# Light and temperature culture conditions impact the metabolome of the cyanobacterium of therapeutic interest Planktothricoides raciborskii PMC 877.14

Inés Seijo Touceda, Justine Demay, Charlotte Duval, Claude Yéprémian, Anita Reinhardt, Benjamin Marie

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Inés Seijo Touceda, Justine Demay, Charlotte Duval, Claude Yéprémian, Anita Reinhardt, et al. Light and temperature culture conditions impact the metabolome of the cyanobacterium of therapeutic interest Planktothricoides raciborskii PMC 877.14. Algal Research - Biomass, Biofuels and Bioproducts, 2024, 84, pp.103738. 10.1016/j.algal.2024.103738. hal-04879104

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# Light and temperature culture conditions impact the metabolome of the cyanobacterium of therapeutic interest *Planktothricoides raciborskii* PMC 877.14

Inés Seijo Touceda<sup>a,b</sup>, Justine Demay<sup>a,b</sup>, Charlotte Duval<sup>a</sup>, Claude Yéprémian<sup>a</sup>, Anita Reinhardt<sup>b</sup>, Benjamin Marie<sup>a,*</sup>

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## ARTICLE INFO

**Keywords:**

Cyanobacteria

Thermal-mud

Culture conditions

Light

Metabolomics

Mass spectrometry

## ABSTRACT

Cyanobacteria are ancient photosynthetic microorganisms with a long evolutionary history that have adapted to inhabit diverse environments, such as thermal waters and muds. To do so, they are known to produce a wide range of bioactive molecules likely involved in adaptative traits such as high light and/or high temperature resistance mechanisms, which makes them particularly interesting for inclusion in thermal treatments and various therapeutic applications.

In this study, the impact of higher temperature and light intensity culture conditions on the metabolome of a promising cyanobacterial strain isolated from the muds of the Balaruc-les-Bains thermal station, *Planktothricoides raciborskii* PMC 877.14, was investigated through liquid chromatography and mass spectrometry analyses. Statistical analyses of the biological data and molecular network construction allowed for the exploration of potential metabolite induction under different culture conditions, in order to drive the production of specific metabolites aimed at coping with cellular stress. A global shift on intracellular metabolic composition was observed over time with increased biomass production. Higher light intensity was found to stimulate both growth and production of antioxidant and/or photoprotective molecules (such as ergothioneine, mycosporine-like amino acids or carotenoids) while an increase in temperature influenced the higher production of phycobilins (such as phycoerythrin and phycocyanin).

These results highlight the importance of culture/environmental conditions in driving intracellular metabolite differentiation and open up new perspectives in selecting of the optimal parameters for the growth and production of bioactive molecules for therapeutic applications in the cyanobacterium *P. raciborskii* PMC 877.14.

## 1. Introduction

Cyanobacteria are ancient photosynthetic prokaryotes that have played a major role in shaping the Earth into the planet we live on [1]. They were responsible for the transition from a primary carbon dioxide-rich atmosphere to the present relatively oxygen-rich terrestrial atmosphere, thanks to their capability to release oxygen through oxygenic photosynthesis. They are among the earliest microorganisms to have inhabited our planet and remain an essential source of atmospheric oxygen [2].

Today, despite their well-known ability to form blooms in numerous freshwater bodies around the world and produce toxins that pose a

threat to human health [3], cyanobacteria are also a prolific source of biologically-active metabolites with interesting applications [2,4]. The current global commercial production of cyanobacteria biomass is mostly focused on obtaining high-value products such as pigments, fluorescent dyes, proteins, essential fatty acids, polysaccharides, minerals, and vitamins, all of which are of interest to both food and pharmaceutical industries [5]. Cyanobacteria have long been known to produce structurally complex and distinct secondary metabolites belonging to 10 structural families including peptides, polyketides, alkaloids, lipids, and terpenes with potent biological properties [4,6]. Among these molecules with great potential for therapeutic and cosmetic applications, photosynthetic pigments such as

<footnote><sup>a</sup> UMR7245 MCAM MNHN-CNRS, Muséum National d'Histoire Naturelle, CP 39, 12 rue Buffon, F-75231 Cedex 05 Paris, France</footnote>

<footnote><sup>b</sup> Thermes de Balaruc-Les-Bains, 1 rue du Mont Saint-Clair BP 45, 34540 Balaruc-Les-Bains, France</footnote>

<footnote>* Corresponding author.
E-mail address: benjamin.marie@mnhn.fr (B. Marie).</footnote>
phycobiliproteins, carotenoids and chlorophyll as well as aeruginosins, scytonemin and mycosporine-like amino acids (MAAs) with potential anti-inflammatory, antioxidant and/or photo-protective activities [7].

Thus, the remarkable therapeutic potential of cyanobacteria can be particularly exploited through the use of beneficial thermal waters and muds, one of their natural ecological niches [8]. This potential was emphasized in previous studies that aimed to characterize the structural and functional diversity of cyanobacterial communities in the muds from the Balaruc-les-Bains thermal station and to explore their therapeutic properties [7]. In the latter, a multi-approach study (genome sequencing, metabolomic analyses and bioassays) revealed that the strain *Planktothricoides raciborskii* PMC 877.14 produces a high number of pigments known for their antioxidant and/or anti-inflammatory properties (such as phycobiliproteins, carotenoids, and chlorophylls). In particular, this strain produced a high concentration of phycoerythrin (PE), phycocyanin (PC) and β-carotene (dried weight, DW), and both its methanol and water extracts showed strong anti-inflammatory activities using cell line assays [7].

Light irradiance and temperature have been observed to have a direct effect on growth rate, biomass composition and metabolite production in different cyanobacterial strains [9,10]. The direct influence of the culture conditions on the diversity and dynamic of metabolite production have already been described [11]. Kultschar and co-workers [12] investigated the intra- and extracellular metabolite production of the cyanobacteria *Chlorogloeopsis fritschii* and observed an increased concentration of carotenoids and of the MAA shinorine under increased light intensity, which is consistent with the hypothesis of their potential antioxidant and photo-protective bioactivity. Taking previous observations into account, influencing bio-active compound production quota using augmented light and temperature constitutes an interesting strategy to study the stress-induced molecular adaptation of the strains through the induction or repression of specific bio-active metabolites.

Here, the dynamics of the intracellular metabolome and the potential of culture conditions to drive the production of specific bio-active metabolites were tested by a chemoinformatic approach (Fig. 1). Therefore, the aims of the present study were 1) to assess the intracellular metabolome variations of a *Plankthricoides raciborskii* strain grown under different light and temperature conditions across a growing cycle; 2) to distinguish the different metabolites whose cellular contents are most impacted by those specific culture conditions; and 3) to propose potential biological/ecophysiological functions linked to the metabolic regulation of those specific compounds.

## 2. Materials and methods

### 2.1. Biological material

The *Planktothricoides raciborskii* (Suda and Watanabe, 2002) [14] strain PMC 877.14 was isolated in 2014 from the thermal mud of Balaruc-les-Bains, France [13,15]. Since then, it has been maintained and cultured in a non-axenic culture at the Paris Museum Collection (PMC) by subculturing every two months in Z8 culture medium, then cultured under experimental conditions as previously described [16]. A large volume (600 mL) of pre-culture was cultivated in a 1-L Duran's bottle for 21 days in Z8 culture medium, with light intensity of and light-dark photoperiod of 12 h:12 h. From this initial culture, final 1:15 diluted cultures were inoculated in triplicates in 1-L bottles (40 mL inoculum in initial volume of 600 mL) for experimentation under "control", higher "light", and higher "temperature" conditions over the next 28 days. When investigating the metabolite production of the strain, great care was taken to maintain cultures in the growth phase to minimize the presence of heterotrophic bacteria. It is assumed that the level of heterotrophic bacteria remains negligible compared to cyanobacteria development, particularly in terms of biovolume, biomass, and metabolite contents, and may not significantly influence the observations on culture condition effects.

### 2.2. Culture conditions and sampling

Cultures were prepared from pre-culture cyanobacterial biomass (21-day old, 1:15 diluted cultures with a starting chlorophyll concentration of 460 µg.L⁻¹). They were then monitored over 28-day period under "control" (24 °C, 12 µmol·m⁻²·s⁻¹), higher "light" (24 °C, 35 µmol·m⁻²·s⁻¹), and higher "temperature" (30 °C, 12 µmol·m⁻²·s⁻¹) conditions (Table 1) [16]. These experimental conditions were chosen based on preliminary tests (at 50 µmol·m⁻²·s⁻¹ and 35 °C, data not

<caption>Table 1</caption>

Conditions tested in triplicates in the experiment (600 mL Z8 in 1-L bottle under constant agitation).

<table><thead><tr><td>Condition</td><td>Light intensity (µmol m<sup>-2</sup> s<sup>-1</sup>)</td><td>Temperature (°C)</td></tr></thead><tbody><tr><td>Control</td><td>12</td><td>24</td></tr><tr><td>Higher light</td><td>35</td><td>24</td></tr><tr><td>Higher temperature</td><td>12</td><td>30</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Analytical framework of the investigation of the heat or light stress-induced metabolomic response in the cyanobacteria *Planktothricoides raciborskii* PMC 877.14.</figcaption></figure>
shown) which indicated that higher temperature and light increases induced significant cellular stress and were not suitable for physiological investigations on biomass and bioactive metabolite production.

The experiments were conducted in triplicates, with a 12 h: light:12 h dark photoperiod and constant homogenization maintained by magnetic agitators. Samples were taken bi-weekly over the 28 days (D0, D3, D7, D10, D14, D17, D21, D24, and D28). Each sample volume (22 mL) was divided for (i) extraction and the untargeted metabolomic analysis of intracellular metabolites (10 mL), (ii) biomass monitoring using extraction and quantification of chlorophyll a pigments and quantification of phycobilin and carotenoid pigments (10 mL), and (iii) optical density absorbance measurements (2 mL). Since this species is filamentous, the cultures were carefully homogenized by mechanical agitation before biomass samplings.

## 2.3. Biomass dynamics

The follow-up on cyanobacterial biomass production was conducted by measuring of the dry biomass weight and optical density at 750 nm (turbidity/cell density) using spectrophotometry (Agilent Cary 60; Santa Clara, CA, USA). Absorbance measurements were converted into a natural logarithm before statistical analyses and growth rate variations were estimated based on the O.D.750nm ratio (h⁻¹).

## 2.4. Pigment quantification

Ten milliliter culture samples were centrifuged (10 min, 3220 g, 4 °C), the supernatant was discarded, and the pellet was stored at -80 °C for at least 24 h. Subsequently, the samples were lyophilized at -50 °C and 0.02 mBar for a minimum of 16 h (Labconco, Kansas) and the dry biomass was weighed. Hydrophilic phycobiliproteins were extracted using a saline buffer following to the method of [17]. Sequentially, lipid-soluble carotenoid and chlorophyll pigments were extracted with ethanol and quantified by spectrophotometry following the protocol of [18].

## 2.5. Metabolite analyses

### 2.5.1. Extractions

Each 10 mL sample take at different time points, corresponding to each of the 3 conditions tested in triplicate, was centrifuged. The resulting pellet was stored at -80 °C for a minimum of 24 h and then lyophilized following the same procedure used for pigment quantification (see Section 2.4). The dried biomass was weighed and kept at -20 °C until metabolite extraction.

At the conclusion of the culture period, two sequential extractions were performed each sample using a solvent ratio of 200 µL per 1 mg of dry biomass weight, ensuring a minimum volume of 200 µL of extraction solvent for samples with a biomass lower than 2 mg at the beginning of the experiment. The first extraction targeted the hydrophilic metabolites and utilized a water/formic acid mixture (0.1 %), while the second extraction focused on hydrophobic metabolites and used with a methanol/acetonitrile/water mixture (2:2:1), acidified with 0.1 % formic acid. Cell lysis was achieved through sonication: 3 cycles of 30 s, with a 10 s break between each cycle, at 75 % of the maximum intensity (SONICS Vibra Cell, Newton, CT, USA; 130 W, 20 kHz). Subsequently, the samples were centrifuged (10 min, 13,400 g, 4 °C), and the resulting supernatants were collected in amber glass vials and stored in the dark at -20 °C until mass spectrometry analyses.

### 2.5.2. Mass spectrometry analyses

Samples were analyzed in positive and negative ionization modes in the pre-culture initial tests. Upon obtaining 1288 ions in the positive ionization mode compared to 327 ions in the negative mode, the positive mode was selected for subsequent metabolome studies on this strain. Extracts were separated using ultra-high performance liquid

chromatography (UHPLC). Each sample, was injected with a volume of 2 µL, and separation was carried out using a Polar Advance II 2.5 pore C18 chromatographic column (Thermo Fisher Scientific, Waltham, MA, USA) at a flow rate of 300 µL.min⁻¹ with a linear gradient of acetonitrile acidified with 0.1 % formic acid (from 5 to 90 % in 15 min). Metabolite contents were analyzed using an electrospray ionization hybrid quadrupole time-of-flight (ESI-qTOF) high-resolution mass spectrometer (Compact, Brucker, Bremen, Germany) in the 50–1500 m/z range in autoMSMS positive mode.

MS data were processed using MetaboScape 4.0 software (Bruker, Bremen, Germany) for recalibration of each sample analysis (according to acetate formate internal standards), peak detection, selection of ions with an intensity >5000 counts in at least 10 % of the samples and peak realignment. Additionally, different states of charge and adducts were grouped together and the “area-under-the-peak” was determined to generate a unique global data matrix containing semi-quantification results for each metabolite. This included peaks for each analyte characterized by the mean mass of its neutral form and its corresponding retention time.

### 2.5.3. Metabolite annotation

The annotation of the metabolites produced by *P. raciborskii* PMC 877.14 was attempted using MS/MS data with the software MetaboScape (Bruker, Bremen, Germany) and NPAtlas V1.0 structural database based on MS (precise mass and isotopic pattern) [19]. This was then confirmed with MetGem 1.3.6 using the GNPS algorithm for MS/MS spectra comparison by querying public spectral databases such as HMDB, NIH, MIADB, ReSpect, MetaboBase, Massbank, EMBL, LipidBlast NIST14 and MS-DIAL [20,21]. The data extraction process was performed with the MetaboScape software in a single step for all samples investigated, including both aqueous and organic phase extracts, where each analyte corresponds to a single entry, considering all adducts merged together.

### 2.5.4. Biosynthetic gene clusters prediction

Biosynthetic gene clusters (BGC) were identified using the anti-SMASH software [22] available through the MicroScope platform. This tool allowed the comparison of homologies between the query and the BGC sequences annotated in the databases, facilitating the characterization of gene cluster organization and the prediction of molecules potentially produced by the strain *P. raciborskii* PMC 877.14. In silico analysis of the whole genome of this strain revealed the presence of at least 7 potential biosynthetic gene pathways, including 3 uncharacterized non-ribosomal peptide synthase (NRPS) and a carotenoid synthesis gene cluster.

## 2.6. Statistical treatments

The spectral data was analyzed using the MetaboAnalyst 5.0 platform [23]. A Pareto normalization of the metabolite data matrices was conducted to reduce the impact of large values while maintaining the data structure. Subsequently, a Partial Least Square Discriminant analysis (PLS-DA), a Principal Component Analysis (PCA), hierarchical clustering and one-way ANOVA were performed.

The statistical analysis of the measured parameters (O.D., biomass concentration and pigment composition) was carried out using RStudio software (version 4.0.5) [24]. An analysis of variance (ANOVA) or the non-parametric Kruskal Wallis test followed by the Dunn post-hoc test with Bonferroni's correction was applied based on normality and homoscedasticity of the data sets. Significant differences between treatments and the control were investigated for both entire culture duration and specific sampling points.
3. Results

**3.1. Effect of culture conditions in growth kinetics**

<caption>Three different parameters (optical density, dry biomass and</caption>

chlorophyll concentration) were monitored as proxy variables for cul-
ture growth (Fig. 2). Higher and lower dry biomass concentrations (524
mg·L⁻¹ and 301 mg·L⁻¹) were respectively observed at the end of the
culture performed under higher “light” and higher “temperature” con-
ditions. Statistically significant differences between conditions (p-value

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Effect of culture conditions on growth kinetics (A, B), pigment production (C, D), and trends in chlorophyll and carotenoid levels (E,F, in mg·L⁻¹) of *P. raciborskii* PMC 877.14. Significant differences between conditions are indicated by different letters at specific time points (Kruskal Wallis tests, *p* < 0.05 followed by Dunn-Bonferroni post-hoc tests). On Day 28, the “light” condition is represented by a purple dot, as only values from two out of three replicates were included in the analyses, the third replicate showing signs senescence. A) Dry biomass, B) optical density absorbance values at 750 nm, C) phycocyanin and D) phycoerythrin concentration evolution are shown throughout the culture time under the three different conditions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
<0.05) were found for the dry biomass and optical density measure-
ments, with both dry biomass and O.D. values being higher under the
higher “light” conditions (Fig. 2A and B). A decrease in the extracted
chlorophyll *a* concentration is observed at the end of the culture time,
for both “control” and higher “light” conditions (Fig. 2E–F).

For the biomass concentration and O.D. monitoring, a characteristic
exponential phase starting from day 14 and day 10, respectively, is
observed, whereas chlorophyll *a* seems to be produced in a more con-
stant manner (Supplementary data S1A, S2A).

3.2. *Effect of culture conditions on the production of pigments of interest*

Out of the three phycobiliproteins, phycoerythrin was produced in
the highest concentration under the higher “temperature” condition
(Fig. 2D), while the maximum concentrations of phycocyanin and
allophycocyanin (APC) were observed at 30 °C (Fig. 2C and Supple-
mentary Fig. S1B). The maximum phycobilin concentrations were
observed under the higher “temperature” and higher “light” conditions
for PE and PC, and APC, respectively (Supplementary Fig. S2A-B-C).

The variation of pigment concentrations throughout the 28 days of
culture followed a global increasing trend. However, this increase ap-
pears more pronounced under the higher “temperature” conditions,
notably from day 14, except for carotenoids for which no positive effect
of the higher “temperature” was detected. At the end of the culture se-
ries, the highest concentration of carotenoids per cellular dry weight
(4.0 µg·mg⁻¹ DW) was measured in cultures performed at 30 °C.

3.3. *Effects of culture conditions on the intracellular metabolome*

3.3.1. *Metabolome variations throughout culture time*

The global variations of both methanol- and water-extracted
metabolomes (i.e., organic and aqueous extracts, representing a total
of 3018 and 1720 ions detected by simple LC-MS experiments, respec-
tively) were initially explored along the time-series with an unsuper-
vised multivariate Principal Component Analysis (PCA) regarding all
three experimental conditions tested (Fig. 3). Both plots show a

progressive evolution over the time of culture of the intracellular con-
centration of metabolites along the principal component 1 regarding
methanol- or water-extracted metabolomes (Fig. 3A–B, respectively).

After conducting the first unsupervised exploration of the global
metabolome dataset, a Partial Least-Squares Discriminant Analysis (PLS-
DA) was performed to highlight the most discriminant variables/me-
tabolites across the time-series considering samples from the three
experimental conditions together. The PLS-DA, like the PCA, aims to
reduce data dimensionality, but it is a discriminant analysis that con-
structs a model based on provided information about sample groups
(time in this case). Variable importance in projection (VIP) plots were
created to observe the 25 variables (with VIP scores >3.6 for organic
extracts and > 2.9 for aqueous extracts) that contribute the most to the
PLS-DA model, and their changes over culture time (Fig. 4A). Overall,
there was a clear increase over time in the relative concentration of the
variables that contribute most to sample discrimination along the time
series (highest VIP scores) as shown on Fig. 4 and supplementary Fig. S3
for aqueous and organic extracts, respectively. However, there were a
few exceptions of variables showing opposite decreasing trends over the
time series (highlighted by a blue rectangle in the aqueous extracts plot,
Fig. 4A and supplementary Table S2). Specific examples of different
time-variation trends within the variables with the most important VIP
scores are displayed in Fig. 4B and C.

3.3.2. *Effect of culture conditions*

A PCA analysis was initially conducted to examine the overall dis-
tribution of intracellular metabolite composition in *P. raciborskii* PMC
877.14 under “control”, higher “light”, and higher “temperature” con-
ditions for both organic and aqueous extracts taking into account all
time-points collectively (Fig. 5). A noticeable distinction between the
higher “light” condition and the other two conditions is evident in the
MeOH:AcN:H₂O extracts along PC2. Meanwhile the clusters formed by
the “control” and “temperature” conditions largely overlap, indicating a
less clear separation among the water extracts obtained from different
cultures compared to the organic extracts. However, PC2 primarily ac-
counts for the variance between the higher “light” condition and the

<figure><img src="image_6.png" /><figcaption>Fig. 3. Principal component analysis (PCA) plots representing the evolution of intracellular metabolite composition of *P. raciborskii* PMC 877.14 over time for organic (left) and aqueous (right) extracts. Each time point observed for every condition (“control”, C, higher “light”, L, and higher “temperature”, T) is included in the analysis.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Partial Least-Squares Discriminant Analysis (PLS-DA) of the variables in the aqueous extracts over time. A) Variable Importance in the Projection (VIP) plot for the aqueous extracts during culture time ($Q_{cum}^2 = 0.91$ and $R_{cum}^2 = 0.87$). The 25 variables that contribute the most to the PLS-DA model are displayed. One un-annotated variable with a decreasing concentration trend is highlighted with a blue rectangle. A list of all variables with a VIP score > 2.5 in the PLS-DA of metabolome composition variation over time can be found in supplementary Table S1. B) Boxplot for two annotated metabolites (the nucleotide 2'-deoxyguanosine 5'-phosphate and the biotin derivative 2-iminobiotin, indicated with red arrows in the VIP plot) from the aqueous extract showing increase concentration over culture time. C) An example of a differential trend observed with the annotated variable oxiglutathione (indicated with a blue arrow in the VIP plot). The boxplots illustrate its evolution over culture time for the three conditions combined (left) and separately (right). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

other two conditions.

In addition, to highlight the differences between conditions, a hierarchical clustering analysis was performed. Dendrogram Euclidean distance trees were built for each group of extracts and culture time to observe their relative proximity or distance with regard to their respective biological observations (Fig. 6). Three distinct main clusters were similarly observed for both hydrophobic and hydrophilic metabolome extracts. For the aqueous extracts, the first branching separates the D0 samples for the three conditions from the rest of the samples. Then, three clusters can be defined: I) from day 10 to day 28 for the "control" and higher "temperature" conditions, II) from day 10 to day 28 for the higher "light" condition and III) from the start of the culture to day 7 for all conditions. The structure of the distance tree for the organic phase reveals that the hydrophobic metabolites follow slightly different diversification time phases than the hydrophilic ones, with a slightly later differentiation of the metabolomes observed under higher "light" and "temperature" conditions. For the organic extracts the first branching separates clusters I (day 10 to 28 for the "control" condition and day 17 to 28 for the higher "temperature" condition) and II (day 14

to day 28 for the higher "light" condition) from cluster III (samples from the early days of cultures). Moreover, cluster III can be further divided into three subgroups: i) samples from the start of the culture to day 3 for all conditions, ii) D7 samples for the "control" and D7 to D14 for the higher "temperature" and iii) D7 to D10 samples for the higher "light" condition.

Overall, these observations allowed for the identification of a specific tipping point of divergence in the metabolite composition (day 10). It was suggested that an analysis restriction on the samples generated during the latter days of the experiments (from day 10 to day 28) may reinforce the initial observation of a metabolite separation in the higher "light" and higher "temperature" metabolic profiles. Indeed, a total of 1239 out of 3012 and 774 out of 1719 metabolites observed for organic and water extracts, respectively, show significant variations induced by the culture conditions at later time points, according to one-way ANOVA tests (supplementary Tables S3–S4).

Therefore, a PLS-DA was conducted using only the samples from late culture days specifically from day 10 to day 28 in order to highlight the differences in metabolite contents between conditions (Fig. 7). This
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Global visualization of the metabolome on a principal component analysis (PCA) plot. The plot represents the distribution of intracellular metabolite composition of *P. raciborskii* PMC 877.14 for organic extracts (on the left) and aqueous extracts (on the right). The conditions include "control", higher "light", and higher "temperature".</figcaption></figure>

analysis clearly demonstrates the distinction between the conditions for both extracts and help identifying the variables that play a significant role in the metabolome variations of *P. raciborskii* PMC 877.14 across the tested experimental conditions.

The capability of culture conditions to increase or decrease the intracellular content of specific metabolites was further demonstrated by boxplots generated using the data of the normalized concentration distribution of two high-VIP score variables. These variables were found to be more prevalent under higher "light" conditions (Fig. 8).

Out of a total of 1720 and 3018 ions detected by simple LC-MS experiments in the aqueous and organic extracts, 638 and 1438, respectively, were automatically selected by the mass spectrometer and further analyzed by LC-MS/MS. Considering MS/MS fragmentation datasets from both aqueous and organic extracts of samples from day 10 to the end of culture time, molecular networks were built and metabolite annotation was attempted (Fig. 9, supplementary Tables S7–S8). This representation advantageously highlights the potential of the different experimental treatments to drive the global production of certain metabolite families.

Each ion was represented by a node in which the diameter is proportional to the respective retention time of the analytes, while the colour pie chart indicates the sum of the relative concentrations retrieved in samples belonging to each culture condition. Metabolite family names were indicated on the figure and nodes of annotated metabolites were encircled in black. Only clusters or nodes that could be annotated were included in Fig. 9 (complete molecular network in supplementary Fig. S4A–B). Different metabolites could then be observed and annotated in the two fractions, with a higher prevalence of hydrophobic molecules in the organic extracts (numerous glycol clusters, phospholipids and glycerolipids) than in the aqueous ones, as could have been expected. A more important number of hydrophilic compounds was observed only in the water extracts (nucleotide sugars, di- and tri-saccharides, glycan trisaccharides). Interestingly, several potentially bioactive metabolites with an interest for therapeutic applications, such as glycerophospho-N-palmitoyl ethanolamine, ergothioneine, 478-Da MAA, or gamolenic and stearidonic acids could be

monitored in both extracts.

# 4. Discussion

## 4.1. Effect of time and conditions in the growth of *P. raciborskii*

The analysis of the growth kinetics of cultures incubated under different conditions showed distinct patterns in terms of optical cell density and biomass production (Fig. 2A and B). An increase from D0 to D28 was documented for the three measured parameters reflecting the overall growth of the strain during culture time. However, maximum optical densities and biomass concentrations were observed in cultures exposed to 35 µmol·m⁻²·s⁻¹ light intensity, whereas the higher temperature (30 °C compared to 24 °C) condition did not seem to favor higher growth of this strain. Similar results were observed in previous studies such as the one carried out by Gonçalves and co-workers [25], who found the optimal growth of four microorganisms (cyanobacteria and microalgae) to be favored at 25 °C rather than the other two tested temperatures (15 and 35 °C). Our present results show that the "control" temperature (set at 24 °C) would be preferred over the higher temperature (30 °C) to obtain a higher biomass in a culture of the cyanobacterium *P. raciborskii* PMC 877.14. Considering chlorophyll *a* production, previous studies have detected a stimulation effect induced by a reduction in light irradiance [25,26]. However, no statistically significant difference in chlorophyll *a* cellular ratio (µg chl *a*·mg⁻¹) could be detected for this strain exposed to the three tested experimental conditions.

## 4.2. Effect of time and culture conditions in photosynthetic pigment production

### 4.2.1. Phycobilin production

Phycobilins are water-soluble, photosynthetic pigment-protein complexes derived from cyanobacteria with various biotechnological applications such as in nutraceuticals, cosmetics, the food industry or biomedical research [27]. Their strong antioxidant and anti-
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Dendrograms of the hierarchical clustering of samples in organic extracts (left) and aqueous extracts (right) based on time and conditions using Euclidean distances. Three distinct clusters are visible for both trees. For the aqueous extracts: I) samples from day 10 to day 28 for the “control” and the higher “temperature” conditions, II) samples from day 10 to day 28 for the higher “light” condition and III) samples from the beginning of the culture to day 7 for all conditions. For the organic extracts: I) samples from day 10 to day 28 for the “control” condition and day 17 to 28 for the higher “temperature” condition, II) from day 14 to day 28 for the higher “light” condition, and III) all samples from the early days of culture. The cluster III for the hydrophobic metabolites can be further divided into i) samples from the start of the culture to day 3 for all conditions, ii) Day 7 samples for the “control” condition and days 7 to 14 for the higher “temperature” condition and iii) Days 7 to 10 samples for the higher “light” condition.</figcaption></figure>

inflammatory bioactivities have been extensively studied and characterized [28–30]. The results of their quantification throughout the 28 days of culture of the strain *P. raciborskii* indicate a positive influence of higher “temperature” and both PC and PE cellular contents. This observation is consistent with the results of previous studies screening phycobiliprotein contents under different environmental conditions in various cyanobacteria. Hemlata and Fatma [31] observed the highest phycobilin production at 30 °C, when testing a temperature range between 20 and 40 °C for *Anabaena* NCCU-9. Similarly, Kumar et al. [9] detected the maximum accumulation of phycobiliproteins at temperature 35 °C, when exploring the effects of various temperatures ranging from 20 to 40 °C on *Arthrospira platensis* (formerly known as *Spirulina platensis*, or spirulina). Taken together, these results suggest that a certain increase in the culture temperature would be globally favorable for phycobiliprotein production by different cyanobacteria. However, a reduction in the global pigment concentration of the culture with a higher increase in temperature (above 40 °C) was also observed in the aforementioned studies, as more extreme temperatures did not support cyanobacterial growth (in line with our observations on the effect of higher temperature in growth in the present case, Section 4.1) and hence

did not favor an increased pigment production, considering the whole culture volume rather than the cellular ratio. Nevertheless, adaptation to temperature changes, tolerance to extreme conditions and optimal growth temperatures vary from one strain to another, therefore the effect of a further increase in temperature on pigment production of *P. raciborskii* remains to be more finely investigated considering a wider temperature range to be tested (e.g., 20–40 °C). Overall, our findings prove that an increase in temperature from 24 to 30 °C has a greater impact on PE and PC production than light for the strain *P. raciborskii* PMC 877.14.

Among the three phycobilins, the highest yield of cellular content was achieved for PE. This is particularly interesting due to the bioactive potential of this red cyanobacterial pigment. For instance, Sonani et al. [32] detected a broader antioxidant activity of PE than that of PC and APC after examining the scavenging effect of the three pigments on α-diphenyl-β-picrylhydrazyl (DPPH) free radicals. They found dose-dependent antioxidant activities for all three phycobiliproteins in decreasing order of PE, PC and APC. In contrast to PE and PC, no statistically significant differences were found for APC production according to the culture conditions investigated in this study.
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Partial Least-Squares Discriminant Analysis (PLS-DA) conducted with samples collected between day 10 and day 28 based on their respective culture conditions ("control", higher "light", and higher "temperature") for both hydrophobic (A) and hydrophilic (B) metabolite extracts. The performance parameters A (accuracy), R² (representativeness) and Q² (reproducibility) for each principal component of the PLS-DA are as follows: 0.94, 0.74 and 0.65 for the organic extracts and 0.94, 0.82, and 0.61 for the aqueous extract, respectively. The supplementary Tables S5–S6 provide a list of variables with a VIP score > 2.5.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** A) Variable Importance in the Projection (VIP) plot for organic extracts comparing different conditions. B) Boxplots showing the concentration of two variables with high VIP scores (indicated by bold dotted lines in the VIP plot) along with their potential metabolite annotations and corresponding structures.</figcaption></figure>

### 4.2.2. *Carotenoid production*

Higher concentrations of carotenoids were observed on day 10 of culture under the higher "light" condition, compared to the "control" and the higher "temperature" conditions. Enhanced carotenoid production with an increase in light intensity could potentially serve as an adaptive mechanism for photoprotection in cyanobacteria according to Kumar et al. [9]. Carotenoids play a role in various photosynthetic processes, such as light harvesting, energy transfer to chlorophyll, photoprotection by quenching triplet state chlorophyll molecules, and scavenging singlet oxygen [26]. This implies that the observed increase

in carotenoid content under higher light intensity and the potential subsequent increase in reactive oxygen species (ROS) production may be linked to the photoadaptation mechanisms of *P. raciborskii* in order to enhance cellular capabilities to mitigate this environmental stress. Additionally, previous studies on various cyanobacteria have shown light inducted carotenoid production without affecting chlorophyll *a* synthesis [33], which aligns with our current observations in *P. raciborskii* cultures.
<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Molecular network representation of the molecules detected in the organic extracts on the left and in the aqueous extracts on the right of samples from day 10 to day 28 of culture time. The colors of the dots correspond to the relative concentration of the detected ions in samples under each of the tested conditions. Metabolite annotations were made using the MetGem 1.3.6 software based on public MS/MS databases. Only clusters with a relevant annotation (cosine score > 0.65) are shown (the complete molecular networks can be founded in supplementary Fig. S4A–B). Dot diameters increase according to the respective retention time (RT) of the corresponding ions ranging from 1 to 16 min.</figcaption></figure>

## 4.3. Variations in the global metabolic composition of *P. raciborskii*

The evolution of metabolic profiles over time and the distribution of metabolite families within culture conditions were examined through LC-MS analyses using various multivariate statistical tests. Results from PCA and hierarchical clustering provided a visual representation of the overall evolution of the metabolome over time by observing to the observation of the distances between samples in relation to their similarities and differences. Additionally, PLS-DA revealed a general trend of intracellular metabolite accumulation of over culture time, following increases in optical density and biomass, with a few exceptions of metabolites showing different trends in their relative concentrations. These trends were observed across all three conditions, although changes in metabolic composition (Fig. 5, PCA by conditions) indicated that “higher” light intensity had a stronger influence on driving intracellular metabolite differentiation compared to the other conditions.

Analysis of metabolites produced by *P. raciborskii* PMC 877.14 through a molecular networking approach reveals that the majority of metabolites and metabolite families were primarily influenced by the extraction solvent based on their respective polarity. Additionally, many

of them displayed distinct distributions under different culture conditions. Among the most hydrophobic compounds, glycols and phospholipids were found in higher quantities in cultures exposed to elevated “light” and “temperature” conditions, compared to control conditions. It is suggested that the secretion of glycosylated polymers and carbohydrates could serve as a universal biological defense mechanism to combat adverse conditions by producing extracellular polymeric substances [34]. This observation may indicate a potential increase in EPS production as a defensive response to stress induced by light and temperature changes. [35]. This hypothesis gains significance when considering that certain bioactive molecules within the glycerophospholipid clusters, such as glycerophospho-N-palmitoyl ethanolamine, the metabolic precursor of N-palmitoyl-ethanolamine, could be involved in anti-inflammatory activities [36].

Our data also shows the presence of common metabolites in the organic and aqueous extracts such as nucleosides and nucleoside diphosphate sugars. Both of these groups of compounds appear to be higher in light-stimulated, conditions, which aligns with the hypothesis of increased photosynthesis efficiency under higher light intensity and a greater demand for metabolism through intermediate metabolites
necessary for cell density and biomass production (as indicated by the higher growth rates observed in the higher “light” condition). Another notable outcome of the culture growth supported by photosynthesis processes is the increase in intracellular sugar accumulation, as evidenced by the higher concentration of di- and tri-saccharides in the water extract in the higher “light” condition extracts.

Light induction of potential antioxidant and photoprotective metabolites was also observed. For example, ergothioneine (Fig. 8B), a potent antioxidant nutraceutical [37], and a 478-Da MAA showed increased concentrations under a light intensity of 35 µmol·m⁻²·s⁻¹. Ergothioneine biosynthesis in cyanobacteria has been linked to coping with oxidative damage caused by oxygenic photosynthesis [38]. Our observations suggest the activation of the production of this antioxidant compound as a response to the increased presence of ROS induced by the “higher” light condition. Similarly, other MAAs, a large family of natural UV-absorbing molecules, have been shown to be triggered by light stresses in cyanobacteria strains [39,40]. The increased light intensity of the higher “light” condition appears to have activated photo-protection mechanisms in *P. raciborskii*. Additionally, gamma-linolenic and stearidonic acid, omega-6 and 3 polyunsaturated fatty acids (PUFAs) known for their potential antioxidant and anti-inflammatory properties [41] were found to be predominantly produced under the higher “light” condition.

Nevertheless, light and temperature appear to have a suppressive effect on the production of long chain unsaturated glycerolipids. Among these lipids are molecules such as mono-galactosyl-mono-acylglycerol, which have potential antibiotic, antifouling and anti-inflammatory properties [42,43]. This observation could be interpreted as a potential adaptation strategy of this *P. raciborskii* strain to cellular stress induced by increased membrane fluidity under higher “light” and “temperature” conditions [44].

## 5. Conclusion

In the present study, we evaluated the effect of higher “light” and “temperature” culture conditions, on the growth and molecular adaptation of a cyanobacterium of therapeutic interest, *Planktothricoides raciborskii* PMC 877.14. We provided insight into the diversity of metabolites produced by this strain and examined the potential of the tested conditions to stimulate the production of molecules of interest. Higher light intensity favored the growth and production of antioxidant (ergothioneine), photoprotective (MAA) and anti-inflammatory (PUFAs) molecules. Conversely, a higher temperature increased phycobilin concentrations (PE and PC). Thus, both light and thermal stresses appeared to potentially stimulate exopolysaccharide (EPS) production as a cellular response to environmental stress. These results represent the first step in selecting optimal parameters for growth and targeted production of therapeutic molecules in the Balaruc's cyanobacterium *Planktothricoides raciborskii* PMC 877.14, with the ultimate goal of integrating them into thermal healing processes profitably.

Future perspectives include further exploration of the effects of other biotic and abiotic factors in the production of bioactive molecules (different light sources, absence of light/dark cycles, coculture of promising strains isolated from Balaruc-les-Bains thermal mud), along with bioactivity assessment through specific bioassays and understanding the biological/physiological function of these components.

Additionally, investigating potential synergistic properties of co-culturing different cyanobacteria strains of interest isolated from mature Balaruc thermal mud (such as *Planktothricoides raciborskii* PMC 877.14 and *Aliinostoc* sp. PMC 882.14) and developing co-culture strategy to enhance the activity spectra of bio-active compounds within therapeutic strategies could be a valuable next step.

## Author declaration

Submission of an article implies that the work described has not been

published previously (except in the form of an abstract or as part of a published lecture or academic thesis), that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work as carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, without the written consent of the copyright-holder. By attaching this Declaration to the submission, the corresponding author certifies that:

- The manuscript represents original and valid work and that neither this manuscript nor one with substantially similar content under the same authorship has been published or is being considered for publication elsewhere.

- Every author has agreed to allow the corresponding author to serve as the primary correspondent with the editorial office, and to review the edited typescript and proof.

- Each author has given final approval of the submitted manuscript and order of authors. Any subsequent change to authorship will be approved by all authors.

- Each author has participated sufficiently in the work to take public responsibility for all the content.

## CRediT authorship contribution statement

Inés Seijo Touceda: Writing – original draft, Visualization, Investigation, Formal analysis. Justine Demay: Writing – review & editing, Supervision, Methodology, Data curation, Conceptualization. Charlotte Duval: Writing – review & editing, Resources. Claude Yéprémian: Writing – original draft, Methodology. Anita Reinhardt: Writing – review & editing, Supervision, Project administration, Conceptualization. Benjamin Marie: Writing – review & editing, Validation, Methodology, Funding acquisition, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

We would like to thank the UMR 7245 MCAM, Muséum National d'Histoire Naturelle, Paris, France for laboratories facilities and the Thermes de Balaruc-les-Bains for funds. This work was supported by the "Société Publique Locale d'Exploitation des Thermes de Balaruc-les-Bains" (SPLETH), the town of Balaruc-les-Bains and the Muséum National d'Histoire Naturelle (MNHN). The MS spectra were acquired at the Plateau technique de spectrométrie de masse bio-organique (MNHN).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.algal.2024.103738.

## Data availability

Data will be made available on request.

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