Journal of Environmental Management: Rugulopteryx Okamurae

Journal of Environmental Management 370 (2024) 122504
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Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman
Research article
Optimizing a biorefinery design for the valorization of Rugulopteryx

okamurae by extracting bioactive compounds and enhancing
methane production
David De la Lama-Calvente a,* , María José Fernández-Rodríguez b, Rafael Borja a
a
Spanish National Research Council (CSIC) - Instituto de La Grasa (IG), Department of Food Biotechnology, Campus Universidad Pablo de Olavide, Edificio 46. Ctra. de
Utrera, Km 1, 41013 Seville, Spain
b
Department of Vegetal Biology and Ecology, Faculty of Biology, University of Seville, 41080 Seville, Spain
A R T I C L E I N F O A B S T R A C T
Keywords: The Mediterranean Sea has suffered recently from the unprecedent invasion of the alien macroalga Rugulopteryx
Biorefinery okamurae due to global warming and climate change putting at risk the natural local ecosystem. Since 2015 this
Rugulopteryx okamurae alga has colonized a great area on the western coast of the Mediterranean Sea and it has been also spotted in
Anaerobic digestion
other areas such as the Azores Islands or the south of France. The arrival of alga tides into the coasts also
Bioactive compounds
Antioxidant
provokes collateral environmental problems that need to be addressed. Seaweed-based biorefineries are
considered a promising alternative within a circular economy model. This study aims to assess the potential of
R. okamurae as raw material for the extraction of reducing sugars (RS) and total polyphenolic compounds (TPC)
with antioxidant capacities, the subsequent production of methane from the extracted residue, and the final use
of the anaerobic digestate as fertilizer. However, the presence of bioactive compounds greatly varies due to
seasonality, location or even natural degradation. In order to provide some insights about these issues, two
different batches were assessed: i.e. natural and ashore R. okamurae. As brown algae are characterised by a cell
wall composed of crystalline cellulose and lignin, the biomasses studied were mechanically pretreated (dried at
100 ◦ C and milled during 60 s) before the single and sequential extraction processes. Results showed that the
extraction of the targeted compounds increased by 30–80 % when the biomasses were extracted sequentially.
Similarly, the biochemical methane potential of the extracted solid residues increased as the RS and TPC content
was reduced (120–150 NLCH4 kg−VS1), with no significant impact regarding the biomass origin (natural or ashore)
or the extraction process order. An increase in the kinetic constant k (first-order model) of 150% and 75% was
observed when the fresh biomass was extracted with water and ethanol, respectively, compared to the value
obtained for the unextracted biomass. Finally, the physicochemical characteristics of the different anaerobic
digestates generated were assessed for their potential use as biofertilizer. In this study, most of the digestate’
liquid fractions (7 out of 10) comply with European regulations for organic fertilizers and could be used directly.
1. Introduction global production surge to approximately 24 million tonnes in 2022

(Ashokkumar et al., 2022). Macroalgae have emerged as promising re
The increasing recognition of the environmental impact of human sources not only for green energy generation but also for obtaining
activities has compelled a shift from traditional extractive industrial value-added compounds (Basile and Dalena, 2019). The diverse
practices towards a more sustainable and eco-friendly approach (Gerdes composition of macroalgae offers a brunch of functional, sensory, and
et al., 2022). Moreover, the environmental repercussions of fossil fuel bioactive chemical compounds that hold potential applications across
reliance have spurred international authorities and the scientific com various industries, including food, cosmetics, and pharmaceuticals
munity to explore and develop new renewable energy alternatives. (Zheng et al., 2022). For instance, several cosmetic formulations use
Second- and third-generation biomasses, particularly macroalgae, have these compounds as additives, excipients, or active ingredients (Zheng
garnered significant attention over the past decade, and have seen a et al., 2022). Similarly, polyphenolic compounds are known for their
* Corresponding author.
E-mail address: dlama@ig.csic.es (D. De la Lama-Calvente).
https://doi.org/10.1016/j.jenvman.2024.122504
Received 24 January 2024; Received in revised form 8 September 2024; Accepted 12 September 2024
Available online 17 September 2024
0301-4797/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
D. De la Lama-Calvente et al. Journal of Environmental Management 370 (2024) 122504
antioxidant activity, with several food applications (Chen et al., 2023), R. okamurae was tested for the α-glucosidase inhibitory activity, showing
and, additionally, they play a fundamental role in the humification an inhibition of 90.9 ± 1.02 % and placing this alga at the fourth highest
process of biomasses (Zhang et al., 2023). The utilization of these position when compared with other 19 species (Jeong et al., 2012).
compounds derived from seaweed in industrial processes underlines Casal-Porras et al. (2021) used an acetone/methanol (1:1 v/v) mixture
their extensive study and documentation in the scientific literature. followed by diethyl ether to extract secondary metabolites (such as
Nevertheless, these industrial applications are mostly based on Dilkamural) and tested their feeding-deterrent activity. Using the same
cultivated seaweed (Zheng et al., 2022) which presents several draw extraction protocol, more than ten new diterpenoids were isolated,
backs, e.g.: it entails the building of specific facilities when cultivated among which some of them showed strong anti-inflammatory behaviour
in-land; it may contribute to eutrophication when cultivated off-shore (Cuevas et al., 2021, 2023). Furthermore, it has been reported that while
within the marine ecosystem; etc. (Khan et al., 2023). Thus, a more the ethanolic (H2O:EtOH 1:4) and the methanolic (H2O:MeOH 1:4) ex
sustainable approach would be to take advantage of naturally produced tracts of R.o. presented some inhibition to some human pathogens
macroalgae biomass, although these systems would be associated with (Staphylococcus aureus and Cutibacterium acnes), with no significant
other challenges as well (e.g. collection costs and logistics, inconsistent differences between them, this effect was not observed with fish path
production, compositional variation, etc). In the last years, the spread of ogens (Vega et al., 2023). Moreover, the aqueous extract showed no
invasive seaweed species and the frequency of the so-called green tides inhibition to either human or fish pathogens (Vega et al., 2023). How
have increased worldwide due to climate change and coastal areas ever, the aqueous extract obtained by microwave-assisted hydrothermal
eutrophication (Ravaglioli et al., 2022; Xia et al., 2022). These phe (MAH) processing showed a 53–65 % (depending on the temperature
nomena are considered ones of the main threats to marine ecosystems’ applied) inhibitory efficacy to cell growth in tumoral cell line HeLa 229
functioning, native wildlife, economics, and human health (Pereira (Ferreira-Anta et al., 2023). Additionally, this MAH method allowed for
et al., 2021; Salimi et al., 2021). Among these macroalgae species, two the extraction of alginate (3.2 % recovery) and phenolic compounds
seaweed have focused the attention due to the extension of their impact: (Ferreira-Anta et al., 2023). Similarly, the microwave hydrodiffusion
the Sargassum invasion in the Caribbean (Lopresto et al., 2022); the and gravity treatment enable the recovery of a gelling fraction and an
Rugulopteryx okamurae (R.o.) invasion in the Mediterranean Sea (Roca tioxidants (López-Hortas et al., 2023). All these evidences proved the
et al., 2022). potential of R. okamurae to provide for bio-active compounds. However,
The brown alga R.o. has colonized a great part of the Mediterranean the use of chemicals (e.g. MeOH or diethyl ether) or temperature during
coasts in Spain and Morocco since 2015, when it was first detected in the extraction process may discourage scaling up to a biorefinery in
Ceuta (El Aamri et al., 2018; García-Gómez et al., 2020). Its rapid and dustry due to the high costs involved.
uncontrolled spread has provoked a severe ecological impact associated Despite the use of the invasive species R. okamurae as a feedstock for
with unprecedented biodiversity loss (García-Gómez et al., 2020). Be the obtention of added-value compounds with bioactive properties,
sides, the loosed biomass ends up on the shores, where is usually left for other studies have highlighted its potential as raw material for the
months, causing environmental pollution and costing millions to local production of compost, bioplastics or aquafeeds for European sea bass
authorities due to waste management and the loss in the fishery sector (Fonseca et al., 2023; Patón et al., 2023; Santana et al., 2022).
(Báez et al., 2023; MITECO, 2022). A sustainable and economically Furthermore, the potential as feedstock for energy production has also
feasible solution is needed in order to palliate the environmental effect been assessed. Hydrogen and short-chain fatty acids production through
of this plague and its impact on anthropogenic activities. dark fermentation after microwave pre-treatment has been considered a
The studies on this invasive species has increased in the last years. promising alternative (Fernández-Medina et al., 2022). Hydrogen is a
Some studies, prior to its appearance in Ceuta, already highlighted the great alternative to fossil energy sources because it has a very high en
presence of secondary metabolites, such as terpenes, and the high ergy content and is a clean and green energy source. In addition, it shows
α-glucosidase inhibitory activity of its ethanolic extracts (Jeong et al., high energy efficiency and compatibility with fuel cells as well as its
2012; Suzuki et al., 2002). More recently, it has been discovered that renewal waste product, i.e. water (Zinatloo-Ajabshir et al., 2020a,
R. okumare produces diterpenoids with a high anti-inflammatory activ 2020b, 2023). In this sense, it has been recently reported that lanthanum
ity. One of them, a chemical weapon namely dilkamural, was presented zirconate ceramic microstructure exhibits an improved electrochemical
as harmful and even lethal to sea urchins (Casal-Porras et al., 2021; hydrogen storage performance compared to the Nd2Zr2O structure due
Cuevas et al., 2021). However, in terms of bioactive compounds to its higher specific surface, as well as its more favorable physical and
extraction methodologies, the reported studies have focused on the chemical nature (Zinatloo-Ajabshir et al., 2023). Other research carried
production and extraction of carbohydrates and antioxidant com out by this same research group also demonstrated the suitability of the
pounds. For instance, the alga has been fermented with the fungus green Nd2Sn2O7 nanostructures, synthesized using palm extract, for
Aspergillus awamori followed by enzymatic hydrolysis allowing the electrochemical hydrogen storage (Zinatloo-Ajabshir et al., 2020a).
production of 240 greducing sugars kg−dw1 (Agabo-García et al., 2023). In a Moreover, Zinatloo-Ajabshir et al. have also shown the benefits of the
different study using only commercial enzymes the highest yield re use of dysprosium stannate nanoparticles using Ficus carica extract as
ported was 13.7 gsugars L− 1 in the hydrolysates (Romero-Vargas et al., photocatalyst for the degradation of organic pollutants (i.e. acid violet 7
2023). From a different perspective, the relevance of drying related to and crystal violet) from water under visible irradiation
the extraction of alginate and antioxidant compounds has also been (Zinatloo-Ajabshir et al., 2020b). However, the production of bio
assessed by comparing the effect of sun drying, conventional oven and methane from this invasive species has been more deeply assessed. For
microwave hydrodiffusion and gravity (MHG) (López-Hortas et al., instance, the use of R. okamurae as co-substrate with olive mill solid
2023). Furthermore, the dried samples were extracted with an ethanol: waste (OMSW) in a 1:3 mixture (VS basis) produced 26.8 % more
water solution (1:1 (v/v)) for 24 h at room temperature and 200 rpm. methane than the mono-digestion of both substrates (De la
Results showed that MHG was the best alternative providing the highest Lama-Calvente et al., 2023a); moreover, a preliminary economic
antioxidant and alginate fraction (López-Hortas et al., 2023). Earlier assessment showed that the zeolite-assisted milling pre-treatment on
experiments carried out by Kurata et al. (1988) showed that the use of R. okumare could potentially increases the income of a full-industrial
alcoholic solvents, such as methanol, was able to extract spatane-type plant, treating only algal biomass, by a 43 % (De la Lama-Calvente
diterpenes with feeding-deterrent activity from the alga R. okamurae. et al., 2023a).
Later, the ester ethyl acetate was used to recover a newly isolated This article attempts, for the first time, to propose a comprehensive
secospastane namely dilkamural, and acetone was used for two novel management system for the disposal and valorization of an invasive
diterpenoids with feeding-deterrent activity (Ninomiya et al., 1999; macroalga (Rugulopteryx okamurae) within a closed-loop circular econ
Suzuki et al., 2002). More recently, the ethanolic extract of the omy approach. Firstly, the extraction efficiency of value-added
2
compounds (i.e. reducing sugars (RS) and total polyphenolic compounds oven-dried at 100 ◦ C for a period of 24 h. The same protocol was
(TPC)) by different solvents (i.e. water and ethanol at room tempera implemented in the second extraction step for the sequentially extracted
ture), and the antioxidant capacity of the obtained extracts were eval tests as it is shown in Fig. 1. Briefly, preliminary studies based on
uated. Furthermore, the biomethane potential of the non-extracted alga response-surface methodology indicated that the selected sample con
and the extracted solid residues have been performed by biochemical ditioning enhances the extraction process. This was mainly due to the
methane potential (BMP) tests, while comparing simultaneously two major cell wall disruption occurring in comparison with less severe
algae batches collected at different stages of their life cycle (i.e. from treatments. Middle temperatures (100–120 ◦ C) proved to reduce
their natural location linked to sea bottom rocks and recently arrived at significantly the cellulose crystallinity of the alga cell wall, which fav
the coast after loosening from its natural location). The effect on the oured the cellulose breakdown during the milling process (De la
kinetic parameters has been also discussed. Finally, the anaerobic Lama-Calvente et al., 2023a). The selection of the two solvents was
digestates have been analyzed and compared with local regulations to based on preliminary studies and literature review: Water is commonly
discuss their potential use as fertilizer or soil amender. the most economical solvent, however, it has limited affinity for some
interesting compounds, such as phenols, terpenes, lipids, etc. On the
2. Materials and methods other hand, ethanol, although more expensive is also widely used as it
could be easily reusable, additionally, it can extract a wider range of
2.1. Seaweed compounds, such as pigments, polyphenols, etc.
The marine algal biomass of R.o. was provided by the Laboratory of 2.3. Anaerobic inoculum biomass
Marine Biology of the University of Seville from two different spots
along the Algeciras coast (36◦ 03′0’’N 5◦ 29′7’’W - 36◦ 03′7’’N The anaerobic inoculum was collected from a brewery wastewater
5◦ 14′8’’W): i) Fresh R.o. (FR) attached to the sea bottom rocks in its anaerobic treatment plant located in Seville (Heineken) and the same
natural environment; ii) Fresh Ashore R.o. (FAR), immediately collected batch was used for all the biomethanization assays. Immediately after
once the loosen biomass reaches the beach. Samples were washed in situ the reception of this inoculum at the laboratory facilities, the sludge was
with seawater to eliminate debris and stored at − 20 ◦ C upon their arrival placed in a water bath at 37 ± 2 ◦ C for 36 h in order to lessen the
at the laboratories. Prior to further use, samples were thawed at 4 ◦ C for endogenous methane production. The correct anaerobic activity of the
2 h and washed again with deionized water in order to eliminate the inoculum was tested by placing positive controls (cellulose; Avicel® PH-
interference of marine salts and other potential contaminants during the 101, Fluka) during the assays and confirming that at least 80 % of the
experiments. Details of the washing effect on the elemental content are theoretical methane yield was achieved (Holliger et al., 2016). The main
provided in Table S1 of the supplementary material. physicochemical parameters of the anaerobic inoculum were as follows:
TS: 54.8 ± 0.4 g kg− 1; VS: 42.0 ± 0.4 g kg− 1; MS: 12.7 ± 0.2; tCOD: 42
2.2. Value-added compounds extraction process ± 4 g O2 kg− 1; pH: 7.59 ± 0.05; TA: 3600 ± 200 mg CaCO3 L− 1.
Based on a preliminary study (De la Lama-Calvente et al., 2024) the 2.4. Biochemical methane potential test
optimum extraction process was set as follows: the algal biomass should
be oven-dried at 100 ◦ C and milled during 60 s. For the single extraction The BMP test was carried out by following the indications described
process, 0.5 gVS of the dried and milled biomass was mixed with 25 ml of by Holliger et al. (2016). Briefly, the assay was performed at mesophilic
solvent (water or ethanol) and stirred (250 rpm) during 1 h at room temperature (37 ± 2 ◦ C) and at an inoculum-to-substrate ratio (ISR) of 2
temperature (25 ◦ C aprox.). Then, the mixture was filtrated (Whatman, (VS) reaching a final concentration of 24 gVS L− 1 in the reactors. As
0.45 μm pore size nylon filter), and the liquid fraction was immediately established in Holliger et al. (2016), the recommended ISR values are
storage at − 20 ◦ C until further use while the solid residue was further between 2 and 4, where higher values are recommended for
Fig. 1. Schematic design of the biorefinery conceptualization carried out.

Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol
and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR) extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or
firstly with ethanol and then with water (FR-EW). BMP: Biochemical methane potential; RS: Reducing Sugars; TPC: Total Phenolic Content; FRAP: Ferric Reducing
Antioxidant Power; DPPHRSA: 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Activity.
3
easy-to-degrade substances and lower values for substrates with recal 2540E were followed in order to obtain the total solids (TS), volatile
citrant components (such as cellulose or lignin). Thus, the ISR was solids (VS), and mineral solids (MS) fractions (APHA, 2017). Chemical
established based on the potential presence of hard-to-degrade compo oxygen demand (COD) was determined by following the indications in
nents in the selected biomass. The 250 mL working volume consisted of Raposo et al. (2008) and the standard method 5220D (APHA, 2017), for
the inoculum, the substrate, and distilled water. The reactor’s headspace total (tCOD) and soluble (sCOD) fractions, respectively. pH and total
was maintained at approximately 10 % of the reactor’s total volume. alkalinity (TA) were determined by using a
Each test, including blanks and positive controls, was carried out in pH meter model Crison and by titration to pH 4.3 as shown in the 2320B
triplicate, and steadily stirred at 300–400 rpm. Prior to the start-up of standard method for TA (APHA, 2017). Total ammonia nitrogen (TAN)
the experiment, nitrogen gas was flushed into each reactor to guarantee was performed by distillation and titration following the standard
an anoxic environment. Finally, reactors were placed in a water bath method 4500-NH3 (APHA, 2017). Metal elements were determined by
where the temperature was maintained at 37 ± 2 ◦ C by using a ther digesting the samples with a Mars Xtraction microwave (CEM, USA) and
mostat (Termotronic, Selecta, Spain). analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS,
The tests were considered finished when the daily measured methane AGILENT 7800, Spain) as described in the U.S. EPA 3051A method
was <2 % of the accumulated value for three days in a row. This period (USEPA, 2007).
was c.a. 28 days. Methane was measured volumetrically after passing The following methods were carried out spectrophotometrically in a
the produced biogas through a 2 N NaOH solution to capture the 96-well microplate using a microplate multiscan reader (Bio-RAD,
generated CO2. The accumulated biomethane yields were then calcu iMark™). Total phenolic content (TPC) was determined by carrying out
lated by subtracting the endogenous methane production from the the Folin-Ciocalteau method using gallic acid (Sigma-Aldrich) as stan
blanks and by normalizing the volume to standard temperature and dard and measuring the absorbance at 750 nm (Magalhães et al., 2010;
pressure conditions (273.15 K; 101.325 kPa). Zhang et al., 2006). Reducing sugars content (RS) was determined by the
In total, 10 different tests were carried out (Fig. 1): (FR) Fresh Anthrone method using glucose (PanReac) as standard and measuring
R. okamurae; (FR-W) Fresh R. okamurae extracted with water; (FR-E) the absorbance at 630 nm (Leyva et al., 2008). The DPPH (stable radical
Fresh R. okamurae extracted with ethanol; (FR-WE) Fresh R. okamurae of 2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity test
extracted with water and then with ethanol; (FR-EW) Fresh R. okamurae (DPPHRAS) was performed by calculating the efficient concentration
extracted with ethanol and then with water; (FAR) Fresh ashore (EC50) of the extract expressed as the amount of antioxidant per mL able
R. okamurae; (FAR-W) Fresh ashore R. okamurae extracted with water; to reduce the DPPH (0.2 mM) (Sigma-Aldrich) initial absorbance at 490
(FAR-E) Fresh ashore R. okamurae extracted with ethanol; (FAR-WE) nm by 50% (Rodríguez et al., 2005). The ferric reducing antioxidant
Fresh ashore R. okamurae extracted with water and then with ethanol; power assay (FRAP) was carried out using Trolox (6-hydroxy-2,5,7,
(FAR-EW): Fresh ashore R. okamurae extracted with ethanol and then 8-tetramethylchroman-2-carboxylic acid) (Sigma, ≥97 %) as standard
with water. and measuring the absorbance at 490 nm; results are given as Trolox mM
equivalents (Benzie and Strain, 1996).
2.5. Analytical methods
2.6. Data analysis
The main physicochemical characteristics of the algal biomasses
before and after the extraction processes and the anaerobic sludge were 2.6.1. Biodegradability
analyzed prior to the biomethanization tests (Table 1). The anaerobic The biodegradability of each substrate was calculated by the CODt
digestates resulted from the biochemical methane potential (BMP) tests analysis as proposed by Nielfa et al. (2015). The theoretical methane
(Table 2) and the liquid fractions obtained after the extraction processes yields and the biodegradability results after each BMP test are displayed
(Table 3) were analyzed as well. For the analysis of soluble parameters, in Table 2.
samples were previously centrifugated (12,000 rpm, 10 min) and fil
trated (Whatman, 0.45 μm pore size nylon fiber filter). All the analyses 2.6.2. Kinetic models
were carried out at least in triplicates. The standard methods 2540B &
2.6.2.1. First-order kinetic model. The kinetic behaviour and the process
Table 1 performance of the AD of studied algal biomasses (fresh and ashore)
Main physicochemical characteristics of the substrates used for the biochemical were analyzed by the following first-order kinetic model:
methane potential tests. Values represent mean ± standard deviation.
G = Gmax⋅[1 – exp (-k⋅t)] (1)
Substrates VS MS TAN tCOD
(% TS) (% TS) (mg N-NH3 kg− 1) (g O2 kg− 1) Where G is the specific cumulative methane production (LCH4 kg−VS1),
FAR 89 ± 1 a,b
11 ± 1 a,b
1140 ± 40a
1160 ± 20 a,b Gmax is the ultimate methane production (LCH4 kg−VS1), k is the specific
FAR-W 90.2 ± 0.9 b 9.8 ± 0.9a 1250 ± 70a,b 1160 ± 50a,b rate constant (days− 1) and t is the digestion time (days). This kinetic
FAR-E 88.4 ± 0.5a,b 11.6 ± 0.5a,b 1400 ± 200a,b 1190 ± 30a,b model is normally applied to assess the kinetics of the batch AD pro
FAR-WE 89.4 ± 0.7a,b 10.6 ± 0.7a,b 1200 ± 100a,b 1150 ± 30a cesses for different types of biodegradable substrates (Li et al., 2012;
FAR-EW 89.9 ± 0.5a,b 10.1 ± 0.5a,b 1420 ± 80a,b 1140 ± 30a
Scarcelli et al., 2020). This model assumes that the microbial cell mass
FR 89 ± 1a,b 11 ± 1a,b 1100 ± 100a 1270 ± 10 b
FR-W 91.05 ± 0.05 b 8.95 ± 0.05a 1130 ± 50a 1336 ± 3c does not affect the methane production, which is treated as
FR-E 87 ± 3a 13 ± 3 b 1340 ± 30a,b 1180 ± 10a,b only-proportional to the amount of substrate (Wang et al., 2017).
FR-WE 90.8 ± 0.6 b 9.2 ± 0.6a 1400 ± 100a,b 1180 ± 30a,b
FR-EW 89.7 ± 0.6a,b 10.3 ± 0.6a,b 1500 ± 200 b 1183 ± 9a,b
2.6.2.2. Modified Gompertz model. The Modified Gompertz model was
Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or assessed as suitable to fit the experimental data recorded by the BMP
ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with tests:
ethanol and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR)
extracted with water (FR-W) or ethanol (FR-E) or with water and then with B = Bmax*exp[-exp(Rmax*exp(1)*(ℷ - t))/Bmax +1)] (2)
ethanol (FR-WE) or firstly with ethanol and then with water (FR-EW).
VS: Volatile Solids; MS: Minerals Solids; TAN: Total Ammonium Nitrogen; Where B (LCH4 kg−VS1)is the cumulative specific methane production,
tCOD: Total Chemical Oxygen Demand. Bmax (LCH4 kg−VS1) is
the ultimate methane production, Rmax is the
Different superscripted letters in the same column mean values are significantly maximum methane production rate (LCH4 (kgVS⋅d)− 1), t (d) is the
different. digestion time and ℷ is the lag time (d). This model assumes that
4
Table 2
Main physicochemical characteristics of the digestates derived from the BMP test. Experimental and theoretical biomethane yields and the biodegradability achieved.
Values represent mean ± standard deviation.
Substrates pH TA TAN TANs TS VS MS tCOD sCOD BMPexp BMPth BDCODt
(mg CaCO3 (mg N-NH3 (mg N- (g kg− 1) (% TS) (% TS) (g O2 (mg O2 (NLCH4 CODt (LCH4 (%)
kg− 1) kg− 1) NH3 L− 1) kg− 1) L− 1) kg−VS1) kg−VS1)
FAR 7.70 ± 3540 ± 20a 640 ± 20a,b 490 ± 20a 23.7 ± 77.4 ± 22.6 ± 29.6 ± 1550 ± 100 ± 6a,b 454 22 %
0.02a,b 0.3a 0.7a 7a 0.1a 80a
b, b b
FAR-W 7.73 ± 3840 ± 70 682 ± 2 560 ± 9 24.2 ± 79 ± 3a,b 21 ± 3a 29.3 ± 2300 ± 100 ± 20 a,
452 22 %
0.00 b c
0.6a 0.9a,b 200 b b,c
c b b b a
FAR-E 7.78 ± 3940 ± 80 690 ± 10 560 ± 10 24.9 ± 78 ± 1 22 ± 1 23 ± 1c 2910 ± 110 ± 10 a,
473 23 %
0.01c 0.4a 80c b,c,d
FAR-WE 7.627 ± 3960 ± 30 b 682 ± 4 b 550 ± 10a, 24.9 ± 78.2 ± 21.8 ± 28.3 ± 2300 ± 150 ± 20c, 451 33 %
0.006 d b
0.1a 0.7 b 0.7a 0.3a,b,d 100 b d
FAR-EW 7.79 ± 4250 ± 90 d 760 ± 20c 650 ± 30c 24.7 ± 77.9 ± 22.1 ± 27.7 ± 2400 ± 150 ± 20 d 443 34 %
0.00c 0.2a 0.6a,b 0.6a 0.3 b,d 100 b
a a,b a,b
FR 7.68 ± 3550 ± 40 630 ± 30 513 ± 9 25 ± 1a 78 ± 5a,b 22 ± 5a 29.5 ± 2800 ± 90 ± 20 a
499 18 %
0.01a 0.5a,b 30c
FR-W 7.70 ± 3650 ± 60a, 590 ± 40a 495 ± 9a 24.2 ± 78 ± 1a,b 22 ± 1a 30.0 ± 2370 ± 115 ± 5a,b, 515 22 %
0.00a b
0.4a 0.6a 80 b c,d
FR-E 7.79 ± 3800 ± 90 b, 670 ± 20 b 530 ± 40a, 24.5 ± 80 ± 1 b 20 ± 1a 28.8 ± 2300 ± 121 ± 3a,b, 479 25 %
0.01c c b
0.6a 0.4a,b,d 100 b c,d
a,b a,b b a
FR-WE 7.81 ± 3700 ± 630 ± 30 513 ± 9 24.8 ± 78 ± 1 22 ± 1 29.0 ± 2100 ± 140 ± 10 454 31 %
0.02c 100a,b 0.5a 0.3a,b,d 100 b b,c,d
FR-EW 7.90 ± 3780 ± 50 b, 660 ± 10 a,b

520 ± 30 a,
24.5 ± 79 ± 1 a,b
21 ± 1 a
27.5 ± 2400 ± 140 ± 10 463 30 %
0.01e c b
0.4a 0.4 d 200 b b,c,d
Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol and
then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR) extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or firstly
with ethanol and then with water (FR-EW).
TA: Total Alkalinity; TANt: Total Ammonium Nitrogen of the whole digestate; TANs: Total Ammonium Nitrogen of the liquid fraction of the digestate; TS: Total Solids;
VS: Volatile Solids; MS: Minerals Solids; tCOD: Total Chemical Oxygen Demand; sCOD: Soluble Chemical Oxygen Demand. Different superscripted letters in the same
column mean values are significantly different.
Table 3
Main physicochemical characteristics of the liquid extracts. Extracted added-value compounds concentration and antioxidant properties. Values represent mean ±
standard deviation.
FAR-W FAR-E FAR-WEa FAR-EWa FR-W FR-E FR-WEa FR-EWa
VS (% TS) 95 ± 8a,b 95 ± 9a,c 100 ± 0c 70 ± 20a,b,c 80 ± 20a,b,c 81 ± 8a,b 99 ± 2a,b,c 90 ± 10 b

pH 7.22 ± 0.02a 7.11 ± 0.05a 7.49 ± 0.08 b 7.57 ± 0.06 b 5.67 ± 0.04c 5.44 ± 0.03 d 5.90 ± 0.06e 5.893 ± 0.006e
TA (g CaCO3 kg¡1) 260 ± 20a 88 ± 9 b 75 ± 1 b 240 ± 20a 240 ± 10a 75 ± 4 b 70 ± 4 b 203 ± 9 b
sCOD (mg O2 L¡1) 1849 ± 30a,b 1700 ± 100a 760 ± 60c 2100 ± 100 b 5700 ± 200 d 4500 ± 200e 2800 ± 100f 6290 ± 40 g
TAN (mg N-NH3 L¡1) n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
RS (gGlucose kgVS¡1) 11.6 ± 0.6a,b 2.3 ± 0.3c 0.7 ± 0.1c 16 ± 2 d 13.3 ± 0.9 b,d 8.0 ± 0.7a,e 4.7 ± 0.6e 32 ± 5f
TPC (gGalic Acid kgVS¡1) 2.90 ± 0.09a 13.3 ± 0.3 b 20.8 ± 0.9c 3.34 ± 0.05a 2.04 ± 0.03a 22 ± 2c 21 ± 2c 2.95 ± 0.08a
FRAP (mM Trolox eq.) 1.50 ± 0.04a 0.76 ± 0.05 b 0.863 ± 1.56 ± 0.01a 1.05 ± 0.04 d 0.89 ± 0.02c 0.91 ± 0.01c 1.16 ± 0.04e
0.009c
DPPHRSA (IC50 – mgTS 0.144 ± 0.007a, 0.15 ± 0.01a 0.12 ± 0.02a,b, 0.098 ± 0.118 ± 0.004 b, 0.29 ± 0.01 d 0.27 ± 0.04 d 0.132 ± 0.006a,
b c
ml¡1) 0.004c c b
Fresh ashore Rugulopteryx okamurae extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol and then
with water (FAR-EW). Fresh Rugulopteryx okamurae extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or firstly with
ethanol and then with water (FR-EW).
VS: Volatile Solids; TS: Total Solids; TA: Total Alkalinity; sCOD: Soluble Chemical Oxygen Demand; TAN: Total Ammonium Nitrogen; RS: Reducing Sugars; TPC: Total
Phenolic Content; FRAP: Ferric Reducing Antioxidant Power; DPPHRSA: 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Activity.
Different superscripted letters in the same row mean values are significantly different.
n.d.: not detected.
a
Results are related to the liquid fraction of the second extraction process.
methane produced in the batch modes is directly related to the specific and values are given by means ± standard deviations. Where corre
methanogenic bacterial growth (Donoso-Bravo et al., 2010; Li et al., sponded, a two-tale Student’s t-test and the one-way analysis of variance
2012). The model was thus used to predict the trend in microbial growth (ANOVA) were carried out. Additionally, the Tukey’s test was per
and simulate the cumulative methane yield. The goodness-of-fit and the formed as a post-hoc analysis. For the purposes of data discussion, a
accuracy of the results for both models have been assessed by the confidence level of α = 0.05 was accepted as not statistically significant.
determination coefficients (R2) and the standard errors of estimates (S.E. All the statistical analyses were carried out by SPSS Statistics software
E.). The kinetic parameters for each experiment and mathematical (v29, IBM, Business Machines Corp.).
adjustment were determined numerically from the experimental data
obtained by non-linear regression using the software Sigma-Plot (v. 11).
2.6.3. Statistical analysis

Analytical tests and BMP tests were at least carried out in triplicates
5
3. Results and discussion Finally, all the tested liquid samples showed no TAN content. This
suggests that the extraction process was not able to extract
3.1. Rugulopteryx okamurae extraction yields protein-derived compounds as these are usually soluble in aqueous so
lution at alkaline pH’s (Veide Vilg and Undeland, 2017).
3.1.1. Physicochemical characteristics of the Rugulopteryx okamurae
extracts 3.1.2. Total phenolic compound yields after single extraction
Table 3 shows the physicochemical characteristics of the different The higher TPC values were obtained when ethanol was used as
extracts assessed after the extraction optimization process. The extrac solvent in contrast with those values obtained from the aqueous liquid
ted organic fraction in terms of sCOD showed significant differences extracts (Table 3). In fact, using water as a solvent would reduce the TPC
among samples. In fact, the extracts from the ashored alga (FAR) pre yield by a 78–90 %, depending on the biomass. These results were in
sented lower values (from 760 ± 60 to 2100 ± 100 mg O2 L− 1) than agreement with other studies showing that alcoholic solvents (such as
those from the natural equivalent (FR) (from 2800 ± 100 to 6290 ± 40 ethanol or methanol) present higher affinity to phenolic compounds
mg O2 L− 1). These results are in accordance with the RS content of the than aqueous solutions (Vega et al., 2023).
extracts, which were higher in the FR samples than in their FAR The phenolic compounds values obtained in the present study (2.04
equivalents. In addition, other compounds not tested, such as pigments, ± 0.03–22 ± 2 ggallic acid kg−VS1) are in the same range of other brown
are expected to be present in FR samples at higher concentrations, as seaweeds (0.2–235 ggallic acid kgdw) (Barciela et al., 2023; Getachew
these compounds are highly sensible to degradation due to light inci et al., 2020) and very similar to those previously reported for R.o. using
dence or temperature (Cikoš et al., 2022). Finally, the organic matter a mixture of H2O:EtOH (1:1) as solvent (≈20 g kg−dw1) (Vega et al., 2023).
detected by the sCOD analysis showed a higher affinity to water than to However, when the alga R.o. was subjected to a hydrothermal pre
ethanol. However, when the accumulated values were compared, it treatment by microwave irradiation (220 ◦ C during 20 min) the
seems that the highest value was obtained when ethanol was used before maximum TPC yield (0.034 ggallic acid kg−VS1) was significantly lower than
water, regardless the sample was the FAR or the FR. In fact, the sum of the reported in the present study (Fernández-Medina et al., 2022).
FAR-E and FAR-EW sCOD values was a 46 % higher than the sum of While there were no significant differences between the aqueous
FAR-W and FAR-WE. Similarly, the sum of FR-E and FR-EW sCOD extract of the two R.o. samples, the ethanolic extracts differed greatly,
values was a 27 % higher than the sum of FR-W and FR-WE, which, being the FR-E sample which showed the highest amount. This sample
support the idea of ethanol as a significant cell disruptor. represents healthy individuals during the summer period, which have
The different elements founded in the mineral fraction are detailed in been pointed as the peak season for the content of phlorotannins in
Table S2 of the supplementary material. Although the content of most of brown algae, concurring with the reproductive season (Connan et al.,
the economically interesting metals (i.e. Cu, Cd, Cr, Ti) were present at 2004; Imbs and Zvyagintseva, 2018). However, the TPC also depends on
low concentrations (≈ppb), their recovery may be a possible path worth the habitat of the algae, the intensity of illumination, or, as shown in this
to investigate. Unfortunately, studies assessing the valorization of study, on the natural degradation, among other factors (Imbs and
macroalgae through metal recovery are scarce. However, the metal re Zvyagintseva, 2018). Although, the reduction of TPC during the natural
covery from other wastewaters or contaminated soils has been widely degradation of marine algae have not been yet assessed, studies on the
studied. For instance, a stable electrocatalyst-coupled with super-stable composting of similar biomasses could provide approximated informa
mineralizer structure showed a recovery yield of 91.1 % for Cu2+ (Liu tion. For example, after the vermicomposting process of oil palm empty
et al., 2024). Other techniques, such as flow-electrode capacitive fruit bunches, the phenolic compounds decreased from 311 ggalic acid
deionization, showed less selectivity and produced low-quality metal kg− 1 to 15 ggalic acid kg− 1, meaning more than a 95 % elimination of
products. However, if coupled with a liquid membrane chamber, then phenolic compounds (Yahaya et al., 2017). Moreover, during the
the outcome improved significantly (Wu et al., 2024). In any case, the senescence of the cells of the brown alga Ectocarpus sp. all the cell or
use of adsorbents is the most spread technique in the removal of heavy ganelles start to degrade as the cell ages up to the cell death when some
metal from wastewaters (Rakib et al., 2021). These results suggest that of the organelles, like the nucleus or the Golgi apparatus, are completely
the valorization of macroalgae through the extraction of heavy metals banished (Oliveira and Bisalputra, 1977). In this study, naturally
could be a promising pathway which need further investigation. degraded FAR samples presented only a 40 % less TPC than FR. This
Regarding the acidity of the extracts, significant differences were could also indicate the freshness of the FAR samples, which were taken
observed between the two samples assessed. While the FAR samples immediately once reached the coasts. And suggests that the TPC of the
showed neutral pH values (from 7.11 ± 0.05 to 7.57 ± 0.06), the FR completely sun-dried algal biomass found in the coasts could be negli
samples showed an acid pH (from 5.44 ± 0.03 to 5.90 ± 0.06). Lesser gible. As a result, extraction with pure water aiming for phenolic com
acute differences were observed between one-step extracts and two-step pounds, except for economic reasons, seems to not be adequate and,
extracts, although they were statistically significant with the pH slightly furthermore, the algae should be as fresh as possible.
increasing, for both FAR and FR, after the second extraction. Similarly,
ethanolic extracts showed slightly lower pH values than the aqueous 3.1.3. Reducing sugars yields after single extraction
equivalents across the board. These findings were also supported by the Opposite to TPC results, water was a better solvent than alcohol for
TA values of the extracts, although, no great differences were observed the RS extraction (Table 3). In fact, the use of ethanol would deplete the
between the two types of algal biomass studied. This acidity difference RS yield by a 40–80 %, depending on the biomass. Similar results have
observed between the fresh and the ashore biomass could also explain been widely reported in the literature stating that RS present higher
the different TPC and RS yields obtained. In fact, the use of diluted acids affinity to aqueous solutions. For example, when the terrestrial plant
improves the oligosaccharides solubilization (Andrade et al., 2022), Vernonia amygdalina was extracted with methanol, ethanol and aqueous
while, by contrast, tend to increase the protein precipitation (Veide Vilg solutions, sugars were only found in the aqueous fraction (Hussen and
and Undeland, 2017). However, these results are in contrast with pre Endalew, 2023). Similarly, the RS extraction of the blue-green micro
vious studies suggesting that the composting process of seaweed tends to algae Oscillatoria limosa was more efficient with water than with meth
reduce the pH value (Walsh and Waliczek, 2020). Reasons behind this anol or ethanol, however, acetone showed the highest affinity (Tunio
may be due to the chemical composition of R. okamurae and the presence et al., 2022).
of specific acidic secondary metabolites (e.g. diterpenes) which are This is the first time that the RS content of R.o. has been reported
eliminated during the natural degradation process. This would also after a sole aqueous or ethanolic extraction process without the appli
explain the lower pH of ethanolic extracts as secondary metabolites, cation of thermal energy. Nevertheless, when the alga R.o. was subjected
such as terpenes, are usually not-water soluble (Jiang et al., 2017). to a hydrothermal pretreatment by microwave irradiation (200 ◦ C
6
during 15 min) the maximum RS yield (0.060 gglucose kg−VS1) brown marine algae, are usually present in a cell vacuole, named
(Fernández-Medina et al., 2022) was significantly lower than the re physode and are accumulated in the outer cortical layer of the thalli
ported in the present study (from 0.7 ± 0.1 to 32 ± 5 gglucose kg−VS1). (Imbs and Zvyagintseva, 2018). Moreover, these compounds tend to
Other studies have assessed the potential of R.o. as a source of mono form a complex with alginic acid, which is a common carbohydrate
saccharides by enzymatic saccharification. In this sense, Agabo-García responsible for the structure of the cell wall (Imbs and Zvyagintseva,
1
et al. (2023) reported values of 1.5–4.27 g L−hydrosilate after a biological 2018). Thus, it could be argued that a first aqueous extraction weakens
treatment (5–12 days) followed by enzymatic hydrolysis (24 h). More this complex, allowing for a higher TPC yield after the use of ethanol.
1
over, values of 12.22 g L−hydrosilate have been reported after the hydro Moreover, as discussed above, the natural degradation of the algae helps
lytically pretreated biomass by CellicCTec2 containing cellulase and reducing the carbohydrate content and increasing the breakdown of
endo-β-1,4-xylanase (Romero-Vargas et al., 2023). Similar results (12 g these compounds, allowing for a greater phenolic release in comparison
1
L−hydrosilate ) were obtained by Fernández-Medina et al. (2022) when the with the alive fresh algae in its natural environment, which could
enzymatic pretreatment with CellicCTec2 was applied after a hydro compensate for the degradation of the phenolic compounds during the
thermal pretreatment by microwave irradiation. In the present study, natural degradation process. This could explain the lack of significant
without a saccharification process of any kind, the concentration of differences in the TPC of both FR and FAR samples when extracted
reducing sugars in the liquid fraction was significantly lower, ranging firstly with water and subsequently with ethanol.
1
from 14 to 640 mg L−extracted liquid. This confirms the great potential of the Regarding the RS yields, from Table 3 it could be observed that the
mentioned pretreatments to enhance the conversion of carbohydrates sequential extraction process of ethanolic and further aqueous extrac
into shorter-chain oligosaccharides. Nevertheless, this study aimed to tion methodology enhance the RS recovery greatly. In fact, the FR-EW
assess the potential of the alga R.o. as a feasible feedstock within a extract yielded 2.4 times more RS than the FR-W extract. Mono and
biorefinery industry based on eco-friendly and low-cost processes, oligo saccharides are relatively soluble in water, which suggests that the
which, as expected, impairs the added-value compound extraction yield. carbohydrate fraction that remains in the solid residue is composed of
However, the energetic valorization of the extracted residue may over higher-complex carbohydrates, such as cellulose or hemicellulose,
come any loss during the extraction process. bonded with other non-soluble compounds, such as phlorotannins (Imbs
Regarding the biomass type, FR samples showed a higher RS content and Zvyagintseva, 2018). The preliminary ethanolic extraction process
in both ethanolic and aqueous extracts. Significantly, the ethanolic could further damages the cell wall allowing for an easier incursion of
extract presented an RS content 2.5 times higher than that found in the the water through the cell. In fact, a recent study on the effect of ethanol
FAR samples, while for the aqueous extract, the increment represented in E. coli showed that ethanol reduced the cell membrane integrity;
only 15 %. Similar to the TPC, the carbohydrate content of algae peaks increased the stress, through the higher production of reactive oxygen
during late spring to middle summer, although its proportion may be species, causing subsequent damage to the DNA; and, generally, dis
altered by several factors, such as: i) temperature; ii) light irradiance; iii) rupted the normal cell behaviour (Cao et al., 2017). Additionally, and
nutrient availability; iv) seawater hydrodynamics; v) day length similar to the single extraction process, the RS content obtained from the
(Schiener et al., 2014). To the best of our knowledge, the effect of nat FR samples after aqueous extraction was higher than that obtained from
ural degradation on the RS content of marine algae has not been re the FAR samples, due, most likely to the natural degradation process
ported earlier. However, several studies on the composting of that takes place when the alga is settled and accumulated on the beach.
macroalgae confirmed that this process reduces and breakdown the
organic matter content, being the short-chain carbohydrates the easier 3.1.5. Antioxidant capacity of the Rugulopteryx okamurae extracts
biodegradable fraction (Oliveira and Bisalputra, 1977; Patón et al., The antioxidant capacity of the extracts was assessed by performing
2023; Walsh and Waliczek, 2020). the FRAP and the DPPHRSA tests. Results are shown in Table 3. Sur
prisingly, FRAP results from the aqueous extract were higher than those
3.1.4. Combined TPC and RS yields in sequential extraction processes from the equivalent ethanolic extracts. For example, while the aqueous
It may be clear that the best methodology should be able to provide extract from the FAR biomass reported a FRAP value of 1.50 ± 0.04 mM
with the highest recovery yield for both TPC and RS in a one step Trolox eq., the ethanolic extract only gave 0.76 ± 0.05 mM Trolox eq.
extraction process. However, while ethanol is presented as the best Although this behaviour was observed regardless of the biomass (i.e. FR
option for the recovery of phenolic compounds, water would be selected or FAR), the differences between the aqueous and the ethanolic extract
to obtain the highest RS yield. These results suggested that a single step were smaller in the FR samples. Moreover, while both aqueous extract
extraction process should be focused on the extraction of one type of (FR-W and FR-EW) showed lower FRAP values than their FAR equiv
compounds, and that a cascade system could be a more recommended alents, the opposite was observed with the ethanolic extracts (FR-E and
option for an efficient extraction of both TPC and RS. Thus, a sequential FR-WE). If the TPC and the RS contents are taken into consideration it
two-step extraction process would be performed in order to simulate a could be concluded that all these results suggested that the antioxidant
more comprehensive biorefinery system where the biomass is exhausted capacity is primary given by a phenolic compound or compounds that
from most of its value-added compounds. shows higher affinity to water than to ethanol or by small oligosaccha
The sequential extractions with water or ethanol have been evalu rides detected by the RS test. Finally, similar to what was observed from
ated and the results are shown in Table 3. Regarding the TPC, no sig the TPC and RS extract yields, both aqueous and ethanolic extracts of the
nificant differences were observed for the FR samples between the first second extraction process showed higher values than their equivalents
extracted fraction and the second extracted fraction, regardless of the single extracts. This supports the idea of cell disruption after a first
solvent. For example, the one-step aqueous extract (FR-W) resulted in a extraction process.
TPC of 2.04 ± 0.03 gglucose kg−VS1, while the second aqueous extract (after This is the first time that the antioxidant capacity of R.o. aqueous and
the ethanolic extraction) (FR-EW) resulted in a TPC of 2.95 ± 0.03 ethanolic extracts is reported by using the FRAP test. Results obtained in
gglucose kg−VS1, with no significant differences between results. Similar this study (0.76 ± 0.05–1.56 ± 0.01 mM Trolox eq.) were in the upper
results were recorded for the FAR aqueous extracts, however, the dif range of other marine seaweed. For instance, Tolpeznikaite et al. (2022)
ferences between the ethanolic extracts were significantly higher. The assessed the FRAP of the ethanol/water (70:30 v/v) extract of the algae
ethanolic extraction of FAR after a first aqueous extraction (FAR-WE) Cladophora rupestris, Cladophora glomerata, Ulva intestinalis and Furcel
process yielded 56 % more TPC than the single ethanolic extraction laria lumbricalis reporting values of 0.077, 0.360, 0.869 and 0.063 mM
(FAR-E). This could be explained by the degree of polymerization of the Trolox eq., respectively. Ummat et al. (2020) also reported the FRAP
phenolic compounds and the strength of the bonds with other molecules. value of 11 brown seaweeds extracted with ethanol/water (50:50).
For example, phlorotannins, as the most common phenolic compound in However, this study reported the FRAP values as mg trolox eq. per gram
7
of dry weight extract (dwe), which do not allow for the comparison with extracts, reporting that the lowest value observed was 0.31 mg ml− 1 for
the previously mentioned article. Nevertheless, the reported values the species Gigartina teedei. On the other side, the brown macroalgae
1
(from 5 to 65 mg Trolox eq. g−dwe ) are lower to those observed in the Padina boergesenii showed values of 36.75 and 42.784 μg ml− 1 for its
1
present study for the alga R.o. (from 97 to 689 mg Trolox eq. g−dwe ). The ethanolic and methanolic extracts, respectively (Kalasariya et al., 2023),
authors are aware that there are more articles reporting the FRAP value and similar results were reported for Padina australis (16.63 μg ml− 1)
of ethanolic and aqueous extract of marine seaweeds, however, the and Turbinaria ornata (43.22 μg ml− 1) (Warouw et al., 2022).
authors would like to highlight the inconsistencies on the FRAP meth
odologies reported and the lack of standardized units which preclude the 3.2. Biochemical methane potential test
comparison between studies when not enough data are provided. In any
case, the aqueous extract of the FAR biomass seems to present one of the The main physicochemical characteristics of the substrates used for
highest FRAP value reported in the literature for marine seaweeds. the BMP tests are described in Table 1. Furthermore, the elemental
Regarding the DPPH radical scavenging activity of the studied ex analysis of the non-extracted biomasses is displayed in Table S1, while
tracts (Table 3), the tendency was similar to that observed for the FRAP for the extracted solid residue is shown in Table S3 of the supplementary
test. DPPHRSA values were lower in the aqueous extracts than in their material. Previously reported studies where the biochemical methane
ethanolic equivalents, which means that less dried extract is needed to potential of the algae Rugulopteryx okamurae were also tested showed a
reduce the DPPH absorbance to half its value. These results confirmed VS/TS ratio around 60–70 % (De la Lama-Calvente et al., 2023a; De la
that the compounds with the highest antioxidant capacity from the alga Lama-Calvente et al., 2023b; De la Lama-Calvente et al., 2021). How
R.o. presented higher affinity to water than to ethanol. Moreover, with ever, the batches tested in this study showed a significant higher ratio
only one exception (i.e. FR-EW), the higher the RS content was, the (87–91.5 %). Subsequently, the mineral fraction of the biomasses used
lower the DPPHRSA. While, by contrast, the higher the DPPHRSA was, the in the present study were significantly lower than those previously re
higher the TPC yield. This, as observed from the FRAP test, strongly ported. Part of this mineral reduction could be related to the washing of
suggested that these antioxidant compounds are phenols or other com the biomasses with tap water (Table S1), which were not done in the
pounds that shows higher affinity to water than to ethanol or small ol previous studies, and a further depletion due to the extraction process
igosaccharides detected by the RS test. Similar to the FRAP, the DPPHRSA (Table S3). However, these processes cannot be accounted as the sole
results from the ethanolic extracts were not significantly different reason. When comparing the results of the un-washed biomasses with
regardless the biomass was extracted once or twice. However, the those reported in De la Lama-Calvente et al. (2023b), some of the macro
DPPHRSA was higher in the FAR-W extract than in the two-step extract elements already showed significant differences. For example, the Ca
FAR-EW, while the opposite was observed for the other two aqueous content of the natural R.o. varied from 19.6 ± 0.7 in the present study to
extracts FR-W and FR-EW. Moreover, the DPPHRSA of the FR extracts 89 ± 3 in De la Lama-Calvente et al. (2023b). This confirms that the
were higher than the equivalent extracts from FAR samples, with the mineral content of natural biomasses varies greatly depending on the
exception of the one-step aqueous extracts which showed no significant several factors, such a seasonality, climatology, etc.
differences. It was also observed, that the contents of the potentially toxic ele
Similar to the FRAP test, different studies reported DPPHRSA values ments were significantly lower than those reported previously for the
in units which are not easily to use for comparison, due to, mainly, the macroalgae R.o. (De la Lama-Calvente et al., 2023a; De la
methodology followed or the expressed units. For example, Vega et al. Lama-Calvente et al., 2023b). In fact, some elements (Pb, Cu and Cd)
(2022), reported values of DPPH for different extracts of R.o. in the that have been directly linked to the inhibition of previously reported
1
range 5–25 μmol Trolox g−dwe . However, López-Hortas et al. (2023) re assays, have been found to be below the reported toxic limit in this
ported the DPPH value as the inhibition percentage (IP) of the ethanolic study. For example, while the toxic limit for
(50 %) extract of R.o., which varied along with the biomass drying Pb have been stablished at 1.27 ppm (Alrawashdeh et al., 2020), the
system (Oven: 4.6 %; MHG: 6.1 %). Table 3 shows the DPPHRSA values as content found in the liquid fraction of the reactors was in the range of
the minimum dried extract required to reduce the DPPH absorbance by 17–65 ppb. However, some other elements (Fe, Ni, Cr, Hg) were found to
half (IC50), which could be interpreted as an IP of 50 %. However, this IP be higher than the stablished toxic limit for AD. For example, the soluble
is dependent on the DPPH solution concentration, the solvent and the Ni content in the reactor was in the range of 57–330 ppb, while the toxic
extract-to-DPPH ratio used during the absorbance measurement. Thus, a limit reported by Alrawashdeh et al. (2020) was stablished at 41 ppb.
direct comparison between the López-Hortas et al. (2023) values and This suggest that although the AD could be potentially inhibited by these
those reported in this study would be not possible. Nevertheless, a rough elements, the methane yield would not be as affected as previously re
comparison could be made if the results are expressed per the amount of ported (De la Lama-Calvente et al., 2023b).
dried extract needed to reduce a certain amount of DPPH. López-Hortas In fact, based on the main physicochemical characteristics of the
1
et al. (2023) reported TS concentration of 0.479 and 1.316 mgTS ml−extract digestates after the BMP test (Table 2) no major disruption of the
for the biomass dried by oven and MHG, respectively. Moreover, the anaerobic performance was observed. pH values (7.64–7.90) were
DPPH assay was performed by measuring the absorbance drop when 50 within the optimum range for AD performance (Holliger et al., 2016).
μl of the extract was added to 2 ml of 0.06 mM DPPH solution. This TA values (3550–4250 mg CaCO3 kg− 1) were below the limit considered
means that, in the case of oven dried R.o., 23.96 ng of dried extract for buffer unbalance (Holliger et al., 2016). Similarly, TAN (590–760
reacted with 2.176 ng of DPPH, and in the case of MHG, 65.8 ng of dried mg N-NH3 kg− 1) were below the established limit (2000 mg N-NH3 L− 1)
extract reacted with 2.886 ng of DPPH. If we normalize this to the to consider a process inhibition due to the presence of free ammonia
amount needed to react with 1 mg of DPPH, then 11.010 and 22.800 mg (Yenigün and Demirel, 2013). The organic fraction, measured as either
of dried extract is requested for the dried biomass by oven or MHG, VS or CODt, showed certain differences between the tests which could be
respectively. In our study, this relation ranged from 12.75 to 37.72 mg of directly linked to the produced methane, indicating that the higher the
dried extract, being the lower value obtained from the aqueous extract methane yield the lower the organic fraction remaining in the final
of the residue previously extracted with ethanol (FAR-EW). digestate.
Other macroalgae also show good antioxidant activity, although, in Although no great significant differences were observed from the
most of the cases, with DPPHRSA values higher than those reported for physicochemical parameters of the digestates, the methane yield
the R.o. For example, the DPPHRSA IC50 of the green algae Caulerpa ra showed a different tendency (Table 2 and Fig. 2). Regardless of the
cemose and Caulerpa sertularioides were 456.1 and 180.9 μg ml− 1, biomass origin (i.e.: FR or FAR), as the alga was further extracted the
respectively (Sanniyasi et al., 2023). Goutzourelas et al. (2023) also methane yield increased. For example, the extraction of bioactive
studied the antioxidant activity of 14 marine macroalgae methanolic compounds of FR using water provoked a methane increase of 28 %,
8
Fig. 2. Accumulated methane of the untreated and extracted algae biomasses versus time. Bars represent standard deviation.
Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol
and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR) extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or
firstly with ethanol and then with water (FR-EW).
Table 4
Methane yield of the algal residue after added-value compounds recovery.
Added-value compound AD Methane Yield of residue (LCH4 Methane Source
System kg− 1 VSadded) Efficiency
Green Macroalgae
Ulva lactuca Sap BMP 206 98 % Mhatre et al. (2019)
Ulvan BMP 226 107 % Mhatre et al. (2019)
Protein BMP 229 109 % Mhatre et al. (2019)
Sap and ulvan BMP 409 195 % Mhatre et al. (2019)
Sap, ulvan and protein BMP 323 154 % Mhatre et al. (2019)
U. rigida Alginic acid, fucoidan, fucoxanthin, laminarin, BMP 73 – Tedesco and Stokes (2017)
mannitol, proteins
Red Macroalgae
Gracilaria Agar BMP 191 85 % Hessami et al. (2019)
manilaensis
G. multipartita Agar BMP 253 131 % Abomohra and Almutairi
Agar and Acid pretreatment BMP 279 144 % (2020)
Abomohra and Almutairi
(2020)
G. persica Agar BMP 148 83 % Hessami et al. (2019)
Brown Macroalgae
Ascophyllum Alginic acid, fucoidan, fucoxanthin, laminarin, BMP 84 – Tedesco and Stokes (2017)
nosodum mannitol, proteins
Ecklonia maxima Polysaccharide BMP 862 – Darko et al. (2022)
Fucus serratus Alginic acid, fucoidan, fucoxanthin, laminarin, BMP 101 – Tedesco and Stokes (2017)
mannitol, proteins
F. vesiculosus Alginic acid, fucoidan, fucoxanthin, laminarin, BMP 103 – Tedesco and Stokes (2017)
mannitol, proteins
Laminaria digitata Succinic Acid BMP 285 100% Alvarado-Morales et al.
(2015)
Alginic acid, fucoidan, fucoxanthin, laminarin, BMP 187 – Tedesco and Stokes (2017)
mannitol, proteins
L. hyperborea Alginic acid, fucoidan, fucoxanthin, laminarin, BMP 195 – Tedesco and Stokes (2017)
mannitol, proteins
Sargassum sp. Sugar BMP 150–166 87–105 % Abomohra et al., 2021
S. muticum Sap, fucoidan, phlorotannin BMP 150 76 % Flórez-Fernández et al.,
2021
R. okamurae TPC, RS BMP 100–150 100–150 % This Study
* VS: Volatile Solids; BMP: Biochemical Methane Potential; TPC: Total Phenolic Compounds; RS: Reducing Sugars.
** Methane efficiency is defined as the value for the extracted residue divided by the value for the non-extracted biomass in percentage.
9
and, moreover, when this residue was further extracted with ethanol, subsequent extraction of fucoidans and phlorotannin by isothermal
the methane increased 56 %. This increment is consistent with the autohydrolysis (120–210 ◦ C) (Flórez-Fernández et al., 2021). The
elimination of inhibitory and toxic compounds throughout the extrac remaining solid residue was also subjected to AD with a significant
tion step. Several attempts have been carried out in this sense, however, improvement concerning the untreated raw algae, and it was concluded
very few studies have assessed the feasibility of an integral biorefinery that the best performance was that obtained after the thermal treatment
where the whole biomass is treated and valorised within a circular- at 150 ◦ C, which allowed for a significant fucoidan extraction despite the
economy perspective. Table 4 summarizes these studies. phenolic fraction which needed higher temperatures (Flórez-Fernández
The extracted residue of the brown macroalga Laminaria digitata, et al., 2021).
obtained after the extraction of succinic acid through enzymatic hy
drolysis and fermentation, did not show any significant difference in 3.2.1. Kinetic performance
terms of methane yield to that obtained from the raw macroalgae The application of kinetic models to the anaerobic digestion exper
(Alvarado-Morales et al., 2015). In contrast, after the extraction of iments of untreated R.o. and extracted R.o. samples is very useful for the
carbohydrates (e.g. alginic acid, fucoidan, fucoxanthin, laminarin, evaluation of the process efficiencies as well as the interpretation of the
mannitol) and proteins of six different macroalgae, the methane yield methane production data (Darko et al., 2022). In this study, the
was on average 20 % lower than that from the untreated alga, with the First-order model (Equation (1)) was applied to estimate the specific or
significant exception of the two algae from the Laminareae species, apparent rate constant, k (days− 1), while the Modified Gompertz model
which showed a 43 % increase (Tedesco and Stokes, 2017). These lower (Equation (2)) was used to estimate the lag phase λ (days), maximum
results were, likely, due to several factors, e.g.: after the extraction the methane production rate Rm (mL CH4 (g VS⋅ d)− 1), and the ultimate
C/N ratio generally increased from ≈10 to 15 to ≈25–50; the total COD methane production Bm (mL CH4 g− 1 VS).
generally dropped to half of the raw algae; the S content increased In general, there was an overall good agreement between the two
(Tedesco and Stokes, 2017). However, Darko et al. (2022) assessed the models and the experimental data. Through the analysis of Tables 5 and
biomethanization potential of an extracted biomass obtained from the 6, it was possible to verify that, between the two tested models, the
enzymatic hydrolysis of the brown seaweed Ecklonia maxima, carried modified Gompertz was the kinetic model that slightly better fitted to
out in order to extract the polysaccharide fraction showing a methane the experimental results for all the assayed biomasses, since it has the
yield of 97 % of the theoretical value. Similar enhancements have been lowest S.E.E. and the highest determination coefficient (R2) values
observed in this study. (>0.9937). However, S.E.E. and R2 values should not be used alone to
Hessami et al. (2019) assessed the effect of Agar extraction of two red validate the suitability of the models and, thus, the percentage of errors
macroalgae (i.e. Gracilaria manilaensis and Gracilariopsis persica) before between the experimental and calculated values of ultimate methane
the AD process. Additionally, the study assessed the effect of acid pre production should also be considered. In this sense, the lowest values of
treatment on the methane yield of both the extracted and the raw algae. these errors (<4.1% in all cases) were also achieved for the Modified
Results showed a carbohydrate, protein and fat loss of 84 %, 68 %, and Gompertz model compared to the First-order model (Tables 5 and 6).
93 %, respectively for Gracilaria manialensis, while for Gracilariopsis
persica the reductions were 88 %, 88 %, and 97 %, respectively. After the 3.2.1.1. First-order model. Table 5 shows the kinetic constant, k, and
extraction, the methane yield was lower (15–19 %) than that obtained ultimate methane production Gm (LCH4 kg−VS1) values obtained with the
from the raw algae in both cases. However, when the extracted algae First-order model for all the tested biomasses. As can be seen, it was
were subjected to an acid pretreatment, the methane yield increased by observed an increase in the k value of 150% and 75% when the FR is
25 % (Gracilaria manilaensis) and 32 % (Gracilariopsis persica) when extracted with water (FR-W) and ethanol (FR-E), respectively,
compared with the untreated raw algae, and by 47 % (Gracilaria man compared to the value observed for unextracted FR. On the other hand,
ilaensis) and 60 % (Gracilariopsis persica) when compared with the un it was appreciated a decrease in the k value when the fresh biomass (FR)
treated extracted algae. The acid treatment increased the protein was doubly extracted first with water and then with ethanol (FR-WE)
content while maintaining the same carbohydrate and lipid fraction. and first with ethanol and then with water (FR-EW) achieving values of
This could be due to the treatment being able to loosen the cell struc 0.06 and 0.04 d− 1, respectively, in relation to the value achieved for the
tures and thus allowing for better protein extraction and subsequent
analysis. Moreover, although the carbohydrate content remained unal
tered, the acid treatment could hydrolyse these compounds into more Table 5
simple molecules and increase the soluble organic fraction, which was Values of the kinetic constant obtained from the First-order model for the
different Rugulopteryx biomasses tested. Figures after ± represent the standard
also confirmed by faster methane production due to the higher avail
deviations.
ability of easily degradable compounds. Similarly, Abomohra and
Almutairi (2020) assessed the effect on the AD process of Gracilaria Substrates Gmax (LCH4 kg−VS1) k (days− 1) R2 S.E.E. Error (%)
multipartita after agar extraction with or without acid pretreatment. FAR 116 ±2 0.09 ± 0.00 0.9977 2.32 11.5
Results showed that in both cases, the extracted biomass reported a FAR-W 139 ±7 0.05 ± 0.00 0.9956 3.08 33.6
FAR-E 138 ±3 0.07 ± 0.00 0.9979 2.40 18.9
higher methane yield, however, the biogas yield was lower. This may be
FAR-WE 171 ±3 0.07 ± 0.00 0.9983 2.76 17.1
because the extraction process reduces the lipid content (as long-chain FAR-EW 188 ±4 0.06 ± 0.00 0.9988 2.43 23.6
fatty acids), which are reported to have inhibitory activity (Salama FR 152 ± 26 0.04 ± 0.01 0.9756 7.52 68.0
et al., 2019). FR-W 121 ±2 0.10 ± 0.00 0.9957 3.29 5.2
Mhatre et al. (2019) assessed the viability of sap, ulvan and protein FR-E 137 ±6 0.07 ± 0.00 0.9912 4.78 13.2
FR-WE 173 ±4 0.06 ± 0.00 0.9982 2.60 27.2
extraction of the green alga Ulva lactuca. Similarly to the results
FR-EW 201 ± 13 0.04 ± 0.00 0.9936 5.23 47.7
observed in the present study, the methane yield after the extraction of
individual compounds and after a complete sequential treatment Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or
improved the methane yield, which could be due to the removal of ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with
ethanol and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR)
inhibitory compounds and the reduction of nitrogen, leading to a sub
extracted with water (FR-W) or ethanol (FR-E) or with water and then with
strate with a higher C/N ratio. In fact, the highest methane yield was
obtained from the sap and ulvan extracted sample, which presented the S.E.E.: Standard Error of Estimate; Gmax: Ultimate methane production; k:
highest C/N ratio and an improvement of 54 % over the untreated specific rate constant or apparent kinetic constant; R2: Determination coeffi
biomass. Similarly, the alga Sargassum muticum was subjected to a cient; Error (%): difference (in percentage) between the experimental and
sequential process for the extraction of sap by pressing and the calculated ultimate methane production.
10
Table 6 anaerobic digestion of the marine green alga Ulva lactuca from 0.15 d− 1
Values of the parameters obtained from the Modified Gompertz model for the (untreated alga) to 0.28 d− 1 when the algal biomass was previously
different Rugulopteryx biomasses studied. Figures after ± represent the standard extracted for recovery sap, ulvan and protein (Mhatre et al., 2019). The
deviations. low value found in the mentioned work was something higher than
Substrate Bm Rm ℷ R2 S.E. Error those observed for the FR and FAR control biomasses studied in the
(LCH4 (d) E. (%) present research (0.04 and 0.09 d− 1, respectively). The reported study
(kgVS⋅d)− 1)
corroborates well with the fact that high protein and sulfate content are
FR 93 ± 6.9 ± 0.3 2.7 ± 0.3 0.9941 3.79 4.1% major inhibitors in AD of Ulva lactuca and their removal leads to
2 improved kinetic constants in anaerobic digestion processes (Mhatre
− 8
FR-W 110 8.9 ± 0.7 1.4⋅10 0.9940 6.46 3.5%
±2
et al., 2019).
FR-E 120 7.2 ± 0.3 1.7⋅10− 11
0.9937 6.63 0.8% On the other hand, considerable increases in the maximum methane
±3 yield, Gm (LCH4 kg−VS1) of 32.2% and 62.1% were observed for FR and
9
FR-WE 140 7.7 ± 0.3 6.4⋅10− 0.9960 3.98 2.2% FAR biomasses when they were sequentially extracted firstly with
±3
ethanol and then with water (FR-EW and FAR-EW, respectively). In the
FR-EW 147 7.6 ± 0.3 0.37 ± 0.9957 4.36 0.8%
±4 0.03 same way, considerable increases in the Gm value from 121 (FR-W) to
FAR 103 7.5 ± 0.3 3.4⋅10− 9
0.9962 3.04 0.9% 201 (FR-EW) and 139 (FAR-W) to 188 (FAR-EW) LCH4 kg−VS1 were
±1 detected when FR and FAR biomasses were extracted only with water
9
FAR-W 108 5.5 ± 0.2 5.5⋅10− 0.9960 2.99 4.3% and doubly extracted firstly with ethanol and then with water, respec
±2
− 9 tively. These considerable increases in the ultimate methane production
FAR-E 116 7.2 ± 0.2 5.4⋅10 0.9972 2.86 0.2%
±2 values can be attributed to the significant rise in the RS extracted in the
FAR-WE 144 9.1 ± 0.3 4.1⋅10 − 9
0.9974 3.46 0.9% sequentially extracted samples compared to the samples extracted only
±2 with water (Table 3).
9
FAR-EW 152 8.8 ± 0.2 1.7⋅10− 0.9976 3.45 0.2%
These values are in accordance with the considerable increase in the
±2
Gm values reported when fresh R.o. biomass was co-digested with olive
Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or mill solid waste (OMSW, waste derived from the two-phase olive-oil
ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with manufacturing process), which is characterized by its high content in
ethanol and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR) easily fermentable compounds (De La Lama-Calvente et al., 2021). In
extracted with water (FR-W) or ethanol (FR-E) or with water and then with
this sense, Gm values of 190 ± 8, 370 ± 11 and 460 ± 15 LCH4 kg−VS1 were
achieved for 100% fresh R.o. biomass, mixture 1 R.o.-1 OMSW and
Bm: is the ultimate methane production; Rm: is the maximum methane produc
tion rate; ʎ is the lag time. S.E.E.: Standard Error of Estimate; R2: Determination mixture 1 R.o.-1.5 OMSW. This was attributed to the appropriate C/N
coefficient; Error (%): difference (in percentage) between the experimental and values of these two mixtures (23.3 and 24.9, respectively), which were
calculated ultimate methane production. very similar to the optimum C/N ratio, thus, avoiding the formation of
inhibitory substances in the process and, therefore, to facilitate the
fresh biomass extracted only with water (FR-W, 0.10 d− 1). This fact can anaerobic process of methane production (De La Lama-Calvente et al.,
be attributed to the decrease in the DPPHRSA of the doubly extracted 2021). In previous biomethanization studies, the high nitrogen content
samples which causes an increase in the inhibition power of these of the macroalgae was indicated as one of the main factors that inhibit
samples (Table 3). Moreover, the higher TPC of the FR-E and FR-WE methane production (Costa et al., 2012). Co-digestion would not only
compared to the value observed for FR-W can also explain these de help to balance the C/N ratio, but it also helps to dilute the presence of
creases in the k values. toxins and inhibitory substances for the anaerobic digestion process.
A recent research (De La Lama-Calvente et al., 2023a) has revealed Additionally, the AD of Ulva lactuca, apart from the presence of
an increase in the kinetic constant, k, of the fresh R.o. biomass of 28.9 %, inhibitory factors like high nitrogen and sulphur, showed lower methane
19.8% and 58.4 % with respect to untreated R.o. biomass when the yields in untreated biomass compared to extracted biomass which could
macroalga is subjected to a thermal pre-treatment to 90 ◦ C, 120 ◦ C and also be attributed to the tough and rigid macroalgal cell wall of
150 ◦ C, respectively. The highest value (0.26 d− 1) was achieved for the U. lactuca and the presence of indigestible polysaccharides which makes
highest temperature used in the pre-treatment (150 ◦ C), while the lowest it resistant to bacterial attacks and hence lead to poor substrate utili
value was found for the untreated macroalgal biomass (0.16 d− 1). The zation. Extraction of these products not only took away inhibitory
high constant rates indicated that higher temperatures increased the components but also fragments or deconstructs the tough and rigid
solubility of biodegradable compounds and that these are consumed in a macroalgal cell wall thus improving the amenability of biomass for rapid
very early stage, even before any inhibition can take place (De La methane production with enhanced yields (Mhatre et al., 2019).
Lama-Calvente et al., 2023a). By contrast to this, the behaviour observed
for the ashore biomasses was different, as, in this case, there was not 3.2.1.2. Modified Gompertz model. The Modified Gompertz model (Eq.
detected an important variation in the k values, which ranged between (2)) was also used for the kinetic modelling for each sample of non-
0.05 and 0.07 d− 1. As was shown in Table 3, the CODs values for these extracted and extracted R.o. biomasses both fresh and ashore. As can
ashore biomasses were lower compared to those obtained for the fresh be seen in Table 6, this kinetic model was found in close agreement with
samples (FR). the obtained cumulative CH4 production data (experimentally) with
On the other hand, the values of the kinetic constant obtained in the high determination coefficient values. As can be seen from this Table,
present research were lower than that reported for AD of the seaweed the lowest R2 value was 0.9937.
Laminaria digitata in batch mode at 35 ◦ C temperature, which was found Similarly to that observed in the first-order model the ultimate
to be 0.45 days− 1 (Membere and Sallis, 2018). This macroalga had a TS methane production, Bm (LCH4 kg−VS1) increased for the extracted bio
content of approximately 94%, and a VS content of content of about masses both fresh and ashore compared to the unextracted samples (FR
65%, giving a fairly high VS/TS ratio of 0.69, and indicating mostly and FAR). Specifically, a rise of 58.1% and 47.5% was observed when
organic digestible matter in the feed. Its C/N ratio was 21.6 which is the biomasses were sequentially extracted firstly with ethanol and then
close to the optimal range (25–30) for a stable AD performance with water, FR-EW and FAR-EW, in relation to the values found for
(Membere and Sallis, 2018). Also, from a comparative point of view, it unextracted biomasses, FR and FAR, respectively. This corroborates
was reported an increase in the first-order kinetic constant of the batch well with the fact that high phenolic contents are major inhibitors in
11
anaerobic digestion of fresh and ashore Rugulopteryx biomasses and their 3.3. Digestates valorization into biofertilizers
removal leads to improved methane yields. Sequential extraction of
these value-added products prior to the anaerobic process not only im The presence of heavy metals in marine macroalgae has been pointed
proves biomass amenability and respective methane yields but also out as being one of the main drawbacks when these species have been
makes the overall anaerobic process more efficient and viable, as was used directly as fertilizer (Rakib et al., 2021). As these elements remain
also reported by Mhatre et al. (2019). present in the digestate generated after the AD process, they may be a
A decrease in Rm value from 8.9 to 7.6 LCH4 (kgVS⋅d)− 1 was observed critical factor that needs to be seriously considered prior its use as bio
when the FR biomass was doubly extracted firstly with ethanol and then fertilizers. Several reports have pointed that the separation of the
water (FR-EW) compared with the FR biomass extracted only with water digestate into their solid and liquid fraction could benefit its application
(FR-W). This diminution can be attributed to the higher TPC reached in as fertilizer or soil amender (Fernández-Rodríguez et al., 2021). Table S4
the doubly extracted biomass as can be seen in Table 3. In the case of the and table S5 of the supplementary material show the elemental analysis
FAR biomasses, the lowest Rm was found for FAR-W, which achieved a of the liquid and solid fraction, respectively, of the resulting digestates
value of 5.5 LCH4 (kgVS⋅d)− 1. This fact can be explained by the higher from this study. The maximum allowable content of heavy metals in
FRAP observed for this simple extracted biomass (1.50 mM Trolox eq.) fertilizers or soil amenders depends on local regulations. In Europe the
(Table 3). main framework is detailed in the Regulation (EU) 2019/1009. This
Similar Rm values (8.6 LCH4 (kgVS⋅d)− 1) to those achieved in the regulation stablishes all the limits and conditions that an anaerobic
present study were reported by Zara et al. (2021) during the anaerobic digestate may comply with in order to be used as a fertilizer or soil
co-digestion of dairy raw materials and Ulva sp. macroalga. A mineral amender. Following this Regulation (EU), and according to the reported
additive medium based on bicarbonate compound was added in this case results, most of the digestate’ liquid fractions are susceptible to be used
to optimize methane yield. Rm values of the same order of magnitude directly as organic fertilizer (i.e. FAR, FAR-W, FAR-E, FAR-EW, FR,
(9.0 LCH4 (kgVS⋅d)− 1) as those obtained in the present research were also FR-W, FR-WE). The others (i.e. FAE-WE, FR-E and FR-EW) presented
obtained by El Nemr et al. (2021) in the anaerobic digestion of the Hg concentrations higher than the allowed limit (1 ppm). Although the
macroalgae Ulva intestinalis. This high value was reduced to 2.1 LCH4 other regulated contaminants (Cd, Cr VI, Ni, Pb, As, Cu and Zn) were
(kgVS⋅d)− 1 when the macroalga was subjected to a sonication below the established limits. Similarly, in a previous study, De la
pre-treatment for 30 min (El Nemr et al., 2021). By contrast, higher Rm Lama-Calvente et al. (2023b) found that only Hg was off-limit when
values (14.6 LCH4 (kgVS⋅d)− 1) to those achieved in the present research assessing the digestate’ liquid fractions derived from the AD process of
were reached by Castro et al. (2022) during anaerobic co-digestion of the R.o. However, the Hg content found in the previous study (19 ± 4
Sargassum spp. and food waste at a ratio of 55%:45%, respectively. ppm) was significantly higher than that obtained in the present inves
Having the Sargassum spp. as part of a feedstock matrix with food waste tigation (0.39 ± 0.01–1.08 ± 0.01 ppm). This could be due to the sea
can provide the versatility required for feedstocks that are not stable in sonal or spatial variation of Hg in macroalgae (Ferreira, 1991). In fact,
their composition. Higher protein and fat content in food waste although results from the present study showed that all the digestate’
compared to Sargassum spp. biomass enriches the media for more solid fractions would need further treatment in order to reduce the high
balanced C/N in a carbohydrate-rich feedstock like Sargassum spp. Hg (>1 ppm) content, in all the studied cases the Hg content were just
(Castro et al., 2022). slightly above the limit (<1.3 ppm). In any case, further treatments of
For the fresh biomasses (FR), a considerable decrease in the ℷ value the digestate should be needed in order to reduce the Hg content and
was achieved for the extracted biomasses compared to the un-extracted thus be allowed for use. These may be solved by removing the heavy
FR in all cases except for FR-EW for which similar values were observed. metals from the algae biomass before its use in the anaerobic digester
These values were very similar to those obtained in the AD of the (Nkemka and Murto, 2010), although, the most widely used methods for
seaweed Ecklonia maxima with and without acclimatized inoculum heavy metals removal in wastewater are bio-adsorbents (Rakib et al.,
under mesophilic conditions (37 ◦ C) for a digestion period of 20 days 2021).
(Darko et al., 2022). Moreover, the lower ℷ values in the extracted FR
biomasses also revealed that the microbes survived better for these 3.4. Environmental and economic impact of a biorefinery scenario
substrates and, as such, commenced methane production on the first
day. In addition, in the present study, the presence of a lower TPC, and In this study, several biorefinery scenarios have been assessed
other inhibitory substances might have led to an increased microbial comprehensively using laboratory-scale methodologies. An economic
activity resulting in shorter lag phases and maximum Rm for doubly and environmental impact based on lab-scale studies may lack of proper
extracted samples compared to extracted biomass only with water or accuracy, however, they may provide with sufficient insights to confi
ethanol. This fact was more pronounced in the case of the FAR dently propose future research and to scale up the system. Section 4 of
biomasses. the supplementary material includes all the details of the economic
Furthermore, the lag phase values obtained in the present study were assessment carried out for the biorefinery scenarios studied.
always much lower than that reported for AD of Sargassum plagiophyllum Although economic assessments are highly susceptible to market
under deionized water and saline water, which were determined around variations, comparisons based on up-to-date prices could provide valu
7.4 and 8.3 days, respectively (Farobie et al., 2023). By contrast, these able information. The most relevant outcome of the economic assess
reported lag phase periods were comparable with the previous work of ment was that the non-extracted (FR and FAR) and single aqueous
Pardilhó et al. (2022), who calculated the lag phase period of 6.6 and 9.8 extraction (FR-W and FAR-W) scenarios were economically viable
days for AD of marine macroalgae waste at different total solid contents without the necessity to sell the TPC or RS extracted. However, if the
of 0.9 and 1.7. In the same way, Allen et al. (2013) reported the lag selling price of TPC or RS is sufficiently high, then the scenario where
phase for Ulva lactuca from 0.4 to 6.3 days according to the harvesting the biomass has been extracted sequentially (FR-WE; FR-EW; FAR-WE;
year and the pre-treatment applied (dried, fresh, wilted or washed). In FAR-EW) could be the most profitable option. This suggests that an
any case, these values were always much higher than those achieved in industrial-scale biorefinery system for the comprehensive valorization
the present study for the extracted biomasses, which reveals the higher of R. okamurae should be built with sufficient robustness to adapt to
biodegradability of the Rugulopteryx biomass after the extraction pro potential fluctuations of by-product prices.
cesses with water and ethanol. Invasive alien species (IAS), such as R. okamurae, present a major
threat to the global biodiversity, forestry, livelihood and human health
(Rai et al., 2023). In fact, the presence of R. okamurae in the Mediter
ranean coasts has provoked the displacement of a great part of the local
12
flora and fauna, and, furthermore, the decomposition of the ashored other compounds, such as oligosaccharides, pigments, etc., not being
biomass on the beaches produced uncontrolled greenhouse gases, such analyzed.
as CO2 and CH4, and unpleasant odours, most likely due to the formation Regarding the biomethanization of the exhausted solid residue,
of SH2. Globally, there are a great number of IAS cases that have pro double extraction process enhanced the methane yield when compared
voked similar problems, for example, the alga Sargassum sp. in the with non-extracted or single-extracted biomass. In fact, according to the
Caribbean (Lopresto et al., 2022), the plants Tithonia diverifolia (Rai first-order model, a decrease in the k value for the doubly extracted
et al., 2023) and Eichhornia crassipes (Nega et al., 2024) in Africa, or even biomass firstly with water and then ethanol (FR-WE) (0.06 d− 1) and the
animals, such as the European green crab (Carcinus maenas) in North doubly extracted firstly with ethanol and then water FR-EW (0.04 d− 1)
America and Australia (Zhang et al., 2022). However, while just in in relation to the value achieved for the biomass only extracted with
Europe there are 88 IAS (47 animals and 41 plants) catalogued as of water (FR-W) (0.10 d− 1) was obtained. From the modified Gompertz
Union Concern (Regulation (EU) 1143/2014), only some of them have model, a decrease in Rm value from 8.9 to 7.6 LCH4 (kgVS⋅d)− 1 was
been extensively studied. Hence, more efforts should be put into observed when passing from FR-W to FR-EW. Finally, no significant
covering diverse IAS and to investigate their invasion biology, envi differences were observed between the natural alga and the ashored
ronmental implications, epidemiology, and health effects. The eradica biomass, and the sequential extraction order. This suggests that the
tion of these IAS from the ecosystem should be the priority, however, sequential extraction process could reduce the impact on the AD process
this may be beyond the limits of current technology and knowledge, as it due to compositional variations between biomass batches.
requires intensive manpower and provoke collateral contamination due This study represents the first integral approach regarding the use of
to the use of chemicals (Rai et al., 2023). In this sense, a biorefinery R. okamurae as a feedstock in a comprehensive biorefinery scenario.
system embedded in a circular economy model is presented as the most However, the conclusions derived from this work may be subjected to
feasible scenario that allows to mitigate the environmental problem further investigations. The major drawback may be the scale of the test
associated with the uncontrolled spread of these IAE. carried out. The scale-up of the extraction and AD processes may present
AD is a well-known technology that has been widely used in different different issues not observed or even considered in this study. For
regions of the world. A recent study revealed that AD technology has a instance, the continuous feed of the assessed macroalga into an anaer
significant impact in terms of economic, environmental and social per obic digester would affect the microbial profile of the inoculum signif
spectives. Indeed, AD contribute to the alleviation of poverty, the icantly, and to some extent, the accumulation of certain compounds
strengthening of economic growth, the improvement of agricultural (such as heavy metals) could inhibit the methane production. Addi
efficiency, the promotion of renewable energy usage, the reduction of tionally, the seasonal and availability variations of the R. okamurae
reliance on fossil fuels, the fostering of inclusive education and gender biomass and its impact on either added-value compounds extraction and
equality, the combating of climate change, the transformation of cities subsequent biomethanization need to be further investigated from its
into sustainable and harmonious environments, and the reduction of technical and economic viability.
environmental pollution (Piadeh et al., 2024). However, the AD process
should be improved to offer better results and significantly reduce the Funding
environmental impact. For example, emissions from transportation,
plant construction, and electricity usage have a negative effect on global The authors wish to express their gratitude to the Spanish Ministry of
warming (Özen-Daş et al., 2024). In the present work, the non-extracted Science and Innovation for providing financial support through the
biomass (FR and FAR) systems appear to be the best scenarios in terms of project PID2020-114975RB-100/AEI/10.13039/5,011,000/11,033. M.
environmental impact. This is primarily due to the utilization of water J. F-R is recipient of a “Juan de la Cierva Formación” grant from the
and ethanol during the extraction process, which will increase the global Spanish State Research Agency (FJC2020-045654-I).
warming potential of the entire system (due to the production, trans
portation and management of the solvents, mainly). Furthermore, it Ethics approval
could potentially pollute the environment as it would generate addi
tional wastewater. The authors declare that in order to fulfil the present review no
From these perspectives, a full-scale biorefinery system should be ethical approval by any organization was needed.
designed taking into consideration these two aspects. If the main goal of
a project is focused on monetary profitability, then a comprehensive
Consent to participate
valorization of the macroalgae based on the extraction of compounds of
commercial interest and the subsequent energetic conversion of the
The authors declare that all the authors mutually agree on this
extracted residue is highly suggested. However, if the objective of a
submission to Science of the Total Environment.
proposal is to reduce the ecological impact associated with the appear
ance of the invasive species R. okamurae, then, the direct utilization of
the untreated biomass in an anaerobic digester may be the most viable Consent for publication
scenario.
The publication of this work is approved by all authors and by the
4. Conclusions responsible authorities of the institution at which the work was carried
out.
This study successfully attempts for the first time to comprehensively
include the invasive algae Rugulopteryx okamurae in a circular man Availability of data and material
agement system where valuable compounds (reducing sugars (RS) and
total phenolic compounds (TPC)) are extracted from the biomass and the All the data and material used to produce the present review is well
exhausted residue is transformed into biogas and a digestate with fer defined and accessible within the text. However, the authors declare that
tilizer potential. any further information would be provided upon request to the corre
Although ethanolic extracts presented higher yields of TPC, aqueous sponding author.
extracts showed higher affinity for RS and for other compounds with
higher antioxidant capacity. These findings could suggest that some Code availability
phenolic compounds with high antioxidant capacity present higher af
finity to water than to ethanol, or that this antioxidant capacity is due to Not applicable.
13
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Journal of Environmental Management: Rugulopteryx Okamurae

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Journal of Environmental Management 370 (2024) 122504

Contents lists available at ScienceDirect

Journal of Environmental Management

Optimizing a biorefinery design for the valorization of Rugulopteryx

1. Introduction global production surge to approximately 24 million tonnes in 2022

Fig. 1. Schematic design of the biorefinery conceptualization carried out.

FR-EW 7.90 ± 3780 ± 50 b, 660 ± 10 a,b

VS (% TS) 95 ± 8a,b 95 ± 9a,c 100 ± 0c 70 ± 20a,b,c 80 ± 20a,b,c 81 ± 8a,b 99 ± 2a,b,c 90 ± 10 b

2.6.3. Statistical analysis

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