Deterioration of concrete is a large societal cost. In the Oslofjord subsea tunnel (Norway), deterioration of sprayed concrete and corrosion of reinforcing steel fibres occur under biofilm formed at sites with intrusion of saline groundwater. In this study, the microbial community structure, in situ environmental gradients and chemical composition of the biofilms were examined at three tunnel sites. Ammonia-and nitrite-oxidising microorganisms, in particular Nitrosopumilus sp., and iron-oxidising bacteria within Mariprofundus sp., were omnipresent, together with a diversity of presumably heterotrophic bacteria. Alpha-and beta diversity measures showed significant differences in richness and community structure between the sites as well as over time and null-models suggested that deterministic factors were important for the community assembly. The superficial flow of water over the biofilm had a strong effect on oxygen penetration in the biofilm and was identified as one major environmental gradient that varied between the sites, likely being important for shaping the microbial communities.

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Summary Bacteria in the order ‘Candidatus Brocadiales’ within the phylum Planctomycetes (Planctomycetota) have the remarkable ability to perform anaerobic ammonium oxidation (anammox). Two families of anammox bacteria with different biogeographical distributions have been reported, marine Ca. Scalinduaceae and freshwater Ca. Brocadiaceae. Here we report evidence of three new species within a novel genus and family of anammox bacteria, which were discovered in biofilms of a subsea road tunnel under a fjord in Norway. In this particular ecosystem, the nitrogen cycle is likely fuelled by ammonia from organic matter degradation in the fjord sediments and the rock mass above the tunnel, resulting in the growth of biofilms where anammox bacteria can thrive under oxygen limitation. We resolved several metagenome‐assembled genomes (MAGs) of anammox bacteria, including three Ca. Brocadiales MAGs that could not be classified at the family level. MAGs of this novel family had all the diagnostic genes for a full anaerobic ammonium oxidation pathway in which nitrite was probably reduced by a NirK‐like reductase. A survey of published molecular data indicated that this new family of anammox bacteria occurs in many marine sediments, where its members presumably would contribute to nitrogen loss.

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