The major scientific objective that was addressed by MOBYDICK is the understanding of the oceanic biological carbon pump by (i) considering end-to-end marine food webs, and (ii) linking biodiversity to biogeochemical fluxes. To address these objectives we have investigated contrasted types of ecosystems in the Southern Ocean (SO): a `Low Biomass Low Export' and a `High Biomass Low Export' ecosystem in the region of Kerguelen Island (Fig. 1). The higher biomass east of Kerguelen Island is due to natural iron fertilization in otherwise High Nutrient Low Chlorophyll (HNLC) waters. The in situ observations and analyses of samples collected during the cruise provided an extensive description of community structures at the different trophic levels in contrasted marine food webs. Compared to previous studies, the originality of MOBYDICK is the concurrent investigation of pelagic biodiversity at all trophic levels and of almost every functional group from bacteria (Bacteria and Archaea) to top predators, together with data acquisition of environmental drivers including dissolved and particulate nutrients.

Fig. 1. Study sites in contrasting ecosystems as defined by their productivity regimes.

In the following, we will briefly describe one major result obtained for each of the trophic levels considered and an integrated view of the associated biogeochemical fluxes at the contrasting sites.

A mosaic of bacterial taxonomy-specific functions in Fe and C utilization

A liter of seawater contains roughly 10¹² bacterial genes (including Bacteria and Archaea), illustrating the immense metabolic potential present in marine microorganisms. But how the associated functions are distributed among bacterial taxa, and how in turn bacterial diversity affects elemental cycling remains a poorly understood question. The MOBYDICK cruise provided an excellent opportunity to collect samples for a comprehensive and in-depth survey of the bacterial community structure and function, using a range of up-to-date ¿omics technologies  (meta-genomics, -transcriptomics and -proteomics). In this context, a relatively recent approach allows to reconstruct individual bacterial taxa from environmental metagenomic sequences. These so-called metagenome assembled genomes (MAGs) provide information on the repertoire and expression of genes at the bacterial `species' level, information that was thus far restricted to cultured strains. The reconstruction of 133 MAGs from the MOBYDICK sequences allowed us to describe the highly diverse strategies of individual bacterial taxa when considering central metabolic pathways in Fe and C-metabolism (Fig. 2)(Sun et al. 2021). Taxonomically closely related MAGs could exhibit contrasting properties of regulating gene expression or distinct gene abundance distribution among sites. This indicates adaptation and specialization to different ecological niches. Our results provide a detailed picture of the functional roles of individual bacterial taxa in contrasting environments of the Southern Ocean and they emphasize the complex interplay between the genetic repertoire of individual taxa and their environment.

Fig. 2. Illustration of the presence, abundance and expression of genes in metagenomic assembled genomes (MAGs) obtainted during the MOBYDICK cruise. From left to right, the panels represent the phylogenetic tree, the genes related to the bacterial Fe-uptake and the genes involved in the tricarboxylic acid cycle (TCA), a central pathway in cellular carbon metabolism. Each square block describes the statistics of a protein family in a MAG. An empty square suggests that no genes in the MAG (y axis) are classified into the corresponding functional group (x axis). A circle in the square block indicates the identification of homologs to a protein family in the MAG, with its size proportional to the number of genes assigned to that family. The square blocks are coloured according to the differential expression patterns of its gene(s). Genes, which are significantly higher expressed in the Fe-fertilized site M2 as compared to the HNLC M3 and M4 sites, are highlighted in orange; vice versa, in blue. From Sun et al. (2021).

CO₂ fixation by small phytoplankton : insights from single-cell observations

Phytoplankton is composed of microorganisms characterized by a wide range of sizes and a great phylogenetic diversity. How much each phytoplankton group contributes to CO₂ fixation in surface waters and to the export of carbon to depth is not well known. The determination of the intra- and inter-group variability of CO₂ fixation is crucial in order to understand the role of different phytoplankton groups to the biological carbon pump. During the MOBYDICK cruise we measured the CO₂ fixation rates of different phytoplankton groups at the single-cell level, using stable isotopes (¹³CO₂) and Nanoscale secondary ion mass spectrometry (NanoSIMS)(Fig. 3)(Irion et al. 2021). We observed that in late Austral summer, when the MOBYDICK cruise took place, small cells (< 20 µm) composed of phylogenetically distant taxa (prymnesiophytes, prasinophytes, and small diatoms) grew faster than larger diatoms. These latter exhibited heterogeneous growth and a considerable proportion of inactive cells (19 ± 13%). As a consequence small phytoplankton had a large contribution to total CO₂ fixation (41-70%). The vertical distribution of pigments indicated that predation could be an important export pathway for carbon fixed by small phytoplankton. Together, our results highlight the important role of small phytoplankton cells in CO₂ fixation in the Southern Ocean. These results complement the seasonal picture in the naturally iron fertilized region of Kerguelen Island, where diatoms were identified as major players in CO₂ uptake and export during spring and summer.

Fig. 3. Sample analysis by NanoSIMS (Nanoscale secondary ion mass spectrometry). The ratio of ¹³C/¹²C, indicated by the color code (y-axis) is used to quantify CO₂ fixation of individual cells sorted by flow cytometry. From Irion Solène (PhD thesis).

Diatoms on their way to the deep ocean

Despite the importance of the export of organic carbon to depth via particles, the composition of the diverse types and size ranges of particles present at different depth layers is still largely unknown. A methodological challenge is the utilization of appropriate instruments to collect particles for their further characterization and quantification. During MOBYDICK, we deployed a recently available device that concentrates and collects deep particles over a pre-determined layer of water, a so-called BottleNet sampler. This first¿time deployment of the BottleNet sampler allowed to provide a detailed description of the particles and individual planktonic cells, including their taxonomy, carbon and lipid content, as well as viability in different depth layers (for example 60-125m, 125-500m and 500-1500m)(Leblanc et al. 2021). Unexpectedly, the majority of the collected particles consisted of single empty diatom frustules, whereas fecal pellets and aggregates were only a minor fraction (Fig. 4). We identified the occurrence of distinct mortality processes of diatom taxa, from parasitic infection to mesozooplankton grazing, and of distinct silicification degrees as well as life-stages, and could thereby evidence distinct export modes to intermediate and deep layers.

Fig. 4. Loose fecal pellet containing recognizable debris of the diatom Fragilariopsis kerguelensis (pink),  and centric diatoms (turquoise). A few coccoliths are also visible (purple). From Leblanc et al. (2021).

Seasonal microbial food web dynamics

Combining the observations made during the MOBYDICK cruise in late summer 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 (Christaki et al. 2021)(Fig. 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 lower as compared to spring. During the post-bloom phase (MOBYDICK), 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. The role of the microbial food web for carbon processing varies during the three stages. 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. The importance of viral lysis in carbon losses results in an inefficient transfer of bacterial biomass to heterotrophic nanoflagellates (HNF). During the postbloom phase, the transfer of bacterial biomass 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, and are thus potentially available to higher trophic levels.

Fig. 5. Mixed layer integrated carbon flow within the microbial food web. From left to right: during early (KEOPS2), late (KEOPS1), and after the diatom bloom (MOBYDICK). A detailed description of the calculations is provided in the publication by Christaki et al. (2021).

Diversity and contribution to C-cycling of metazoans

While the effect of natural iron fertilization on biogeochemical cycles and the structure of the lower level food-web has been intensively studied over the past decade in the Kerguelen region, higher trophic levels have thus far not been included for a complete ecosystem approach. Using macro- and micro-scopic taxonomic identification of organisms collected with nets and trawls, and acoustic measurements (18 and 38 kHz) we could describe the structure and function of the pelagic food webs up to micronecton. The communities of macrozooplankton and micronekton differed between distinct productivity regimes despite a conspicuous three-layer vertical system detectable in the upper 800m at all sites that are shallow (10 to 200 m), mid-depth (200 to 500 m), and deep (500 to 800m) scattering layers (Cotté et al. 2021)(Fig. 6). While salps (Salpa thompsoni) dominated the biomass over the productive Kerguelen plateau, they were scarce under HNLC conditions. In addition, crustaceans (mainly Euphausia vallentini and Themisto gaudichaudii) were particularly abundant over the plateau, representing a large, although varying carbon stock in the 0-500 m water layer. Mesopelagic fish were prominent below 500m and formed permanent or migrant layers accounting for the main source of C. Our high-frequency observations allowed to identify spatial and temporal patterns in micronekton vertical distributions and associated carbon content. We further estimated the respiratory carbon fluxes mediated by migratory myctophids and these provided insights to the main components and mechanisms of active carbon export in the region and how they are modulated by complex topography and land mass effects.

Used stable isotopes (delat¹⁵N, delta¹³C) combined with taxonomic identifications revealed that the food chain length, calculated from a standardized subset of species and size classes, was 1 trophic level shorter in iron fertilized Kerguelen plateau waters than in HNLC waters. Predator prey mass ratios (PPMR) were one order of magnitude lower in the HNLC region (PPMR = 170) as compared to the plateau (PPMR > 2000), indicating  high internal recycling in the more stable HNLC waters. The overall transfer efficiency of carbon and nitrogen was estimated to be roughly 2-fold higher (21 %) in the HNLC region than in the ecosystem sustained by natural iron fertilisation (7-10 %)(Hunt et al. 2021). It is interesting to note that these observations from higher trophic levels are in line with those for the microbial food webs (Christaki et al. 2021), illustrating a different functioning of the entire food web in high and low productive ecosystems of the Southern Ocean.

Fig. 6. Acoustics of daily Red Green Blue (RGB) composites of volume backscattering strength values (dB re m^-1) from 12 to 800m for the four MOBYDICK stations. From Cotté et al. (2021).