Bioresource Technology Reports 25 (2024) 101807

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Bioresource Technology Reports

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Microbial bioethanol production from Agave tequilana leaves juice sugars:

Process variable optimization and kinetic modeling
Eleazar Máximo Escamilla-Silva a, 1, Giovanni Alexander Escamilla-García a,
Filiberto Rocha-Arriaga a, Miriam Granados-Vallejo b, 1, David Antonio Flores-Méndez a, *
a
Departamento de Ingeniería Química-Bioquímica, Tecnológico Nacional de México en Celaya, Antonio García Cubas #600 Esq. Av. Tecnológico, 38010 Celaya,
Guanajuato, Mexico
b
Departamento de Química, Universidad Tecnológica de Salamanca, Av. Universidad Tecnológica #200, 36766 Salamanca, Guanajuato, Mexico

A R T I C L E I N F O A B S T R A C T

Keywords: This study focuses on the valorization of Agave tequilana leaves through bioethanol production from juice sugars.
Agave tequilana leaves Optimal cultivation conditions were determined using a Taguchi orthogonal array design to maximize bioethanol
Bioethanol production by Saccharomyces cerevisiae BEL6 61003. The optimal conditions included reducing sugars at 120 g/L,
Optimization
air flow at 0.5 vvm; agitation at 300 rpm, and pH at 4.5. Under these conditions, the maximum bioethanol
Kinetic model
concentration reached 43.26 ± 2.20 g/L, with a productivity (rP) of 2.4 ± 0.12 g/L⋅h and a yield of 43 %. A
Thermal hydrolysis
Saccharomyces cerevisiae mathematical model was developed to describe the dynamics of the fermentation process under optimal con­
ditions, comparing the kinetic equations of Moser-Boulton and Moser-Loung. The simulations strongly fit the
experimental data, with coefficient of determination (R2) values reaching 0.96 for both models. The selection of
the best model was assessed using the Akaike Information Criterion (AIC), favoring the Moser-Boulton equation
as the more effective model.

(Sawarkar et al., 2022; Flores-Gómez et al., 2018). Agave leaves,

1. Introduction
In recent years, global research efforts have intensified in pursuing
bioethanol production as a sustainable alternative to fossil fuels, driven
by the environmental imperative to reduce their negative impact (Kamal
et al., 2022). Bioethanol, characterized by a lower boiling point, high
octane rating, and increased oxygen content compared to gasoline, fa­
cilitates cleaner combustion, thereby reducing soot and hydrocarbon
emissions (Sawarkar et al., 2022). Presently, the United States and Brazil
lead in large-scale first-generation bioethanol production, primarily
utilizing sugarcane (25 %) and corn (60 %), among other resources.
However, this approach raises ethical concerns regarding using food
crops for fuel production, leading to resource scarcity and increased
food prices (Ortíz-Méndez et al., 2017; Althuri and Venkata Mohan,
2022).
A promising solution lies in using lignocellulosic biomass from agro-
industrial waste, avoiding direct competition with food crops. Such
waste includes corn stover, sugarcane bagasse, corn straw, forestry
residues, switchgrass, and agave waste products like bagasse and leaves
comprising 40 % of the plant's total weight, amounting to one million
tons annually, are a notable by-product of the tequila industry (CRT,
2023; Flores-Méndez et al., 2023).
Studies by Corbin et al. (2016) and Oyarzábal Saldaña et al. (2009)
reveal that Agave Tequilana leaves contain fermentable sugars (glucose,
fructose, sucrose) and a substantial amount of fructans (39.54 mg/g of
dry weight tissue). However, due to the complex structure of fructans,
only limited Saccharomyces cerevisiae strains can metabolize them
directly into ethanol is limited (Yuan et al., 2013). Thermal hydrolysis,
proven effective in agave heads, offers a viable and straightforward
alternative to increase fermentable sugars in leaves without the need for
additional catalysts (Waleckx et al., 2008). With the growing interest in
industrial alcoholic fermentation, kinetic models play a crucial role in
understanding and optimizing the process. Common models like Monod,
Logistic, and modified Gompertz equations, while useful, may not cap­
ture all phenomena governing batch fermentation kinetics (Salakkam
et al., 2023; Zentou et al., 2019; Dodić et al., 2012). This study aims to
evaluate conditions maximizing bioethanol production from heat-
treated Agave tequilana leaves and to provide a comparative analysis

* Corresponding author at: Departamento de Ingeniería Química, Tecnológico Nacional de México en Celaya, Antonio García Cubas #600 Esq. Av. Tecnológico,
38010 Celaya, Guanajuato, Mexico.
E-mail address: david.flores@itcelaya.edu.mx (D.A. Flores-Méndez).
1
These authors contributed to the work equally and should be regarded as co-first authors.

https://doi.org/10.1016/j.biteb.2024.101807
Received 11 January 2024; Received in revised form 5 March 2024; Accepted 5 March 2024
Available online 7 March 2024
2589-014X/© 2024 Elsevier Ltd. All rights reserved.
E.M. Escamilla-Silva et al. Bioresource Technology Reports 25 (2024) 101807

Nomenclature YX/N biomass yield per nitrogen consumed (g/g)

Kp mass transfer rate constant (1/h)
S/N signal to noise ratio Eti interface ethanol concentration (g/L)
n number of experiments carried out R2 coefficient of determination
i experimental run number SSV sum of square of a variable
y output result of the experiment TSS total sum of squares
YX/S,max maximum biomass yield per substrate consumed (g/g) AICc corrected Akaike Information Criterion
YP/S,max maximum bioethanol yield per substrate consumed (g/g) ne number of points
Kd cell death constant (g/L) k number of estimated parameters
X concentration of biomass (g/L) OF objective function
m Loung model parameter
KS substrate saturation constant (g/L) Greek letters
KE ethanol inhibition constant (g/L) μ specific growth rate (1/h)
S reducing sugars concentration (g/L) μmax maximum specific growth rate
E bioethanol concentration (g/L) ε Moser model parameter
YX/S biomass yield per substrate consumed (g/g) ηmax maximum specific reaction constant
N ammoniacal nitrogen concentration (g/L) α growth-associated product formation coefficient,
KN nitrogen saturation constant (g/L) β non-growth-associated product formation coefficient

of kinetic models under optimal cultivation conditions.
2. Materials and methods
2.1. Materials
The leaves of seven-year-old Agave tequilana were generously pro­
vided by the Institute of Agricultural Sciences of the University of
Guanajuato (ICA, Irapuato, México).
2.2. Thermal hydrolysis
Agave leaves were cut into approximately 15 cm lengths, underwent
thermal hydrolysis with steam in a tubular autoclave (3870E, Tuttnauer
Brinkmann) at 105 ◦ C for 12 h. Cooked leaf pieces were shredded and
pressed in a roller mill with added water for sugar extraction. This
process was applied to three 50 kg batches of leaves. The juice obtained
was characterized for reducing sugars (Miller, 1959), ammoniacal ni­
trogen (Solórzano, 1969), phosphorus (AASGP Committee Report,
1958), and suspended solids (dry weight).
2.3. Microorganism and inoculation
Saccharomyces cerevisiae BEL6 61003 was obtained from the Center
for Research and Advanced Studies of the National Polytechnic Institute
(CINVESTAV, Irapuato, Mexico). The yeast was sub-cultured on yeast
extract peptone dextrose (YPD, 1245 ATCC) agar at 27 ◦ C for 48 h. A 10
% v/v inoculum was prepared using sterile saline (0.9 % m/v), and a
liquid culture medium was formulated for incubation on an orbital
shaker (IRO-65, LumistellMR) at 280 rpm and 28 ◦ C for 16 h (Thatipa­
mala et al., 1992).
2.4. Fermentation medium
The bioethanol production medium, formulated with the juice, was
supplemented with MgSO4⋅7H2O (1 g/L), KH2PO4 (3 g/L), and a
micronutrient solution. The medium was sterilized at 121 ◦ C for 15 min.
determined the optimal conditions.
/ ( )
1∑ n
1
S N = − 10log (1)
n i=1 y2i
where n is the number of experiments carried out; i is the experimental
run number, and y is the output result of the experiment.
All experimental runs, each conducted with a replica, took place at
28 ◦ C in a glass bioreactor (Applikon®, JG Delft, Netherlands) with a
total medium volume of 3 L. The pH was meticulously maintained by
adding 1 M solutions of NaOH and HCl. Essential bioreactor operating
variables, including temperature, pH, aeration, and agitation, were
closely monitored and controlled using the Bio Controller (ADI 1030,
Applikon®, JG Delft, Netherlands) and Bio Console (ADI 1035, Appli­
kon®, JG Delft, Netherlands).
Concurrently, culture samples were collected systematically at six-
hour intervals throughout the cultivation period. Kinetic data obtained
from these samples facilitated the calculation of maximum productivity
and maximum yield for each experimental run. Biomass yield (YX/S,max)
and ethanol yield (YP/S,max) were determined by assessing the ratio of
the produced biomass or ethanol to the consumption of reducing sugars
(g products/g substrate). The comprehensive data analysis, including
ANOVA and surface response, was performed using STATISTICA 7.0.
2.6. Analytical methods
Culture broth samples underwent filtration using a 0.22 μm mem­
brane (Millipore). The retained biomass was utilized for growth deter­
mination employing the dry weight method. For further analysis, the
filtered broth was subjected to assessing reducing sugar concentrations
using the DNS method (Miller, 1959), as well as ammoniacal nitrogen
levels (Solórzano, 1969). Ethanol concentrations were quantified using a
gas chromatography system (3800, Varian®) equipped with an auto­
sampler (8200, Varian®), a flame ionization detector, and an Alltech
Econo-Cap EC-WAX analytical column (30 m × 0.53 mm). The injection
volume was set at 1 μL, with the injector and detector temperatures were
200 and 230 ◦ C, respectively.

2.5. Optimization of bioethanol production conditions 2.7. Mathematical model and kinetic parameters

Optimization was conducted using a Taguchi orthogonal array A mathematical model, based on material balances for a batch-
design L9 (34) with factors such as initial reducing sugars concentration, operated system was developed to describe the fermentation process
air flow, agitation, and pH. The bioethanol concentration was the dynamics under optimal conditions.
response evaluated. Signal-to-Noise (S/N) ratio, calculated using Eq. (1), The biomass growth rate is given by:

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E.M. Escamilla-Silva et al. Bioresource Technology Reports 25 (2024) 101807

dX Table 1
= (μ − Kd )X (2)
dt Characterization of the leaf must.

Here μ is the specific growth rate, Kd is the cell death constant, and X Juice Volume Reducing Ammoniacal Phosphates Suspended
sample (L) sugars nitrogen (mg/ (mg/L) solids (g/
is the biomass concentration.
(g/L) L) L)
Different Monod-type kinetic laws have been successfully proposed
to describe the specific growth rate (μ) of Saccharomyces cerevisiae under 1 43 95 1.99 14.26 41.3
2 35 147 1.08 5.67 23.7
substrate-limited conditions. An example is the Moser model, or in the 3 38 120 1.33 12.6 76.7
case of ethanol inhibition, models like Bulton or Loung have been Mean 38.7 ± 120.7 ± 1.47 ± 0.47 10.8 ± 4.56 47.23 ±
combined to obtain the Moser-Boulton and Moser-Loung kinetic models 4.04 26.01 26.99
described by Eqs. (3) and (4), respectively (Arellano-Plaza et al., 2007).
( )( )
Sε KE culture medium in alcoholic fermentation. As shown in Table 1, the
μ = μmax (3)
KS + S ε KE + E obtained results reveal an average concentration of reducing sugars at
( )( ( )m ) approximately 120 g/L. This underscores the substantial potential of
Sε E agave leaves as a by-product for obtaining fermentable sugars. The rich
μ = μmax 1− (4)
KS + S ε KE sugar content in agave leaves can be attributed to crassulacean acid
metabolism (CAM). Through photosynthesis, glucose is directly pro­
where ε and m are parameters of the Moser and Loung models; μmax is duced in the leaves, and subsequent biological reactions generate fruc­
the maximum specific growth rate; KS is the substrate saturation con­ tose for sucrose synthesis (Michel-Cuello et al., 2008).
stant, and KE is the ethanol inhibition constant. S is the reducing sugars Reports indicate that fresh juice extracted from Agave tequilana
concentration, and E is the bioethanol concentration. leaves typically contains soluble sugars within the range of 30 to 40 g/L
The rate of reducing sugar consumption was described by Eq. (5), (Flores-Méndez et al., 2023; Corbin et al., 2015; Rijal et al., 2016; Yang
with YX/S is a biomass yield per substrate consumed. et al., 2015). The thermal treatment applied to the leaves significantly
dS μX increased the sugar content in the juice to more than twice that of the
= − (5) fresh juice. Waleckx et al. (2008) reported that the thermal hydrolysis
dt YX/S
process of Agave tequilana head fructans yields “cooking honeys” con­
On the other hand, the nitrogen consumption rate was written as, taining between 57.3 and 145.6 g/L of reducing sugars. However, it is
dN ηX noteworthy that this process also produces growth-inhibiting by-prod­
= − (6) ucts such as hydroxymethylfurfural (HMF), whose adverse effects are
dt YX/N
observed between 1000 and 5000 mg/L. According to Waleckx et al.
For ethanol biosynthesis, nitrogen availability plays an important (2008), HMF levels reached 50 mg/kg during the initial 15 h of heat
role, and its consumption dynamics in the fermentation process were treatment of agave heads. Considering the amount of leaves processed
described by David et al. (2010) with a model similar to Michaelis- (50 kg) and the average recovered volume (38.7 L), it has been deter­
Menten, represented by Eq. (7). mined that the HMF concentration remains below inhibitory levels for
N yeast in this process. Further exploration of the thermal hydrolysis
η = ηmax (7) process may uncover nuances in sugar content and by-product formation
KN + N
that impact the overall fermentability of agave leaves.
where N is the ammoniacal nitrogen concentration, ηmax is the maximum In terms of reducing sugar recovery, the traditional thermal hydro­
specific reaction constant, KN is the nitrogen saturation constant, and lysis process demonstrates effectiveness for both agave leaves and heads.
YX/N is a biomass yield per nitrogen consumed. Similar success has been reported for other agave species using different
The ethanol production rate (Eq. (8)) was written in terms of the hydrolysis methods. For instance, Villegas-Silva et al. (2014) increased
Leudeking-Piret equation, and a term referring to the ethanol evapora­ reducing sugars concentration from 40 to 75 g/L by hydrolyzing fresh
tion rate was added [Kp (E - Eti)] (Corona et al., 2019). juice from Agave fourcroydes leaves using sulfuric acid. Using inulinase
enzymes, González-Llanes et al. (2017) achieved a range of 68.42 to
dE 137.46 g/L of reducing sugars after hydrolyzing the fresh juice from
= (αμ + β)X − Kp (E − Eti ) (8)
dt Agave salmiana leaves.
where α is a growth-associated product formation coefficient, β is a non-
growth-associated product formation coefficient, Kp is the mass transfer 3.1.1. Nutrient composition of juice
rate constant, and Eti is the interface ethanol concentration. Nitrogen and phosphorus are crucial for microbial development,
The Muser-Boulton and Muser-Loung Eqs. (2)–(3) were substituted participating in metabolic processes associated with the synthesis of
independently into the system of differential equations. The kinetic amino acids, proteins, and nucleic acids (DNA or RNA). The average
parameters (μmax, KS, Kd, ε, m, KE, YX/S, ηmax, KN, YX/N, α, β, Kp, and Eti) amounts of ammoniacal nitrogen and phosphates in the analyzed juice
were estimated using the ‘ode23tb’ function to solve the system of were 1.47 and 10.84 mg/L, respectively. These nutrient levels, though
equations, coupled with the Nelder-Mead method implemented in the essential, are relatively lower compared to sugars. The proportion of
‘fminsearch’ function in MATLAB, which minimizes the sum of the these nutrients is influenced by factors such as soil fertilization levels,
squared error between the experimental and predicted data. The model crop age, and nitrogen assimilation by endophytic bacteria (Beltran-
fit was evaluated with the coefficient of determination (R2). Garcia et al., 2014; Sánchez-Mendoza et al., 2022). Therefore, supple­
menting the culture medium with nitrogen and phosphorus sources is
3. Results and discussion deemed crucial to ensure the yeast's development and growth from the
inoculum preparation stage to the production phase.
3.1. Characterization of juice
3.2. Statistical analysis of bioethanol production
Following the cooking, shredding, and pressing of agave leaves, the
extracted juice was analyzed to determine its nutrient composition, 3.2.1. Taguchi method for process optimization
including carbon, nitrogen, and phosphorus, for its potential use as a The Taguchi method is employed in this study to determine the
optimal combination of variable factors through an analytical approach.

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E.M. Escamilla-Silva et al. Bioresource Technology Reports 25 (2024) 101807

Table 2 values into a Signal-to-Noise (S/N) ratio, where the “signal” represents
Experimental matrix with the response variable and the value of the S/N ratio. the desirable value, the “noise” signifies the undesirable value, and the
Run Reducing Air flow Agitation pH Ethanol S/N S/N ratio expresses the dispersion around the desired value (Kiran,
sugars (g/L) (vvm) (rpm) (g/L) Ratio 2017; Rout et al., 2021). The S/N is categorized into three groups:
9 120 1.5 700 8.5 19.29 ± 25.69 nominal is better, the-smaller-the-better, and the-larger-the-better (Rout
0.89 et al., 2021). In this study, the-larger- the-better is employed, aiming for
4 90 1 700 4.5 15.67 ± 23.89 high ethanol production in batch fermentation.
0.72 Based on this approach, the variability of the resulting concentration
8 90 0.5 500 8.5 3.04 ± 9.65
0.08
concerning noise factors should be minimal, while variability concern­
6 60 1 300 8.5 6.26 ± 15.80 ing signal factors should be maximized. Table 2 provides an analysis of
0.91 the S/N ratio considering the factors of fermentation and ethanol pro­
3 60 1.5 500 4.5 12.04 ± 21.58 duction (g/L). Additionally, Table 3 reveals that the optimal conditions
0.86
for ethanol production align with those obtained using the main effects
1 60 0.5 700 6.5 17.20 ± 24.69
1.07 plot, where the peak of each variable is chosen, as depicted in Fig. 1. This
2 120 1 500 6.5 17.50 ± 24.85 alignment serves as evidence that the ratio is the-larger-the-better.
0.90 Consequently, the optimum conditions obtained through this approach
7 90 1.5 300 6.5 21.98 ± 26.84 were reducing sugars at 120 g/L, air flow at 0.5 vvm, agitation at 300
0.15
5 120 0.5 300 4.5 42.04 ± 32.47
rpm, and pH at 4.5.
0.88
3.2.2. Analysis of variance (ANOVA) for ethanol production
ANOVA is employed to investigate the relationship between the
Table 3
concentration output and input variables in the ethanol production
Response for the signal to noise ratio. process. The impact of four factors was tested in nine replicate runs, as
outlined in Table 2. The ethanol concentration ranged from 3.04 to
Level Reducing sugars Air flow Agitation pH
(g/L) (vvm) (rpm)
42.04 g/L, reflecting the combined effect of the four factors within their
specific ranges. ANOVA, detailed in Table 4, was utilized to assess the
1 26.28 20.76 23.44 23.25
2 13.57 13.15 10.86 18.90
3 11.84 17.78 17.39 9.53
Table 4
Rank 1 4 3 2
Optimum 1 1 1 1
ANOVA analysis and percentage contribution of each variable.
Source Sum of DF Mean F- p-Value %
squares square value Prob>F contribution
By measuring significant factor-level variances, the Taguchi method
Reducing 826.71 2 413.36 624.90 < 0.0001 42.53
predicts the ideal factor level for optimal outcomes (Rout et al., 2021). sugars
The unique technique used in the Taguchi method involves the Signal- Air flow 176.45 2 88.23 133.38 < 0.0001 9.08
to-Noise (S/N) ratio for process optimization. This approach is crucial Agitation 365.77 2 182.89 276.48 < 0.0001 18.82
to determine the influence of each variable on the output. The S/N ratio pH 568.05 2 284.03 429.38 < 0.0001 29.22
Residual 5.29 8 0.6615
of each experiment is calculated to assess the impact of each variable on Cor Total 1943.76 17
the output. This involves transforming the variability of concentration

Fig. 1. The signal main effect plot value to noise ratio (S/N Ratio) and the corresponding fermentation process variables.

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E.M. Escamilla-Silva et al. Bioresource Technology Reports 25 (2024) 101807

Table 5
Maximum yields and productivities for biomass and ethanol.
Run Maximum yields Maximum productivity

YX/S,max (g/g) YP/S,max (g/g) rX,max (g/L⋅h) rP,max (g/L⋅h)

1 0.451 ± 0.06 0.446 ± 0.10 0.774 ± 0.18 1.165 ± 0.36

2 0.198 ± 0.01 0.232 ± 0.04 0.484 ± 0.01 1.459 ± 0.07
3 0.168 ± 0.05 0.316 ± 0.09 0.406 ± 0.13 1.003 ± 0.07
4 0.382 ± 0.02 0.246 ± 0.03 0.797 ± 0.04 0.871 ± 0.04
5 0.197 ± 0.01 0.407 ± 0.01 1.128 ± 0.06 2.336 ± 0.05
6 0.288 ± 0.02 0.320 ± 0.08 0.474 ± 0.03 1.044 ± 0.15
7 0.264 ± 0.01 2.656 ± 0.84 0.651 ± 0.03 3.664 ± 0.02
8 0.733 ± 0.13 0.156 ± 0.01 2.729 ± 0.12 0.507 ± 0.01
9 0.856 ± 0.10 0.454 ± 0.01 1.423 ± 0.14 1.072 ± 0.05

Fig. 2. Response-surface representation for the concentration of ethanol.

Fig. 4. The ratio between the amounts produced of biomass and ethanol.

values in combination with a low pH.

To validate the optimal conditions, the best parameters for ethanol
production (reducing sugars:120 g/L, air flow: 0.5 vvm, agitation: 300
rpm, and pH: 4.5) were selected, resulting in a maximum ethanol con­
centration of 43.26 ± 2.20 g/L. This way, 43 % of the sugars were
converted into ethanol, with a productivity of 2.4 g/L⋅h, similar to that
observed in Table 2.

3.3. Kinetic analysis and modeling of bioethanol production

Fig. 3. Kinetic profiles of ethanol production.

The kinetic profiles of ethanol production for all experimental runs

contribution of each fermentation variable in the batch study of ethanol are depicted in Fig. 3. Experiment 5 exhibited a higher ethanol pro­
production according to the Eq. (9) (Taiwo et al., 2020): duction (42.06 g/L), reaching an ethanol yield of 0.407 g/g, as shown in
SSV Table 5.
%Contribution = (9) In all fermentations, 6 to 18 h were required to reach peak ethanol
TSS
production (Fig. 3), achieving ethanol productivities ranging from 0.507
Here, SSV represents the sum of the squares of a variable, and TSS is
to 3.366 g/L⋅h (Table 5). However, during the rest of the cultivation
the total sum of squares. The p-value being <0.05 indicates the signifi­
time, a loss of ethanol is observed, potentially attributed to its high
cance of the factors. The results highlight that reducing sugar had the
volatility leading to migration from the culture broth to the gas phase
most substantial influence on ethanol production, as indicated by the
and subsequently discharge with the fermenter exhaust gases (Löser
contribution percentage.
et al., 2005).
Fig. 4 illustrates the relationship between net biomass generation
3.2.3. Analysis of response surface and validation of optimal conditions
and ethanol production. In most experiments, except for 3 and 5,
The 3D response surface graph illustrates interaction effects among
biomass production exceeds ethanol production, resulting in ethanol
the significant factors on ethanol production. Fig. 2 reveals that the
yield relative to biomass (YP/X, g/g) being <1. The highest value cor­
interaction between reducing sugars and pH had the most significant
responds to experiment 5 (YP/X = 2.07 g/g).
impact on ethanol concentration. The region with the highest ethanol
Operational parameters of a bioreactor, such as pH, temperature, and
production was observed when reducing sugars were at their peak
dissolved oxygen, directly influence cellular metabolism. The Crabtree

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E.M. Escamilla-Silva et al. Bioresource Technology Reports 25 (2024) 101807

Table 6 Moser-Boulton and Moser-Loung, were applied and compared. The

Estimated kinetic parameters. estimated values of kinetic parameters (Table 6) were very similar for
Parameters Moser-Boulton Moser-Loung Units both models, except for differences in one yield (YX/N) and some con­
stants (KS, KE, and KN). The fit of the models concerning the experi­
μmax 0.0543 0.0504 1/h
KS 7.9404 23.9688 g/L mental data was very similar, describing 96 % of the fermentation
Kd 0.0198 0.0199 1/h process. A slight difference was observed in the simulation of ethanol
ε 1.4082 1.5959 dimensionless production (Fig. 5). Both models showed a high coefficient of determi­
m – 2.6476 dimensionless nation (R2) of 0.961 and 0.962 for Moser-Boulton and Moser-Loung,
KE 191.64 126.4046 g/L
YX/S 0.3188 0.3209 g/g
respectively, suggesting their ability to explain and predict the
ηmax 0.0006 0.0005 1/h observed data.
KN 1.5391 5.1799 g/L To determine the “best” model, the Akaike Information Criterion
YX/N 1.1945 0.3468 g/g (AIC) was applied, considering the model with the lowest AIC as the
1.3123 1.6512 g/g
α
most accurate (Rigdon et al., 2023). The corrected Akaike Information
β 0.0369 0.0126 g/g⋅h
Kp 0.0691 0.051 1/h Criterion (AICc) is recommended for small sample sizes, where AICc is
Eti 0.1891 0.331 g/L calculated using Eq. (10) (Sene et al., 2023).
( )
OF 2k(k + 1)
AICc = ne⋅ln + 2k + (10)
effect, where competition for substrate occurs between alcoholic ne ne − k − 1
fermentation and aerobic metabolism, is associated with Saccharomyces
Here ne is the number of points concerning the independent time
cerevisiae (Panikov, 2011). In this study, fermentations predominantly
variable, k is the number of estimated parameters, and OF is the objec­
exhibited the aerobic metabolism associated with this effect.
tive function.
Several experimental strategies have recently been studied to pro­
The AICc values for the models with the Moser-Boulton and Moser-
duce bioethanol from Agave tequilana leaves using Saccharomyces cer­
Loung kinetic equations were − 65.22 and − 53.76, respectively.
evisiae. For example, Avila-Gaxiola and Avila-Gaxiola (2022) carried out
Following the analysis scheme Sene et al. (2023) proposed for small
dehydration and pulverization of leaves, followed by enzymatic hy­
samples, the “best” model is chosen based on the lowest AICc value. In
drolysis and fermentation. This methodology achieved a sugars-to-
this case, the model with the Moser-Boulton equation is deemed more
ethanol conversion yield of 41 % (YP/S = 0.41 g/g) and a production
suitable for describing the ethanol production process using agave
of 12.2 g/L. Rijal et al. (2016) extracted the juice from the leaves, pro­
leaves. The number of kinetic parameters can notably impact model
ceeding to its hydrolysis using dilute sulfuric acid, reaching maximum
selection based on the AICc criterion. According to this criterion, the
fermentation productivity of 1.77 g/L⋅h and a concentration of 12.4 g/L
Moser-Boulton equation demonstrates superior performance, providing
ethanol. Comparisons with previous studies indicate consistent values in
a more accurate representation of the observed data.
terms of ethanol production despite variations in the methodology.
Modeling of biomass growth, ethanol production, and substrate
consumption (sugars and nitrogen) aimed to describe the batch culti­
vation of S. cerevisiae under optimal conditions. Two kinetic laws,

Fig. 5. Comparison between experimental data (reducing sugars, ▴; nitrogen, ■; ethanol, ◆; biomass, ●) and simulations (model with the Moser-Boulton equation,
“continuous line”; model with the Moser-Loung equation, “discontinuous line”).

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E.M. Escamilla-Silva et al.
4. Conclusions
This study highlights the potential of agave leaves as a valuable raw
material for obtaining fermentable sugars through heat treatment. The
Taguchi method was effectively employed to optimize fermentation
process variables, with the initial concentration of reducing sugars
identified as the most influential factor. Under optimal conditions, the
study achieved ethanol concentrations of 43.26 g/L, yielding 43 %,
aligning with reported literature values for agave leaves. The developed
models successfully described cellular growth, substrate consumption
(sugars and nitrogen), and ethanol production under optimal conditions,
exhibiting good fits with R2 values exceeding 0.95. The model com­
parison using the Akaike Information Criterion (AICc) favored the
Moser-Boulton equation, establishing its suitability for describing
ethanol production from agave leaves. These findings contribute to
developing an operational scheme for assessing and predicting ethanol
production in continuous and fed-batch cultivation scenarios.
CRediT authorship contribution statement
Eleazar Máximo Escamilla-Silva: Writing – review & editing, Re­
sources, Methodology, Investigation, Conceptualization. Giovanni
Alexander Escamilla-García: Supervision, Resources, Investigation,
Conceptualization. Filiberto Rocha-Arriaga: Methodology, Investiga­
tion. Miriam Granados-Vallejo: Writing – review & editing, Method­
ology, Investigation. David Antonio Flores-Méndez: Writing – original
draft, Validation, Methodology, Investigation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgement
Financial support for Filiberto Rocha-Arriaga originated from Con­
sejo Nacional de Ciencia y Tecnología (CONACyT) through a master's
scholarship.
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