BioEnergy Research

https://doi.org/10.1007/s12155-020-10197-6

Optimization of Chemical Pretreatments Using Response Surface

Methodology for Second-Generation Ethanol Production from Coffee
Husk Waste
J. L. Morales-Martínez 1 & M. G. Aguilar-Uscanga 2 & E. Bolaños-Reynoso 1 & L. López-Zamora 1

Received: 23 June 2020 / Accepted: 23 September 2020

# Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
Within the strategies of substitution of energy from fossil fuels by renewable energies, the research is based on second-generation
ethanol production (2G ethanol). One of the raw materials considered for this is residual biomass of the coffee industry, being the
subject of study here. The cellulose contained in the coffee husk (coffee husk or coffee skin or coffee exocarp or pericarp) was
maximized using pretreatment processes. In dilute acid hydrolysis (DAH), using a fixed 1:6 w:v solid to liquid ratio (SLR),
process times (35, 45, 55 min) and H2SO4 concentrations (3, 4, 5% v/v) were evaluated, achieving 53.63% hemicellulose
removal. A delignification process resulted in 58.82% lignin removal, evaluating the effect of process times (30, 35, 40 h) and
SLR (1:8, 1:10, 1:12 w:v) at a fixed concentration of 8% v/v H2O2. A 115.59 g/L glucose concentration was obtained with an
interaction of fixed concentrations of 4–6% w/w Cellic CTec3 enzyme and 6:1 to 1:12 v:w SLR. The fermentation process
considered the composition variation of the culture medium (enriched culture C1 and non-enriched culture C2), generating
ethanol at 48.19 and 29.02 g/L concentrations, respectively. Fermentation efficiency (ηf) was improved from 21.99 to 81.74%
with the addition of inorganic nutrients (KH2PO4, (NH4)2SO4, and MgSO4·7H2O). These results confirmed that the optimization
of the pretreatments in coffee husk waste favored the cellulose production and facilitated the enzymatic process to produce a high
glucose concentration, revealing these residues as a carbon source promising for second-generation ethanol production.

Keywords Response surface methodology . Glucose . Enzymatic hydrolysis . Renewable source . Cellulose

Introduction to the increase in population and new consumer habits.

In the early 1970s, rising oil prices initiated an energy crisis
which together with global warming and the depletion of oil
reserves [1–3] aroused great interest in the study of renewable
resources in order to produce alternative energy, allowing
decarbonization in the environment and reducing the emis-
sions of greenhouse gases by ~ 43% [1, 4, 5]. Different re-
sources used to produce alternative energy have been studied.
Currently, the world produces a large amount of waste daily
(municipal, food, beverages, agricultural, and industrial) due
* L. López-Zamora
llopezz@orizaba.tecnm.mx
1
Graduate Studies and Research Division, National Institute of
Technology of Mexico/Orizaba Institute of Technology, Oriente 9
852, 94320 Orizaba, Veracruz, Mexico
2 ronmental problems such as water eutrophication, acidifica-
Food Research and Development Unit, National Institute of
Technology of Mexico/Veracruz Institute of Technology, Calz. M.A.
de Quevedo 2779, 91860 Veracruz, Mexico
Dumping the waste directly to the landfills may cause serious
environmental problems due to the existence of organic com-
pounds that demand excessive amounts of oxygen to be de-
graded [6–11]. Utilization of the lignocellulosic agro-residues
which are non-food sources, since they are cheap, unutilized,
and locally available most of the time, has been used for the
production of bioethanol, which allows it to become a renew-
able fuel, capable of replacing crude oils because of its posi-
tive net energy balance. Such residues, such as rice or coffee
husks, are chemically made up of cellulose (35–50%), hemi-
celluloses (25–35%), and lignin (10–15%) [1, 3, 7, 12–15].
Green coffee beans are deemed to be a second commodity
only to petroleum in terms of currency traded worldwide.
World exports reached 10.49 million 60-kg bags, being the
third largest volume registered in May [16, 17]; it is estimated
that 1 ton of coffee generates more than 600 kg of waste [3,
18]. The improper disposal of coffee husk can generate envi-
tion and salinization of soils, and the toxic effects on some
biological processes, aspects which have limited its

application in agriculture [19–21]. Coffee husk (CH) is a
promising alternative biomass resource for bioethanol produc-
tion, given the large amount annually produced [22, 23]. CH is
rich in fermentable sugars, accounting for approximately 37–
42% of waste [24], which can be utilized as a carbohydrate
source for bioethanol production. CH has a high carbohydrate
content (70%), of which 7–29% are in the form of hemicellu-
lose, 20–43% cellulose, and 6–10% lignin [4, 7, 17, 25].
Despite the high carbonate content compared with other bio-
masses, information on the use of CH in ethanol production is
limited, excluding previous studies regarding their use as an-
tioxidants or as an activated carbon resource [22, 26–30].
One of the main limitations for bioethanol production from
agricultural residues is the low biodegradability of lignocellu-
losic materials [31]. Efficient utilization of lignocellulose re-
quires an initial pretreatment step to minimize lignin and
hemicellulose for effective enzymatic hydrolysis [22,
32–35]. The production of bioethanol utilizing coffee husk
involves pretreatment, enzymatic hydrolysis, and fermenta-
tion [21, 36]. The pretreatment process must separate the pen-
toses without degrading them, does not generate residues, and
should be highly efficient in the removal of lignin. The chem-
ical treatments used to cover these requirements, however,
tend to increase process costs, so chemical agents must be
selected that allow maximum use, subsequent recovery, and
use of generated byproducts. Acid and alkaline pretreatments
hydrolyze lignin and make the cellulose and hemicellulose
easily available for the hydrolytic enzymes to produce fer-
mentable sugars [1, 3, 7, 12–14].
After pretreatment, the resulting material is enzymatically
hydrolyzed, using cellulase enzymes. Hydrolysis also helps
verify the efficacy of the pretreatment processes by demon-
strating the success of conversion of cellulose to fermentable
sugars. The enzymatic hydrolysis of cellulose is a multi-step
reaction that is carried out in a heterogeneous system, in which
insoluble cellulose is fractioned at the solid-liquid interface.
The majority of cellulases come from fungi and consist of
three types of enzymes, i.e., endo-1,4-β-glucanase, exo-
1,4-β-glucanase, and β-glucanase [37–42].
However, the lack of information on optimal pretreatments
to CH necessitates the use of methodologies for designing
experiments to find the optimum response values of interest.
Response surface methodology (RSM) is an experimental
technique and analysis strategy where optimal operating con-
ditions of a process are re-pinpointed, using a limited experi-
mental range for each factor. The experimentation of the cen-
tral composite design (CCD) type is an optimization method-
ology applied using two or more factors, generally efficient in
terms of the number of experiments. It is a rotatable design
extrapolating the experimental region [43, 44].
The objective of this study was to use RSM, to maximize
hemicellulose and to remove lignin in CH residues by chem-
ical hydrolysis, as a pretreatment for glucose production. The
Bioenerg. Res.
cellulose obtained from the delignification process was sub-
jected to a saccharification process using the commercial en-
zyme Cellic CTec3, and the reducing sugars produced were
used in both enriched and unenriched fermentative cultures in
order to find maximum productivity using Saccharomyces
cerevisiae ITV-01.
Methods and Materials
Materials
CH was obtained from La Fortuna coffee processing plant,
located in the municipality of Huatusco, Veracruz, Mexico,
from their October 2018 to March 2019 harvest. It was dried
by solar radiation for 48 h, prior to storage. Saccharomyces
cerevisiae ITV-01 was isolated from sugarcane in the
Veracruz Institute of Technology by Ortiz-Zamora [45] and
kept in refrigeration at 4 °C for the maintenance.
Chemical Hydrolysis
CH was subjected to dilute acid hydrolysis (DAH) to destroy
the hemicellulosic structure; pretreatment times (35, 45, and
55 min) and H2SO4 concentrations (3, 4, and 5% v/v) were the
independent variables considered at three levels in a CCD, the
result being hemicellulose removal (%). The pretreatment was
conducted at SLR 1:6 w/v in samples of 20 g, carried out in an
autoclave (AESA brand, model CV-250, Mexico) at pressure
of 1.2 kg/cm2 [37, 46].
The CH obtained from DAH was subjected to a peroxide-
alkaline pretreatment (PAT) [37, 47]. A central composite
design with three levels was used, in samples of 20 g, and
the independent variables were the pretreatment times (20,
35, and 40 h) and SLR (1:8, 1:10, and 1:12 w/v), using 8%
v/v H2O2 solution, adjusting to pH 11.5 with 10 M NaOH.
The response variable was % lignin removal.
Enzymatic Hydrolysis Study
Once the cellulose was obtained, the study of the enzymatic
hydrolysis [37] was carried out, using 5 g of CH pretreated
using Cellic CTec3 (Novozymes, Mexico City, Mexico) as
the enzyme and CH3COONa 0.05 M at pH 5 as a buffer
solution. A design 22 with one replica was used; the indepen-
dent variables were enzyme concentration (4 and 6% w/v) and
SLR (1:6 and 1:12 w/v) being the glucose concentration (g/L)
response variable. The process was carried out in an incubator
(STIK, model Shaking PS-B2125, Mexico) at 200 rpm, 50 °C
for 72 h.
The validation of the experimental conditions obtained
from the factorial design was carried out in a my-Control
bioreactor (Applikon, model H87-PZ, Netherlands) using
Bioenerg. Res.

176.1 g of lignocellulosic fiber with an enzyme concentration Biomass Analysis

of 6% w/w and SLR of 1:6 w/v.
Biomass determination was carried out using a correlation
between optical density (620 nm) measured in a UV-Vis spec-
Inoculum and Fermentation Conditions
trophotometer (Thermo Spectronic, Genesys 20, Mexico) and
dry weight of the samples. The correlation was verified using
The inoculum and fermentation conditions were performed
direct cell counting with a Neubauer chamber (Blau Brand,
based on the methodology of Diaz-Nava et al. [48], where
Germany).
yeast culture was maintained at 4 °C in Petri dishes containing
an agar medium with the following composition: 20 g/L agar-
Substrate and Product Analysis
agar, 100 g/L glucose, and 10 g/L yeast extract. For inoculum
preparation, cells were transferred from the maintenance me-
To evaluate substrates and products, the samples were centri-
dium to a 250-mL Erlenmeyer flask containing 100-mL cul-
fuged for 10 min at 10,000 rpm (Eppendorf Centrifuge 5424,
ture medium composed of 5 g/L KH2PO4, 2 g/L (NH4)2SO4
Germany) and the supernatant was stored at − 5 °C until its
g/L, 0.4 g/L MgSO4·7H2O, and 1 g/L yeast extract. The solu-
analysis. Glucose, ethanol, acetic acid, and glycerol concen-
tion was aseptically mixed to obtain the desired concentration
trations were determined by high-performance liquid chroma-
of each nutrient in the culture medium. The inoculated flasks
tography Waters 600 TSP Spectra System (Waters, Milford,
were incubated in a shaking incubator (STIK, model PS-
MA, USA) using a Shodex SH1011 column (8 × 300 mm)
B2125, Mexico) at 200 rpm, 30 °C for 12 h. The fermentations
specific for sugars, organic acids, and alcohols; the column
were carried out in 250-mL flasks with an operation volume of
was maintained at 55 °C, mobile phase was 0.05 N sulfuric
160-mL fermentable sugars and an initial cell concentration of
acid, with a flow rate of 0.600 mL/min, and the components
3 × 106 cell/mL. Two culture types were used, i.e., enriched
were detected by an index refraction detector (Waters 2414,
culture (C1) composed of 5 g/L KH2PO4, 2 g/L (NH4)2SO4,
TSP Refracto Monitor V, Waters). The software used was
1 g/L MgSO4·7H2O, and 2 g/L yeast extract and unenriched
Empower (Waters), which directly reports the concentration
culture (C2) with only 1 g/L yeast extract. The fermentation
of the sample through a correlation with previously made
process was carried out in a shaking incubator (STIK, model
standards (anhydrous glucose, food grade Golden Bell
PS-B2125, Mexico) at 30 °C, 200 rpm for 32 h.
Reagents in concentrations of 100 and 0.33 g/L; anhydrous
glycerol, reagent grade, baked in concentrations of 10 and
Analytical Methods 0.03 g/L; glacial acetic acid, reagent grade, baked in concen-
trations of 30 and 0.1 g/L; and anhydrous absolute ethyl alco-
Lignocellulosic Composition Analysis hol, reagent grade, baked in concentrations of 100 and 0.03 g/
L). All the samples were subjected to detoxification process
The lignocellulosic composition analysis of the CH lignocel- with 5% (w/v) zinc sulfate and 0.3 M barium oxide before
lulosic fiber was performed using a double acid hydrolysis HPLC analysis.
[49], the first hydrolysis carried out for 60 min using a 72%
H2SO4 solution at 30 °C (Thermo Haake C10-B3 water bath, Statistical Analysis
Germany), and the second hydrolysis carried out using a 4%
H2SO4 solution in an autoclave (AESA, CV-250, Mexico) at An analysis of variance (ANOVA) with a p value of 0.05 was
121 °C and 1.1 atm for 60 min. Glucose, xylose, acetic acid, performed over the data, using NCSS 2007 statistical software
furfural, and 5-hydroxymethylfurfural (5-HMF) were ana- (7.1.20 version, NCSS, LLC, Kaysville, UT, USA) to predict
lyzed by HPLC (Waters 600 TSP Spectra System, Waters, the mathematical model and generate statistical graphs.
Milford, MA, USA) using a Shodex SH1011 8 Å–300 mm Kinetic modeling was carried out using Matlab software
column (H203153, Japan), at 50 °C, a 5-mM sulfuric acid (R2018b version, The MathWorks, Inn).
mobile phase, a 0.6 mL/min flow rate, and a refractive index
detector (Waters 2414, TSP Refracto Monitor V, Waters,
Milford, MA, USA). Acid-soluble lignin was analyzed in a Results and Discussion
UV-Vis spectrophotometer with a diode array detector
(Hewlett-Packard 8452 A, Germany); insoluble lignin mois- CH Structural Composition
ture was determined according to the AOCS methodology
(Ab 2-49) using a vacuum oven (Thermo Scientific Lab- Lignocellulosic composition of the CH used in the present
Line, 3608-1CE, USA), and ash determination was carried study is shown in Fig. 1; the results obtained for cellulose
out using the AOCS methodology (Ba 5ª-49) in a furnace (34.55%) were higher than those found by Ulloa et al. [50]
(Thermo Scientific Thermolyne, model FB1310M-33, USA). which was 28.6% and Mussatto et al. [7] which was 8.6%, but

Fig. 1 Lignocellulosic
composition of CH in initial, post-
DAH, and post-PAT conditions
the hemicellulose content (13.23%) was lower (36.7%). Elias
[51] reported values of 17.7% cellulose, which was lower than
that obtained in this work. de Carvalho et al. [52] reported
values of 25.5% hemicellulose and 33.5% lignin, which were
higher than those obtained in this work. Salmones et al. [53]
found concentrations of 24.5% cellulose, 17.1% hemicellu-
lose, and 26% lignin. The composition of the CH usually
varies based on factors such as the crop variety, cultivation
conditions, and the chosen method of processing [54], which
explains the differences in the composition reported by other
authors [21, 55]. This indicates that the solid residue seems
quite promising for ethanol production, given the high per-
centage of sugars available for fermentation. Cellulose present
in CH, after PAT, is the most important component in this
work because it is the specific carbon source for its conversion
to glucose and subsequent biosynthesis to ethanol, while the
hemicellulose fraction remains as a secondary component by
rising to a byproduct of added value such as xylose [56, 57].
Chemical Pretreatments
Acid Hydrolysis
The hemicellulose removal obtained by the established CCD
is shown in Table 1. Hemicellulose removed ranged from
15.78 to 53.48%, about four times higher than that reported
by [1] and 5.4% higher than that reported by [52]. The DAH
generated products of biological and commercial interest (fer-
mentable sugars) such as xylose in a concentration of 18.19 g/
Bioenerg. Res.
L in a time of 45 min and concentration of H2SO4 of 5.68% v/
v and a maximum concentration of glucose of 10.13 g/L under
conditions of 55 min and concentration of H2SO4 of 5% v/v
(Fig. 2). Dessie et al. [58] reported 6.65% lower than the
xylose concentration, Dadi et al. [59] obtained 6.06 g/L of
glucose 76.61% less with respect to this work, and Urbaneja
et al. [60] reported 468% and 60.5% lower than xylose and
glucose concentrations, respectively.
The acid hydrolysis process did not present formation of
inhibitors such as furfural and 5-hydroxymethylfurfural (5-
HMF); however, a maximum concentration of acetic acid of
3.01 g/L was obtained when evaluating an equiradial point of
the CCD (run 11) in a time of 45 min and concentration of
H2SO4 of 5.68% v/v (Fig. 2), presenting concentrations below
the limit of allowed inhibitors (less than 6 g/L) for acetic acid
reported by Parajo et al. [61].
RSM application generates a regression polynomial
(Eq. 1), which represents the empirical relationship between
the response (hemicellulose removal) and the variables
evaluated.
Y 1 ¼ −414:2124 þ 12:0959X 1
þ 92:6338X 2 −0:1069X 21 −7:8631X 22 −0:5781X 1 X 2 ð1Þ
The analysis of variance (ANOVA) (Table 2) showed that
the experimental regression is not significant at a significance
level of 95%. The model showed a good fit with experimental
Bioenerg. Res.

Table 1 Levels and results of the DAH process and CH delignification using CCD

Run X1 (time, min) X2 (H2SO4,% v/v) Y1 (hemicellulose X3 (time, h) X4 (SLR, w/v) Y2 (lignin removal, %)
removal, %)

1 −1 −1 35.88 −1 −1 47.70
2 1 −1 47.06 1 −1 40.98
3 −1 1 45.35 −1 1 42.34
4 1 1 33.41 1 1 48.09
5 0 0 53.62 0 0 58.82
6 0 0 49.75 0 0 57.36
7 0 0 53.48 0 0 58.74
8 − √2 0 15.78 − √2 0 46.27
9 √2 0 25.33 √2 0 53.61
10 0 − √2 16.47 0 − √2 51.49
11 0 √2 40.53 0 √2 42.28

data given that the coefficient of determination (R2aj ) has a
value of 0.8252, implying that 82.52% of the variable data
conforms to the model.
The response surface analysis (Fig. 3a) shows that the
hemicellulose removal process is effective at long operating
times ranging from 30 to 50 min with removals greater than
40%, while at low acid concentrations, there is no significant
change in the polysaccharide structure of the lignocellulosic
complex, reaching removals similar to those of run 10
(Table 1).
The model was validated through a canonical analysis (Eq.
2) whose negative eigenvalues guarantee process maximiza-
tion. The model predicts a theoretical removal of 54.19%, at
the stationary points of 45.11 min (X1) and 4.23% v/v (X2) that
are within the range defined in the contour graph (Fig. 3b) as
the optimum region of maximization of hemicellulose

Fig. 2 Concentration of
metabolites generated from CH
DAH
 Bioenerg. Res.

Table 2 ANOVA on hemicellulose content after DAH Table 3 ANOVA on lignin content after PAT

Effect SS DF MS F value p value Effect SS DF MS F value p value

Regression 1573.9830 5 314.7966 4.72 0.0570 Regression 379.3605 5 75.8721 112.18 0.8518
Total error 333.2033 5 66.6404 Total error 31.99284 5 13.2002 0.1481
Lack of fit 323.5559 3 107.8520 22.36 0.43 Lack of fit 64.64848 3 21.5494 31.86 0.1451
Pure error 9.6463 2 4.8231 Pure error 1.352733 2 0.67636 0.0030

removal (52.03%), which comprises a range in H2SO4 con-
centration of 3.6 to 4.38% v/v and a process time of between
40 and 51 min.
by1 ¼ −414:21−7:87W 21 −0:09W 22 ð2Þ
The resulting conditions were experimentally validated,
obtaining a hemicellulose reduction of 53.63%, 2.56% greater
than the removal found by the CCD, while the degradation of
pentoses contained in the lignocellulosic fibers generated xy-
lose and glucose concentrations of 18.19 g/L and 6.10 g/L,
respectively, with a presence of acetic acid of 2.98 g/L. The
lignocellulosic composition of CH after DAH is observed in
Fig. 1, obtaining an increase in cellulose of 25.55% compared
with the initial content of the substrate.
Peroxide-Alkaline Pretreatment
The lignin removed (Y2) after PAT is presented in Table 1.
Lignin present in the CH pretreated with acid was reduced
between 40.98 and 58.82%. In order to determine the optimal
pretreatment parameters, it was necessary to carry out a statis-
tical analysis, showing that there is no significant variation
between the experiments performed when evaluating the
ANOVA (Table 3) with a significance level of 0.05.
Equation 3 represents the empirical relationship between
the response (lignin removed (%)) and the evaluated variables.
The model shows a good fit with the experimental data since
the determination coefficient (R2aj ) has a value of 0.8518,
implying that 85.18% of the data of the variables adjusted to
the model and only 14.82% of the total variation could not be
explained by it. The lack of adjustment is not significant,
indicating that the model is well adapted to the response.
Therefore, the model is suitable for predicting lignin (%) ac-
cording to the established conditions.
Y 2 ¼ −504:9038 þ 21:9050X 3
þ 35:7088X 4 −0:2643X 23 −1:2756X 24 −0:3117X 3 X 4 ð3Þ
The analysis of the response surface (Fig. 4a) shows the
behavior of the different combinations of effects, where the
central points (runs 5, 6, and 7) produced the highest removal
effect in the process, finding an average decrease in the lignin
content of 58.30%. The PAT results found in this work im-
proved the delignification effect 5.7 times more than what was
presented by Niglio et al. [62], who removed only 10.16% of
the lignin content present in coffee residues.
An optimization analysis determined by maximization of
lignin removal theoretical of 49.79% was obtained at the sta-
tionary points of 35.75 h (X3) and 9.62:1 v/w (X4) that are

Fig. 3 RSM of hemicellulose removal process; time (h) vs. sulfuric acid (% v/v). a Response surface graph (RSG). b Contour plot delimiting DAH
process operation boundaries
Bioenerg. Res.
Fig. 4 RSM of lignin removal process; time (h) vs. sulfuric acid (% v/v). a Response surface graph (RSG). b Contour plot delimiting PAT process
operation boundaries
within the range defined in the contour graph (Fig. 4b), which
comprises a process time of between 33.8 and 37.85 h and
SLR of 1:8.75 to 1:10.52 w/v. The model was validated
through a canonical analysis (Eq. 4) whose negative eigen-
values guarantee process maximization.
by2 ¼ −504:90−1:2991w23 −0:24w24 ð4Þ
The resulting conditions were experimentally validated,
obtaining a lignin reduction of 58.82%, being slightly higher
(0.89%) than the removal found by the CCD, while the ligno-
cellulosic composition after PAT leaves exposed a content of
85.47% of cellulose available for the next saccharification pro-
cess, in addition to a hemicellulose content of 5.02% and a final
lignin content of 6.57%, as observed in Fig. 1. Menezes et al.
[40] studied the alkaline pretreatment of coffee pulp using a
mixture of NaOH (4% w/v) and Ca(OH)2 (4% w/v) at 121 °C
for 25 min and obtained a lignocellulosic composition of
45.57% hemicellulose and 39.34% of lignin, many times
higher than those found in this work, while cellulose was
7.7% higher than that found by them (79.37%). Guarneros
et al. [37] performed peroxide-alkaline pretreatment of sweet
sorghum bagasse with H2O2 solution (4% v/v), 16:1, SLR, for
45 h, yielding 79.03% useful cellulose, 8.13% lower than in
Table 4 Glucose production from
a factorial design 22 Run (X5) Enzyme
concentration (% w/w)
1 −1
2 +1
3 −1
4 +1
this work. Xu et al. [63] studied the treatment of grasses using
concentrations of 0.5 to 2% NaOH at 121 °C with incubation
times of 15 min, 30 min, and 1 h and obtained maximum
removal of lignin at 85.8% (lignin 14.2%) using 2% NaOH
for 1 h; this is slightly better than the highest result
obtained in this work. Wang and Cheng [64] performed pre-
treatment of coastal Bermuda grass with sodium hydroxide
(1%) and calcium hydroxide (0.1 g Ca(OH)2/g dry biomass)
at 121 °C for 30 min and obtained approximately 75% and less
than 20% lignin removal, respectively. Silverstein et al. [65]
investigated the chemical treatment of cotton stalks and stems,
and reported that among the four methods tested (NaOH,
H2SO4, H2O2, and ozone), pretreatment with NaOH showed
the highest level of delignification (65.63% using 2% NaOH
for 90 min at 121 °C).
Alkaline pretreatments increase the digestibility of cellu-
lose and are more effective in solubilizing the lignin, and
besides have a limited effect on the solubilities of the cellulose
and hemicellulose, when compared with acid or hydrothermal
pretreatments [66]. To get a successfully enzymatic hydrolysis
of the cellulose present in the lignocellulosic biomass, it is
very important to use a suitable pretreatment in order to re-
move the lignin and hemicellulose [67] because hemicellu-
loses and lignin form a physical barrier involving cellulose.
(X6) SLR (w/v) (Y3) Glucose (g/L)
−1 44.99 ± 0.28
−1 112.30 ± 0.179
+1 32.86 ± 0.144
+1 54.88 ± 0.096
 Bioenerg. Res.

Fig. 5 Glucose production. a

Effect of factors and their levels in
the 22 design. b Enzymatic
kinetics for glucose production
from CH

Glucose Production polynomial (Eq. 6). The proposed model has a determination
coefficient (R2aj ) of 0.9991, implying that 99.91% of the var-
To verify whether the cellulose present in the pretreated CH iable data conforms to the model and only 0.09% of total
was available, an enzymatic hydrolysis was performed with variation could not be explained.
Cellic CTec3 (Novozymes, Mexico City, Mexico). The results
of the factorial design 22 (Table 4) allowed us to know the g 
interaction and influence between enzymatic load and SLR on Glucose =L ¼ −0:024t 2 þ 3:3194t−0:2541 ð6Þ
the production of glucose from CH previously treated by
DAH and PAT. Figure 5a shows that low levels do not present Experimentally, a 115.59 g/L glucose concentration
a significant influence, obtaining product concentrations be- was obtained, representing an overall yield of 69.35%.
low 54.98 g/L, while the highest glucose production was Scully et al. [68] report a glucose production from coffee
112.48 g/L from an enzyme concentration of 6% w/w and residues to 120 h, a 4.97-g substrate, and an enzymatic
an SLR of 1:6 w/v, whose conditions were validated at the load of 1246 μL using commercial cellulases (Celluclast)
reactor level, exceeding by 2.69% (115.59 g/L) the glucose reaching a glucose concentration of 26.05 g/L, while
obtained in the 22 study. Shankar et al. [1] using coffee cherry husk only reached
The saccharification process generated a first-order model 20.11 g/L of reducing sugars in 24 h at 50 °C and pH of
(Eq. 5) based on the ANOVA (Table 5); it is observed that 9, using an enzymatic cocktail. Martin et al. [69] studied
both factors are significant with a significance level of 0.05, the pretreatment of sugarcane bagasse by steam explosion
evidencing the existence of a maximum concentration of using temperatures of 205 and 215 °C, and hydrolyzed
product in levels (+ 1, − 1), so the enzymatic load must be using Celluclast 2L (75 FPU/g and 12 IU/g for cellobiase)
optimized, as it shows a generation of glucose in its high levels and Novozym 188 (392 IU/g for cellobiase) and obtained
while the SLR did not show an upward behavior in the glu- 23.5 and 21 g glucose/L hydrolysate, respectively, after
cose concentration as seen in Fig. 5a. enzymatic hydrolysis. These results are lower than those
found in this work (115.59 g glucose/L). Menezes et al.
Y 3 ¼ −128:53 þ 51:357X 5 þ 11:428X 6 −3:7746X 5 X 6 ð5Þ [40] studied the pretreatment of coffee pulp with 4%
NaOH and 0% Ca(OH)2 for 25 min in an autoclave at
121 °C using enzyme Celluclast 1.5L (69.106 FPU/mL,
Figure 5b shows the kinetic behavior of the saccharification
Novozymes, Brazil) and obtained 27.02 g/L of glucose in
kinetics of experimental results versus the regression
all cases, four times less than that found in this study. CH
is shown as a competitive substrate to improve efficiency
with respect to the saccharification process of sweet sor-
Table 5 ANOVA of the saccharification process from the design 22
ghum juice bagasse, obtaining a yield of 42.87%, higher
Effect SS DF MS F value p value than the 48.54% reported by Guarneros et al. [37] from
optimal process conditions with a 5% w/w enzyme con-
Regression 7434.53 4 1858.63 28,428.49 0.0000
centration, 51 h, and SLR of 1:5 w:v.
X5 2418.25 1 2418.25 36,988.14 0.0000
Figure 6 shows the general balance of material and valida-
X6 3990.37 1 3990.37 61,034.29 0.0000
tion conditions applied to 1 kg of raw material (CH) generat-
Total error 1125.82 7 1125.82 0.0000
ing 0.420 L of substrate with a final concentration of glucose
Pure error 0.20 3 0.07 0.0000
of 115.59 g/L, reaching a use of 42% of the final product
Bioenerg. Res.
Fig. 6 Balance of the process of glucose production from optimization DAH and PAT by enzymatic saccharification
obtained from chemical treatments and enzymatic
biotransformation.
Fermentation of Hydrolyzed CH
To confirm that the glucose released during enzymatic hydro-
lysis was available for the yeast, fermentation was performed.
The consumption of hydrolyzed CH substrate varies accord-
ing to the culture (Fig. 7). In culture C1, substrate consump-
tion was 100%, reaching the maximum ethanol concentration
(48.19 g/L ± 0.70) in 20 h, 66.05% higher than that reached by
crop C2 at 12 h (29.02 g/L ± 0.43), where only 78.71% initial
substrate was consumed. Productivities for C1 and C2 were
2.40 and 2.41 g/L, respectively.
Culture C1 fermentation kinetics presented a specific con-
sumption speed (Vs) of 1.35 gg−1 h−1, reaching a maximum
cell growth (μmax) of 0.27 h−1 at 12 h; a similar effect took
place with culture C2, with a μmax of 0.25 h−1, requiring 15 h
of fermentation at a Vs of 1.72 gg−1 h−1. Both cultures showed
Fig. 7 Kinetics of lignocellulosic
ethanol production from coffee
residues. (a) C1 and (b) C2. Blue-
filled circle represents substrate.
White-filled circle represents
EtOH
an average biomass concentration, 5.36 and 3.38 g/L for C1
and C2 respectively.
Menezes et al. [40] carried out a batch fermentation using a
sugar concentration of 34.23 g/L (2.3 times lower than the
fermentable sugar concentration available in this study), sup-
plemented with 0.1 g/L KH2PO4, 0.1 g/L (NH4)2SO4, and
0.2 g/L MgSO4·7H2O, generating an ethanol concentration
of 11.99 g/L, 301% lower than the enriched culture and 83%
lower than the unenriched culture. The 0.416 g/g maximum
YE/S value was 18.59% higher than the theoretical yield
(0.511 g/g) and 26.92% higher than that obtained with the
C2 crop (YE/S = 0.112 g/g), this being evidence of the need
for nutrimental contribution through inorganic salts, while
Shankar et al. [1] report 12.19 g/L of ethanol from coffee
cherry husk hydrolysates using Saccharomyces cerevisiae,
from 20.11 g/L of sugars in a time of 72 h, four times less
than that obtained in this study.
Mussatto et al. [70] performed fermentative kinetics of cof-
fee hydrolysates using 3 g/L yeast extract (S. cerevisiae, RL-
11), generating a YE/S of 0.26 g/g, this being 57.69% lower
 Bioenerg. Res.

Table 6 Comparison of ethanol

concentrations, productivities, Author Substrate EtOH (g/L) Q (g/L/h) YE/S (g/g)
and yields of lignocellulosic crops
Martín et al. [69] Sugarcane 9.9 1.26 0.37
Wang et al. [71] Sweet sorghum 38.00 1.28 -
Kleingesinds et al. [72] Corn cob 26.60 1.02 0.37
Govumoni et al. [73] Wheat straw 24.40 0.67 0.44
Sathendra et al. [74] Palm wood 22.90 0.27 -
This study Coffee cherry husk 48.19 2.40 0.41

than the yield reported in this study for the enriched culture
(0.416 g/g), despite having used a 50% higher concentration
of yeast extract. The fermentation efficiencies (ηf) of culture
C1 and C2 were 81.74% (62.82% lower than that reported by
Mussatto et al. [70] of 50.2%) and 21.99%, respectively. The
difference in ƞf values highlights the need for inorganic nutri-
ent substances for cell substrate metabolization; in culture C1,
0.112 g ethanol was produced for each gram of glucose.
Gouvea et al. [20] fermented whole coffee husk in water
(13%, w/v) with commercial baker’s yeast S. cerevisiae and
achieved a final ethanol concentration equal to 13.6 g/L.
These results are 3.5 times less than those obtained in this
work.
The production of ethanol from lignocellulosic materials
allows the use of feedstocks rich in carbohydrates; in this
study, the use of pretreated CH presents better results than
those reported using substrates from different lignocellulosic
fibers (wheat straw, corn cob, palm wood, and sweet sor-
ghum), as shown in Table 6, in terms of ethanol concentration
(EtOH, g/L) that occurred up to 110% obtained with palm
wood and 25.26% for sweet sorghum. Using the yeast
Saccharomyces cerevisiae, this study exceeds the productivity
Q (g/L h) obtained by Wang et al. [71] and Martín et al. [69]
by 87.5% and 90.4%, respectively, improving the yield YE/S
(g/g) up to 11%, according to that obtained by Kleingesinds
et al. [72] with corn cob residues and Martin et al. [69] with
sugarcane bagasse.
Conclusions
The use of the response surface methodology in the CH,
through the CCD, led to the optimal conditions in the chem-
ical pretreatments (DAH and PAT), to maximize the removal
of hemicellulose (53.62%) and lignin (58.30%), respectively,
achieving an increase in available cellulose from 34.55 to
72.16%. The second-order mathematical model estimated a
maximum hemicellulose removal of 54.19% at 45.11-min
treatment time and 4.23% v/v H2SO4 concentration, whose
validation showed an increase of 2.56% greater than the re-
moval found by the CCD. The optimal analysis of maximiza-
tion of lignin removal theoretically at 35.75 h and 1:9.62 w/v
of SLR generated a removal of 58.82%, whose experimental
validation obtained a lignin reduction of 58.82%. The optimi-
zation of both treatments permitted an increase in available
cellulose from 34.55 to 72.16%, which allowed obtaining a
high glucose concentration of 115.59%; hence, the process
becomes novel, as there are no reports of saccharification
using coffee residues, which report close values.
The study of the fermentative glucose process of CH
pinpointed the need for enrichment by inorganic salts. Its
use in conjunction with Saccharomyces cerevisiae ITV-01
generated a YE/S of 0.416 g/g and a fermentative efficiency
(ƞf) of 81.74%. CH residue is an agro-industrial waste of great
potential for the production of second-generation ethanol or
biofuel, obtaining productivities (Q) higher than 23.75% com-
pared with other waste.
Acknowledgments The authors wish to thank the Bioengineering labo-
ratory of the Veracruz Institute of Technology for the access to its facil-
ities to carry out experimental tests, and the critical reading of Patricia
Margaret Hayward-Jones, M Sc, and Dulce María Barradas-Dermitz, M
Sc.
Funding The authors also wish to thank SAGARPA-CONACyT for fi-
nancing the project entitled “Production of 2nd generation bioethanol,
from agroindustrial waste and enzymes obtained from autochthonous
microorganisms”, No. 291143.
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