The mystery of the ice cold rose—Microbiome of an Arctic winter frost flower

Abstract Under very cold conditions, delicate ice‐crystal structures called frost flowers emerge on the surface of newly formed sea ice. These understudied, ephemeral structures include saline brine, organic material, inorganic nutrients, and bacterial and archaeal communities in their brine channels. Hitherto, only a few frost flowers have been studied during spring and these have been reported to be dominated by Rhizobia or members of the SAR11 clade. Here we report on the microbiome of frost flowers sampled during the winter and polar night in the Barents Sea. There was a distinct difference in community profile between the extracted DNA and RNA, but both were dominated by members of the SAR11 clade (78% relative abundance and 41.5% relative activity). The data further suggested the abundance and activity of Cand. Nitrosopumilus, Nitrospinia, and Nitrosomonas. Combined with the inference of marker genes based on the 16S rRNA gene data, this indicates that sulfur and nitrogen cycling are likely the major metabolism in these ephemeral structures.


| INTRODUCTION
Frost flowers arise under very cold and calm conditions when water vapor from brine expelled from white or black nilas ice crystallizes on the sea ice surface. As these ephemeral structures grow, they wick up brine, quickly becoming highly saline (up to~10% salinity). Frost flowers can also contain high concentrations of sulfate (~50 μM), nitrate (~22 mM), dissolved organic carbon, and colored dissolved organic matter Douglas et al., 2012). They have been suggested to be the main source of salinity in aerosols, a hotspot for photochemical reactions, and foster ocean-sea iceatmosphere interactions Douglas et al., 2012).
Due to the fragile and short-lived nature of frost flowers, as well as their formation only on thin ice, which is difficult to sample, very few studies have addressed sea ice frost flowers before. Most of which are from coastal or land-fast ice, and never from open ocean sea ice.
Bacterial and archaeal numbers are 3-6 times elevated in frost flowers as compared to the underlying ice as they are wicked into the frost flower during flower growth (Bowman & Deming, 2010;Eronen-Rasimus et al., 2014). This was shown for frost flowers in MicrobiologyOpen. 2023;12:e1345.
www.MicrobiologyOpen.com Barrow, Alaska, where the cell abundance was 1.28 × 10 5 mL −1 in the frost flowers as compared to 0.28-3.83 × 10 5 mL −1 in the underlying ice. Frost flowers from the central Arctic Ocean harbored even more cells with 3.46 × 10 6 mL −1 (Bowman & Deming, 2010). This abundance is correlated with higher salinity and results in high concentrations of cryoprotectant exopolymers in the frost flowers (Bowman & Deming, 2010). The process of bacterial and archaeal acquisition is selective and leads to communities with a different composition than sea ice or water (Bowman & Deming, 2010;Bowman et al., 2013). In coastal frost flowers from spring in Barrow, Alaska, a dominance of Rhizobia (Bowman et al., 2013) or Propionibacterineae ( (Quast et al., 2013). Thereafter, ASVs of mitochondria, chloroplast, or eukaryote origin were removed. The genetic potential was inferred using PiCRUST2 (Douglas et al., 2020) to detect the occurrence of marker genes, thereby indicating specific metabolisms.
Other marker genes were not inferred or were in very low abundance. The carbohydrate-active enzyme (CAZyme) classes glycosyl hydrolases (GH) and glycosyl transferases (GT) were inferred for nearly all ASVs, but specifically for Alpha-, Gammaproteobacteria, Bacteroidia, and Verrucomicrobiae.

| DISCUSSION
The bacterial and archaeal community of the frost flowers from the Barents Sea, sampled during the dark polar night, differs substantially from the communities reported previously. The cell abundance in the surface waters (10 m depth) sampled at one of the closest by stations (~50 km distance), was relatively similar to the cell abundance in the frost flower with 2.31 × 10 5 mL −1 , while at the other close by station, it was lower with 1.48 × 10 5 mL −1 (Thiele, pers. communication). This is much lower than the numbers reported from summer samples by Bowman and Deming (2010). The difference in the season might lead to differences in surface water cell abundance and also the growth behavior of frost flowers might explain these differences. Another factor could be the age of the frost flowers, with fewer bacteria in young frost flowers. Even though a high relative abundance of members of the SAR11 clade was shown before (Barber et al., 2014), the relative abundance was three times higher in the frost flower community analyzed here. In comparison, the abundance of members of the SAR11 clade at the closest stations was lower (37.8 ± 10.6% relative abundance), although the relative abundance of Cand.
The relative activity of all members of the SAR11 clade was lower than the relative abundance. The inference of the aprA gene is due to the abundance of members of SAR11 clades, which are suspected to perform sulfur oxidation for energy and detoxification of sulfite (Meyer & Kuever, 2007;Thiele et al., 2017). This would imply high sulfate concentrations in the frost flower Douglas et al., 2012). Cand. Nitrosopumilus is reported for the first time in frost flowers but has previously been found in Arctic sea ice and seawater during winter (Thiele et al., 2022;Wilson et al., 2017). Cand.

Nitrosopumilus and Nitrosomonas oxidize ammonia to nitrite and
Nitrospina might be capable of complete nitrification (Könneke et al., 2005;Koops et al., 1991;Lücker et al., 2013), hence the abundance of amoA predicted for these genera. Under anaerobic conditions, all of these taxa could rely on denitrification, as indicated by the abundance of nirS/nirK genes (Cantera & Stein, 2007). Nitrospina has F I G U R E 1 (a) Relative abundance and relative activity of the total bacterial and archaeal community on class level (<0.1% merged into "Other classes"), the black lines mark genera within the class. (b) Relative abundance and relative activity of the 20 most abundant genera, encompassing 94% of DNA sequences and 76% of RNA sequences in the sample. THIELE ET AL.
| 3 of 5 been found in spring frost flowers, while Cand. Nitrosopumilus might have been absent at that time due to photoinhibition (Merbt et al., 2012). Taken together our results indicate that nitrogen and sulfur cycling are the predominant metabolisms in polar night frost flowers.
Another factor shaping the bacterial and archaeal community might be the cell size and the size of the brine channels which range from μm to cm scale (Krembs et al., 2000). As frost flowers are intricate, the brine channels are very small and might act as a sieve that only allows entry of small cells, such as those of Cand. Nitrosopumilus or members of the SAR11 clade are among the smallest cells in marine environments with average cell diameters of 0.12-0.22 μm (Könneke et al., 2005;Rappé et al., 2002). The inference of GH and GT indicates carbon cycling activities by the main marine carbon degraders Gammaproteobacteria, Bacteroidia, and Verrucomicrobiae (Teeling et al., 2012). However, the low abundance of these classes, suggests that it is not organic carbon cycling, but nitrification which is the main metabolism in sea ice frost flowers in the polar night.

ACKNOWLEDGMENTS
This work was funded by the Research Council of Norway through the projects The Nansen Legacy (RCN #276730) and CLIMAGAS (RCN #294764). We thank the captain and crews of the RV Kronprins Haakon, Oliver Müller for the FCM data, and Tobias Sammet for the inspiration.

CONFLICT OF INTEREST STATEMENT
None declared.