Benthic Carbon fixation and cycling in diffuse hydrothermal 1 and background sediments in the Bransfield Strait , 2 Antarctica 3

12 Sedimented hydrothermal vents are likely to be widespread compared to hard substrate hot vents. They host 13 chemosynthetic microbial communities which fix inorganic C at the seafloor, as well as a wide range of 14 macroinfauna, including vent-obligate and background non-vent taxa. There are no previous direct observations 15 of Carbon cycling at a sedimented hydrothermal vent. We conducted C isotope tracing experiments at 3 16 sedimented sites in the Bransfield Strait, Antarctica, which showed different degrees of hydrothermalism. Two 17 experimental treatments were applied, with C added as either algal detritus (photosynthetic C), or as 18 bicarbonate (substrate for benthic C fixation). Algal C was taken up by both bacteria and metazoan 19 macrofaunal, but its dominant fate was respiration, as observed at deeper and more food limited sites elsewhere. 20 Rates of C uptake and respiration suggested that the diffuse hydrothermal site was not the hotspot of benthic 21 C-cycling that we hypothesised it would be. Fixation of inorganic C into bacterial biomass was observed at all 22 sites, and was measurable at 2 out of 3 sites. At all sites, newly fixed C was transferred to metazoan macrofauna. 23 Fixation rates were relatively low compared to similar experiments elsewhere, thus C fixed at the seafloor was a 24 minor C source for the benthic ecosystem. However, as the greatest amount of benthic C fixation occurred at the 25

2 off vent (non-hydrothermal) site (0.077±0.034 mg C m -2 fixed during 60 h), we suggest that benthic fixation of 26 inorganic C is more widespread than previously thought, and warrants further study.

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Sedimented hydrothermal vent (SHV) sites are those where hydrothermal fluid diffuses through soft sediment (Table 1) using cooled incubators. Experiments were initiated by addition of isotopically enriched substrates.

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Cores were then sealed and incubated for ~60 h, during which core-top water was continuously stirred.

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Duplicate cores were subjected to each of two treatments. In the 'algae' treatment, lyophilized algal cells 118 (Chlorella, Cambridge Isotope Laboratories, CNLM-455-1) enriched in 13 C and 15 N (both ~100 at %) were 119 allowed to settle on the sediment surface, giving a final dose of 436±30 mg C m -2 . This was equivalent to ~1.6% 120 of total OC in the surface 1 cm of sediment, or ~9% of annual OC input (Bell et al., 2017b). It is recognised that 121 such organic detritus is less degraded than the sinking photosynthetic material which normally reaches the 122 depths of our study sites. This is a limitation of the method common to all such experiments in the literature, and 123 means that rates for processing of added C in 'algae' experiments should be considered maximal. Further, 124 diatom detritus would have been more representative of local photosynthetic material, but was unfortunately not 125 available.

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In the 'Bicarbonate' treatment a solution of 100 % 13 C labelled sodium bicarbonate and 100 % 15 N labelled 127 ammonium chloride was injected in the surface 5 cm of sediment porewater, to give a dose of 306 mg C m -2 and 128 2.52 mg N m -2 , and an estimated porewater bicarbonate concentration of 1 mM. samples onto ISOLUTE SPE columns, washing with chloroform and acetone, and eluting with methanol. After washing with 4:1 isohexane:chloroform. Samples were dried and then taken up in isohexane for analysis on a Trace Ultra GC, connected via a GC Combustion III to a Delta V Advantage IRMS (Thermo Finnigan, 145 Bremen). The isotopic signature of each PLFA was measured against a CO2 reference gas which is traceable to 146 IAEA reference material NBS 19 TS-Limestone, with a precision of ± 0.31 ‰, and corrected for the C atom 147 added during derivatization.

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Sediment horizons between 0 and 10 cm preserved in formalin were sieved over a 300m mesh. Macrofauna

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were extracted under a binocular microscope, identified to broad taxonomic level, air dried in pre-weighed tin 150 capsules, and weighed. In some cases multiple individuals were pooled to create samples large enough for 151 analysis. Fauna were de-carbonated by dropwise addition of 0.1M HCl, followed by air drying at 50°C.

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Calcareous foraminifera and bivalves which were too small for manual removal of shells were de-carbonated 153 with 6N HCl. Fauna were analysed for their C contents and isotopic signature using a Flash EA 1112 Series

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Elemental Analyser connected via a Conflo III to a Delta Plus XP isotope ratio mass spectrometer (all Thermo 155 Finnigan, Bremen). Carbon contents was quantified using the area under the mass spectrometer response curve,

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against National Institute of Standards and Technology reference material 1547 peach leaves (repeat analysis 157 gave precision ± 0.35 %). Isotopic data were traceable to IAEA reference materials USGS40 and USGS41 (both 158 L-glutamic acid), with a precision ± 0.13 ‰.

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Respiration of added algal C was calculated for cores subjected to the algae treatment. The amount of excess 161 DI 13 C in each sample was calculated by first subtracting the natural abundance of 13 C in DIC. This was scaled 162 up to give the total amount of DIC from the added algae at each sample timepoint, and corrected for water 163 removed and added during sampling. Respiration rate was calculated for each core by placing a line of best fit 164 through the amount of added 13 C over time, and normalised to surface area.

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Bacterial incorporation of 13 C was calculated by first subtracting the natural abundance of 13 C from the isotopic the fact that the addition of 13 C bicarbonate was calculated to result in a porewater DIC pool that was 22 atom % 173 13 C.

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Faunal uptake of added 13 C was calculated by subtracting 13 C attributable to its natural abundance in the     In the algae addition experiments, total bacterial uptake of C throughout the experiment was maximal at Middle 186 Sister and Hook Ridge (1.30-1.91 and 1.25 mg C m -2 , respectively), and minimal at the off vent site (0.25-0.77 187 mg C m -2 , Fig. 3). In bicarbonate addition experiments, in which incorporation of 13 C into bacterial PLFAs 188 represents chemosynthesis, bacterial incorporation of bicarbonate was maximal at the off vent site (0.05-0.10 189 mg C m -2 ), and was also detectable in one of the replicates at Middle Sister (0.003 mg C m -2 , close to detection 190 limits, so this value is treated with caution), however it was not detectable at Hook Ridge.

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Small size of individuals meant that organisms had to be pooled for isotopic analysis, limiting the taxonomic 210 resolution of the faunal uptake data. Although limited in this way, the data show that faunal uptake of 13 C in 211 both algae and bicarbonate addition experiments was mostly carried out by either polychaetes, or 'mixed 212 macrofauna' (Fig. 6). This latter category contained variously bivalves, crustaceans, echinoderms, nematodes 213 and foraminifera, in cases where those groups were not present in sufficient numbers for separate reporting of 214 their C uptake. When a group was present in sufficient quantity it was analysed separately. As with total 215 macrofaunal 13 C uptake, there was considerable variability between replicate cores in the most abundant 216 taxonomic groups. In addition, meiofaunal organisms took up 13 C at Middle Sister, and the bicarbonate 13 C that 217 was transferred to macrofauna at Hook Ridge was mostly observed in amphipod crustaceans.   The results of bicarbonate addition experiments show evidence for occurrence of benthic C-fixation at all sites, 221 and transfer of that C to the macrofauna, in the form of isotopic enrichment of bacterial PLFAs at the off-vent quantities of bicarbonate 13 C detected in bacterial and faunal biomass were low, and tended to be 1 to 2 orders of 224 magnitude smaller than equivalent values for algae addition experiments (Table 2). We have confidence that the 225 values reported are above detection limits, in that data were only used where the enrichment of organisms or 226 PLFAs above their natural background signatures was greater than the analytical precision of the method. The 227 greatest quantities of bacterial uptake were measured at the off-vent site (Fig. 3), and the greatest quantity 228 transferred to the fauna was measured at Hook Ridge (Fig. 5), however, due to the low values measured and the 229 evident patchiness of faunal communities we do not feel these differences are suitable for further discussion.

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The most striking result of the bicarbonate addition experiments was that evidence for benthic C fixation was 231 found at all sites, not only at the hydrothermally influenced Hook Ridge. Further, the site showing the largest 232 amount of incorporation of bicarbonate 13 C into bacterial PLFAs was the off-vent 'control' site (

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This is consistent with the occurrence of siboglinids at all sites. These host chemosynthetic endosymbionts most 234 of which conduct sulphide oxidation (Thornhill et al., 2008;Georgieva et al., 2015). It should be noted that the 235 evidence for inorganic C fixation comes from PLFAs in the bulk sediment, while isotopic signatures of 236 siboglinids did not show enrichment above background values. Therefore the occurrence of benthic C fixation is 237 not only associated with siboglinids.

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Experiments were designed to replicate natural conditions as far as practically possible, while being limited to 239 shipboard rather than in situ methods. One result of this is that the sediment contained in cores was detached

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The evidence suggests that while the amount of benthic C-fixation was always low, it was not restricted to 249 environments typically thought of as chemosynthetic (sedimented or hard substrate hydrothermal vents, methane 250 seeps, or organic falls (Bernardino et al., 2012)). Thus, benthic C-fixation appears to play a role in benthic C-251 cycling at a much wider range of sites and over a much larger area of the seafloor than previously thought. This 252 is supported by linear inverse modelling of C-cycling at the sites in this study, which led Bell et al. (2017b) to suggest that chemosynthetic support for ecosystems may have a far greater spatial extent than previously 254 thought, extending beyond those which are directly hydrothermally influenced. Similar results have also been 2019). In addition, in situ observations of benthic C fixation have also been made at mesotrophic, abyssal sites 258 in the eastern equatorial Pacific, which were not associated with hydrothermal or methane seep activity 259 (Sweetman et al. 2018). In that study incorporation of 13 C labelled bicarbonate into bacterial PLFAs was 260 observed at 2 sites separated by 100's of kilometres, at rates similar to bacterial assimilation of phytodetritus C 261 at the same sites. Together with global scale modelling completed by Middelburg (2011), these studies suggest 262 that chemoautotrophic C fixation may be considerably more widespread than previously thought. It is therefore 263 deserving of further study so that it can be quantitatively incorporated into our understanding of the marine C-264 cycle.

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In their study using linear inverse modelling of the benthic food web and C cycle, based on natural isotopic and

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The maximal rate of benthic C-fixation measured in this study was 0.050 mg C m -2 d -1 , which occurred in one 278 core at the off-vent site. This remains considerably lower than the 0.24-1.02 mg C m -2 d -1 measured by Molari et    (Fig. 2). Temperature is often recognised as a dominant control on benthic respiration rates (e.g. sites may have been driven by differences in bacterial biomass (Table 1) It has been suggested that reducing benthic environments are often hotspots of faunal biomass and 368 biogeochemical cycling due to the increased availability of labile food sources supplied by chemosynthesis 369 (Bernardino et al., 2012). In this study, the hydrothermally active site Hook Ridge showed rates of respiration hotspots, as in algae addition experiments the overall amount of added C processed by the benthic community 375 was not greater than that observed at non-hydrothermal sites (Fig. 8). In line with this, biological processing of 376 added C in the algae addition experiments did not show a major role for faunal C uptake as we hypothesised, but 377 was instead dominated by respiration, as is typically observed at relatively deep, cold sites (Woulds et al., 2009).

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The Middle Sister site showed the greatest amount of biological processing of added algal C, which was 379 probably attributable to it having the greatest bacterial biomass and organic carbon concentrations, and the fact 380 that the macrofaunal community, composed mostly of ambient Southern Ocean taxa, will have been functioning 381 without the stress imposed by hydrothermal fluid.

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The main fate of photosynthetic C was respiration in common with other deeper and more food limited sites.

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The rates of respiration and C uptake by both macrofaunal and bacteria that we measured were comparatively 385 low, and this is attributable to the low temperature of the experiments, and the toxicity and thermal stress caused 386 by hydrothermal fluid. The hydrothermal site (Hook Ridge) in this study did not show more rapid C-cycling 387 than other similar experiments, as we hypothesised it would.

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Benthic fixation of inorganic was observed at all sites, and quantified at 2 out of 3 sites. While the rates were 389 low compared to other similar experiments, the fact that the greatest amount of benthic C fixation occurred at 390 the off vent site suggests that benthic C fixation may not be restricted to hydrothermal and other reducing 391 settings. We suggest that it could be an important aspect of the marine C-cycle, and warrants further study.