Warming-induced shifts in phytoplankton carbon release are species-dependent

Warming-induced shifts in phytoplankton carbon release are species-dependent | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Warming-induced shifts in phytoplankton carbon release are species-dependent Israel Cassiano-Oliveira, Inessa Lacativa Bagatini, Hugo Sarmento This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8058037/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Dissolved organic matter (DOM) is the most abundant labile form of carbon in aquatic environments, comprising a wide range of biologically-derived molecules. It plays a central role in the global carbon cycle by impacting water turbidity, thus primary production, and serving as the main energy source to bacteria. Phytoplankton is a major DOM source, however, a comprehensive understanding of how global warming impacts the quality, production and accumulation of phytoplankton-derived DOM (DOMp) is still a challenge. Here we subjected axenic cultures of the bloom-forming cyanobacteria Microcystis aeruginosa and the diatom Cyclotella sp. to warming assays (+ 4°C), with and without acclimation. Samples at different growth stages were analyzed for dissolved organic carbon (DOC) concentration, flow cytometry and fluorescence spectroscopy. Bioavailability and DOC release rates consistently increased with warming in M. aeruginosa exudates. In contrast, Cyclotella sp. showed a decline in bioavailability with rising temperature, while DOC release rates appeared to increase only in the short term (i.e., without the long acclimation exposure). These findings point to potential shifts in DOM quantity and composition that may affect the microbiome structure and functioning in freshwaters as a consequence of global warming. Acclimation had an effect on some parameters, illustrating the importance of this procedure in warming experiments. Also, the detection of humic-like components in exudates from axenic cultures highlights the need for caution when attributing a terrestrial origin to fluorescence data. Fluorescent Dissolved Organic Matter Parallel Factor Analysis Phytoplankton Exudate Carbon Cycle Climate Change Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Dissolved organic matter (DOM) is a broad concept, as it refers to a complex mixture of numerous biologically-derived aromatic and aliphatic molecules with chemical and spatial-temporal variability, which represents the largest pool of organic carbon in aquatic (Stedmon et al. 2003 ; Coble 2007 ; Li and Hur 2017 ; Kelso et al. 2020 ; Monteiro et al. 2021). Phytoplankton is one of the primary sources of autochthonous DOM by fixing carbon dioxide by photosynthesis, producing a huge range of organic molecules (Bertilsson and Jones 2003 ; Myklestad 2005; Morana et al. 2014 ; Sarmento et al. 2016 ) and releasing part of it into the environment through exudation (Thornton 2014 ; Mühlenbruch et al. 2018 ) – these so-called “exudates” are known to be the preferred source of energy for micro-heterotrophs such as bacteria (Romera-Castillo et al. 2011 ; Sarmento et al., 2013; Bagatini et al. 2014 ; Eigemann et al. 2022 ). In this paper, we refer to them as “phytoplankton-derived DOM” (DOMp) (eg. Tada et al. 2017 ; Eigemann et al. 2022 ). The DOMp composition often change with species composition – with similarities found among phylogenetically related taxa – and also throughout the phytoplankton growth stages (Myklestad 2005; Romera-Castillo et al. 2009; Becker et al. 2014 ; Thornton 2014 ). In addition, the composition, bioavailability and release of exudates is altered by biotic factors such as cell senescence and lysis, predation (“sloppy feeding”) and viral lysis, which cause cytoplasm leakage (Bertilsson and Jones 2003 ; Suttle 2007 ; Jiao et al. 2010 ; Xiao et al. 2021 ) and by abiotic factors such as nutrient availability, lighting conditions, pH and temperature (Thornton 2014 ). Projections by the Intergovernmental Panel on Climate Change (IPCC) estimate an increase of up to 4ºC in the average global surface temperature by the end of the 21st century compared to current values (IPCC 2021, 2022). To identify the impacts of these changes on the dynamics and composition of both phytoplankton and the entire aquatic microbiome, an increasing number of studies have been conducted in mesocosms within temperate ecosystems, often combining warming and trophic state changes (Diehl et al., 2022 ; Engel et. al. 2011 ; Feuchtmayr et al. 2019 ; Urrutia-Cordero et al. 2020 ; Vijayaraj et al. 2022 ; Yvon-Durocher, Schaum and Trimmer 2017 ). There is also extensive literature on the effects of temperature on phytoplankton cell size and growth rates, resulting from numerous investigations conducted over the past 50 years (Eppley 1972 ; Hillebrand 2021; Mousing, Ellegaard, and Richardson 2014 ; Peter and Sommer 2012; Sherman et al. 2016 ; Yvon-Durocher et al. 2011 ; Zohary, Flaim, and Sommer 2021 ). However, the impact of rising environmental temperatures on DOMp production still requires further elucidation. By using axenic phytoplankton cultures, we can eliminate interference from other DOM sources and decomposers, allowing us to test the impact of warming on DOMp in a controlled environment. Therefore, in this work, we aimed to investigate the effects of temperature increase upon the composition and concentration of fluorescent DOM (FDOM) released by two freshwater phytoplankton species, Microcystis aeruginosa and Cyclotella sp., at different growth stages, using axenic cultures. 2. Materials and Methods 2.1. Microalgae isolates Axenic strains of Microcystis aeruginosa and Cyclotella sp. were obtained at the Collection of Freshwater Microalgae Cultures (CCMA) of the Laboratory of Phycology, Universidade Federal de São Carlos (register numbers 666 and BB041, respectively). These two species were elected because they are culturable model organisms, thereby used in many studies (e.g. Amano et al. 2011; Wang et al. 2017 ; Mesquita et al. 2020 ; Cui et al. 2022 ); are freshwater cosmopolitans (e.g. Paerl et al. 2001 ; Faustino et al. 2016 ; Li et al. 2016 ); are adequate for flow cytometry, as they rarely or never form large colonies or filaments in cultures; and genome sequences for those genera are publicly available at genome databases. 2.2. Culturing Conditions and Growth Monitoring The strains were maintained in WC sterile medium (Guillard and Lorenzen 1972 ) pH 7.0, which was found not to interfere with fluorescence spectroscopy (unpublished data). LED panels were manually crafted and installed into incubators, providing diffuse warm white light from below at the intensity of 200 ± 20 µmol photons s -1 m -2 , with a 12:12h light:dark cycle. The incubators were programmed for continuous temperatures of 24°C (experiment control), 26°C (first acclimation step) or 28°C (final acclimation step and experiment treatment), with minimal temperature fluctuation (± 0.2°C), creating a stable temperature-controlled environment. More details on temperature choice and acclimation process can be found in Supporting Information. 2.3. Experiment Measurements Daily absorbance measurements at 680 nm were taken from day zero up to the stationary stage, from which point measurements were taken every two days until culture decline. The same procedure was priorly performed with pilot cultures to establish reference growth curves (figure S1 ), which served as a guide for identifying the sampling points during the assays. Cultures were destructively sampled (in triplicate) for analysis within ~ 24h from culture inoculation and during the exponential and stationary stages (figure S2). Each culture was filtered through a pre-combusted (450°C for 4 hours) 25 mm diameter glass fiber membrane (Whatman, GF/F). Immediately prior to filtration, the membrane was flushed with ultrapure water. The filtration was carried out using a pre-washed (10% HCl and ultrapure water) plastic syringe coupled with a plastic filter holder. The filtrate was stored in equally pre-combusted amber glass bottles and kept refrigerated in the dark at 4°C for a maximum of 168h before fluorescence spectroscopy analysis. Fluorescence and absorbance data were obtained using an Edinburgh FS5 Spectrofluorometer and a 1 cm pathlength quartz cuvette, previously decontaminated with 10% HCl and ultrapure water. For Excitation-Emission Matrices (EEMs), an aliquot at room temperature (25°C) was exposed to a 5 nm stepwise excitation spectrum from 240 to 450 nm, with 0.25s permanence time, and the emission spectrum was scanned in 2 nm steps from 300 to 560 nm. The absorbance spectrum was measured from 200 to 800 nm with 0.2 s permanence time and 1 nm steps (Nieto-Cid et al. 2006 ). These same parameters were used to obtain the absorbance spectrum and EEM of ultrapure water, used as a blank sample for subtraction. The inner filter correction was made by subtracting the Raman dispersion fluorescence intensity (Larsson et al. 2007 ; Kothawala et al. 2013 ). After fluorescence spectroscopy, the aliquot was returned to the amber glass bottle, preserved with 10 ­µL of 85% phosphoric acid and kept at 4°C until the DOC measurements were carried out on a Shimadzu TOC-VCPH. As the equipment requires at least 15 mL per reading, 2 mL of each sample were diluted in 18 mL ultrapure water, and a sample of ultrapure water alone was also measured for value correction. Prior to filtration, an aliquot of each sample was both screened for bacterial contamination and used for culture cell counts in a FACSCalibur™ flow cytometer (BD Biosciences). Further details on this procedure can be found in the Supporting Information. 2.5. Statistical Analysis. Absorbance spectra and EEMs were used to calculate Fluorescence and Humification indexes (FI and HIX, respectively) (Gabor et al. 2014 ; Begum et al. 2023 ) and to perform Parallel Factor Analysis (PARAFAC) (Stedmon et al. 2003 ) in MATLAB software (Mathworks, v. R2018a), using the DOMFluor tool (Murphy et al. 2014 ), with a total of 72 samples. The PARAFAC model obtained comprised the components described below and was interpreted according to established reference peaks (Stedmon et al. 2003 ; Coble 2007 ). It was also matched against the OpenFluor database ( https://openfluor.lablicate.com ) (Murphy et al. 2014 ), selecting the correspondences with similarity ≥ 97% (with exception of fourth component, for which was used a value of ≥ 95% due to few matches with the higher cutoff). In addition, the fluorescence intensity values for each component were divided by cell concentration to account for the variations in cell density across growth stages and between species. To analyze DOC increase over time, linear regressions were performed. The DOC concentration data (in mg C L -1 ) was plotted against the number of days from the 24h sampling to stationary growth stage, and the slope values were resampled by bootstrap (1000 iterations) to calculate a median DOC release rate. The absolute and relative fluorescence for each PARAFAC component (in Raman Units and percentage, respectively), DOC release rates and optical indices were compared between treatments at the same growth stage. ANOVA tests followed by Tukey tests, or Kruskal-Wallis tests followed by Dunn tests, were used for the comparisons, depending on the data distribution and variances. Data normality was determined by Shapiro-Wilk tests, and variance homogeneity was assessed using Levene tests. To evaluate the influence of time (growth stage change) versus temperature on the shifts in FDOM composition and concentration, coefficients of variation were calculated for both variables. For all FDOM figures on the main text, each PARAFAC component was divided by cell concentration in order to minimize the influence of population growth on the measured values. All of these analysis were performed using the R programming language (R Core Team 2025 ). 3. Results and Discussion 3.1. DOC Release is Species-Dependent and Varies with Warming Exposure Times Warming triggered a 165% rise in M. aeruginosa DOC release rates, which could represent a transition to a new metabolic state (Fernández-González et al. 2020; Moreno et al. 2023) with greater carbon export (figure 1a). On the other hand, Cyclotella sp. DOC release rates almost doubled, but only in non-acclimated strains (figure 1b). This might suggest a photosynthetic overflow as a short-term response to temperature rise (Thornton 2014), but as warming persists (as in the acclimated strains), trade-offs may come into play and the DOC release rates decrease. These findings are in line with what is known about the ecology of each group: diatoms thrive in colder environments and are particularly abundant in polar waters, while cyanobacteria prefer warmer conditions. This is linked to the contrasting effects of warming on the photosynthetic capacity of these two groups (Staehr and Birkeland 2006; Tan 2011; Chen 2015; Miettinen 2018). This corroborates the idea that phytoplankton DOC release increases as a function of temperature (Thornton 2014; Lønborg et al. 2020), at least in the short term. Also, it reiterates the importance of considering acclimation procedures, as well as their duration, in studies that are interested in evaluating this and other responses of phytoplankton to temperature (Staehr and Sand-Jensen 2006). Figure 1 DOC release rates (slopes from the linear regression models) for each treatment. Distinct letters represent significant differences between strains (p < 0,05) from higher to lower values in alphabetical order, given by a Kruskal-Wallis test (n = 3000). An outlier in Cyclotella sp. control strain with value of ~ 2.5 is omitted for improved readability. Slope values here presented are a result of a resampling by bootstrap (1000 iterations) 3.2. Warming Affects FDOM Composition and Bioavailability. The PARAFAC 4-component model (Fig. 2) was cross-referenced with the 267 public models of fluorescent components in the OpenFluor database, which returned 11, 29, 12 and 4 matching components for the components 1 to 4, respectively. The description given by this analysis, together with the peak classification (Stedmon et al. 2003; Coble 2007), is shown on Table 1. Interestingly, a quarter of the corresponding components are from studies conducted in polar regions, where DOM is known to be primarily autochthonous, once vascular plants are absent and algal DOM becomes prevalent (Pointing et al. 2015; Berggren et al. 2020). Table 1 Description of the four components according to peak classification and OpenFluor cross-referencing. λ ex max. (nm) λ em max. (nm) Literature peaks correspondence OpenFluor component matches description C1 270 348 T (Tryptophan-like) DL-tryptophan and indoles (Wünsch et al. 2015), protein-like, from microbes, algae and/or bacteria (Cory and Mcknight, 2005; Lambert et al. 2017) protein-bound or free amino acids (DERRIEN et al. 2019). C2 275 300 B (Tyrosine-like) L-tyrosine and p-cresol (Wünsch et al. 2015), tannin-like and protein-like (D’Andrilli et al. 2017), freshly produced or freshly degraded proteinaceous material (D’Andrilli et al. 2019), protein-bound or free (Chen et al. 2017). C3 < 240(350) 468 A + C (Fulvic and humic-like) Terrestrial humic-like (Dainard and Guéguen 2013; Williams et al. 2013), microbially processed (Osburn et al. 2012), photo-refractory (Kida et al. 2019). C4 < 240(315) 396 A + M (Marine/Microbial humic-like) Microbial humic-like (Lapierre and Del Giorgio 2014; Wang et al. 2020), present in algae cultures (Søndergaard et al. 2011). Among the 4 fluorophores detected in this set of axenic phytoplankton cultures, three are more commonly recognized as microbially-derived (components 1, 2 and 4) and one is rarely attributed to this source (component 3). This highlights a relevant aspect for interpreting the occurrence of humic substances in natural waters: many studies indicate that such compounds are also produced in the absence of terrestrial inputs, and thus the assumption of an allochthonous origin must be made with caution (Vines and Terry 2020; Amaral et al. 2021). Also related to this finding are the low FI values found in the exponential and stationary samples of Cyclotella sp. (1.34–1.36, see figure S4), below the threshold for DOM of mixed sources (~ 1.4) and approaching the reference value for terrestrial DOM (~ 1.2) (Gabor et al. 2014; Hansen et al. 2016; Melo et al. 2020).Fluorescent DOMp concentration and composition exhibited shifts in response to both warming and acclimation time (Fig. 3 and figure S5). In M. aeruginosa cultures, contrasts were only observed in the stationary growth stage. The component 2 mean relative fluorescence (%C2) is significantly higher at 28°C than at 24°C. Conversely, the component 3 mean relative fluorescence (%C3) is higher in the control than in the acclimated cultures, both with no significant difference to non-acclimated cultures, which show intermediate values. The humic-like compounds associated to C3 are considered less bioavailable, while the protein-like compounds associated to C2 are highly labile (Cory and Kaplan 2012; Yan et al. 2022), thus the observed changes indicate higher exudate bioavailability. Changes in HIX values for stationary M. aeruginosa samples endorse these results. In control cultures, HIX values (2.54) are greater than in acclimated cultures (1.77), with non-acclimated cultures showing an intermediate value (2.22) (see figure S4). High HIX values are indicative of further polycondensation and reduced hydrogen/carbon ratio of organic molecules, characteristics of more recalcitrant DOM (Fellman et al. 2010; Gabor et al. 2014; Yan et al. 2022), so the decrease in HIX values with temperature rise also points to an increase in bioavailability in response to warming. Now looking at absolute fluorescence data for stationary M. aeruginosa , an almost twice fold rise in all components happened in acclimated cultures. That could indicate an increase in the proportion of fluorophores in the DOMp when this cyanobacteria becomes acclimated to a higher temperature (see figure S5 for further comparisons). In turn, trends in FDOM from Cyclotella sp. cultures suggest a less labile exudate in warmer conditions. Both C1 in the stationary stage and %C1 in the exponential stage decreased with higher temperature and warming exposure time. Simultaneously, both %C4 in the stationary stage and C3 in the exponential stage tended to increase. Considering the protein-like and more labile (Cory and Kaplan 2012; Yan et al. 2022) characteristics of C1 and the humic-like and possibly less labile features (Cory and Kaplan 2012; Yan et al. 2022) of C3 and C4, these changes represent a reduction in bioavailability of FDOM from Cyclotella sp. exudates under warmer conditions. Again, HIX values support this interpretation, at least partially: a higher mean value is observed for non-acclimated cultures (0,81), significantly greater than the value for control cultures (0,55) but not than the values for acclimated ones (0,66) (figure S4). 3.3. Warming affects FDOM composition and concentration as much as growth state change When evaluating whether changes in growth stage or temperature were the main drivers of variation in fluorescent DOMp, an overall pattern of similar influence emerged (Fig. 4). In almost all cases, fluorescent DOMp concentration and composition was determined by the joint action of growth state change and temperature. The only exception in the general pattern was observed for FDOM concentration in M. aeruginosa cultures. This could be due to the sharp variation in cell density observed from the exponential to the stationary stage, approximately threefold, which represents a strong contrast to the nearly constant values observed in Cyclotella sp. cultures (Figure S6). Here it is worth noting that the present investigation did not set out to estimate FDOM degradation by phytoplankton, but once the concentration of all components has increased over time, it is assumed that the processes of production, release and transformation outweigh those of absorption and degradation. Also, it is fundamental to frame into context the findings reported here, as they come from relatively short assays with isolated species under very controlled conditions – also, the analytical window of fluorescence spectroscopy is limited and there is a persistent challenge to make progress towards a molecular comprehension of DOM (Murphy et al. 2010; Stubbins et al. 2014; Catalán et al. 2020). Thus, long-term phytoplankton evolutionary adaptations, ecological interactions with other microbial communities – especially bacteria (Amin et al. 2015; Johansson et al. 2019; Shibl et al. 2020) – and the influence of synergistic effects with other environmental shifts associated with climate change (such as eutrophication and rise in atmospheric CO 2 levels) were beyond the scope of this work, and extrapolations must be made wisely. Nonetheless, the outcomes here reported allow us to set some expectations on the response of aquatic microbiomes to climate change. The competitive advantages of phytoplankton taxa with better fitness to warmer conditions can influence microbiome species composition (Bagatini et al. 2014), which added to metabolic changes that affect the release and bioavailability of DOMp, even at small scales, can trigger disruptions in microbial communities and in the structure of the (osmo)trophic web, impacting the speed of efficiency and intensity of carbon flows in aquatic trophic webs (Sarmento and Gasol 2012; Landa et al. 2016; Tisserand et al. 2020), especially in the so-called “microbial loop”. Declarations Author Contributions Conceptualization and Methodology: Israel Cassiano-Oliveira, Inessa Lacativa Bagatini and Hugo Sarmento. Data curation, Formal analysis and Investigation: Israel Cassiano-Oliveira; Funding acquisition: Israel Cassiano-Oliveira and Hugo Sarmento; Project administration: Hugo Sarmento; Visualization and writing - original draft: Israel Cassiano-Oliveira; Supervision, Resources and Validation and writing - review & editing: Inessa Lacativa Bagatini and Hugo Sarmento. Competing Interests: the authors have no competing interests to declare that are relevant to the content of this article. Funding: ICO was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [grant number 2022/16279-3]. HS acknowledges continuous funding through Research Productivity Grants provided by the Brazilian Research Council (CNPq) [grant number 303906/2021-9]. Data availability: data will be made available at Zenodo upon publication. ACKNOWLEDGMENTS The authors thank Cristina Romera Castillo and Michaela Ladeira de Melo for the contributions to this work presented as an undergraduate thesis; Adriana Miwa and Maria do Carmo Calijuri from the Laboratório de Biotoxicologia de Águas Continentais e Efluentes – USP for the support on DOC measurements. Thanks to all members of both Laboratory of Microbial Processes and Biodiversity and the Laboratory of Phycology for the support throughout the execution of this research. References Aiken, G (2014) Fluorescence and Dissolved Organic Matter: A Chemist’s Perspective. In: PG Coble, J Lead, A Baker, DM Reynolds, and RGM Spencer (eds), Aquatic Organic Matter Fluorescence. Cambridge University Press, New York, pp. 35–74. Alho LDOG, Gebara RC, Paina KDA, et al (2019) Responses of Raphidocelis subcapitata exposed to Cd and Pb: Mechanisms of toxicity assessed by multiple endpoints. 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11:09:12","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9970,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinegroupimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/4fe93fdbf0753255ed0757c2.png"},{"id":96623235,"identity":"741e1568-ff52-4afc-8820-d65f02df4a52","added_by":"auto","created_at":"2025-11-24 11:09:13","extension":"xml","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":193822,"visible":true,"origin":"","legend":"","description":"","filename":"1541b87a7ad84addacda525e979e185a1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/713beacf27090a6aecdec1cf.xml"},{"id":96623232,"identity":"9d5a6743-5c6b-4f09-b3fc-449936e2ed63","added_by":"auto","created_at":"2025-11-24 11:09:12","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":208274,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/4053b6439409c27aac06bc1d.html"},{"id":96708750,"identity":"d517b0fc-5c72-4269-8b68-b7d29db13d49","added_by":"auto","created_at":"2025-11-25 10:05:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":254330,"visible":true,"origin":"","legend":"\u003cp\u003eDOC release rates (slopes from the linear regression models) for each treatment. Distinct letters represent significant differences between strains (p\u0026lt;0,05) from higher to lower values in alphabetical order, given by a Kruskal-Wallis test (n=3000). An outlier in \u003cem\u003eCyclotella\u003c/em\u003e sp. control strain with value of ~2.5 is omitted for improved readability. Slope values here presented are a result of a resampling by bootstrap (1000 iterations)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/c9788d4cca928db7bb6a0dcb.png"},{"id":96623214,"identity":"9a6fe148-8e61-4839-977b-93a507779449","added_by":"auto","created_at":"2025-11-24 11:09:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163581,"visible":true,"origin":"","legend":"\u003cp\u003eEEMs for the four-component model provided by PARAFAC. Excitation wavelengths are displayed on the x-axis, emission wavelengths are displayed on the y-axis and color scale indicates fluorescence intensity.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/f64c9d624a5ab306d49737c9.png"},{"id":96623219,"identity":"a83f1e27-db56-46e0-8a50-65c02bfad0a3","added_by":"auto","created_at":"2025-11-24 11:09:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":605847,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in FDOM composition for the two microalgae species in exponential and stationary growth stages, shown by relative (upper graphs) and absolute fluorescence (lower graphs). Distinct letters represent significant differences between strains (p\u0026lt;0,05) from higher to lower values in alphabetical order, and when absent there are no statistically significant differences. Graphs with\u003csup\u003e ‡\u003c/sup\u003e show results for Kruskal-Wallis tests, and those without them show results for ANOVA tests. Error bars represent standard deviation. In all cases, for \u003cem\u003eM. aeruginosa\u003c/em\u003e n=9 and for \u003cem\u003eCyclotella\u003c/em\u003e sp. n=8. Absolute fluorescence graphs are shown in supplementary material.\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/6da9d4b4ac7bb6b503678997.png"},{"id":96709990,"identity":"5b8b7b45-a48e-49a1-8c37-128b9fc34905","added_by":"auto","created_at":"2025-11-25 10:09:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":186982,"visible":true,"origin":"","legend":"\u003cp\u003eCoefficients of variation for absolute (“concentration”, in R.U.) and relative (“contribution”, in %) fluorescence, for both phytoplankton species. Grey bars represent the average of three values (one for each strain/treatment) of coefficient of variation across growth stages (24h to stationary), in other words, variation due to time. Yellow bars represent the average of three values (one for each growth stage) of coefficient of variation between strains/treatments (acclimated, non-acclimated and control), essentially variation due to rise in temperature. The * denotes significant differences between pairs of values (p\u0026lt;0,05). For all comparisons, n=6.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/a6366ce1b3b53dd20091f71b.png"},{"id":96712705,"identity":"5754f2b6-6f2c-4cfb-aebf-c52a67c38fc8","added_by":"auto","created_at":"2025-11-25 10:16:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1919949,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/c7484752-8cde-48d9-9709-905e40584130.pdf"},{"id":96623220,"identity":"8a11ad46-01de-496d-a00a-b93e305db2ae","added_by":"auto","created_at":"2025-11-24 11:09:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":863321,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationforarticleWarminginducedshiftsinphytoplanktoncarbonreleasearespeciesdependent.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8058037/v1/4d8ccea62b14ba6daffb133d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Warming-induced shifts in phytoplankton carbon release are species-dependent","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDissolved organic matter (DOM) is a broad concept, as it refers to a complex mixture of numerous biologically-derived aromatic and aliphatic molecules with chemical and spatial-temporal variability, which represents the largest pool of organic carbon in aquatic (Stedmon et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Coble \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Li and Hur \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kelso et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Monteiro et al. 2021). Phytoplankton is one of the primary sources of autochthonous DOM by fixing carbon dioxide by photosynthesis, producing a huge range of organic molecules (Bertilsson and Jones \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Myklestad 2005; Morana et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sarmento et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and releasing part of it into the environment through exudation (Thornton \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; M\u0026uuml;hlenbruch et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) \u0026ndash; these so-called \u0026ldquo;exudates\u0026rdquo; are known to be the preferred source of energy for micro-heterotrophs such as bacteria (Romera-Castillo et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sarmento et al., 2013; Bagatini et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Eigemann et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this paper, we refer to them as \u0026ldquo;phytoplankton-derived DOM\u0026rdquo; (DOMp) (eg. Tada et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Eigemann et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe DOMp composition often change with species composition \u0026ndash; with similarities found among phylogenetically related taxa \u0026ndash; and also throughout the phytoplankton growth stages (Myklestad 2005; Romera-Castillo et al. 2009; Becker et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Thornton \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In addition, the composition, bioavailability and release of exudates is altered by biotic factors such as cell senescence and lysis, predation (\u0026ldquo;sloppy feeding\u0026rdquo;) and viral lysis, which cause cytoplasm leakage (Bertilsson and Jones \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Suttle \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Jiao et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Xiao et al. \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and by abiotic factors such as nutrient availability, lighting conditions, pH and temperature (Thornton \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eProjections by the Intergovernmental Panel on Climate Change (IPCC) estimate an increase of up to 4\u0026ordm;C in the average global surface temperature by the end of the 21st century compared to current values (IPCC 2021, 2022). To identify the impacts of these changes on the dynamics and composition of both phytoplankton and the entire aquatic microbiome, an increasing number of studies have been conducted in mesocosms within temperate ecosystems, often combining warming and trophic state changes (Diehl et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Engel et. al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Feuchtmayr et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Urrutia-Cordero et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Vijayaraj et al. \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yvon-Durocher, Schaum and Trimmer \u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). There is also extensive literature on the effects of temperature on phytoplankton cell size and growth rates, resulting from numerous investigations conducted over the past 50 years (Eppley \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1972\u003c/span\u003e; Hillebrand 2021; Mousing, Ellegaard, and Richardson \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Peter and Sommer 2012; Sherman et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yvon-Durocher et al. \u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zohary, Flaim, and Sommer \u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the impact of rising environmental temperatures on DOMp production still requires further elucidation.\u003c/p\u003e\u003cp\u003eBy using axenic phytoplankton cultures, we can eliminate interference from other DOM sources and decomposers, allowing us to test the impact of warming on DOMp in a controlled environment. Therefore, in this work, we aimed to investigate the effects of temperature increase upon the composition and concentration of fluorescent DOM (FDOM) released by two freshwater phytoplankton species, \u003cem\u003eMicrocystis aeruginosa\u003c/em\u003e and \u003cem\u003eCyclotella\u003c/em\u003e sp., at different growth stages, using axenic cultures.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Microalgae isolates\u003c/h2\u003e\u003cp\u003eAxenic strains of \u003cem\u003eMicrocystis aeruginosa\u003c/em\u003e and \u003cem\u003eCyclotella\u003c/em\u003e sp. were obtained at the Collection of Freshwater Microalgae Cultures (CCMA) of the Laboratory of Phycology, Universidade Federal de S\u0026atilde;o Carlos (register numbers 666 and BB041, respectively). These two species were elected because they are culturable model organisms, thereby used in many studies (e.g. Amano et al. 2011; Wang et al. \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mesquita et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e); are freshwater cosmopolitans (e.g. Paerl et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Faustino et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); are adequate for flow cytometry, as they rarely or never form large colonies or filaments in cultures; and genome sequences for those genera are publicly available at genome databases.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Culturing Conditions and Growth Monitoring\u003c/h2\u003e\u003cp\u003eThe strains were maintained in WC sterile medium (Guillard and Lorenzen \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1972\u003c/span\u003e) pH 7.0, which was found not to interfere with fluorescence spectroscopy (unpublished data). LED panels were manually crafted and installed into incubators, providing diffuse warm white light from below at the intensity of 200\u0026thinsp;\u0026plusmn;\u0026thinsp;20 \u0026micro;mol photons s\u003csup\u003e-1\u003c/sup\u003e m\u003csup\u003e-2\u003c/sup\u003e, with a 12:12h light:dark cycle. The incubators were programmed for continuous temperatures of 24\u0026deg;C (experiment control), 26\u0026deg;C (first acclimation step) or 28\u0026deg;C (final acclimation step and experiment treatment), with minimal temperature fluctuation (\u0026plusmn;\u0026thinsp;0.2\u0026deg;C), creating a stable temperature-controlled environment. More details on temperature choice and acclimation process can be found in Supporting Information.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Experiment Measurements\u003c/h2\u003e\u003cp\u003eDaily absorbance measurements at 680 nm were taken from day zero up to the stationary stage, from which point measurements were taken every two days until culture decline. The same procedure was priorly performed with pilot cultures to establish reference growth curves (figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which served as a guide for identifying the sampling points during the assays.\u003c/p\u003e\u003cp\u003eCultures were destructively sampled (in triplicate) for analysis within ~\u0026thinsp;24h from culture inoculation and during the exponential and stationary stages (figure S2). Each culture was filtered through a pre-combusted (450\u0026deg;C for 4 hours) 25 mm diameter glass fiber membrane (Whatman, GF/F). Immediately prior to filtration, the membrane was flushed with ultrapure water. The filtration was carried out using a pre-washed (10% HCl and ultrapure water) plastic syringe coupled with a plastic filter holder. The filtrate was stored in equally pre-combusted amber glass bottles and kept refrigerated in the dark at 4\u0026deg;C for a maximum of 168h before fluorescence spectroscopy analysis.\u003c/p\u003e\u003cp\u003eFluorescence and absorbance data were obtained using an Edinburgh FS5 Spectrofluorometer and a 1 cm pathlength quartz cuvette, previously decontaminated with 10% HCl and ultrapure water. For Excitation-Emission Matrices (EEMs), an aliquot at room temperature (25\u0026deg;C) was exposed to a 5 nm stepwise excitation spectrum from 240 to 450 nm, with 0.25s permanence time, and the emission spectrum was scanned in 2 nm steps from 300 to 560 nm. The absorbance spectrum was measured from 200 to 800 nm with 0.2 s permanence time and 1 nm steps (Nieto-Cid et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). These same parameters were used to obtain the absorbance spectrum and EEM of ultrapure water, used as a blank sample for subtraction. The inner filter correction was made by subtracting the Raman dispersion fluorescence intensity (Larsson et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kothawala et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAfter fluorescence spectroscopy, the aliquot was returned to the amber glass bottle, preserved with 10 \u0026shy;\u0026micro;L of 85% phosphoric acid and kept at 4\u0026deg;C until the DOC measurements were carried out on a Shimadzu TOC-VCPH. As the equipment requires at least 15 mL per reading, 2 mL of each sample were diluted in 18 mL ultrapure water, and a sample of ultrapure water alone was also measured for value correction.\u003c/p\u003e\u003cp\u003ePrior to filtration, an aliquot of each sample was both screened for bacterial contamination and used for culture cell counts in a FACSCalibur\u0026trade; flow cytometer (BD Biosciences). Further details on this procedure can be found in the Supporting Information.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.5. Statistical Analysis.\u003c/b\u003e Absorbance spectra and EEMs were used to calculate Fluorescence and Humification indexes (FI and HIX, respectively) (Gabor et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Begum et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and to perform Parallel Factor Analysis (PARAFAC) (Stedmon et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) in MATLAB software (Mathworks, v. R2018a), using the DOMFluor tool (Murphy et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), with a total of 72 samples. The PARAFAC model obtained comprised the components described below and was interpreted according to established reference peaks (Stedmon et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Coble \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). It was also matched against the OpenFluor database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://openfluor.lablicate.com\u003c/span\u003e\u003cspan address=\"https://openfluor.lablicate.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Murphy et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), selecting the correspondences with similarity\u0026thinsp;\u0026ge;\u0026thinsp;97% (with exception of fourth component, for which was used a value of \u0026ge;\u0026thinsp;95% due to few matches with the higher cutoff). In addition, the fluorescence intensity values for each component were divided by cell concentration to account for the variations in cell density across growth stages and between species.\u003c/p\u003e\u003cp\u003eTo analyze DOC increase over time, linear regressions were performed. The DOC concentration data (in mg\u003csub\u003eC\u003c/sub\u003e L\u003csup\u003e-1\u003c/sup\u003e) was plotted against the number of days from the 24h sampling to stationary growth stage, and the slope values were resampled by bootstrap (1000 iterations) to calculate a median DOC release rate.\u003c/p\u003e\u003cp\u003eThe absolute and relative fluorescence for each PARAFAC component (in Raman Units and percentage, respectively), DOC release rates and optical indices were compared between treatments at the same growth stage. ANOVA tests followed by Tukey tests, or Kruskal-Wallis tests followed by Dunn tests, were used for the comparisons, depending on the data distribution and variances. Data normality was determined by Shapiro-Wilk tests, and variance homogeneity was assessed using Levene tests.\u003c/p\u003e\u003cp\u003eTo evaluate the influence of time (growth stage change) versus temperature on the shifts in FDOM composition and concentration, coefficients of variation were calculated for both variables. For all FDOM figures on the main text, each PARAFAC component was divided by cell concentration in order to minimize the influence of population growth on the measured values. All of these analysis were performed using the R programming language (R Core Team \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1. DOC Release is Species-Dependent and Varies with Warming Exposure Times\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWarming triggered a 165% rise in \u003cem\u003eM. aeruginosa\u003c/em\u003e DOC release rates, which could represent a transition to a new metabolic state (Fernández-González et al. 2020; Moreno et al. 2023) with greater carbon export (figure 1a). On the other hand, \u003cem\u003eCyclotella\u003c/em\u003e sp. DOC release rates almost doubled, but only in non-acclimated strains (figure 1b). This might suggest a photosynthetic overflow as a short-term response to temperature rise (Thornton 2014), but as warming persists (as in the acclimated strains), trade-offs may come into play and the DOC release rates decrease. These findings are in line with what is known about the ecology of each group: diatoms thrive in colder environments and are particularly abundant in polar waters, while cyanobacteria prefer warmer conditions. This is linked to the contrasting effects of warming on the photosynthetic capacity of these two groups (Staehr and Birkeland 2006; Tan 2011; Chen 2015; Miettinen 2018). This corroborates the idea that phytoplankton DOC release increases as a function of temperature (Thornton 2014; Lønborg et al. 2020), at least in the short term. Also, it reiterates the importance of considering acclimation procedures, as well as their duration, in studies that are interested in evaluating this and other responses of phytoplankton to temperature (Staehr and Sand-Jensen 2006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure\u0026nbsp;1\u003c/strong\u003e DOC release rates (slopes from the linear regression models) for each treatment. Distinct letters represent significant differences between strains (p \u0026lt; 0,05) from higher to lower values in alphabetical order, given by a Kruskal-Wallis test (n = 3000). An outlier in \u003cem\u003eCyclotella\u003c/em\u003e sp. control strain with value of ~ 2.5 is omitted for improved readability. Slope values here presented are a result of a resampling by bootstrap (1000 iterations)\u003c/p\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e3.2. Warming Affects FDOM Composition and Bioavailability.\u003c/h2\u003e\n \u003cp\u003eThe PARAFAC 4-component model (Fig. 2) was cross-referenced with the 267 public models of fluorescent components in the OpenFluor database, which returned 11, 29, 12 and 4 matching components for the components 1 to 4, respectively. The description given by this analysis, together with the peak classification (Stedmon et al. 2003; Coble 2007), is shown on Table 1. Interestingly, a quarter of the corresponding components are from studies conducted in polar regions, where DOM is known to be primarily autochthonous, once vascular plants are absent and algal DOM becomes prevalent (Pointing et al. 2015; Berggren et al. 2020).\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eDescription of the four components according to peak classification and OpenFluor cross-referencing.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eλ\u003csub\u003eex\u003c/sub\u003e max. (nm)\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eλ\u003csub\u003eem\u003c/sub\u003e max. (nm)\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eLiterature peaks correspondence\u003cbr\u003e\u003c/th\u003e\n \u003cth align=\"left\"\u003eOpenFluor component matches description\u003cbr\u003e\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eC1\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003e270\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e348\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eT (Tryptophan-like)\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eDL-tryptophan and indoles (Wünsch et al. 2015), protein-like, from microbes, algae and/or bacteria (Cory and Mcknight, 2005; Lambert et al. 2017) protein-bound or free amino acids (DERRIEN et al.\u0026nbsp;2019).\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eC2\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003e275\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e300\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eB (Tyrosine-like)\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eL-tyrosine and p-cresol (Wünsch et al. 2015), tannin-like and protein-like (D’Andrilli et al. 2017), freshly produced or freshly degraded proteinaceous material (D’Andrilli et al. 2019), protein-bound or free (Chen et al.\u0026nbsp;2017).\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eC3\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026lt; 240(350)\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e468\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eA + C (Fulvic and humic-like)\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eTerrestrial humic-like (Dainard and Guéguen 2013; Williams et al. 2013), microbially processed (Osburn et al. 2012), photo-refractory (Kida et al.\u0026nbsp;2019).\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003eC4\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026lt; 240(315)\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"char\"\u003e396\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eA + M (Marine/Microbial humic-like)\u003cbr\u003e\u003c/td\u003e\n \u003ctd align=\"left\"\u003eMicrobial humic-like (Lapierre and Del Giorgio 2014; Wang et al.\u0026nbsp;2020), present in algae cultures (Søndergaard et al. 2011).\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003eAmong the 4 fluorophores detected in this set of axenic phytoplankton cultures, three are more commonly recognized as microbially-derived (components 1, 2 and 4) and one is rarely attributed to this source (component 3). This highlights a relevant aspect for interpreting the occurrence of humic substances in natural waters: many studies indicate that such compounds are also produced in the absence of terrestrial inputs, and thus the assumption of an allochthonous origin must be made with caution (Vines and Terry 2020; Amaral et al. 2021). Also related to this finding are the low FI values found in the exponential and stationary samples of \u003cem\u003eCyclotella\u003c/em\u003e sp. (1.34–1.36, see figure S4), below the threshold for DOM of mixed sources (~ 1.4) and approaching the reference value for terrestrial DOM (~ 1.2) (Gabor et al. 2014; Hansen et al. 2016; Melo et al. 2020).Fluorescent DOMp concentration and composition exhibited shifts in response to both warming and acclimation time (Fig. 3 and figure S5). In \u003cem\u003eM. aeruginosa\u003c/em\u003e cultures, contrasts were only observed in the stationary growth stage. The component 2 mean relative fluorescence (%C2) is significantly higher at 28°C than at 24°C. Conversely, the component 3 mean relative fluorescence (%C3) is higher in the control than in the acclimated cultures, both with no significant difference to non-acclimated cultures, which show intermediate values. The humic-like compounds associated to C3 are considered less bioavailable, while the protein-like compounds associated to C2 are highly labile (Cory and Kaplan 2012; Yan et al. 2022), thus the observed changes indicate higher exudate bioavailability.\u003cp\u003eChanges in HIX values for stationary \u003cem\u003eM. aeruginosa\u003c/em\u003e samples endorse these results. In control cultures, HIX values (2.54) are greater than in acclimated cultures (1.77), with non-acclimated cultures showing an intermediate value (2.22) (see figure S4). High HIX values are indicative of further polycondensation and reduced hydrogen/carbon ratio of organic molecules, characteristics of more recalcitrant DOM (Fellman et al. 2010; Gabor et al. 2014; Yan et al. 2022), so the decrease in HIX values with temperature rise also points to an increase in bioavailability in response to warming.\u003c/p\u003e\n \u003cp\u003eNow looking at absolute fluorescence data for stationary \u003cem\u003eM. aeruginosa\u003c/em\u003e, an almost twice fold rise in all components happened in acclimated cultures. That could indicate an increase in the proportion of fluorophores in the DOMp when this cyanobacteria becomes acclimated to a higher temperature (see figure S5 for further comparisons).\u003c/p\u003e\n \u003cp\u003eIn turn, trends in FDOM from \u003cem\u003eCyclotella\u003c/em\u003e sp. cultures suggest a less labile exudate in warmer conditions. Both C1 in the stationary stage and %C1 in the exponential stage decreased with higher temperature and warming exposure time. Simultaneously, both %C4 in the stationary stage and C3 in the exponential stage tended to increase. Considering the protein-like and more labile (Cory and Kaplan 2012; Yan et al. 2022) characteristics of C1 and the humic-like and possibly less labile features (Cory and Kaplan 2012; Yan et al. 2022) of C3 and C4, these changes represent a reduction in bioavailability of FDOM from \u003cem\u003eCyclotella\u003c/em\u003e sp. exudates under warmer conditions. Again, HIX values support this interpretation, at least partially: a higher mean value is observed for non-acclimated cultures (0,81), significantly greater than the value for control cultures (0,55) but not than the values for acclimated ones (0,66) (figure S4).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\"\u003e\n \u003ch2\u003e3.3. Warming affects FDOM composition and concentration as much as growth state change\u003c/h2\u003e\n \u003cp\u003eWhen evaluating whether changes in growth stage or temperature were the main drivers of variation in fluorescent DOMp, an overall pattern of similar influence emerged (Fig. 4). In almost all cases, fluorescent DOMp concentration and composition was determined by the joint action of growth state change and temperature. The only exception in the general pattern was observed for FDOM concentration in \u003cem\u003eM. aeruginosa\u003c/em\u003e cultures. This could be due to the sharp variation in cell density observed from the exponential to the stationary stage, approximately threefold, which represents a strong contrast to the nearly constant values observed in \u003cem\u003eCyclotella\u003c/em\u003e sp. cultures (Figure S6).\u003c/p\u003e\n \u003cp\u003eHere it is worth noting that the present investigation did not set out to estimate FDOM degradation by phytoplankton, but once the concentration of all components has increased over time, it is assumed that the processes of production, release and transformation outweigh those of absorption and degradation. Also, it is fundamental to frame into context the findings reported here, as they come from relatively short assays with isolated species under very controlled conditions – also, the analytical window of fluorescence spectroscopy is limited and there is a persistent challenge to make progress towards a molecular comprehension of DOM (Murphy et al. 2010; Stubbins et al. 2014; Catalán et al. 2020). Thus, long-term phytoplankton evolutionary adaptations, ecological interactions with other microbial communities – especially bacteria (Amin et al. 2015; Johansson et al. 2019; Shibl et al. 2020) – and the influence of synergistic effects with other environmental shifts associated with climate change (such as eutrophication and rise in atmospheric CO\u003csub\u003e2\u003c/sub\u003e levels) were beyond the scope of this work, and extrapolations must be made wisely.\u003c/p\u003e\n \u003cp\u003eNonetheless, the outcomes here reported allow us to set some expectations on the response of aquatic microbiomes to climate change. The competitive advantages of phytoplankton taxa with better fitness to warmer conditions can influence microbiome species composition (Bagatini et al. 2014), which added to metabolic changes that affect the release and bioavailability of DOMp, even at small scales, can trigger disruptions in microbial communities and in the structure of the (osmo)trophic web, impacting the speed of efficiency and intensity of carbon flows in aquatic trophic webs (Sarmento and Gasol 2012; Landa et al. 2016; Tisserand et al. 2020), especially in the so-called “microbial loop”.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization and Methodology: Israel Cassiano-Oliveira, Inessa Lacativa Bagatini and Hugo Sarmento. Data curation, Formal analysis and Investigation: Israel Cassiano-Oliveira; Funding acquisition: Israel Cassiano-Oliveira and Hugo Sarmento; Project administration: Hugo Sarmento; Visualization and writing - original draft: Israel Cassiano-Oliveira; Supervision, Resources and Validation and writing - review \u0026amp; editing: Inessa Lacativa Bagatini and Hugo Sarmento.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e the authors have no competing interests to declare that are relevant to the content of this article. \u003cstrong\u003eFunding:\u003c/strong\u003e ICO was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [grant number 2022/16279-3]. HS acknowledges continuous funding through Research Productivity Grants provided by the Brazilian Research Council (CNPq) [grant number 303906/2021-9]. \u003cstrong\u003eData availability:\u003c/strong\u003e data will be made available at Zenodo upon publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Cristina Romera Castillo and Michaela Ladeira de Melo for the contributions to this work presented as an undergraduate thesis; Adriana Miwa and Maria do Carmo Calijuri from the Laborat\u0026oacute;rio de Biotoxicologia de \u0026Aacute;guas Continentais e Efluentes \u0026ndash; USP for the support on DOC measurements. Thanks to all members of both Laboratory of Microbial Processes and Biodiversity and the Laboratory of Phycology for the support throughout the execution of this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAiken, G (2014) Fluorescence and Dissolved Organic Matter: A Chemist’s Perspective. In: PG Coble, J Lead, A Baker, DM Reynolds, and RGM Spencer (eds), Aquatic Organic Matter Fluorescence. Cambridge University Press, New York, pp. 35–74.\u003c/li\u003e\n \u003cli\u003eAlho LDOG, Gebara RC, Paina KDA, et al (2019) Responses of Raphidocelis subcapitata exposed to Cd and Pb: Mechanisms of toxicity assessed by multiple endpoints. 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Hydrobiologia 848:143–155. https://doi.org/10.1007/s10750-020-04246-6\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"aquatic-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aeco","sideBox":"Learn more about [Aquatic Ecology](http://link.springer.com/journal/10452)","snPcode":"10452","submissionUrl":"https://submission.nature.com/new-submission/10452/3","title":"Aquatic Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fluorescent Dissolved Organic Matter, Parallel Factor Analysis, Phytoplankton Exudate, Carbon Cycle, Climate Change","lastPublishedDoi":"10.21203/rs.3.rs-8058037/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8058037/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDissolved organic matter (DOM) is the most abundant labile form of carbon in aquatic environments, comprising a wide range of biologically-derived molecules. It plays a central role in the global carbon cycle by impacting water turbidity, thus primary production, and serving as the main energy source to bacteria. Phytoplankton is a major DOM source, however, a comprehensive understanding of how global warming impacts the quality, production and accumulation of phytoplankton-derived DOM (DOMp) is still a challenge. Here we subjected axenic cultures of the bloom-forming cyanobacteria \u003cem\u003eMicrocystis aeruginosa\u003c/em\u003e and the diatom \u003cem\u003eCyclotella\u003c/em\u003e sp. to warming assays (+\u0026thinsp;4\u0026deg;C), with and without acclimation. Samples at different growth stages were analyzed for dissolved organic carbon (DOC) concentration, flow cytometry and fluorescence spectroscopy. Bioavailability and DOC release rates consistently increased with warming in \u003cem\u003eM. aeruginosa\u003c/em\u003e exudates. In contrast, \u003cem\u003eCyclotella\u003c/em\u003e sp. showed a decline in bioavailability with rising temperature, while DOC release rates appeared to increase only in the short term (i.e., without the long acclimation exposure). These findings point to potential shifts in DOM quantity and composition that may affect the microbiome structure and functioning in freshwaters as a consequence of global warming. Acclimation had an effect on some parameters, illustrating the importance of this procedure in warming experiments. Also, the detection of humic-like components in exudates from axenic cultures highlights the need for caution when attributing a terrestrial origin to fluorescence data.\u003c/p\u003e","manuscriptTitle":"Warming-induced shifts in phytoplankton carbon release are species-dependent","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 11:09:07","doi":"10.21203/rs.3.rs-8058037/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-13T14:45:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-12T00:58:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T14:14:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-02T02:54:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"210357412073958147722676152375847791822","date":"2025-11-20T19:42:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"188504308556717856227710532407763679373","date":"2025-11-20T07:12:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"57141525232987672722689737352434433621","date":"2025-11-13T02:18:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-12T18:56:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-12T10:01:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-11T14:40:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Aquatic Ecology","date":"2025-11-07T14:21:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"aquatic-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aeco","sideBox":"Learn more about [Aquatic Ecology](http://link.springer.com/journal/10452)","snPcode":"10452","submissionUrl":"https://submission.nature.com/new-submission/10452/3","title":"Aquatic Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"80adcb51-5a90-4909-9422-678ef5efbf41","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T15:23:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 11:09:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8058037","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8058037","identity":"rs-8058037","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}