Applied Biochemistry and Biotechnology (2021) 193:1338–1350

https://doi.org/10.1007/s12010-020-03387-7

Pretreatment of Mango (Mangifera indica

L. Anacardiaceae) Seed Husk for Bioethanol Production
by Dilute Acid Treatment and Enzymatic Hydrolysis

Francis Dave C. Siacor, et al. [full author details at the end of the article]

Received: 5 December 2019 / Accepted: 16 July 2020 /

Published online: 5 September 2020
# Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
One of the targets of the Sustainable Development Goals is clean and affordable energy.
This is also the aim of the Biofuels Act of 2007 in the Philippines. However, this law is
confronted with challenges such as the limitation of lignocellulosic feedstock, specifically
available for bioethanol production. The present study sought to address the issue by
exploring the potential of mango seed husk (MSH), a by-product of the mango industry, in
bioethanol production. MSH is considered a waste material and its utilization also permit
value-addition as this can serve as an alternative and affordable source of feedstock in
energy production. Two pretreatment strategies are employed to exploit the cellulose and
hemicellulose content of MSH, namely, dilute acid treatment and enzymatic hydrolysis.
Results show that the %H2SO4 resulting in the highest glucose concentration and yield is
4% v/v at 95 °C hydrolysis temperature, 1:10 (w/v) solid-to-solvent ratio, and 60-min
hydrolysis time. For enzymatic hydrolysis using a commercial enzyme preparation, the
reaction time up to 72 h did not affect glucose concentration and yield at the following
conditions: 50 °C hydrolysis temperature, 150 rpm, pH 5.0, 10% solids loading, and 4%
enzyme loading. This could be attributed to the lignin and non-structural compounds
present in MSHs. However, a combined process strategy of dilute acid pretreatment
followed by enzymatic hydrolysis in the pretreatment of MSH contributes to an increased
concentration and yield of sugars in the hydrolysates, which is advantageous for
bioethanol production.

Keywords Bioethanol . Dilute acid treatment . Enzymatic hydrolysis . Lignocellulosic biomass .

Mango seed husk . Pretreatment strategies

Highlights
• Mango seed husk as a lignocellulosic material is a potential feedstock for bioethanol production.
• Chemical and enzyme-assisted pretreatment of mango seed husk generated sugar-rich hydrolysates.
• Dilute acid pretreatment using 4% H2SO4 at 95 °C for 1 h produces sugar-rich hydrolysates with minimum
inhibitor content.
• Enzyme hydrolysis using a cellulase mixture at 50 °C for up to 72 h resulted in a hydrolysate having optimal
sugar concentration.
• The combination of both dilute acid and enzymatic hydrolysis in the pretreatment process of mango seed husks
significantly increased the sugar concentration of the resulting hydrolysates.
The presenting author in ACB2019 is F.D.C.S.
Applied Biochemistry and Biotechnology (2021) 193:1338–1350 1339

Introduction

The Sustainable Development Goal (SDG) No. 7 specifies to target “Clean and Affordable
Energy.” In the Philippines, the Biofuels Act (Republic Act 9367) in 2007 mandated 10% and
5% ethanol and biodiesel blends in transport fuel, respectively, to alleviate issues concerning
energy. In 2017, the capacity of fuel ethanol in the country is 282 million liters and this is
expected to increase up to 365 million liters in 2018 due to the addition of local ethanol
manufacturing facilities. However, this volume is still inadequate at present to meet the 10%
blend mandate in the next 3 to 5 years. This is partly attributed to the limitation of domestic
feedstock that can be exploited for ethanol production. Currently, sugarcane remains the most
dominant source for first-generation bioethanol in the country, but this has consequently
ushered competition with sugar production, which affected both domestic and international
demands for sugar [1]. Because of this, a shift toward second-generation bioethanol derived
from lignocellulosic biomass is seen as a potential solution to address this concern. Agricul-
tural and forestry residues are examples of lignocellulosic materials that are widely available in
the Philippine archipelago in different forms and types [2, 3].
Lignocellulosic biomass is structurally made up of cellulose, hemicellulose, and lignin, with
considerable amounts of extractives and pectin, in its cell wall. The cellulose molecules,
consisting of linear homopolysaccharide, are arranged regularly and determines the framework
of the cell wall [4]. Hemicelluloses, which are heterogeneous polysaccharides, fill the cellulose
fibers through hydrogen bonds [5]. Lignin, on the other hand, forms a protective coating to the
cellulose–hemicellulose matrix through hydrogen and chemical bonds. Lignocelluloses are
compact and their structure dictates the specific processes that can be employed in order to
utilize their cellulose and hemicellulose content for bioethanol production [6].
One of the most abundant agricultural residues in the Philippines is the by-products from
mango processing, namely, the peels and seeds. It is known that the country is among the top
producers of the fruit in the world with an annual production of over 700,000 MT in 2017 [7].
The seeds account for almost half the amount of the waste materials produced annually, which
is mostly made up of mango seed husks (MSH’s). Several studies have found that the chemical
constituents of MSH comprise cellulose (52–58%), hemicellulose (23–29%), and lignin (16–
24%) with traces of ash [8, 9]. Thus, it is suggested that MSH can be an alternative feedstock
for second-generation bioethanol production with these compositions [10, 11].
Pretreatment processes are necessary to properly utilize MSH for bioethanol production via
fermentation. These processes dictate the accessibility of cellulose and hemicellulose to the
fermenting microorganisms [5] since they hydrolyze the latter components, which are poly-
mers of hexoses and pentoses, to form a sugar-rich hydrolysate [12]. There are several
pretreatment techniques available for lignocellulosic biomass; however, the most common
methods are chemical and enzymatically catalyzed hydrolysis.
Chemically catalyzed hydrolysis, particularly the use of dilute acid, enables cellulose and
hemicellulose polymers to be broken down because of the disruption of β-1-4-linkages in the
β-D-glucopyranose structure of cellulose and the bonds found within the linear and branched
heteropolymers of sugars and sugar derivatives in hemicellulose [4, 5]. Various acids at lower
concentrations can be used for this process, such as phosphoric, hydrochloric, and sulfuric
acids. In a study by Kou et al. [13], four types of lignocellulosic grasses, namely, giant reed,
switch grass, silver grass, and Pennisetum, were treated with ≤ 2.5% sulfuric acid at 60 to
120 °C for up to 60 min, and hydrolysates were produced with glucose and xylose concen-
trations at 14–18 and 2–5 g/L, respectively. Acid hydrolysis takes advantage of its rate of
1340 Applied Biochemistry and Biotechnology (2021) 193:1338–1350

hydrolysis since it is much faster than enzymatic hydrolysis, and that the acid is able to
infiltrate lignin without prior pretreatment. However, this process requires conditions at
elevated temperature and the product glucose deteriorates immediately under acidic conditions.
Furthermore, inhibitory compounds such as furfural and acetic acid that are undesirable during
fermentation can be formed during the process [14].
Enzyme-catalyzed hydrolysis, on the other hand, is a heterogeneous reaction in which
hydrolytic and oxidative enzymes synergistically break down structural components of ligno-
cellulosic materials. In most cases, a concoction of cellulase and hemicellulase enzymes
consisting of endoglucanases, exoglucanases (cellobiohydrolases), β-glucosidases, and
glycosylhydrolases are employed under mild conditions to degrade cellulose and hemicellu-
lose in the process, leading to the production of sugar-rich hydrolysates [15]. Enzymatic
hydrolysis was tried on bamboo and wood chips using commercially available cellulase
enzyme (52 to 116 FPU/mL activity) at 50 °C for 72 to 96 h with an enzyme loading of up
to 9600 mg protein/mg glucan. The study achieved the highest enzyme conversion of glucan
equal to 46% [16]. The advantages of enzymatic hydrolysis over other pretreatment processes
include higher yields and selectivity, a lower energy costs, and milder operating conditions.
However, this process is known to have a lower reaction rate and can incur higher material
costs since a higher glucose yield may entail an increase in enzyme loading [17].
In the present study, the potential of MSH as feedstock for bioethanol production was
explored. Two pretreatment strategies were considered: dilute acid hydrolysis using sulfuric
acid and enzymatic hydrolysis using cellulase–xylanase system. Specifically, the effects of
acid concentration (for dilute acid treatment) and reaction time (in the case of enzymatic
hydrolysis) on the glucose concentration and the corresponding sugar yield of the hydrolysate
were investigated. Furthermore, a combination of both pretreatment process strategies was
demonstrated in order to explore their effect on sugar concentration and yield in the resulting
hydrolysate.

Experimental

Materials

Mango seeds were kindly provided by Green Enviro Management Systems (GEMS) Inc.,
Lapu-Lapu City, Cebu, Philippines. Glucose, sulfuric acid, furfural, sodium citrate dihydrate,
citric acid, phenol, furfural, 5-hydroxymethylfurfural, acetonitrile, and acetic acid were pur-
chased from Sigma-Aldrich Pte. Ltd. (Singapore). All chemicals were reagent grade and used
without further purification. Cellulase enzyme preparation Cellic CTec3 HS (20–30% active
enzyme protein) was provided by Novozymes A/S (Denmark).

Preparation of MSH

Mango seeds were thoroughly washed with tap water and dried in a local solar drying facility
at 60–70 °C until the seed husks became brittle. The seeds were then cracked open to remove
the seed kernels inside, obtaining the husks or MSHs. Afterwards, the MSH was cut into strips
and further dried at 60 °C for 4 to 6 h to remove residual moisture. Dried MSH was then
ground in a Wiley mill (Thomas Scientific, USA) to a particle size of ≤ 1 mm. MSH powders
were stored in polyethylene containers at controlled atmospheric conditions until use. MSH
Applied Biochemistry and Biotechnology (2021) 193:1338–1350 1341

was characterized for their holocellulose, α-cellulose, and extractives content, as well as their
proximate composition and calorific value.

Dilute Acid Hydrolysis of MSH

Dilute acid hydrolysis of MSH was done using 1–5% v/v aqueous H2SO4 at 95 °C and
a solid-to-solvent ratio of 1:10 (w/v) for 60 min. Approximately 2 g of MSH was
treated with 20 mL of H2SO4 solution through incubation in a water bath (Memmert
GmbH, Germany) at 95 °C for 60 min with intermittent mixing every 30-min interval.
Afterwards, the hydrolysis mixture was directly quenched in an ice bath to stop the
reaction. The mixture was then subjected to vacuum filtration to separate the hydroly-
sate from the solid residues. The residues were washed with generous amounts of water
and the washings were pooled with the hydrolysate. The pH of the final hydrolysate
was measured prior to analysis for its total carbohydrate concentration, as well as
furfural and 5-HMF contents as inhibitory compounds.

Enzymatic Hydrolysis of MSH

Enzymatic hydrolysis of MSH was carried out using Cellic CTec3 HS cellulase enzyme
preparation at 50 °C and 150 rpm for 24 to 72 h. About 5 g of MSH was mixed with
50 mL of citrate buffer solution (pH 5.0) maintaining a solids loading of 10%. The enzyme
was then added to the mixture at 4% enzyme loading based on the amount of feedstock. The
resulting mixture was incubated in an orbital incubator shaker (SI-64; Hanyang Scientific
Equipment Co., Ltd., Korea) at 50 °C for 24 to 72 h. Afterwards, the hydrolysis mixture was
allowed to cool and then subjected to vacuum filtration to separate the hydrolysate from the
solid biomass. The biomass was washed with generous amounts of water and the washings
were pooled with the hydrolysate. The pH of the final hydrolysate was measured prior to
analysis for its total carbohydrate content.

Acid Treatment and Enzymatic Hydrolysis of MSH

MSH was sequentially treated via dilute acid treatment and then enzyme hydrolysis to evaluate
its improvement in terms of glucose concentration and yield of the hydrolysate. The process
conditions and procedures for both strategies were similar to the previous sections, except that
the acid concentration was set at 4% for dilute acid treatment and the reaction time was 3 days
for enzymatic hydrolysis. The solid residue after acid hydrolysis was washed with water until
pH 5.0 prior to enzymatic hydrolysis.

Analytical Methods

Determination of Total Carbohydrates The total carbohydrate content of hydrolysates was

determined using the colorimetric method (phenol-sulfuric method) based on Nielsen [18].
About 0.1 mL hydrolysate was diluted with water to 25 mL. A 2-mL aliquot of the diluted
sample was mixed with 2 mL of 5% phenol solution. Then, 5 mL of concentrated H2SO4 was
added rapidly directly to the liquid surface. The solution was allowed to stand for 10 min before
mixing. Subsequently, the solution was incubated at 30 °C for 2 min. Absorbance readings at
490 nm were taken using a spectrophotometer (Spectroquant Pharo 100; Merck, USA). A
1342 Applied Biochemistry and Biotechnology (2021) 193:1338–1350

calibration curve using glucose as standard was also prepared to obtain the total carbohydrate
content of the hydrolysate samples expressed as grams of glucose per liter of hydrolysate.

Determination of Inhibitory Compounds Furfural and 5-HMF content in the hydrolysates

were determined using high-performance liquid chromatography (HPLC) equipped with a C-
18 reverse-phase column (ODS-3V; Inertsil, Europe), LC 10AT high-pressure pump, CTO-
10A oven, and UV–Vis detector (SPD-10AV; Shimadzu, Japan). Acetonitrile–water–acetic
acid solution at 11:88:1 (in volume) ratio was prepared and used as a mobile phase. Before
analysis, samples were filtered thoroughly to remove suspended solids using a sterilized
0.45-μm membrane filter. A 20-μL filtered sample was injected into the HPLC system at
30 °C, with elution at a constant flow rate equal to 1 mL/min, and absorbance was measured at
276 nm. The inhibitor concentrations were calculated from calibration curves (peak area vs.
concentration of analyte) obtained from the response detection of the standard furfural and 5-
HMF.

Determination of Chemical and Proximate Composition The proximate composition

(moisture, ash, volatile matter, and fixed carbon) of MSH was determined following ASTM
D1762-84 [19]. The hexane, water, and ethanol extractives were quantified following
NREL/TP-510-42619 [20]. The lignin content of MSH was analyzed following NREL/TP-
510-42618 [21] while the holocellulose and α-cellulose contents were determined based on the
modified TAPPI T19m-54 procedure by Trindade et al. [22]. The calorific value of MSH was
measured using bomb calorimetry.

Results and Discussion

Chemical Characteristics of MSH

The chemical and proximate composition of MSH are summarized in Table 1. It can be noted
that MSH is largely composed of α-cellulose, which is higher than most agricultural residues
like corn cobs [23]. The cellulose chain consists of repeating units of disaccharide cellobiose in
which glucose units are held together by extensive intramolecular and intermolecular hydrogen
bonding networks. It is desired to have a feedstock with high cellulose content for bioethanol
production since glucose and ethanol yields are consequently higher [24]. The hemicellulose
content of MSH is found to be similar to several fruit processing by-products such as banana
waste [25]. The hemicellulose structure is a group of several heteropolymers made up of β-
1,4-linked xylose and other pentose sugars. The presence of hemicellulose can positively
impact sugar and ethanol yield since yield is directly related not only to cellulose but also to
hemicellulose and other sugars present in the feedstock [26]. Lignin, on the other hand, is
antagonistic in bioethanol production because it forms a barrier to microorganism during
fermentation; hence, lignin content should be at a minimum [27]. The lignin content of MSH is
lower than most lignocellulosic residues, for example, silver grass, Napier grass, and other
forages, which is about 17–20% [13].
On the other hand, extractives refer to the non-structural components in the feedstock that
are soluble in various solvents. Some of the components of extractives include lipids or waxes,
nitrogenous materials, and phenols that are soluble in hexane, water, and ethanol, respectively,
Applied Biochemistry and Biotechnology (2021) 193:1338–1350 1343

Table 1 Characteristics of mango seed husks

Constituents % (w/w)

Chemical compositiona
Total lignin 11.91 ± 2.34
Holocellulose 74.08 ± 5.55
α-Cellulose 55.93 ± 4.58
Hemicellulose 18.14 ± 5.07
Total extractives 14.01 ± 0.83
Hexane extractives 6.66 ± 0.22
Water extractives 6.53 ± 0.97
Ethanol extractives 0.82 ± 0.50
Proximate compositiona
Moisture 5.57 ± 0.17
Volatile matter 75.14 ± 0.11
Ash 1.04 ± 0.06
Fixed carbon 18.24 ± 0.13
Calorific valueb 20.38 ± 0.17
a Dry-matter basis
b Dry-matter basis (kJ/g)

or a mixture of these solvents. MSH contains 14% total extractives, which is within the
average extractives content (7.3–23.5%) of most lignocellulosic biomass. A higher extractives
concentration may negatively impact cellulose digestibility during hydrolysis [28]. Moreover,
the volatile matter is highest in MSH based on its proximate composition. This is expected
since biomass typically contains high levels of volatile matter which influences fuel reactivity
of the feedstock. The ash content of MSH, denoting mineral content, is lower than most
agricultural crop residues such as rice husk. Similar to lignin, high levels of ash can be
undesirable during bioethanol production [27]. Furthermore, the calorific value represents
the energy content of feedstock and is found to directly correlate with fixed carbon content.
Materials with high fixed carbon are expected to have a high heating value [29]. The fixed
carbon, as well as calorific value, of MSH is found to be similar to non-wood lignocellulosic
biomasses such as hazelnut and walnut shells [30].

Dilute Acid Hydrolysis of MSH

The results obtained on the dilute acid treatment of MSH using 1 to 5% (v/v) sulfuric acid are
presented in Figs. 1, 2, and 3. It is shown in Fig. 1 that an increase in H2SO4 concentration led
to a significant increase in glucose concentration of hydrolysate up to 4%. A further increase in
acid concentration to 5% did not impart a significant increase in glucose concentration;
instead, a steady trend is observed. Thus, 4% H2SO4 concentration was chosen as the optimum
acid concentration for dilute acid hydrolysis of MSH at 95 °C and 1:10 (w/v) solid-to-solvent
ratio for 60-min reaction time. This resulted in a hydrolysate with a glucose concentration of
14.64 ± 1.21 g/L and a sugar yield equivalent to 10.58% (± 0.20). This result is in agreement
with Kumar et al. [31], wherein ≤ 4% H2SO4 has been the widely used acid concentration
during dilute acid treatment of an extensive range of feedstocks. The presence of acid degrades
and depolymerizes cellulose and hemicellulose chains in the lignocellulosic structure, thereby
releasing and solubilizing hexose and pentose sugars. In addition, an increase in concentration
would lead to an enhancement in cellulose and hemicellulose digestibility but is also limited
because of the presence of lignin and other non-structural components.
1344 Applied Biochemistry and Biotechnology (2021) 193:1338–1350

Fig. 1 Glucose concentration (g/L) as a function of acid concentration (v/v) in the dilute acid treatment of mango
seed husks at 95 °C and a solid-to-solvent ratio of 1:10 (w/v) for 60 min (values represent the mean values for
three replicates; error bars represent SD)

Figure 2 presents the concentration of inhibitors, specifically furfural and 5-HMF, in

hydrolysates from the acid treatment of MSH using 1 to 5% (v/v) H2SO4. It can be seen that
there is an insignificant difference in the formation of 5-HMF while there is a significant
increase in the formation of furfural as acid concentration is increased. When hemicellulose is
degraded, it releases xylose; however, xylose simultaneously breaks down to furfural. Simi-
larly, glucose is further converted to 5-HMF during hydrolysis [32]. At 4% H2SO4, the 5-HMF
and furfural concentration of the hydrolysate are at 0.009 ± 0.002 g/L and 0.052 ± 0.003 g/L,
respectively. This is approximately ≤ 0.3% of the amount of glucose being formed, and these
concentrations are below toxicity levels that would lead toward impairment of the fermentation

Fig. 2 Inhibitor concentration (g/L) as a function of acid concentration (v/v) in the dilute acid treatment of mango
seed husks at 95 °C and a solid-to-solvent ratio of 1:10 (w/v) for 60 min (values represent the mean values for
three replicates; error bars represent SD)
Applied Biochemistry and Biotechnology (2021) 193:1338–1350 1345

Fig. 3 pH as a function of acid concentration (v/v) in the dilute acid treatment of mango seed husks at 95 °C and
a solid-to-solvent ratio of 1:10 (w/v) for 60 min (values represent the mean values for three replicates; error bars
represent SD)

process [33]. Moreover, the pH of hydrolysate from the treatment of MSH using 1 to 5% (v/v)
H2SO4 is shown in Fig. 3. It can be noted that the pH continues to decrease and it even reached
a pH value of zero as the concentration is increased. This pH value indicates that the
concentration of hydronium ions in the solution is 1 M due to its dissociation, leading to an
elevated acidic nature of hydrolysates.
The production of inhibitory compounds and the pH level of the hydrolysates are
some of the major drawbacks of the dilute acid hydrolysis. These disadvantages have
implications for the overall process of bioethanol production because of the need for
detoxification and/or neutralization steps [34]. In a study by Zahed et al. [35], rice
straw hydrolysate was further treated with a series of adsorption steps using activated
carbon and ion exchange resins before microbial fermentation. Adsorption processes
were necessary to reduce the inhibitor content and to increase the pH of hydrolysate
through the action of neutralization. However, these steps would result in an additional
cost of production and the impending risk of sugar degradation in acidic medium
resulting in much lower sugar concentration [14, 31].

Enzymatic Hydrolysis of MSH

Enzymatic hydrolysis of lignocellulosic biomass generally entails the use of a concoction of

enzymes with a synergistic and cooperative effort. Usually, the mixture is composed of
cellobiohydrolase, endoglucanase, β-glucosidase, and hemicellulase. Cellobiohydrolase tar-
gets the ends of cellulose chains to produce cellobiose, while cleaving within cellulose chains
are caused by endoglucanase; this reduces the degree of polymerization of cellulose. Cellobi-
ose is further converted into glucose through the action of β-glucosidase. Due to the hemi-
cellulose structure, hemicellulase is needed to convert xylose from xylan [36]. In the present
study, a concoction of enzymes largely composed of cellulase with xylanase (hemicellulase)
was used to hydrolyze MSH for bioethanol production.
1346 Applied Biochemistry and Biotechnology (2021) 193:1338–1350

The results obtained on enzymatic hydrolysis of MSH using an enzyme preparation at

50 °C for a period of 24 to 72 h are presented in Figs. 4 and 5. Figure 4 shows the glucose
concentration of the hydrolysate at different hydrolysis periods. A steady trend of glucose
concentration can be noted throughout 72 h, which is on average equal to 2.40 ± 0.56 g/L with
a sugar yield of 2.38% (± 0.56). This yield is much lower compared with the results of Silvello
et al. [37] and Pengilly et al. [38] wherein sugarcane and sweet sorghum bagasse, respectively,
were enzymatically hydrolyzed using an enzyme preparation that is similar to the present
study. The low yield obtained in the present study could be attributed to the low digestibility of
cellulose and hemicellulose of MSH probably due to a limited to non-removal of lignin and
extractives before hydrolysis. The access of enzymes to the cellulose surface may have been
hindered by the presence of these components [16]. The higher yield in Silvello et al. [37] and
Pengilly et al. [38] is, in part, due to the inclusion of treatment steps before hydrolysis, such as
ultrasonic and steam treatment; these led to the removal of lignin, thereby promoting cellulose
digestibility.
Furthermore, the pH of the hydrolysate did not significantly change for 72 h as presented in
Fig. 5. The average pH is 4.51 ± 0.16, which is within the recommended optimum pH range
(4.0–5.0) for bioethanol production using yeasts. The pH is an important parameter during
fermentation because it affects yeast growth and fermentation rate, as well as by-product
formation. A pH level below 4.0 increases the incubation period, whereas a pH > 5.0 reduces
ethanol concentration considerably [39].

Dilute Acid Treatment and Enzymatic Hydrolysis of MSH

Generally, dilute acid treatment can recover hemicelluloses and celluloses as dissolved sugars
with certain limitations because of the presence of residual hemicellulose and cellulose
fractions that were not solubilized to a certain extent. In addition, the removal of lignin
presents a challenge. This is also the case when enzymatic hydrolysis alone is employed as
a pretreatment technique [40]. In order to maximize recovery of sugars from the recalcitrant

Fig. 4 Glucose concentration (g/L) as a function of time (hours) in the enzymatic hydrolysis of mango seed
husks at 50 °C hydrolysis temperature, 150 rpm, pH 5.0, 10% solids loading, and 4% enzyme loading (values
represent the mean values for three replicates; error bars represent SD)
Applied Biochemistry and Biotechnology (2021) 193:1338–1350 1347

Fig. 5 pH as a function of time (h) in the enzymatic hydrolysis of mango seed husks at 50 °C hydrolysis
temperature, 150 rpm, pH 5.0, 10% solids loading, and 4% enzyme loading (values represent the mean values for
three replicates; error bars represent SD)

structure of lignocellulosic biomasses, a combination of pretreatment methods was employed

in several studies which are usually a sequence of chemical treatments prior to enzymatic
hydrolysis [16, 41]. In the present study, MSH was pretreated with dilute acid followed by
enzymatic hydrolysis according to the process conditions obtained in the previous sections,
and the results are shown in Table 2. It can be noted that there is an almost threefold increase in
glucose concentration and yield in the hydrolysate compared with the hydrolysate obtained
when enzymatic hydrolysis was only employed. This shows that the enzymes were able to
benefit from the application of dilute acid treatment since the latter enhanced the access of
enzymes into the recalcitrant structure of MSH [41].

Conclusions and Recommendations

In the present study, MSH’s are shown to be a potential feedstock for second-generation
bioethanol production based on its chemical composition. Two pretreatment strategies were
demonstrated to hydrolyze the cellulose and hemicellulose components of MSH. In dilute acid
treatment, the acid concentration producing the highest sugar concentration and yield is 4%
H2SO4 at the following conditions: 95 °C, 1:10 (w/v) solid-to-solvent ratio, and 60-min
hydrolysis time. The reaction time insignificantly affected enzymatic hydrolysis using a
specific enzyme preparation at 50 °C. However, the sugar concentration and yield for both
strategies are comparably low, especially for enzymatic hydrolysis, which is attributed to the

Table 2 Sequential dilute acid–enzyme hydrolysis of mango seed husks

Hydrolysate origin Glucose concentration (g/L) Sugar yield (%) pH

Dilute acid treatment 14.45 ± 0.88 10.82 ± 0.39 0

Enzymatic hydrolysis 6.75 ± 0.50 5.08 ± 0.80 4.60 ± 0.01
1348 Applied Biochemistry and Biotechnology (2021) 193:1338–1350

limitation brought about by the presence of lignin and other non-structural compounds. On a
similar note, the sequential dilute acid treatment and enzymatic hydrolysis pretreatment
strategy was able to dramatically improve the sugar content and yield of the hydrolysate.
It is recommended to consider other pretreatment strategies before dilute acid treatment or
enzymatic hydrolysis to enhance cellulose and hemicellulose digestibility. In addition, the
hydrolysates should be subjected to further characterization to elaborate their composition in
preparation for the subsequent step of microbial fermentation during bioethanol production.
Furthermore, a sequential pretreatment strategy combining dilute acid and enzymatic hydro-
lysis is a good potential approach to investigate more deeply the overall process including
bioethanol production.

Acknowledgments The authors are genuinely indebted to the Department of Science and
Technology—Engineering Research and Development for Technology (ERDT) for F.D.C.S.’s scholarship and
research grant. Likewise, Novozyme A/S, Denmark is acknowledged for the enzyme samples. The authors would
also like to recognize Ms. Pearly Jane Mendoza and Mr. Marvin Comendador for invaluable assistance during
the conduct of this research.

Author’s Contributions Conceptualization: F.D.C.S., C.F.Y.L., and E.B.T. Methodology: F.D.C.S. Formal
analysis and investigation: F.D.C.S. Writing—original draft preparation: F.D.C.S. Writing—review and editing:
F.D.C.S., C.F.Y.L., and E.B.T. Funding acquisition: F.D.C.S., C.F.Y.L., and E.B.T. Resources: F.D.C.S.,
C.F.Y.L., and E.B.T. Supervision: F.D.C.S., C.F.Y.L., and E.B.T. All authors read and approved the final
manuscript.

Funding This work was supported by the Department of Science and Technology—Engineering Research and
Development for Technology (ERDT) program through the scholarship granted to F.D.C.S.

Data Availability All data generated or analyzed during this study are available from the corresponding author
on reasonable request.

Compliance with Ethical Standards

Competing Interests The authors declare that they have no competing interests.

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Affiliations

Francis Dave C. Siacor 1 & Camila Flor Y. Lobarbio 1 & Evelyn B. Taboada 1

* Francis Dave C. Siacor

[email protected]

Camila Flor Y. Lobarbio

[email protected]
Evelyn B. Taboada
[email protected]

1
BioProcess Engineering and Research Center, Department of Chemical Engineering, School of Engineering,
University of San Carlos, Talamban, 6000 Cebu City, Philippines