biology

Article
Microalgae to Bioenergy: Optimization of Aurantiochytrium
sp. Saccharification
Joana Oliveira 1,2,† , Sara Pardilhó 2,3,† , Joana M. Dias 2,3, * and José C. M. Pires 1,2, *

1 Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE), Department of
Chemical Engineering, Faculty of Engineering, University of Porto, 4200-465 Porto, Portugal;
up201904660@edu.fe.up.pt
2 ALiCE—Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto,
4200-465 Porto, Portugal; spardilho@fe.up.pt
3 Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE),
Department of Metallurgical and Materials Engineering, Faculty of Engineering, University of Porto,
4200-465 Porto, Portugal
* Correspondence: jmdias@fe.up.pt (J.M.D.); jcpires@fe.up.pt (J.C.M.P.)
† These authors contributed equally to this work.

Simple Summary: Over recent years, microalgae have gained attention as a potential source for
bioethanol production due to their fast growth rates, ability to grow in several environments and
carbohydrate content, mainly in starch form. Biomass pretreatment is considered a critical step
in bioethanol production since fermentable sugars are required for yeast growth. Although many
studies have been conducted on the thermal acid hydrolysis of microalgae, the use of systematic
methodologies, such as the response surface methodology, to obtain a comprehensive view of the
influence of the studied parameters is relatively unexplored. This study evaluates the influence
of acid concentration, hydrolysis time and biomass/acid ratio on the thermal acid hydrolysis of
Aurantiochytrium sp. through the response surface methodology, aiming at further bioethanol produc-
tion. For each of the developed models, the maximum sugar concentration and yield were 18.05 g/L
and 12.86 g/100 g, respectively. This study provides insights into the hydrolysis of microalgae for
bioethanol production and suggests that Aurantiochytrium sp. microalgae could be a promising
Citation: Oliveira, J.; Pardilhó, S.; feedstock for bioethanol production.
Dias, J.M.; Pires, J.C.M. Microalgae to
Bioenergy: Optimization of Abstract: Microalgae are a promising feedstock for bioethanol production, essentially due to their
Aurantiochytrium sp. Saccharification.
high growth rates and absence of lignin. Hydrolysis—where the monosaccharides are released
Biology 2023, 12, 935. https://
for further fermentation—is considered a critical step, and its optimization is advised for each
doi.org/10.3390/biology12070935
raw material. The present study focuses on the thermal acid hydrolysis (with sulfuric acid) of
Academic Editors: John Korstad, Aurantiochytrium sp. through a response surface methodology (RSM), studying the effect of acid
Bhaskar Singh, Kuldeep Bauddh and concentration, hydrolysis time and biomass/acid ratio on both sugar concentration of the hydrolysate
Aidyn Mouradov and biomass conversion yield. Preliminary studies allowed to establish the range of the variables to
Received: 28 April 2023 be optimized. The obtained models predicted a maximum sugar concentration (18.05 g/L; R2 = 0.990)
Revised: 22 June 2023 after 90 min of hydrolysis, using 15% (w/v) biomass/acid ratio and sulfuric acid at 3.5% (v/v),
Accepted: 28 June 2023 whereas the maximum conversion yield (12.86 g/100 g; R2 = 0.876) was obtained using 9.3% (w/v)
Published: 29 June 2023 biomass/acid ratio, maintaining the other parameters. Model outputs indicate that the biomass/acid
ratio and time are the most influential parameters on the sugar concentration and yield models,
respectively. The study allowed to obtain a predictive model that is very well adjusted to the
experimental data to find the best saccharification conditions for the Aurantiochytrium sp. microalgae.
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
Keywords: thermal acid hydrolysis; microalgae; Aurantiochytrium sp.; response surface methodology;
This article is an open access article
bioethanol production
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Biology 2023, 12, 935. https://doi.org/10.3390/biology12070935 https://www.mdpi.com/journal/biology

Biology 2023, 12, 935 2 of 14

1. Introduction
The increasing concerns over the use of fossil fuels reinforce the need to explore
renewable alternatives to change the paradigm regarding the current energy crisis, such as
the production of liquid biofuels [1,2]. Bioethanol is a renewable fuel produced through
the fermentation of biomass [3]. The use of this biofuel is aligned with the United Nations
Sustainable Development Goals, namely Goals 7 (Affordable and Clean Energy) and
13 (Climate Action), since it (i) can be produced from renewable sources, (ii) reduces net
CO2 emissions—as the production of the feedstock usually involves CO2 absorption and
(iii) increases energy security, since this fuel can be produced locally [1,2,4,5].
The most used feedstocks for bioethanol production are food crops (e.g., sugarcane,
corn and wheat) as they are rich in fermentable sugars [1]. However, their use leads to
competition with food production and arable land [6]; thus, alternative feedstocks such as
residual lignocellulosic biomass have recently been used to produce bioethanol. However,
high lignin content requires additional pretreatments to ferment the biomass, which are
generally associated with high processing costs, making the search for alternative non-food
raw materials relevant [7].
Over recent years, microalgae have gained attention as a potential source for bioethanol
production due to their fast growth rates and ability to grow in various environments,
including wastewater and seawater [2]. These microorganisms have a short harvesting
cycle (approximately 1–10 d) [8], reaching high biomass productivity. Furthermore, the
absence of lignin and its high carbohydrate content (which can reach 60 wt.%) indicate
that such biomass can be easily converted into fermentable monosaccharides [7,9–12].
In particular, Aurantiochytrium sp. shows a carbohydrate content between 20 wt.% and
60 wt.% [7,13,14], depending on the species and growth conditions, which explains the
interest in its use for bioethanol production.
Microalgae contain complex carbohydrates such as starch and cellulose and simpler
sugars such as glucose, fructose and sucrose. Depending on the microalgal species and
growth conditions, the carbohydrate content and composition can vary significantly [15];
therefore, the optimal hydrolysis conditions depend on the carbohydrate profile of each
feedstock. No studies in the literature related to Aurantiochytrium sp. as a feedstock for
bioethanol production or the optimization of saccharification were found.
Bioethanol production generally comprises three steps: (i) biomass pretreatment,
(ii) alcoholic fermentation and (iii) bioethanol purification. The pretreatment step involves
breaking down the complex carbohydrates into simple sugars that can be fermented by
yeast [7]. This can be achieved through acid, alkaline or biological pretreatment [5]. Even
though enzymatic pretreatment is highly specific and does not require harsh conditions,
the high enzyme cost makes it unattractive to implement at an industrial scale [3,12]. On
the other hand, the chemical process is associated with significantly lower costs [16]. In this
case, after the pretreatment, the pH of the solution needs to be adjusted to 4.5–5.0 before
adding the yeast and performing fermentation (e.g. with Saccharomyces cerevisiae) [6].
Biomass pretreatment is considered a critical step in bioethanol production since fer-
mentable sugars are required for yeast growth [10]. Regarding chemical hydrolysis, acid
pretreatment is most commonly used compared with alkali, leading to higher hydrolysis
efficiency [8]. In that sense, biomass hydrolysis with dilute acid solutions at high tempera-
tures has been demonstrated to be a simple and low-cost method to release those sugars
without compromising the economic feasibility of the process [9,17].
An extensive literature has been published concerning the dilute thermal acid hydrol-
ysis of microalgae for bioethanol production [1,2,6,8,9,11,16–19]. Miranda et al. [17] studied
the use of Scenedesmus obliquus microalgae for bioethanol production, achieving the best re-
sults through acid hydrolysis with H2 SO4 (2.8% (v/v)) and 5% (w/v) biomass concentration
at 120 ◦ C (autoclave) for 30 min leading to a sugar yield of 28.6 gglucose equivalent per 100 g
dry algae and a sugar concentration of 14.4 g/L. For the sugar concentration, the optimized
conditions obtained included a much higher biomass concentration—50% (w/v).
Biology 2023, 12, 935 3 of 14

Castro et al. [18] used a full factorial experiment to optimize saccharification condi-
tions for bioethanol production of a mixture of microalgae species (Scenedesmus, Chlorella,
Ankistrosdemus, Micromonas and Chlamydomonas) by dilute acid hydrolysis. The authors
found that the optimal conditions were: 10% (w/v) solid loading, 2.8% (v/v) H2 SO4 solution
and 120 min hydrolysis at 80–90 ◦ C, which led to a sugar yield of 16.61 g/100 g dry algae.
The response surface methodology (RSM) is a statistical technique used to optimize a
process considering the modeling of the influence of several input variables on a selected
response. This tool lowers the number of experiments necessary and makes reliable
predictions [20]. No studies were found regarding the study of microalgae saccharification
using the RSM. Therefore, the aim of this work was to gain a comprehensive view of
how thermal acid hydrolysis reaction conditions (time, acid concentration and amount of
biomass) influence the saccharification of Aurantiochytrium sp. biomass, considering the
use of the RSM to model their impact, as response variables, on both sugar concentration
of the hydrolysates and sugar yield, keeping in mind further bioethanol production.

2. Materials and Methods

2.1. Preliminary Studies: Definition of Variable Ranges
Aurantiochytrium sp. microalgal biomass was provided by Allmicroalgae—Natural
Products S.A. Cultivation. The biomass was obtained from industrial-scale fermentation
in a heterotrophic industrial medium with control of sugar content during fermentation.
Fermentation conditions favored lipid accumulation (the main product to obtain with
this microalga). Preliminary studies were carried out in duplicate to define the range
of parameters (hydrolysis time, biomass/acid ratio and acid concentration) to be used
in further optimization of the hydrolysis process. The reactions were conducted in a
water bath (~90 ◦ C) in 250 mL flasks using 100 mL H2 SO4 solution (concentration ranging
2.5–4% (v/v)), with different biomass/acid ratios (1, 2.5 and 10% (w/v)) and hydrolysis
times (30, 60 and 90 min). Based on the literature review [6,9,21], it was not considered
relevant to perform preliminary studies at temperatures lower than 90 ◦ C. Preliminary
assays were conducted to evaluate the influence of the temperature in the sugar release in
harsher conditions, namely with an autoclave (121 ◦ C; Uniclave 88, AJC, Cacém, Portugal)
and a thermoreactor (100 ◦ C for 60 min plus 148 ◦ C for 30 min). The other parameters, kept
in the range previously mentioned, were selected based on the results obtained.

2.2. Experimental Design

For the optimization process, different hydrolysis times, biomass/acid ratios and
H2 SO4 concentrations were used in the thermal acid hydrolysis (water bath at ~90 ◦ C)
considering results obtained in the preliminary studies. Since the RSM usually involves
the study of the behavior of relevant parameters to reduce the number of experiments that
need to be conducted, the number of factors considered was 3, namely time, biomass/acid
ratio and acid concentration, based on the preliminary experiments.
A central composite design (CCD; Face Centered) was used to evaluate the influence
of the studied variables on sugar concentration and yield through JMP (SAS Institute; Cary,
NC, USA) and Minitab (Minitab Inc., State College, PA, USA) software. Being the most
commonly used response surface design of experiments, this methodology allows second-
order polynomial models to be built [22]. The use of other standard designs for second-order
models, such as the Box–Behnken design (BBD), was considered. Despite requiring fewer
experiments and accurately estimating main effects and two-factor interactions, the BBD
shows poorer performance in estimating quadratic effects, since it is not possible to check
the adequacy of the quadratic model [23] and therefore was not chosen for the current
work. Furthermore, Czyrski et al. [24] and Zolgharnein et al. [25] compared the use of the
CCD, BBD and Doehlert design in the RSM and found that the CCD showed the best-fitting
results. The three factors were studied at three levels, coded as −1, 0 and 1, with the ranges
reported in Table 1. The independent variables were defined as x1 (time), x2 (biomass/acid
ratio) and x3 (acid concentration).
Biology 2023, 12, 935 4 of 14

Table 1. Range of studied variables and code levels used in the central composite design.

Range and Level

Parameter Unit
−1 0 1
x1 Time min 10 50 90
x2 Biomass/acid ratio % (w/v) 5 10 15
x3 Acid concentration % (v/v) 0.5 2 3.5

The experimental data were analyzed to fit a second-order polynomial model

(Equation (1)), considering the linear, quadratic and interaction effects of the exploratory
variables. Two models were developed, considering the sugar concentration (y1 ) and the
yield (y2 ) as the dependent variables.

y = β 0 + ∑ βi xi + ∑ βii xi2 + ∑ βij xi x j (1)

where y is the predicted response; β0 , βi , βii and βij are regression coefficients; and i and j
are indexes of the number variables, where i, j ∈ {1,2,3} and i 6= j.
Statistical analysis was performed to validate the model results. In that sense, the
obtained models were evaluated through analysis of variance using Minitab. The models’
parameters were analyzed considering the p-value obtained in the analysis of variance
and were considered statistically significant at a 95% confidence level. The variables with
no statistical significance (p > 0.05) were removed in descending order of p in a stepwise
algorithm. In that sense, the achieved models considered only statistically significant
parameters (p < 0.05). The adjusted model and the respective coefficients of determination
(R2 ) were used to evaluate the model performance in training and test sets. The training
set was used to determine the model parameters, while the test set was used to validate
the model (evaluation of the model predictive performance with new observations). In
detail, R2 provides information regarding the fraction of the variance of the output variable
explained by the model [26], whereas the predicted and adjusted R2 values are related
to the performance of the model in predicting new data (test set) and provide a more
conservative measure of model fit that avoids overfitting, since it takes into consideration
the number of significant elements in the model [27,28]. The lack of fit and the analysis of
residues were also considered to evaluate model performance.
JMP software was used to define the critical points of the models. Microsoft Excel
Solver was used to determine the conditions where the responses were maximized.
Considering the obtained results, a hydrolysis assay with conditions corresponding to
the maximum output value (namely 90 min; 10% (w/v) biomass/acid ratio and 3.5% (v/v)
acid concentration) was performed to validate the achieved models. The experimental
value was then compared with the model given by the model, and the predictive error
was calculated.

2.3. Sugars Quantification

The samples were hydrolyzed and cooled to room temperature, and the supernatant
was centrifuged at 4000 rpm for 10 min (HERMLE Z 200 A). The total sugar content was
determined spectrophotometrically (UV mini-1240, UV–Vis spectrophotometer, Shimadzu,
Kyoto, Japan) at 490 nm, through the phenol-sulfuric acid method as described by Con-
nan [29] with minor modifications (0.25 mL of the sample was used instead of 0.5 mL).
Determinations were made in triplicate, and the results were presented as g/L.
The produced sugars and the amount of biomass used in the hydrolysis were consid-
ered when calculating the biomass conversion yield (g sugar/100 g of dry algae).

3. Results and Discussion

According to the supplier, microalgae biomass is composed of around 30% (w/w)
carbohydrates, which include neutral sugars, amino sugars and uronic acids [30].
Biology 2023, 12, 935 5 of 14

3.1. Preliminary Results

The reaction conditions and results from the preliminary studies are presented in
Table 2.

Table 2. Preliminary assays performed for different durations (30–90 min), biomass/acid ratio
(1–10% (w/v)) and acid concentrations (2.5–4% (v/v)). Assays at 90 ◦ C and 121 ◦ C were performed in
a water bath and autoclave, respectively.

[H2 SO4 ] Time Biomass/Acid Ratio Yield Sugar Concentration

Temperature (◦ C)
(% (v/v)) (min) (% (w/v)) (g/100 g) (g/L)
2.5 30 2.5 13 ± 1 3.2 ± 0.3
2.5 60 2.5 13.2 ± 0.6 3.3 ± 0.1
2.5 90 2.5 14 ± 1 3.6 ± 0.4
90 2.5 90 1 24 ± 3 2.4 ± 0.3
2.5 90 10 13.3 ± 0.7 13.3 ± 0.7
3 90 2.5 13.7 ± 0.2 3.48 ± 0.05
4 90 2.5 14 ± 1 3.7 ± 0.4
121 2.5 90 2.5 18 ± 1 4.6 ± 0.3
100–148 * 3 90 2.5 13.2 ± 0.7 3.3 ± 0.2
Note: Experimental values are presented as mean value ± standard deviation. * 100–148 ◦ C corresponds to the
temperature of the thermoreactor, considering 100 ◦ C for 60 min plus 148 ◦ C for 30 min.

Under the studied conditions, acid concentration does not seem to have a clear effect
on sugar concentration or yield, since similar results were obtained for the studied con-
centrations. Regarding hydrolysis time, this variable showed a positive impact on both
sugar concentration and biomass conversion yield. Keeping constant the acid concentration
(2.5% (v/v)) and the biomass/acid ratio (2.5% (w/v)), an increase of 19% was verified by
increasing the reaction time from 30 to 90 min.
Results showed that the biomass/acid ratio has the opposite effect on sugar concentra-
tion and yield. As expected, an increase in the ratio (algae amount) resulted in an increase
in sugar concentration, since there is a larger amount of biomass available for hydrolysis.
However, it led to a decrease in biomass conversion yield. Namely, by increasing the ratio
from 1% (w/v) to 10% (w/v), an increase of around 6-fold in sugar concentration and a
decrease of 44% in yield was verified. In that sense, this parameter seems to be much more
preponderant regarding the sugar concentration compared to the process yield. Further-
more, despite having a high yield, the use of 1% (w/v) biomass/acid ratio led to a very low
sugar concentration. Since the final objective is bioethanol production, optimizing the sugar
concentration for further fermentation is crucial. Even though the biomass conversion
yield is an important parameter for the industrial feasibility of the process, using such a
low biomass/acid ratio (1% (w/v)) seems unfeasible. Comparing the use of 2.5% (w/v)
and 10% (w/v) biomass/acid ratio, the results show an increase in sugar concentration (by
270%) without a significant decrease in yield (7.5%).
Temperature is a relevant parameter for the dilute thermal acid hydrolysis of microal-
gae biomass. From the literature review, temperatures between 90 and 130 ◦ C are usually
employed in this process [6,9,21]. Therefore, two different temperatures (90/121 ◦ C) were
used in the preliminary assays. Higher hydrolysis temperature led to an increase of 22% in
both sugar concentration and yield, keeping the other parameters constant. However,
the increase in this variable can lead to a 46–90% rise in process costs [18], compromising
the industrial feasibility of the process. Furthermore, Castro et al. [18] found the optimal
hydrolysis temperature to be 80–90 ◦ C. Additionally, the use of a thermoreactor led to
a slight decrease both in sugar concentration and yield of around 5%, compared to the
water bath assay for the same time, biomass/acid ratio and acid concentration. Taking
into consideration the higher cost associated with a higher hydrolysis temperature, the
differences were not considered relevant, so the use of the water bath was considered for
the following studies.
Biology 2023, 12, 935 6 of 14

The preliminary assays performed in the current study aimed to screen the most
relevant variables for the hydrolysis of the biomass aiming further bioethanol production.
The industrial and economic feasibility of the hydrolysis process are relevant factors to also
take into consideration besides the yield to ensure the possibility of producing bioethanol
from this biomass at a large scale. In that sense, the time, biomass/acid ratio and acid
concentration were considered the most relevant parameters to optimization studies using
the CCD.
Minh Thu et al. [6] optimized the hydrothermal acid pretreatment conditions for
the microalgae Chlamydomonas reinhardtii, considering a biomass/acid ratio of 5% (w/v).
Results show that longer hydrolysis times led to a higher sugar concentration and yield,
which is in agreement with the present study. On the other hand, for an established
hydrolysis time and temperature, higher acid concentrations increased the release of sugars
(studied range: 1–5% (v/v)); however, for higher concentrations (3–5% (v/v)), the difference
was much less significant. The optimal conditions obtained by the authors were 3% acid
concentration for 30 min at 110 ◦ C, which led to a sugar concentration of 28.5 g/L and
a sugar conversion yield of 58 g/100 g. The optimal conditions the authors found were
greater than those obtained in this study; however, the feedstock was different (different
microalgae species), and the process temperature was higher. In the present study, only
sulfuric acid concentrations over 2.5% (v/v) were studied, and no significant influence of
acid concentration on sugar release was found.
Miranda et al. [17] studied the influence of acid concentration and biomass/acid ratio
on the saccharification of Scenedesmus obliquus by autoclaving (120 ◦ C). Even though a direct
comparison of the result cannot be performed (different feedstocks and temperature), the
biomass/acid ratio had a positive effect on the sugar concentration, which is in agreement
with the verified result in the present study. Furthermore, for acid concentrations of
2.8% (v/v) and 5.6% (v/v), sugar yields of around 25 g/100 g and 27 g/100 g, respectively,
were obtained. These findings corroborate the preliminary assays under analysis, where
there were no significant differences in the sugar yield for acid concentrations between
2.5 and 4% (v/v).
Considering the obtained results, for the process optimization using the RSM design
of experiments, the range of variables was defined as follows: the biomass/acid ratio
was increased (5–15% (w/v)) to evaluate the point where mass transfer limitations start to
occur, lowering the process yield, the acid concentration was lowered (0.5–3.5% (v/v)), to
minimize the acid required since higher concentrations did not show a positive effect in the
release of the sugars and a wider range of time (10–90 min) was used to determine whether
a shorter time period would still lead to efficient hydrolysis.

3.2. Model Fitting

The results from the CCD are summarized in Table 3, where the responses obtained (sugar
concentration and biomass conversion yield) are presented for the 32 performed assays.
Using the RSM, an empirical second-order polynomial model was obtained for the
two response parameters. The regression models showed high fitting performance, R2 of
99.0% and 90.2% for the sugar concentration and yield, respectively. However, the results
from the variance analysis (data not presented) showed that some of the variables had no
statistical significance (p > 0.05), and, therefore, a reduced model with only statistically
significant variables with a 95% confidence level (p < 0.05) was obtained through a stepwise
algorithm, where the statistically non-significant variables were removed in descending
order of p. For the yield model (y2 ), even though the amount of biomass (x2 ) was not a
statistically significant variable (p = 0.126), it was kept in the reduced model for hierarchical
reasons, as the quadratic term (x2 2 ) and interaction with time (x1 ·x2 ) were statistically sig-
nificant. The empirical relationship between the responses (sugar concentration and yield)
and the variables under study in the reduced model are presented in Equations (2) and (3).

y1 = 10.678 + 1.196x1 +5.195x2 +0.433x3 − 0.542x22 +0.268x1 x2 +0.412x1 x3 +0.405x2 x3 (2)

Biology 2023, 12, 935 7 of 14

y2 = 10.662 + 1.343x1 +0.185x2 +0.353x3 − 0.642x22 +0.378x1 x2 +0.412x1 x3 (3)

Table 3. Central composite design and the corresponding responses (sugar concentration—y1 ; yield—y2 ).

Biomass/Acid [H2 SO4 ]

Run. Time (min) x1 x2 x3 y1 (g/L) y2 (g/100 g)
Ratio (% (w/v)) (% (v/v))
1 50 10 0.5 0 0 −1 10.55 10.52
2 50 5 2 0 −1 0 5.54 11.05
3 50 15 2 0 1 0 15.57 10.38
4 10 5 0.5 −1 −1 −1 4.32 8.57
5 10 10 2 −1 0 0 9.13 9.12
6 10 15 3.5 −1 1 1 14.92 9.95
7 90 5 0.5 1 −1 −1 5.11 10.16
8 10 10 2 −1 0 0 8.72 8.72
9 10 5 3.5 −1 −1 1 3.98 7.92
10 90 5 0.5 1 −1 −1 5.52 10.92
11 10 15 0.5 −1 1 −1 13.63 9.07
12 10 5 0.5 −1 −1 −1 4.24 8.44
13 90 10 2 1 0 0 11.78 11.76
14 50 5 2 0 −1 0 4.91 9.80
15 90 15 3.5 1 1 1 18.89 12.54
16 50 10 2 0 0 0 10.84 10.82
17 90 15 3.5 1 1 1 17.32 11.53
18 10 5 3.5 −1 −1 1 3.59 7.14
19 50 10 0.5 0 0 −1 10.23 10.22
20 90 15 0.5 1 1 −1 15.08 10.05
21 50 10 2 0 0 0 10.85 10.83
22 90 5 3.5 1 −1 1 5.98 11.92
23 90 5 3.5 1 −1 1 6.23 12.43
24 10 15 3.5 −1 1 1 13.77 9.18
25 50 10 2 0 0 0 10.28 10.26
26 10 15 0.5 −1 1 −1 13.54 8.98
27 50 10 2 0 0 0 11.70 11.68
28 50 15 2 0 1 0 15.02 10.00
29 50 10 3.5 0 0 1 10.41 10.39
30 50 10 3.5 0 0 1 11.39 11.36
31 90 15 0.5 1 1 −1 15.59 10.38
32 90 10 2 1 0 0 12.26 12.26
Note: Design of experiments considers two replicates for each condition and four replicates for central point (0, 0, 0).

The analyses of variance of the model for sugar concentration and yield are presented
in the Supplementary Materials (Tables S1 and S2, respectively). Regarding the sugar
concentration (y1 ), the model was found to be statistically significant with a p lower
than 0.001, demonstrating a maximum probability of 0.1% that the model can assume
due to noise, validating the current model. In the same sense, the high coefficient of
determination (R2 = 0.990) obtained shows that the model explains 99.0% of the variation
in sugar concentration [20]. Finally, the high predicted R2 (0.981) indicates that the sugar
concentration can be very well predicted by Equation (2). These results are also confirmed
by the non-significant lack of fit (p = 0.565), which means that the data variation can be
explained by the experimental error (pure error). In that sense, the model can accurately
predict the sugar concentration in the studied variable ranges.
Similarly, the yield model was found to be statistically significant (p < 0.001) and to
have an R2 value of 0.876. The model-predicted R2 value was 0.794, indicating that the
model is expected to perform adequately in terms of prediction. The lack-of-fit test was
not significant (p = 0.289), indicating that the model fits the data. Despite having a lower
statistical significance than the sugar concentration model, the yield model can nonetheless
properly adjust to experimental values and predict yield results.
 significant lack of fit (p = 0.565), which means that the data variation can be explained by
the experimental error (pure error). In that sense, the model can accurately predict the
sugar concentration in the studied variable ranges.
Similarly, the yield model was found to be statistically significant (p < 0.001) and to
have an R2 value of 0.876. The model-predicted R2 value was 0.794, indicating that the
Biology 2023, 12, 935 model is expected to perform adequately in terms of prediction. The lack-of-fit 8 of 14test was
not significant (p = 0.289), indicating that the model fits the data. Despite having a lower
statistical significance than the sugar concentration model, the yield model can nonethe-
less properly adjust to experimental values and predict yield results.
Figure 1 shows the sugar concentration and process yield predicted by the model in
Figure 1 shows the sugar concentration and process yield predicted by the model in
relation to the obtained experimental values. Results show that the prediction capability
relation to the obtained experimental values. Results show that the prediction capability
of the models is relatively good, especially for the sugar concentration. Additionally, the
of the models is relatively good, especially for the sugar concentration. Additionally, the
residuals (distance of the points to the line) seem to have high homoscedasticity. Therefore,
residuals (distance of the points to the line) seem to have high homoscedasticity. There-
the models appear to models
fore, the be an accurate representation
appear to be of the experimental
an accurate representation values, which
of the experimental values, which
confirms the statistical results previously discussed.
confirms the statistical results previously discussed.

Figure 1. Predicted sugar

Figure concentration
1. Predicted (a) (R2 = 0.990)
sugar concentration (a) and
(R2 =yield
0.990)(b)
and
2 = 0.876) versus
(Ryield the
(b) (R2 = 0.876) experi-
versus the experi-
mental values obtained in the same conditions. Line corresponds to the absencethe
mental values obtained in the same conditions. Line corresponds to ofabsence
error. of error.

In that sense, In
thethat sense,for
models thesugar
models for sugar concentration
concentration and yield and yield high
showed showed high statistical
statistical
significance
significance and and strong
strong predictive predictive capability.
capability.

3.3. Surface Plots and Respective Analysis

Figure 2 shows the response surface plots for the models obtained for sugar con-
centration and yield response variables. Table 4 summarizes the β coefficients from
Equations (2) and (3).
By analyzing Figure 2 and Table 4, it is possible to understand the behavior of the
system in the studied parameter ranges.

3.3.1. Sugar Concentration Model

Regarding the model for sugar concentration, the biomass/acid ratio appears to be
the parameter that influences the response variable the most (β2 = 5.195). As expected, the
amount of biomass has a positive effect on the sugar concentration, since there are more
sugars in the system available to be hydrolyzed. In the same way, the linear influence of
time seems to be relevant (β1 = 1.196). This variable shows a positive effect on the sugar
concentration, as it results in an increase in contact time between the acid and the biomass
at high temperatures. In that sense, there is a higher probability that the acid can catalyze
the hydrolysis process. Regarding the linear effects, acid concentration is the parameter
with less influence on the sugar concentration (β3 = 0.433). These results are in accordance
with the literature, where a study from Miranda et al. [17] revealed that there is a positive
effect of both biomass/acid ratio and acid concentration on the sugar concentration in the
studied ranges.
It should be highlighted that some studies suggest that exposing the biomass to
extreme acid and temperature conditions for long periods might degrade the hydrolyzed
monosaccharides to other compounds not fermentable by yeast, compromising further
bioethanol production [17]. In that sense, to avoid monosaccharide degradation and reduce
production costs (sulfuric acid has a high cost [6]), using low acid concentrations in the
hydrolysis might be an interesting approach from an industrial point of view.
 3.3. Surface Plots and Respective Analysis
Figure 2 shows the response surface plots for the models obtained for sugar concen-
Biology 2023, 12, 935 tration and yield response variables. Table 4 summarizes the β coefficients from Equations
9 of 14
(2) and (3).

Figure 2. Response surface for sugar concentration (a,c,e) and yield (b,d,f): (a,b) 50 min hydrolysis
Figure
time; 2. Response
(c,d) surface
10% (w/v) for sugarratio;
biomass/acid concentration
(e,f) 2.0%(a,c,e) and yield
(v/v) acid (b,d,f): (a,b) 50 min hydrolysis
concentration.
time; (c,d) 10% (w/v) biomass/acid ratio; (e,f) 2.0% (v/v) acid concentration.

Table 4. Results concerning β coefficients for the reduced sugar concentration and yield models.

Model
β Coefficients
Yield Sugar Concentration
β0 10.662 10.678
β1 1.343 1.196
β2 0.185 * 5.195
β3 0.353 0.433
β12 0.378 0.268
β13 0.412 0.412
β23 - 0.405
β2 2 −0.642 −0.542
* Statistically insignificant parameter at a 95% confidence level, kept in the reduced model for hierarchical reasons.
Biology 2023, 12, 935 10 of 14

Regarding the features of the JMP software, it was not possible to determine the
optimal point for neither of the models, since a singular value was obtained. However, the
maximum point for the studied range was determined with the optimization tool Microsoft
Excel Solver. The maximum value for the sugar concentration model was found to be
18.05 g/L, obtained for x1 , x2 , x3 of 1, 1, 1 at the positive extreme of the variables, which
corresponds to a hydrolysis time, biomass/acid ratio and acid concentration of 90 min,
15% (w/v), 3.5% (v/v).

3.3.2. Yield Model

The developed model for the process yield shows a different behavior compared to
sugar concentration. Even though the linear effect of time (β1 = 1.343) is higher when
compared to the acid concentration (β3 = 0.353)—confirming the relevance of lowering acid
concentration instead of process time if milder hydrolysis conditions are required—the
behavior of the system regarding the biomass/acid ratio is significantly different. The
linear effect of this parameter is much less prominent (β2 = 0.185), and it is not statistically
significant (p = 0.126), whereas the influence of the quadratic effect is clear (β22 = −0.642)
in Figure 2b,f. This shows that the process yield increases with an increase in biomass/acid
ratio until it reaches a point where mass transfer is compromised, and, even though there is
more biomass in the system (therefore more sugars available to be hydrolyzed), the system
in those conditions is not able to completely hydrolyze the microalgae polysaccharides.
These results are in accordance with the study of Ho et al. [2], where the sulfuric
acid concentration showed a positive effect on the sugar yield. The study also reported
a significant decrease in hydrolysis efficiency after reaching a point of mass transfer or
reaction kinetics limitations. However, that point is reached at a biomass/acid ratio of
6% (w/v), a lower value compared to the current study, which can be justified by the
different microalgae species used (Scenedesmus obliquus).
As occurred for the sugar concentration model, the maximum value for yield was
obtained through Microsoft Excel Solver. The maximum value (12.86 g/100 g) was obtained
for x1 , x2 , x3 of 1, −0.15, 1, corresponding to a hydrolysis time, biomass/acid ratio and acid
concentration of 90 min, 9.3% (w/v) and 3.5% (v/v).

3.3.3. Model Validation

To validate the model prediction, laboratory experiments were performed in conditions
close to the optimal yield conditions, and the obtained experimental results are presented
in Table 5. The low error (<15%) obtained for both models confirms the adequate prediction
capacity of the models.

Table 5. Results for the validation essay and differences between the prediction and experimental
results.

[H2 SO4 ] Time Biomass/Acid Yield Sugar Concentration

(% (v/v)) (min) Ratio (% (w/v)) (g/100 g) (g/L)
Prediction 12.84 12.72
3.5 90 10
Experimental 11.41 ± 0.04 11.45 ± 0.03
Prediction error (%) ≈11 ≈13
Note: The experimental values are presented as average ± error (two replicates, expressed by the absolute
difference between measured values and average). Prediction error was calculated by the relative difference (%)
of the prediction relative to the experimental value.

The sugar concentration and yield models indicate the biomass/acid ratio and time
as key variables, respectively. Optimal conditions were determined, and finally, model
validation confirmed the models’ predictive capability.
Biology 2023, 12, 935 11 of 14

3.4. Critical Analysis

Even though it was impossible to obtain an optimal value in the studied variables
intervals, studying points outside the defined ranges was considered of low relevance for
further research developments. Regarding the process time, increasing this variable indefi-
nitely, despite potentially increasing sugar concentration and yield, results in a decrease
in productivity and an increase in energy costs (to maintain the reactor at the operational
temperature of 90 ◦ C). In that sense, the literature concerning thermal acid hydrolysis of
microalgae uses process times in the studied range [2,17]. Higher temperatures might be
unfeasible due to economic constraints, and temperatures lower than 90 ◦ C are known as
generally ineffective; however, the evaluation of this variable remains relevant for future
studies. Furthermore, long residence times at high temperatures can be associated with
the degradation of glucose [6], which would decrease the amount of sugars available for
fermentation, although that was not possible to detect in this study (total sugars quantifi-
cation was employed). Focusing on the acid concentration, despite verifying a positive
effect of this variable on the studied responses, high acid concentrations are usually asso-
ciated with yeast growth inhibition in the fermentation process after neutralization. For
instance, Markou et al. [31] found that using H2 SO4 at 7% (v/v) inhibits yeast growth
and metabolism, further compromising bioethanol production. Finally, although high
biomass/acid ratios result in high sugar concentrations, there is a decrease in process yield,
most likely due to mass transfer limitations that occur for high solid contents [2]. In that
sense, increasing this variable outside the studied range would compromise the process
yield and the economic feasibility of the process.
No studies in the literature were found concerning the optimization of saccharification
of microalgae through thermal acid hydrolysis using RSM. Thus, no direct comparison
with the literature can be carried out. However, Castro et al. [18] performed a full facto-
rial optimization of the process and found that the optimal conditions were 10% (w/v)
biomass/acid ratio, 2.8% (v/v) H2 SO4 concentration, 120 min process time and 80–90 ◦ C
temperature. In these conditions, a sugar yield of 16.6 g/100 g was found. Although using
different species and methodologies, the authors obtained similar optimal conditions to
those obtained in the present study (90 min process time; 9.3% (w/v) biomass/acid ratio;
3.5% (v/v) H2 SO4 concentration; 90 ◦ C), also resulting in a similar yield (12.86 g/100 g).
In addition, the results indicate that the Aurantiochytrium sp. species requires a similar
temperature to hydrolyze the sugars to the mixture of microalgae species used in the
mentioned study (Scenedesmus, Chlorella, Ankistrosdemus, Micromonas and Chlamydomonas).
The production costs are one of the major hurdles for large-scale bioethanol production
from microalgae [11]. Considering the economic feasibility of the process, a biorefinery
approach seems to be interesting to explore for this kind of biomass [11], as it can obtain
several compounds with industrial and economic value. More specifically, pigments, lipids
and other compounds can be successively extracted, and the remaining biomass (rich in
carbohydrates) can be used for renewable energy production, namely through bioethanol
production. As an example, Aurantiochytrium sp. biomass is rich in docosahexaenoic acid
(DHA), an omega-3 lipid with high value for human health [32], which can be extracted, and
the remaining biomass can be further processed as a feedstock for bioethanol production.
A techno-economic analysis of the studied process was not performed; however, dilute
thermal acid hydrolysis is widely reported as a low-cost method [33] compared to using
enzymes. In fact, enzymatic hydrolysis is usually less cost-effective (due to high enzyme
cost and low process velocity [34,35]), which could be a disadvantage at the industrial
scale, and it has been reported that the enzyme can comprise about 20% of the total
cost of bioethanol production [36]. In that sense, it is expected that the studied process
represents a low-cost method for the saccharification of microalgae biomass. Furthermore,
the valorization of other biomass compounds following a biorefinery approach can be
interesting from an economic point of view.
Biology 2023, 12, 935 12 of 14

4. Conclusions
The thermal acid hydrolysis of microalgae biomass to release sugars using sulfuric
acid was assessed. Response surface methodology was employed to model the process and
understand the impact of relevant parameters (time; biomass/acid ratio; acid concentration)
on sugar concentration and process yield. A statistically significant quadratic model was
obtained for each response variable (sugar concentration: R2 = 0.990; yield: R2 = 0.876).
Regarding sugar concentration, biomass/acid ratio was the variable with the most influence
in the system, whereas, for yield, time was the most influential parameter. Furthermore,
the conditions that maximized the responses in the studied ranges were found to be
90 min, 15% (w/v) biomass/acid ratio, 3.5% (v/v) acid concentration 90 min and 9.3% (w/v)
biomass/acid ratio and 3.5% (v/v) acid concentration for sugar concentration and yield,
respectively. Further studies should be conducted to evaluate bioethanol production under
the obtained optimal hydrolysis conditions and its integration with a biorefinery approach.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/biology12070935/s1, Table S1: Variance analysis for the
response surface reduced model for sugar concentration (y1 ); Table S2: Variance analysis for the
response surface reduced model for yield (y2 ).
Author Contributions: Conceptualization, J.M.D. and J.C.M.P.; methodology, J.O., S.P., J.M.D. and
J.C.M.P.; investigation, J.O. and S.P.; writing—original draft preparation, J.O.; writing—review
and editing, S.P., J.M.D. and J.C.M.P.; visualization, J.O.; supervision, J.M.D. and J.C.M.P.; project
administration, J.C.M.P.; funding acquisition, J.M.D. and J.C.M.P. All authors have read and agreed
to the published version of the manuscript.
Funding: This research was funded by (i) LA/P/0045/2020 (ALiCE) and UIDB/00511/
2020-UIDP/00511/2020 (LEPABE) funded by national funds through FCT/MCTES (PIDDAC);
(ii) Project Power2Biofuel (Ref. IJUP2021-GALP-17) funded by Galp, Portugal, in the scope of
IJUP-Companies 2021 Edition; (iii) Project PhotoBioValue (ref. PTDC/BTA-BTA/2902/2021), funded
by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacional-
ização (POCI) and by national funds (PIDDAC) through FCT/MCTES and (iv) project “HyGreen &
LowEmissions—Tackling Climate Change Impacts: the role of Green Hydrogen production, storage
and use, together with low emissions energy systems”, with the reference NORTE-01-0145-FEDER-
000077, supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the
PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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