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Process optimization of the extraction of reducing sugars and total

phenolic compounds from the invasive alga Rugulopteryx
okamurae by response surface methodology (RSM)

David De la Lama-Calvente, María José Fernández-Rodríguez,

Antonio Garrido-Fernández, Rafael Borja
PII: S2211-9264(24)00112-7
DOI: https://doi.org/10.1016/j.algal.2024.103500
Reference: ALGAL 103500

To appear in: Algal Research

Received date: 2 February 2024

Revised date: 19 March 2024
Accepted date: 6 April 2024
Please cite this article as: D. De la Lama-Calvente, M.J. Fernández-Rodríguez, A.
GarridoFernández, et al., Process optimization of the extraction of reducing sugars and
total phenolic compounds from the invasive alga Rugulopteryx okamurae by response
surface methodology (RSM), Algal Research (2023),
https://doi.org/10.1016/j.algal.2024.103500

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© 2024 Published by Elsevier B.V.

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Process optimization of the extraction of reducing sugars and total phenolic

compounds from the invasive alga Rugulopteryx okamurae by response surface

methodology (RSM)

David De la Lama-Calventea*, María José Fernández-Rodríguezb, Antonio Garrido-

Fernándeza, Rafael Borjaa

a
Spanish National Research Council (CSIC) - Instituto de la Grasa (IG), Department

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of Food Biotechnology, Campus Universidad Pablo de Olavide, Edificio 46. Ctra. de

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Utrera, km 1, 41013 Seville, Spain.

Department of Vegetal Biology and Ecology, Faculty of Biology, University of

* corresponding author: dlama@ig.csic.es

Seville, 41080 Seville, Spain.

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Abstract

The brown marine alga Rugulopteryx okamurae (R. okamurae) has quickly spread

through the Mediterranean coasts since it was firstly spotted in the area in 2015. This

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alien species has already colonized a great part of the Mediterranean western coasts

and displaced the local biota, disrupting the natural and autochthonous marine

ecosystem. Moreover, the related ‘green’ tides also provoke environmental problems

ashore that need to be addressed. Comprehending the potential of this unwanted

biomass could be of vital importance to lessen its ecological impact. In this study, the

ashore R. okamurae was assessed for its potential as raw material for the extraction of

reducing sugars and polyphenolic compounds with antioxidant capacities. The

extraction process was evaluated and optimized using the response to surface

methodology (RSM) combined with a modified Box-Behnken design. Initially, 2 key

operational factors were identified as the most influential parameters (i.e. drying

temperature and milling time). However, the type of solvent and the use of zeolite were

included in the design as factors that potentially could have a significant impact on the

process. The analyzed responses were the extraction yields for total phenolic

compounds (TPC) and reducing sugars (RS). For the extraction of TPC the optimized

methodology was based on the use of ethanol as solvent, a drying temperature of 60

ºC and a zeolite-assisted milling time of 60 s, reaching a maximum yield of 15.33 g gallic acid

kg-1. For the extraction of RS the optimized methodology was based on the use of water

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as solvent, a drying temperature of 100 ºC and a zeolite-absence milling time of 50s,

reaching a maximum yield of 10.42 gglucose kg-1.

Keywords: Biorefinery; Rugulopteryx okamurae; Process optimization; Total

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phenolic compounds; Reducing sugars; Response surface methodology.

1. Introduction

The increasing environmental problems related to anthropogenic activities have

led the scientific communities towards the search for new methods to provide

sustainable petroleum-like products aiming to lessen the associated environmental

impact. Moreover, the global demand for nutraceuticals, functional foods or cosmetics

based on natural ingredients has also increased, pushing further the exploration of

novel natural sources (Cassani et al., 2024). Third-generation biomasses, such as algae,

have drawn attention in the last years due to the significant content of bioactive

compounds like dietary fiber, fatty acids, polyphenols, terpenoids, polysaccharides and

minerals (Zhang et al., 2023).

While the commercialization of polysaccharides (e.g. alginates) from algae has led

this industry, the market for other metabolites (e.g. phenolic compounds such as

phlorotannins) is currently growing (Thiyagarasaiyar et al., 2020). Furthermore, the

biorefinery conceptualization applied to these biomasses has proven their potential as

feedstocks to provide the above-mentioned biocompounds, produce bio-fuels (e.g.

bioethanol, biomethane,…) and capture CO2 (Johnston et al., 2023). In these scenarios,

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the use of naturally produced biomasses that are considered invasive and harmful to

the ecosystem presents a promising opportunity. This is the case with the invasive alien

species Rugulopteryx okamurae (R. okamurae). A brown macroalga that has colonized

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most of the southern coasts of Spain in recent years and is spreading through the

Mediterranean Sea uncontrolled (El Madany et al., 2024; Terradas-Fernández et al.,

2023).

Earlier experiments carried out by Kurata et al. (1988) showed that the use of

alcoholic solvents, such as methanol, was able to extract spatane-type diterpenes with

feeding-deterrent activity from the alga R. okamurae. Later, the ester ethyl acetate was

used to recover a newly isolated secospastane namely dilkamural, and acetone was

used for two novel diterpenoids with feeding-deterrent activity (Ninomiya et al., 1999;

Suzuki et al., 2002). More recently, the ethanolic extract of R. okamurae was tested for

the αglucosidase inhibitory activity, showing an inhibition of 90.9 ± 1.0 % and placing

this alga at the fourth highest position when compared with other 19 species (Jeong et

al., 2012).

To the best of our knowledge, only the above-mentioned studies dealt with the

extraction of active compounds and metabolites from the phaeophyte R. okamurae

prior to its appearance on the coast of Ceuta and its fast spread through the

Mediterranean Sea. Since then, the number of research articles has increased

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exponentially. For instance, Casal-Porras et al. (2021) used an acetone/methanol (1:1

v/v) mixture followed by diethyl ether to extract secondary metabolites (such as

Dilkamural) and tested their feeding-deterrent activity. Using the same extraction

protocol, more than ten new diterpenoids were isolated, among which some showed

strong anti-inflammatory behaviour (Cuevas et al., 2021; Cuevas et al., 2023).

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Furthermore, it has been reported that while the ethanolic (H 2O:EtOH 1:4) and the

methanolic (H2O:MeOH 1:4) extracts of R. okamurae presented some inhibition to

human pathogens (Staphylococcus aureus and Cutibacterium acnes), with no significant

differences between them, this effect was not observed with fish pathogens (Vega et

al., 2023). Moreover, the aqueous extract showed no inhibition to either human or fish

pathogens (Vega et al., 2023). However, the aqueous extract obtained by

microwaveassisted hydrothermal (MAH) processing showed a 53 to 65 % (depending

on the temperature applied) inhibitory efficacy to cell growth in tumoral cell line HeLa

229 (Ferreira-Anta et al., 2023). Additionally, this MAH method allowed for the

extraction of alginate (3.2 % recovery) and phenolic compounds (Ferreira-Anta et al.,

2023). Similarly, microwave hydrodiffusion and gravity treatment enable the recovery

of a gelling fraction and antioxidants (López-Hortas et al., 2023).

All these evidences proved the potential of R. okamurae to provide bioactive

compounds. However, using chemicals (e.g. MeOH or diethyl ether) or temperature

during the extraction process may dissuade scaling up to a biorefinery industry due to

the high cost associated. Moreover, the different variables affecting the extraction
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efficiency have not been fully assessed through a comprehensive and systematic

methodology. Thus, an economical and efficient method is yet to be established and

proposed as a viable alternative. This research attempts to comprehensively and

systematically understand the potential of the alga Rugulopteryx okamurae as a

feedstock for extracting added-value compounds. Specifically, phenolic and reducing

sugar compounds. The extraction process was evaluated using economical

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methodologies (i.e. water and ethanol solvents at room temperature) and essential

factors (i.e. drying temperature, milling time, effect of zeolite) and optimized using

response surface methodology (RSM).

2. Materials and Methods

2.1 Analytical methods

For the analysis of soluble parameters, samples were previously centrifugated

(9000 rpm, 10 min) and filtrated (Whatman, 1.2 µm pore size glass fiber filter). All the

analyses were carried out at least in triplicates. The standard methods 2540B & 2540E

were followed in order to obtain the total solids (TS), volatile solids (VS), and mineral

solids (MS) fraction (APHA, 2017). Total (CODt) and soluble (CODs) chemical oxygen

demand were determined by following the indications in Raposo et al. (2008) and the

standard method 5220D (APHA, 2017), respectively. pH and Total Alkalinity (TA) were

determined by using a pH meter model Crison and by titration to pH 4.3 as shown in

the 2320B standard method for TA (APHA, 2017). Total ammonia nitrogen (TAN) was

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performed by distillation and titration following the standard method 4500-NH 3 (APHA,

2017). The milling process was carried out by using a commercial blender (120 W,

Aromatic FP905S,

Taurus, Spain).
The following methods were carried out spectrophotometrically in a 96-well

microplate using a Microplate Multiscan reader (Bio-RAD, iMark TM). Total phenolic

content (TPC) was determined by carrying out the Folin-Ciocalteau method using gallic

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acid (Sigma-Aldrich) as standard and measuring the absorbance at 750 nm (Magalhães

et al., 2010; Zhang et al., 2006). Reducing sugar content (RS) was determined by the

Anthrone method using glucose (PanReac) as standard and measuring the absorbance

at 630 nm (Leyva et al., 2008).

2.2 Seaweed

The marine algal biomass of Rugulopteryx okamurae was provided by the

Laboratory of Marine Biology of the University of Seville and collected from the

Algeciras coast (36º03’0’’N 5º29’7’’W - 36º03’7’’N 5º14’8’’W). Samples consisted of

fresh ashore R. okamurae (FAR) immediately collected once the loosened biomass

reached the beach. Samples were washed in situ with seawater to eliminate debris and

stored at -20 ºC upon their arrival at the laboratories. Before further use, samples were

thawed at 4 ºC for 2 h and washed again with deionized water in order to eliminate the

interference of marine salts during the experiments. Fresh R. okamurae (FR), which was

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directly collected from its natural habitat, was also used to confirm the optimized

extraction method.

2.3 Experimental design for added-value compounds extraction methodology

Based on preliminary experiments, 3 variables were identified as the main factors

influencing the extraction of the targeted compounds (levels included in brackets): 1.-

Solvent (water; ethanol); 2.- Drying temperature (0 ºC; 60 ºC; 100 ºC); and 3.- Milling

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time (5 s; 10 s; 20 s; 30 s; 60 s). Additionally, a previous study showed that the use of

zeolite during the milling process of the alga R. okamurae reduced significantly the

crystallinity index of the cellulose present in the cell wall, allowing a higher methane

yield due to easier access to the hydrolytic enzymes released by the microbial

communities involved in the anaerobic digestion process (De la Lama-Calvente et al.,

2023). This behaviour could also facilitate as well the release and extraction of the

targeted compounds. On the other hand, zeolites could act as a catalyst to reduce the

TPC content, and could also influence the RS content (Orege et al., 2022) . For these

reasons, it was included as a fourth variable in the optimization process to elucidate if

this material had any effect on the extraction of the targeted compounds (4.- Use or not

of zeolite during the milling process). The design (summarized in Figure 1) consisted of

a user-defined randomized response surface (Table 1). The responses were the RS

content of the extracted fraction (for the analysis, transform square root of

concentration, as recommended by the Box-Cox plot for power transformation) or the

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extracted TPC (for the analysis, the inverse of the square root). The experiment

included 223 and 248 randomized combinations of the control variables for the

extraction with water and ethanol, respectively. Table 2 reproduces all the randomized

tests carried out and the associated responses. Considering the levels of the continuous

variables the data could then be fit as up to a cubic model (Table 3).

Initially, the data were subjected to a sequential model sum of squares (Type I),

which sequentially evaluated the significance of the ampliation of the linear model to

two-way interactions, quadratic and cubic terms. The significant model with a higher

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level of non-aliased terms is then suggested. This previous analysis detected that the

cubic model was aliased. Consequently, the following analysis was adapted up to only

quadratic models for both RS and TPC (Table 3). Then, an ANOVA (Type III-Partial sum

of squares) for the suggested model was performed, and the significant terms (and

those necessary for maintaining the hierarchical character of the model) were retained.

During the stepwise selection process, the criteria for entering and removing variables

were set at p ≤ 0.10. The model’s ANOVA also provided the model fit significance and

lack of fit. Other parameters, such as adjusted R-square, precision, or standard errors of

coefficients, were also examined to assess the model quality. The equations, in terms of

actual components (in g kg-1VS), were deduced and plotted as a response surface to

facilitate the interpretation of the models’ outcomes. Besides, the interactions were

also evaluated to assess the effects regarding the levels of the others. Finally, a

numerical and graphical optimization was studied to deduce the conditions for
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maximum yield. At this point, the desirability function was considered for factor

optimization. This function is based on the transformation of all the obtained responses

from different scales into a scale-free value, thus, its value oscillates between 0 and 1.

Being 0 a representation of an undesirable response and 1 the optimal performance of

the studied factor (Amdoun

et al., 2018).
2.4 Data analysis

Analytical tests were at least carried out in triplicates and values are given by

means ± standard deviations. Where corresponded, a two-tale Student's t-test and the

oneway analysis of variance (ANOVA) were carried out. Additionally, the Tukey’s test

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was performed as a post-hoc analysis. For the purposes of data discussion, a

confidence level of α = 0.05 was accepted as not statistically significant, except when

otherwise specified. All the statistical analyses were carried out by SPSS Statistics

software (v29, IBM, Business Machines Corp.). The optimization analysis was conducted

using DesignExpert

v.13.0.15.0 (Stat-Easy, Inc., MN, USA).

3. Results and discussion

3.1 Total phenolic compounds extraction

For the TPC, the sequential sum of squares suggested a quadratic model by which

only significant terms (p < 0.05) were included (Table 3). The transformation applied in

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this case, as suggested by the Box-Cox plot for power, was the inverse of the square

root of each value. The different terms were highly significant (Table 4). The variance

inflation factor (VIF) values were close to one, implying no multiple collinearity.

Additionally, the adjusted and predicted R 2 were 0.9068 and 0.9031, respectively. They

were, then, very similar and indicated good predictive power for biological samples

(Lundstedt et al., 1998). Finally, the precision was rather high (52.70). Furthermore, the

normal plot of the externally studentised residual was also in reasonable agreement

with a typical distribution. As a result, the model was appropriate to navigate through

the region of the experimental design.

In addition, the adequate precision of the model (33.43), which measures the

signal to noise ratio, was also highly above the established limit (4). As a result, the

model was appropriate to navigate through the region of the experimental design

space.

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Solvent: Water; Zeolite: Used

1 = 0.84650 + 0.000035𝐴 − 0.005247𝐵 + 0.000023𝐵2 (Eq. 1)

√𝑦

Solvent: Water; Zeolite: Not Used

1 = 0.854438 − 0.001758𝐴 − 0.004684𝐵 + 0.000023𝐵2 (Eq. 2)

√𝑦

Solvent: Ethanol; Zeolite: Used

1 = 0.464509 − 0.001207𝐴 − 0.003290𝐵 + 0.000023𝐵2 (Eq. 3)

√𝑦

Solvent: Ethanol; Zeolite: Not Used

1 = 0.528647 − 0.00300𝐴 − 0.002728𝐵 + 0.000023𝐵2 (Eq. 4)

√𝑦

Where: A is related to the term milling time; B to drying temperature

The equations, in terms of the initial units, were as follows: i)

ii)

iii)

iv)

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The equations did not have significant time-square term. In its absence and

bearing in mind the relatively low coefficient for temperature squared, the model

should resemble almost a plane. However, despite this circumstance, its interpretation

without plotting is challenging. Figure 2 and Figure 3 illustrate the response surface

obtained for the phenols extracted with water and ethanol, respectively. First, the

concentrations for the extraction with water (Fig. 2, A and B) are relatively low, with

unappreciable effects of temperature, milling time and zeolite presence. On the

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contrary, the extraction with ethanol (Fig. 3, A and B) yielded higher concentrations and

showed an incline hill with the crest around half the interval of temperature (45-65 ºC

approx.) with a peak at the highest milling period. The presence of zeolite slightly

increases the concentrations at the lowest milling times. As a result, extraction with

water, except for economic reasons, would not be adequate, and the incorporation of

zeolite would be slightly beneficial at the longer milling period.

The study of interactions may help to illustrate the effects of the variables in the

presence of the others. In the case of nominal variables (i.e. solvent and presence of

zeolite) it would be necessary to extend the study to the four levels of combinations

(Figure 4). Using water as a solvent, the milling time has no practical effect and drying

at 100 ºC only increased the extracted phenols in a very low proportion (Fig. 4, A and

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B). Furthermore, the incorporation of zeolite (Fig. 4 B vs Fig. 4 A) did not produce any

appreciable improvement, according to the graphs. On the contrary, when the solvent

was ethanol, the extraction led to higher yields, which increased to about double as the

milling time increased and the temperature reached 100 ºC, obtaining a peak at a

milling time of 60 s. The incorporation of zeolite, more than increasing the extraction,

decreased the variability in the responses. Finally, the optimization process was

relatively simple since the peak of phenol extraction followed an increasing trend at

approximately 60 ºC as the milling time was longer, reaching the maximum at the

highest milling time (60 s). At this point, desirability (Figure 5) was maximum (1.00), as

well as the extraction yield (15.33 ggallic acid kg-1).

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These combinations clearly showed that the use of ethanol was one of the most

influential factors, as it appears as the unique option across the board. This was in

agreement with other studies showing that alcoholic solvents (such as ethanol or

methanol) present higher affinity to phenolic compounds than aqueous solutions (Vega

et al., 2023). Similarly, the highest milling time (60 s) was selected as the best option.

Although this is the first time that the effect of milling time on the release of phenolic

compounds is assessed on marine seaweed, this relation is well known in other

biomasses, such as microalgae. Loureiro et al. (2023) reported that the cell disruption

of the microalgae Coelastrella sp. was maximum by bead milling using a time interval of

6 min, above which no significant effect was observed. This system allowed for a

phenolic extraction yield similar to that obtained by high-speed homogenization and

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higher than those obtained by microwave or sonication (Loureiro et al., 2023). On a

different study, the phenolic profile of milled hulless barley was determined and linked

to the milling time. It was observed that the highest free and bound phenolic content

was found in the lesser milled fraction, in fact, several phenolic compounds (e.g. p-

cumaric, ferulic and sinapic acids) were not found in any other fraction (Xia et al.,

2022).

The other two factors, i.e. the use of zeolite during the milling process and the

drying temperature, showed a high correlation. Zeolite can be used as an abrasive

during the milling process diminishing the crystallinity of cellulose, a main component

of the cell wall, and thus increasing the cell wall disruption and improving the release of

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organic matter, such as phenolic compounds (De la Lama-Calvente et al., 2023). This

supports the finding of higher total phenolic compounds’ yield when zeolite is used.

However, the necessity of higher temperatures during the drying process suggests that

the zeolite, a porous aluminosilicate, may also act as an adsorbent and thus hinder the

phenolic release, so higher temperatures are needed during the drying step to assure a

higher disengagement of phenolic compounds. This is also in agreement with other

studies, for example, it has been demonstrated that increasing temperatures (from 50

to 70 ºC) improved the phenolic content of avocado powder produced by foam-mat

drying (Santos et al., 2023). However, the relationship of total phenolic compounds’

release and drying temperature is not always direct, and the biomass characteristics

performs a significant role. In fact, when the seaweed Caulerpa lentillifera was dried at
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different temperatures (40 - 80 ºC), the highest phenolic content was obtained at 50 ºC,

which diminished as the temperature rose (Fakhrulddin et al., 2022). The TPC values

obtained in the present study (1.32 ± 0.02 - 12.4 ± 0.7 g gallic acid kg-1VS) were in the same

range of other brown seaweeds (0.2 – 23.5 ggallic acid kgdw) (Barciela et al., 2023;

Getachew et al., 2020). Although, they were slightly lower than those previously

reported for the alga R. okamurae using a mixture of H2O:EtOH (1:1) as solvent (≈ 20 g

kg-1dw) (Vega et al., 2023). However, when the alga R. okamurae was subjected to a

hydrothermal pretreatment by microwave irradiation (220 ºC during 20 min) the

maximum TPC yield (0.034 ggallic acid kg1VS) was significantly lower than the reported in

the present study (Fernández-Medina et al., 2022).

3.2 Reducing sugars extraction

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For the reducing sugars’ recovery, the sequential sum of squares suggested a

quadratic model (Table 3) where only significant terms with a p-value < 0.05 were

included plus those needed to maintain the model hierarchy. The quadratic model was

significant at p < 0.0001 (Table 4) and had an adjusted and predicted R 2 of 0.9083 and

0.9036, respectively, which were significantly above the value to accept the accuracy of

the model for biological samples (0.7) (Lundstedt et al., 1998). However, it exhibited a

significant lack of fit, which is not desirable. Nevertheless, the precision was 50.54, far

above the value of 4, considered convenient, indicating that the noise to signal ratio

was adequate.

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All the retained terms were significant except D-D (Table 4), which was introduced

for maintaining the hierarchical character of the model and was the only one to include

zero within its confidence limits. The coefficients mean the change in response per unit

change in factor when the remaining ones are unchanged. Additionally, the VIFs are

very close to one, indicating the absence of multiple collinearity. The equations in

terms of actual units were as follows:

i) Solvent: Water; Zeolite: Used

(Eq. 5)

ii) Solvent: Water; Zeolite: Not Used

(Eq. 6)

iii) Solvent: Ethanol; Zeolite: Used

iv)
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Solvent: Ethanol; Zeolite: Not Used
(Eq. 7)

(Eq. 8)

Where: A is related to the term milling time; B to drying temperature

The equations indicate that the only terms changing among the different

combinations of the nominal variables were the intercept and the linear terms, while

the quadratic terms remained similar. Thus, the surfaces for the four combinations

were parallel and preserve their curvature. In any case, their interpretation is visualised

better using response to surface plots (Figure 6 and Figure 7). Using water as solvent

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(Fig. 6), zeolite had a scarce effect since its absence only produced a slight increase in

the sugar yield at 100 ºC and the highest milling time (Fig. 6 B). When ethanol was used

as solvent (Fig. 7), the surfaces were markedly below those obtained using water, and

the effect of temperature was low but opposed to that observed in water (slightly

higher yield at low temperature).

In any case, the effect of time of milling was hardly identifiable in both solvents,

except in water at a high temperature in the absence of zeolite. However, the effects of

the quantitative and nominal variables could be better illustrated through the

interaction plots (Figure 8). Fig. 8 illustrates the effect of the temperature (100 ºC vs 0

º
C) on the RS extraction using water. The yield was higher at high temperatures than at

zero degrees. The extraction in the absence of zeolite slightly increases the sugar

recuperation. With ethanol, the sugar recuperation was fairly low, being at high

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temperatures even lower than at 0 oC, but the zeolite had a similar trend than in water.

Therefore, Fig. 8 showed clearly that water was a better solvent than ethanol for the RS

extraction and that zeolite slightly increased the sugar extraction yield at 100 ºC and 60

s milling time.

This result was expected, as it has been widely reported in the literature that RS

present higher affinity to aqueous solutions. For example, when the terrestrial plant

Vernonia amygdalina was extracted with methanol, ethanol and aqueous solutions,

sugars were only found in the aqueous fraction (Hussen & Endalew, 2023). Similarly, the

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RS extraction of the blue-green microalgae Oscillatoria limosa was more efficient with

water than with methanol or ethanol, however, acetone showed the highest affinity

(Tunio et al., 2022).

The use of zeolite during the milling process enhances the crystallinity reduction of

cellulose and the release of bioactive compounds from the inner cell due to the

occasioned cell wall disruption (De la Lama-Calvente et al., 2023). However, results

from this study plead for the absence of zeolite during the milling process, as the

increased yield was almost negligible. In any case, these findings support the idea of

zeolite affecting only morphologically the cellulose structure, rather than breaking

down the chemical structure to smaller oligosaccharides, which should have been

detected in the RS analysis. Regarding the drying temperature, it seems clear that the

higher the temperature the higher the RS recovery. In contrast with the effect of

zeolite, thermal energy is able to both reduce the crystallinity and hydrolyse to some

extent long-chain carbohydrates (Grams, 2022). Finally, the RS yield increases with

milling time, which is in accordance with most of the data reported in the literature

(Barakat et al., 2013). However, the optimal time determined by the model was 50 s

approximately (Figure 9). This may be due to the potential formation of 5-

hydroxymethylfurfural (5-HMF) which can occur during the milling process as the

cellulose structure is amorphized, and acidic compounds presented in the biomass

could catalyze the formation of 5-HMF from the released RS (Shapla et al., 2018; Shen

et al., 2020).
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To the best of our knowledge, this is the first time that the RS content of R.

okamurae has been reported after a sole aqueous or ethanolic extraction process

without the application of thermal energy. Nevertheless, when the alga R. okamurae

was subjected to a hydrothermal pretreatment by microwave irradiation (200 ºC during

15 min) the maximum RS yield (0.060 g glucose kg-1VS) was significantly lower than the

reported in the present study (from 2.3 ± 0.3 to 13.3 ± 0.9 g glucose kg-1VS)

(FernándezMedina et al., 2022). Other studies have assessed the potential of R.

okamurae as a source of monosaccharides by enzymatic saccharification. In this sense,

Agabo-García et al. (2023) reported values of 1.5 - 4.27 g L -1hydrosilate after a biological

treatment (5-12 days) followed by enzymatic hydrolysis (24 h). Moreover, values of

12.22 g L-1hydrosilate have been reported after the hydrolytically pretreated biomass by

CellicCTec2 containing cellulase and endo-β-1,4-xylanase (Romero-Vargas et al., 2023).

Similar results (12 g L-1hydrosilate) were obtained by Fernández-Medina et al. (2022) when

the enzymatic pretreatment with CellicCTec2 was applied after a hydrothermal

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pretreatment by microwave irradiation. In the present study, without a saccharification

process of any kind, the concentration of reducing sugars in the liquid fraction was

significantly lower, ranging from 17 to 208 mg L -1extracted . This confirms the great
liquid

potential of the mentioned pretreatments to enhance the conversion of carbohydrates

into shorter-chain oligosaccharides. Nevertheless, this study aimed to assess the

potential of the alga R. okamurae as a feasible feedstock within a biorefinery industry

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based on eco-friendly and low-cost processes, which, as expected, impairs the

addedvalue compound extraction yield.

3.3 Confirmation of the optimized extraction method

In order to confirm the optimized extraction process, two different batches of R.

okamurae were assessed. FAR samples corresponded to fresh ashore R. okamurae, as

used during the optimization study, and FR samples corresponded to fresh R. okamurae

directly collected from its natural habitat in the deep waters.

The theoretical TPC for the optimum process using ethanol as solvent (Eq. 3) was

12.91 ggalic acid kg-1VS while for water as solvent (Eq. 1) it was 3.13 ggalic acid kg-1VS. The FAR

sample extracted with ethanol (13.3 ± 0.3 ggalic acid kg-1VS) differed only a 3 % from the

theoretical value. Additionally, when extracted with water (2.90 ± 0.09 g galic acid kg-1VS) the

yield was only an 8 % less than expected. These results confirmed the method to be

adequate for the optimization of the extraction of TPC. Similarly, the differences

between the experimental and theoretical values for the RS extraction were very

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similar. In fact, no significant differences were observed with the ethanolic extract (2.3

gglucose kg-1VS). However, the experimental water extract showed a content 26 % higher

than expected (11.6 ± 0.6 and 9.23 g glucose kg-1VS, respectively). This discrepancy could be

due to the above-mentioned formation of 5-HMF, which may be linked to several

factors not controlled in this study and related to the nature of the biomass. A

biological entity whose composition could vary significantly between batches but also

22
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between individual specimens of the same batch. These results strongly encourage

further investigations regarding the formation of 5-HMF during the milling process and

the naturally occurring catalytic reactions. In any case, the results obtained confirm the

effectiveness of the proposed methodology and model.

From a more general perspective, and although outside of the principal scope of

this study, it was interesting to gain with some insight into the potential discrepancies

between biomasses collected at different stages of their natural life cycle. In fact, the

results of the FR samples were significantly different from those obtained of the FAR

samples. For TPC, the response from the ethanolic extract of the FR sample (22 ± 2 gallic

acid kg-1VS) was 70 % higher than the theoretical value, and the response from the

aqueous extract of the FR sample (2.04 ± 0.03 ggalic acid kg-1VS) was 35 % lower than the

expected. FR samples were composed of healthy individuals collected during the

summer period, which have been pointed as the peak season for the content of

phlorotannins in brown algae, concurring with the reproductive season (Connan et al.,

2004; Imbs and Zvyagintseva, 2018). However, the TPC also depends on the habitat of

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the algae, the intensity of illumination, or, as shown in this study, the natural

degradation, among other factors (Imbs and Zvyagintseva, 2018). Although, to the best

of our knowledge, the reduction of TPC during the natural degradation of marine algae

have not been yet assessed, studies on the composting of similar biomasses could

provide approximated information. For example, after the vermicomposting process of

oil palm empty fruit bunches, the total phenolic compounds decreased from 311 g galic
23
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acid kg-1 to 15 ggalic acid kg-1, meaning more than 95 % elimination of total phenolic

compounds (Yahaya et al., 2017). In this study, naturally degraded FAR samples

presented a 40 % less TPC than FR. This could also indicate the freshness of the FAR

samples, which were taken immediately once reached the coasts, and suggests that the

TPC of the completely sun-dried algal biomass found in the coasts could be negligible.

Similar behaviour was observed in the RS analysis. The ethanolic extract of the FR

sample showed an RS content 362 % higher than that obtained from Eq. 6 (8.0 ± 0.7

and 1.73 gglucose kg-1VS, respectively). Thus, the ethanolic extract of FR presented an RS

content 2.5 times higher than that found in the FAR samples, while for the aqueous

extract, the increase was only 15 %. Similar to the TPC, the carbohydrate content of

algae peaks during late spring to middle summer, although its proportion may be

altered by several factors, such as: i) temperature; ii) light irradiance; iii) nutrient

availability; iv) seawater hydrodynamics; v) day length (Schiener et al., 2014). To the

best of our knowledge, the effect of natural degradation on the RS content of marine

algae has not been reported earlier. However, several studies on the composting of

macroalgae confirmed that this process reduces and breakdown the organic matter

content, being the short-chain carbohydrates the easier biodegradable fraction

(Oliveira and Bisalputra, 1977; Patón et al., 2023; Walsh and Waliczek, 2020).

24
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4. Conclusions

This study aimed to further understand the potential of the invasive alga

Rugulopteryx okamurae as a feedstock for the obtention of added-value compounds

such as total phenolic compounds (TPC) and reducing sugars (RS). The response surface

methodology allowed for the optimization of the extraction of TPC and RS. The main

difference between variables was related to the used solvent. While ethanol was shown

as the most efficient for TPC extraction, water was selected for the obtention of RS. This

suggests that a cascade system where the biomass is firstly extracted with ethanol and

then with water, or vice versa, could be the most appropriate approach to increase the

efficiency of the system. Regarding the studied drying temperatures, lower

temperatures (60 ºC) enhanced the TPC extraction, while higher values (100 ºC) were

found optimum for the extraction of RS. The other two studied variables did not show

significant differences. In both cases, the optimum milling time was circa to 60 s and

while the use of zeolite during the milling process could increase the TPC recovery, this

improvement was negligible, and its presence showed no impact on the RS recovery.

Declarations

Funding. The authors wish to express their gratitude to the Spanish Ministry of

Science and Innovation for providing financial support through the project

PID2020114975RB-100/AEI/10.13039/5011000/11033. M.J. F-R is recipient of a “Juan

25
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de la Cierva Formación” grant from the Spanish State Research Agency (FJC2020-

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045654-I).

Conflicts of interest/ Competing interests. The authors declare that they have no

known competing financial interests or personal relationships that could have appeared

to influence the work reported in this paper.

Ethics approval. The authors declare that in order to fulfil the present review no

ethical approval by any organization was needed.

Consent to participate. The authors declare that all the authors mutually agree on

this submission to Algal Research.

Consent for publication. The publication of this work is approved by all authors

and by the responsible authorities of the institution at which the work was carried out.

Availability of data and material. All the data and material used to produce the

present review is well defined and accessible within the text. However, the authors

declare that any further information would be provided upon request to the

corresponding author.

Code availability. Not applicable.

Authors’ contributions. David De la Lama-Calvente: Conceptualization, Data

curation, Methodology, Supervision, Visualization, Writing – original draft. María José

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Fernandez-Rodríguez: Conceptualization, Data curation, Methodology, Supervision,

Writing – original draft. Antonio Garrido-Gutierrez: Data curation, Supervision, Writing

– original draft. Rafael Borja: Conceptualization, Data curation, Funding acquisition,

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Methodology, Resources, Supervision, Writing – original draft.

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Figure 1. Schematic description of the experimental design conceptualization. (MUST BE IN COLOR)

Biomass Journal Pre-proof Rugulopteryx okamurae

Temperature
Drying Process
0 ºC; 60 ºC; 100 ºC

Time Assisting Zeolite

Milling Process
5s; 10s; 20s; 30s; 60s Yes / No

Solvent
Extraction Process
Ethanol / Water

Analyses Phenolic Compounds Reducing Sugars

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Figure 2. Extraction of phenols by solvents. Response surface using water as a function

of temperature (ºC) and milling time (s), for (A) absence of zeolite and (B) presence of
zeolite. (MUST BE IN COLOR)

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Figure 3. Extraction of phenols by solvents. Response surface using alcohol as a

function of temperature (ºC) and milling time (s), for (A) absence of zeolite and (B)
presence of zeolite. (MUST BE IN COLOR)

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Figure 4. Extraction of
phenols by
Interaction
(s) and
(ºC) for the
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milling time
temperature
different
combinations of the
nominal variables as a
function of milling time
and temperature
(oC). (A) water and
presence of zeolite. (B)
water and absence of
zeolite. (C) ethanol and
presence of zeolite. (D)
ethanol and absence of
zeolite. Drying
temperatures of 0oC and
100oC are represented
by black and red lines,
respectively. Model fit is
indicated by continuous
lines (ending in triangles
when visible), with
confidence limits of the
fitted model shown as
discontinuous lines in corresponding colours. Experimental design points for 100, 60 (non-fitted), and 0 oC are denoted by red, green, and black
dots, respectively. (MUST BE IN COLOR)

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Figure 5. Extraction of phenols by solvents. Optimisation of the extraction as a function of milling time (s) and temperature (ºC), using alcohol as
solvent and zeolite as extraction aid or catalyser. (A) Desirability. (B) TPC recovery. (MUST BE IN COLOR)
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Figure 6. Extraction of sugars by solvents. Response surface using water as a function of

temperature (ºC) and milling time (s), for the presence (A) and absence (B) of zeolite.
(MUST BE IN COLOR)

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Figure 7. Extraction of sugars by solvents. Response surface using alcohol as a function

of temperature (ºC) and milling time (s), for the presence (A) and absence (B) of zeolite.
(MUST BE IN COLOR)
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Figure 8. Extraction of reducing sugars by solvents. Interaction milling time (s) and temperature ( oC) for the different combinations of the
nominal variables. (A) water in the presence of zeolite, (B) water in the absence of zeolite, (C) ethanol in the presence of zeolite, and (D) ethanol
in the absence of zeolite. Drying temperatures of 0 oC and 100oC are represented by black and red lines, respectively. Model fit is indicated by
continuous lines (ending in triangles when visible), with confidence limits of the fitted model shown as discontinuous lines in corresponding
colours. Experimental design points for 100, 60 (non-fitted), and 0 oC are denoted by red, green, and black dots, respectively. (MUST BE IN
COLOR)
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Figure 9. Extraction of reducing sugars (RS) by solvents. Optimisation of the extraction as a function of milling time (s) at a temperature of 100
ºC, using water as solvent. (A) Desirability. (B) RS recovery. Red line: use of zeolite. Green line: absence of zeolite. (MUST BE IN COLOR)
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Table 1. Experimental design. Studied variables and their codes for the optimisation of
the extraction process.
Variable Symbol
Milling Time (s) A 5 10 20 30 60
Drying Temperature (ºC) B 0 60 100
Solvent C Water Ethanol
Use of Zeolite D Yes No

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Table 2.

Experimental design used for the optimization of total phenolic compounds (TPC) and
reducing sugars (RS) extraction and their response results. Values are given as mean ±
standard deviation.
VS: Volatile solids.

Table 3. Sequential sum of squares (Type I) for selecting the non-aliased higher-order
polynomial model to be fitted to the sugar (square root) and phenol extracts (inverse
square root). Note: ANOVA was performed on the coded variable.

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 Reducing Sugars Content (RS)

Source Sum of Squares df Mean Square F-value p-value Observations

Mean vs Total 845.89 Journal
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845.89
Linear vs Mean 31.57 4 7.89 38.53 < 0.0001
2FI vs Linear 36.15 6 6.02 150.01 < 0.0001
Quadratic vs 2FI 1.87 2 0.9357 29.58 < 0.0001 Suggested
Cubic vs Quadratic 4.22 11 0.3836 31.5 < 0.0001 Aliased
Residual 2.42 199 0.0122
Total 922.12 223 4.14
Total Phenolic Content (TPC)

Source Sum of Squares df Mean Square F-value p-value Observations

Mean vs Total 66.92 1 66.92
Linear vs Mean 5.78 4 1.45 284.97 < 0.0001
2FI vs Linear 0.4297 6 0.0716 21.31 < 0.0001
Quadratic vs 2FI 0.1608 2 0.0804 29.85 < 0.0001 Suggested
Cubic vs Quadratic 0.1488 11 0.0135 6.29 < 0.0001 Aliased
Residual 0.4733 220 0.0022
Total 73.92 244 0.3029
Table 4. ANOVA (Type III – Partial) regarding model fit to experimental data for total
phenolic compounds (TPC) and reducing sugar (RS) recovery.
Source Sum of Squares Df Mean Square F-value p-value Observations
TPC response

Model 6.14 11 0.5584 138.82 < 0.0001 significant

A-Time 0.2068 1 0.2068 51.41 < 0.0001
B-Temperature 1.06 1 1.06 262.37 < 0.0001
C-C 3.96 1 3.96 985.64 < 0.0001
D-D 0.0166 1 0.0166 4.12 0.0435
AC 0.0394 1 0.0394 9.79 0.0020

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AD 0.0844 1 0.0844 20.97 < 0.0001
BC 0.3347 1 0.3347 83.21 < 0.0001
BD 0.0332 1 0.0332 8.26 0.0044
CD 0.0418 1 0.0418 10.40 0.0014
A2 0.0439 1 0.0439 10.92 0.0011
B2 0.2454 1 0.2454 61.01 < 0.0001
Residual 0.9453 235 0.0040
Lack of Fit 0.5064 18 0.0281 13.91 < 0.0001 significant
Pure Error 0.4389 217 0.0020
Total 7.09 246
RS response

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Model 69.52 9 7.72 245.2 < 0.0001 significant

A-Time 2.01 1 2.01 63.92 < 0.0001
B-Temperature 3.11 1 3.11 98.75 < 0.0001 741.26
C-C 23.35 1 23.35 < 0.0001
D-D 0.0056 1 0.0056 0.1776 0,6739
AD 0.8132 33.24 1 0.8132 25.81 < 0.0001
BC 0.1506 1 33.24 1055.25 < 0.0001
BD 0.2272 1 0.1506 4.78 0,0299
A2 1.79 1 0.2272 1.79 7.21 0,0078
B² 6.71 1 0.0315 56.85 < 0.0001
Residual 5.02 213 0.2511
Lack of Fit 1.69 20 0.0087 significant
28.72 < 0.0001
Pure Error 76.23 193
Cor Total 222

TPC: Total phenolic content; RS: Reducing sugars.

HIGHLIGHTS

• Extraction of R. okamurae compounds optimized via response surface

methodology

• Aqueous extracts presented higher reducing sugar extraction yields

• Ethanol extracts presented higher total phenolic compound extraction yields

• Biomass drying temperature and milling time are the most influential factors

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