Final Report Summary - NITRICARE (Nitrification Reloaded - a Single Cell Approach)
Nitrification, the microbially catalyzed oxidation of ammonia via nitrite to nitrate, is of immense ecological importance. Nitrification (i) forms most of the nitrate in the world’s oceans, which constitutes an impressive 88% of the marine bioavailable N pool; (ii) impairs dramatically the efficiency of N fertilization in agriculture by causing severe eutrophication of natural waters; (iii) is a crucial step of biological wastewater treatment; and (iv) produces considerable amounts of N2O, a highly potent greenhouse gas contributing to the depletion of atmospheric ozone. The goal of this project was to develop and apply innovative Raman and NanoSIMS-based single cell tools in combination with classical cultivation and molecular biology approaches to investigate diversity, physiological versatility and evolution of nitrifying microbes. Since the discovery of nitrifying microbes more than a century ago it was believed that these microbes either oxidize ammonium to nitrite (ammonia-oxidizers) or nitrite to nitrate (nitrite-oxidizers) and cooperate to collectively convert ammonia to nitrate.
In the framework of this project we discovered microbes of the genus Nitrospira that can catalyze both steps (so-called comammox microbes) and thus changed dramatically the perception of nitrification (Daims et al., 2015 Nature). Comammox microbes are diverse within the genus Nitrospira, widespread in the environment, and occur at considerable abundances in drinking water and waste water plants and in various soils. We obtained the first pure culture of a comammox organism (Nitrospira inopinata) and performed a comparative kinetic characterization of it. The comammox microbes showed the highest yield per mol substrate of all ammonia oxidizers, demonstrating efficient coupling of ammonia- and nitrite-oxidation in a single cell. Unexpectedly, the comammox organism also has a higher affinity for ammonia than several ammonia-oxidizing archaea that we tested for comparison. We also showed that the long-held dogma in the field that ammonia-oxidizing archaea generally have a higher affinity for ammonia than ammonia-oxidizing bacteria is incorrect.
Another major finding of this project was that the archaeal ammonia-oxidizer Nitrososphaera gargensis is the first known microbe that grows with cyanate as only energy and nitrogen source and uses a cyanase for this purpose (Palatinszky et al. 2015, Nature). This cyanase is a member of a new clade within the cyanase family, which is frequently detected in metagenomic data sets from various environments. Interestingly, we could demonstrate that nitrite-oxidizers are also capable of cyanate conversion and produce ammonia by its degradation. By teaming up with ammonia-oxidizers that produce nitrite from ammonia, nitrite-oxidizers are thus able to thrive on cyanate by this reciprocal feeding mechanism. Subsequently, we demonstrated that cyanate-degradation is actually performed in the environment by nitrifiers by performing in collaboration with Marcel Kuyper’s team (MPI Bremen, Germany) isotope-labeling studies and NanoSIMS analyses in marine oxygen minimum zone samples. Furthermore, the research team financed by this project contributed to several studies demonstrating that nitrifying microbes are physiologically much more versatile than previously thought. For example, some nitrite-oxidizers can grow aerobically unconnected from the nitrogen cycle on hydrogen (Koch et al. 2014, Science) or on formate (Koch et al., 2015 PNAS).
As proposed, we also developed new techniques for in situ labeling (Berry et al. 2013, PNAS) and sorting of physiologically active microbes from complex microbial communities in the framework of this project. For example, we designed a Raman-based approach that uses the incorporation of deuterium from heavy water as activity marker of individual microbial cells and managed to specifically sort activity-labeled cells for subsequent single-cell genomics (Berry at al. 2015 PNAS). This approach was applied to metagenomically investigate many nitrifying microcolonies from activated sludge and to identify their heterotrophic interaction partners. Interestingly, we also discovered a new giant virus by this sorting approach that possesses an expanded translation machinery, but nevertheless very likely did not evolve from a cellular ancestor, but rather was derived from a much smaller virus through the extensive gain of host genes (Schulz et al. 2017, Science). While in the initial phase of the project, sorting of isotope-labeled cells was done manually with the help of an optical tweezer, we managed to replace this slow procedure by designing in collaboration with the team from Prof Roman Stocker (ETH Zürich, Switzerland) a fully automated microfluidic Raman-device. This device is now used by several teams in the Department and has already proven very useful for other applications like the analysis of microbial communities thriving in the gut of mice. Excitingly, our new single cell techniques developed in the framework of this project were also successfully used in several collaborations ranging from the characterization of anaerobic methane oxidizers (Milucka et al. 2012, Nature) to following the fate of platinum anticancer compounds in tumor cells (Legin et al. 2014 and 2016, Chemical Science)
In total 26 manuscripts including two Nature, two Science and three PNAS papers (all acknowledging the ERC funding) have been published in the framework of this project.
In the framework of this project we discovered microbes of the genus Nitrospira that can catalyze both steps (so-called comammox microbes) and thus changed dramatically the perception of nitrification (Daims et al., 2015 Nature). Comammox microbes are diverse within the genus Nitrospira, widespread in the environment, and occur at considerable abundances in drinking water and waste water plants and in various soils. We obtained the first pure culture of a comammox organism (Nitrospira inopinata) and performed a comparative kinetic characterization of it. The comammox microbes showed the highest yield per mol substrate of all ammonia oxidizers, demonstrating efficient coupling of ammonia- and nitrite-oxidation in a single cell. Unexpectedly, the comammox organism also has a higher affinity for ammonia than several ammonia-oxidizing archaea that we tested for comparison. We also showed that the long-held dogma in the field that ammonia-oxidizing archaea generally have a higher affinity for ammonia than ammonia-oxidizing bacteria is incorrect.
Another major finding of this project was that the archaeal ammonia-oxidizer Nitrososphaera gargensis is the first known microbe that grows with cyanate as only energy and nitrogen source and uses a cyanase for this purpose (Palatinszky et al. 2015, Nature). This cyanase is a member of a new clade within the cyanase family, which is frequently detected in metagenomic data sets from various environments. Interestingly, we could demonstrate that nitrite-oxidizers are also capable of cyanate conversion and produce ammonia by its degradation. By teaming up with ammonia-oxidizers that produce nitrite from ammonia, nitrite-oxidizers are thus able to thrive on cyanate by this reciprocal feeding mechanism. Subsequently, we demonstrated that cyanate-degradation is actually performed in the environment by nitrifiers by performing in collaboration with Marcel Kuyper’s team (MPI Bremen, Germany) isotope-labeling studies and NanoSIMS analyses in marine oxygen minimum zone samples. Furthermore, the research team financed by this project contributed to several studies demonstrating that nitrifying microbes are physiologically much more versatile than previously thought. For example, some nitrite-oxidizers can grow aerobically unconnected from the nitrogen cycle on hydrogen (Koch et al. 2014, Science) or on formate (Koch et al., 2015 PNAS).
As proposed, we also developed new techniques for in situ labeling (Berry et al. 2013, PNAS) and sorting of physiologically active microbes from complex microbial communities in the framework of this project. For example, we designed a Raman-based approach that uses the incorporation of deuterium from heavy water as activity marker of individual microbial cells and managed to specifically sort activity-labeled cells for subsequent single-cell genomics (Berry at al. 2015 PNAS). This approach was applied to metagenomically investigate many nitrifying microcolonies from activated sludge and to identify their heterotrophic interaction partners. Interestingly, we also discovered a new giant virus by this sorting approach that possesses an expanded translation machinery, but nevertheless very likely did not evolve from a cellular ancestor, but rather was derived from a much smaller virus through the extensive gain of host genes (Schulz et al. 2017, Science). While in the initial phase of the project, sorting of isotope-labeled cells was done manually with the help of an optical tweezer, we managed to replace this slow procedure by designing in collaboration with the team from Prof Roman Stocker (ETH Zürich, Switzerland) a fully automated microfluidic Raman-device. This device is now used by several teams in the Department and has already proven very useful for other applications like the analysis of microbial communities thriving in the gut of mice. Excitingly, our new single cell techniques developed in the framework of this project were also successfully used in several collaborations ranging from the characterization of anaerobic methane oxidizers (Milucka et al. 2012, Nature) to following the fate of platinum anticancer compounds in tumor cells (Legin et al. 2014 and 2016, Chemical Science)
In total 26 manuscripts including two Nature, two Science and three PNAS papers (all acknowledging the ERC funding) have been published in the framework of this project.