Enrichment and characterization of novel, marine comammox.

Oceans cover the vast majority of our planets surface and harbor a great biodiversity. From microorganisms to the great blue whales, all marine organisms require nitrogen in order to survive and grow. Nitrification, the oxidation of ammonia to nitrate via nitrite, is a critical process as it supplies the majority of bioavailable nitrogen required to sustain marine ecosystems. In the ocean, nitrification is believed to be mainly performed by the well -orchestrated cooperation of two functionally separate groups of microorganisms (ammonia-oxidizing archaea and nitrite-oxidizing bacteria) that are physiologically adapted to survive in these environments. However, the recent discovery that complete ammonia oxidizers (comammox) are able to perform both nitrification steps in a single cell, highlighted our limited understanding of marine nitrification. To date, comammox microorganisms have been solely identified within the bacterial genus Nitrospira and although they have been found in a variety of ecosystems, they curiously still elude identification in the ocean. The MarCo project aims to develop an innovative approach for the selective enrichment and study of marine comammox microorganisms. Novel comammox species will be identified based on their ability to oxidize ammonia and nitrite in a single cell using a combination of two activity-based, fluorescent staining methods. For the first time, these methods will be optimized and applied on living cells. Subsequently, phylogenetically diverse, active comammox cells will be enriched via fluorescence-activated cell sorting. Their ability to oxidize ammonia, nitrite as well as a variety of different substrates will be studied using a combination of state-of-the-art metagenomics, activity assays and microrespirometry, providing an in-depth understanding of the driving forces shaping the nitrifying communities in the ocean. Overall, with the MarCo project aims to discover the previously overlooked diversity involved in the comammox process and answer fundamental questions on their diversity and ecophysiology while shedding light in the nature of the marine nitrogen cycle.

Nitrification is a critical process within the marine nitrogen cycle. Nitrate, the end-product of nitrification, is essential for the functioning of marine ecosystems. To date, marine nitrification is believed to be performed by the well-orchestrated collaboration of ammonia-oxidizing Archaea and nitrite-oxidizing bacteria. Since their discovery, complete ammonia oxidizers (comammox) have been identified as key players within the global nitrogen cycle. Curiously, despite their ubiquitous presence these oligotrophic nitrifiers haven't been identified in marine environments. This indicates our lack of comprehensive understanding of the overall comammox environmental distribution but also our limited grasp of marine nitrification. The project TAI-621 "Enrichment and characterization of novel, marine comammox" successfully developed an innovative method for the activity-based detection of comammox bacteria. The developed tool is based on the differential labelling of comammox microorganisms. For this, a combination of an activity-based method for the specific detection of microbial cells able to perform ammonia oxidation and bioorthogonal, non-canonical amino acid tagging (BONCAT) for the detection of translationally active cells under nitrite-feeding conditions was optimized and applied. This innovative approach was able to specifically detect and selectively enrich comammox microorganisms present in mock microbial communities, as well as nitrifying enrichments. Combination of the method developed in the TAI-621 project with downstream fluorescence-activated cell sorting and metagenomic sequencing allowed the identification of novel comammox species and the study of their metabolic potential through metagenomics. Finally, when applied on marine (surface) water column samples from the Mediterranean the developed method was able to detect canonical nitrifiers. However, no comammox microorganisms were identified. That confirmed the initial hypothesis formulated in TAI-621 project that deep marine water column and sediment environments represent the potential habitats of marine comammox microorganisms. Overall, the project TAI-621 was successful in developing and optimizing the first activity-based method for the specific detection comammox microorganisms. This groundbreaking technique was already proven sufficient in detecting novel comammox species. Furthermore, it represents an invaluable addition to our toolbox that can facilitate further the detection and study of comammox in multiple environments providing the means to expand our understanding of the global nitrogen cycle.