ABSTRACT: Inhibition of Nitrospira and Nitrotoga by paracetamol: Evidence for the rapidly start-up and long-term stable operation of partial nitrification in municipal wastewater
Project description:Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidizing either ammonia or nitrite. No known nitrifier carries out both steps, although complete nitrification should be energetically advantageous. This functional separation has puzzled microbiologists for a century. Here we report on the discovery and cultivation of a completely nitrifying bacterium from the genus Nitrospira, a globally distributed group of nitrite oxidizers. The genome of this chemolithoautotrophic organism encodes the pathways both for ammonia and nitrite oxidation, which are concomitantly activated during growth by ammonia oxidation to nitrate. Genes affiliated with the phylogenetically distinct ammonia monooxygenase and hydroxylamine dehydrogenase genes of Nitrospira are present in many environments and were retrieved on Nitrospira contigs in new metagenomes from engineered systems. These findings fundamentally change our picture of nitrification and point to completely nitrifying Nitrospira as key components of nitrogen-cycling microbial communities.
Project description:Nitrite-oxidizing bacteria are vital players in the global nitrogen cycle that convert nitrite to nitrate during the 2nd step of nitrification. Within this functional guild, the genus Nitrospira is among the most widespread and phylogenetically and physiologically diverse nitrite oxidizers and its members drive nitrite oxidation in many natural and biotechnological ecosystems. Despite their ecological and biotechnological importance, our understanding of Nitrospira’s energy metabolism is still limited. The main bottleneck for a detailed biochemical characterization of Nitrospira is biomass production, since they are slow-growing organisms and fastidious to culture. In this study, we cultured Nitrospira moscoviensis in a continuous stirred tank reactor system (CSTR) allowing constant biomass harvesting. Additionally, this cultivation setup enabled accurate control of physicochemical parameters and thus avoided fluctuating levels of nitrite and accumulation of nitrate. We performed transcriptome analysis and confirmed constant gene expression profiles in the chemostat culture over a period of two weeks. The transcriptomic data supports the predicted core metabolism of N. moscoviensis, including the reductive TCA cycle as a CO2 fixation pathway, the novel bd-like oxidase as terminal oxidase and the octaheme nitrite reductase involved in nitrogen assimilation. Additionally, the expression of multiple copies of respiratory complexes suggests functional differentiation of these copies within the respiratory chain. Transcriptome analysis also suggests a soluble and a membrane-bound gamma subunit as part of the nitrite oxidoreductase (NXR), the enzyme catalyzing nitrite oxidation. Overall, the transcriptome data provided novel insights into the metabolism of Nitrospira supporting the genome-based prediction of key pathways. Moreover, the application of a CSTR to cultivate Nitrospira is an important foundation for future proteomic and biochemical characterizations, which are crucial for a better understanding of canonical and complete nitrifying microorganisms.
Project description:The ecophysiology of complete ammonia oxidizing Nitrospira (CMX) and their widespread occurrence in groundwater suggests that CMX bacteria have a competitive advantage over ammonia-oxidizing bacteria (AOB) and archaea (AOA) in these environments. However, the relevance of their activity from the ecosystem-level process perspective has remained unclear. We investigated oligotrophic carbonate rock aquifers as a model system to assess the contribution of CMX, AOA and AOB to nitrification and to identify the environmental drivers of their niche differentiation at different levels of ammonium and oxygen. CMX accounted for up to 95% of the ammonia oxidizer communities. Nitrification rates were positively correlated to CMX clade A-associated phylotypes and AOB affiliated with Nitrosomonas ureae. Surprisingly, short-term incubations amended with the nitrification inhibitors allylthiourea and chlorate suggested that AOB contributed more than 90% to overall ammonia oxidation, while metaproteomics analysis confirmed an active role of CMX in both ammonia and nitrite oxidation. Ecophysiological niche differentiation of CMX clades A and B, AOA and AOB was linked to their requirements for ammonium, oxygen tolerance, and metabolic versatility. Our results demonstrate that despite numerical predominance of CMX, the first step of nitrification in oligotrophic groundwater is primarily governed by AOB. Higher growth yields at lower NH4+ turnover rates and energy derived from nitrite oxidation most likely enable CMX to maintain consistently high populations. Activity measurements combined with differential inhibition allowed a refined understanding of ammonia oxidizer coexistence, competition and cooperation beyond the insights from molecular data alone.