org/10.1128/AEM.03163-15) - (2017). Oxidation kinetics and inverse isotope effect of marine nitrite-oxidizing isolates. Aquatic Microbial Ecology, 80(3), 289-300
Jacob, J, Nowka, B, Merten, V, Sanders, T, Spieck, E, Dähnke, K
(See online at https://doi.org/10.3354/ame01859) - (2018). The draft genome sequence of "Nitrospira lenta" strain BS10, a nitrite oxidizing bacterium isolated from activated sludge. Stand Genomic Sci, 13, 32
Sakoula, D, Nowka, B, Spieck, E, Daims, H, Lücker, S
(See online at https://doi.org/10.1186/s40793-018-0338-7) - (2019). Low Temperature and Neutral pH Define Candidatus Nitrotoga sp. as a Competitive Nitrite Oxidizer in Co-Culture with Nitrospira defluvii. Appl Environ Microbiol.
Wegen, S, Nowka, B, Spieck, E
(See online at https://doi.org/10.1128/AEM.02569-18) - (2020). Taxonomic and functional profiling of nitrifying biofilms in freshwater, brackish and marine RAS biofilters. Aquacultural Engineering
Hüpeden, J, Wemheuer, B, Indenbirken, D, Schulz, C, Spieck, E
(See online at https://doi.org/10.1016/j.aquaeng.2020.102094)
