3 Biotech (2020) 10:432

https://doi.org/10.1007/s13205-020-02426-8

ORIGINAL ARTICLE

Saccharification of water hyacinth biomass by a combination of steam

explosion with enzymatic technologies for bioethanol production
L. A. Figueroa‑Torres1 · M. A. Lizardi‑Jiménez2   · N. López‑Ramírez3 · E. C. Varela‑Santos1   · F. Hernández‑Rosas4   ·
E. Favela‑Torres3   · R. Hernández‑Martínez5 

Received: 27 April 2020 / Accepted: 4 September 2020 / Published online: 15 September 2020
© King Abdulaziz City for Science and Technology 2020

Abstract
In the present work, bioethanol was produced by sugar fermentation obtained from water hyacinth using a novelty hybrid
method composed of steam explosion and enzymatic hydrolysis, using hydrolytic enzymes produced by solid-state fermen-
tation and water hyacinth as substrate. The highest activity, 42 U for xylanase and 2 U for cellulase per gram of dry matter,
respectively, was obtained. Steam explosion pretreatment was performed at 190 ℃ for 1, 5, and 10 min, using water hya-
cinth sampled from the Maria Lizamba Lagoon, the Arroyo Hondo and the Amapa River. The highest amounts of reducing
sugars of water hyacinth were obtained form the samples from the lagoon (5.4 g/50 g of dry matter) after 10 min of treat-
ment. Steamed biomass was hydrolysed using the enzymes obtained by solid-state fermentation, obtained reducing sugars
(maximum 15.5 g/L); the efficiency of enzymatic hydrolysis was 0.51 g of reducing sugars per gram of water hyacinth.
Finally, reducing sugars were fermented using Saccharomyces cerevisiae for conversion to ethanol, with the highest ethanol
concentration (7.13 g/L) and an ethanol yield of 0.23 g/g of dry matter.

Keywords  Lignocellulosic biomass · Solid-state fermentation · Fermentable sugars · Hydrolytic enzymes · Alcoholic
fermentation

Introduction

Water hyacinth (WH) is an important aquatic plant high in

hemicellulose and low in lignin content. It contains about
48% hemicellulose, 18% cellulose and 3% lignin, although
* R. Hernández‑Martínez the reported composition varies (Aswathy et  al. 2010).
odracirhema@gmail.com
The species is an important source of fermentable sugars
1
Instituto Tecnológico Superior de Tierra Blanca, Av. for bioethanol production to substitute fossil fuels, and its
Veracruz S/N Esq., Héroes de Puebla, Colonia Pemex, residual biomass can also be converted into other value-
C.P. 95180 Tierra Blanca, Veracruz, Mexico added chemicals in a well-integrated biorefinery facility. Its
2
CONACYT-Universidad Autónoma de San Luis Potosí, further advantages are that it does not compete with food
Sierra Leona 550, Lomas 2da Secc., 78210 San Luis Potosí, crops for arable land, proliferates in clear water and waste-
Mexico
water and is highly reproducible (Singh and Bishnoi 2013;
3
Departamento de Biotecnología, Universidad Autónoma Yan et al. 2015). However, the production of bioethanol from
Metropolitana, Unidad Iztapalapa, C.P. 09340 Mexico D.F.,
Mexico WH requires the recovery of fermentable sugars using pre-
4 treatments that destroy covalent bonds between hemicellu-
Colegio de Postgraduados-Campus Córdoba, Carretera
Federal Córdoba‑Veracruz Km 348, Congregación Manuel lose and lignin but not between cellulose and hemicellulose
León, Municipio Amatlán de los Reyes, 94946 Veracruz, within the complex physical mixture of lignocellulose (Sun
Mexico et al. 2016). Hemicellulose and lignin are strongly inter-
5
CONACYT- Colegio de Postgraduados-Campus Córdoba, twined and linked by covalent bonds that hinder saccharifi-
Carretera Federal Córdoba‑Veracruz Km 348, Congregación cation, presenting a bottleneck for enzymatic hydrolysis or
Manuel León, Municipio Amatlán de los Reyes, fermentation (Gütsch et al. 2012). Also, there are technical
94946 Veracruz, Mexico

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challenges in the pretreatment of biomass and in enzy- flasks containing liquid YPD medium (20 g L− 1 of yeast
matic saccharification, especially the sourcing of enzymes extract, 20 g L− 1 of peptone, and 40 g L− 1 of glucose) and
(Aswathy et al. 2010). maintained at 30 ℃ for 24 h; mycelial growth was consid-
Regular ethanol production from lignocellulosic biomass ered as inoculum in SSF.
is realised by three major steps: pretreatment to disrupt the Saccharomyces cerevisiae was propagated in culture
recalcitrant structures and to facilitate polysaccharide acces- medium with glucose (50  g  L−  1), ­K2HPO4 (5  g  L−  1),
sibility due to the increase of the surface area, enhancing ­(NH4)2SO4 (2 g L− 1), ­MgSO4∙7H2O (0.4 g L− 1) and yeast
accessibility for enzymatic attacks in the hydrolysis step. extract (1 g L− 1) and was maintained at 30 ℃ for 36 h. Bio-
Enzymatic hydrolysis is then performed to hydrolyse the mass was used as inoculum for ethanol production. Both
polysaccharides into fermentable sugars, followed by their strains were conserved in distilled water.
fermentation into bioethanol. Hydrothermal pretreatment as
a steam explosion has been used as a promising method to Sampling and collection of water hyacinth
enhance cellulose availability and hemicellulose recovery
without requiring any chemicals; as an environmentally The sampling area for WH (Eichornia crassipes) was
friendly method, it only uses compressed hot water as a sol- defined on the Papaloapan Hydrological Basin reported by
vent and maintains cellulose and hemicellulose availability the National Commission for the Knowledge and Use of Bio-
for the enzymes, resulting in lower downstream detoxifi- diversity (CONABIO 2016). Samples were obtained from
cation costs compared to other pretreatment techniques the Maria Lizamba Lagoon located at 18° 29′ N 70′’ and 96°
(Batista et al. 2019; Pratto et al. 2020). 01′ 42′’ W, the Amapa River located at 18° 18′ 88′’ N and
The steam explosion has been classified as a green and 96° 18′ 19′’ W, and the Arroyo Hondo River located at 18°
competitive technology as it only contains lignocellulosic 27′ 31′’ N and 96° 20′ 28′’ W.
feedstock and water, preventing corrosion problems and the
formation of neutralisation sludge; this pretreatment has Water hyacinth conditioning
successfully been used in delignification and the removal
of hemicellulose (Ibrahim et al. 2010; Oliveira et al. 2013; Once sampled, WH was washed to remove impurities. Sub-
Martin-Sampedro et al. 2014a), facilitating hemicellulose sequently, leaves, stem, and root were separated, followed
removal and lignin transformation because of the increase by drying in the sun for approximately 60 h at an average
in the surface area for cellulose hydrolysis (Singh et al. temperature of 35 ± 2 ℃. Finally, the dried biomass was cut
2015). Hence, de-lignification can substantially improve into pieces of uniform size (1 cm) and stored until use as a
biomass enzymatic saccharification. Pretreatment of WH via substrate in SSF and steam explosion.
steam explosion is an effective delignification strategy (Das
et al. 2015). Ferro et al. (2015) indicate that pretreatment Solid‑state fermentation
enhances enzymatic accessibility of cellulose and increases
the level of saccharification. In this context, we evaluated the Water hyacinth (from the Maria Lizamba Lagoon) was
steam explosion for subsequent enzymatic hydrolysis using used to support hydrolytic enzyme production (xylanase
hydrolytic enzymes produced by solid-state fermentation and cellulose) in packed bed columns (2.5 cm in diameter
(SSF) for the recovery of fermentable sugars for bioethanol and 20 cm long) with oxygen supply by forced aeration.
production, using Saccharomyces cerevisiae. Prior to SSF, WH was impregnated (pretreated) with ­H2SO4
(0.25 M), homogenised and sterilised in an autoclave at
120 ℃ for 15 min. After sterilization, WH was impregnated
Methods with a culture medium containing the following macronu-
trients (g L− 1): glucose (50), ­KH2PO4 (5), ­NH4NO3 (5),
Microorganisms and inoculum Urea (2), ­MgSO4∙7H2O (0.42), ­CaCl2 (1), NaCl (5) and
peptone (5), as well as 1 mL of micronutrients contain-
Trichoderma harzianum PBLA (Lopez-Ramirez et al. 2018) ing (g/100 mL): F ­ eSO4∙7H20 (0.5), M­ nSO4∙7H2O (0.61),
was used as inoculum for xylanase and cellulase production ­ZnSO4∙7H2O (0.1) and C ­ oCl2∙H2O (0.036) (Mekala et al.
by SSF. Saccharomyces cerevisiae was used as inoculum 2008). The inoculum was adjusted to 2 × 107 spores/mL at
in alcoholic fermentation. Both strains were provided by 65% of initial moisture (quantified in a gravimetric balance
Plant Pilot of Solid-State Fermentation of the Autonomous Ohaus, Model MB45), and the initial pH value was adjusted
Metropolitan University, Mexico. Trichoderma harzianum at 5.5. Columns were packed with the inoculated mixture,
PBLA was grown in 250-mL Erlenmeyer flasks with potato oxygen was supplied with water-saturated air at a flow rate
dextrose agar (PDA) (BIOXON, Mexico) for seven days of 50 mL/min, and the packed columns were maintained at
at 30 ℃. Generated spores were inoculated in Erlenmeyer 30 ℃. The concentration of produced ­CO2 (respirometry)

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was measured online connecting the outflow air to a gas sugars was monitored using the DNS method (Miller et al.
analyser (Ávila-Cisneros et al. 2014). 1960).

Enzymatic extract Ethanol production using water hyacinth

hydrolysed by a combination of methods
The enzyme extracts consisted of the recuperated mixed fer-
mented medium from SSF with distilled water in a ratio of Ethanol production was carried out using the product of WH
1:10 (w/v), vortexed for 1 min (Lopez-Ramirez et al. 2018) pretreated by steam explosion combined with the enzymatic
The extract was separated by centrifugation at 10,000 rpm method as a single carbon source. Alcoholic fermentation
for 15 min at 4 ℃. The supernatant was decanted and used as was performed by adding the culture medium containing
an enzyme source for xylanase and cellulase assays. yeast extract 1 g L− 1, ­NH4SO4 2 g L− 1 and ­MgSO4∙7H2O;
inoculum concentration was adjusted at 1 × 106 cellules/mL
Xylanase and cellulase activity and maintained for 72 h at 30 ℃ with agitation at 125 rpm.
After fermentation, the broth was centrifuged at 10,000 rpm
Xylanase activity was determined by mixing 0.1 mL of the for 15 min and the obtained supernatant was filtered through
enzymatic extract with 0.9 mL of 0.25% (w/v) birchwood 0.45-μm filters and used for ethanol quantification.
xylan solution in 0.1 M sodium citrate buffer pH 5.2. The
mixture was incubated at 40 °C for 15 min. The enzymatic Ethanol quantification
reaction was stopped by adding 1.5 mL of 3,5-dinitrosali-
cylic acid (DNS); reducing sugars were estimated by the The ethanol content was analysed via the spectrometric
DNS method (Miller 1959) using xylose as standard. Cel- method for ethanol quantification (Isarankura-Na-Ayudhya
lulase activity was determined by mixing 0.1 mL of the et al. 2007), using 0.1 M solution of potassium dichromate in
enzymatic extract with 0.9 mL of 0.25% (w/v) solution of 5 M sulphuric acid. The reaction was performed by mixing
carboxymethyl cellulose in 0.1 M sodium citrate buffer pH 300 μL of alcoholic samples with 3 mL of dichromate solu-
5.2; the mixture was incubated at 40 ℃ for 30 min. The tion, maintained at room temperature for 30 min. Absorb-
enzymatic reaction was stopped by the addition of 1.5 mL ance was measured at 590 nm, and ethanol concentration
of DNS; reducing sugars were estimated by the DNS method was determined using a standard curve of ethanol.
(Miller et al. 1960) using glucose as standard.
One enzymatic unit (U) was defined as the amount of
enzyme required to liberate 1 μmol of reducing sugars from Results and discussion
the substrate per minute. Enzyme activities are reported as
units per gram of dry matter (U/g dm). All assays were per- Steam explosion
formed in duplicate.
Results of the steam explosion of samples of WH obtained
Steam explosion from the Maria Lizamba Lagoon, the Arroyo Hondo River
and the Amapa River are shown in Fig. 1. Based on these
Pretreatment of WH was carried out using the emerging results, the time of the steam explosion treatment is impor-
technology (steam explosion) at 190 ℃ with retention times tant, and maximum quantities of reducing sugars from WH
of 1, 5, and 10 min. Batch processing was used for 50 g of sampled from all three sites were obtained after 10 min
WH samples. (maximum time assayed in this research) at 190  ℃. It
should be noted that the highest level of reducing sugars
Enzymatic hydrolysis of water hyacinth pretreated (fermentable sugars) was obtained from WH sampled from
with steam explosion the Maria Lizamba Lagoon (5.4 g/50 g of water hyacinth),
in contrast to the results obtained for the Arroyo Hondo
Enzymatic saccharification was carried out by mixing 20 g River (4.3 g/50 g of water hyacinth) and the Amapa River
of exploited WH and 200 mL of liquid generated from the (3.4 g/50 g of water hyacinth). Tukey´s test showed a signifi-
steam explosion with the enzymatic extract (cellulase and cant difference (p > 0.05) between reducing sugar levels, and
xylanase), adjusting the enzymatic activity in the reaction for this reason, WH from the Maria Lizamba Lagoon was
at 36 U/g of pretreated WH. The enzymatic reaction was used for hydrolytic enzyme production by SSF.
performed in 500-mL Erlenmeyer flasks with gentle agita- Steam explosion is classified as a green technology due
tion (125 rpm) in a water bath; the pH was adjusted to 5.2 to the reaction medium, which contains only lignocellu-
with 0.1 M citrate buffer and maintained at 50 ℃ for 48 h. losic feedstock and water; this pretreatment has effectively
Samples were taken every 12 h, and the release of reducing been used in delignification and hemicellulose removal

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Fig. 1  Release of reducing
sugars from samples of water
hyacinth pretreated by steam
explosion at 190 ℃ and different
treatment times

(Martin-Sampedro et  al. 2014a, b; Ibrahim et  al. 2011; and delignified after pretreatment by steam explosion, as
Oliveira et  al. 2013) through lignin transformation and can be seen in Fig. 1 (release of fermentable sugars) and
increases the surface area for cellulose hydrolysis (Singh Fig. 2a. Our results were similar to those found by Oliveira
et al. 2015). In our study, WH was obviously hydrolysed et  al. (2013), who indicated that the steam explosion

Fig. 2  Effect of steam explosion on lignin present in water hyacinth a Water hyacinth from the Maria Lizamba lagoon pretreated by steam explo-
sion, b Schematic representation of the steam explosion effect on water hyacinth delignification

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degraded lignin (Fig. 2b) or transformed/hydrolysed it by

the explosion-produced rapid decompression and high
temperatures to hemicelluloses (auto-hydrolysis) and free
mono- and oligosaccharides. The sugar yields obtained in
these work were lower than those obtained in other works,
such a 19.7 g/100 g of dry olive leaves at 180 ℃ for 10 min
(Romero-Garcia et al. 2016) and 46% of glucose after of two
cycles of the steam explosion at 183 ℃ for 5 min for Euca-
lyptus globulus (Martin-Sampedro et al. 2014b). Similar
results have been obtained for rice husk by Piñeros-Castro
et al. (2011) at 190°℃ for 10 min. However, it is important
to mention that the lignocellulosic raw materials pretreated
with steam explosion were different from those obtained Fig. 4  Cellulase production profil by T. harzianum PBL4 during SSF
fromWH, since this is the first report.
On the other hand, it is important to note that Piñeros-
Castro et al. (2011) indicated that in the steam explosion the highest xylanolytic and cellulolytic activity was observed
process, the amounts of furfural and hydroxymethylfur- after 108 h of cultivation, similar to the results reported by
fural increase at exposure times of more than 10 min. The Lopez-Ramirez et al. (2018) (48 h) under similar conditions.
presence of these compounds decreases both the specific The time needed to obtain the highest enzymatic activity in
growth rate and ethanol production in alcoholic fermentation this work was shorter than that reported in other studies that
(Taherzadeh et al. 1999); similar results have been observed used the same substrate in SSF. In a study be Deshpande
by Sun et al. (2016). For this reason, no longer periods were et al. (2008), the time required to obtain the highest cel-
assayed in the present work. lulase and xylanase activity using other supports and fungi
was 9 days, while fir cellulase activity, Zhao et al. (2011)
Hydrolytic enzyme production by SSF reported 7 days under optimized conditions. The activity
levels found in the present study were lower for cellulase
The results of hydrolytic enzyme production in packed bed and xylanase activity produced via WH as a substrate in
columns using T. harzianum PBLA as inoculum and WH SSF. It should, however, be noted that only a few reports are
from the María Lizamba Lagoon as substrate are shown in available on cellulase and xylanase production using WH
Figs. 3 and 4. The highest xylanase activity was obtained as a substrate in SSF (Lopez-Ramirez et al. 2018; Arana-
after 96 h (42 U/g of dry matter) of cultivation in SSF. How- Cuenca et al. 2019).
ever, after 72 h, a first activity peak occurred (40 U/g of dry Maximum ­CO2 production occurred between 54 and 62 h
matter). Maximum cellulase activity was observed after 68 h in all analysed columns in SSF (Fig. 5). The respirometric
of cultivation (2 U/g of dry matter). These results indicate results showed that xylanase and cellulase production started
that the time required for maximum xylanolytic and cel- after reaching the maximum C ­ O2 production. Respirometric
lulolytic activity was shorter than that for the production of
hydrolytic enzymes by SSF, using the same support and fun-
gus, reported by Arana-Cuenca et al. (2019); in their study,

Fig. 5  Profile of C
­ O2 production rate by T. harzianum PBL4 in SSF
performed in packed bed columns. C1 and C2 were SSF blanks
(without T. harzianum PBL4), and C3 to C6 were SSF columns mon-
Fig. 3  Xylanase production profil by T. harzianum PBL4 during SSF itored over 110 h

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analysis suggests that for the production of the hydrolytic obtained in this work is highly important; according to
enzyme by T. harzianum PBLA in SSF, it is necessary that Zhang et al. (2016), the use of enzymes in the saccharifi-
the maximum rate of ­CO2 production has been reached; cation of water hyacinth is larger for ethanol production;
approximately after 12 h, maximum xylanase (first maxi- however, when the hydrolytic enzymes were produced by
mum, Fig. 3) and cellulase activity (Fig. 4) will be obtained, SSF from WH (present work), the costs of ethanol produc-
indicating that the SSF must be stopped. Respirometry anal- tion were reduced. Also, the efficiency of saccharification
ysis is a useful tool for the microbial study of physiology and obtained in the present research (0.46 and 0.51) is higher
metabolism (Lopez-Ramirez et al. 2018; Pliego-Sandoval than that obtained when WH was saccharified using acid
et al. 2012; Méndez-González et al. 2020). pretreatment followed by enzymatic hydrolysis using com-
mercial enzymes (0.40 g of reducing sugars per g of WH),
Enzymatic hydrolysis of water hyacinth pretreated even when the enzymatic reaction conditions were optimised
with steam explosion (Zhang et al. 2016). The result obtained after 48 h of enzy-
matic hydrolyses corresponds to the theoretical maximum
Lignocellulosic material, such as WH, may provide ferment- of reducing sugars from WH (0.51 g/g) reported by Xia
able sugars for ethanol production. However, recalcitrant et al. (2013). We conclude that the combination of steam
structures of WH biomass are difficult to convert. For that explosion pretreatment and enzymatic hydrolysis with WH-
reason, it is necessary to use combinations of methods, as produced enzymes is adequate to obtain higher amounts of
described by Singh et al. (2015), especially when enzymes fermentable sugars. The yields after 24 h of enzymatic reac-
are used for cellulose hydrolysis. In this case, pretreatment is tion were similar to those obtained by Satyanagalakshmi
required to make cellulose more accessible to the hydrolytic et al. (2011), who reported 0.48 g/g after 24 h of incubation.
enzymes, facilitating its conversion to glucose (fermentable However, it is important to mention that in the cited works,
sugars). Martín-Sampedro et al. (2014b) indicated that the commercial enzymes for WH hydrolysis were used, and the
steam explosion facilitates enzymatic saccharification, and results obtained by these authors after 48 and 72 h were
for this reason, we used enzymatic hydrolysis as a pretreat- similar to those obtained after 24 h in the present research.
ment method; additionally, hydrolytic enzymes (cellulases In contrast to previous findings, in our study, the efficiency
and xylanases) were produced via SSF using WH as a sub- of enzymatic reaction was superior after 48 h increasing
strate because the specificity by substrates may be greatest. reducing sugars in 5% compared to the results obtained after
Enzymatic hydrolysis of WH from the María Lizamba 24 h of the enzymatic reaction. The yields of reducing sugars
Lagoon pretreated by steam explosion showed that this pro- were lower in our study than in the studies by Aswathy et al.
cess improved enzymatic saccharification, as can be seen in (2010) (0.73 g/g) and Sukumaran et al. (2009) (0.71 g/g),
the enzymatic kinetics shown in Fig. 6. The results indicate who used enzymes produced by SSF, and in Das et al. (2015)
that the maximum reducing sugars were obtained after 48 h (0.567 g/g), who used commercial enzymes. The amount of
of enzymatic reaction (15.5 g/L); however, after 24 h, a level fermentable sugars was higher than that reported in other
of 14.5 g/L of reducing sugars was obtained. The efficiency works, probably because enzyme production using WH as
of enzymatic hydrolysis was 0.46 and 0.51 g of reducing support by SSF increases the specificity for the same sub-
sugars per gram of WH (dry matter) after 24 and 48 h of the strate. This is the first report that used enzymes produced by
enzymatic reaction, respectively. The enzymatic efficiency SSF, using the substrate for saccharification.
Comparing the results on fermentable sugars obtained
from WH saccharified by steam explosion followed by enzy-
matic hydrolysis with those found for other lignocellulosic
material, such as sugar cane bagasse (Bunterngsook et al.
2018; with 0.79 g/g), our results were lower. However, it
should be noted that we used recombinant enzymes; how-
ever, our yield was higher than that reported for saccharified
wheat bran, where a yield of reducing sugars of 0.19 g/g was
obtained (Jiang and Guo 2016). It is important to mention
that the yield of fermentable sugars depends on the recalci-
trant strength of the substrate to be saccharified and on the
amount of fermentable sugars they contain.
On the other hand, as mentioned by Singh et al. (2015)
and Martín-Sampedro et al. (2014b), the steam explosion
Fig. 6  Kinetics of enzymatic hydrolysis of Maria Lizamba Lagoon
water hyacinth biomass using xylanases and cellulases produced by improved enzymatic saccharification. After the steam
SSF explosion, cellulose was more accessible to the enzymes.

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than that obtained (1.289 g/L) when acid hydrolysis followed

by enzymatic hydrolysis was used (Zhang et al. 2016). Our
results indicate that combined pretreatment may improve
bioethanol production from WH. It is, however, important
to indicate that this is the first report on the use of steam
explosion and enzymatic saccharification (using enzymes
produced in the same substrate). The yield of ethanol may
be increased if S. cerevisiae is replaced by microorganisms
that use hexoses and pentoses since the former can only use
hexoses (Das et al. 2015; Ganguly et al. 2013).
Comparing our results with those obtained using other
lignocellulosic materials showed that the yields of etha-
nol obtained in the present research (23%) are higher, even
Fig. 7  Ethanol production by Saccharomyces cerevisiae using reduc- when the same methods of saccharification were used (steam
ing sugars obtained from water hyacinth enzymatic saccharification
explosion followed by enzyme hydrolysis). However, it
should be noted that the enzymes used in previous stud-
We used WH as the source of cellulases and xylanases for ies were commercial enzymes. For example, in reports on
SSF to reduce ethanol production costs. This is the first ethanol production from bagasse of cane, cane leaves and
report on this combination of pretreatments for saccharifi- sugarcane straw, ethanol yields were 16.2, 20.3 and 5.7%,
cation, namely the steam explosion followed by enzymatic respectively, as reported by Mokomele et al. (2018) and
hydrolysis. Pratto (2020).
In general, the processes for obtaining ethanol from lig-
Ethanol production nocellulosic biomass present technical–economic problems
such as energy costs and the costs of the enzymes used, as
To verify the effect of pretreatments on ethanol production, well as enzyme specificity: in this work, the cost of hydro-
reducing sugars obtained after enzymatic hydrolysis of WH lytic enzymes was kept low by producing them on the sub-
were used as carbon sources for alcoholic fermentation by strate to be hydrolysed, thus solving not only the cost prob-
Saccharomyces cerevisiae. We obtained the highest ethanol lem, also obtaining highly specific enzymes (Madadi et al.
concentration (7.13 g/L) after 72 h of fermentation, with 2017). Regarding the operating costs generated by the steam
an ethanol yield of 0.23 g/g of dry matter (WH) or 0.46 g/g explosion, as commented by Pratto et al. (2020), the pro-
of reducing sugars with 80% of reducing sugars consumed. jected operating costs of an economic analysis in an ethanol
The kinetics of ethanol production from WH biomass sac- production process from lignocellulosic biomass are reduced
charified with SSF-produced enzymes can be seen in Fig. 7. by up to 20%, even using steam explosion. This is because
The ethanol yield was higher than that reported by Ma et al. it is a process carried out for only a few seconds to a few
(2010) (0.192 g/g of dry matter), who used reducing sug- minutes, requiring low amounts of energy (Tu et al. 2017;
ars obtained from WH pretreated with acid after enzymatic Kumari and Singh 2018; Kucharska et al. 2018).
hydrolysis. Likewise, the volumetric ethanol yield in our
study was higher those of previous studies, e.g. 3.49 g/L
(Ganguly et  al. 2013), 4.25  g/L, (Aswathy et  al. 2010),
Conclusions
9.8 g/L (Singh et al. 2013), while it was lower than several
previously found values (10.1 g/L (Mishima et al. 2008),
The combination of pretreatments proposed in the present
10.44 g/L (Das et al. 2016), 13.6 g/L (Ganguly et al. 2013))
investigation allowed better recovery of reducing sugars
and comparable with the 0.46 g/g of reducing sugar reported
from WH than other proposed pretreatments. Also, the
by Kumar et al. (2009), who used different pretreatments
steam explosion allows higher efficiency of enzymatic
for WH saccharification. Ethanol production from cellulosic
hydrolysis by making the substrates more accessible for
materials greatly depends on the disruption of the lignocellu-
enzymes. Likewise, the recovered reducing sugars have a
losic structure; different pretreatments including acid, alkali,
higher ethanol conversion rate than reported previously for
microwave, liquid hot water and compound pretreatments
water hyacinth. In this sense, SSF is an excellent process for
have been used for this purpose. For ethanol production
hydrolytic enzyme production, reducing the costs of ethanol
using WH as a substrate, an effective method is an acid pre-
production.
treatment (Zhang et al. 2016). However, the ethanol content
obtained in the present work (7.13 g/L), when steam explo- Acknowledgements  Figueroa-Torres L. A. thanks Consejo Nacional de
sion followed by enzymatic hydrolysis was used, was higher Ciencia y Tecnología, México for the scholarship No. 446317.

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Author contributions  RH conceptualization. LAF and NL and ECV did Gütsch JS, Nousiainen T, Sixta H (2012) Comparative evaluation
the analytical work and methodology. RH, LAF and EF Data analysis. of autohydrolysis and acid-catalyzed hydrolysis of Eucalyp-
MAL validation. RH and MAL helped in preparing and editing the MS. tus globulus wood. Bioresour Technol 109:77–85. https​://doi.
org/10.1016/j.biort​ech.2012.01.018
Ibrahim MM, Agblevor FA, El-Zawawy WK (2010) Isolation and
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