# Seasonal dynamics of DOM diversity and molecular composition in the NW Mediterranean Sea

Nawal Bouchachi, Hélène Lavanant, Carlos Afonso, Isabelle Schmitz, Barbara

Marie, Ingrid Obernosterer, Eva Ortega-Retuerta

► To cite this version:

Nawal Bouchachi, Hélène Lavanant, Carlos Afonso, Isabelle Schmitz, Barbara Marie, et al. Seasonal dynamics of DOM diversity and molecular composition in the NW Mediterranean Sea. Marine Chemistry, In press, 10.1016/j.marchem.2025.104562. hal-05262521

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Submitted on 16 Sep 2025

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Distributed under a Creative Commons Attribution 4.0 International License
1 Seasonal dynamics of DOM diversity and molecular composition in the
2 NW Mediterranean Sea.

3 Bouchachi, Nawal¹; Lavanant, Hélène², Afonso, Carlos², Schmitz, Isabelle², Marie, Barbara¹, Obernosterer, Ingrid¹, Ortega-Retuerta, Eva¹

5 ¹Laboratoire d'Océanographie Microbienne, LOMIC UMR7621, CNRS, Sorbonne Université, Banyuls-sur-Mer, France

7 ² INSA Rouen Normandie, CNRS, Normandie Université, COBRA UMR 6014, INC3M FR 3038, 76000 Rouen, France

9 Graphical Abstract

<figure><img src="image_2.png" /></figure>

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11 Abstract

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In the Mediterranean Sea, an accumulation of dissolved organic matter (DOM) is observed during the stratified summer period, which is decoupled from the spring phytoplankton maxima. This has been classically explained by nutrient limitation, that hampers prokaryotic DOM uptake of the labile DOM released by phytoplankton. However, relatively high heterotrophic prokaryotic activity in summer led us to hypothesize that the accumulated DOM might be recalcitrant and derived from these microorganisms. To test this, we tracked the seasonality of DOM molecular composition and chemical diversity using FT-ICR MS from 2019 to 2021 in the NW Mediterranean Sea, and framed it using a broad suite of environmental and biological parameters. Our results reveal a clear seasonal variation in DOM molecular composition and diversity in the surface mixed layer. Changes in composition reflected a higher proportion of molecular formulae containing CHO in spring and a higher proportion of molecular formulae containing CHOS and CHONS in summer. Proxies of DOM recalcitrance, such as aromaticity, unsaturation, and molecular size, were higher in summer, confirming our
hypothesis of an accumulation of recalcitrant DOM in summer. In parallel, an increase in DOM diversity (as number of total molecular formulae, their relative intensity, their average distance (Dist) in the van Krevelen space and their functional diversity) was observed, suggesting that the accumulated DOM in summer is likely the result of the interplay between different processes including dissolved primary production, photodegradation and prokaryotic activity. Our results may have significant implications for carbon sequestration through the microbial carbon pump in the Mediterranean Sea as this DOM accumulated in the surface is likely to be stored once exported into deep layers.

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## Keywords

Dissolved organic matter, FT-ICR MS, molecular composition, Mediterranean Sea, seasonal dynamics

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## 1. Introduction

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Dissolved organic matter (DOM) in the ocean is one of the largest reservoirs of reduced carbon on earth (662 Pg., D. A. Hansell, 2013). It consists of a complex mixture of thousands of compounds of diverse composition at different concentrations. While DOM originates from different sources, phytoplankton primary production is the main source of DOM in the open ocean. Heterotrophic prokaryotes (Bacteria and Archaea) transform about 50 % of this DOM to biomass (prokaryotic production) or to CO₂ (remineralization) (Cole et al., 1988). However, a part of this DOM escapes from degradation and accumulates in the surface ocean before being exported into deeper layers and accumulates as recalcitrant DOM (RDOM) (Hansell et al., 2009; Hansell, 2013). The microbial carbon pump (MCP) hypothesis states that heterotrophic prokaryotic metabolism leads to successive transformation of labile DOM into RDOM resulting in long term carbon storage (Jiao et al., 2010), being thus an important carbon sequestration mechanism (Legendre et al., 2015). Understanding the mechanisms of prokaryotic DOM release and its persistence in the ocean is key to better constrain carbon sequestration through the microbial carbon pump.

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To explain RDOM persistence in the ocean, two key hypotheses have been advanced: the intrinsic stability, which attributes DOM recalcitrance to its molecular characteristics (Shen
and Benner, 2020), and the dilution or diversity hypothesis, which states that RDOM is composed of various but highly diluted labile molecules (Arrieta et al., 2015). One of the biggest challenges to study DOM dynamics in the ocean is its characterization due to the concurrence of thousands of different compounds. Over the last years, FT-ICR MS (Fourier Transform Ion Cyclotron Resonance) has revealed to be a powerful tool to trace DOM cycling through heterotrophic prokaryotes thanks to the identification of large numbers of individual molecular formulas and chemical compounds groups in DOM (Cooper et al., 2022; Nebbioso and Piccolo, 2013; Qi et al., 2022) that provide information about its potential recalcitrance. Several molecular structures and properties have been proposed through FT-ICR MS as intrinsically RDOM. Namely, carboxyl-rich alicyclic molecules (CRAM; Hertkorn et al., 2006), one of the most abundant constituents of deep ocean DOM, are considered to be biologically resistant and are widely used as a proxy for RDOM (Lechtenfeld et al., 2015; Li et al., 2019; Lian et al., 2021; Martínez-Pérez et al., 2017). Molecular data are also used to separate labile versus recalcitrant DOM based on their intrinsic properties such as the unsaturation degree (D'Andrilli et al., 2015; Li et al., 2019; Medeiros et al., 2015). As for the dilution hypothesis, despite the lack of knowledge about the concentration of individual molecules, DOM molecular diversity based on FT-ICR MS analyses has been estimated both in field and incubation studies, including molecular richness (number of formulas) (e.g., Lechtenfeld et al., 2015; Schmidt et al., 2009), signal-based diversity (Gini-Simpson index) and functional molecular diversity (Landa et al., 2014; Mentges et al., 2017; Noriega-Ortega et al., 2019). In this line, some studies have reported an increase in chemical and structural diversity of DOM in microbial incubations (Lechtenfeld et al., 2015; Noriega-Ortega et al., 2019; Osterholz et al., 2015).

Characterizing the temporal evolution of DOM composition and properties using FT-ICR MS together with environmental and microbial variables in the field can give us information about its main sources and sinks, and its changes in lability. The Mediterranean Sea is a semi-enclosed oligotrophic basin, known to be limited in phosphorus. Characterized by pronounced seasonality, the Mediterranean Sea experiences surface stratification and enhanced
84 nutrient limitation in the upper mixed layer during the summer period (Laghdass et al., 2012).
85 During this period, an accumulation of dissolved organic carbon (DOC) is recurrently observed
86 (Laghdass et al., 2010; Romera-Castillo et al., 2013; Sánchez-Pérez et al., 2020, 2016), as in
87 other oligotrophic ocean areas (Carlson et al., 2002; Church et al., 2002), which is decoupled
88 from the phytoplankton maximum, which takes place in late winter-early spring (D'Ortenzio
89 and Ribera d'Alcalà, 2009; Michaels et al., 1994). The proposed reason for this decoupling is
90 the malfunctioning of the microbial loop, that is, that nutrient limitation prevents heterotrophic
91 prokaryotes from uptaking the DOM released by phytoplankton, therefore promoting its
92 accumulation (Thingstad et al., 1997; Thingstad and Rassoulzadegan, 1999). Should this be the
93 case, the DOM accumulated during summer would be labile and therefore degraded when
94 inorganic nutrients are available (e.g. when reaching deeper waters after winter mixing).
95 However, nutrient addition experiments in oligotrophic oceanic areas showed contrasting results
96 (Carlson et al., 2002; Church, 2008; Laghdass et al., 2012; Obernosterer et al., 2003).
97 Heterotrophic prokaryotic production in the NW Mediterranean Sea is usually higher in summer
98 when inorganic nutrients are depleted in surface waters (Alonso-Sáez et al., 2008; Laghdass et
99 al., 2010). This suggests that at least a part of the accumulated DOM is derived from
100 heterotrophic prokaryotes through the consumption of labile DOM and the release of
101 recalcitrant DOM through the microbial carbon pump (Bouchachi et al. 2022).

102 In this study, we hypothesized that DOM composition in the surface NW Mediterranean
103 Sea changes from labile to recalcitrant compounds from spring to summer leading to its
104 accumulation in summer. In addition, heterotrophic prokaryotes might play a significant role in
105 its formation, i.e., the MCP would be enhanced under oligotrophic summer conditions. To test
106 this hypothesis, we tracked the seasonality of DOM molecular composition and chemical
107 diversity using FT-ICR MS during 3 years of monthly sampled time series in a NW
108 Mediterranean Sea station (Fig. S1). The generated dataset was compared to a broad suite of
109 environmental parameters, prokaryotic abundances and richness as well as DOM optical
110 properties in order to better understand the potential sources and drivers of DOM dynamics.

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## 2. Material and methods

### 2.1.1 Study site and sampling strategy

The MOLA Station (Microbial Observatory Laboratoire Arago) is located on the edge of the continental shelf in the NW Mediterranean Sea (42°27′205 N – 03°32′565 E, 550 m bottom depth, Fig. S1). MOLA is part of the MOOSE network (Mediterranean Ocean Observing System for the Environment: https://www.moose-network.fr/) and is sampled monthly since 2003.

Several biochemical measurements, including inorganic nutrients, chlorophyll a (Chl a) and flow cytometry analyses, are routinely determined in the upper 500 m water column as part of MOOSE. From January 2019 to August 2022, 6 depths (5, 20, 40, 80, 200 and 500 m) were additionally sampled for dissolved organic carbon (DOC); colored and fluorescent DOM (CDOM and FDOM), details can be found in supplementary information. Samples for FT-ICR MS and microbial community composition (16s rRNA) were also taken at 5 and 500m. The station could not be sampled in December due to meteorological conditions and between March and June 2020 due to COVID restrictions. For convenience, only one deep (500 m) sample per season was analyzed by FT-ICR MS (Jun 2020, Aug 2020, Nov 2020, Feb 2021, Mar 2021). We chose 500m as our deep sample reference since it is the deepest sample taken in the MOLA timeseries. All samples were collected using 12L Niskin bottles. Temperature and salinity were measured in the whole vertical profile using a CTD (SEA BIRD SBE 19plus).

MOLA is characterized by a marked seasonal cycle with a succession of water mixing and stratification. In our study years, during the mixing period from winter to early spring, the mixed layer depth (MLD) deepened (>35-74 m, Table S1, Fig. S2) allowing nutrient inputs to the surface, up to 2.3 µmol L⁻¹ NO₃⁻ and 0.08 µmol L⁻¹ PO₄³⁻ (Fig. S3). These nutrients sustain a spring phytoplankton bloom where, during the study period, Chl a concentration reached up to 0.94 µg L⁻¹ (March 2021, Fig. S4a). In the summer stratification period, the MLD did not exceed 32 m depth (Table S1) and nutrient levels were low (0.16 µmol L⁻¹ NO₃⁻ and 0.04 µmol L⁻¹ PO₄³⁻, Table S1, Fig. S3). DOC accumulates in the surface mixed layer at this period of the
year (Fig. S4b). During the study period we observed abnormally high nutrient levels, in particular PO₄³⁻, in August 2020 (0.26 µmol L⁻¹ NO₃⁻ and 0.20 µmol L⁻¹ PO₄³⁻, Table S1, Fig. S3).

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## 2.1. Chemical and biological analyses

### 2.1.1. SPE-DOM samples

Water samples for solid phase extracted DOM (SPE-DOM) from 5 and 500 m were transferred to HDPE carboys (acid washed and rinsed with MilliQ water) until their processing in the lab, within 2 hours. The samples were successively filtered through 3 then 0.2 µm polycarbonate filters using a peristaltic pump. The first 1 L, used to rinse the filters and tubing, was discarded. The filtered sea water was then recovered in 2 L combusted glass bottles and acidified to pH 2 with 37% HCl. DOM was extracted using styrene divinyl benzene polymer cartridges (Agilent Bond Elut PPL, 1 g) as described in (Dittmar et al., 2008). After extraction, cartridges were rinsed with acidified MilliQ water (200 mL, pH 0.01N). The cartridges were then dried with pure air using sterile air-filters and stored at -20 °C until elution. Elution was done by gravity using 6 mL HPLC grade methanol (MeOH). Extracts aliquots were dried under pure air (sterile air-filtered) until complete evaporation of MeOH for mass spectrometry analysis. However, the presence of chloride ions from the rinsing process caused DOM signal suppression. To resolve this, all samples underwent a second SPE process: dried DOM extracts were resuspended in 5 mL of LCMS water and run through 1 g PPL cartridges. After this second extraction step the cartridges were rinsed with 3 mL of LCMS water and dried with N₂. Elution was done using 2 mL of LCMS MeOH in amber glass vials and stored at -20 °C.

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### 2.1.2. FT-ICR MS analysis

Fourier transform ion cyclotron resonance mass spectrometry FT-ICR MS analyses were performed at the COBRA Laboratory (Infranalytics FR2054 research infrastructure) using a 12 T Solarix XR mass spectrometer (Bruker Daltonics). SPE-DOM MeOH extracts were diluted in LCMS grade MeOH and ran in FIA (flow injection analysis) in analytical triplicates
and several MeOH blanks were randomly run with each sample set. Sample replicates analysis order was randomized to rule out running order effects. A quality control sample consisting of an equi-volume mixture of all samples was also analyzed. An electrospray ionization source was used in negative mode. Mass spectra were acquired in a mass range of *m/z* 98.2-1200. Accumulation time was set to 0.02 s and 200 scans were accumulated. Samples Jul 2019, Sep 2019, Nov 2019 and Aug 2021 were discarded from the data set due to contamination problems during the extract evaporation process.

The instrument was first externally calibrated with sodium trifluoroacetate (NaTFA), and then internally calibrated with a calibration list of CₓHᵧOz molecular formulas (MFs, n=90). This internal list of calibrants was made using a test sample spiked with an Agilent tune mix mass calibration solution. A series of ions repeatedly observed in previous DOM studies (*m/z* 339.10854, 369.11911, 411.12967, 469.13515, 541.15628 (Flerus et al., 2011)) were also identified and checked based on their fine isotopic structure. These ions were then added to the calibrants list. The alkylate series of these ions were then identified and included in the calibrants list.

MFs were assigned using the Bruker DataAnalysis software in a mass range of *m/z* 98.3-1200 respecting the following restrictions: CₓHᵧN₀.₂O₀.₁₅S₀.₁, double bond equivalent (DBE) ≤ 80, 0.4≤ H/C ratio≤ 3, only peaks with signal to noise ratio (S/N) > 9 were considered and a 0.5 ppm maximum mass error tolerated. These parameters ensure the reliable determination of the molecular formulas, which, like in most molecular assignment softwares (Fu et. al. 2020, Tolic et. al. 2017; Merder et. al. 2020), relies on filtering criteria as described by Kind and Fiehn (2006, 2007). The maximum number of possible formulas was set to 50; even with this very high number of accepted formulas, the software yielded only one possibility per *m/z* value, thus indicating the assignment was unambiguous. It should be noted that in order to assign low intensity ions, for which several peaks in the isotopic distribution have low signal to noise ratio, we did not take into account the isotopic fine structure (the msigma value set to 1000), but we manually checked the isotopic fine structures of intense ions to confirm their assignments. The obtained MF attribution lists were exported and further processed under R
3.6.1 (R Core Team, 2019). Since there was very low signal at *m/z* above 850, attributed formulas were filtered to keep only *m/z* values ≤ 850. An intense peak at *m/z* 265.1479 (assigned to C₁₂H₂₅SO₄⁻, dodecyl sulfate); likely contamination; was removed from all samples. Formula assignment was reproducible among analytical replicates sharing between 65 - 84 % common MFs. Only the MFs identified in all analytical replicates were kept for each sample replicate and used for multivariate analysis (see statistical analyses). With these cleaning steps, we obtained a set of 3789 of MFs identified across all samples (Fig. S5). For each sample, a mean MF list was created by merging the analytical replicates.

All peak intensities were normalized to the summed total intensity (i.e., relative intensity). For each compound we calculated the modified aromaticity index AI<sub>mod</sub> (eq. 1), the DBE (eq. 2) (Koch and Dittmar, 2016).

$$AI_{mod} = \frac{1 + 0.5(2C - O - H - 2S - N)}{C - 0.5O - S - N} \quad (1)$$

$$DBE = C + \frac{N}{2} - \frac{H}{2} + 1 \quad (2)$$

The mean lists were used for diversity measures (see below), average intensity weighted (denoted with ‘_w’) molecular parameters (Sleighter et al., 2010), building compound groups and sorting MFs into heteroatoms combinations classes: CHO, CHONS, CHON, CHOS, CHNS. The compound groups considered here are: polyphenols 0.5 < AI<sub>mod</sub> < 0.66; highly unsaturated (HU) AI<sub>mod</sub> < 0.5, H/C < 1.5, O/C < 0.9; unsaturated aliphatic (UA) 1.5 ≤ H/C < 2, O/C < 0.9, N = 0; peptides 1.5 < H/C < 2, O/C ≤ 0.9 and N > 0 (Martínez-Pérez et al., 2017); carboxyl-rich alicyclic molecules (CRAM) 0.3 < DBE/C < 0.68, 0.2 < DBE/H < 0.95, 0.77 < DBE/O < 1.75 (Hertkorn et al., 2006); carbohydrates 1.6 < H/C < 2.0, 0.7 < O/C < 1.0 and N = 0 (Li et al., 2019).
## 2.2. DOM chemical diversity measures

We assessed DOM chemo-diversity using several diversity indices that are traditionally used in biological diversity studies. We calculated signal-based diversity (Gini-Simpson) and functional diversity (F). These indices were used in previous studies and have been considered to be robust against variation in sample preparation and instrument settings, allowing thus comparison among samples and studies (Mentges et al., 2017). Gini-Simpson (eq. 3) considers the relative ion intensities and describes the probability that two randomly chosen molecular formulas differ. In biological diversity studies, F (eq. 4) is interpreted as the variability of a biological trait in the community. Similarly, F here quantifies how a chemical property (e.g., H/C, C number, DBE etc.) varies (Mentges et al., 2017) and it is calculated based on Rao's Quadratic Entropy.

$$Gini-Simpson = 1 - \sum_{i=1}^{N} I_{i}^{2} \quad (3)$$

$$F(p) = \sum_{i=1}^{N} \sum_{j=1}^{N} I_{i} I_{j} |p_{i} - p_{j}| \quad (4)$$

N is the total number of MFs, $I_{i(j)}$ is the relative intensity of $i^{th}$ ($j^{th}$) MF, p the considered molecular parameter.

F can be viewed as the expected difference between two molecules in terms of a chosen chemical parameter. In this study we assessed functional diversity of DOM lability based on the H/C ratio (F(H/C). H/C ratio reflects the saturation state of the sample and its decrease has been associated to a decrease in DOM lability (D'Andrilli et al., 2015; Lechtenfeld et al., 2014; Medeiros et al., 2015).

We also measured chemical differences between molecules in each sample using the average distance (Dist, eq. 5) in van Krevelen space between each MF and all other MFs of the sample as described in Landa et al. (2014). In a van Krevelen diagram, molecules with similar chemical properties are situated close to each other leading to low Dist values, while high Dist values reflect more chemically different molecules in a given sample.
$$Dist = \frac{1}{N^2} \sum_{j=1}^{N} \sum_{i=1}^{N} \sqrt{(OC_j - OC_i)^2 + (HC_j - HC_i)^2} \quad (5)$$

Molecular richness (MR), i.e., the number of MFs in a sample, and evenness were also considered. Pielou's evenness depicts how MFs are distributed in terms of their relative intensity (Pielou, 1966).

$$P = - \frac{\sum_{i=1}^{N} pi \cdot Ln(pi)}{Ln(MR)} \quad (6)$$

## 2.3.Indices for evaluating overall DOM recalcitrance

To assess DOM recalcitrance, we used a combination of average intensity weighted molecular parameters that have been described as indices of the recalcitrance /bioavailability of DOM: AI<sub>mod_w</sub>, DBE_w, H/C_w, nominal oxidative state of carbon (NOSC_w), C_w, m/z_w. AI<sub>mod</sub> and DBE values are indicators of aromatic rings and double bonds presence in a molecule and their increase has been associated to an increase in DOM recalcitrance (Li et al., 2019; Martínez-Pérez et al., 2017; Medeiros et al., 2015). The NOSC (eq. 6), that reflects the oxidative state of a molecule regardless of its chemical structure, is an indicator of overall DOM reactivity. High NOSC compounds are thermodynamically more profitable to degrade (LaRowe &. Van Cappellen, 2011).

$$NOSC = 4 - \frac{(4C + H - 3N - 2O - 2S)}{C} \quad (7)$$

C number and m/z represent molecular size and weight that are related to DOM reactivity (Benner and Amon, 2015). In the FT-ICR MS analysis acquisition window (commonly below 1500 Da), different field and experimental studies show that an increase in
molecular weight is associated to an increase in DOM recalcitrance (Cai et al., 2019; Li et al., 2019; Martínez-Pérez et al., 2017; Medeiros et al., 2015).

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269 **2.4. Statistical analyses**

To visualize dissimilarities in the molecular composition between deep and surface samples we used a non-metric multidimensional Scaling (NMDS) based on Bray Curtis dissimilarity matrices (package vegan, 2.5.7; Oksanen et al., 2020).

To visualize mean seasonal trends for one year at 5 m, a loess smoothed curve was fitted to the data using *geom.smooth* function from package *ggplot2* (v3.3.6 Wickham, 2016).
A principal coordinate analysis (PCoA) based on Bray Curtis dissimilarity matrix was used to visualize seasonal dissimilarities in surface samples. Environmental variables were fitted to the PCoA axes using *envfit* function (package vegan, 2.5.7; Oksanen et al., 2020).

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279 **3. Results**

3.1. *Comparison of DOM composition between deep and surface samples*

The NMDS analysis revealed a clear separation between deep and surface samples (figure 1, Fig. S6). Surface samples showed a larger spread than 500m samples, reflecting higher seasonal and interannual variability in DOM composition in the mixed layer than in deep waters (see section 2).

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<figure><img src="image_3.png" /><figcaption>Figure 1 NMDS based on Bray-Curtis distance using relative intensities of the selected 3789 MFs.</figcaption></figure>

In terms of chemo-diversity, both the Gini-Simpson and Dist indices were higher in surface than in deep samples (figure 2a, b). Deep and surface samples had similar number of MFs, but a decrease in Pielou's evenness indicated that MFs intensities are less evenly distributed in deep samples (Fig. S7). Deep samples were also more recalcitrant than the surface counterparts as shown by higher aromaticity, CRAM proportion, and carbon number (figure 2c-e).
<figure><img src="image_4.png" /><figcaption>**Figure 2** Chemodiversity and Molecular characteristics of deep and surface samples. Gini-Simpson diversity index (a). Average distance in van Krevelen space (Dist) between MFs (b). Weighted modified aromaticity index (Almod_w) (c). Relative intensity (%) of CRAM (d), and weighted carbon number C_w (e), relative intensity (%) of C, H, O, N and S heteroatoms formulas (f-h), weighted average O/C ratio.</figcaption></figure>

CHO containing molecules had a lower relative intensity (%) in 5m samples compared to 500m samples (figure 2f). CHOS, CHON and CHONS molecules, however, were higher in surface samples compared to deep (figure 2g-i). Weighted average of O/C ratio ranged between 0.47 and 0.50 with minimum values corresponding to deep samples (figure 2j). Taken together, this suggests that deep DOM in MOLA was overall less oxygenated and less enriched in S, N containing molecules compared to surface DOM.

## 3.2. Temporal trends in DOM chemo-diversity and DOM recalcitrance indicators in surface waters

DOM chemo-diversity, as expressed by the different diversity indexes, showed a seasonal variation in surface samples (figure 3). The Gini-Simpson diversity had a relatively stable mean trend in winter and spring and then increased in summer, reaching a maximum in July-August (figure 3a). A higher chemical diversity in summer was also supported by the increase of the average distance (Dist) in the van Krevelen space in this period (figure 3b). The functional diversity F(HC) index was relatively stable from January to May and then increased
314 from May to a maximum in summer (figure 3c), indicating that the DOM pool in summer
315 contained both high and low H/C molecules.

<figure><img src="image_5.png" /></figure>

316

317 **Figure 3** Seasonal changes of DOM chemodiversity descriptors in surface. Gini-Simpson diversity index
318 (a). Average distance (Dist) in van Krevelen space between MF (b). H/C ratio functional diversity F(HC)
319 (c). Each dot represents a sample based on mean lists of analytical replicates. Blue lines represent mean
320 trends and shaded bands correspond to 0.95 confidence level intervals.

321

322 We observed a decrease in DOM aromaticity (AI<sub>mod_w</sub>), unsaturation (DBE_w) and
323 molecular size (C and *m/z*) from winter to spring, reaching a minimum in April-May (figure 4
324 a-c). In parallel to this, H/C, an indicator of DOM lability, reached a maximum also in April-
325 May (figure 4e). Recalcitrant DOM proxies (DOM aromaticity, unsaturation and molecular
326 size) increased again in summer, reaching a maximum in July-August; while HC_w fell to its
327 minimum (figure 4 a-e). Nominal oxidative state of carbon (NOSC) values slightly decreased
328 in spring but were maximal in summer (figure 4f), further indicating the increase of the overall
329 oxidation state of DOM and its recalcitrance in summer.

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<figure><img src="image_6.png" /><figcaption>**Figure 4** Seasonal change of intensity-weighted molecular indices in surface. Modified aromaticity index, Almod_w (a). Double bound equivalent, DBE_w (b). Carbon atom number, C_w (c). Mass to charge ratio, m/z_w (d). Hydrogen to carbon ratio, HC_w (e). Nominal oxidative state of carbon NOSC_w (f). Each dot represents a sample based on mean lists of analytical replicates. Blue lines represent mean trends and shaded bands correspond to 0.95 confidence level intervals.</figcaption></figure>

We also observed a higher relative intensity of CHOS and CHONS containing molecules in summer, while molecules containing CHO were at their relative minimum (figure 5a-c). Compounds groups also showed seasonal trends, with peptides peaking in spring while the highly aromatic polyphenols were at their minimum and increased in summer, showing a maximum in July (figure 5d, e). CRAM, that constitute a large proportion of marine recalcitrant RDOM, showed a stable trend in winter-spring before decreasing in summer and reaching a minimum in July-August (figure 5f).
<figure><img src="image_7.png" /><figcaption>**Figure 5** Seasonal change of different heteroatom classes (a-c) and compound groups (d-f) in surface water. Each dot represents a sample based on mean lists of analytical replicates. Blue lines represent trend lines and shaded bands correspond to 0.95 confidence level intervals. CRAM = carboxyl-rich alicyclic molecules.</figcaption></figure>

### 3.3. Linking DOM molecular composition to DOM optical properties and prokaryotic activity

Similar to DOM molecular composition, trends of DOM optical properties and microbial metrics followed a seasonal pattern. Humic-like specific fluorescence (this is, humic-like fluorescence, peaks M A and C, divided by DOC concentration) and SUVA<sub>254</sub> (DOM absorbance at 254 nm divided by DOC) were higher in winter-spring and decreased in summer (figure 6), while CDOM spectral slopes increased in summer and protein-like specific fluorescence showed a less clear seasonal trend (figure 6). DNA prokaryote abundances (total cells and high and low nucleic acid cells separately) were high in winter and decreased from winter to the end of spring then increased again in summer (Fig. S8).
<figure><img src="image_8.png" /><figcaption>Figure 6 Seasonal change of DOM optical properties in the surface mixed layer. Dots represents monthly measurements. Blue line represents trend line, shaded bands correspond to 0.95 confidence level interval. spM, marine humic-like. spA, humic-like. spC, terrestrial humic-like. spT, tryptophan-like. S<sub>275-295</sub>, CDOM spectral slope. SUVA<sub>254</sub>, Specific ultraviolet absorbance at 254 nm (L mg C<sup>-1</sup> m<sup>-1</sup>). FDOM. Peaks are reported as specific fluorescence (sp, RU L mg C<sup>-1</sup>).</figcaption></figure>

To better understand the seasonal dynamics of DOM in the surface and to identify potential explanatory drivers, we used a PCoA. The drivers included FT-ICR MS derived measures (chemo-diversity, compounds groups, AI<sub>mod_w</sub>, m/z, C, etc), DOM optical properties (specific fluorescence of FDOM peaks, humification index (hix), spectral slope S<sub>275-295</sub>, SUVA<sub>254</sub>), microbial metrics (prokaryotic diversity based on 16s rRNA, total prokaryotes, low and high DNA prokaryote abundances, viral abundances, heterotrophic nanoflagellates (HNF) abundances) and bulk environmental parameters (DOC, Chl *a*, NO<sub>3</sub><sup>-</sup>, PO<sub>4</sub><sup>3-</sup>, Salinity, Temperature). Only significantly related variables are plotted in the PCoA space (figure 7). The first two PCoA components explained 48.9% of the variance. The analysis showed a clear separation among samples by seasons, although we could observe interannual variability particularly in early summer samples (figure 7). June and July samples from different years were not grouped together (figure 7). For instance, in June 2019, we observe a higher H/C ratio (figure 5), higher protein-like specific fluorescence (figure 7) with higher Chl *a* (Fig. S4) than in June 2020.
<figure><img src="image_9.png" /><figcaption>**Figure 7** PCoA of the molecular composition of surface samples based on Bray Curtis distances calculated from normalized intensities of MFs. The arrows correspond to fitted variables: environmental variables (a). Optical properties (b). Microbial metrics (c). Molecular parameters (d). Only significant drivers are plotted. Simpson= 16srRNA Simpson index of free-living fraction, Richness = 16srRNA richness of free-living fraction (nASVs). Total HP, HNA HP and LNA HP are respectively total, high DNA and low DNA abundances in the surface mixed layer (Cell mL⁻¹). hix=humification index. spA, spM, spC, spB are respectively specific fluorescence of fluorescent DOM peaks A (humic-like), peak M (marine humic-like), peak C (terrestrial humic-like), peak B (protein tyrosine-like), S275_295=CDOM spectral slope between 275-295 nm.</figcaption></figure>

Among the environmental variables significantly associated to the ordination (figure 7a), temperature and nitrate had a strong relationship with the ordination (r2>0.6), while DOC, salinity, Chla and phosphate showed moderate to very weak relationship to the ordination (r2≤0.4) (figure 7b). The microbial parameters, including prokaryotic abundances, 16s rRNA Simpson diversity, and richness, showed weak to moderate associations to the chemical composition ordination, with 0.2<r²<0.5 (figure 7c). The summer samples, however, were closely related to prokaryotic abundances (figure 7c).

## Discussion

### Comparison of deep and surface DOM
Overall, our results indicate that, in the NW Mediterranean Sea, deep (500m) SPE-DOM is more degraded and recalcitrant, but less diverse than surface SPE-DOM. Consistently with previous results from the Mediterranean Sea (Martínez-Pérez et al., 2017) and other ocean areas (Li et al., 2019; Medeiros et al., 2015), the higher recalcitrance of deep SPE-DOM is shown by the higher aromaticity, CRAM proportion, and carbon number, of deep samples. SPE-DOM diversity, based on the Gini-Simpson index and the distance between MFs in the Van Krevelen space, was higher in surface than in deep SPE-DOM samples, indicating a decrease not only in the number of MFs but in their chemical characteristics and the intensities distribution in deep samples. Mentges et al. (2017), similar to our results, reported high diversity in surface waters and low diversity in deep waters from the Atlantic Ocean. They also showed a relationship between the diversity decrease and the increase of the degradation state of SPE-DOM with depth.

Higher SPE-DOM diversity at the surface may reflect the coexistence of different DOM sources, including continuous phytoplankton DOM release, either directly or indirectly after zooplankton feeding, photochemically degraded DOM (Bercovici et al., 2023), allochthonous DOM inputs (see discussion in section 2), while in deep samples several DOM fractions have already been consumed before reaching a depth where the persistent DOM accumulates, explaining the lower SPE-DOM diversity.

Our results also suggest that deep SPE-DOM is less oxygenated compared to the surface DOM. In line with this, previous studies conducted in the Northern Pacific Ocean and the Mediterranean Sea showed that compounds at depth had smaller O number and lower O/C ratios compared to those at the surface (Martínez-Pérez et al., 2017; Medeiros et al., 2015). A decrease in the O/C ratio has been associated to more degraded DOM as Kim et al., (2006) showed that biodegradation results in a preferential removal of high O/C molecules, suggesting that deep DOM have been subject to successive degradation by microorganisms.

## Summer accumulation of recalcitrant DOM in the upper mixed layer

Our results reveal a clear seasonal variation in SPE-DOM molecular composition and diversity in the surface layer, according to recent observations in other oligotrophic oceans
432 (Longnecker et al., 2024). In the spring period, we observed a lower aromaticity and
433 unsaturation degree along with an increase in peptides proportion, concordant with the presence
434 of fresh phytoplanktonic DOM as indicated by higher *Chla* during spring. By contrast, SPE-
435 DOM aromaticity, unsaturation, and size, all indicators of recalcitrant DOM (Cai & Jiao, 2022
436 and references within), were higher in summer, thus confirming our hypothesis of an increase in
437 DOM recalcitrance in the upper mixed layer during the stratified period. Noteworthy, CHOS
438 compounds in SPE-DOM were suggested to be rather unreactive as they cannot be further
439 oxidized or hydrolyzed (Pohlabeln, 2013) which is consistent with the accumulation of
440 recalcitrant DOM in surface in summer (figure 5a). However, the nature of sulfur-containing
441 DOM compounds accumulated in summer is intriguing: A broad suite of sulfur-containing
442 metabolites have been described in the ocean, many produced by phytoplankton and bacteria.
443 Well-known examples are DMSP, gonyol, S-containing amino acids like methionine,
444 sulfonated polysaccharides or sulpholipids (Moran et. al. 2019). In particular, DMSP and
445 glycine betaine are S-containing DOM compounds that have been described to be peaking in
446 summer in surface ocean waters (Vila-Costa et. al. 2008, Airs et. al. 2023), although their high
447 turnover rates described in the literature (Kiene et. al. 1998, Vila-Costa et. al. 2007) are not
448 consistent with summer accumulation of recalcitrant DOC. The increase in the oxidative state of
449 DOM in summer, however, may indicate the increase of its reactivity and thermodynamic
450 favorability for biodegradation (LaRowe and Van Cappellen, 2011). The carbon oxidative state
451 and oxygen content of DOM may give ambiguous information about its recalcitrance. Increases
452 in NOSC have been reported in light exposed estuarine DOM resuspended from sediments
453 (Harfmann et al., 2021) but also in dark microbial incubations (LaBrie et al., 2022) or in
454 microbial incubations after 49 days (Li et. al. 2025). These two last studies suggested that the
455 increase in NOSC indicates the creation of recalcitrant compounds. By contrast, Lechtenfeld et
456 al. (2014) did not support a correlation between NOSC and DOM degradation rate of DOM but
457 they reported a positive correlation between NOSC and the residence time of molecules. This is
458 in line with the assumption that more oxidized molecules have a low energetic value for
459 prokaryotes because they require energy-efficient uptake systems (Kattner et al., 2011; Perdue,
2009). This may explain the increase and persistence of high NOSC observed in summer in our study. The use of NOSC to evaluate DOM reactivity is more often used in studies involving sediment-extracted DOM and or anoxic environments (Boye et al., 2017; Zhang et al., 2021). Thus, further data of DOM might be needed to understand NOSC relationship to DOM bioavailability.

However, the proportion of CRAM, a major constituent of recalcitrant DOM (Hertkorn et al., 2006), decreased from spring to summer. CRAM are shown to be sensible to photobleaching (Stubbins et al., 2010) which may explain their removal at surface in the summer period. In this line, we also detected summer increases in the CDOM spectral slope $S_{275-295}$ in the upper mixed layer, indicative of the prevalence of low molecular weight compounds, suggesting photobleaching (Hansen et al., 2016; Helms et al., 2008). The likely photodegradation of CRAM in summer in surface was further supported by the negative correlation between their relative abundance and the $S_{275-295}$ (Table S2). Generally, photodegradation of recalcitrant CRAM results in enrichments in relatively more labile compounds with high H/C ratios such as unsaturated aliphatic (UA) (Chen et al., 2014; Medeiros et al., 2015). We did not observe increases in H/C in summer, supporting further that DOM accumulated is enriched in recalcitrant compounds (D'Andrilli et al., 2015).

A possible explanation for the increase in DOM recalcitrance during the summer accumulation period is the prokaryotic DOM reprocessing during this period: the changes in SPE-DOM quality that we observe in our study (i.e. summer increases in unsaturation, aromaticity, *m/z*, and SN heteroatom content) were also reported during prokaryotic incubations (Cai et al., 2019; Lechtenfeld et al., 2015; Lian et al., 2021). Likewise, the observed increase of SN heteroatom content in summer has been suggested to be a good indicator for microbial alteration (Koch et al., 2014). Harir et al. (2022) showed that photodegradation has also a significant effect on DOM heteroatom composition with S and N compounds increased during photo-exposure incubations. The release of likely recalcitrant DOM by heterotrophic prokaryotes under P limited condition was also showed in incubation studies (Bouchachi et al., 2022).
In summer, several processes such as photodegradation, phytoplankton extracellular release and prokaryotic degradation and release occur simultaneously, explaining the increase in SPE-DOM diversity in summer. Even when the relative importance of each process is difficult to detangle, photochemical degradation of DOM could influence its prokaryotic transformation into recalcitrant DOM (Bittar et al., 2015; Miranda et al., 2020; Obernosterer and Benner, 2004; Rossel et al., 2013). The presence of phytoplankton-derived DOM is also supported by a high dissolved primary production in oligotrophic summer conditions (Alonso-Sáez et al., 2008; López-Sandoval et al., 2011). Evidence from D-L amino acids analyses corroborates the contribution of prokaryotes to the DOM pool (Kaiser and Benner, 2008; Kawasaki and Benner, 2006) with a higher concentration of D-amino acids in summer but a presence of phytoplankton-derived DOM at this period (Von Jackowski et al., in 2024).

A high chemical diversity with low individual concentrations were suggested as one of the reasons behind DOM persistence in the ocean, i.e., the dilution hypothesis (Dittmar, 2015, Dittmar and Lennartz, 2024). Within this context, DOM accumulation in summer would result not only in the accumulation of intrinsically recalcitrant compounds (as seen by the increases in recalcitrance indices) but also in the increase of its diversity, that might result in lower degradability given the need of more diverse metabolic pathways for its degradation. The increase in the functional and abundance-based diversity of SPE-DOM in summer might be interpreted as an accumulation of a high number of chemically and functionally different molecules, with a myriad of potential isomers (Zark et al., 2017), each at low individual concentrations hampering their uptake (Arrieta et al., 2015).

## Conclusion and implication for carbon sequestration

Our results provide the first analysis of seasonal changes in molecular composition of SPE-DOM in the Mediterranean Sea. We observed that during summer stratification, recalcitrant and diverse SPE-DOM accumulates in the surface mixed layer. We suggest a combined effect of prokaryotic processing and photodegradation on DOM resulting in a diverse pool enriched in recalcitrant compounds. This recalcitrant SPE-DOM accumulated in the
surface will be exported to deep layers in the autumn-winter mixing where it may undergo more
reprocessing and likely be accumulated and stored. Testing bioavailability of this summer
surface DOM to deeper microbial communities will give more insights about its potential
recalcitrance and transformations. With carbon sequestration becoming an increasingly
important issue, it is worthwhile to explore whether the predicted increases in the length of the
summer stratification period would enhance both photodegradation and nutrient limitation,
therefore increasing the pool of recalcitrant DOM. In such a case, the MCP might play an
important role in carbon sequestration in the Mediterranean Sea.

## 524 Acknowledgements

This study was funded by INSU through the project ODISEA (LEFE-CYBER 2021)
and the French national science agency through the project ANR MicroPump (ANR-20-
CE01-0007) to E.O.-R.. We thank the crew of the R/V Nereis II and the Banyuls
Observation Service, specially Eric Maria and Paul Labatut, for sample collection;
Olivier Crispi for inorganic nutrient analyses, and David Pecqueur and Christophe
Salmeron from the Imaging and Flow Cytometry (BioPIC) platform of the Oceanology
Observatory of Banyuls-sur-Mer (OOB), whose French state funds are managed by the
ANR within the Investments of the Future program under reference ANR-10-INBS-02,
for flow cytometric data. The authors would like to thank the National Observing
Service (SNO) MOOSE, coordinated by the Research Infrastructure ILICO (CNRS-
IFREMER) dedicated to coastal ocean observations. Financial support from the IR
INFRANALYTICS FR2054 for the FT-ICR analyses is gratefully acknowledged. N.B
was supported by a doctoral fellowship of the Doctoral School 129 from Sorbonne
University. We thank Dr. Bianchi and two anonymous reviewers for helpful comments
on the manuscript.

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