# Effects of Phosphorus Limitation on the Bioavailability of DOM Released by Marine Heterotrophic Prokaryotes

Nawal Bouchachi, Ingrid Obernosterer, Cécile Carpaneto Bastos, Franck Li, Lorenzo Scenna, Barbara Marie, Olivier Crispi, Philippe Catala, Eva Ortega-Retuerta

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## To cite this version:

Nawal Bouchachi, Ingrid Obernosterer, Cécile Carpaneto Bastos, Franck Li, Lorenzo Scenna, et al. "Effects of Phosphorus Limitation on the Bioavailability of DOM Released by Marine Heterotrophic Prokaryotes." Microbial ecology, 2023, 10.1007/s00248-023-02201-1. hal-04117517

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# Effects of phosphorus limitation on the bioavailability of DOM released by marine heterotrophic prokaryotes

**Nawal Bouchachi**

CNRS/Sorbonne Université, UMR7621 Laboratoire d'Océanographie Microbienne, Banyuls sur Mer

**Ingrid Obermosterer**

CNRS/Sorbonne Université, UMR7621 Laboratoire d'Océanographie Microbienne, Banyuls sur Mer

**Cécile Carpaneto Bastos**

CNRS/Sorbonne Université, UMR7621 Laboratoire d'Océanographie Microbienne, Banyuls sur Mer

**Franck Li**

CNRS/Sorbonne Université, UMR7621 Laboratoire d'Océanographie Microbienne, Banyuls sur Mer

**Lorenzo Scenna**

Università Politecnica delle Marche

**Barbara Marie**

CNRS/Sorbonne Université, UMR7621 Laboratoire d'Océanographie Microbienne, Banyuls sur Mer

**Olivier Crispi**

CNRS/Sorbonne Université, UMR7621 Laboratoire d'Océanographie Microbienne, Banyuls sur Mer

**Philippe Catala**

CNRS/Sorbonne Université, UMR7621 Laboratoire d'Océanographie Microbienne, Banyuls sur Mer

**Eva Ortega-Retuerta** (✉ ortegaretuerta@obs-banyuls.fr)

CNRS/Sorbonne Université, UMR7621 Laboratoire d'Océanographie Microbienne, Banyuls sur Mer

## Research Article

**Keywords:** Dissolved organic matter, heterotrophic prokaryotes, protein-like fluorescence, humic-like fluorescence, prokaryotes diversity

**Posted Date:** November 23rd, 2022

**DOI:** https://doi.org/10.21203/rs.3.rs-2288825/v1

**License:** © This work is licensed under a Creative Commons Attribution 4.0 International License.
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**Additional Declarations:** No competing interests reported.
**Version of Record:** A version of this preprint was published at Microbial Ecology on March 13th, 2023. See the published version at https://doi.org/10.1007/s00248-023-02201-1.
**Abstract**

Heterotrophic prokaryotes (HP) contribute largely to dissolved organic matter (DOM) processing in the ocean, but they also release diverse organic substances. The bioavailability of DOM released by HP under varying environmental conditions are not fully elucidated. In this study, we investigated the effect of P-limitation on the bioavailability of DOM released by a single bacterial strain and 2 natural HP communities. These HP were grown under P-replete and P-limited conditions, and their DOM (HP-DOM) was used as a substrate for natural prokaryotic communities from a coastal site in the Northwestern Mediterranean Sea. We followed changes in prokaryotic growth, enzymatic activity, diversity and community composition together with the consumption of HP-DOM fluorescence (FDOM). HP-DOM produced under P-replete and P-limited conditions promoted both significant and similar prokaryotic growth. The humic-like fluorescence, commonly considered recalcitrant, was consumed during the incubations when this peak was initially dominating the FDOM pool, and this consumption coincided with higher alkaline phosphatase activity. HP-DOM supported growth of diverse prokaryotic communities and P-driven differences in HP-DOM quality selected for different indicator taxa in the degrading communities. Taken together, our findings emphasize that HP-DOM lability is dependent on both the DOM quality, shaped by P availability, and the composition of the consumers community.

**Introduction**

Dissolved organic matter (DOM) is a large pool of reduced carbon consisting of diverse molecules with various degrees of reactivity. A significant portion of this DOM is recalcitrant to microbial degradation, being thus a carbon sequestration mechanism [1]. Heterotrophic prokaryotes (HP) play an important role in DOM processing in the ocean [2] and are well known to uptake DOM for biomass production and respiration. But besides their role as DOM consumers, HP metabolism leads also to the generation of DOM. This process, termed the microbial carbon pump (MCP) [3], was suggested to make an important contribution to carbon sequestration [4]. The MCP encompasses different pathways [5] including successive microbial transformation of labile DOM into recalcitrant DOM and direct release of recalcitrant DOM by HP [6–11]. These processes sustain a large DOM pool, including DOM that can be refractory depending on the environmental context, i.e. compounds that are refractory in specific biogeochemical condition but labile in others; and DOM that is concentration-constrained refractory, i.e. comprising a variety of molecules each present at concentrations that would be below the uptake limit by prokaryotes [12]. DOM bioavailability to prokaryotes can be then viewed as a relative concept that can be challenging to address. Additional information on DOM released by HP, and the factors influencing its release and its further bioavailability, are crucial for a better understanding of carbon sequestration through the MCP.

DOM lability in the ocean can be controlled by the composition and metabolic capacities of the degrading community [13–16], but is also shaped by environmental variables such as inorganic nutrients or temperature [17, 18]. Nutrient availability, for example, can alter both the suite of DOM molecules released to the environment and their uptake. Many areas in the ocean are subject to phosphorus (P) limitation [19, 20], particularly in the Mediterranean Sea [21]. Previous studies showed that the condition in which
DOM is released can influence its availability to bacteria [22, 23]. Using exudates of diatoms grown under P enriched and P depleted conditions, [23] showed that bacterial growth was significantly lower in the exudates from P depleted diatoms. However, while previous studies showed that P-limitation can influence the HP extracellular metabolome [24] and HP-derived DOM (HP-DOM) optical properties [25, 26], no studies have investigated the effects of P-limitation on the bioavailability of DOM released by HP.

In this study, we aimed to determine the bioavailability of DOM released by HP (a single bacterial strain and 2 natural HP communities from different seasons) grown under P-limitation and P-repletion. We have shown previously that HP-DOM released under P-limitation was enriched in humic-like fluorescence [25] largely considered refractory [27–30], while under P-repletion protein-like fluorescence, considered labile [31–33], prevailed. We hypothesize that (i) DOM released under P-limitation would thus promote lower prokaryotic growth and that (ii) the labile protein-like fluorescence would decrease during the incubation contrary to the likely refractory humic-like DOM. To test these hypotheses, we carried out short-term incubations of prokaryotic communities from the NW Mediterranean Sea with HP-DOM collected during the stationary phase from different culturing conditions. HP-DOM consumption was followed as changes in DOM fluorescence spectroscopy (FDOM). The response of the prokaryotic communities to the different type of DOM was investigated using flow cytometry enzymatic activity and 16S rRNA sequencing.

# Methods

## Experimental design

To evaluate the bioavailability of DOM released by HP under P-repletion and P-limitation, we conducted two-step experiments. First (Fig. 1a), a single bacterial strain (*Sphingopyxis alaskensis*) and 2 natural HP communities from different seasons (SOLA-fall and SOLA-spring) were grown in minimum media with glucose (200 µMol C L⁻¹) as the sole carbon source under P-replete and P-limited conditions (coded balanced “B” and unbalanced “U”). Cultures were harvested at stationary phase, and DOM in the media was separated from the cells by 0.2 µm filtration. Since little glucose (1.8 to 23.4 µmol C L⁻¹) was remaining at the end of the cultures of the 1st step, measured dissolved organic carbon (DOC) was assumed to reflect that released by the HP. This released DOC quantification and DOM characterization is described elsewhere [25]. In the 2nd step of the experiment, the DOM derived from the bacterial strain and the HP communities was used as substrate for biodegradation experiments (Fig. 1b). Inocula of *in situ* prokaryotic communities from the Mediterranean Sea were added to the DOM collected in the release experiments, i.e. DOM released by the strain and the two HP communities under P-limited (U-DOM) or P-replete (B-DOM). *In situ* DOM (IS-DOM) alone or *in situ* DOM supplemented with 20 µmol L⁻¹ of glucose (G-DOM) were used as additional culture media in each of the experiments. The G-DOM treatment served as a control to rule out that HP grew only on glucose leftovers of the first step of the experiment. IS-DOM was collected from the same location and date than the prokaryotes inoculum of the respective experiment (Fig. 1, table S1).
To prepare the inocula, sea water collected at the SOLA observatory (Northwestern Mediterranean Sea, 42°29'300N-03°08'700E) was first prefiltered through 0.8 µm to remove phytoplankton and grazers. Two to 3 L of the prefiltered water was then reduced to 200-250 mL by filtration through 0.2 µm filters using a vacuum pump, to concentrate the inocula (Table S1). We then stopped the filtration and resuspended the cells stuck to the filter by pipetting out and in several times. This volume was then distributed to the different treatments as inoculum and an aliquot was reserved for 16S rRNA gene sequencing.

The incubations consisted of 10% (vol/vol) of the inocula and 90 % of DOM from each treatment. Inorganic nutrients ($NH_4^+$ and $PO_4^{3-}$) were added to force carbon limitation (Table S2). The incubations were performed in triplicates at *in situ* temperature in the dark. Cell abundance was followed through the incubation and samples for FDOM, exoenzymes activity, and diversity were taken at the onset ($T_0$) and end of the incubations ($T_f$).

## Chemical and biochemical analysis

Prokaryotic cell abundances were followed by flow cytometry using a BD Biosciences FACSCanto. Samples were fixed using Glutaraldehyde (0.5 % final concentration) and preserved at -80°C until analysis. Samples were thawed and stained with SYBR Green. Fluorescent beads were added as internal standards and prokaryotic cells were identified in plots of side scattered light vs. green fluorescence.

Samples for FDOM were filtered through 0.2 µm polycarbonate filters and analysed within few hours upon sampling. Excitation emission matrices (EEMs) were recorded using a JASCO FP-8500 Spectrofluorimeter in a 10 mm quartz cell at a scan speed of 5000 nm min⁻¹. Excitation scans ranged between 240 to 450 nm at 5 nm intervals and emission scans were recorded between 300 to 560 nm at 2 nm intervals. MilliQ EEM blanks were recorded in every batch of analysis. Fluorescence intensities were reported in Raman units (RU) obtained by dividing the fluorescence units by the MilliQ blank peak area (Raman scatter) excited at 350 nm. EEMs were separated into 6 distinct components using parallel factor analysis (PARAFAC) under the drEEM toolbox in MATLAB (Murphy et al., 2013). These results are described elsewhere [25]. We used the protein-like component $C_{340}$ (ex 275/em 340) as an indicator of labile DOM and the microbial humic-like $C_{398}$ (ex 314/em 398) as an indicator of recalcitrant DOM.

Extracellular enzyme activities were quantified using fluorogenic substrates in a plate reader. Each sample was pipetted in triplicate into 96-well black plates and added to the following substrates: 4-methylumbelliferyl phosphate (for alkaline phosphatase APA), and L-leucine-7-amido-4-methyl coumarin (for leu-aminopeptidase AMA) (final concentration of 125 µmol L⁻¹). Fluorescence was measured immediately after addition of the substrates, after 1.5 hour, and after 3 to 4-hour incubations in the dark at the same temperature as for the experimental incubations. Fluorescence readings were done with a JASCO FP-8500 Spectrofluorimeter at 365/450 nm ex/em wavelengths. The increase of fluorescence units during the period of incubation was converted into enzymatic activity (µM h⁻¹) with standard curves prepared with 7-amido-4-methylcoumarin for AMA and 4-methylumbelliferone for APA.
Samples for HP diversity (70 to 140 mL) were filtered onto 0.2 µm polycarbonate filters that were stored at -80°C until analysis. Frozen filters were then cut with sterilized scissors. Cell lysis was conducted in 2 steps: adding 50 µL of lysozyme (20 mg mL⁻¹) and incubating for 45 min at 37 °C followed by a 1-hour incubation at 55 °C after adding 10 µL proteinase K (20 mg mL⁻¹). DNA was then purified using Quick DNA™ fungal/bacteria miniprep kit (Cat. No.: D6005 Zymo Research). Amplification and sequencing were done at LGC Genomics GmbH (Germany). 16sRNA gene amplicon sequencing (300bp paired end read Illumina Miseq V3) was done using the primer pair 515FY-926Rjed [34]. Samples demultiplexing using Illumina bcl2fastq, as well as adapter and primer clipping were performed by the sequencing company. Further processing, including filtering and trimming, dereplication, sample inference, merging pair reads and removing chimeras, were done under R using the DADA2 package (v1.18.0, Callahan et al., 2016). Obtained amplicon sequence variants (ASVs) were taxonomically assigned using the Genome Taxonomy Database (GTDB) [36].

## Data analyses

Differences in cell abundance between treatments were tested using repeated measures ANOVA and post hoc pairwise t-tests (package rstatix in R, v0.7.0; Kassambara, 2021). Differences in exoenzymatic activities were tested using a two-way ANOVA and post-hoc pairwise t-tests (package rstatix in R, v0.7.0; Kassambara, 2021). To see dissimilarities in prokaryotic communities' structure we used a Non-metric Multidimensional Scaling (NMDS), based on Bray Curtis dissimilarity matrices, then differences in communities' structure were tested by ANOSIM tests (package vegan, 2.5.7; Oksanen et al., 2020). This was performed on ASVs relative abundance obtained after rarefaction to the minimum number of reads per sample, using *rrarefy* function package vegan, 2.5.7; Oksanen et al., 2020). An indicator ASVs analysis was run using *indval* function from package labdsv (v2.0.1 Roberts, 2019) based on the absolute abundance after rarefaction. Only ASVs with more than 100 reads were considered for this analysis.

# Results

## Growth on DOM released by HP

Prokaryotes from the Mediterranean Sea grown on B-DOM and U-DOM (DOM released under P-repletion and P-limitation respectively) from *S. alaskensis* reached significantly higher abundances than if grown on IS-DOM and on G-DOM (Fig. 2a; repeated measurement ANOVA and post hoc pairwise t-test in Table S3, Fig S1). In contrast, DOM released by natural HP communities SOLA-fall and SOLA-spring in both B- and U-DOM treatments supported lower prokaryotic carrying capacities as compared to the IS-DOM and the G-DOM (Fig. 2b, c; Table S3, Fig S1). No clear differences in cell abundances between B and U-DOM treatments were observed in the *S. alaskensis* DOM biodegradation experiment. Cells grew slightly higher in the B-DOM treatment during the exponential phase but tended to be similar to the U-DOM treatment in the stationary phase. However, significantly higher cell numbers (Table S3, Fig S1) on U-DOM as compared to B-DOM were detected in both SOLA-fall and SOLA-spring experiments (Fig. 2b, c).
## Fdom Consumption And Exoenzyme Activity

Whenever the protein-like component C₃₄₀ was initially dominant, this component was largely consumed over the course of the incubations. Similarly, the humic-like C₃₉₈ was consumed in those treatments where this component dominated at the onset of the experiments.

At the onset of the degradation experiments, the protein-like component C₃₄₀ was dominant in the B-DOM treatments in the SOLA-fall and SOLA-spring experiments with FDOM > 4 10⁻² RU (Table 1). The C₃₄₀ component was generally lower in both *S. alaskensis* DOM treatments (B-DOM and U-DOM) and in U-DOM treatments in SOLA-fall and SOLA-spring experiments (Table 1). The humic-like component C₃₉₈, however, was high in both B and U-DOM in *S. alaskensis* and in the U-DOM SOLA-fall experiments (initial values ≥ 1.7 10⁻² RU) but lower in both B- and U-DOM treatments of SOLA-spring (Table 1). In the *S. alaskensis* DOM degradation experiment, the protein-like component C₃₄₀ remained stable both in the B-DOM and U-DOM treatments, while the humic-like component C₃₉₈ decreased (Fig. 3a, b). In SOLA-fall, a pronounced decrease in C₃₄₀ was observed in the B-DOM treatments, while it was less depleted in U-DOM (Fig. 3a). The humic-like C₃₉₈ was only consumed in the U-DOM treatment (Fig. 3b). In SOLA-spring, a C₃₄₀ consumption was detected in the B-DOM treatments but not in the U-DOM and there was no decrease of C₃₉₈ (Fig. 3a, b).

In parallel, APA was slightly higher in the *S. alaskensis* B-DOM than in the U-DOM, however this difference was not statistically significant. APA was higher in the SOLA-fall U-DOM, where humic-like FDOM C₃₉₈ was consumed, than in B-DOM (Fig. 3b). AMA did not differ among treatments or experiments (Fig. 3b).

## Changes In Prokaryotic Community Composition

Samples for community composition were only taken in *S. alaskensis* and SOLA-fall DOM experiments. Changes in diversity and composition were observed in all treatments (Table 2, Fig. 4). At T₀, prokaryotic communities growing on *S. alaskensis* and SOLA-fall were both highly dominated by *Vibrionaceae* (92% and 56–95%, respectively) and minor contributions from *Flavobacteriaceae*, *Pelagibacteriaceae* and *Rhodobacteraceae* were observed (Fig. 4a, b). The dominance of *Vibrionaceae* at the onset of the experiments could be due to the concentration procedure of the inocula, that would favour the presence of larger cells such as *Vibrio* species (See experimental design). However, the prokaryotic taxa that were initially present at low relative abundances made up the majority of the community composition at T_f. This led to an increase in diversity (either expressed as Shannon or evenness) between T₀ and T_f in both experiments (Table 2). At T_f, there were no significant differences in diversity (evenness, Shannon) between the B-DOM and U-DOM treatments (p-value > 0.05). The *S. alaskensis* derived DOM led to a lower diversity compared to its *in situ* DOM treatment while HP community derived DOM (SOLA-fall) led to similar diversity as *in situ* DOM (Table 2).
<table><caption>Table 1
Initial (T₀) values of component C₃₄₀ (protein-like) and C₃₉₈ (microbial-humic fluorescence), expressed in x 10⁻² Raman units (RU). B-DOM: DOM produced under P-repletion. U-DOM: DOM produced under P-limitation</caption><thead><tr><th>DOM source</th><th>Treatment</th><th>C<sub>340</sub></th><th>C<sub>398</sub></th></tr></thead><tbody><tr><td rowspan="2">S. alaskensis</td><td>B-DOM</td><td>1.82</td><td>2.34</td></tr><tr><td>U-DOM</td><td>3.45</td><td>5.71</td></tr><tr><td rowspan="2">SOLA-fall</td><td>B-DOM</td><td>4.87</td><td>0.62</td></tr><tr><td>U-DOM</td><td>2.44</td><td>1.72</td></tr><tr><td rowspan="2">SOLA-spring</td><td>B-DOM</td><td>5.71</td><td>1.08</td></tr><tr><td>U-DOM</td><td>2.98</td><td>0.46</td></tr></tbody></table>

In the *S. alaskensis* DOM degradation experiment, the B-DOM treatment was dominated by *Alteromonadaceae* (> 20%) and *Methylophagaceae* (> 30%), except for replicate 2 where *Methylophagaceae* were not present. In the U-DOM treatment, *Vibrionaceae* and *Rhodobacteraceae* were the dominant families, contributing each to more than 29% and 23% to the community composition, respectively (Fig. 4a). In the SOLA-fall experiment, both B-DOM and U-DOM showed relatively high proportions of *Alteromonadaceae*, *Flavobacteraceae* and *Rhodobacteraceae* but the U-DOM treatment was characterized by the presence of *Methylophilaceae* (Fig. 4b). In both experiments, the G-DOM treatment was dominated by *Vibrionaceae* at the end of the incubation (24 to 83%). The NMDS ordination showed that T₀ samples of both experiments grouped together, but showed separation among experiments at T<sub>f</sub> (ANOSIM, p-value < 0.05) and separation among treatments as replicates grouped together (ANOSIM, p-value < 0.05) (Fig. 4c). Therefore, we further studied the two experiments independently to identify which taxa are specifically associated to each treatment.

To identify which taxa were specifically associated to a DOM source, an indicator taxa analysis was run at the ASV level for each experiment separately. In the *S. alaskensis* experiment, the number of indicator ASVs in the B-DOM and U-DOM was 3 and 2 ASVs respectively while the G- and IS-DOM treatment had a higher indicator ASV number (4 and 27 respectively). B-DOM indicator ASVs accounted for 32%, in average, of the community in this treatment and consisted of 3 families: *Alteromonadaceae*, *Marinomonadaceae* and a few *Rhodobacteraceae* (Fig. 5a). Indicator ASVs of U-DOM represented up to 23% of the U-DOM community, mainly belonging to the family *Rhodobacteraceae* and few *Flavobacteriaceae* (Fig. 5b). G-DOM indicator ASVs belonged to *Vibrionaceae*, *Flavobacteriaceae* and *Sphingomonadaceae* while the IS-DOM indicators were representative of several families including *Pelagibacteraceae* and *Flavobacteraceae* (Fig. 5c, d).
Indicator taxa analysis for the SOLA-fall experiment showed significant indicator ASVs for all treatments. The indicator ASVs of B-DOM (8 ASVs) represented up to 44% of the community in this treatment and were dominated by *Rhodobacteraceae* and *Alteromonadaceae* with a lower contribution from *Methylophilaceae* and *Saccharospirillaceae* (Fig. 5e). Indicator ASVs of U-DOM accounted for 19%, in average, of the community of this treatment. U-DOM indicators belonged to *Methylophilaceae*, *Rhodobacteraceae*, *Vibrionaceae* and *Sphingomonadaceae*. Indicator ASVs of G-DOM treatment consisted of mainly *Vibrionaceae* and showed a high contribution to the other treatment communities at T<sub>f</sub> (14 to 45%). Even though many families were repeatedly shown as indicators in different treatments of both experiments (e.g. *Rhodobacteraceae*, *Methylophilaceae*, etc), the ASVs were different as illustrated by the genus level taxonomy (Fig. S2). For example, *Rhodobacteraceae* were respectively represented by *Litoreibacter* and *Pelagimonas* in *S. alaskensis* U-DOM and B-DOM while it was rather *Thalassobacter* and *Pseudophaeobacter* in SOLA-fall U-DOM and B-DOM, respectively.

Discussion

Our experiments allowed us to confirm that a fraction of DOM released by heterotrophic prokaryotes is
labile, promoting significant prokaryotic growth in all incubations, in line with previous studies [10, 31]. In
contrast to our hypothesis, clear differences in DOM lability released under P-repletion and P-limitation (B-
DOM and U-DOM, respectively) could not be evidenced based on the cell growth, and P-limitation was not
demonstrated to decrease the lability of the released DOM.

Fdom Consumption

Although clear differences in cell growth were not observed between U-DOM and B-DOM, different
patterns in FDOM consumption were detected. The protein-like component C<sub>340</sub>, usually considered as a
proxy of labile DOM [33, 37], was only partially used on the time scale of our incubations. Lønborg et al.
[31], using long term incubations, suggested that a fraction of bacterial derived protein-like FDOM could
be refractory. However, in contrast to our initial hypothesis, the microbial humic-like component C<sub>398</sub> was
consumed by the communities from the Mediterranean Sea in some of the incubations. This component
has been considered refractory that resists bacterial degradation [28, 29], however we evidence that the
microbial humic-like FDOM fluorophore released by HP is not always refractory and can be partially used
when this DOM component dominates. Previous studies in marine and freshwater environments reported
bacterial consumption of similar humic-like fluorescence (summarised in Table 3).
<table><caption>Table 3
Comparison to previous studies reporting the consumption of microbial humic like fluorescence originating from different sources.</caption><thead><tr><th>Study</th><th>FDOM peak</th><th>DOM Producer / source</th><th>Consumer</th><th>% of consumed FDOM</th></tr></thead><tbody><tr><td></td><td>Ex Em</td><td></td><td></td><td></td></tr><tr><td rowspan="4">[38]</td><td rowspan="4">310 392</td><td>Single Phytoplankton</td><td rowspan="4">Natural HP community</td><td>30.3%</td></tr><tr><td><i>Skeletonema cotatum</i></td><td>5%</td></tr><tr><td><i>Chaetoceros sp.</i></td><td>10%</td></tr><tr><td><i>Micromonas pusilla</i></td><td></td></tr><tr><td rowspan="2">[39]</td><td rowspan="2">310 420</td><td><i>Stream water</i></td><td rowspan="2">Stream water Bacteria</td><td rowspan="2">16%</td></tr><tr><td>(white Clark Creek)</td></tr><tr><td rowspan="6">[10]</td><td rowspan="6">320 410</td><td>Single bacteria strains</td><td>HP communities collected in:</td><td></td></tr><tr><td rowspan="4"><i>Photobacterium angustum</i></td><td>500 m, March</td><td>20%</td></tr><tr><td>5 m, March</td><td>30%</td></tr><tr><td>5 m, April</td><td>59%</td></tr><tr><td>5 m, December</td><td>0%</td></tr><tr><td rowspan="2"><i>S. alaskensis</i></td><td>5 m, April</td><td>57%</td></tr><tr><td>5 m, December</td><td>11%</td></tr><tr><td rowspan="3">[40]</td><td rowspan="3">320 410</td><td>Mesopelagic North Atlantic water fractionated to low (L) and high (H) molecular weight DOM</td><td rowspan="3">Natural HP community</td><td>0%</td></tr><tr><td>LDOM</td><td>2%</td></tr><tr><td>L + H DOM</td><td></td></tr><tr><td>[41]*</td><td>330 386</td><td>Coastal HP</td><td>Coastal HP</td><td>52%</td></tr><tr><td rowspan="4">Actual study</td><td rowspan="4">315 398</td><td>Single bacteria strains</td><td>HP communities collected in</td><td></td></tr><tr><td><i>S. alaskensis</i> under P replete condition</td><td>5 m, February</td><td>32.5%</td></tr><tr><td><i>S. alaskensis</i> under P limited condition</td><td></td><td>60.9%</td></tr><tr><td>Fall HP communities under P-replete</td><td>5 m, November</td><td>0%</td></tr></tbody></table>
<table><thead><tr><th>Study</th><th>FDOM peak</th><th>DOM Producer / source</th><th>Consumer</th><th>% of consumed FDOM</th></tr></thead><tbody><tr><td></td><td>Ex Em</td><td>Fall HP communities under P-limited</td><td></td><td>78.3%</td></tr><tr><td></td><td></td><td>Spring HP communities under P-replete</td><td>5 m, May</td><td>0%</td></tr><tr><td></td><td></td><td>Spring HP communities under P-limited</td><td></td><td>0%</td></tr></tbody></table>

* in the present studies fluorescence consumption was estimated as the decrease from the maximum of fluorescence along the same incubation.

Based on these studies, the humic-like fluorescence was used to different extent (Table 3) depending on its molecular weight [40], source [38] and or the degrading community [10]. In our incubations, only 32.5–78.3% of the humic-like DOM was consumed. The percentages of prokaryotic degradation of microbial humic-like FDOM derived either from bacteria or phytoplankton (Table 3) are higher than the degradation of DOM directly isolated from natural waters [39, 40]. This is most likely because humic-like DOM isolated from natural systems is more processed than DOM produced over short time incubations. Together with our results, this also suggests that similar humic-like FDOM peaks could be of different bioavailability and likely different chemical composition since fluorescence spectroscopy captures only a bulk signal from DOM. Further DOM analyses (e.g. high-resolution mass spectrometry) would be needed to elucidate this point.

Our results indicate that humic-like FDOM use is also related to the lack of other DOM compounds. In our experiment, the humic-like FDOM component was consumed only when the presumably labile protein-like component was initially not prevailing, implying that heterotrophic prokaryotes are able to use less labile carbon sources when the presumably labile ones are scarce. This is in line with the results by [43], that followed the bioavailability of *Synechococcus*-derived DOM to estuarine and coastal bacteria for 180 days and showed that a similar humic-like fluorophore can be produced by some bacterial groups when labile *Synechococcus*-derived DOM is available, then re-used by other bacterial groups after the labile DOM is depleted. Microbial humic-like consumption was also accompanied by a consistently high alkaline phosphatase activity. Although high alkaline phosphatase activity could be interpreted as a response to P-limitation, this was not the case in our incubations since inorganic P was added (Table S2). Studies demonstrated that prokaryotes can use alkaline phosphatases to unbind bioavailable organic carbon [44]. We hypothesize that alkaline phosphatase in our experiments was used to break P-containing DOM polymers to use them as a substrate.

## Selection Of Community Composition According To Hp-dom Quality

HP-DOM quality driven by P availability appeared to change the taxonomic composition of the Mediterranean Sea communities growing on it. Both HP-DOM released under P-repletion and P-limitation (B- and U-DOM) lead to the growth of diverse communities, suggesting that HP-DOM is composed of a
multitude of substrates. Previous studies using ultra-high resolution mass spectroscopy revealed a large number of molecules in bacterially derived DOM [24, 45]. Landa et al., [46] discussed different effects of DOM generated by diatoms and cyanobacteria on the composition and diversity of bacterial communities. According to their results, the more chemically diverse the DOM is, the higher the diversity of the community growing on it.

Even though prokaryotic diversity did not differ between B- and U-DOM, community composition analyses revealed different taxonomic groups growing on the different HP-DOM sources, indicating the different composition of the HP-DOM released under P-repletion vs. P-limitation. Few families, belonging mainly to *Alphaproteobacteria* and *Gammaproteobacteria* and few *Bacteroidia*, dominated the HP-DOM incubations. However, the ASVs differed among the experiments and the treatments, suggesting that community composition plays an important role in determining DOM lability. Members of *Rhodobacteraceae*, *Alteromonadaceae* (*Pseudophaeobacter*, *Alteromonas*) were the main indicators of the protein-like rich treatment in SOLA-fall B-DOM. Some members of the Alteromonadales class have already been shown to be related to the protein-like peak T, corresponding to our component C<sub>340</sub>, in bacterial incubations of high and low molecular weight DOM suggesting their preference for labile DOM [40]. DOM rich in humic-like components, namely *S. alaskensis* DOM and SOLA-fall U-DOM, selected different indicator taxa, including mainly *Methylophilaceae*, *Marinomonadaceae*, *Rhodobacteraceae* and *Alteromonadaceae*. A coupling between FDOM Peak M (corresponding to our C<sub>398</sub>) and several members of *Rhodobacterales*, was observed in communities growing on low molecular weight DOM [40] or on viral lysates of cyanobacteria (Xiao et al., 2021). Members of *Marinomonadaceae*, particularly *Marinomonas*, have been shown to degrade complex organic matter such as terrestrial DOM inputs in high latitudes [48]. But they are also known to be associated with Cyanobacteria lysates [49]. This suggests that they can respond positively to various carbon sources including complex DOM or humic-like DOM as shown here. In addition, Alteromonadales, *Burkholderiales* (*Methylophilaceae*), and *Rhodobacterales* are among most abundant heterotrophic marine bacteria that might hydrolyse dissolved organic phosphorus outside the cytoplasmic membrane using alkaline phosphatase [50], in line with our results showing high alkaline phosphatase activities in these treatments.

Overall, our results illustrate how P availability shapes HP-DOM degradation patterns, suggesting that P would play a key role shaping prokaryote-mediated DOM fluxes in the ocean. Although the growth of Mediterranean Sea communities did not differ between P-replete and P-limited derived DOM, different metabolisms and taxonomic groups were selected for the different DOM conditions. We could show that a considerable portion of humic-like DOM might be bioavailable when this DOM pool prevails as a substrate for HP. This bioavailability is also controlled by the releasing conditions, and the consumers community composition. Our results emphasize that DOM lability is context dependent, and implies that the impact of P-limitation on DOM fluxes via the MCP should be placed on its local environmental context rather than generalized to a global context.

## Statements And Declarations
Acknowledgements

This work was supported by the Caramba (European commission, H2020-MSCA-IF-2015-703991), ODISEA (Institut National des Sciences de l'Univers LEFE/CYBER 2019) and MicroPump (Agence Nationale de Recherche ANR-20-CE01-0007) projects to EOR. Flow cytometric analyses were performed at the BioPic cytometry and imaging platform (Sorbonne University/CNRS). The authors thank the crew of R/V 'Nereis II' and the technicians of the Banyuls observation service for their assistance in getting Mediterranean Sea samples for the natural communities' isolation.

Funding

This work was supported by the Caramba (European commission, H2020-MSCA-IF-2015-703991), ODISEA (INSU LEFE/CYBER 2019) and MicroPump (ANR JCJC 2020) projects to EOR.

Competing interests

The authors have no competing interests to disclose.

Authors contributions

NB, EOR and IO conceived the experimental work. NB, FL, CCB, LS and EOR performed the experiments with active contribution from OC, BM and PC to chemical and biological analyses. NB analysed data with contribution from FL, CCB, LS and EOR. NB wrote the manuscript with significant contributions from IO and EOR. All authors reviewed and approved the final manuscript.

Data availability

The datasets generated during and/or analysed during the current study are available from the
corresponding author on request. Sequence data will be uploaded in the EMBL-ENA data repository.

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Table

Table 2 is not available with this version

Figures

<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>

Scheme displaying the experimental design. HP-DOM: DOM released by heterotrophic prokaryotes. B-DOM: DOM produced under P-repletion. U-DOM : DOM produced under P-limitation. IS-DOM: in situ DOM collected at the same time than the natural HP inocula. G-DOM: in situ DOM added with 20 µmol L⁻¹ of glucose. SOLA: a coastal observation station in the Northwestern Mediterranean Sea
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

Changes in HP abundance over incubation time in the three biodegradation experiments: (a) S. alaskensis DOM, (b) SOLA-fall DOM, (c) SOLA-spring DOM. Dots correspond to triplicates. B-DOM: DOM produced under P-repletion. U-DOM : DOM produced under P-limitation. IS-DOM: in situ DOM collected at the same time than the natural HP inocula. G-DOM: in situ DOM added with 20 µmol L⁻¹ of glucose
<figure><img src="image_5.png" /></figure>

pwc: T test; pzejust: None

<caption>Figure 3</caption>

FDOM consumption and enzymatic activities in the B-DOM and U-DOM treatments of each experiment:
Difference between T~f~ and T~0~ (T~f~ - T~0~) fluorescence of (a) the protein-like component C~340~ and (b) the humic-like component C~398~. Values < 0 correspond to consumption. (c) Aminopeptidase activity (AMA) and (d) alkaline phosphatase activity (APA) at T~f~. Dots represent triplicates
HP community composition (≥1%)

<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>

HP community composition based on 16s rRNA sequencing in (a) the S. alaskensis and (b) SOLA-fall experiments. (c) NMDS plot of all replicates and treatments based on Bray Curtis dissimilarity. Note that samples at the onset of the experiment (T₀) were taken for the inoculum (T0 Inoc), B-DOM (T0B), U-DOM (T0U), G-DOM (T0G), IS-DOM (T0IS) for the SOLA-fall experiment while in S. alaskensis experiment only an inoculum (T0 Inoc) sample was taken
<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>

Contribution of B-DOM, U-DOM, G-DOM and IS-DOM indicator ASVs to T₀ and T_f communities
composition of the different treatments and their taxonomy at the family level, in *S. alaskensis* DOM
degradation experiment (a-d) and SOLA-fall DOM degradation experiment (e-h).

**Supplementary Files**
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