In oceanic nitrogen cycle, denitrification was considered only known pathway of fixed nitrogen loss. However, after 1990s anaerobic ammonium oxidation (anammox) process was discovered and detected in a wide range of natural environments. Several researches tried to estimate anammox contribution to fixed nitrogen loss. However, even now oceanic nitrogen budget is still poorly constrained.

To estimate the global marine nitrogen budget, measurement of N and O stable isotope ratios of fixed nitrogen compounds has long been used. In order to use natural isotopic distribution to infer the pathways of microbial N transformation, it is important to know the N and O isotope effect for each of the relevant process. However, despite significant importance of anammox, nitrogen isotope effect was determined only for freshwater anammox strain “Ca. Kuenenia stuttgartiensis”. In addition, O isotope effect of anammox has never been determined yet.

One of the obstacles to progress is difficulty of cultivation. No anammox pure cultures are still available. In addition, development of enriched planktonic cell culture requires an enormous amount of time and effort. In our laboratory, we developed a method for cultivating planktonic anammox cells, and successfully developed continuous MBR anammox-enrichment cultures of three anammox species: ‘Ca. Scalindua japonica’, ‘Ca. Jettenia caeni’, and ‘Ca. Brocadia sinica’. “Ca. Scalindua japonica” is a putative marine species.

In this study, we reported the N isotope effect (¹⁵ε) and combined O isotope effect (¹⁸E) of nitrite and nitrate associated with anammox metabolism by three anammox species. We believe that the N and O isotope effects provide significant insights into the relative contribution of anammox bacteria to the fixed N loss and nitrite re-oxidation (recycling N) in various natural environments.

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