# Integrative assessment of biohydrogen and biomethane production from Brittany and Caribbean Sargassum (Ochrophyta, Fucales)

Yiru Zhao, Nathalie Bourgougnon, Jean-Louis Lanoisellé, Thomas Lendormi

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Yiru Zhao, Nathalie Bourgougnon, Jean-Louis Lanoisellé, Thomas Lendormi. Integrative assessment of biohydrogen and biomethane production from Brittany and Caribbean Sargassum (Ochrophyta, Fucales). Biomass and Bioenergy, 2026, 206, pp.108579. 10.1016/j.biombioe.2025.108579. hal-05368703

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Integrative assessment of biohydrogen and biomethane production from
Brittany and Caribbean *Sargassum* (Ochrophyta, Fucales)

Yiru Zhao<sup>a</sup>, Nathalie Bourgougnon<sup>b, c</sup>, Jean-Louis Lanoisellé<sup>a, 1</sup>, Thomas Lendormi<sup>a, * d</sup>

A B S T R A C T

Dark fermentation (DF) and anaerobic digestion (AD) are promising approaches for energy recovery and waste management, yet the process performance and operational feasibility of Caribbean *Sargassum* under semi-continuous conditions remain uncertain. This study provides a holistic evaluation of the biochemical hydrogen potential (BHP) and biochemical methane potential (BMP) of two brown algae species: *Sargassum muticum* from Brittany and a mixture of Caribbean *Sargassum* species. Batch tests showed that *S. muticum* exhibited the highest hydrogen yield (32 NL H₂·kg VS⁻<fnref n="1" />), while methane yields across all samples ranged from 57 to 140 NL CH₄·kg VS⁻<fnref n="1" />. HCl-assisted pretreatment enhanced energy recovery in Caribbean biomass, with gains of 66.6 % for beached samples. For the first time, semi-continuous DF-AD was implemented in 2 L bioreactors with powdered Brittany and Caribbean *Sargassum* as substrates. Under these conditions, *S. muticum* produced four times more hydrogen (12.7 vs. 3.0 NL H₂·kg VS⁻<fnref n="1" />) and twice as much methane (59.9 vs. 29.5 NL CH₄·kg VS⁻<fnref n="1" />) compared with Caribbean biomass. These findings demonstrate the limited suitability of Caribbean *Sargassum* for hydrogen production alone but confirm its viability for methane recovery, providing new insights for the valorization of invasive seaweed through integrated bioenergy systems.

**1. Introduction**

The 21st Conference of Parties (COP21) established a universal and
sustainable climate agreement in 2015, aiming to limit global warming
to 1.5-2 °C above pre-industrial levels [1]. Since then, annual United
Nations Climate Change Conferences have been held to refine, imple-
ment, monitor, and strengthen commitments made under this historic
accord [2]. In response, the International Energy Agency (IEA) pub-
lished initial and updated versions of the Net Zero Emissions by 2050
(NZE) Scenario in its 2021 and 2023 benchmark reports. According to
the latest report, fossil fuels accounted for approximately 125 million
tonnes (Mt) of methane emissions in 2022, while global carbon dioxide
(CO₂) emissions from the energy sector reached a record high of 37
billion tonnes (Gt), exceeding pre-pandemic levels by 1 %. Methane
alone has accounted for about 30 % of global temperature increases
since the Industrial Revolution. Substantial reductions in both CO₂ and
methane emissions are therefore essential to achieve the 1.5 °C target,
with a goal of reducing methane emissions from fossil fuels by 75 %
within this decade [3]. Meeting these objectives requires the adoption of
cost-effective alternatives to natural gas, including the promotion of
biogas and biomethane. In parallel, the development of technologies for
carbon capture, utilization, and storage, as well as advances in

hydrogen, hydrogen-based fuels, and sustainable bioenergy, are
considered essential [4].

Dark fermentation (DF) has emerged as a promising pathway for
biohydrogen generation, supported by progress in reactor engineering,
process optimization, and the discovery of efficient hydrogen-producing
microorganisms [5,6]. Recent research has focused on enhancing
hydrogen yields by regulating the reducing power [7], optimizing pro-
cess parameters [8], incorporating inorganic nanoparticles [9], and
applying effective pretreatment strategies [10]. In contrast, anaerobic
digestion (AD) has already achieved a relatively high technology read-
iness level, with current efforts directed toward process intensification.
Key strategies include substrate selection [11], combinations of pre-
treatment methods [12], and the integration of conductive materials
[13].

Marine biomass is estimated to possess an energy potential exceeding 100 EJ per year—approximately five times greater than that of terrestrial biomass [14]. Brown seaweeds, which contains 30–60 % carbohydrates on a dry weight basis, represent a promising feedstock for biogas production due to the absence of lignin-like components in their cell walls [15,16]. Studies on species such as *Macrocystis* (Laminariales) have reported biochemical methane potential (BMP) values comparable to those of terrestrial crops, ranging from 0.39 to 0.41 m³ CH₄·kg⁻<fnref n="1" />

<footnote>ª Univ. Bretagne Sud, UMR CNRS 6027, IRDL, F-56300, Pontivy, France</footnote>

<footnote>ᵇ Univ. Bretagne Sud, EMR CNRS 6076, LBCM, F-56000, Vannes, France</footnote>

<footnote>* Corresponding author.
E-mail address: thomas.lendormi@univ-ubs.fr (T. Lendormi).</footnote>

<footnote n="1">¹ Deceased author.</footnote>
Volatile Solids (VS) [17]. Brown seaweas also act as effective biological carbon sinks, contributing both short-term carbon assimilation and long-term carbon sequestration [18]. Through photosynthesis, they help mitigate greenhouse gas (GHG) emissions. Large-scale cultivation is feasible without arable land or fertilizers, further enhancing their potential for sustainable bioenergy. However, their biochemical composition varies significantly, making some species unsuitable for AD. Such variability is influenced by species, geographical origin, harvesting season, and biotic or abiotic parameters [19].

For *Sargassum* spp., available data indicate extremely low methane (CH₄) yields from Caribbean-origin biomass [20]. Thompson et al. [21] reported less than 50% of the theoretical biochemical methane potential (TBMP) under monodigestion in lab-scale study, while Milledge et al. [20] attributed this poor performance to phenolic compounds that inhibit hydrolysis during the initial stages of AD. Additional limiting factors include the recalcitrant structure of *Sargassum*, its high ash content (31–37 % dw), and substantial fiber content (34–47 % dw). With respect to hydrogen production via DF, the literature is scarce. To date, Costa et al. [22] conducted the only study on this species, using batch-mode assays with *Caldicellulosiruptor saccharolyticus* in a 160 mL bioreactor, which produced results of limited industrial relevance. Laboratory-scale DF research on algal has emphasized pretreatment strategies to disrupt the polysaccharide matrix. Chemical pretreatments such as acids, alkalis, or surfactants are commonly used to enhance substrate accessibility and improve hydrogen yields [10,23–25]. Additionally, energy-intensive methods such as thermal treatments [23,24, 26,27] and autoclaving have been applied to facilitate biomass breakdown [22,27,28].

Interest in using *Sargassum muticum* for biofuel and biorefinery applications has increased since the early 2010s [29–31]. Native to Japan and widely available in Europe, particularly in Brittany, this brown alga has been identified as a promising feedstock for AD-based biomethane production [32,33]. Since 2011, however, other *Sargassum* species have been linked to recurring “golden tide” events in the Caribbean, Gulf of Mexico, and West Africa [34]. Satellite images has estimated these tides contribute nearly 1 million tonnes (wet weight) of biomass annually in the Atlantic [35]. The holopelagic species *S. fluitans* and *S. natans* are the main contributors to these events. Their decomposition releases hydrogen sulfide (H₂S), a harmful gas that causes respiratory irritation and corrosion, creating environmental and socio-economic crises for coastal communities [36]. Large beach-cast deposits also accumulate debris, contributing to pollution and waste management challenges.

The French Agency for Ecological Transition (ADEME) estimates that nearly 99 % of the *Sargassum* collected in Guadeloupe is currently spread without any valorization, contributing to the saturation of spreading sites. Direct spreading is discouraged because of the risks of soil salini-zation (7g·kg⁻¹ of wet *Sargassum*), the presence of contaminants, and limited fertilizing effect whether used fresh or stored [37]. At present, the only feasible valorization pathway is biomaterial production.

A review of the literature indicates that most studies on DF and AD have focused on food waste and lignocellulosic biomass. In contrast, the limited studies on macroalgae have generally relied on cost-prohibitive and environmentally unsustainable pretreatment methods, particularly problematic in Caribbean regions where reducing chemical inputs is essential. Moreover, investigations of *Sargassum* have been restricted to batch mode experiments; to the best of our knowledge, no studies have evaluated semi-continuous systems, likely due to their technical complexity.

This study addresses this gap by introducing an integrative framework that transitions from batch-mode BHP and BMP assays to semi-continuous coupled experiments with *Sargassum*. The approach provides practical insights for AD systems design and lays the groundwork for the development of marine-based biogas plants. Ultimately, this work advances bioenergy valorization strategies in coastal areas, thereby supporting informed decision-making for seaweed waste management. Within this context, the present study investigates the

hydrogen and methane potential of *Sargassum* species in batch mode, as well as the stability and performance of semi-continuous systems, with particular emphasis on conditions that promote hydrogen production.

## 2. Materials and methods

### 2.1. Algal samples

The brown alga *S. muticum* (Sm) was collected (June 2022) from the coast of Kercambre (47°29'22.5"N, 2°49'15.4"W) (Saint-Gildas-de-Rhuys, France). The harvested seaweed was rinsed with seawater to remove epiphytes, then transported to the laboratory in seawater to slow down degradation and minimize losses of mannitol and polyphenol. All Sm samples were dried at 40 °C for 16 h to achieve a dry matter above 85 %.

In December 2022, a collection campaign for Caribbean *Sargassum* (a mixture of *S. fluitans* and *S. natans*) was conducted at Anse Salabouelle in Guadeloupe (N 16° 19' 45.88", W 61° 18' 33.66"). Three types of biomass were selected to represent different stages of seaweed decomposition: coastal, beached, and composed *Sargassum*.

Caribbean coastal *Sargassum* (Cc): Due to limited submerged biomass, collection took place within 3 m of the shore. Macro-debris (e. g., shoes, plastic) were removed, and the seaweed was drained and lightly air-dried in sunlight for approximately 30 min (Fig. S1a).

Caribbean beached *Sargassum* (Cb): This site contained large amounts of *Sargassum* at various drying and decomposition stages. Sampling focused on recently arrived, partially dried biomass. Despite sorting, the collected material contained sand and was infested with sea fleas (Fig. S1b).

Caribbean composted *Sargassum* (Cp): Collected from backshore stockpiles where beach-cast algae had been deposited without treatment. The composted biomass, mixed with soil, estimated to be < 5 months old (Fig. S1c).

The three batches were packed in insulated transport crates and delivered to the laboratory one week after collection. Upon arrival, part of each sample was stored at -20 °C, while the remainder was dried under the same conditions as Sm (40 °C, 16 h), achieving a dry matter content greater than 85 %. After drying, the seaweed was ground using a kitchen blender (Magimix Power Blender 4). The powdered *S. muticum* (PSm) and powdered Caribbean costal seaweed (PCC) (<5 mm) were sealed in zip-lock plastic bags and stored at 4 °C until further use.

### 2.2. Inoculum samples

The inoculum was sourced from a mesophilic methanization unit (Liger, Locminé, France) processing manure, municipal and food industry residues. After retrieval, the inoculum was sieved at 400 µm to ensure homogeneity. For the BHP and coupling tests, the inoculum was thermally pre-treated at 90 ± 2 °C for 15 min to select spore-forming hydrogen producing bacteria [38]. Further details of inoculum preparation are provided in the Supplementary Data.

### 2.3. Analytical methods

Total solids (TS) were determined by oven drying at 105 °C, and Volatile Solids (VS) by calcination at 550 °C for 2 h.

SCOD was measured according to DIN ISO 15705 (analogous to US EPA 410.04). 25 g of the sample was centrifuged at 8000 g for 10 min 1 mL of supernatant was analyzed using Spectroquant® tubes (500–10000 mg/L; Merck KGaA, Darmstadt, Allemagne). The sealed tubes were heated to 148 °C for 2 h in a Spectroquant TR 320 (Merck, Darmstadt, Allemagne). and SCOD was subsequently quantified using a NOVA 60 spectrophotometer (Merck, Germany).

For biochemical composition, dried and ground samples were hydrolyzed using a three-step sequential protocol with distilled water, HCl, and NaOH [39]. Neutral sugars were quantified by the Dubois method
[40], uronic acids by the Blumenkrantz and Asboe-Hansen method [41], proteins by the Smith method [42], total phenolics by the Folin-Ciocalteu method [43], and sulfated groups by the Jaques method [44]. All methods are described in detailed by Hardouin et al. [45].

Elemental composition (C, H, N, S, O) of the dried seaweed biomass was measured using a FLASH 2000 CHNS/O analyzer (Thermo Fisher Scientific). Based on substrate composition, biogas production was estimated using the modified stoichiometric Buswell's Eq. (1) [46]. Theoretical methane yields (TBMP) can then be determined using the Eq. (2) [47]. Additionally, the oxygen demand required to oxidize 1 mol of the substrate was calculated using Eq. (3) for COD determination:

$$C_n H_a O_b N_c S_d + \left(n - \frac{a}{4} - \frac{b}{2} + \frac{3c}{4} + \frac{d}{2}\right) H_2 O \rightarrow \left(n - \frac{a}{8} + \frac{b}{4} + \frac{3c}{8} + \frac{d}{4}\right) CO_2 + \left(n + \frac{a}{8} - \frac{b}{4} - \frac{3c}{8} - \frac{d}{4}\right) CH_4 + c NH_3 + d H_2 S \quad (1)$$

$$TBMP \left( \frac{NL}{gVS} \right) = \frac{22.415 \left( \frac{n}{2} + \frac{a}{8} - \frac{b}{4} - \frac{3c}{8} - \frac{d}{4} \right)}{12n + a + 16b + 14c + 32d} \quad (2)$$

$$C_n H_a O_b N_c S_d + \left(n + \frac{a}{4} - \frac{b}{2} - \frac{3c}{4} + d\right) O_2 \rightarrow n CO_2 + \left(\frac{a}{2} - \frac{3c}{2}\right) H_2 O + c NH_3 + d SO_2 \quad (3)$$

## 2.4. Experiment set-up

A schematic overview of the experimental workflow, from feedstock preparation to biohydrogen and biomethane production, is presented in Fig. 1. BMP tests were performed on all five samples (PSm, PCc, Cc, Cb and Cp), whereas BHP tests were carried out specifically on Cc, Cb, Cp, and PSm. The semi-continuous DF-AD process was performed using two

powdered samples, PSm and PCc.

## 2.5. BMP and BHP evaluation

The AMPST II® system (Bioprocess Control, Lund, Sweden) was used to perform BMP and BHP evaluation. All hydrogen and methane production experiments were conducted in triplicate under strictly controlled conditions, with gas production (methane or hydrogen) monitored continuously throughout the process. The BMP assay lasted 60 days, whereas the BHP assay was carried out over 7 days. The bio-reactors had a working volume of 0.45 L and a headspace of 0.05 L, and were maintained at 37 ± 1 °C with a continuous stirring. The inoculum to substrate ratio (ISR), calculated on a volatile solids basis, was set at 2 for AD and 1 for DF. During the process, the biogas produced was passed through bottles containing 3M NaOH solution to remove acid gases (CO₂ and H₂S). Net gas production was determined by subtracting the average endogenous gas production from inoculum-only controls. Detailed experimental conditions are provided in the Supplementary data.

## 2.6. Coupling DF-AD experiment

The coupling experiment was conducted in duplicate using 2 L reactors under mesophilic conditions (37 ± 1 °C) in an anaerobic sequencing batch reactor (ASBR) system. The first reactor was designated for hydrogen production, and its effluent was subsequently transferred to the second reactor for methane production. Both reactors operated independently and were connected to NaOH traps within the automated AMPTS II system for continuous gas production monitoring. The operational conditions are summarized in Table 1.

For the first phase of the coupling process (DF), previous batch experiments demonstrated optimal H₂ production conditions at an initial pH of 6.2 ± 0.1. The pH was set at this level and allowed to decrease to 5 during the experiment [48]. After each feeding, pH was corrected as necessary with 5M NaOH solution to ensure it remained above the target

<figure><img src="image_2.png" /><figcaption>Fig. 1. Schematic of experiments.</figcaption></figure>
<table><caption>**Table 1**
Operational conditions of semi-continuous experiment.</caption><thead><tr><th rowspan="2">Substrate</th><th colspan="2">Brittany</th><th colspan="2">Caribbean</th></tr><tr><th colspan="2">Sargassum</th><th colspan="2">Sargassum</th></tr><tr><th>Phase</th><th>1</th><th>2</th><th>1</th><th>2</th></tr></thead><tbody><tr><td>Working volume (L)</td><td>1.8</td><td>1.8</td><td>1.8</td><td>1.8</td></tr><tr><td>Initial substrate concentration (kg VS·m<sup>-3</sup>)</td><td>10.4</td><td>5.2</td><td>9.6</td><td>4.8</td></tr><tr><td>Organic Loading Rate (kg VS·m<sup>-3</sup>·d<sup>-1</sup>)</td><td>3.6</td><td>-</td><td>3.3 (5.9)</td><td>-</td></tr><tr><td>Hydraulic Retention Time (d)</td><td>8</td><td>18</td><td>9</td><td>31</td></tr></tbody></table>

value (5.5 or 6). The feed concentration for both experiments was set at 30 g VS·L<sup>-1</sup> H<sub>2</sub>O. Given the historically low BMP values reported for Caribbean-origin *Sargassum*, a low hydrogen yield was anticipated. Therefore, a two-step feed strategy was applied, increasing the organic loading rate (OLR) from 3.3 kg VS·m<sup>-3</sup>·d<sup>-1</sup> for the first four injections to 5.9 kg VS·m<sup>-3</sup>·d<sup>-1</sup> for subsequent injections. In the second phase (AD), a two-week acclimation period was required to deplete residual organic matter in the inoculum and substrate. Thereafter, digested sludge from phase 1 was added according to the set hydraulic retention time (HRT). Manual mixing was performed daily to ensure uniformity throughout the process.

Volatile fatty acid (FOS in German), and total inorganic carbon (TAC) were measured using a HACH AT1000 potentiometric titrator following protocol DOC316.52.93087, based on Nordmann's method [49]. Results are expressed as mg·L<sup>-1</sup> of acetic acid equivalents.

Measurements of pH, FOS, TAC, and SCOD were taken during each batch cycle to assess reactor performance throughout the process. At the end of the experiment, biogas composition was measured using a Geotech BIOGAS 5000 gas analyzer (Garches, France), which quantified the exact proportions (in %) of CH<sub>4</sub>, CO<sub>2</sub>, and O<sub>2</sub>. H<sub>2</sub> and H<sub>2</sub>S contents were indicative only and could be calculated further.

## 2.7. Kinetic analysis

Hydrogen production curves were fitted using Eq. (4) [50], with key parameters estimated using the Solver function in Microsoft® Excel.

$$BHP_t = BHP_0 \cdot \exp \left\{ - \exp \left[ \frac{R_m \cdot e}{BHP_0} (\lambda - t) + 1 \right] \right\} \quad (4)$$

Where: $BHP_0$ (NL H<sub>2</sub>·kg VS<sup>-1</sup>) is the biochemical hydrogen potential of the substrate, $R_m$ (NL H<sub>2</sub>·kg VS<sup>-1</sup>·h<sup>-1</sup>) is the maximum hydrogen production rate, $\lambda$ (hours) is the time of the lag phase, and $e = \exp(1) = 2.71828$.

## 2.8. Data analysis methods

Analyses of variance (ANOVA) was conducted to assess differences in compositional content, as well as the BMP and BHP values obtained. Tukey's test was used to determine the significance of differences in BMP across conditions. Results were considered statistically significant when the *p*-value was less than 0.05. The adjusted coefficient of determination $R^2_{adjusted}$, Eq. (5), was calculated to evaluate the discrepancy between observed and predicted values and to assess model quality. To examine linear relationships between continuous, normally distributed variables, the Pearson correlation coefficient was calculated using the Eq. (6):

$$R^2_{adjusted} = 1 - \frac{(n-1) \cdot \sum_{i=1}^{n} (y_i - \hat{y}_i)^2}{(n-k-1) \cdot \sum_{i=1}^{n} (y_i - \bar{y})^2} \quad (5)$$

Where: n is the number of measurements, k is the number of factors, $y_i$ is the value of the ith measurement, $\hat{y}_i$ is the corresponding predicted value, $\bar{y}_i$ is the average of the measurements.

$$r = \frac{\text{Cov}(X, Y)}{\sigma_X \cdot \sigma_Y} = \frac{\sum_{i=1}^{n} (X_i - \bar{X})(Y_i - \bar{Y})}{\sqrt{\sum_{i=1}^{n} (X_i - \bar{X})^2 \sum_{i=1}^{n} (Y_i - \bar{Y})^2}} \quad (6)$$

Where: n is the number of paired values, $X_i$ and $Y_i$ are the values of the two variables, $\bar{X}$ and $\bar{Y}$ are the mean values.

Principal Component Analysis (PCA) is widely employed to explore and synthesize multidimensional datasets [51]. In this study, this method was applied to investigate the relationships between kinetic parameters ($R_m$, $\lambda$, BHP<sub>0</sub>) and pH values at the beginning and end of each batch cycle. The goal was to identify potential correlations among these parameters and to determine favorable conditions for hydrogen production.

All statistical analyses were performed using RStudio® software.

# 3. Results and discussion

## 3.1. Characterization of Brittany *S. muticum* and Caribbean *Sargassum* spp

Table 2 presents the averages and standard deviations (triplicates) of TS, VS, VS/TS ratio, and ash content for the five algae samples (Cc, Cb, Cp, PSm, PCc) used in this study.

Comparative results for Cc were reported by Milledge et al. [20], who found a TS content of 18 % and a VS content of 9.56 % from a 'Mixed *Sargassum*' sample collected in Shark Bay. In the present study, the VS content of Caribbean seaweed increased following beach exposure (108 g VS·kg<sup>-1</sup> for Cc versus 153 g VS·kg<sup>-1</sup> for Cb). Such exposure typically promotes decomposition and evaporation of organic material. The observed difference should be explained by moisture loss due to sunlight exposure, as reflected in the VS/TS ratio and ash content during this process (Table 2). For the Brittany sample (PSm), a TS content of 914 g kg<sup>-1</sup> was consistent with the value reported by Jard et al. [32] (908 g kg<sup>-1</sup> under identical drying conditions at 40 °C). Additionally, the VS content was slightly higher in the present study (683 g kg<sup>-1</sup> vs. 634 g kg<sup>-1</sup>), while the ash content was notably lower (232 g kg<sup>-1</sup> vs. 366 g kg<sup>-1</sup>). Significantly higher levels of TS and VS were observed in the Brittany seaweed (PSm) compared with Caribbean seaweed (PCc), despite being dried under the same mild conditions. These differences are attributable to the inherent biochemical composition of the algal species.

Two powder samples, PSm and PCc, were used for the semi-continuous coupling DF-AD experiment, their chemical compositions are presented in Table 3. All values are expressed as the mean and standard deviation (SD) of triplicate measurements.

Compared to the Caribbean sample, the Brittany-originated PSm contained significantly higher levels of sugar, polyphenols, and sulfate content, while displaying lower protein and ash contents. It should be noted that carbohydrate content is generally correlated with hydrogen production [52,53]. Attention should be given to the low sugar content (approximately 10 % dw) observed in both samples, along with the notably high uronic acid content in PSm (5.2 % dw) and PCc (7.3 % dw).

<table><caption>**Table 2**
Characterization of the substrates.</caption><thead><tr><th rowspan="2">Samples</th><th>Total Solids</th><th>Volatile Solids</th><th>VS/TS</th><th>Ash</th></tr><tr><th>g·kg<sup>-1</sup></th><th>g·kg<sup>-1</sup></th><th>%</th><th>g·kg<sup>-1</sup></th></tr></thead><tbody><tr><td>Cc</td><td>161 ± 9</td><td>108 ± 7</td><td>67.1 ± 1.5</td><td>53 ± 3</td></tr><tr><td>Cb</td><td>262 ± 10</td><td>153 ± 7</td><td>58.3 ± 0.4</td><td>109 ± 3</td></tr><tr><td>Cp</td><td>190 ± 2</td><td>86 ± 1</td><td>45.3 ± 0.2</td><td>104 ± 1</td></tr><tr><td>PSm</td><td>914 ± 1</td><td>683 ± 1</td><td>74.7 ± 0.1</td><td>232 ± 1</td></tr><tr><td>PCc</td><td>871 ± 1</td><td>578 ± 2</td><td>66.4 ± 0.2</td><td>293 ± 2</td></tr></tbody></table>

Cc: Caribbean costal seaweed, Cb: Caribbean beached seaweed, Cp: Caribbean composted seaweed, PSm: Powdered *Sargassum muticum*, PCc: Powdered Caribbean costal seaweed.
<table><caption>**Table 3**

Characteristics of two powder samples.</caption><thead><tr><th>Parameters</th><th>Units</th><th>PCc</th><th>PSm</th></tr></thead><tbody><tr><td>Protein content</td><td>g/100 g dry weight</td><td>36.6 ± 2.8</td><td>27.2 ± 4.3</td></tr><tr><td>Uronic acid content</td><td></td><td>7.3 ± 2.7</td><td>5.2 ± 1.5</td></tr><tr><td>Total sugar content</td><td></td><td>9.8 ± 2.9</td><td>12.9 ± 1.6</td></tr><tr><td>Polyphenols content</td><td></td><td>5.4 ± 0.3</td><td>6.6 ± 1.5</td></tr><tr><td>Ash</td><td></td><td>29.7 ± 0.6</td><td>22.7 ± 1.6</td></tr><tr><td>Sulfate content</td><td></td><td>4.0 ± 0.2</td><td>6.7 ± 0.1</td></tr><tr><td>Others</td><td></td><td>14.4 ± 6.8</td><td>23.8 ± 9.1</td></tr><tr><td>C</td><td></td><td>32.4 ± 0.2</td><td>34.7 ± 0.4</td></tr><tr><td>H</td><td></td><td>3.8 ± 0.1</td><td>4.3 ± 0.1</td></tr><tr><td>O</td><td></td><td>34.3 ± 0.6</td><td>37.5 ± 0.7</td></tr><tr><td>N</td><td></td><td>1.6 ± 0.1</td><td>1.6 ± 0.1</td></tr><tr><td>S</td><td></td><td>2.0 ± 0.1</td><td>1.3 ± 0.1</td></tr><tr><td>C:N</td><td>—</td><td>19.9</td><td>21.2</td></tr><tr><td>C:S</td><td>—</td><td>16.2</td><td>26.7</td></tr><tr><td>TBMP</td><td>NL CH<sub>4</sub>-kg VS<sup>-1</sup></td><td>371</td><td>381</td></tr><tr><td>COD</td><td>g COD·g VS<sup>-1</sup></td><td>1.10</td><td>1.11</td></tr></tbody></table>

Uronic acids, as key structural components of alginates, are difficult to break down without pretreatment, even in AD processes [20], and their conversion through DF may be highly restricted, as DF is considered a partial degradation stage of AD. Stiger-Pouvreau et al. [54] reviewed the composition of the *Sargassum* genus, emphasizing that composition can be influenced by abiotic factors such as geographical location, season, water temperature, and salinity, as well as by the growth and maturity of the algal thallus. Overall, the total cell wall polysaccharide content (neutral sugars + uronic acids + sulfate groups) observed here aligns with the literature. Protein levels, typically ranging from 6 to 20 % in brown seaweed [54], were higher in our samples, while ash content were within reported values. The C:N ratios of 19.9 and 21.2 are consistent with the findings of Thompson et al. [55] (21.7 ± 0.2 for pelagic *Sargassum*) and Milledge et al. [20] (16.1–22.1 for Caribbean *Sargassum*). Generally, the AD process requires a C:N ratio between 20 and 30 [56]. Regarding TBMP, a range of 119–380 L CH₄·kg VS⁻¹ has been reported by Thompson et al. [21], and our values remain high in comparison. The sulfur content aligns with the study of Jard et al. [32] (1.3 % for *S. muticum*). Based on the predictive model of Peu et al. [57] for H₂S levels estimation in biogas, a high H₂S content is anticipated in our case. The model indicates that a minimum C:S ratio of 40 is required to maintain H₂S concentrations below 2 % in the biogas. Finally, regarding COD content, no significant differences were observed

between the two samples.

## 3.2. BMP and BHP evaluation

The BMP and BHP of five algae samples are shown in Fig. 2.

The highest BMP value of 140 ± 7 NL CH₄·kg VS⁻¹ was obtained for PSm, consistent with previously reported values of 130–208 L CH₄·kg VS⁻¹ for *S. muticum* [32,33]. Tukey's test revealed that the BMP of coastal Caribbean seaweed Cc (138 ± 25 NL CH₄·kg VS⁻¹) was significantly higher than that of stranded (75 ± 9 NL CH₄·kg VS⁻¹) and composted seaweed (57 ± 11 NL CH₄·kg VS⁻¹). Notably, The BMP of Cc at 138 ± 25 NL CH₄·kg VS⁻¹ remained relatively high compared to literature values. Milledge et al. [20] reported a range of 66–145 L CH₄·kg VS⁻¹ for freeze-dried *Sargassum*. Thompson et al. [55] recorded the highest BMP value of 117 ± 2 L CH₄·kg VS⁻¹ after hydrothermal treatment. In contrast, their untreated *Sargassum* (sun-dried for two weeks and heat-treated at 80 °C for 15 h) exhibited a much lower BMP of 42 ± 3 L CH₄·kg VS⁻¹. In our study, following a drying process at 40 °C for 16 h, powdered Caribbean coastal seaweed (PCc) retained only 42 % of the BMP of the thawed Cc sample (58 ± 12 NL CH₄·kg VS⁻¹ vs. 138 ± 25 NL CH₄·kg VS⁻¹). A similar reduction was observed in *S. muticum*, where BMP values dropped from 172 to 219 NL CH₄·kg VS⁻¹ (thawed, unpublished data) to 140 ± 7 NL CH₄·kg VS⁻¹. Since no notable differences in VS/TS ratio were detected between Cc and PCc (67.1 % vs. 66.4 %, Table 2), the reduction in BMP is likely attributable to alterations in the organic matrix. This may result from the loss of easily fermentable components, particularly those contained in the algal juice, during the drying process. Another possible explanation is the loss of hydrolytic bacteria naturally present on the surface of the algae [58]. These results emphasize the importance of selecting appropriate preparation methods prior to AD. For an accurate assessment of the substrate's energy potential, frozen samples may be preferred to minimize BMP loss.

The BMP of PCc (58 NL CH₄·kg VS⁻¹) from this study is relatively low when compared to terrestrial crops like sorghum (260–390 NL CH₄·kg VS⁻¹) and sugarcane (230–300 NL CH₄·kg VS⁻¹) [59]. It is also significantly lower than that of common substrates such as food waste, which typically range from 200 to 670 NL CH₄·kg VS⁻¹ [60]. Nevertheless, PCc may represent a valuable co-substrate in co-digestion processes (e.g., with pig slurry), as it demonstrates a higher methane potential on a fresh-weight basis (33.5 NL CH₄·kg FW⁻¹ versus 10 NL CH₄·kg FW⁻¹) [61]. Such a strategy can help mitigate the high salt content of *Sargassum*, while simultaneously balancing the C:S and C:N ratios,

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** BMP and BHP of all algae samples (in triplicate). No BHP test was performed on PCc due to the low BHP values obtained from Cc.</figcaption></figure>
thereby potentially improving the overall efficiency of digestion system [20,62].

Regarding the BHP test, the highest value of 32 ± 5 NL H₂·kg VS⁻¹ was obtained from PSm. Although published data on BHP values for *Sargassum* are scarce, our results align with those reported for another brown macroalgae species, *S. japonica*, which contains a higher carbohydrate content (55.6 % dw; [27]). BHP values of 10 ± 1.21 NL H₂·kg TS⁻¹ (approximately 18 NL H₂·kg VS⁻¹) [27] and 35.7 ± 0.3 NL H₂·kg VS⁻¹ [63] have been reported for dried *S. japonica* samples (105 °C) processed without additional pretreatment. In contrast, the three Caribbean-originated *Sargassum* samples yielded extremely low BHP values, with a significant portion attributed to endogenous hydrogen production of inoculum (29 NmL out of 49 NmL for Cc). These results are far below the exceptionally high BHP 91.3 ± 3.3 NL H₂·kg VS⁻¹ reported by Costa et al. [22], where *Sargassum* was autoclaved and fermented with *Caldicellulosiruptor saccharolyticus*. More efficient pretreatment methods seem necessary for enhanced COD solubilization and improve hydrogen recovery.

### 3.3. Two-stage AD in single bioreactor

This section focuses on the three Caribbean-originated *Sargassum* samples (Cc, Cb, Cp), representing three stages of decomposition, each with potential industrial relevance. The duration of the BHP test (7 days) was extended to over four months due to the occurrence of a two-stage AD process: an initial phase of DF for hydrogen production, followed by a secondary methanogenesis phase characterized by several degradation stages over time (Fig. S2).

In our study, pH measurements taken before and after the batch experiments showed an increase for all three samples (e.g., from 6.48 to 7.32 for Cp), suggesting that the initial thermal pre-treatment was insufficient to completely eliminate methanogenic bacteria [64]. Besides, the lack of medium renewal favored methanogen growth, ultimately leading to methane production. Pardilho et al. [65] associated the emergence of multiple lag phases with the substrate's characteristics (e.g., organic matter availability, chemical composition) and the accumulation of VFAs that are not readily converted. Panigrahi et al. [66] observed multi-stage behavior during the co-digestion of food and yard waste when the feedstock-to-inoculum ratio (F/M) was high (2.0 and 2.5). In their study, the first inflection point corresponded to a VFAs concentration of approximately 4500 mg·L⁻¹, which further decreased with varying slopes. This could provide a plausible explanation for the inflection points observed in the present study, which may indicate the

<table><caption>**Table 4**
Assessment of the energy gain of three varieties of Caribbean seaweed.</caption><thead><tr><th></th><th>Classical AD</th><th>Two-stage AD</th></tr></thead><tbody><tr><td></td><td colspan="2">Coastal seaweed (Cc)</td></tr><tr><td>BMP (NL CH<sub>4</sub>·kg VS<sup>-1</sup>)</td><td>138 ± 25<sup>a</sup></td><td>126 ± 8<sup>a</sup></td></tr><tr><td>BHP (NL H<sub>2</sub>·kg VS<sup>-1</sup>)</td><td>-</td><td>8 ± 0.8</td></tr><tr><td>Energy potential (kJ·kg VS<sup>-1</sup>)</td><td>5492</td><td>5116</td></tr><tr><td>Energy gain</td><td>✓ -6.8 %</td><td></td></tr><tr><td></td><td colspan="2">Stranded seaweed (Cb)</td></tr><tr><td>BMP (NL CH<sub>4</sub>·kg VS<sup>-1</sup>)</td><td>75 ± 9<sup>b</sup></td><td>123 ± 11<sup>a</sup></td></tr><tr><td>BHP (NL H<sub>2</sub>·kg VS<sup>-1</sup>)</td><td>-</td><td>6 ± 0.7</td></tr><tr><td>Energy potential (kJ·kg VS<sup>-1</sup>)</td><td>2985</td><td>4972</td></tr><tr><td>Energy gain</td><td>✓ 66.6 %</td><td></td></tr><tr><td></td><td colspan="2">Composted seaweed (Cp)</td></tr><tr><td>BMP (NL CH<sub>4</sub>·kg VS<sup>-1</sup>)</td><td>57 ± 11<sup>b</sup></td><td>77 ± 1<sup>b</sup></td></tr><tr><td>BHP (NL H<sub>2</sub>·kg VS<sup>-1</sup>)</td><td>-</td><td>4.9 ± 0.1</td></tr><tr><td>Energy potential (kJ·kg VS<sup>-1</sup>)</td><td>2269</td><td>3127</td></tr><tr><td>Energy gain</td><td>✓ 37.8 %</td><td></td></tr></tbody></table>

Letters indicate Tukey's test results performed on BHP test. A common letter between samples means the BMP is not significantly different (p-value >0.05). A Higher Heating Value of 39.8 kJ·NL⁻¹ for CH₄ and 12.7 kJ·NL⁻¹ for H₂ is considered here (0 °C, 1 bar).

presence of VFAs that degrade at different rates.

A two-way ANOVA was conducted to analyze the BMP and energy gains achieved through the classical AD process and two-stage AD configuration. The results are summarized in Table 4.

Notably, for methane production alone, the two-stage AD enabled Cb to increase BMP by 64 % (123 ± 11 NL CH₄·kg VS⁻¹ versus 75 ± 9 NL CH₄·kg VS⁻¹). When including the contribution of hydrogen, an overall energy gain of 66.6 % was achieved. Composted algae also showed an energy gain of 37.8 %, although this value should be interpreted cautiously due to the significant influence of the standard deviations associated with the BMP of Cp. A lower initial pH, potentially acting as an acid treatment and thereby enhancing enzymatic hydrolysis of the algae, appeared to promote the release of organic material from the matrix [27]. However, no significant improvement in BMP was observed for coastal seaweed (126 ± 8 NL CH₄·kg VS⁻¹ versus 138 ± 25 NL CH₄·kg VS⁻¹). These values suggests that the loss of easily fermentable compounds during sun exposure likely contributed to the reduced BMP (138 ± 25 NL CH₄·kg VS⁻¹ versus 75 ± 9 NL CH₄·kg VS⁻¹). Additionally, transformations in the recalcitrant organic compounds may have occurred, whereby components initially resistant to degradation became fermentable under acid pretreatment. This shift could partly explain the higher BMP values observed (123 ± 11 NL CH₄·kg VS⁻¹) for the stranded seaweed (Cb) in the two-stage AD process. For further optimization of pretreatment of recalcitrant biomass such as Caribbean species, harsher conditions such as acid combined with heat or additional physical pretreatments may be required.

### 3.4. Coupling DF-AD experiment

#### 3.4.1. Coupling experiment with *S. muticum*

The cumulative hydrogen production curves are illustrated in Fig. S3a. During the first three injections, hydrogen yields comparable to those observed in the BHP test (32 ± 5 NL H₂·kg VS⁻¹) were obtained, although they exhibited a declining trend. This decline was accompanied by a drop in pH from 6.18 to 5.1 by day 5, and a decrease in TAC from 1550 mg·L⁻¹ to undetectable levels. pH adjustments were subsequently performed after each batch cycle to maintain values above 5.5. Despite these corrections, hydrogen yield continued to decline, with an extended lag phase until production eventually ceased in one of the two reactors (Fig. S3a). This temporary failure in hydrogen production could be attributed to insufficient alkalinity in the medium. In AD systems, HCO₃⁻/CO₂⁻³ species play a crucial role in providing buffering capacity, stabilizing the pH, and maintaining volatile fatty acids (e.g., acetate, propionate, and butyrate) in their ionic forms. The distribution of HCO₃⁻/CO₂⁻³ depends strongly on pH; at pH 5.5, only about 10 % of HCO₃⁻ remains in solution, with approximately 90 % converted to CO₂ [67]. Wan et al. [68] reported that buffering capacity is disrupted when TAC levels fall below 1000 mg L⁻¹, as was observed in our case. To address this, the reference pH was raised to 6, ensuring that approximately 30 % of HCO₃⁻ was retained in the medium. Following this adjustment, hydrogen yields in both reactors were partially recovered, stabilizing at 25–40 % of the BHP level by the end of the experiment. Throughout the entire experiment, FOS concentrations fluctuated between 200 and 2300 mg L⁻¹ (Fig. S3c).

Regarding the second phase of the coupling experiment, six injections were performed using the effluent from the first phase to further determine methane production. As shown in Fig. S3d and Fig. S3e, a slight pH decrease was noted from 7.5 to 7.3, while TAC dropped from 7500 to 4000 mg L⁻¹, and FOS remained around 2000 mg L⁻¹. Consequently, the FOS/TAC ratio fluctuated between 0.3 and 0.4 during the first five injections, before rising to 0.5 at the end. The FOS/TAC ratio is a widely used diagnostic tool in industrial-scale AD processes [69], with typical values ranging from 0.2 to 0.6 [68] and an optimum range between 0.3 and 0.4 [70]. According to Riau et al. [71], a FOS/TAC value exceeding 0.5 may indicate an imbalance between acidogenic and methanogenic microbiota. In this experiment, no extreme deviations
were observed. With respect to alkalinity, Khanal et al. [72] emphasized the importance of maintaining an adequate buffering capacity (1000–5000 mg L⁻¹ as CaCO₃) to meet the microbial population's requirements. Although TAC values were initially above this range, they progressively converged toward the recommended levels as the experiment advanced.

### 3.4.2. Coupling DF-AD experiment with coastal Caribbean Sargassum spp

The cumulative hydrogen production curves are illustrated in Fig. S4a. Less than 20 % of the hydrogen potential was achieved compared with assay using *S. muticum* during the first three injections, with low hydrogen volumes observed (around 75 mL for the second and third batch cycle). To improve representativeness, the OLR was increased from 3.3 to 5.9 kg VS·m⁻³·d⁻¹, resulting in a higher potential of 4.2 ± 1.3 NL H₂·kg VS⁻¹ during the fifth injection. Thereafter, production stabilized at approximately 2 NL H₂·kg VS⁻¹ (Fig. S4a). In this experiment, the reference pH was set at 6 to ensure sufficient buffering capacity, and no NaOH adjustments were required. The feed mixture of powdered algae and water allowed the pH to rise after each batch cycle. During the first half of the experiment, the FOS concentration remained around 2000 mg L⁻¹, similar to the assay with *S. muticum*. The lower hydrogen production may have resulted from diversion into competitive and inhibitory pathways, such as the accumulation of lactate and pro-pionate [73]. A notable increase in FOS concentration, from 2000 mg L⁻¹ to 5000 mg L⁻¹, was observed following the higher OLR application. Alkalinity fluctuated from 600 to 1200 mg L⁻¹ throughout the experiment (Fig. S4b and S4c).

For methane production in the second phase, four injections were performed. Parameter trends were comparable to those observed in the PSm assay, including a pH decrease from 7.44 to 7.27 and TAC reduction from 6800 to 4800 mg L⁻¹. This led to an increase in the FOS/TAC ratio from 0.3 to 0.6 (Fig. S4d and S4e).

### 3.5. Parameters of production and biogas composition

The hydrogen and methane production parameters are presented in Table 5.

The average hydrogen production rate and yield of the PSm sample (45.6 10⁻³ NL H₂·L⁻¹·Reactor·d⁻¹, 12.7 NL H₂·kg VS⁻¹·Added) were 3.6 and 4.3 times higher, respectively, than those of the PCc sample (13.8 NL H₂·L⁻¹·Reactor·d⁻¹, 3.0 NL H₂·kg VS⁻¹·Added). Compared with the batch test, PSm sample achieved 40 % efficiency in hydrogen production and 42.8 % in methane production, whereas Caribbean-origin biomass reached 37.5 % efficiency for hydrogen and 50.9 % for methane. Notably, PSm sample showed a significantly higher methane production rate, with a 3.5-fold increase in rate (100.4 10⁻³ NL H₄·L⁻¹·Reactor·d⁻¹ versus 28.5

<table><caption>**Table 5**

Hydrogen and methane production parameters.</caption><thead><tr><th rowspan="2">Parameter</th><th colspan="2">PSm</th><th colspan="2">PCc</th></tr><tr><th>Phase - 1</th><th>Phase - 2</th><th>Phase - 1</th><th>Phase - 2</th></tr></thead><tbody><tr><td>H₂ production rate (10<sup>-3</sup> NL H₂·L<sup>-1</sup>·Reactor·d<sup>-1</sup>)</td><td>45.6 ± 3.7</td><td>-</td><td>13.8 ± 0.9</td><td>-</td></tr><tr><td>H₂ yield (NL H₂·kg VS<sup>-1</sup>·Added)</td><td>12.7 ± 1.0</td><td>-</td><td>3.0 ± 0.2</td><td>-</td></tr><tr><td>CH₄ production rate (10<sup>-3</sup> NL CH₄·L<sup>-1</sup>·Reactor·d<sup>-1</sup>)</td><td>-</td><td>100.4 ± 7.9</td><td>-</td><td>28.5</td></tr><tr><td>CH₄ yield (NL H₄·kg VS<sup>-1</sup>·Added)</td><td>-</td><td>59.9 ± 4.7</td><td>-</td><td>29.5</td></tr><tr><td>Efficiency compared to batch test (%)</td><td>40</td><td>42.8</td><td>37.5</td><td>50.9</td></tr><tr><td>CH₄ (%)</td><td>0.5</td><td>56.8</td><td>0</td><td>-</td></tr><tr><td>CO₂ (%)</td><td>49.5</td><td>38.3</td><td>84.9</td><td>-</td></tr><tr><td>O₂ (%)</td><td>0.1</td><td>0.2</td><td>2</td><td>-</td></tr><tr><td>H₂ (%)</td><td>&lt;48.9</td><td>0</td><td>&lt;12</td><td>-</td></tr><tr><td>H₂S (%)</td><td>&gt;1</td><td>0.2</td><td>&gt;1</td><td>-</td></tr></tbody></table>

10⁻³ NL H₄·L⁻¹·Reactor·d⁻¹) and a twofold increase in methane yield (59.9 NL H₄·kg VS⁻¹·Added versus 29.5 NL H₄·kg VS⁻¹·Added) compared with PCc sample (Table 5).

Concerning the composition of the produced biogas, the methane content in the second phase of the PSm assay (56.8 %) was comparable to that reported in previous studies with food waste (56–61 %), although higher methane yields were achieved in those studies [74–76]. It should be noted that the H₂/CO₂ ratio was close to 1 for the PSm sample but below 1/7 for the PCc sample in the DF phase, reflecting differences in the metabolic pathways commonly observed in *Clostridium* and Enterobacteriaceae species [77]. As shown in Eq. (7) and Eq. (8) (ΔG₀ values expressed at 25 °C and pH 7), the acetate pathway leads to an H₂/CO₂ ratio of 2, whereas the butyrate pathway results in a ratio of 1. However, the undesired production of secondary metabolites such as ethanol, succinate, or acetone can lead to significant CO₂ production [78], thereby reducing this ratio. High hydrogen productivity is generally associated with an H₂/CO₂ ratio close to 1, as observed in the PSm sample. By contrast, the metabolic activity in the PCc sample appears less favorable for hydrogen production, instead shifting toward greater by-product accumulation (e.g., ethanol, Eq. (9)) and hydrogen-consuming pathways such as propionate formation (Eq. (10)) [73].

$$C_6H_{12}O_6 + 2 H_2O \rightarrow 2 CH_3COOH + 2 CO_2 + 4 H_2 \quad \Delta G'_{0} = -215 \text{ kJ·mol}^{-1} \quad (7)$$

$$C_6H_{12}O_6 \rightarrow CH_3CH_2CH_2COOH + 2 CO_2 + 2 H_2 \quad \Delta G'_{0} = -264 \text{ kJ·mol}^{-1} \quad (8)$$

$$C_6H_{12}O_6 \rightarrow 2 CH_3CH_2OH + 2 CO_2 \quad \Delta G'_{0} = -235 \text{ kJ·mol}^{-1} \quad (9)$$

$$C_6H_{12}O_6 + 2 H_2 \rightarrow 2 CH_3CH_2COOH + 2 H_2O \quad \Delta G'_{0} = -359 \text{ kJ·mol}^{-1} \quad (10)$$

A high concentration of H₂S (>10,000 ppm) was recorded. This could originally be attributed to the natural presence of sulfated polysaccharides in seaweed, or the sulfate-rich environmental conditions in which it grows [79], underscoring the importance of rinsing the biomass prior to AD [58]. Two main metabolic pathways are responsible for H₂S production: (i) the reduction of sulfates by sulfate-reducing bacteria [80] and (ii) the reduction of sulfur-containing amino acids (notably cysteine and methionine) [81]. H₂S concentrations ranging from 10,000 ppm to 31,000 ppm have been reported during the co-digestion of pig slurry with *Ulva* sp., a sulfur-rich seaweed [82,83]. Peu et al. [83] indicated that the H₂S concentration threshold specified by co-generator manufacturers is approximately 150 mg/m³ (≈100 ppm). Therefore, monitoring H₂S formation is essential, and biogas post-treatment (e.g., activated carbon, chemical oxidation, biofiltration, etc.) appears indispensable for any potential application of the biogas produced in this study. Veluchamy et al. [69] indicated that the increase in H₂S could result from an increment in OLR, causing the hydrolysis and acidogenesis rates to exceed those of acetogenesis and methanogenesis. The elevated hydrogen availability drives an increase in H₂S production. This explanation is plausible in our case, as FOS values increased during the first stage of the semi-continuous process (Fig. S3c and Fig. S4c), which may simulate the impact of a higher loading rate.

### 3.6. Kinetic analysis

Gas production curves were generated using the modified Gompertz model for both samples and across all batch cycles, as illustrated in Table S1. The mean values and standard deviations of the kinetic parameters (R<sub>m</sub>, λ, BHP₀) are presented.

The R<sup>2</sup><sub>adjusted</sub> values ranged from 0.952 to 0.999, suggesting a good fit between the experimental points and the applied model. For the Brittany algae (PSm), λ varied consistently with the experimental conditions: it
<figure><img src="image_4.png" /><figcaption>Fig. 3. The correlation matrix with the Pearson coefficients.</figcaption></figure>

acquisition. **Jean-Louis Lanoisellé:** Validation, Supervision, Resources, Project administration, Investigation, Funding acquisition. **Thomas Lendormi:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## Disclosure statement

No potential conflict of interest was reported by the authors.

## Funding

This research was funded by Regional Council of Brittany, France (grant number ARED 2020-1858).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biombioe.2025.108579.

## Data availability

Data will be made available on request.

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