Low Temperature and Neutral pH Define “ Candidatus Nitrotoga sp.” as a Competitive Nitrite Oxidizer in Coculture with Nitrospira defluvii

Abstract

Nitrification is an essential process for N removal in activated sludge to avoid toxicity of ammonium and nitrite. Besides Nitrospira, “Candidatus Nitrotoga” has been identified as a key nitrite-oxidizing bacterium (NOB) performing the second step of nitrification, nitrite oxidation to nitrate, in wastewater treatment plants (WWTPs). However, the driving forces for the dominance of Nitrotoga in certain plants have often remained unclear and could not be explained solely by temperature effects. In this study, we characterized the physiology of the ammonium-dependent Nitrotoga sp. BS with regard to temperature and pH variations and evaluated its competitiveness against Nitrospira defluvii. Both NOB originated from the same WWTP and shared a comparable pH optimum of 7.3. Based on these results, coculturing experiments with these NOB were performed in batch reactors operated at either 17°C or 22°C to compare their abundances under optimal (pH 7.4) or suboptimal (pH 6.4) conditions using 1 mM nitrite. As revealed by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), and 16S amplicon sequencing, Nitrotoga sp. BS was clearly favored by its optimal growth parameters and dominated over Ns. defluvii at 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 of NOB is influenced by a complex interaction of environmental parameters and has to be evaluated for single species. IMPORTANCE “Ca. Nitrotoga” is a NOB of high environmental relevance, but physiological data exist for only a few representatives. Initially, it was detected in specialized niches of low temperature and low nitrite concentrations, but later on, its ubiquitous distribution revealed its critical role for N removal in engineered systems like WWTPs. In this study, we analyzed the competition between Nitrotoga and Nitrospira in bioreactors and identified conditions where the K strategist Ns. defluvii was almost replaced by Nitrotoga sp. BS. We show that the pH value is an important factor that regulates the composition of the nitrite-oxidizing enrichment with a dominance of Nitrotoga sp. BS versus Ns. defluvii at a neutral pH of 7.4 in combination with a temperature of 17°C. The physiological diversity of novel Nitrotoga cultures improves our knowledge about niche differentiation of NOB with regard to functional nitrification under suboptimal conditions.

Defining Culture Conditions for the Hidden Nitrite-Oxidizing Bacterium Nitrolancea

“…hollandica depends on ammonium as N-source and accordingly its genome did not encode any enzymes for nitrite reduction to ammonium ( Sorokin et al, 2012 ). This absence of assimilatory nitrite reduction pathways was recently also observed in other thermophilic NOB within the phylum Chloroflexi ( Spieck et al, 2020 ), and some Nitrotoga species are apparently dependent on ammonium addition, too ( Ishii et al, 2017 ; Wegen et al, 2019 ). In contrast, the early Nitrolancea enrichments obtained in this study were able to oxidize nitrite also without ammonium addition, although no assimilatory nitrite reductase genes were detected in their genomes.…”

Physiological and genomic characterization of a new ‘Candidatus Nitrotoga’ isolate

“…Members of 'Ca. Nitrotoga' were initially assumed to be psychrophilic NOB, but recent Nitrotoga enrichments have shown a more versatile temperature dependence (Hüpeden et al, 2016;Boddicker and Mosier, 2018;Wegen et al, 2019) (Table 1). Notably, the 'Ca.…”

“…Some NOB members require ammonium addition for growth and stable nitrite oxidation (Sorokin et al, 2012;Ishii et al, 2017;Wegen et al, 2019;Spieck et al, 2020). The nitrite oxidation activity of the strain AM1P was also stimulated in the presence of 1.4 mM NH 4 Cl (Supporting Information Fig.…”

“…These findings suggest that the response to ammonium may determine the niche differentiation between 'Ca. Nitrotoga' and Nitrospira, beside responses to temperature and pH (Wegen et al, 2019).…”

Genomic and Physiological Characteristics of a Novel Nitrite-Oxidizing Nitrospira Strain Isolated From a Drinking Water Treatment Plant

“…N. japonica reduced transcripts of the gene likely encoding octaheme cytochrome c nitrite reductase (NSJP_2412) in medium amended by nitrite and ammonium compared to medium with nitrite as a sole nitrogen source (Ushiki et al, 2018). Ammonium addition can also promote growth and allow cultivation of NOB from other genera (Sorokin et al, 2012;Sayavedra-Soto et al, 2015;Ishii et al, 2017;Wegen et al, 2019;Spieck et al, 2020). Therefore, some types of NOB might utilize ammonium from their surroundings instead of assimilatory nitrite reduction.…”