Progress 07/01/23 to 06/30/24

Outputs
Target Audience: The target audience is the international scientific community, as well as local stake holders, such as farmers, environmental stewards, students, and the general public. Changes/Problems:No major changes of project approach were necessary. Based on reviewer suggestions on the original proposal we expanded on the technical approach of enrichment/cultivation of novel AOA lineages by implementing a enrichment strategy withing soil matrix. Although this did not allow us to make as much progress on cultivation of individual lineages, it gave us new insights into the nature of the different AOA lineages and their growth preferences, that substantially informed our current cultivation approaches. What opportunities for training and professional development has the project provided?In Year 1 the main focus was on recruiting a fulltime postdoctoral scientist and a graduate student. Both start at the beginning of Year 2. In the meantime, we trained a part-time postdoctoral scientist, a graduate student intern, and an undergraduate student intern. Each of them made significant contributions to the project, learned new laboratory techniques, and will co-author pending publications and conference presentations. How have the results been disseminated to communities of interest?Draft publications for academic journals and conference presentations are being prepared to be disseminated during Year 2. What do you plan to do during the next reporting period to accomplish the goals?In Year 2, we plan to systematically expand on our soil AOA cultivation and physiological characterization. We further plan to conduct dedicated plant-microbe interaction experiments and plant to present initial results at conferences and in publications.

Impacts
What was accomplished under these goals? In Year 1 of the project activities focused on the most challenging parts of the project, namely on Objective 1 and Objective 3. Objective 1. In preliminary experiments aiming to enrich novel archaeal lineages from soil, we obtained a Nitrosocosmicus strain. Nitrosocosmicus sp. have been noted for the broad range of temperature- and pH tolerance. However, predominant lineages in soil have been characterized as having rather narrow temperature- and pH ranges, leading to substantial diversification. Therefore, we adopted two approaches. We further refined our growth media by establishing a CO2/carbonate-based buffering system that allows us to reduce pH fluctuations and maintain stable growth conditions over months of time between pH 4 and 8. Using a combination of CO2 headspace and bicarbonate buffer, this system now allows us to enrich organisms more efficiently at desired target pH. Using this approach we test chemostat growth in Nitrososphaera viennensis, a soil ammonia-oxidizing archaeon deemed to be less competitive and having comparatively high half-saturation constant for ammonia. Our results demonstrate that N. viennensis grows well under chemostat conditions in our newly designed media formulation and pH buffering system. Intriguingly, kinetic measurements showed that N. viennensis adapts is kinetic properties, reducing its half-saturation constant for ammonia by 50% relative to batch culture growth. These results demonstrate substantial potential for oligotrophic growth within soil AOA. For Year 2 we now plan to adapt this approach for the enrichment of novel soil AOA lineages. While improving our cultivation approach itself, we further expanded our enrichment strategy based on reviewer comments, by implementing a solid-state enrichment methodology based on a soil disturbance approach. Theoretical models propose that biodiversity enhances resilience of natural ecosystems by enabling asynchronous taxonomic responses to disturbances. Imposing disturbances and measuring responses of soil nitrifiers could then inform physiological characteristics particularly of thus far uncultivated AOA. Using controlled soil microcosms, we examine the nitrifier responses to population-reducing disturbance. The results of these experiments revealed that growth patterns varied taxonomically, exhibiting a continuum of fast to slow species growth that was inversely related to population density. In other words, strains of highly abundant soil AOA lineages grew slowest and rare Nitrososphaera- and Nitrosocosmicus-related strains grew fastest, only outpaced by AOB and comammox, where nutrient levels were high enough. As a result, low-abundance ammonia-oxidizing bacteria (AOB) and complete ammonia oxidizers (comammox) initially took over the dominant role of ammonia-oxidizing archaea (AOA) after disturbance with an enhanced nitrogen flux. Competitive interactions persisted in these enrichments after disturbance, shaping the community and functional dynamics of ammonia-oxidizers. Notably, post-disturbance nitrogen cycling was impaired when AOB regrowth was absent. These findings have important implications for our understanding of soil AOA and soil nitrification in general. The data demonstrate that disturbances reconfigure functional redundancy through resource reallocation and competitive restructuring, transforming nutrient flux and microbial assembly in soil. These data further provide strong indication that the predominant soil AOA lingeages in soil may harbor some of the most oligotrophic soil microorganisms. A manuscript describing the approach and results has been submitted for publication. Objective 3. In order to characterize physical and chemical interactions between AOA, NOB, AMF, and plants, we first focused on establishing a laboratory model system that allows us to track and trace such interactions. Many studies suggest that the rhizosphere microbiome engages in intricate interactions with plants and imparts specific functions, such as modulating plant nutrient uptake, development, and stress resistance. Recent studies indicate that some AOA lineages may interact with plant roots with potential to influence plant nutrient uptake, plant fitness, and health, as well as nitrous oxide emissions. However, information is limited on how archaeal nitrification may be linked to plant nutrition, and ultimately how reactive N species are acquired and shared among microbial partners and plants. We therefore developed a novel hydroponic media capable of supporting growth of nitrifiers and plants in co-culture using Nitrosocosmicus sp. R2, Nitrospira moscoviensis, and Solanum lycopersicum, to characterize interactions between nitrification and plant nutrition. We formulated a new media recipe through the stepwise removal of phytotoxic ingredients from Synthetic Freshwater Crenarchaeota Medium (SFCM), our standard AOA cultivation media. Briefly, salts in the form of chlorides and sodium bicarbonate were removed and replaced with PIPES that was titrated to desired pH with KOH. Ammonium sulfate and potassium nitrate were supplemented as nitrogen sources. Lastly, concentrations of trace elements were increased to support plant growth and development. Performance of Solanum lycopersicum growing in the new hydroponic media was phenotypically indistinguishable from Hoaglands solution, and growth of nitrifiers was based on ammonia consumption and nitrate accumulation was indistinguishable from SFCM media. These results suggest that our new hydroponic media enables cocultivation of nitrifiers and Solanum lycopersicum absent of any growth defects. This hydroponic media platform now allows us to systematically investigate nitrogen partitioning between plants and nitrifiers in Year 2 and 3. A manuscript describing the results of these experiments is in preparation.

Publications