Novel and unusual genes for nitrogen and metal cycling in Planctomycetota- and KSB1-affiliated metagenome-assembled genomes reconstructed from a marine subsea tunnel.

The Oslofjord subsea road tunnel is a unique environment in which the typically anoxic marine deep subsurface is exposed to oxygen. Concrete biodeterioration and steel corrosion in the tunnel have been linked to the growth of iron- and manganese-oxidizing biofilms in areas of saline water seepage. Surprisingly, previous 16S rRNA gene surveys of biofilm samples revealed microbial communities dominated by sequences affiliated with nitrogen-cycling microorganisms. This study aimed to identify microbial genomes with metabolic potential for novel nitrogen- and metal-cycling reactions, representing biofilm microorganisms that could link these cycles and play a role in concrete biodeterioration. We reconstructed 33 abundant, novel metagenome-assembled genomes (MAGs) affiliated with the phylum Planctomycetota and the candidate phylum KSB1. We identified novel and unusual genes and gene clusters in these MAGs related to anaerobic ammonium oxidation, nitrite oxidation, and other nitrogen-cycling reactions. Additionally, 26 of 33 MAGs also had the potential for iron, manganese, and arsenite cycling, suggesting that bacteria represented by these genomes might couple these reactions. Our results expand the diversity of microorganisms putatively involved in nitrogen and metal cycling, and contribute to our understanding of potential biofilm impacts on built infrastructure.

Geochemical and morphological characterization of particles originating from tunnel construction.

Rock particles from drilling and blasting during tunnel construction (DB particles) are released to the aquatic environment where they may cause negative toxicological and ecological effects. However, there exists little research on the difference in morphology and structure of these particles. Despite this DB particles are assumed to be sharper and more angular than naturally eroded particles (NE particles), and in consequence cause greater mechanical abrasion to biota. Moreover, morphology of DB particles is assumed to depend on geology, thus depending on where construction takes place different morphologies may be emitted. The objectives in the current study were to investigate the morphological differences between DB and NE particles, and the influence of mineral and elemental content on DB particles. Particle geochemistry and morphology were characterized by inductively coupled plasma mass spectrometry, micro-X-ray fluorescence, X-ray diffraction, environmental scanning electron microscope interfaced with energy dispersive X-ray, stereo microscope, dynamic image analysis and coulter counter. DB particles (61-91% < 63 µm) collected from five different tunnel construction locations in Norway were 8-15% more elongated (lower aspect ratio) than NE particles from river water and sediments, although their angularity was similar (solidity; diff 0.3-0.8%). Despite distinct mineral and elemental characteristics between tunnel construction locations, DB morphology was not explained by geochemical content since only 2-2.1% of the variance was explained. This suggests that particle formation mechanisms during drilling and blasting are more influential of morphology than mineralogy, when working in granite-gneiss terrain. When tunnelling in granite-gneiss terrain, particles with greater elongation than natural particles may enter aquatic systems.

Metagenomic evidence of a novel family of anammox bacteria in a subsea environment.

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.

Subsea tunnel reinforced sprayed concrete subjected to deterioration harbours distinct microbial communities.

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.