# Dissolved organic matter offsets the detrimental effects of climate change in the nitrogen-fixing cyanobacterium Crocosphaera

Alba Filella, Jacqueline Umbricht, Angelina Klett, Angela Vogts, Thomas

Vannier, Olivier Grosso, Maren Voss, Lasse Riemann, Mar Benavides

► To cite this version:

Alba Filella, Jacqueline Umbricht, Angelina Klett, Angela Vogts, Thomas Vannier, et al.. Dissolved organic matter offsets the detrimental effects of climate change in the nitrogen-fixing cyanobacterium Crocosphaera. Limnology and Oceanography Letters, 2024, 9 (3), pp.296-306. 10.1002/lol2.10380 .
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## LETTER

# Dissolved organic matter offsets the detrimental effects of climate change in the nitrogen-fixing cyanobacterium *Crocosphaera*

Alba Filella,<sup>1,2</sup> Jacqueline Umbricht,<sup>3</sup> Angelina Klett,<sup>3</sup> Angela Vogts,<sup>3</sup> Thomas Vannier,<sup>2</sup> Olivier Grosso,<sup>1</sup> Maren Voss,<sup>3</sup> Lasse Riemann,<sup>4</sup>
Mar Benavides<sup>1,2*</sup>

<sup>1</sup>Aix Marseille Univ, Université de Toulon, CNRS, IRD, MIO UM 110, Marseille, France; <sup>2</sup>Turing Center for Living Systems, Aix-Marseille University, Marseille, France; <sup>3</sup>Biological Oceanography, Leibniz Institute for Baltic Sea Research, Warnemünde, Germany; <sup>4</sup>Marine Biological Section, Department of Biology, University of Copenhagen, Copenhagen, Denmark

### Scientific Significance Statement

Diazotrophs fix dinitrogen into bioavailable nitrogen, sustaining ocean primary productivity. Increasing anthropogenic CO₂ emissions decrease seawater pH and induce warming enhancing water column stratification. This will reduce inorganic nutrient availability, forcing microbial communities to obtain resources from dissolved organic matter instead. While the source of nitrogen for diazotrophs is inexhaustible, their ability to use dissolved organic matter (DOM) may influence their performance in the future warm and acidified ocean. Here, we examine the response of *Crocosphaera* to DOM additions under three climate change scenarios. Our findings show that the availability of dissolved organic matter helps diazotrophs facing energetic stress under high temperature and low pH, allowing a more balanced metabolism and a winning strategy to survive in the future warm and acidified ocean.

### Abstract

Diazotrophs provide a significant reactive nitrogen source in the ocean. Increased warming and stratification may decrease nutrient availability in the future, forcing microbial communities toward using dissolved organic matter (DOM). Not depending on reactive nitrogen availability, diazotrophs may be “winners” in a nutrient-depleted ocean. However, their ability to exploit DOM may influence this success. We exposed cultures of the widespread *Crocosphaera* to low (26°C, pH 8.1), moderate (28°C, pH 8.0), and extreme (30°C, pH 7.9) climate

**Author Contribution Statement:** AF, JU, MV, LR, and MB designed the experiments. AF acclimated the cultures prior to the experiments. AF, JU, and AK performed the experiments and sampling. OG ran MIMS analyses. JU and AV analyzed nanoSIMS samples. TV did the bioinformatic analyses. AF did the formal data analyses, statistics, and graphs. AF and MB wrote the manuscript with input from all co-authors.

**Data Availability Statement:** All the data generated by this experiment are available in the Dryad and Figshare repositories at https://doi.org/10.5061/dryad.70rxwdc5j and www.doi.org/10.6084/m9 figshare.22773503, respectively. The RNAseq data are deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE229037 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229037). Additional Supporting Information can be found in the online version of this article. An accompanying paper evaluates the potential changes in dissolved inorganic nitrogen uptake by *Crocosphaera* under the same treatment conditions (Umbricht et al. in prep.).

Additional Supporting Information may be found in the online version of this article.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

<footnote>*Correspondence: mar.benavides@ird.fr</footnote>

<footnote>Associate editor: Angelique White</footnote>
change scenarios, under control or DOM-amended conditions. Growth was suboptimal in the low and extreme treatments and favored in the moderate treatment. DOM was preferred as a carbon source regardless of the treatment and promoted N₂ fixation in extreme conditions. This was reflected in the increased expression of photosynthesis genes to obtain energy. DOM provides *Crocosphaera* with a key ecological advantage, possibly dictating diazotroph-derived nitrogen inputs in the future ocean.

Increasing anthropogenic CO₂ emissions induce ocean warming and acidification, affecting ecosystems and biogeochemical cycling (Gruber 2011; Cheng et al. 2019; Rossi 2023). Stratification inhibits the vertical supply of nutrients impacting biological productivity and consequently biomass transfer and carbon export (Passow and Carlson 2012). In the future ocean, reduced nutrient availability may force microbial communities to depend on regenerated compounds such as dissolved organic matter (DOM; Thornton 2014; Lønborg et al. 2020). Increased CO₂ also alters the seawater carbonate system, weakening the ocean's buffering ability and reducing pH (Orr et al. 2005; Doney et al. 2009). While low pH has been generally observed to decrease phytoplankton growth (Burkhardt et al. 1999; Hinga 2002), increased CO₂ concentrations can improve the affinity of the RuBisCO enzyme enhancing primary productivity (Riebesell et al. 2007; Wu et al. 2010). In contrast, heterotrophic bacteria are more resistant to low pH and their metabolic rates tend to increase with temperature (Grossart et al. 2006; Kirchman et al. 2009; Morán et al. 2017). Thus, ecosystem functioning in the future ocean will be significantly driven by the sensitivity of different planktonic groups to changes in CO₂, pH, and temperature (Riebesell and Tell 2011; Dutkiewicz et al. 2015; Sunagawa et al. 2015).

Prokaryotic cells called “diazotrophs” replenish the nitrogen oceanic reservoir by fixing dinitrogen (N₂), sustaining primary productivity (Zehr 2011). As the main suppliers of reactive nitrogen, the response of diazotrophs to future ocean conditions can have profound implications in productivity and the ocean’s role as a climate buffer (Hutchins and Capone 2022). Diazotrophic cyanobacteria, including *Trichodesmium* and *Crocosphaera* predominate in (sub)tropical waters where temperatures range between ~20°C and 27°C (Breitbarth et al. 2007; Sohm et al. 2011), while the symbiotic UCYN-A and noncyanobacterial diazotrophs (NCDs) can be found across wider latitudinal ranges (polar to tropical waters; 4 to > 25°C; Martínez-Pérez et al. 2016; Harding et al. 2018; Turk-Kubo et al. 2022). Despite the diversity of diazotrophs, most climate change research has focused on *Trichodesmium* and *Crocosphaera* (e.g., Kranz et al. 2009; Garcia et al. 2013a; Hutchins et al. 2013). The different temperature optima of *Trichodesmium* and *Crocosphaera*, 24–28°C and 26–31°C, respectively (Webb et al. 2009; Sohm et al. 2011; Fu et al. 2014) will likely influence their biogeography and N₂ fixation activity as the ocean becomes warmer (Breitbarth et al. 2007; Hutchins et al. 2007).

Models predict altered nutrient availability changing inter-
species competition, favoring diazotrophs (Dutkiewicz

et al. 2021). Under increased CO₂ levels, the energy saved in intracellular carbon concentration allows diazotrophs to invest more energy in fixing N₂ (Levitan et al. 2007; Kranz et al. 2011). Previous work examined the response of *Trichodesmium* and *Crocosphaera* cultures to partial pressure of CO₂ (*pCO₂*) levels between 150 and 1500 ppm (e.g., Hutchins et al. 2007, 2015; Fu et al. 2008; Kranz et al. 2011). Some studies observed increased N₂ fixation from 380 to 750 ppm *pCO₂* (Hutchins et al. 2007; Levitan et al. 2007; Boatman et al. 2017), but others only observed enhanced N₂ fixation when comparing much larger *pCO₂* ranges (e.g., 190 vs. > 350 ppm; Kranz et al. 2009; Garcia et al. 2013a). These positive responses only seem to hold under high iron or light conditions (Fu et al. 2008; Garcia et al. 2013b). However, culture-based studies do not reflect field studies, where diazotroph assemblages show a high resilience to short-term *pCO₂* perturbations (Böttjer et al. 2014; Gradoville et al. 2014a). In contrast, when temperature and *pCO₂* increase are combined, *Trichodesmium* growth decreases if temperature exceeds the strain optimum (Boatman et al. 2017), suggesting that the interlinked climate change stressors will affect diazotrophic performance in the future ocean.

Although CO₂ and photosynthesis provide the main
sources of carbon and energy for marine cyanobacteria—
including diazotrophs—DOM may supply an alternative
source of carbon, energy, and nutrients when facing stress
(e.g., Muñoz-Marín et al. 2013). For example, the unicellular
cyanobacterium *Cyanothecе* can grow on glycerol (Feng
et al. 2010), the filamentous *Nodularіа* can assimilate leucine
(Hietanen et al. 2002), and UCYN-A and *Crocosphaera* may use
sugars when photosynthesis is energy-limited (Moisander
et al. 2012). *Trichodesmium* and *Crocosphaera* are able to use
phosphoesters (Dyhrman et al. 2002; Orchard et al. 2003), but
only *Trichodesmium* obtains phosphorus from phosphonates
(Dyhrman et al. 2006). *Crocosphaera* can obtain both phos-
phorus and energy from phosphoanhydrides (Filella
et al. 2022). These studies point toward an overlooked role of
osmotrophy, the uptake of DOM, for cyanobacteria survival
in the future ocean. In this study, we sought to understand
the potential of DOM as an alternative carbon and/or energy
source for *Crocosphaera* when subjected to future temperature
and pH conditions (IPCC 2014; Pachauri et al. 2014). Com-
bining molecular, physiological, and biogeochemical tech-
niques, we find that DOM is an important carbon source for
*Crocosphaera*, compensating for the energy and stress imbal-
ances faced under future climate change conditions. These
results call for a reassessment of the role of DOM for dia-
zotrophic cyanobacteria, as nitrogen inputs in the future strat-
ified ocean may be closely linked to these interactions.

Methods

To provide a comprehensive assessment of the behavior of *Crocosphaera* under future ocean conditions, we combined temperature and pH levels simulating representative concentration pathways (RCP) 2.6 (26°C and pH 8.1), 4.5 (28°C and pH 8.0), and 6.0 (30°C and pH 7.9) (IPCC 2014), here called “low,” “intermediate,” and “extreme” treatments (10.6084/m9.fishshare.22773503). Cultures of *Crocosphaera* were acclimated to the different treatments for 1 month in 500-mL bottles before the start of the experiment in larger volumes (40 liters; duplicate low and triplicate intermediate and extreme treatments) with a starting concentration of ~1.5 × 10⁴ cells mL⁻¹ (Fig. 1A). The experiment was run for 8 d (here days –2 to 5) under a 12-h light : dark cycle with a light intensity of ~250 µmol photons s⁻¹. The temperature

and pH levels of each treatment were maintained constant along the experiment using an IKS aquastar system (Supporting Information). Temperature and salinity were measured daily with a YSI Pro30 probe, while pH was measured spectrophotometrically (Supporting Information). Cell abundance and nutrient concentrations were measured daily using standard microscopy and colorimetry methods (Supporting Information). On alternate days (days 0, 2 and 4), subvolumes were sampled from each treatment and incubated to measure single-cell N₂ and dissolved inorganic carbon (DIC) fixation using dissolved ¹⁵N₂ and ¹³C-HCO₃, respectively. Similarly, a mix of ¹³C-labeled organic compounds representative of marine DOM including the amino acids alanine, leucine, and arginine, and the carbohydrates glucose and N-acetylglucosamine was used to measure DOM uptake rates and RNA expression responses (see Supporting Information). RNA was sequenced using an Illumina NovaSeq 6000 platform generating more than 30 million paired end reads per sample (2 × 50 bp). RNA reads were annotated using Diamond and the databases Kofam UniProt's Swissprot, trEMBL, and NCBI's RefSeq (see Supporting Information).

<figure><img src="image_2.png" /><figcaption>**Fig. 1.** (A) *Crocosphaera* cell abundance in the low, intermediate, and extreme treatments along the experiment, where each dot represents the average, and the error bars the standard deviation of analytical duplicates in each treatment replicate. (B) Single-cell DIC and (C) N₂ fixation rates in the different treatments on days 0, 2, and 4, where each dot represents the uptake rate for single cells and boxplots show the 25th to 75th percentile, error bars the minimum and maximum percentile and the black dot and crossbar the median and mean values, respectively. Negative day values (days −2 to 0) correspond to the post-acclimation period, when *Crocosphaera* cells are transferred from concentrated cultures to the larger tanks, while positive day values (days 0–5) correspond to the experimental period.</figcaption></figure>

Results and discussion

Responses to combined temperature and pH treatments

Temperature and pH remained close to the target values in all treatments (10.6084/m9.fishshare.22773503). Nutrient conditions were similar between treatments and favorable for diazotrophic growth, indicating that any responses in growth, DIC and N₂ fixation can be confidently attributed to the different treatments (Supporting Information). Cells grown in batch incubations without continued nutrient additions, slowly consume limiting elements contained in the culture medium (YBC-II), including vitamins and iron which cause cultures to end collapsing.

*Crocosphaera* grew exponentially from day −2 until day 0 in all treatments, increasing from ~ 1.5 to >2.7 × 10⁴ cell mL⁻¹ (Fig. 1A). Growth in the low and extreme treatments reached a plateau between days 0 and 2, then decreased until day 5 (Fig. 1A). Conversely, cells in the intermediate treatment continued growing until day 4 and only started decaying after. The intermediate treatment thus was the most beneficial for *Crocosphaera*, in agreement with previous studies where the thermal range of *Crocosphaera* oscillates between 26°C and 31°C and optimal growth is ~ 28°C (Waterbury and Willey 1988; Falcón et al. 2005; Webb et al. 2009; Fu et al. 2014).

DIC and N₂ fixation rates differed among treatments (ANOVA; *p* < 0.001; Supporting Information Fig. S1), ranging between 0 and 34.4 fmol C cell⁻¹ d⁻¹ and between 0 and 10.3 fmol N cell⁻¹ d⁻¹, respectively (Fig. 1B), in the range of previous field measurements at similar temperatures (i.e., ~ 26–30°C; Foster et al. 2013; Bonnet et al. 2018; Benavides et al. 2022). While neither DIC nor N₂ fixation rates varied significantly with time in the low treatment, rates increased
on day 2 in the other two treatments (Fig. 1B,C). However, on day 4 both rates increased in the intermediate treatment where cells were actively growing but decreased in the other two treatments (Fig. 1A,B). Our results agree with previous reports of enhanced DIC and N₂ fixation with increased temperature or pCO₂ during exponential growth (Fu et al. 2014; Gradoville et al. 2014a,b). The drop in DIC and N₂ fixation rates observed in the extreme treatment on day 4 suggests that cells cannot withstand these harsh conditions for experimental periods of time. Similarly, natural assemblages of picocyanobacteria increase their photosynthetic rates with elevated pCO₂ initially but are unable to maintain elevated rates after 3 d of incubation (Lomas et al. 2012). A closer look at the intrapopulation variability confirms that while extreme conditions may enhance DIC and N₂ fixation momentarily, they became detrimental over time as suggested by the decrease of metabolically active cells between days 2 and 4 (Supporting Information Fig. S4). Similar intra-population variability has been observed in wild and cultured *Crocosphaera* (Foster et al. 2013, Masuda et al. 2020), which questions previous interpretations of increased pCO₂ having a positive effect on *Crocosphaera*.

Comparative transcriptomics captures changes in gene expression identifying regions involved in the regulation of gene expression (Ozsolak and Milos 2011, Wilhelm and Landry 2009). Although gene expression is a complex process regulated at many cellular levels (e.g., Buccitelli and Selbach 2020), it is generally defined as the production of an observable phenotype by a gene, ultimately directing the synthesis of a protein, thus showing how cells attempt to regulate their metabolism (Alberts et al. 2002). Here, we use transcriptomics to investigate differential gene expression of *Crocosphaera* under three future temperature and pH treatments in control or DOM-amended conditions. We screened for the most differentially expressed genes among treatments and experimental time points, covering many important metabolic pathways of this species (Supporting Information Table S1).

Connecting physiological responses to gene expression, we observed that the DIC fixing gene *rbcL*, encoding for part of the RuBisCO enzyme, was least expressed in the extreme treatment and most expressed in the low treatment on day 4 (Supporting Information Fig. S3), as previously observed in *Synechocystis* (Price et al. 2008). This could be due to the thermal damage of RuBisCO, as seen in *Trichodesmium* (Boatman et al. 2017). Increased pCO₂ is thought to alleviate the energy investment that concentrating carbon involves, in turn promoting both DIC and N₂ fixation (Hutchins et al. 2007; Levitan et al. 2007). N₂ fixation (*nif*) genes expression were not mirrored with N₂ fixation rates at any time point of the experiment (Supporting Information Fig. S3; Fig. 1B), which may be due to post-translational modifications and/or differing energy availability for nitrogenase activity (Kranz et al. 2011; Levitan et al. 2010). Alternatively, the time of RNA sampling

at sunrise might not reflect the effect of treatments on N₂-fixation genes, as *nif* gene expression in *Crocosphaera* peaks at sunset (Wilson et al. 2017).

Through 1st steps of photosynthesis, light is harvested and transferred to generate energy and reductants that keep most metabolic processes active including DIC and N₂ fixation (Steward 1980; Kranz et al. 2011). The lower expression of the light-harvesting machinery coincided with lower DIC and N₂ fixation rates in the low and extreme treatments (Fig. 1B; Supporting Information Fig. S3), reflecting a close link between the expression of energy-producing genes and *Crocosphaera*'s main metabolic activity. Stress-induced protein genes such as heat shock chaperones were highly expressed in the low and extreme treatments (Supporting Information Fig. S3). Molecular chaperones allow cyanobacteria to maintain protein conformation homeostasis, which is key for temperature stress adaptability (Blondin et al. 1993). *Synechocystis* and *Synechococcus* upregulate about 90 different heat shock proteins after 1 h exposure to heat stress (Slabas et al. 2006) and similar responses are observed when exposed to cold temperatures (Porankiewicz and Clarke 1997). Moreover, osmotic and oxidative stresses promote heat shock expression in cyanobacteria (Apte et al. 1989), suggesting that these genes regulate essential cellular functions (Rajaman et al. 2014). Collectively, our comparative transcriptomics analysis confirms that cells in the low and extreme treatment were more stressed than in the intermediate treatment where the higher expression of light-harvesting genes allowed cells to keep balanced DIC and N₂ fixation activity (Supporting Information Fig. S3). Moreover, the high expression of stress-induced proteins maintained over time only in the extreme treatment suggests a strong effect of low pH and high temperature, negatively affecting all cellular functions despite potential energetic benefits of higher pCO₂.

## DOM as a source of carbon when facing environmental stress

*Crocosphaera* obtained at least twice more carbon from DOM as from DIC, independently of the treatment (ANOVA; *p* = 0.12; Fig. 2). DOM thus provided a preferred source of carbon under all conditions tested here, suggesting that osmometry is an important trait of *Crocosphaera*. Given the lability of the amended ¹³C-DOM, these results should be carefully extrapolated to natural environments where most DOM is generally refractory. Considering that 1% of the background DOM in our cultures was labile, DOM uptake rates decreased by 94% and were two times lower than DIC fixation rates in all treatments on day 0, and remained lower (*t*-test, *p* < 0.01) in the intermediate but similar (*t*-test, *p* > 0.1) in the low and extreme treatments for the rest of the experiment. In nature, DOM uptake rates by *Crocosphaera* may be in the above ranges. Furthermore, despite the competitiveness of *Crocosphaera* against other community components for these labile substrates is unknown, this diazotroph shows
<figure><img src="image_3.png" /><figcaption>**Fig. 2. (A)** Single-cell DIC fixation and (B) DOC uptake rates in unamended and DOM-amended conditions, respectively, for each sampling time point. Each dot represents the uptake rate for single cells.</figcaption></figure>

significant uptake of organic carbon derived from a mixture of representative natural carbohydrates and amino acids (see Supporting Information). Diazotrophic cyanobacteria may use DOM as an alternative source of carbon, nutrients and/or energy (Dhyrman et al. 2006; Filella et al. 2022), for example *Trichodesmium* can obtain as much carbon from amino acids as from DIC (Benavides et al. 2017). In contrast, the non-diazotrophic cyanobacterium *Prochlorococcus* obtains more carbon from DIC than from DOM uptake but can assimilate glucose in culture and field conditions (Moisander et al. 2012; Muñoz-Marín et al. 2013; Duhamel et al. 2018). Direct glucose assimilation is more energetically favorable than producing it through the Calvin Cycle (Muñoz-Marín et al. 2013). Moreover, while DIC is tightly coupled to the reactive carbonate system and hence susceptible to pH changes, the chemical composition of DOM does not change significantly with varying pH, providing a more stable carbon source under changing environmental conditions (Zark et al. 2015). The cellular machinery to assimilate DOM may also be more resistant to pH changes than that required to fix DIC, as observed by similar gene expression of the main carbohydrate degradation pathways regardless of pH changes (Supporting Information Fig. S3). In all, DOM seems a more stable source of carbon for cyanobacteria under low pH conditions.

Diazotrophic cyanobacteria are able to obtain carbon and phosphorus from DOM, which can enhance their N₂ fixation

activity (Benavides et al. 2017, 2020; Dyhrman et al. 2007). However, organic nitrogen molecules contained in DOM may instead inhibit N₂ fixation (Mulholland and Capone 1999; Walworth et al. 2018). The DOM mix used here contained ala-nine, arginine and leucine, but average N₂ fixation rates were similar under DOM-amended or unamended conditions with few exceptions (Fig. 3). Again, single-cell analyses showed beneficial responses to DOM additions, masked if treating the population as whole (Fig. 3; Supporting Information Fig. S2). On day 4, N₂ fixation rates were close to zero in the low and extreme treatments (Fig. 3), where only ~18% and 21% of the cells were actively fixing N₂, respectively (Supporting Information Fig. S2), while upon DOM amendments ~30% and 54% of the measured cells actively fixed N₂ (Supporting Information Fig. S2; Fig. 3). In contrast, the percentage of cells actively fixing N₂ remained similar over time in the interme-diate treatment where cells were healthier (Fig. 1A; Supporting Information Fig. S2). Our results support previous field experi-ments arguing that DOM aids cyanobacterial diazotrophs fix-ing N₂ when facing environmental stress (Benavides et al. 2018; Bonnet et al. 2013).

Differential gene expression was observed in response to DOM additions, with changes among growth phases and treatment conditions (Fig. 4). Most genes increased their expression upon DOM additions on day 4, while only a few differences were measured in the intermediate treatment on day 2 (Fig. 4).
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Single-cell N₂ fixation rates in unamended and DOM-amended conditions, for each experimental treatment and time. Each colored dot represents the uptake rate for single cells and the centered gray dot the average value of these. Differences in the averages between conditions were tested with paired *t*-test (*p*-values are shown for each comparison).</figcaption></figure>

DOM amendments did not influence the expression of DIC fixation genes (Fig. 4). Expression of N₂ fixation genes increased slightly in all treatments on day 4, and in the extreme treatment on day 2 upon DOM additions (Fig. 4), providing inconclusive trends and uncoupled patterns with the measured rates as seen above (Supporting Information Fig. S3). Conversely, DOM additions enhanced the expression of light-harvesting genes in the low treatment and components of the photosystem II in all treatments on day 2 (Fig. 4). In *Crocosphaera*, photosynthesis genes are expressed before sunrise (Wilson et al. 2017) and their expression depends on cellular resources produced on the previous day (Dron et al. 2012). Therefore, cells can easily face energy limitation if metabolic processes are not balanced throughout the diel cycle. For example, *Crocosphaera* shows a temporal delay in cell division of approximately 1 h after sunrise reflecting the need to recover cellular reserves with light-derived energy after night-time activity (Wilson et al. 2017). Hence, DOM could provide an additional and energetically cheaper source of carbon for restoring the light-harvesting components when cells are energetically deprived after the dark period. Overall, DOM enhanced the expression of the machinery required to

produce cellular resources to fix DIC and N₂, improving cell performance specially under stress conditions including late growth stage, non-optimal temperature, and low pH.

*Crocosphaera* in the future ocean

In a future ocean, decreased nutrient supply may limit non-diazotrophic phytoplankton growth, benefitting diazotrophs as long as iron and phosphorus remain available (Weber and Deutsch 2010; Hutchins and Capone 2022). Phosphorus concentrations, unlike N₂ and carbon, will decrease considerably with intensified stratification, while the enhancement of desertification and dust deposition could increase iron and phosphorus inputs regionally, creating diazotroph niches (Jickells et al. 2005; Moulin and Chiapello 2006; Hamilton et al. 2020).

We show that DOM uptake was higher and more constant than DIC fixation regardless of the treatment, providing *Crocosphaera* with an extra and secure carbon source. DOM additions allowed cells in the low and extreme treatments to maintain a greater number of cells actively fixing N₂ than in unamended conditions, which coincided with increased expression of genes involved in the 1st steps of photosynthesis.
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Differentially expressed genes between DOM-amended and unamended samples (*p* < 0.01) for each treatment on days 2 and 4. Genes are grouped by common pathway or function (see Supporting Information Table S1 for gene full names and grouping). Positive fold changes (purple dots) indicate genes that were more expressed in DOM-amended samples than in unamended samples for each treatment. Negative fold changes and genes showing no differences between both conditions are colored in gray. The dot size increases with increasing fold change.</figcaption></figure>

Overall, DOM became especially important in treatments or at growth stages where cells were more limited, presumably lacking stored resources to maintain a balanced metabolism over time.

Osmotrophic *Crocosphaera* may maintain balanced growth in a future stratified, warm, and acidic ocean, contributing to nitrogen inputs where they predominate. Warming may favor *Crocosphaera* relative to other coexisting diazotrophs in (sub) tropical waters like *Trichodesmium* (Fu et al. 2014; Breitbarth et al. 2007; Yang et al. 2021), while increasing *pCO₂* may

similarly affect both species (Hutchins et al. 2007; Fu et al. 2008). Our results indicate that warming and acidification are more adverse when combined, and that beyond thermal and pH limits, metabolic versatility to exploit DOM is likely to influence the expansion of diazotrophic niches, potentially dictating nitrogen inputs in the future. Therefore, investigating osmotrophy in other prominent diazotrophs such as *Trichodesmium*, UCYN-A and NCDs should be prioritized if we are to understand their role in the future ocean.
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## Acknowledgments

This research was supported by the NOTION project by the BNP Paribas Foundation for Climate and Biodiversity (MB). The MIMS equipment used in this study was obtained with European FEDER Funds. The NanoSIMS at the Leibniz-Institute for Baltic Sea research in Warnemuende (IOW) was funded by the German Federal Ministry of Education and Research (BMBF), grant identifier 03F0626A. The bioinformatic platform has been funded from France 2030, the French Government program managed by the French National Research Agency (ANR-16-CONV-0001) and from the Excellence Initiative of Aix-Marseille University—A*MIDEX. The authors would like to thank M. Camps and S. Bonnet for their comments and valuable advice on diazotroph culture work. F. Van Wambeke and S. Duhamel for advice regarding the experimental setup and osmotrophy studies, T. Wagener and F. Gazeau for carbonate system measurements advice, and E. Seris, J. Berdun, C. Ducrocq, and J-C. Cancel for their help during pilot experiments. We are indebted to M. Lafont, D. Malengros, K. Idri, N. Bhairy of the SAM platform of the MIO and to L. Thyssen and the Endoume marine station for facilitation and assistance in setting up the experimental infrastructure and advice in handling large volumes. The captain and crew of R/V *Antedon II* are warmly thanked for the collection of seawater and, D. Lefèvre, O. Pringault and S. Alliouane for training and assistance in light, oxygen respiration and total alkalinity measurements, respectively. The PRECYM Flow Cytometry Platform (https://precym.mio.osupytheas.fr/) and S. Nunige are acknowledged for flow cytometry and nutrient analyses respectively, and the Centre de Calcul Intensif d'Aix-Marseille for granting access to its high-performance computing resources.

Submitted 11 May 2023
Revised 20 December 2023
Accepted 24 January 2024