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Seasonal microbial food web dynamics in contrasting Southern Ocean productivity regimes

Spatial and seasonal dynamics of microbial loop fluxes were investigated in contrasting productivity regimes in the Indian sector of the Southern Ocean. Observations carried out in late summer (February–March 2018; project MOBYDICK) revealed higher microbial biomasses and fluxes in the naturally iron‐fertilized surface waters of Kerguelen island in comparison to surrounding off‐plateau waters. Differences were most pronounced for bacterial heterotrophic production (2.3‐fold), the abundance of heterotrophic nanoflagellates (HNF; 2.7‐fold). Independent of site, grazing by HNF was the main loss process of bacterial production (80–100%), while virus‐induced mortality was low (< 9%). Combining these results with observations from previous investigations during early spring and summer allowed us to describe seasonal patterns in microbial food web fluxes and to compare these to carbon export in the iron‐fertilized and high‐nutrient, low‐chlorophyll (HNLC) Southern Ocean. Our data suggest an overall less efficient microbial food web during spring and summer, when respiration and viral lysis, respectively, represent important loss terms of bacterially‐mediated carbon. In late summer, primary production is more efficiently transferred to bacterial biomass and HNF and thus available for higher trophic levels. These results provide a new insight into the seasonal variability and the quantitative importance of microbial food web processes for the fate of primary production in the Southern Ocean.

around the Kerguelen and Crozet islands provide an excellent environment to investigate the spatial and temporal heterogeneity of planktonic C-fluxes in the Southern Ocean (Blain et al. 2007;Pollard et al. 2007Pollard et al. , 2009). The extensive phytoplankton blooms occurring in iron-fertilized waters, mainly characterized by diatom communities (e.g., Poulton et al. 2007;Lasbleiz et al. 2016), have higher primary production rates Cavagna et al. 2015) and higher carbon export in contrast to the surrounding HNLC waters (Savoye et al. 2008;Planchon et al. 2015). The Kerguelen bloom, -despite being a major carbon sink with high CO 2 sequestration per unit of iron (Blain et al. 2007), follows the "High Biomass Low Export" paradigm (Lam and Bishop 2007) of the Southern Ocean (Mosseri et al. 2008;Savoye et al. 2008;Cavagna et al. 2015;Planchon et al. 2015).
Microbial food web processes determine to a large extent what fraction of primary production is remineralized or available for higher trophic levels or export. The fraction of primary production consumed by heterotrophic bacteria is highly variable in polar oceans (Kirchman et al. 2009), likely resulting from variable degrees of phytoplankton-bacteria coupling (Morán et al. 2006). In Kerguelen plateau waters, heterotrophic bacteria respond rapidly to the phytoplankton bloom and process a considerable fraction of the primary production Christaki et al. 2014). Protists channel prokaryotic carbon to higher trophic levels through bacterivory while viruses return dissolved and small particulate prokaryotic carbon forms to the water column via the viral shunt. Theoretically, grazing should therefore enhance carbon export while viral lysis would enhance respiration and reduce prokaryote growth efficiency. Viral activity in the Southern Ocean could be crucial for iron regeneration, especially in HNLC areas (Strzepek et al. 2005;Evans and Brussaard 2012). This said, integrative studies of microbial stocks (viruses, heterotrophic bacteria, heterotrophic and phototrophic flagellates) and fluxes (bacterial production, respiration, grazing and viral lysis) along with community production and C-export measurements are necessary to describe the role of bacteria in C-flux variability.
The present study was undertaken in the framework of the MOBYDICK project (Marine Ecosystem Biodiversity and Dynamics of Carbon around Kerguelen: an integrated view). MOBYDICK's objective was to trace C from its biological fixation, cycling within and across trophic levels, and its export to depth, under different productivity regimes of the Southern Ocean in late summer. Our aim here was to compare C-fluxes mediated by microbes at the surface to carbon export to deeper layers and carbon availability for higher trophic levels at the surface. Seasonal dynamics of bacterial carbon processing in iron-fertilized and HNLC waters and its implications for ecosystem functioning is described during three periods, separated by 7 to 13 yr: early spring (onset of the bloom, October-November 2011, KEOPS2 cruise), summer (decline of the bloom, January-

Sample collection
Samples were collected at four stations (M1, M2, M3, and M4) in the region of Kerguelen island in the Indian sector of the Southern Ocean during late austral summer (19 February to 20 March 2018; MOBYDICK cruise) (Fig. 1). The timing of the visits corresponded to about one month after the seasonal maximum in chlorophyll a (Chl a) (Figs. S1, S2). Sta. M2 was located above the Kerguelen plateau and is the "historical" A3 station of the previous KEOPS1 and KEOS2 cruises (Figs. 1, S1). This site is located in naturally iron-fertilized waters and characterized by high primary production during spring and summer Mosseri et al. 2008;Cavagna et al. 2015). Sta. M1, M3, and M4 were located off the plateau characterized by lower seasonal primary production (Fig. S1). Sta. M2 was sampled three times with an eight day interval between visits (M2-1, M2-2, M2-3; Table 1). Sta. M3 (M3-1, M3-3) and M4 (M4-1, M4-2) were both sampled twice with a 2-week interval each, and Sta. M1 was visited once (Table 1). All water samples were collected with 12L Niskin bottles mounted on a rosette equipped with CTD (SeaBird 911-plus). Microbial parameters were sampled at selected depths throughout the water column (Table 2).

Viral and microbial abundance
Pico-and nanophytoplankton, virus-like particles, heterotrophic bacteria (sensus-stricto includes Archaea and Bacteria), and heterotrophic nanoflagellates (HNF) were enumerated  Pauthenet et al. (2018). The polar front is the dark, thick, blue line, gray zone around the polar front indicates variations in its trajectory.
from surface to 450-1000 m. Samples for virus-like particles and heterotrophic bacteria (2.5 mL) were fixed with 1% v/v glutaraldehyde. Samples for HNF were fixed with 1% v/v paraformaldehyde prepared on board. Samples were stored at 4 C for 40 min, flash frozen in liquid nitrogen and then kept at −80 C until analysis. Counts for heterotrophic bacteria were made after staining with SYBRGreen I by flow cytometric analysis on a BD FACS Canto. Two heterotrophic bacteria populations were discriminated, one with high fluorescence called "high nucleic acid" and one with low fluorescence called "low nucleic acid". Pico-nanophytoplankton and cyanobacteria of the genus Synechococcus were discriminated based on their side scatter and red fluorescence. HNF and virus-like particles were enumerated after staining with SYBRGreen I on a Cytoflex Beckman-Coulter flow cytometer, following Christaki et al. (2011) andBrussaard (2004), respectively. As for bacteria, two populations of virus-like particles could be distinguished based on their fluorescence intensity. Ciliates and dinoflagellates were counted in acid Lugol's (2% v/v) fixed samples (500 mL) under an inverted microscope (Nikon Eclipse TE2000-S ×400).

Bacterial production
Heterotrophic bacterial production was measured from surface to 500 m with the 3 H-leucine incorporation method. At each depth 20 mL triplicate samples and a formalin killed control (2% v/v) were spiked with a mixture of nonradioactive (19 nM) and L-[4.5-3 H] leucine (Perkin Elmer,120 Ci/mmol). The concentration of radioactive leucine was 1 nM in the mixed layer and 2 nM below the mixed layer. Concentration kinetics were checked at 25 and 250 m with 4, 6, 12, 20, and 40 nM of leucine, to ensure that there was no isotopic dilution. The incubation times were 2 h in the 0-150 m layer, 4 h in the 150-200 m layer, and 8 h in the 250-500 m layer. Time kinetics were tested for 10, 125, 250, and 500 m (0, 3, 6, 8, 10 h) and were found linear for the applied incubation times. Samples were incubated in the dark at in situ temperature. Incubations were terminated with formalin (2% v/v). Samples were filtered onto 0.22 μm Nuclepore filters, after filtration ice-cold 5% trichloroacetic acid (10 mL) was added on the filters for 10 min. The filters were rinsed 3 times with 4 mL ice-cold 5% trichloroacetic acid, placed in scintillation vials where 8 mL of scintillation cocktail (Ultima Gold-Packard) was added and radioassayed on board. Incorporation rates of leucine into carbon were calculated using a theoretical conversion factor of 1.55 kg of C mol −1 (Kirchman 1993).
Dark community and bacterial respiration, net community production, and gross community production Rates of dark community respiration, bacterial respiration and net community production were determined from changes in the concentration of dissolved oxygen (O 2 ) during 24 h light and dark incubations as described previously ). For dark community respiration and net  community production, seawater was collected at four depths corresponding to 1%, 4%, 25%, and 50% of photosynthetically active radiation levels of surface values. Unfiltered seawater was transferred to 125 mL borosilicate glass bottles and incubated in an on-deck incubator at the respective photosynthetically active radiation levels using optical density filters (Nickel screens). The outdoor incubators were connected with a running seawater system to maintain the incubation bottles at the temperature of the mixed layer zone. The 1% light depth (euphotic depth) was at the bottom of the mixed layer at Sta. M2, but the euphotic depth was well below the mixed layer at the off-plateau sites (Table 1). Gross community production was calculated as the sum of dark community respiration and net community production. For bacterial respiration, seawater from two depths corresponding to the 1% and 50% surface photosynthetically active radiation levels were filtered through 0.8 μm polycarbonate filters (Nuclepore) and the subsequent filtrate was incubated in 125 mL borosilicate bottles in the dark at in situ temperature for 1-3 days . All incubations were done in five replicates. Dissolved oxygen (O 2 ) concentration was determined by spectrophotometric detection of iodine following the Winkler reaction, using a Hitachi U-3010 Spectrophotometer equipped with a sipper system.

Grazing induced bacterial mortality
Bacterial mortality due to protists was evaluated in the mixed layer (30 m) using the principle of fluorescently labeled bacteria (FLB) as prey analogues (Sherr et al. 1987). The FLB were prepared with a culture of Brevundimonas diminuta (CECT 313) following the protocol of Sherr et al. (1987). Triplicate glass Schott bottles of 2 L were filled with 60 μm screened water directly from the Niskin bottle and one control with 0.2 μm filtered sea water were inoculated with FLB at a final concentration of approx. 0.7 × 10 5 mL −1 in a refrigerated room (4 C). The expected FLB to bacteria ratio-based on bacterial abundances during previous cruises was expected to be around 20%. Because of increased bacteria abundances during MOBYDICK, this ratio was of 6-14%. After addition of labeled preys in all bottles, samples for FLB, heterotrophic bacteria and HNF counts were immediately withdrawn for T 0 counts. Incubations were performed in an on-deck incubator with surface seawater circulation, covered with a screen providing 50% light attenuation. Grazing on bacteria was measured by counting both the number of ingested FLB in food vacuoles by microscopy and FLB disappearing by flow cytometry. The food vacuole observation approach allows visual observation of the proportion and the morphology of protists that ingested FLB. Both approaches are based on several assumptions which will be further discussed in the paper.

Food vacuole analysis
After 4 h of incubation, 45 mL samples were retrieved from the bottles, fixed with glutaraldehyde (1% final conc.) and filtered on 0.8 μm Nuclepore filters before being stained with DAPI (4 0 ,6-diamidino-2-phenylindole). They were then kept frozen at −80 C until they were observed with a Zeiss AX10 epifluorescence microscope. Prokaryotes were identified by their blue fluorescence when excited with UV radiation, while DTAF (4,6-dichlorotriazinyl-aminofluorescein) stained FLB were identified by their yellow-green fluorescence when excited with blue light. The ingestion rate (IR: FLB HNF −1 h −1 ) was calculated according to Dolan and Šimek (1998) as: where VC is vacuole content at steady state, and k is a conservative digestion rate of 0.92% cell content min −1 , which corresponds to a t 1/2 digestion time of 75 min. For comparison, a higher k of 1.1% was also calculated (t 1/2 digestion time of 63 min).
Depth ( described above. Triplicate counts by flow cytometry were performed for each sample. Bacterial grazing rates were obtained by measuring the rate of disappearance of FLB after subtracting the FLB disappearance in the "control." The abundance of FLB was plotted vs. time, and the calculation of grazing rates (g, h −1 ) of FLB corresponds to the slope of the abundance of FLB as a function of time. The decrease of FLB abundance was linear for the first 40-48 h, at the exception of M2-3 where it was linear only for the first 24 h. The "control" bottles showed no decrease of FLB during the incubations. In the experimental bottles, the abundances of bacteria and nanophytoplankton did not show significant increases or decreases for the first 40 h. The impact of protists on bacteria was calculated as the % of the bacterial production consumed, assuming that FLB were grazed at the same rates as natural bacteria (Sherr et al. 1987). For comparison with FLB disappearance and vacuole content analysis, the potential consumption of bacteria by HNF and ciliates was also estimated assuming a clearance rate of 10 5 cell volume h −1 (Fenchel 1982). For all approaches, the % of bacterial production consumed was calculated from the number of bacterial cells produced as determined by leucine incorporation (see above).

Virus-induced bacterial mortality
Triplicate 4.5 mL samples, collected at each depth (Table 2), were fixed and stored as for flow cytometry (see above).
Upon analysis, they were pooled and ultracentrifugated in order to harvest the bacterial cells for transmission electron microscope observations (detailed in Christaki et al. 2014). Virus induced bacterial mortality (VIBM) was calculated as (Eq. 2, Binder 1999;Eq. 3, Weinbauer et al. 2002): where FIC is the frequency of infected cells, and FVIC is the frequency of visibly infected cells (i.e., cells that contain five or more phages).

Statistical analyses
Principal component analysis (PCA) and hierarchical clustering on principle components was performed on environmental variables with R-software, version 3.5.1 using the package FactoMineR (Lê et al. 2008). Before these analyses, environmental variables were standardized to zero mean using function "decostand" in the package vegan (Oksanen 2017).

Environmental context
The hydrological context of the stations was characterized by the location of the polar front during the MOBYDICK cruise. Sta. M1, situated in Antarctic waters, was influenced by the highly dynamic polar front (Pauthenet et al. 2018 Fig. 2). Sta. M3 was situated in the sub-Antarctic zone outside the influence of the polar front (mixed layer depth , 65-79 m). Temperature in the mixed layer was lowest at Sta. M4 (4.5 C) and highest at Sta. M3 (5.6 C, Table 1). The mixed layer deepened at all stations after a storm that occurred on the 10 th of March 2018. The greatest increase in the mixed layer depth induced by the storm was observed at Sta. M4 (77%, from 49 to 87 m) while at stations M2 and M3 the increase of the mixed layer depth was moderate (10% and 20%, respectively, Table 1).
Average Chl a concentrations in the mixed layer ranged from 0.14 (M3-3) to 0.58 μg L −1 (Sta. M2-3). Chl a in the mixed layer doubled from the first to the third visit at Sta. M2 3 weeks later (from 0.27 to 0.58 μg L −1 Table 1, Fig. 2). Phosphate and nitrate were abundant in the mixed layer at all stations, while the highest ammonium concentrations were observed on the plateau at Sta. M2 (0.75-1.12 μM). Silicic acid concentration was overall higher off the plateau (2.3-8.4 μM) than in surface waters on the Kerguelen plateau (1.4-2.8 μM, Table 1). The first two components of the principal component analysis (PCA) of environmental parameters in the mixed layer explained 76% of the variability of the data (Fig. S3). Phosphate, nitrate and ammonium were the most important variables for the principal component axis formation. Sta. M2 was associated with higher ammonium concentrations while Sta. M4 presented the highest nitrate, phosphate and silicic acid concentrations, and Sta. M1 was associated to high dissolved oxygen concentrations (Fig. S3).

Stocks and fluxes of microbial components
During the late Austral summer, abundances of virus-like particles, heterotrophic bacteria, bacterial production, Table 3. Mean AE SD values of all visits at the M2 plateau station and the off-plateau stations (M1, M3, and M4). heterotrophic bacteria (HB), % of high and low DNA bacteria as defined by flow cytometry (%"high nucleic acid", %"low nucleic acid"), virus like particles (VLP), % of low and high green fluorescence VLP (VLP1 VLP2), heterotrophic nanoflagellates (HNF), pico-and nanophytoplankton (PNP) and Synechococcus spp. at on-plateau (M2) and the off-plateau stations (M1, M3, M4).   heterotrophic nanoflagellates (HNF), and phototrophic picoand nanophytoplankton were higher on the plateau (Sta. M2) than at the off-plateau stations (M1, M3, and M4, Fig. 3a-e). These differences were more pronounced in the mixed layer and were attenuated at depth. The proportion of "high nucleic acid" bacteria was slightly higher on the plateau (56%) than off the plateau (44%, Table 3). The proportion of the two virus-like particles populations identified based on their fluorescence was similar at all sites, with low fluorescence viruslike particles dominating throughout the water column (84-90%) ( Table 3). Synechococcus was found at low abundances of a magnitude of 10 4 cells L −1 (Table 3). Bacterial production, averaged over the three visits, was about 2-fold higher in the mixed layer at Sta. M2 than the off-plateau stations (Table 3). Bacterial respiration in the mixed layer on the plateau (mean 0.45 AE 0.14 μmol O 2 L −1 d −1 ) was not different to that determined off the plateau (mean 0.38 AE 0.26 μmol O 2 L −1 d −1 ), with the exception of one high value recorded during the first visit of Sta. M2 (Table 4). As a consequence, bacterial growth efficiency was substantially higher on the plateau during the 2 nd and 3 rd visit (mean 20 AE 6%) than off the plateau at Sta. M3 and M4 (mean 7 AE 3%). Bacterial growth rates were also higher during the 2 nd and 3 rd visit of Sta. M2 (0.12 AE 0.03 d −1 ) as compared to the off-plateau stations (0.05 AE 0.00 d −1 ) ( Table 4).

Short-term temporal variability
The higher microbial stocks and fluxes measured on the plateau at Sta. M2 were also accompanied by a more pronounced variability between the three visits as compared to off-plateau waters. Concentrations of Chl a and the abundance of pico-and nanophytoplankton in the mixed layer increased between the first to the third visit by factors of 2.0 and 2.6, respectively. A similar pattern was observed for bacterial production (3-fold increase, Fig. 3e), while the abundance of heterotrophic bacteria decreased by 1.9-fold during this production loss due to grazing (b) and proportion (%) of bacterial production loss due to viral lysis (c). FLB: based on FLB disappearance, error bars for FLB indicate standard deviation (SD) between replicates. The estimation based on vacuole content (VC) analysis was calculated for digestion times k = 1.1 and k = 0.9. The estimation based on cell volume (CV) was calculated assuming a clearance rate of 10 5 cell volume h −1 of grazers (for more details see M&M section). Error bars indicate SD between replicates for grazing and between different depths in the mixed layer for viral lysis. (Fig. 3b). The inverse temporal pattern in bacterial production and heterotrophic bacteria abundance resulted in a 3.7 and 2-fold increase in the bacterial growth rate (μ) and cell-specific bacterial production, respectively (Table 4). Concomitant with the decrease in heterotrophic bacteria abundance, bacterial grazers (HNF) and virus-like particles increased by 2-and 1.5-fold between the first and the second visit of Sta. M2 (Fig. 3a-c). All three bacterivory estimation approaches used in this study revealed higher grazing during the first visit (> 100%) and grazing decreased to 50-75% of the bacterial production during the third visit (Fig. 4b). Off the Kerguelen plateau, the short-term variability was much less pronounced. Noticeable decreases between consecutive visits were observed for the abundance of heterotrophic bacteria and pico-and nanophytoplankton at Sta. M3.

3-week time period
Net community production, dark community respiration, and gross community production Above the Kerguelen plateau, at Sta. M2, net community production fluxes integrated over the euphotic zone varied between 30 and 44 mmol O 2 m −2 d −1 during the 1 st and 3 rd visit. Net community production was substantially higher during the 2 nd visit (100 mmol O 2 m −2 d −1 ) (Fig. 5), which could explain the observed increase in the Chl a concentrations about 1 week later (Table 1). Off-plateau, highest net community production fluxes were determined at Sta. M4 (88-106 mmol O 2 m −2 d −1 ). Euphotic zone integrated fluxes of dark community respiration varied between 75 and 113 mmol O 2 m −2 d −1 during the first two visits at Sta. M2 and dark community respiration decreased to 39 mmol O 2 m −2 d −1 during the 3 rd visit. Fluxes of dark community respiration off-plateau ranged between 69 and 117 mmol O 2 m −2 d −1 , with the exception of the last visit at Sta. M4 (23 mmol O 2 m −2 d −1 ) (Fig. 5), which corresponded to a deepening of the mixed layer following the storm event. Highest depth-integrated fluxes of gross community production were recorded during the 2 nd visit at Sta. M2 (215 mmol O 2 m −2 d −1 ) while gross community production ranged between 121 and 187 mmol O 2 m −2 d −1 off-plateau (Fig. 5). Comparison of the volumetric rates of oxygen consumption in unfiltered and < 0.8 μm filtered seawater revealed that above the Kerguelen plateau bacterial respiration accounted for 41 AE 2% and 87 AE 24% of community respiration at 10 and 60 m, respectively. Off-plateau, bacterial respiration accounted for 100% of community respiration during both visits.

Late summer microbial food web
We observed that the microbial standing stocks and fluxes, apart from microbial respiration, were higher in the mixed layer of the iron-fertilized area above the Kerguelen plateau as compared to surrounding HNLC waters, even one month after the decline of the phytoplankton bloom. Our results thereby complement previous observations during the onset and the decline of the Kerguelen bloom (e.g., Table 4 in Christaki et al. 2014). A comparison of the different phases of the bloom reveals, however, distinct features at the end of summer. While the enhancement of Chl a and bacterial production on-vs. off-plateau ratios exceeded by 2-4-fold that of the other parameters in early spring and summer, their on-vs. offplateau ratios were in a similar range during the post bloom period (Table S1, Fig. 6). Pico-and nanophytoplankton were characterized by marked increases in their abundance (about 15-fold) and contribution to total Chl a (10-53%) between the onset and the post bloom periods in Kerguelen plateau waters (Table S1). In addition, the ratio of pico-and nanophytoplankton in fertilized vs. HNLC waters was < 1 during the onset and decline of the bloom and > 1 after the bloom (Fig. 6). This observation is in line with previous studies in the Kerguelen area indicating that pico-and nanophytoplankton dominate communities on the plateau before ) and after the bloom (Uitz . Furthermore, estimates of mean annual primary production reveal that pico-and nanophytoplankton (2.5 Gt C yr −1 ) have a higher contribution as compared to microphytoplankton (0.9 Gt C yr −1 , Uitz et al. (2010), underlying their importance in Southern Ocean waters.
Heterotrophic bacteria reached 1.0 × 10 9 cells L −1 during the post bloom period, and their biomass was about 2 to 3-fold higher as compared to those recorded at this station during the onset and decline of the bloom (Table S1). The most important heterotrophic bacterial mortality factors HNF and virus-like particles revealed opposite seasonal trends. While HNF had higher on-/off-plateau ratios during early spring and late summer, virus-like particles revealed highest on-/off-plateau differences in summer (Fig. 6). These observations are coherent with estimates of grazing and viral lysis of heterotrophic bacteria. Grazing was identified as the dominant loss process of heterotrophic bacteria during late summer, and a similar scenario has been observed in early spring ). The intense bacterial grazing seemed to co-occur with the NH 4 increase between the first and the second visit at Sta. M2 (Table 1, Figs. 4, S3). The methods assessing grazing are based on several assumptions. In the present study, we used three experimental and theoretical approaches to better frame the lower and upper limits of bacterial mortality attributed to grazing. The grazing estimations based on volume specific clearance rates (Fenchel 1982) or digestion times (Dolan and Šimek 1998) were determined in cultures, and even though they are not a direct measure of grazing, they provide representative patterns of the potential of HNF to ingest bacteria. At Sta. M2-1, the obtained values from the different approaches showed the highest variability, however, all values had a consistent pattern in that grazing exceeded bacterial production (Sta. M2-1, CV 26%, Fig. 4). The mean ingestion rates obtained by all methods (1-3 bact. grazer −1 h −1 ) varied within a narrow range, being consistent at all stations and similar to previous observations (e.g., Christaki et al. 2008Christaki et al. , 2014Gast et al. 2018). This mean ingestion rate corresponds to the ingesting capacity of HNF since they were one order of magnitude more abundant than ciliates and Gymnodinium spp. despite the higher number of cells they ingest. Few studies have considered grazing by protists and viral mortality simultaneously, particularly in the Southern Ocean (Guixa-Boixereu et al. 2002;Malits et al. 2014;Vaqué et al. 2017). Prey consumption by grazers is expected to be more important than viral activity during early stages of phytoplankton blooms, which could explain the delay between bacterial production and the increase in abundances (Brum et al. 2016). Confirming this hypothesis, in our study, predation was the dominant cause of bacterial mortality during the onset and after the bloom, while viral lysis dominated during the decline of the bloom. During summer (decline of the bloom), virus induced mortality accounted on average 72 AE 72 and 27 AE 19% of bacterial production inside and outside the bloom, respectively. Lysogenic infected bacteria could be detected only in 7 out of 15 essays and ranged from 0 to 31% of infected bacteria on the plateau and from 0 to 4% in HNLC waters, without any significant difference between the two areas (Malits et al. 2014). In the Southern Ocean, a number of studies taking into account viral infection have found consistent high viral lytic production (e.g., Guixa-Boixereu et al. 2002;Evans and Brussaard 2012;Vaqué et al. 2017 and references therein) while lysogeny was varying greatly (e.g., Evans and Brussaard 2012;Vaqué et al. 2017).
Mixotrophy of unicellular eukaryotes is widespread (e.g., Zubkov and Tarran 2008;Stoecker et al. 2017), including in the Southern Ocean (Moorthi et al. 2009;Gast et al. 2018), but the possible triggering factors such as light or nutrient limitation remain largely unresolved. Even though pico-and nanophytoplankton were abundant at all stations during MOBYDICK, we observed insignificant uptake of FLB by nanophytoplankton. It was therefore impossible to evaluate accurately mixotrophic activity in the present study. In oligotrophic systems, pico-and nanophytoplankton can account for up to 95% of bacterivory (Zubkov and Tarran 2008). In this case, phagocytosis might be advantageous to photosynthetic organisms as it provides an increased access to limiting nutrients. This could suggest that the nutrient and light conditions were optimal for pico-and nanophytoplankton growth during late summer in the Kerguelen region, and phototrophic cells did not need to complement their diet by ingesting bacteria. However, in a study in the eastern Mediterranean (Christaki et al. 1999) it was found that 12% of nanophytoplankton ingested small spherical FLB called "minicells" (E. coli X-1488, 0.65 μm in diameter), while only 6% ingested larger elongated FLB (approx. 0.8 μm ESD: equivalent spherical diameter) as the ones used here (Fig. S4). Thus, selection against the FLB is also a possibility and it most likely depends on the prevailing taxonomic groups. During MOBYDICK, nanophytoplankton were dominated at all stations by the prymnesiophyte Phaeocystis antarctica (> 70% based on pigment and microscopic data; Irion et al. 2020), which is known to be widespread in the Southern Ocean Gast et al. 2018). Contrary to many prymnesiophytes, mixotrophic activity has not been reported so far for P. antarctica (Gast et al. 2018). We therefore suggest one of the following possibilities, or a combination of them, to explain our results on mixotrophy: (i) a selection against the FLB used in this study; (ii) environmental conditions leading to low bacterivory by phototrophs; and (iii) the dominance of non-mixotrophic cells such as P. antarctica.

Seasonal microbial dynamics and ecosystem functioning
Combining the observations made during late summer (MOBYDICK project) with those from previous projects carried out in early spring (KEOPS2) and summer (KEOPS1) has allowed us to draw a seasonal scenario for microbial food web fluxes in iron-fertilized and HNLC waters. In Kerguelen plateau waters, the three observation periods corresponded to the onset, decline, and post phytoplankton bloom phase (Fig. 7, Table 5). In early spring, the system is highly productive, with low carbon export and carbon export efficiency, resulting in a large amount of phytoplankton carbon potentially available for higher trophic levels. During the decline of the bloom, gross community production decreases by 3-fold as compared to early spring. The system is characterized by high carbon export and carbon export efficiency while phytoplankton carbon potentially available for higher trophic levels is 10-fold Arrows in gray indicate rates of bacterial respiration (BR) and dark community respiration (DCR). Bacterial carbon demand (BCD) defined as BCD = bacterial production (BP) + bacterial respiration (BR), dissolved inorganic carbon (DIC), gross community production (GCP), biomass available for higher trophic levels (HTL), defined as HTL = GCP-(DCR+BCD+export). MOBYDICK export data were calculated based on the 234 Th approach (Planchon pers. comm., the method is described in detail in Planchon et al. 2015). Late bloom (KEOPS1) data from Carlotti et al. (2008), Christaki et al. (2008), Lefèvre et al. (2008, Savoye et al. (2008) and Malits et al. (2014). Early bloom data (KEOPS 2) from Planchon et al. (2015) and Carlotti et al. (2015), Christaki et al. (2014).
lower as compared to spring. During the post-bloom phase, gross community production is similar to that determined during the declining bloom phase. However, carbon export and export efficiency are again low, resulting in biomass available for higher trophic levels roughly 2-fold higher as compared to the bloom decline (Fig. 7, Table 5).
Heterotrophic bacteria contribute substantially to carbon processing during all stages. The fraction of total biomass available for higher trophic levels accounted for by bacteria is minor during early spring (< 1%) and it increases up to 14% during the declining and post-bloom phases (Fig. 7, Table 5). In addition, bacteria have a substantial contribution to microbial community respiration (42% and 85% of dark community respiration). The role of the microbial food web for carbon processing varies during the three stages (Table 5). In early spring, a moderate fraction of primary production is channeled through bacteria (21%) and due to a low bacterial growth efficiency (9%) this organic matter is mainly respired. During the decline of the bloom, heterotrophic bacteria process a higher fraction of primary production (44%) with a more efficient bacterial biomass production as compared to spring (bacterial growth efficiency of 18%, Table S1). The importance of viral lysis in carbon losses results in an inefficient transfer of bacterial biomass to HNF. During the post bloom phase, organic carbon is processed with a bacterial growth efficiency (17%) similar to the declining bloom phase, and the transfer to HNF dominates over the loss by viral lysis. Taken together, these observations suggest an overall less efficient microbial food web during early spring and summer with respiration and viral lysis, respectively, representing important loss terms. During the post bloom period, roughly 30% of the gross community production are efficiently transferred to bacterial biomass and HNF, suggesting a good functioning of the microbial loop.
In HNLC waters, a seasonal pattern similar to that in ironfertilized waters can be depicted, though with fluxes that are less variable among periods (Fig. 7, Table 5). In contrast to iron-fertilized waters, most of the gross community production is remineralized, leading to lower export and biomass available for higher trophic levels. Heterotrophic bacteria dominate microbial community respiration in early spring and summer, and their contribution decreases to 18% at the end of the season. Similarly, bacteria process large fractions of primary production in early spring (45%) and in summer (100%), and this fraction substantially decreases in late summer (17%). Despite the low bacterial growth efficiency (3-8%), the carbon processed by bacteria supports the microbial food web as grazing by HNF dominates over viral lysis. These Table 5. Seasonal ecosystem characteristics and microbial food web dynamics above the Fe-fertilized Kerguelen plateau and in HNLC waters. Productivity regime refers to gross community production (GCP), export efficiency is the ratio of carbon exported below 200 m to GCP, bacterial growth efficiency (BGE), heterotrophic nanoflagellates (HNF). Early spring and summer BGE and microbial food web fluxes from Lefèvre et al. 2008;Obernosterer et al. 2008Malits et al. 2014 (see also Fig. 7). observations illustrate the quantitative importance of microbial food web processes across seasons and productivity regimes in the Southern Ocean. What are the potential consequences of climate-driven environmental changes on the role of microbes in food webs of cold-water environments? The Southern Ocean is a major storage reservoir of heat, thus the warming of surface waters is expected to affect microbial communities (Morán et al. 2010;Kim and Ducklow 2016). Temperature and the supply of bioavailable dissolved organic matter were identified as key factors for bacterial biomass and growth in polar waters (Kirchman et al. 2009). Warming is expected to increase total and dissolved phytoplankton primary production (Morán et al. 2006;Boyd et al. 2016;Deppeler and Davidson 2017), which could result in a stronger coupling to bacteria through the utilization of dissolved organic matter (Vaqué et al. 2019). These observations suggest an increasingly important role of heterotrophic bacteria. In this context, our data provide two interesting observations. Firstly, the high contribution of bacteria to community respiration, and secondly the amount of carbon transferred in the form of bacterial biomass to HNF, which is in the same range as the carbon export (2--7 mmol C m −2 d −1 ) in early spring and late summer. Thus, the predicted changes could lead to an increase in the amount of phytoplankton-derived organic matter consumed by bacteria. Temperature effects on the bacterial growth efficiency are uncertain (López-Urrutia and Morán 2007), leaving the question on the quantitative importance of organic carbon remineralization open (Sarmento et al. 2010). The increased flow of carbon to bacterial biomass and HNF could support higher trophic levels, but in turn lead to a weakening of the Southern Ocean capacity to sequester organic carbon.