# Low-molecular-weight reduced sulfur substances: A major component of nonvolatile dissolved organic sulfur in the Pacific Ocean

Pierre Fourrier, Gabriel Dulaquais

► To cite this version:

Pierre Fourrier, Gabriel Dulaquais. Low-molecular-weight reduced sulfur substances: A major component of nonvolatile dissolved organic sulfur in the Pacific Ocean. Limnology and Oceanography Letters, 2024, 10.1002/lool.2.10417. hal-04655345

HAL Id: hal-04655345

https://hal.univ-brest.fr/hal-04655345v1

Submitted on 22 Jul 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
## LETTER

# Low-molecular-weight reduced sulfur substances: A major component of nonvolatile dissolved organic sulfur in the Pacific Ocean

Pierre Fourier¹,², Gabriel Dulaquais³*

¹Conservatoire National des Arts et Métiers, Intechmer, Cherbourg-en-Cotentin, France; ²LUSAC, Laboratoire Universitaire des Sciences Appliquées de Cherbourg, University Normandie, Cherbourg-en-Cotentin, France; ³Laboratoire des Sciences de l'Environnement Marin CNRS UMR 6539, Institut Universitaire Européen de la Mer, Université de Bretagne Occidentale, Plouzané, France

### Scientific Significance Statement

The role of sulfur (S) regarding the stability of marine dissolved organic matter remains unclear. Direct measurements of dissolved organic S (DOS) compounds are scarce in the ocean, making it difficult to decipher the bioavailability and dynamics of DOS. A combination of size exclusion chromatography and cathodic stripping voltammetry highlights the possibility of such quantification in a new way. Our results show that low-molecular-weight hydrophilic reduced sulfur substances (RSS) are major contributors to the DOS pool. RSS are produced by phytoplankton to prevent oxidative and metal stresses. These compounds are partly remineralized by microheterotrophs to fulfill their S requirement. This microbial alteration of DOM produces a very stable pool of secondary metabolites hydrophilic LMW DOS possibly composed of thioamidated RSS. In deep waters, the homogenous concentration of RSS highlights their recalcitrance to long-term mineralization.

### Abstract

The low-molecular-weight (LMW) reduced sulfur substances (RSS) composition of dissolved organic matter (DOM) was examined along the GEOTRACES US-GP15 section in the Pacific Ocean. We demonstrate that LMW RSS constitutes a significant fraction of nonvolatile dissolved organic sulfur (DOS). While thiols such as glutathione were below our detection limit (300 pM), RSS containing two carbon (C) sulfur (S) bonds were present at concentrations in the hundreds of nM range. RSS accumulation was observed in subtropical waters. The most likely source of these RSS is microbial alteration of sulfurized DOM with production of secondary thioamidated metabolites. RSS are initially produced by cyanobacteria to mitigate copper and oxidative stress induced by UV-B irradiance. A preferential remineralization of RSS over dissolved organic carbon (DOC) in the upper 350 m suggests a partial lability of LMW DOS. Deeper, homogeneous concentrations and C : S ratio indicate increasing stability of this LMW DOS.

<footnote>*Correspondence: gabriel.dulaquais@univ-brest.fr</footnote>

<footnote>Associate editor: Sarah Fawcett</footnote>

<footnote>Author Contribution Statement: Each of the authors contributed equally to the data processing and writing of this manuscript.</footnote>

<footnote>Data Availability Statement: Size exclusion chromatography data and cathodic stripping voltammetry data from this project are available in Dryad (https://doi.org/doi:10.5061/dryad.d51c5b0bq).</footnote>

<footnote>Additional Supporting Information may be found in the online version of this article.</footnote>

<footnote>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>
Among the ~660 Gt of carbon (C) contained in marine dissolved organic matter (DOM), ~640 Gt are refractory to bacterial remineralization and persists for thousands of years in the ocean (Hansell et al. 2009). Several hypotheses explaining the high stability of DOM over time were proposed, notably the concept of intrinsic recalcitrance, which suggests that reactivity is determined by the molecular properties and composition of DOM (Jiao et al. 2010; Lechtenfeld et al. 2015; Baltar et al. 2021). It was shown that a low-molecular-weight C to nitrogen ratio reflects a high lability of DOM but it is still not clear regarding sulfur (S). In marine waters, dissolved S is mainly under its oxidized form: sulfate. However, several oceanic microheterotrophs cannot reduce sulfate (Tripp et al. 2008; Dupont et al. 2012), despite a high S demand (Matrai and Eppley 1989). These microorganisms depend on a pool of ~6.7 Gt of dissolved organic S (DOS, Ksionzek et al. 2016) to fulfill their metabolism. Consequently, microheterotrophs play a key role in controlling the dynamic of DOS in the ocean (Ksionzek et al. 2016; Levine 2016) and S content in DOM may reflect a certain level of lability. However, in the deep ocean, DOS seems refractory to microbial degradation and only scarce data suggest that S is selectively remineralized compare to C in refractory DOM (Koch et al. 2017; Longnecker et al. 2020; Ibrahim and Tremblay 2023). Abiotic sulfurization of DOM in sulfate-reducing sediments and its subsequent release into the ocean was proposed as a pathway of production of refractory DOS (Pohlabeln and Dittmar 2015). During early diagenesis, in sulfide-bearing sediments, organic matter reacts abiotically (reactions additions during H and/or O removal/addition) with inorganic species (sulfides, polysulfides), forming organic S compounds (Damsté et al. 1989; Vairavamurthy et al. 1995; Schmidt et al. 2009). Abiotic sulfurization is a well-known process contributing to the protection of labile organic matter from microbes in fossil deposits (Sinninghe et al. 1998; Kok et al. 2000; Amrani et al. 2007; Bushnev and Burdel'Naya 2009). The release of refractory DOS from superficial sediments (Pohlabeln and Dittmar 2015) could balance the slow and continuous mineralization of DOS in refractory DOM (Ksionzek et al. 2016) and induces a change in the chemical nature of DOS as observed by Longnecker et al. (2020). Phillips et al. (2022) indicate that the diffusion of sulfurized dissolved organic matter (DOM) plays a negligible role in the overall distribution of DOS. This is in line with the recent findings from Ibrahim and Tremblay (2023) that the primary origins of water-column and sedimentary DOS are distinct.

The above opposing hypotheses are based on a limited amount of data as the determination of organic S in DOM is complex. On the one hand, total organic S concentrations can be calculated as the difference between total S determined by reductive pyrolysis and sulfate seawater concentration (Cutter et al. 2004; Longnecker et al. 2020). On the other hand, solid-phase extractions (SPE) along with mass spectrometry also provide an estimation of DOS concentrations

(Ksionzek et al. 2016; Phillips et al. 2022). However, this may lead to a bias since SPE fractionate DOM and cannot access the fraction of LMW hydrophilic DOM (Dulaquais et al. 2023). Alternatively, direct determination of reduced sulfur substances (RSS), such as thiourea, cysteine and/or glutathione, can be assessed either directly using cathodic stripping voltammetry technics (Al-Farawati and van den Berg 1997, 2001; Laglera and van den Berg 2003; Pernet-Coudrier et al. 2013) or after SPE and subsequent derivitization in line with high-pressure liquid chromatography (HPLC, Swarr et al. 2016). These RSS are present in the environment at a wide range of concentrations (pM to µM) and protect eukaryotic cells for from oxidative damage and exposure to toxic metals (Mopper and Kieber 1991; Leal and Van Den Berg 1998; Ercal et al. 2001; Rijstenbil 2002; Sunda et al. 2002; Jacob et al. 2003; Laglera and van den Berg 2003; Dupont et al. 2006; Dryden et al. 2007; Tilliette et al. 2023). RSS, especially thiols, may be exuded by phytoplankton (Leal et al. 1999; Swarr et al. 2016) but they seem mostly originate from heterotroph metabolism (Laglera and Tovar-Sánchez 2012; Mahanta et al. 2019; Moran and Durham 2019). Although RSS play a major role in biogeochemical interactions between S and marine microorganisms (autotrophs and heterotrophs) and have broader ecological roles such as for metal detoxification and microbial signaling (Moran and Durham 2019), their contribution to the pool of DOS in open ocean has, to our knowledge, been quantified only twice by Swarr et al. (2016) and Whitby et al. (2018).

The objective is to evaluate the spatial and vertical contribution of RSS detected by adsorptive cathodic stripping voltammetry (Ad-CSV) to the nonvolatile DOS pool in the Pacific. We study the sources and fate of thiols at large scale. A combination of Ad-CSV and size exclusion chromatography with organic carbon detection (SEC-OCD) datasets contributes to a better understanding of the role of low-molecular-weight (LMW) RSS in the oceanic DOS cycle.

## Experimental section

Samples were collected during the cruise GEOTRACES US-GP15 (Fig. 1a). Bulk, high-molecular-weight (HMW) and LMW DOC concentrations were measured by SEC-OCD (DOC-Labor®) (Huber et al. 2011; Dulaquais et al. 2018; Fourrier et al. 2022a, 2022b). The analysis of RSS was performed on bulk samples by Ad-CSV at a deposition potential of 0 V (vs. Ag/AgCl) with a deposition time of 180 s (Pernet-Coudrier et al. 2013). Further details on the methods used in this study are provided in the Supporting Information.

## Results and discussion

### DOC concentrations and fractionation in the Eastern Pacific

Bulk DOC concentrations ranged from 34.9 ± 1.0 µMC at 2633 m depth (20°S; 152°W) to 71.2 ± 2.2 µMC in the
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** (a) Location of the transect overlaid on a composite background map of chlorophyll a distribution (Chl a, µg L⁻¹; 8 daily 4-km over September–November 2018, MODIS-Aqua Satellite). Yellow boxes show Chl a data by week based on boat advance for the GEOTRACES US-GP15 cruise. (b) Time-averaged map of solar irradiance (310 nm) in the study area (mW m⁻² nm⁻¹; daily 1°). Figure generated using Giovanni (giovanni.gsfc.nasa.gov).</figcaption></figure>

euphotic layer at 55 m depth in the northern subtropical gyre (17.5°N; 152°W). DOC concentrations measured in this study were compared with data from CLIVAR P16N cruise (150°W; OCADS database, 2006). The vertical distributions of DOC concentrations as a function of depth (m) presented in Fig. 2 include data from the CLIVAR P16N cruise as well as data measured in this study (GEOTRACES transect US-GP15). Data were paired and the distribution clearly shows an overlap between the two datasets along the water column. The scatter plot of the paired data set (n = 271) shows a distribution along a 1 : 1 line taking into account the measurement uncertainties (Fig. 2b). A strong correlation (p < 0.05) with a R² equal to 0.93 between DOC from CLIVAR P16N and our measurements demonstrates that concentrations of DOC measured are consistent with the existing literature. Deviations from the 1 : 1 line mainly correspond to samples from surface waters (15–185 m) and mesopelagic waters (200–1000 m). These differences between the two datasets could be due to temporal changes in biological production or bacterial degradation (Cherrier et al. 1996; Thingstad et al. 1997; Morris et al. 2005; Carlson et al. 2007). This result confirms the relevance of using SEC for DOC determination in oceanic environment (Fourrier et al. 2022a). Preliminary results of DOC size fractionation along the section are presented in Fig. 2a. It clearly shows that HMW DOC was only significant (3% ± 2% of bulk DOC) in the upper 100 m of the water columns. The LMW hydrophilic DOC, that mostly escapes SPE using styrene divinyl benzene polymer resin (PPL) sorbent (Dulaquais et al. 2023), accounted for 28.8% ± 5.8% of bulk DOC all along the water column. All the RSS that can be detected by the Ad-CSV method we used (glutathione, cysteine, N-acetyl

cysteine, thioacetamide, thiourea) eluted in this fraction. These results imply that RSS are LMW hydrophilic compounds and may not be accurately recovered from seawater samples after SPE using PPL sorbent.

## Reduced sulfur species in the Eastern Pacific

Electrochemical analyses of RSS using 180 s of deposition time revealed the systematic occurrence of a peak at –0.193 V (vs. Ag/AgCl) in all analyzed samples (n = 395). This peak is typical of the thioacetamide (TA) electrochemical response and can be related to thioamidated compounds with thioamide and thiocarbonyl functional groups. Quantification of this peak using TA as external standard (referred as TA-like in the next sections) revealed concentrations ranging from 117.6 ± 14.1 nMeqTA at 125 m in the equatorial area to 1140.4 ± 136.8 nMeqTA at 25 m in the north subtropical gyre (Fig. 3). The TA-like distribution showed high concentrations in the upper 100 m (652 ± 213 nMeqTA, n = 48), decreasing in the upper mesopelagic zone (343 ± 180, n = 95 at depth deeper than 100 m) and reaching stable concentration (235 ± 41 nMeqTA; n = 252 at depths deeper than 600 m) (Fig. 3). In deep waters (> 600 m), neither significant variation or correlation with hydrographic (e.g., temperature, salinity, O₂) nor biogeochemical (nutrients) parameters were observed. The distribution of TA-like compounds we report in this study suggests a biogenic production of these RSS in surface and particularly in oligotrophic waters (Fig. 3). The rapid decrease of TA-like at the 100-m horizon indicates a partial lability of freshly produced TA-like compounds. The homogenous concentration in the deep sea (Fig. 3) indicates that a fraction of the TA-like pool becomes refractory to long-term
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** (a) Vertical profiles of DOC (µMC) vs. depth (m) for the campaigns CLIVAR P16N (Swan et al., 2009; deep blue dots), and GEOTRACES USGP15 (this study; gray dots). High-molecular-weight (HMW) DOC (> 10 kDa; green dots) and low-molecular-weight (LMW) DOC (< 0.3 kDa; yellow dots) are also represented for this study. (b) Comparison between the two datasets for global DOC. The observed data were paired with historical data by station and depth.</figcaption></figure>

remineralization during microbial alteration of DOM in the mesopelagic zone. Biologic origin of TA-like compounds is in agreement with the previous work of Leal et al. (1999). Direct production/excretion may not be the main production pathway of TA-like compounds. These compounds are probably thioamidated secondary metabolites from S-containing molecules such as, but not restricted to, methanobactin (Kenney et al. 2018), thioholgamide (Kjaerulff et al. 2017), and thioalbamide (Frattaruolo et al. 2019).

Differently to TA-like compounds, electrochemical signal of cysteine-like and glutathione-like compounds were only detected one and three times at subnanomolar levels. The low-detection rate of these thiols may be related to their subnanomolar to picomolar concentrations in oceanic

waters (Swarr et al. 2016; Whitby et al. 2018) as our limit of detection was 0.3 nM at the deposition time we used (Pernet-Coudrier et al. 2013). The difference of abundance between TA-like and these thiols can be related to their specific reactivity and stability.

This difference may partly originate from the internal C–S bonds of these compounds with TA-like compounds having a double C–S bound (among others thioamide and thiocarbonyl functional groups), while cysteine-like and glutathione-like have single C–S bound. The stability of C–S single bonds (< 300 kJ mol⁻¹) is low in oxygenated environments. Indeed, radical substitutions can lead to dissociation of the C–S bond within thiols (Rudyk and Eaton 2014). Among TA-like compounds, thioamide have a C–S double bond

<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Spatial and vertical distribution of sulfur compounds of thioacetamide type (TA; nMeqTA L⁻¹) along the GEOTRACES US-GP15 section (152° W).</figcaption></figure>
conjugated to a nonbonding doublet of the nitrogen atom
making them more stable, potentially explaining their detec-
tion in all the analyzed samples.

Surface waters (< 100 m) of the subtropical gyres and equa-
torial area showed RSS concentrations significantly higher
than in the subpolar gyre displaying a north to south meridio-
nal gradient of concentration (Fig. 3). Subpolar, north sub-
tropical, equatorial, and south subtropical surface waters
displayed concentrations of 537 ± 168 (n = 10), 640 ± 256
(n = 13), 646 ± 199 (n = 18) and 853 ± 41 (n = 7) nMeqTA,
respectively. The mean irradiance at 310 nm along the
section during the cruise (Fig. 1b) shows a north to south gra-
dient of surface waters exposition to UV-B. These observations
are in line with the biological importance of RSS in the regula-
tion of oxidative (Ercal et al. 2001; Rijstenbil 2002; Sunda
et al. 2002) and copper (Whitby et al. 2018) stresses. Further-
more, it is likely that high levels of TA-like observed in the
subtropical gyres reflect high production of RSS by cyano-
bacteria combined with continuous alteration and accumula-
tion in these stratified domains.

Cellular oxidative damages on lipids, proteins, and DNA result from direct UV-B solar exposure or by indirect oxidative damages from reactive oxygen species (ROS) induced by UV-B irradiance (He and Häder 2002). Inside the phytoplankton cell, thiols can react with ROS lowering the intracellular concentration of hydroxyl, peroxyl and superoxide radicals (Weiss 2005; Regoli and Giuliani 2014). Production of RSS compounds is a possible way for microorganisms to protect themselves from post-transcriptional oxidative modification of proteins (Rudyk and Eaton 2014). Large phytoplankton (diatoms) develop mostly in the subpolar gyre (Harrison et al. 1999) and the equatorial zone (Chavez et al. 1990) of the Pacific, while cyanobacteria (mainly *Prochlorococcus* and *Synechococcus*) dominate phytoplankton in the subtropical gyres (Winn et al. 1995; Ras et al. 2008). The surface gradient of RSS we measured from north to south (Fig. 3) may indicate the meridional variation in how the various phytoplankton communities respond to mitigating the production of ROS induced by UV-B radiation. On the one hand, for the subtropical gyres, increased sunlight exposure would lead to higher levels of ROS (Mopper and Kieber 2000). Consequently, there would be an increase in the biological production of RSS as a response to the induced oxidative stress (Latifi et al. 2009). This cellular response was observed in various thiol-related studies in the literature (Apel and Hirt 2004; Dupont et al. 2004; Bertini 2007; Fahey 2013). On the other hand, cloud cover and less sunlight in the subpolar gyre would not catalyze as much ROS production (Scully et al. 1996; Johannsson et al. 2017) resulting in a lower RSS production. Furthermore, diatoms that develop in the subpolar gyre and the equatorial zone may be less sensitive to these stresses compared to cyanobacteria from the subtropical gyres. These latter prokaryotic species have a single membrane that would make them more sensitive to oxidative stress and solar irradiation

compared to diatoms (Murphy 1983; Tyrrell 1991; Lesser 2006;
Llabrés and Agustí 2006; Agustí and Llabrés 2007).

Cyanobacteria are also likely major producers of RSS for
metal detoxification and acquisition (Singh et al. 1999;
Tilliette et al. 2023). Among RSS, thiols are a major class of
ligand particularly for the complexation of dissolved copper
(dCu). Thiols limits the toxicity of this element to phyto-
plankton by lowering ambient copper ion (Cu²⁺) concentra-
tions (Florence and Stauber 1986; Rijstenbil et al. 1998;
Morelli and Scarano 2004; Dulaquais et al. 2020). Alterna-
tively, they can increase the bioavailability of dCu (Walsh
et al. 2015). Thereby, thiols enhance acquisition or reduce
toxicity of Cu²⁺ depending on phytoplankton species. The
distribution of total dCu and labile dCu along GP15
section reported by Moriyasu et al. (2023) provides further
perspective onto the links between RSS, Cu, and UV irradia-
tion. According to authors, labile dCu (the more bioavailable
fraction) accumulates in the stratified subtropical waters. This
accumulation is probably induced by a continuous photo-
chemical decomposition of an inert fraction of dCu. In these
waters, cyanobacteria that have a low resistance to Cu toxicity
(Brand et al. 1986) may enhance their production of RSS to
keep Cu²⁺ at subpicomolar levels. This mechanism is in agree-
ment with the high RSS concentrations we report in the sub-
tropical domains. In addition, the previous works from
Whitby et al. (2018) and Ruacho et al. (2020) in the Pacific
showed higher dCu and Cu²⁺ in the surface waters of the sub-
polar gyre than in the equatorial waters. In these conditions,
phytoplankton may experience a higher copper stress, in
terms of scarcity, in the equatorial waters than in the subpolar
waters. This would have induced the higher production of RSS
for Cu²⁺ acquisition in the equatorial waters compared to the
subpolar gyre resulting in the difference of concentration of
RSS we observed (Fig. 3).

Contribution of RSS to DOS in the Eastern Pacific

To compare the DOS contained in RSS to bulk DOS, TA-like
concentrations in nMeqTA were directly converted into nM of
equivalent nonvolatile reduced DOS (RSS-DOS) by assuming
1 mol eq-TA = 1 mol DOS. This assumption relies on the
mechanism of TA quantification by the CSV method we used
(see Supporting Information) and consider that almost all S
functional groups in RSS are electroactive. Briefly during CSV
analysis, the sulfur is adsorbed on the working electrode, for-
ming a S–Hg complex. Then, during the voltammetric strip-
ping scan, the complex is reduced creating a quantitative
current proportional to the amount of electroactive S atoms
adsorbed. A scheme is proposed in Supporting Information
Fig. S2. The resulting TA-like DOS (117.6–1140.4 nM RSS-
DOS, Fig. 4a) concentrations are lower than the scarce data
reported in the literature for the West Atlantic total organic
sulfur (43.8–1275.1 nM, TOS; Longnecker et al. 2020) and
higher than DOS isolated by SPE using PPL (SPE-DOS) in the
East Atlantic (59–201 nM-SPE-DOS; Ksionzek et al. 2016). Our
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Compiled data along with depth of (a) organic sulfur concentrations for total (black dots), LMW RSS (white dots) and SPE (orange diamonds) in the Atlantic and the Pacific. (b) Carbon : sulfur (C : S) elemental ratios for total (black dots), LMW RSS (white dots) and SPE (orange diamonds, green triangles) in the Atlantic and the Pacific.</figcaption></figure>

result indicates that RSS-DOS (LMW hydrophilic) is a major
contributor to bulk DOS and is at least as large as the SPE-DOS
reservoir (LMW hydrophobic). The lower SPE-DOS concentra-
tions than TA-like DOS is probably due to the incapacity of
SPE using PPL to isolate LMW thiols and other RSS (Pohlabeln
and Dittmar 2015; Gomez-Saez et al. 2017), resulting in a low
recovery of hydrophilic DOS by the SPE technic.

We then calculated the C : S ratios of LMW hydrophilic
DOM (e.g., LMW DOC : RSS-DOS). Ratios ranged from
15 ± 10 to 121 ± 50 (Fig. 4b). These C : S are significantly
lower than those reported for the West Atlantic TOS (38.0–
1472.6, Longnecker et al. 2020) and DOM isolated by SPE
(SPE-DOM) in the East Atlantic (92.7–351.4; Ksionzek
et al. 2016) and the Pacific (160–303; Phillips et al. 2022) fur-
ther suggesting LMW hydrophilic compounds as a major res-
ervoir of DOS. The C : S ratios of LMW DOM were low in the
shallowest waters and reached a threshold of 50 at 350 m.
The sharp increase of C : S from the euphotic layer to the mid
mesopelagic zone indicates a selective consumption of S com-
pared to C in LMW DOM by microheterotrophs to fulfill
their S requirement. Deeper C : S ratios kept a quasi-
homogenous values of 56 ± 9 (n = 240) down to the most
abyssal waters (Fig. 4). It indicates a strong stability of this
DOS reservoir, without preferential remineralization of S
over C with time at these depths. This apparent discrepancy
with Ksionzek et al. (2016) and Longnecker et al. (2020) may
indicate difference of lability between the DOS pools and
oceanic basins. It could also reflect the long-term removal of
nonlabile DOS (Ksionzek et al. 2016). According to size reac-
tivity continuum, LMW DOM is often consider as less labile
than high-molecular-weight compounds (Benner and
Amon 2015; Walker et al. 2016). Our data suggest that LMW

hydrophilic DOS is an important pool of bioavailable DOS
for microheterotrophs. This study suggests that the refrac-
tory nature of LMW DOM is not intrinsic but emerges from
biological processes occurring in the mesopelagic zone in
line with Dittmar et al. (2021).

References

Agustí, S., and M. Llabrés. 2007. Solar radiation-induced mor-
tality of marine pico-phytoplankton in the oligotrophic
ocean. Photochem. Photobiol. **83**: 793–801. doi:10.1111/j.
1751-1097.2007.00144.x

Al-Farawati, R., and C. M. van den Berg. 1997. The determina-
tion of sulfide in seawater by flow-analysis with
voltammetric detection. Mar. Chem. **57**: 277–286. doi:10.
1016/S0304-4203(97)00014-5

Al-Farawati, R., and C. M. van den Berg. 2001. Thiols in
coastal waters of the western North Sea and English Chan-
nel. Environ. Sci. Technol. **35**: 1902–1911. doi:10.1021/es0
00073i

Amrani, A., J. W. Turner, Q. Ma, Y. Tang, and P. G. Hatcher.
2007. Formation of sulfur and nitrogen cross-linked macro-
molecules under aqueous conditions. Geochim.
Cosmochim. Acta **71**: 4141–4160. doi:10.1016/j.gca.2007.
06.051

Apel, K., and H. Hirt. 2004. Reactive oxygen species: Metabo-
lism, oxidative stress, and signal transduction. Annu. Rev.
Plant Biol. **55**: 373–399. doi:10.1146/annurev.arplant.55.
031903.141701

Baltar, F., X. A. Alvarez-Salgado, J. Arístegui, R. Benner, D. A.
Hansell, G. J. Herndl, and C. Lønborg. 2021. What is
refractory organic matter in the ocean? Front. Mar. Sci. **327**: 642637. doi:10.3389/fmars.2021.642637

Benner, R., and R. M. Amon. 2015. The size-reactivity continuum of major bioelements in the ocean. Ann. Rev. Mar. Sci. **7**: 185–205. doi:10.1146/annurev-marine-010213-135126

Bertini, I. 2007. *Biological inorganic chemistry: Structure and reactivity*. Univ. Science Books. doi:10.1002/anie.200785504

Brand, L. E., W. G. Sunda, and R. R. Guillard. 1986. Reduction of marine phytoplankton reproduction rates by copper and cadmium. J. Exp. Mar. Biol. Ecol. **96**: 225–250. doi:10.1016/0022-0981(86)90205-4

Bushnev, D. A., and N. S. Burdel'Naya. 2009. Kerogen: Chemical structure and formation conditions. Russ. Geol. Geophys. **50**: 638–643. doi:10.1016/j.rgg.2008.12.004

Carlson, C. A., P. A. Del Giorgio, and G. J. Herndl. 2007. Microbes and the dissipation of energy and respiration: From cells to ecosystems. Oceanography **20**: 89–100. doi: 10.5670/oceanog.2007.52

Chavez, F. P., K. R. Buck, and R. T. Barber. 1990. Phytoplankton taxa in relation to primary production in the equatorial Pacific. Deep Sea Res. A Oceanogr. Res. Pap. **37**: 1733–1752. doi:10.1016/0198-0149(90)90074-6

Cherrier, J., J. E. Bauer, and E. R. M. Druffel. 1996. Utilization and turnover of labile dissolved organic matter by bacterial heterotrophs in eastern North Pacific surface waters. Mar. Ecol. Prog. Ser. **139**: 267–279. doi:10.3354/meps139267

Cutter, G. A., L. S. Cutter, and K. C. Filippino. 2004. Sources and cycling of carbonyl sulfide in the Sargasso Sea. Limnol. Oceanogr. **49**: 555–565. doi:10.4319/lo.2004.49.2.0555

Damsté, J. S. S., W. I. C. Rijpstra, A. C. Kock-van Dalen, J. W. De Leeuw, and P. A. Schenck. 1989. Quenching of labile functionalised lipids by inorganic sulphur species: Evidence for the formation of sedimentary organic sulphur compounds at the early stages of diagenesis. Geochim. Cosmochim. Acta **53**: 1343–1355. doi:10.1016/0016-7037 (89)90067-7

Dittmar, T., S. T. Lennartz, H. Buck-Wiese, D. A. Hansell, C. Santinelli, C. Vanni, B. Blasius, and J. H. Hehemann. 2021. Enigmatic persistence of dissolved organic matter in the ocean. Nat. Rev. Earth Environ. **2**: 570–583. doi:10.1038/s43017-021-00183-7

Dryden, C. L., A. S. Gordon, and J. R. Donat. 2007. Seasonal survey of copper-complexing ligands and thiol compounds in a heavily utilized, urban estuary: Elizabeth River, Virginia. Mar. Chem. **103**: 276–288. doi:10.1016/j.marchem.2006.09.003

Dulaquais, G., J. Breitenstein, M. Waeles, R. Marsac, and R. Riso. 2018. Measuring dissolved organic matter in estuarine and marine waters: Size-exclusion chromatography with various detection methods. Environ. Chem. **15**: 436–449. doi:10.1071/EN18108

Dulaquais, G., M. Waeles, J. Breitenstein, J. Knoery, and R. Riso. 2020. Links between size fractionation, chemical speciation of dissolved copper and chemical speciation of

dissolved organic matter in the Loire estuary. Environ. Chem. **17**: 385–399. doi:10.1071/EN19137

Dulaquais, G., P. Fourrier, J. F. Maguer, C. Denis, M. Waeles, and R. Riso. 2023. Size exclusion chromatography and stable carbon isotopes reveal the limitations of solid phase extraction with PPL to capture autochthonous DOM production. Mar. Chem. **249**: 104213. doi:10.1016/j.marchem.2023.104213

Dupont, C. L., T. J. Goepfert, P. Lo, L. Wei, and B. A. Ahner. 2004. Diurnal cycling of glutathione in marine phytoplankton: Field and culture studies. Limnol. Oceanogr. **49**: 991–996. doi:10.4319/lo.2004.49.4.0991

Dupont, C. L., J. W. Moffett, R. R. Bidigare, and B. A. Ahner. 2006. Distributions of dissolved and particulate biogenic thiols in the subartic Pacific Ocean. Deep-Sea Res. I Oceanogr. Res. Pap. **53**: 1961–1974. doi:10.1016/j.dsr.2006.09.003

Dupont, C. L., and others. 2012. Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage. ISME J. **6**: 1186–1199. doi:10.1038/ismej.2011.189

Ercal, N., P. Yang, and N. Aykin. 2001. Determination of biological thiols by high-performance liquid chromatography following derivatization by ThioGlo maleimide reagents. J. Chromatogr. B Biomed. Sci. Appl. **753**: 287–292. doi:10.1016/S0378-4347(00)00560-0

Fahey, R. C. 2013. Glutathione analogs in prokaryotes. Biochim. Biophys. ActaGen. Subj. **1830**: 3182–3198. doi:10.1016/j.bbagen.2012.10.006

Florence, T. M., and J. L. Stauber. 1986. Toxicity of copper complexes to the marine diatom *Nitzschia closterium*. Aquat. Toxicol. **8**: 11–26. doi:10.1016/0166-445X(86)90069-X

Fourrier, P., G. Dulaquais, C. Guigue, P. Giamarchi, G. Sarthou, H. Whitby, and R. Riso. 2022a. Characterization of the vertical size distribution, composition and chemical properties of dissolved organic matter in the (ultra) oligotrophic Pacific Ocean through a multi-detection approach. Mar. Chem. **240**: 104068. doi:10.1016/j.marchem.2021.104068

Fourrier, P., G. Dulaquais, and R. Riso. 2022b. Influence of the conservation mode of seawater for dissolved organic carbon analysis. Mar. Environ. Res. **181**: 105754. doi:10.1016/j.marenvres.2022.105754

Frattaruolo, L., and others. 2019. Thioalbamide, a thioamidated peptide from *Amycolatopsis alba*, affects tumor growth and stemness by inducing metabolic dysfunction and oxidative stress. Cells **8**: 1408. doi:10.3390/cells8111408

Gomez-Saez, G. V., A. M. Pohlabeln, A. Stubbins, C. M. Marsay, and T. Dittmar. 2017. Photochemical alteration of dissolved organic sulfur from sulfidic porewater. Environ. Sci. Technol. **51**: 14144–14154. doi:10.1021/acs.est.7b03713

Hansell, D. A., C. A. Carlson, D. J. Repeta, and R. Schlitzer. 2009. Dissolved organic matter in the ocean: A controversy
stimulates new insights. Oceanography **22**: 202–211. doi: 10.5670/oceanog.2009.109

Harrison, P. J., P. W. Boyda, D. E. Varela, S. Takeda, A. Shiomoto, and T. Odate. 1999. Comparison of factors controlling phytoplankton productivity in the NE and NW subarctic Pacific gyres. Prog. Oceanogr. **43**: 205–234. doi: 10.1016/S0079-6611(99)00015-4

He, Y. Y., and D. P. Häder. 2002. UV-B-induced formation of reactive oxygen species and oxidative damage of the cyano-bacterium *Anabaena* sp.: Protective effects of ascorbic acid and N-acetyl-L-cysteine. J. Photochem. Photobiol. B Biol. **66**: 115–124. doi:10.1016/S1011-1344(02)00231-2

Huber, S. A., A. Balz, M. Abert, and W. Pronk. 2011. Characterisation of aquatic humic and non-humic matter with size-exclusion chromatography—organic carbon detection—organic nitrogen detection (LC-OCD-OND). Water Res. **45**: 879–885. doi:10.1016/j.watres.2010.09.023

Ibrahim, H., and L. Tremblay. 2023. Origin of dissolved organic sulfur in marine waters and the impact of abiotic sulfurization on its composition and fate. Mar. Chem. **254**: 104273. doi:10.1016/j.marchem.2023.104273

Jacob, C., G. I. Giles, N. M. Giles, and H. Sies. 2003. Sulfur and selenium: The role of oxidation state in protein structure and function. Angew. Chem. Int. Ed. **42**: 4742–4758. doi:10.1002/anie.200300573

Jiao, N., and others. 2010. Microbial production of recalcitrant dissolved organic matter: Long-term carbon storage in the global ocean. Nat. Rev. Microbiol. **8**: 593–599. doi:10.1038/nrmicro2386

Johannsson, O. E., D. S. Smith, H. Sadauskas-Henrique, G. Cimprich, C. M. Wood, and A. L. Val. 2017. Photo-oxidation processes, properties of DOC, reactive oxygen species (ROS), and their potential impacts on native biota and carbon cycling in the Rio Negro (Amazonia, Brazil). Hydrobiologia **789**: 7–29. doi:10.1007/s10750-016-2687-9

Kenney, G. E., and others. 2018. The biosynthesis of methanobactin. Science **359**: 1411–1416. doi:10.1126/science.aap9437

Kjaerulff, L., A. Sikandar, N. Zaburannyi, S. Adam, J. Herrmann, J. Koehnke, and R. Müller. 2017. Thioholgamides: Thioamide-containing cytotoxic RPP natural products. ACS Chem. Biol. **12**: 2837–2841. doi:10.1021/acschembio.7b00676

Koch, B. P., K. B. Ksionzek, O. J. Lechtenfeld, S. L. McCallister, P. Schmitt-Kopplin, J. K. Geuer, and W. Geibert. 2017. Response to comment on “Dissolved organic sulfur in the ocean: Biogeochemistry of a petagram inventory”. Science **356**: 813. doi:10.1126/science.aam6328

Kok, M. D., S. Schouten, and J. S. S. Damsté. 2000. Formation of insoluble, nonhydrolyzable, sulfur-rich macromolecules via incorporation of inorganic sulfur species into algal carbohydrates. Geochim. Cosmochim. Acta **64**: 2689–2699. doi:10.1016/S0016-7037(00)00382-3

Ksionzek, K. B., O. J. Lechtenfeld, S. L. McCallister, P. Schmitt-Kopplin, J. K. Geuer, W. Geibert, and B. P. Koch. 2016. Dissolved organic sulfur in the ocean: Biogeochemistry of a petagram inventory. Science **354**: 456–459. doi:10.1126/science.aaf7796

Laglera, L. M., and C. M. van den Berg. 2003. Copper complexation by thiol compounds in estuarine waters. Mar. Chem. **82**: 71–89. doi:10.1016/S0304-4203(03)00053-7

Laglera, L. M., and A. Tovar-Sánchez. 2012. Direct recognition and quantification by voltammetry of thiol/thioamide mixes in seawater. Talanta **89**: 496–504. doi:10.1016/j.talanta.2011.12.075

Latifi, A., M. Ruiz, and C. C. Zhang. 2009. Oxidative stress in cyanobacteria. FEMS Microbiol. Rev. **33**: 258–278. doi:10.1111/j.1574-6976.2008.00134.x

Leal, M. F. C., and C. M. Van Den Berg. 1998. Evidence for strong copper (I) complexation by organic ligands in seawater. Aquat. Geochem. **4**: 49–75. doi:10.1023/A:1009653002399

Leal, M. F. C., M. T. S. Vasconcelos, and C. M. van den Berg. 1999. Copper-induced release of complexing ligands similar to thiols by *Emiliania huxleyi* in seawater cultures. Limnol. Oceanogr. **44**: 1750–1762. doi:10.4319/lo.1999.44.7.1750

Lechtenfeld, O. J., N. Hertkorn, Y. Shen, M. Witt, and R. Benner. 2015. Marine sequestration of carbon in bacterial metabolites. Nat. Commun. **6**: 6711. doi:10.1038/ncomms7711

Lesser, M. P. 2006. Oxidative stress in marine environments: Biochemistry and physiological ecology. Annu. Rev. Physiol. **68**: 253–278. doi:10.1146/annurev.physiol.68.040104.110001

Levine, N. M. 2016. Putting the spotlight on organic sulfur. Science **354**: 418–419. doi:10.1126/science.aai8650

Llabrés, M., and S. Agustí. 2006. Picophytoplankton cell death induced by UV radiation: Evidence for oceanic Atlantic communities. Limnol. Oceanogr. **51**: 21–29. doi:10.4319/lo.2006.51.1.0021

Longnecker, K., L. Oswald, M. C. Kido Soule, G. A. Cutter, and E. B. Kujawinski. 2020. Organic sulfur: A spatially variable and understudied component of marine organic matter. Limnol. Oceanogr. Lett. **5**: 305–312. doi:10.1002/lol2.10149

Mahanta, N., D. M. Szantai-Kis, E. J. Petersson, and D. A. Mitchell. 2019. Biosynthesis and chemical applications of thioamides. ACS Chem. Biol. **14**: 142–163. doi:10.1021/acs.chembio.8b01022

Matrai, P. A., and R. W. Eppley. 1989. Particulate organic sulfur in the waters of the Southern California Bight. Global Biogeochem. Cycles **3**: 89–103. doi:10.1029/GB003i001p00089

Mopper, K., and D. J. Kieber. 1991. Distribution and biological turnover of dissolved organic compounds in the water column of the Black Sea. Deep Sea Res. A Oceanogr. Res. Pap. **38**: S1021–S1047. doi:10.1016/S0198-0149(10)80022-6
Mopper, K., and D. J. Kieber. 2000. Marine photochemistry and its impact on carbon cycling, p. 101–129. In S. De Mora, S. Demers, and M. Vernet [eds.], *The effects of UV radiation in the marine environment*, v. **10**. Cambridge Univ. Press. doi:10.1017/CBO9780511535444.005

Moran, M. A., and Durham, B. P. 2019. Sulfur metabolites in the pelagic ocean. Nat. Rev. Microbiol. **17**(11): 665–678. doi:10.1038/s41579-019-0250-1

Morelli, E., and G. Scarano. 2004. Copper-induced changes of non-protein thiols and antioxidant enzymes in the marine microalga *Phaeodactylum tricornutum*. Plant Sci. **167**: 289–296. doi:10.1016/j.plantsci.2004.04.001

Moriyasu, R., S. G. John, X. Bian, S. C. Yang, and J. W. Moffett. 2023. Cu exists predominantly as kinetically inert complexes throughout the interior of the Equatorial and North Pacific Ocean. Global Biogeochem. Cycl. **37**: e2022GB007521. doi:10.1029/2022GB007521

Morris, R. M., K. L. Vergin, J. C. Cho, M. S. Rappé, C. A. Carlson, and S. J. Giovannoni. 2005. Temporal and spatial response of bacterioplankton lineages to annual convective overturn at the Bermuda Atlantic Time-series Study site. Limnol. Oceanogr. **50**: 1687–1696. doi:10.4319/lo.2005.50.5.1687

Murphy, T. M. 1983. Membranes as targets of ultraviolet radiation. Physiol. Plant. **58**: 381–388. doi:10.1111/j.1399-3054.1983.tb04198.x

Pernet-Coudrier, B., M. Waeles, M. Filella, F. Quentel, and R. D. Riso. 2013. Simple and simultaneous determination of glutathione, thioacetamide and refractory organic matter in natural waters by DP-CSV. Sci. Total Environ. **463**: 997–1005. doi:10.1016/j.scitotenv.2013.06.053

Phillips, A. A., and others. 2022. Novel sulfur isotope analyses constrain sulfurized porewater fluxes as a minor component of marine dissolved organic matter. Proc. Natl. Acad. Sci. USA **119**: e2209152119. doi:10.1073/pnas.2209152119

Pohllabeln, A. M., and T. Dittmar. 2015. Novel insights into the molecular structure of non-volatile marine dissolved organic sulfur. Mar. Chem. **168**: 86–94. doi:10.1016/j.marchem.2014.10.018

Ras, J., H. Claustre, and J. Uitz. 2008. Spatial variability of phytoplankton pigment distributions in the Subtropical South Pacific Ocean: Comparison between in situ and predicted data. Biogeosciences **5**: 353–369. doi:10.5194/bg-5-353-2008

Regoli, F., and M. E. Giuliani. 2014. Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms. Mar. Environ. Res. **93**: 106–117. doi:10.1016/j.marenvres.2013.07.006

Rijstenbil, J. W. 2002. Assessment of oxidative stress in the planktonic diatom *Thalassiosira pseudonana* in response to UVA and UVB radiation. J. Plankton Res. **24**: 1277–1288. doi:10.1093/plankt/24.12.1277

Rijstenbil, J. W., S. Haritonidis, P. Malea, M. Seferlis, and J. A. Wijnholds. 1998. Thiol pools and glutathione redox ratios

as possible indicators of copper toxicity in the green macroalgae Enteromorpha spp. from the Scheldt Estuary (SW Netherlands, Belgium) and Thermaikos Gulf (Greece, N Aegean Sea). Hydrobiologia **385**: 171–181. doi:10.1023/A:1003502428466

Ruacho, A., R. M. Bundy, C. P. Till, S. Roshan, J. Wu, and K. A. Barbeau. 2020. Organic dissolved copper speciation across the US GEOTRACES equatorial Pacific zonal transect GP16. Mar. Chem. **225**: 103841. doi:10.1016/j.marchem.2020.103841

Rudyk, O., and P. Eaton. 2014. Biochemical methods for monitoring protein thiol redox states in biological systems. Redox Biol. **2**: 803–813. doi:10.1016/j.redox.2014.06.005

Schmidt, F., M. Elvert, B. P. Koch, M. Witt, and K. U. Hinrichs. 2009. Molecular characterization of dissolved organic matter in pore water of continental shelf sediments. Geochim. Cosmochim. Acta **73**: 3337–3358. doi:10.1016/j.gca.2009.03.008

Scully, N. M., D. J. McQueen, and D. R. S. Lean. 1996. Hydrogen peroxide formation: The interaction of ultraviolet radiation and dissolved organic carbon in lake waters along a 43–75 N gradient. Limnol. Oceanogr. **41**: 540–548. doi:10.4319/lo.1996.41.3.0540

Singh, N., R. K. Asthana, A. M. Kayastha, S. Pandey, A. K. Chaudhary, and S. P. Singh. 1999. Thiol and exopolysaccharide production in a cyanobacterium under heavy metal stress. Process Biochem. **35**: 63–68. doi:10.1016/S0032-9592(99)00033-3

Sinninghe, D. J., M. D. Kok, J. Koster, and S. Schouten. 1998. Sulfurized carbohydrates: An important sedimentary sink for organic carbon? Earth Planet. Sci. Lett. **164**: 7–13. doi:10.1016/S0012-821X(98)00234-9

Sunda, W. K. D. J., D. J. Kieber, R. P. Kiene, and S. Huntsman. 2002. An antioxidant function for DMSP and DMS in marine algae. Nature **418**: 317–320. doi:10.1038/nature00851

Swan, C. M., Siegel, D. A., Nelson, N. B., Carlson, C. A., and Nasir, E. 2009. Biogeochemical and hydrographic controls on chromophoric dissolved organic matter distribution in the Pacific Ocean. Deep Sea Research Part I: Oceanographic Research Papers. **56**: 2175–2192. doi:10.1016/j.dsr.2009.09.002

Swarr, G. J., T. Kading, C. H. Lamborg, C. R. Hammerschmidt, and K. L. Bowman. 2016. Dissolved low-molecular weight thiol concentrations from the US GEOTRACES North Atlantic Ocean zonal transect. Deep Sea Res. I Oceanogr. Res. Pap. **116**: 77–87. doi:10.1016/j.dsr.2016.06.003

Thingstad, T. F., Å. Hagström, and F. Rassoulzadegan. 1997. Accumulation of degradable DOC in surface waters: Is it caused by a malfunctioning microbialloop? Limnol. Oceanogr. **42**: 398–404. doi:10.4319/lo.1997.42.2.0398

Tilliette, C., and others. 2023. Influence of shallow hydrothermal fluid release on the functioning of phytoplankton communities. Front. Mar. Sci. **10**: 1082077. doi:10.3389/fmars.2023.1082077
Tripp, H. J., J. B. Kitner, M. S. Schwalbach, J. W. Dacey, L. J. Wilhelm, and S. J. Giovannoni. 2008. SAR11 marine bacteria require exogenous reduced sulphur for growth. Nature **452**: 741–744. doi:10.1038/nature06776

Tyrrell, R. M. 1991. UVA (320–380 nm) radiation as oxidative stress, p. 57–83. *In* H. Sies [ed.], *Oxidative stress: Oxidants and antioxidants*. Academic Press. doi:10.1113/expphysiol. 1997.sp004024

Vairavamurthy, M. A., Schoonen, M. A., Eglinton, T. I., Luther III, G. W., & Manowitz, B. 1995. *Geochemical transformations of sedimentary sulfur ACS Symposium Series*. American Chemical Society. doi:10.1021/bk-1995-0612.fw001.

Walker, B. D., S. R. Beaupré, T. P. Guilderson, M. D. McCarthy, and E. R. Druffel. 2016. Pacific carbon cycling constrained by organic matter size, age and composition relationships. Nat. Geosci. **9**: 888–891. doi:10.1038/nge02830

Walsh, M. J., S. D. Goodnow, G. E. Vezeau, L. V. Richter, and B. A. Ahner. 2015. Cysteine enhances bioavailability of copper to marine phytoplankton. Environ. Sci. Technol. **49**: 12145–12152. doi:10.1021/acs.est.5b02112

Weiss, N. 2005. Mechanisms of increased vascular oxidant stress in hyperhomocysteinemia and its impact on endothelial function. Curr. Drug Metab. **6**: 27–36. doi:10.2174/1389200052997357

Whitby, H., A. M. Posacka, M. T. Maldonado, and C. M. van Den Berg. 2018. Copper-binding ligands in the NE Pacific. Mar. Chem. **204**: 36–48. doi:10.1016/j.marchem.2018.05.008

Winn, C. D., L. Campbell, J. R. Christian, R. M. Letelier, D. V. Hebel, J. E. Dore, L. Fujieki, and D. M. Karl. 1995. Seasonal variability in the phytoplankton community of the North Pacific Subtropical Gyre. Global Biogeochem. Cycles **9**: 605–620. doi:10.1029/95GB02149

## Acknowledgments

This work is part of the BioDOMPO project (PI. Gabriel Dulaquais), funded by the French National program LEFE (Les Enveloppes Fluides et l'Environnement) Cyber of the CNRS and ISblue (www.isblue.fr). The Ph. D. grant of Pierre Fourrier was funded by ISblue and Région Bretagne. This work was conducted in the framework of the GEOTRACES program. The authors thank the ODF water catchers and the support of Karen Casciotti for providing samples. We are grateful to the Université de Bretagne Occidentale for funding the publication fees of the manuscript.

Submitted 19 June 2023

Revised 08 April 2024

Accepted 01 June 2024