Total nitrate uptake by an invasive benthic foraminifer in marine sediments

1: UMR 6112 LPG BIAF, Univ. Angers, Univ. Nantes, CNRS, France 2: Department of Geology, Lund University, Sweden 3: Department of Geosciences, Aarhus University, Denmark 4: Ifremer, IRD, Univ. Nouvelle‐Calédonie, Univ. La Réunion, CNRS, UMR 9220 ENTROPIE, New Caledonia 5: Université de Nantes, Mer Molécules Santé, EA 2160, France 6: BioISI – Biosystems & Integrative Sciences Institute, Campo Grande, University of Lisbon, Faculty of Sciences, Portugal


Introduction
Hypoxic water ([O2] < 2 mg L -1 or < 63 µmol L -1 ) occurs frequently in bottom-waters of shallow coastal seas, due to 25 remineralization of organic matter and water stratification (e.g. Diaz et al., 2008;Breitburg et al., 2018). Hypoxia may have large ecological effects (Levin et al., 2009;Rabalais et al., 2010;Zhang et al., 2010), such as an increase of fauna mortality (Diaz et al., 2001). However, certain microorganisms, e.g. bacteria and foraminifera, can perform denitrification by respiring nitrate (Risgaard-Petersen et al., 2006) and thereby to survive in depleted oxygen environments. The effects of decreasing dissolved oxygen availability at spatial and temporal scales will impact biogeochemical cycles such as the nitrogen cycle 30 The nitrogen cycle occurring in marine sediments is dependent on the bottom-water oxygenation. In oxic bottom water conditions (Fig. 1a), ammonium (NH4 + ) produced from remineralization of particulate organic nitrogen (PON) in 35 sediments, diffuses toward the oxic sediment-superficial layer and through the water-sediment interface. Nitrification can occur in the oxic sediment and in the oxic water column through the conversion of NH4 + to nitrate (NO3 -) (Rysgaard et al., 1995;Thamdrup and Dalsgaard, 2008). Conversely, denitrification occurs in sediment when oxygen is scarce (below 5 µmol L -1 , Devol et al., 2008) and organic carbon and nitrate are available. Denitrification named "canonical denitrification" (NO3 - NO2 - NO  N2O  N2) is an anoxic process whereby nitrate is used as the terminal electron acceptor in the oxidation of 40 organic matter by facultative anaerobic metabolisms when oxygen is exhausted. Denitrification participates in the loss of the fixed Nitrogen to N2 gas (Brandes et al., 2007 and references within). Another process can contribute to this loss of N2 gas: Anammox (anaerobic ammonia oxidation) (Engström et al., 2005;Brandma et al., 2011). According to Brandes et al. (2007 and references within) the "total denitrification" can be defined as the sum of the canonical denitrification plus the anammox.
Nitrification and denitrification are thus strongly coupled, and denitrification can be enhanced by adjacent sedimentary 45 nitrification zones or by direct NO3diffusion from the overlying water towards the sediment (Kemp et al., 1990;Cornwell et al., 1999). When bottom water turns hypoxic, the nitrogen cycle occurring in the sediment is strongly affected (Fig. 1 b).
Nitrate production is reduced since nitrification cannot process under low oxygen conditions. However, deeper into reduced sediment, nitrification can occur through secondary reactions with NH4 + oxidation by Mn and Fe oxides (Luther et al., 1997;Mortimer et al., 2004). Denitrification is the dominant process of nitrate reduction in coastal marine sediments (Thamdrup and 50 Dalsgaard, 2008;Herbert, 1999). However, dissimilatory nitrate reduction to ammonium (DNRA) can also contribute to nitrate depletion in reduced sediment leading to NO3converstion into NH4 + instead of nitrogen (N2) (Christensen et al., 2000) and compete denitrification.
Benthic foraminifera were the first marine eukaryotes found to perform denitrification (Risgaard-Petersen et al., 2006), but not all foraminifera species can denitrify (Piña-Ochoa et al., 2010). Denitrifying foraminifera species are defined in 55 our study as species able to perform denitrification proved by denitrification rate measurements. These denitrifying species have a facultative anaerobic metabolism and nitrate-storing foraminifera can use either environmental oxygen or nitrate to respire (Piña-Ochoa et al., 2010). Nonionella cf. stella and Globobulimina turgida were identified as the first denitrifying https://doi.org/10.5194/bg-2020-287 Preprint. Discussion started: 5 August 2020 c Author(s) 2020. CC BY 4.0 License.
can be averaged within the volume of a sediment slice. Moreover, sediment core slicing or centrifugation can induce cell lysis, which can induce a bias in porewater nitrate concentrations (Risgaard-Petersen et al., 2006). To characterize these 85 microenvironments at submillimeter/ millimeter scales, new approaches have to be used. Recently, a 2D-DET (Diffusive Equilibrium in Thin-film) technique combining colorimetry and hyperspectral imagery was developed to obtain the distribution of nitrite and nitrate in sediment porewater at millimeter resolution in two dimensions (Metzger et al., 2016). This method avoids mixing of intracellular nitrate and nitrate contained in the sediment porewater.
The present study aims to examine how the invasive Nonionella sp. T1 and the other denitrifying species affect the 90 nitrogen cycle by comparing two stations with contrasting oxygen and nitrate environments subjected to hypoxic events. The objectives of the paper are: (1) to characterize the density of the living benthic foraminifera at two contrasted stations; (2) to measure the denitrification rate of the invasive Nonionella sp. T1 and (3) to quantify its contributions to benthic denitrification; (4) to discuss the probable future impact of the invasive Nonionella sp. T1 on the foraminifera fauna and the nitrogen cycle in the Gullmar Fjord. 95
Two sampling cruises were conducted in the Gullmar Fjord on board R/V Skagerak and Oscar von Sydow,110 respectively. The first cruise (GF17) took place between 14 th and 15 th November 2017 and two stations were sampled (GF17-3 and GF17-1, Fig. 2 c and d) to define the living foraminifera fauna and the sediment geochemistry at two contrasted stations.
The second cruise (GF18) took place on the 5 th September 2018 with the focus to collect living Nonionella sp. T1 for O2 respiration and denitrification rates measurements. Only one station (at the same position as GF17-3) was sampled.
GF17-3 (50 m water depth) is located closest to the mouth of the fjord (58°16'50.94"N/ 11°30'30.96"E) with bottom 115 waters from Skagerrak (blue diamond, Fig. 3) and GF17-1 (117 m depth) close to the deepest part of the fjord (58°19'41.40"N/11°33'8.40"E) near Alsbäck monitoring station in the middle of the stagnant basin (red square, Fig. 3). In November 2017, CTD profiles indicated the water mass structures at both stations (Fig. S1). Bottom water at GF17-3 station was oxic with a dissolved oxygen content of 234 µmol L -1 . The dissolved oxygen content decreased strongly with depth at the GF17-1 station reaching 9 µmol L -1 at the seafloor, which is below the severe hypoxia threshold. 120

Foraminifera sampling and processing
During the first cruise, two sediment cores per station (1A, 1C and 3A, 3C for GF17-1 and GF17-3 stations respectively) were immediately subsampled with a smaller cylindrical core (Ø 8.2 cm) and sliced every 2 mm up to 2 cm and every 5 mm from 2 to 5 cm to study living foraminifera distribution. The samples were incubated without light for 10-19 hours in ambient 125 seawater with Cell Tracker Green (CMFDA, 1 mM final concentration) at in situ temperatures (Bernhard et al., 2006) and then fixed with ethanol 96°. Fixed samples were sieved and the > 100 µm fraction was examined using an epifluorescence microscope equipped for fluorescein detection (i.e., 470 nm excitation; Olympus SZX13). In the present study, the foraminifera distribution will be described highlighting the invasive species Nonionella sp. T1. One core from the shallow GF17-3 station was reserved for O2 microelectrode profiling. Oxygen concentration was measured in the dark with a Clark electrode (50 µm tip diameter, Unisense ®, Denmark) within the first 5 mm depth at a 100 µm vertical resolution. Due to technical problems, no oxygen profiling was done at the GF17-1 station.
One core per station was dedicated for geochemical analyses, they were carefully brought to Lund University 135 (Sweden) and stored at the sampling site temperature (10°C) until further analysis the next day. Overlaying water of the GF17-3 core was gently air bubbled to maintain the oxygenated conditions recorded at this station. Overlaying water of the GF17-1 core was bubbled with N2 gas passed through a solution of carbonate/bicarbonate to avoid pH rise due to degassing of CO2 by N2 bubbling.
A summary of the NO2 -/ NO3 -2D gel method is presented in Figure 4 (details see, Metzger et al., 2016). For each 140 core, a DET (Diffusive Equilibrium in Thin films) gel probe (16 cm x 6.5 cm and 0.1 cm thickness, Fig. 4 a) was hand-made prepared (Metzger et al., 2016). The gel probe was inserted into the sediment and left for 5 hours to allow for a diffusive equilibration time between the gel and porewaters (Fig. 4 b). After equilibration, the equilibrated gel was removed of the core and was laid on a first NO2reagent gel (Fig. 4 c). A reflectance analysis photograph of the nitrite gels fauna was taken with a hyperspectral camera (HySpex VNIR 1600). The next step was to convert existing nitrate into nitrite with the addition of a 145 reagent gel of vanadium chloride (VCl3) (Fig. 4 d). After 20 min at 50°C, a pinkish coloration appeared revealing porewater nitrate concentration (Fig. 4 e). Followed by the acquisition of another hyperspectral image and converted into false colours through a calibrated scale of concentrations, the final image was cropped to avoid border effects (Fig. 4 f). Each pixel (190 µm x 190 µm) was decomposed as a linear combination of the logarithm of the different end-member spectra using ENVI software (unmixing function) (Cesbron et al., 2014;Metzger et al., 2016). Nitrite and nitrate detection limit is 1.7 µmol L -1 (Metzger et 150 al., 2016). Nitrate production/consumption zones for each station were estimated by extracting the average and standard deviation of the 290 vertical 1D profiles ((5.5 cm width x 1 pixel) / 0.019 cm for 1-pixel size) on the 2D gels and modelling using PROFILE software (Berg et al., 1998).

Oxygen respiration and denitrification rates measurements of the invasive Nonionella sp. T1
The two cores sampled in the 2 nd cruise (GF18, September 2018) at the shallower GF17-3 station were carefully transported at in situ temperature (8 °C) and stored for three days at the Department of Geosciences, Aarhus University (Denmark). Nonionella sp. T1 specimens were picked under in situ temperature and collected in a Petri dish, containing a thin layer of sediment (32 µm) to check their vitality. Only living, active Nonionella sp. T1 specimens were picked and cleaned several times using a brush with micro-filtered, nitrate-free artificial seawater. 160 Oxygen respiration rates were measured, following the method developed by Høgslund et al. (2008) using a Clark type oxygen microsensors (50 µm tip diameter, Unisense ®, Denmark) (Revsbech, 1989) calibrated by a two-point calibration using air-saturated water at in situ temperature (8 °C) and sodium ascorbate solution (to strip O2 out of the system) as zero.
Then, a pool of 5 living Nonionella sp. T1 was transferred into a glass microtube (inner diameter 0.5 mm, height 7.5 mm) that was fixed inside a 20 ml test tube mounted in a glass-cooling bath (8 °C). A motorized micromanipulator was used to measure 165 O2 concentration profiles along a distance gradient that ranged from 200 µm of the foraminifera to 1200 µm using 100 µm steps. Seven O2 concentration profiles were generated with one incubation containing the pool of Nonionella sp. T1. Negative controls were done by measuring O2 rates from microtube with empty foraminifera shells and blanks with empty microtube.
Oxygen respiration rates were calculated with Fick's first law of diffusion, J = -D * dC/dx, where J is the flux, dC/dx is the concentration gradient obtained by profiles and D is the free diffusion coefficient of oxygen at 8 °C for a salinity of 34 (1.382 170 x 10 -5 cm -2 s -1 , Ramsing and Gundersen, 1994). The seven O2 respiration rates were calculated as the product of the flux by the cross section area of the microtube (0.196 mm 2 ). Then, the average O2 respiration rate was divided by the 5 Nonionella sp. T1 presented in the microtube to obtain the respiration rate per individual.
The same pool of Nonionella sp. T1 specimens as for the O2 respiration rates was used for denitrification rate 175 measurements. Denitrification rates were measured as it is described in Risgaard-Petersen et al., (2006). In this method, denitrification is stopped at the N2O production by acetylene inhibition that can be measured with a N2O microprobe (50 µm tip diameter, Unisense ®, Denmark). Thus, N2O was measured as the end product instead of N2 (Risgaard-Petersen et al., 2006). https://doi.org/10.5194/bg-2020-287 Preprint. Discussion started: 5 August 2020 c Author(s) 2020. CC BY 4.0 License.
Nitrous oxide flux was estimated from the chemical gradient profiled from the pool of Nonionella sp. T1 inserted in 180 a microchamber. The N2O production was multiplied by two because two moles of NO3are required for the production of one mole of N2O (Risgaard-Petersen et al., 2006). The microchamber is porous to gases and is bathed in a sodium ascorbate solution that maintains oxygen concentration at zero within the microchamber. The microchamber was filled with an oxygen/nitratefree solution of artificial seawater saturated with acetylene (to inhibit N2O transformation into N2) containing 5 mM of Hepes buffer (to maintain the pH stable). Calibration was performed using the standard addition method by successive injections of 185 a N2O saturated solution in order to have 14 µM steps of final concentration. Negative controls were done by checking the absence of O2 from microchamber with empty foraminifera shells and blanks with empty microchamber. Then, the pool of Nonionella sp. T1., was transferred to the microchamber with a micropipette. The N2O concentration profiles were repeated seven times on the pool of Nonionella sp. T1. The source of nitrate during denitrification comes from intracellular nitrate storage of Nonionella sp. T1 (not measured in this study). 190 Since O2 respiration and denitrification rates are linked to cytoplasmic volume or biovolume (BV) Glock et al., 2019), the specimens from the pool of Nonionella sp. T1 were measured (width (a) and length (b) Fig. 5) using a micrometer mounted on a Leica stereomicroscope (MZ 12.5) to estimate the average BV. The volume of the shells was estimated by using the best resembling geometric shape, a spheroid prolate (V = 4 3 π ( a 2 ) 2 ( b 2 )). Then, according to Hannah et al., (1994) 75 % of the measured entire volume of the shell was used corresponding to the estimated cytoplasmic volume. To 195 compare the size of the Nonionella sp. T1 sampled in the 1 st cruise (GF17, study of the fauna) with the Nonionella sp. T1 samples in the 2 nd cruise (GF18, denitrification rate measurements), 5 specimens sampled in the 1 st cruise were also measured.

Contributions of the invasive Nonionella sp. T1 to diffusive oxygen and nitrate uptake
The following estimated contributions to sediment diffusive oxygen and nitrate uptake were performed mainly on the 200 dominant denitrifying species, Nonionella sp. T1. The size of the Nonionella sp. T1 specimens sampled during the two cruises differed markedly (Table S1). Thus, we need to correct the denitrification rate of Nonionella sp. T1 specimens from the 1 st cruise to take into account the difference of shell size. Thus, the measured Nonionella sp. T1 denitrification rate (2 nd cruise) was normalized by specimen BV (1 st cruise) using the relationship: ln (y) = 0.68 ln (x) -5.57, where y is the denitrification https://doi.org/10.5194/bg-2020-287 Preprint. Discussion started: 5 August 2020 c Author(s) 2020. CC BY 4.0 License. rate (pmol ind -1 d -1 ) and x is the shell BV (µm 3 ) ( Glock et al., 2019;Equation S1). The corrected Nonionella 205 sp. T1 denitrification rate is multiplied by the Nonionella sp. T1 specimens counted found in each denitrifying zones defined by PROFILE modelling. Then, two calculation approaches were discussed to estimate Nonionella sp. T1 contributions to benthic denitrification: (A) to divide the Nonionella sp. T1 denitrification rate by the nitrate porewater denitrification rate estimated from PROFILE modelling, then the second calculation (B) to divide the Nonionella sp. T1 denitrification rate by the total denitrification from PROFILE plus the Nonionella sp. T1 denitrification rate. In the first approach (A) we suggest 210 Nonionella sp. T1 use only the nitrate in the sediment porewater. In the second approach (B) we suggest that the foraminifera only use both intracellular and porewater nitrate pool for denitrification.
interface to the OPD. Nitrate concentration decreased strongly after the OPD from 11.7 ± 3.4 to 2.8 ± 0.9 µmol L -1 until 4.0 cm depth. From 4.0 to 5.0 cm depth NO3concentration was very low with an average value of 2.7 ± 0.9 µmol L -1 (Fig. 6 c ,  230 d). The PROFILE parameters (Berg et al., 1998) used on laterally averaged nitrate porewater vertical distribution of both stations are available in Table S2. Thus, the PROFILE modelling of the averaged nitrate porewater profiles revealed one nitrification zone from 0 to 1.2 cm depth and two denitrifying zones (red line, Fig. 6 d). The first denitrification zone occurred between 1.2 to 3.6 cm depth with a nitrate consumption of 3.39 10 -07 µmol m -2 d -1 and the second smaller consumption zone was from 3.6 to 5 cm depth (1.32 10 -08 µmol m -2 d -1 ). The total denitrification rate from 1.2 to 5 cm depth was 3.52 10 -07 µmol 235 m -2 d -1 (Fig. 6 d).
The total densities of living foraminifera were similar between the cores GF17-3A and 3C (Ø 8.2 cm, 5 cm depth) with 1256 individuals and 1428 individuals, respectively (Fig. 6 a and b; Table S3, GF17-3A and 3C). Nonionella sp. T1 was the main denitrifying species, accounting for 34 % of the total living fauna in the core GF17-3A and 74 % in GF17-3C (Fig. 6 a, b; Table S4). One other candidate to denitrification, Stainforthia fusiformis, was found in the core GF17-3A and 3C in minority: 240 1 % of the total fauna in both cores (Fig. 6 a, b; Table S4,  and Leptohalysis scotti (11 and 9 %). 245 The density and the micro-distribution of Nonionella sp. T1 differed between the two cores ( Fig. 6 a and b; Table S3, GF17-3A and 3C). In the core GF17-3A and 3C respectively, Nonionella sp. T1 density showed large variability from the water-sediment interface to 1.2 cm depth (Table S3, GF17-3A and 3C) where Nonionella sp. T1 relative abundance accounted for 18 % and 50 % of the fauna in the nitrification zone (Table S4, GF17-3A and 3C). In the first denitrifying zone from 1.2 cm to 3.6 cm the Nonionella sp. T1 relative abundance represented 27 % and 78 % of the fauna. In the second denitrifying 250 zone, the Nonionella sp. T1 relative abundance increased from 3.6 to 5 cm depth and dominated the fauna by 60 % and 98%.

260
Due to severe hypoxia at the GF17-1 station, oxygen was assumed to be below detection limit within the sediment. No nitrite was detected at this station (< 1.7 µmol L -1 ). Average NO3concentration in the bottom water reached 5.7 ± 1.0 µmol L -1 (Fig. 6 g and h). Nitrate concentrations decreased from the sediment surface (4.2 ± 1.0 µmol L -1 ) to 1.6 cm (1.8 ± 0.6 µmol L -1 ) and then average nitrate concentration remained below the detection limit (1.7 µmol L -1 ). However, a patch with higher nitrate concentration was visible on the left part of the gel between 2.0 and 3.0 cm depth. A 1D vertical profile passing through 265 this patch (white line, Fig. 6 g) was extracted from the 2D image and the maximal nitrate concentration of the patch was above the detection limit with a value of 6.5 µmol L -1 at 2.3 cm depth (blue squares profile, Fig. 6 h). The PROFILE modelling (parameter details Table S2) of the laterally averaged nitrate vertical distribution revealed at the sampling time one denitrifying zone from the surface to 1.6 cm depth with a nitrate consumption of 2.34 10 -07 µmol m -2 d -1 (red line, Fig. 6 h). Below 1.6 cm depth, nitrate concentration was below the detection limit (hatched grey zone, Fig. 6 h), thus no PROFILE modelling was done 270 after this depth.

Towards a change in living foraminifera fauna of the Gullmar Fjord?
The presence and relative abundance of Nonionella sp. T1 in the Gullmar Fjord and in the Skagerrak-Kattegat strait has been documented during the last decades. The earliest SEM observations of specimens resembling Nonionella sp. T1 morphotype in the deepest part of the fjord date back to summer 1993 (identified as Nonionella turgida, Gustafsson and 295 Nordberg, 2001). The invasive characteristic of Nonionella stella was firstly demonstrated by Polovodova Asteman and Schönfeld, (2015). Then, Nonionella stella was named Nonionella sp. T1 morphotype also described as invasive by Deldicq et al. (2019). The estimated introduction date of the invasive species into the deepest part of the fjord is 1985 according to Polovodova Asteman and Schönfeld, (2015). The relative abundance of the invasive species in the deepest fjord station was less than 5 % between 1985 and 2007 (Polovodova Asteman and Schönfeld, 2015 and references within). At the GF17-1 300 hypoxic station, the Nonionella sp. T1 relative abundance was between 1-5 % (Table S4, GF17-3A and 3C). Thus, the Nonionella sp. T1 relative abundance in the deepest part of the fjord seems to remain stable. Whereas, at the GF17-3 oxic station, closest to the mouth of the fjord, the relative abundance of Nonionella sp. T1 varied between 34 and 74 % (Table S4,  (Nordberg et al., 2000). However, the fauna changed. Stainforthia fusiformis and Bolivina pseudopunctata became the major species (Nordberg et al., 2000;Filipsson and Nordberg, 2004). Further studies by Polovodova Asteman and Nordberg, (2013) (Table S4, GF17-1A and 1C), B. pseudopunctata reached only 2 % in the core GF17-1C (Table S4, GF17-1C) and T. earlandi was a minor species < 1 %. Then, in November 2017 Bulimina marginata, Cassidulina laevigata and Leptohalysis scotti were the dominant species in the fjord, ranging between 5-64 %, 5-16 % and 4-37 % of the total fauna. The Elphidium clavatum-selseyensis species complex (following the definition from Charrieau et al., 2018), Hyalinea baltica, Nonionellina 320 labradorica, and Textularia earlandi were present in low relative abundance (< 5 %, Table S4). Namely, Globobulimina turgida reached 37 % of the foraminifera fauna in August 2005 at the deepest station (Risgaard-Petersen et al., 2006); whereas in November 2017 this species was minor. The decreasing in relative abundance of Stainforthia fusiformis and Bolivina pseudopunctata must be interpreted with caution since our study used the > 100 µm fraction whereas some of the previous studies used > 63 µm. We also wet picked the specimens and used Cell Tracker Green to identify living foraminifera, which 325 might affect the results compared to Rose Bengal studies of dry sediment residuals. The relative abundance of the invasive Nonionella sp. T1 has increased since the study of Polovodova Asteman and Schönfeld, (2015) in the oxic part of the fjord.
Kattegat fauna (Filipsson and Nordberg, 2004) have again increased markedly in the fjord. It is evident that the foraminifera fauna in the Gullmar Fjord is presently very dynamic with considerable species composition shifts. 330

The invasive Nonionella sp. T1 ecology considering the nitrate micro-distribution at the oxic station
Our study showed, for the first time, Nonionella sp. T1 dominated the foraminifera fauna in the Gullmar Fjord, this at the GF17-3 oxic station despite some spatial variability (Fig. 6 a, b; Table S3; S4, GF17-3). Nonionella sp. T1 density increased with sediment depth below the oxic zone (Fig. 6 a -d; Table S3, GF17-3), which could be explained by its preference to 335 respire nitrate rather than oxygen. This would be following the hypothesis of using nitrate as a preferred electron acceptor suggested by Glock et al., (2019). Nonionella sp. T1 distributions could be explained by its capacity to store nitrate intracellularly before porewater nitrate was denitrified by other organisms such as bacteria. At this station, Nonionella sp. T1 distributions may be explained as: following the oxic zone (Fig. 6 c, d; from the surface to OPD) Nonionella sp. T1 respires oxygen (169 ± 11 pmol O2 indiv -1 d -1 ). Deeper in the hypoxic zone containing nitrate (Fig. 6 c, d; from OPD to 3.6 cm depth), 340 Nonionella sp. T1 accumulates intracellular nitrate and respires nitrate (38 ± 8 pmol N indiv -1 d -1 ). In the hypoxic zone where the nitrate porewater is depleted (Fig. 6 c, d; from 3.6 to 5 cm depth) Nonionella sp. T1 respires on its intracellular nitrate reserves to survive (Fig. 6 a, b; from 3.5 to 5 cm depth). When the intracellular nitrate reserve runs out, Nonionella sp. T1 can migrate to an upper zone where nitrate is still present in the sediment to regenerate its intracellular nitrate reserve (Fig. 6 a, b; from 1.2 to 3.5 cm depth). 345

The foraminifera ecology considering the nitrate micro-distribution at the hypoxic station
Hypoxia occurred approximately at least one month before the sampling cruise in the deepest part of the fjord (Fig. 3).
When hypoxia is extended to the water column, nitrification both in the water column and the sediments is reduced or even stopped, as oxygen is almost absent (Fig. 1 b; Childs et al., 2002;Kemp et al., 2005;Conley et al., 2007;Jäntti and Hietanen, 350 2012). Under this condition, the coupled nitrification-denitrification processes are strongly reduced (Kemp et al., 1990). At the GF17-1 station, no nitrification in superficial sediment was showed by our data and nitrate was low but still detectable in the https://doi.org/10.5194/bg-2020-287 Preprint. Discussion started: 5 August 2020 c Author(s) 2020. CC BY 4.0 License. bottom water. Nitrate can diffuse from the water column into the sediment, and thereby generate the denitrification zone as modelled by PROFILE between the surface and 1.6 cm depth (Fig. 6 h).
The rare presence of the invasive Nonionella sp. T1 and other denitrifying species as Globobulimina auriculata, Bolivina 355 pseudopunctata and Stainforthia fusiformis in the hypoxic station indicate that sediment chemical conditions turned unfavorable towards denitrification during prolonged hypoxia. Instead, the non-denitrifying species Bulimina marginata, Cassidulina laevigata, and Leptohalysis scotti dominated in this hypoxic environment. Their survival could be due to seasonal dormancy (Ross and Hallock, 2016;LeKieffre et al., 2017). The suspected deep nitrification zone (blue square profile, Fig. 6 h) could explain the presence of nitrate micro-niches deeper in the sediment and might explain the patchy distribution of 360 Nonionella sp. T1 also at the hypoxic site (see Fig. 6 e; Table S3, GF17-1A). Therefore, deep nitrate production in these microenvironments could favor the presence of Nonionella sp. T1, which can be attracted by this nitrate source as a electron acceptor to respire (Nomaki et al., 2015;Koho et al., 2011). This deep nitrification zone could be a result of an aerobic or anaerobic process. An aerobic nitrification zone in deep sediment can be formed by macrofaunal activity (burrowing activity) that introduce some oxygen deeper into anoxic sediment (Aller, 1982;Karlson et al., 2007;Nizzoli et al., 2007;Stief, 2013;Maire 365 et al., 2016). This nitrification zone could also be due to an anaerobic process. The Gullmar Fjord is Mn-rich (Goldberg et al., 2012) and metal-rich particles can be bio-transported into the anoxic sediment, thus allowing ammonium oxidation into NO3by Mn and Fe-oxides in the absence of oxygen deeper in the sediment (Aller, 1994;Luther et al., 1997).

Fjord
If we consider that Nonionella sp. T1 is denitrifying the nitrate from sediment porewater (approach A, Table 1; see method 2.5) its contribution to benthic denitrification in the oxic station would be 46 % in the core GF17-3A and would reach 100 % in the core GF17-3C. If we consider that Nonionella sp. T1 also uses its intracellular nitrate pool for denitrification (approach B), its contribution to benthic denitrification would be 32 % in the core GF17-3A and would reach 50 % in the core 375 GF17-3C (Table 1). These two calculation approaches highlight the difficulties and the importance of knowing the concentration of environmental nitrate and foraminifera intracellular nitrate at the same time to estimate at best the https://doi.org/10.5194/bg-2020-287 Preprint. Discussion started: 5 August 2020 c Author(s) 2020. CC BY 4.0 License.
contributions of foraminifera to benthic denitrification. Moreover, in this study there is no data on anammox process which contributes also in the total denitrification (Brandes et al., 2007). The results reported in previous studies as Engström et al., (2005) do not allow us to extrapolate their data at our oxic station, located at the entrance to the fjord. Thus, we assume that 380 our estimate of denitrification is conservative, since the possible contribution of anammox is not included in the calculation.
However, despite these uncertainties Nonionella sp. T1 contributions to benthic denitrification support the hypothesis that this invasive denitrifying foraminifer play a major role in the benthic nitrogen cycle for sediments showing nitrification processes.
At the hypoxic station, the opposite was shown where the estimated contribution of Nonionella sp. T1 to benthic denitrification was below 1 % whatever the calculation approach. The estimated contributions of the other denitrifying foraminifera found in 385 the hypoxic station were low. Stainforthia fusiformis did not exceed 5 %, Globobulima auriculata and Bolivina pseudopunctata were scarce and their contributions to benthic denitrification were negligible. Foraminifera contributed to almost 5 % of benthic denitrification in the hypoxic station. Compared to the oxic station, the invasive Nonionella sp. T1 and the other denitrifying species contributions to benthic denitrification were small in a prolonged hypoxic station of the Gullmar Fjord.
Overall, the Gullmar Fjord is well oxygenated except for the deepest basin where oxygen goes down when there is no deep water exchange (Fig. 3 c). Therefore, the GF17-3 oxic station could be considered more representative of the Gullmar Fjord benthic ecosystem. Nonionella sp. T1 is not the most efficient denitrifying species compared to Globobulimina turgida (42 pmol N ind -1 d -1 , with BV = 1.3 10 +06 µm 3 ) and also less efficient than Nonionella cf. stella from Perú. However, Nonionella sp. T1 high density could accelerate sediment denitrification and participate to increase the contrast between the two 395 hydrographic conditions. Indeed, an increase in contrast due to oxygenation conditions: oxic vs severe hypoxia induced a gap in the availability of nitrate for anaerobic facultative metabolisms in the sediment. In the oxygenated part of the fjord, high contribution to benthic denitrification (estimated between 50 and 100%) by Nonionella sp. T1 could contribute to the deeutrophication of the system by increasing the N2 loss. Thus, the high densities of denitrifying foraminifera as Nonionella sp. T1 would be rather beneficial. Whereas, in the hypoxic parts of the fjord, nitrate and nitrite rapidly exhausted become scarce, 400 resulting in a decrease in denitrification. The consequence is a decrease of denitrifying foraminifera fauna. The increase of ammonium in anoxic sediment resulting by a decrease in nitrification, denitrification and anammox processes does not allow https://doi.org/10.5194/bg-2020-287 Preprint. Discussion started: 5 August 2020 c Author(s) 2020. CC BY 4.0 License. the nitrogen elimination from the sediment to the water column, thus potentially promoting eutrophication of the fjord in parts subjected to prolonged severe hypoxia (Fig. 1). Moreover, the low availability of nitrate in the sediment would possibly increase the benthic transfer towards the water column of reduced compounds such as manganese and iron produced deeper 405 in the sedimentary column by other anaerobic metabolisms (Hulth et al., 1999). These new results demonstrate that the role of denitrifying foraminifera is underestimated in the nitrogen cycle and overlooking this part of the meiofauna may lead to a misunderstanding of environments subject to hydrologic changes.

Conclusion 410
This study revealed a drastic change in living foraminifera fauna due to several hypoxic events that occurred in the last decennium in the Gullmar Fjord. For the first time, the invasive Nonionella sp. T1 dominated up to 74 % the foraminifera fauna at a station with oxygenated bottom waters. This invasive species can denitrify up to 50-100 % of the nitrate porewater sediment under oxic conditions in the fjord. Whereas, under prolonged hypoxia, nitrate depletion turns environmental conditions unfavorable for foraminifera denitrification, resulting in a low density of Nonionella sp. T1 and other denitrifying 415 species. Thus, foraminifera contribution to benthic denitrification was negligible (~ 5 %) during prolonged seasonal hypoxia in the fjord. Moreover, the invasive denitrifying Nonionella sp. T1 could impact the nitrogen cycle under oxic conditions by increasing the sediment denitrification and could counterbalance potential eutrophication of the fjord. Thus, our study demonstrated that the role of denitrifying foraminifera is underestimated in the nitrogen cycle especially in oxic environments.      https://doi.org/10.5194/bg-2020-287 Preprint. Discussion started: 5 August 2020 c Author(s) 2020. CC BY 4.0 License. Table 1. Summary of the invasive Nonionella sp. T1 contributions to benthic denitrification in the Gullmar Fjord. The porewater denitrifications zones come from PROFILE modelling (Fig. 6 d, h). To estimate the contributions of Nonionella sp. T1 the counted specimens per zones was used. Two different approaches were used to estimate the contribution of Nonionella to hyperspectral camera treatments and scientific discussions and manuscript rewriting.