Patterns of (trace) metals and microorganisms in the Rainbow hydrothermal plume at the Mid-Atlantic Ridge 2

16 Hydrothermal vent fields found at mid-ocean ridges emit hydrothermal fluids which disperse as neutrally 17 buoyant plumes. From these fluids seafloor massive sulfides (SMS) deposits are formed which are being 18 explored as possible new mining sites for (trace) metals and rare earth elements (REE). It has been 19 suggested that during mining activities large amounts of suspended matter will appear in the water column 20 due to excavation processes, and due to discharge of mining waste from the surface vessel. Understanding 21 how hydrothermal plumes can be characterised by means of geochemistry and microbiology as they 22 spread away from their source and how they affect their surrounding environment may help in 23 characterising the behaviour of the dilute distal part of chemically enriched mining plumes. 24 This study on the extensive Rainbow hydrothermal plume, observed up to 25 km downstream from the vent site, enabled us to investigate how microbial communities and (trace) metal composition change in a natural plume with distance. The (trace) metal and REE content of suspended particulate matter (SPM) was determined using HR-ICP mass spectrometry and the microbial communities of the neutrally buoyant 28 plume, above plume-, below plume-, and near-bottom water and sediment were characterised by using 29 16S rRNA amplicon sequencing methods. Both vertically in the water column and horizontally along the 30 neutrally buoyant plume, geochemical and biological changes were evident as the neutrally buoyant 31 plume stood out by its enrichments in (trace) metals and REEs as e.g. Fe, Cu, V, Mn and REE were 32 enriched by factors of up to ~80, ~90, ~52, ~2.5 and ~40 respectively, compared to above plume water 33 samples taken at 1000 m water depth. The concentrations of these elements changed as the plume aged 34 shown by the decrease of element/Fe molar ratios of chalcophile elements (Cu, Co, Zn), indicative of 35 rapid removal from the hydrothermal plume or removal from the solid phase. Conversely, increasing 36 REE/Fe molar ratios imply uptake of REE from the ambient seawater onto Fe-oxyhydroxides. This was 37 also reflected in the background pelagic system as Epsilonproteobacteria started to dominate and 38 univariate microbial biodiversity declined with distance away from the Rainbow hydrothermal vent field. 39 The Rainbow hydrothermal plume provides a geochemically enriched natural environment, which is a 40 heterogeneous, dynamic habitat that is conducive to ecological changes in a short time span. This study of a hydrothermal plume provides a baseline study to characterize the natural plume before the 42 interference of deep-sea mining.

This study on the extensive Rainbow hydrothermal plume, observed up to 25 km downstream from the 25 vent site, enabled us to investigate how microbial communities and (trace) metal composition change in 26 a natural plume with distance. The (trace) metal and REE content of suspended particulate matter (SPM) 27 was determined using HR-ICP mass spectrometry and the microbial communities of the neutrally buoyant 28 plume, above plume-, below plume-, and near-bottom water and sediment were characterised by using 29 16S rRNA amplicon sequencing methods. Both vertically in the water column and horizontally along the little is known about changes in chemical composition and microbial assemblages in the hydrothermal 94 plume after its initial rise, when it becomes neutrally buoyant and is dispersed by currents, remaining 95 traceable in particulate form to at least 50 km away from its source (Severmann et al., 2004), and even up 96 to 4000 km is dissolved form (Resing et al., 2015). Considering the majority of microbial growth is 97 predicted to occur in the neutrally buoyant portion of the plume (Reed et al., 2015), further efforts should 98 be concentrated on sampling this portion of the plume.

99
In order to address this gap, water column and sediment samples from the Rainbow hydrothermal vent 100 area were investigated during the TREASURE cruise. Geochemical and biological changes were explored 101 vertically in the water column and horizontally along the neutrally buoyant plume using HR-ICP mass 102 spectrometry to determine the (trace) metal and REE content of the SPM. Next generation sequencing 103 methods were used to quantify the microbial diversity in the pelagic system that was influenced by the

127
The vent field consists of 10 active, high temperature (365 °C) black smokers and emits an extensive 128 plume with a distinct chemical composition compared to the ambient seawater (Severmann et al., 2004).

129
The plume is considered the largest and widest spreading in the region (German et al., 1996), rising up to 130 200 m above its source and was traceable over at least 50 kilometres (Severmann et al., 2004    were tested with Pearson correlation and hierarchical clustering to identify broad response groups. Temperature, salinity and density plots indicated that the water column at each location had similar 264 physical traits, whereby three main different water masses could be distinguished (Supplement Fig. S1).

265
The surface Eastern North Atlantic Central Water (ENACW) was characterised by a temperature, salinity  with stations further downstream displaying greater dissimilarity (Fig. 9, Fig. S3).

353
Group average cluster analysis showed high level of dissimilarity, i.e. large community variation, between plume diversity was low (Fig. 10), but further differences were not statistically significant, likely due to 364 limited replication and intra biotope variation.

365
The plume microbial community at sites upstream of Rainbow and at the immediate downstream sites 366 (stations 28, 16 and 27) showed similar and relatively high biodiversity (>4.5) (Fig 11). Plume  Epsilonproteobacteria accounted for about 20 % of the plume community at stations near the vent. Beyond 384 the near vent stations, an increase in relative abundance of Epsilonproteobacteria with distance from vent 385 was observed, accounting for 64 % of the community at the distant station 46 (Fig. 12).

386
Alphaproteobacteria, Deltaproteobacteria and Gammaproteobacteria appeared to become less dominant 387 with distance from the plume source (Fig. 12). The communities at distant stations 47 and 49 were less in the plume were evident and appeared class specific (Fig. S4). The hierarchical clustering revealed eight 393 broad response groups, which displayed different relationships with the environmental variables.

394
Using a multidisciplinary approach in which physical, geochemical and ecological data were collected 396 from the Rainbow vent neutrally buoyant plume and its underlying sediment, we aimed to expand 397 knowledge and characteristics of the background (i.e. before impact) state of a hydrothermal vent. Such 398 knowledge is deemed essential to be able to assess (potential) impacts of future deep-sea SMS mining, as 399 it may help in characterising the behaviour of the dilute distal part of chemically enriched mining plumes. 400 We found geochemical and microbial differences between the above-plume, plume, below plume and no-

405
The plume was observed within the NADW mass, constrained to an isopycnal density envelope of 27.82 406 kg m -3 ( Fig. 2 and 3). The apparent continuity of this turbid water layer, especially to the NE of the  (Table S2).   The microbial plume community composition and diversity altered with distance from the plume source 525 showcasing a horizontal heterogeneity within the plume. Despite dilution, the vent associated group 526 community composition. This is likely due to its flexibility to exploit mainly sulfur compounds as electron 528 donors, and oxygen and nitrate as acceptors (Nakagawa et al., 2005), making them suitable inhabitants of 529 dynamic environments (Huber et al., 2003). From the relative abundance data presented here it cannot be . This is further supported by the increasing uniqueness of the plume community with 537 distance from the source, suggesting that mixing and entrainment between downstream biotopes is 538 negligible.

539
The neutrally buoyant plume is likely too chemically enriched for non-adapted microbial taxa to thrive, 540 and consequently are outcompeted by groups that can benefit from or tolerate the chemical nature of the 541 plume. Therefore, it is likely that less specialised groups die out due to lack of appropriate resources and within and between microbial classes and water properties, i.e. ranging from chemical to physical 553 variables (Fig. S4), indicates a complex array of community drivers within the plume.