Journal of Cleaner Production 339 (2022) 130702

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Journal of Cleaner Production

journal homepage: www.elsevier.com/locate/jclepro

Efficient ethanol production from rice straw through cellulose restructuring

and high solids loading fermentation by Mucor indicus
Maryam Molaverdi a, Safoora Mirmohamadsadeghi a, Keikhosro Karimi a, b, **,
Mortaza Aghbashlo c, Meisam Tabatabaei d, e, f, *
a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran
b
Department of Chemical Engineering, Vrije Universiteit Brussel, 1050, Brussels, Belgium
c
Department of Mechanical Engineering of Agricultural Machinery, Faculty of Agricultural Engineering and Technology, College of Agriculture and Natural Resources,
University of Tehran, Karaj, Iran
d
Higher Institution Centre of Excellence (HICoE), Institute of Tropical Aquaculture and Fisheries (AKUATROP), Universiti Malaysia Terengganu, 21030 Kuala Nerus,
Terengganu, Malaysia
e
Henan Province Engineering Research Center for Biomass Value-added Products, School of Forestry, Henan Agricultural University, Zhengzhou, 450002, China
f
Biofuel Research Team (BRTeam), Terengganu, Malaysia

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

Handling Editor: Cecilia Maria Villas Bôas de The production of high concentrations of ethanol from N-methylmorpholine N-oxide- (NMMO) and phosphoric
Almeida acid-pretreated rice straw was investigated at high solid loadings (solid-state) using filamentous fungus Mucor
indicus. The impacts of most influential factors on ethanol production by simultaneous saccharification and
Keywords: fermentation (SSF), i.e., enzyme loadings (2.5, 5, 10, and 20 FPU/g substrate), solids loadings (15, 20, and 30%
Dry fermentation
w/w), and SSF time (72 and 120 h), were assessed. The highest glucose concentrations were 106.5 and 94.1 g/L
Ethanol
gained from 72-h hydrolysis of the straw pretreated with NMMO and phosphoric acid at 15% (w/w) solids
Lignocellulose fractionation
Mucor indicus loadings, respectively. The highest ethanol concentrations were 63.4 and 69.0 g/L, achieved by 30% solids
N-methyl morpholine N-Oxide loading from 72-h SSF of the straw pretreated with NMMO and phosphoric acid at 20 and 10 FPU celluloses per g
Phosphoric acid substrate, respectively. A prolonged SSF process at enzyme loadings less than 5 FPU/g straw could not improve
ethanol concentration from NMMO-pretreated straw, while ethanol concentrations above 40 g/L (61.0 and 59.9
g/L) were achieved through 120-h fermentation of the straw pretreated with phosphoric acid at 30% solid
loading. The highest ethanol yields were 73.7 and 100% of the theoretical yield gained from the straw pretreated
with phosphoric acid and NMMO, respectively, at the lowest solids loading (15%) and the highest enzyme
loading (20 FPU/g substrate).

with the SHF method is contamination. In the hydrolysis process, which

1. Introduction
Nowadays, many aspects of converting lignocellulosic materials to
biofuels and other valuable products have been investigated (Bhatia
et al., 2017). Two common processes for ethanol production from
lignocellulosic materials are the separate hydrolysis and fermentation
(SHF) and the simultaneous saccharification and fermentation (SSF)
(Kazemi Shariat Panahi et al., 2020). In SHF, hydrolysis and fermenta­
tion occur in their optimal conditions. The inhibition effect of released
sugars, mainly cellobiose and glucose, on cellulase activity is a major
drawback of the SHF method (Khounani et al., 2019). Another problem
is a relatively long process (1–4 d), a dilute sugar solution is produced
that could be easily contaminated with microbes even at relatively high
temperatures of 45–50 ◦ C. However, in SSF, cellulose and glucose pro­
duced from hydrolysis are consumed simultaneously by microorganisms
(Kazemi Shariat Panahi et al., 2022). Therefore, they pose no inhibitory
effect on the enzyme function because the concentration of these sugars
always remains low during the operation. It has been shown that the
efficiency of ethanol production using the SSF method is higher than the
SHF, while this method also requires less enzyme content (Kazemi
Shariat Panahi et al., 2019). The risk of contamination in SSF is lower
than in SHF due to ethanol’s presence. Furthermore, the number of

* Corresponding author. Henan Province Engineering Research Center for Biomass Value-added Products, School of Forestry, Henan Agricultural University,
Zhengzhou, 450002, China.
** Corresponding author. Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran.
E-mail addresses: karimi@cc.iut.ac.ir (K. Karimi), meisam.tabatabaei@umt.edu.my, meisam_tab@yahoo.com (M. Tabatabaei).

https://doi.org/10.1016/j.jclepro.2022.130702
Received 30 September 2021; Received in revised form 13 January 2022; Accepted 26 January 2022
Available online 29 January 2022
0959-6526/© 2022 Elsevier Ltd. All rights reserved.
M. Molaverdi et al.
Abbreviations
CI Crystallinity index
FPU Filter paper unit
HPLC High-performance liquid chromatography
NMMO N-methyl morpholine N-Oxide;
NREL: National Renewable Energy Laboratory
SHF Separate hydrolysis and fermentation
SSF Simultaneous saccharification and fermentation
TCI Total crystallinity index
containers needed for SSF is less than SHF, resulting in reduced process
investment costs (Taherzadeh and Karimi, 2007). With all, the economy
of ethanol production from lignocelluloses is still one of the important
process bottlenecks (Amid et al., 2021). The main reasons are the low
concentration of ethanol from lignocelluloses, increasing energy con­
sumption for the distillation step, and the price of cellulose hydrolytic
enzymes (Zhang et al., 2009).
Enzymatic hydrolysis and fermentation at high solids loadings and
very low enzyme loading are a solution with several advantages. Higher
final sugar concentrations and, consequently, higher ethanol concen­
tration could reduce energy for the distillation process and minimize
capital and production costs (Molaverdi et al., 2013). Water use in this
type of process is reduced, and fewer reactors and smaller equipment
can be applied. Besides, downstream and wastewater processing and
environmental impacts are minimized due to low water levels (Mola­
verdi et al., 2019a). However, heat and mass transfer limitations are the
main technological problems in high solid loading processes (Kristensen
et al., 2009; Olofsson et al., 2008). Therefore, the efficiency of ethanol
productivity would be the main challenge at high solids loading (Lu
et al., 2010). Although the high solid loading process is essential to
improve and modify the economy of second-generation bioethanol
production, an increase in solids loading leads to a reduction in ethanol
yield. Therefore, high titer ethanol production with high conversion
efficiency using low enzyme loading is still the main challenge (Nguyen
et al., 2016).
The choice of pretreatment can also affect the effectiveness of the
overall conversion process (Phwan et al., 2018; Soltanian et al., 2020). A
useful method for pretreating lignocellulosic materials is using cellulose
solvents. Their mechanism is often based on the dissolution of the
lignocellulose components in a solvent under certain circumstances.
Then, all or some components are deposited and recovered by adding an
anti-solvent. In the case of retrieving all the components after adding
anti-solvent, breaking the recalcitrant structure of lignocellulose im­
proves the hydrolysis yield. However, in the case of depositing some
components, the function of enzymes is enhanced by removing some
inhibitors and reshuffling the structure (Shafiei et al., 2010).
N-methylmorpholine N-oxide (NMMO) is an industrial cellulose
solvent that dissolves cellulose by defeating intermolecular forces
without changing the material composition considerably. Today,
NMMO is used as a direct solvent for cellulose in the industrial Lyocell
process. The Lyocell process is an eco-friendly and modern technique for
fiber production based on the dissolution of cellulose in NMMO. This
solvent does not create any toxic substance, and almost all of it (more
than 98%) can be recovered and reused. Cellulose dissolution in NMMO
causes changes in its crystallinity; thus, it is presented as a promising
agent for the pretreatment of lignocellulosic materials (Shafiei et al.,
2010). It should be noted that NMMO with a concentration between 83
and 87% is a cellulose solvent, and this concentration range is the most
suitable one for lignocelluloses pretreatment (Kuo and Lee, 2009). In
NMMO solutions with concentrations ranging between 76 and 82%,
cellulose swells and slightly dissolves, while lower concentrations
(70–75%) make only cellulose swell. At concentrations below 70%, no
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Journal of Cleaner Production 339 (2022) 130702
change occurs in cellulose fibers (Kuo and Lee, 2009).
Concentrated phosphoric acid is another cellulose solvent that is
recognized as one of the most effective pretreatments at mild conditions
for improving the hydrolysis of lignocelluloses (Moradi et al., 2013).
This process is called cellulose-solvent and organic-solvent-based
lignocellulose fractionation (COSLIF). In this method, cellulose type I
in lignocellulosic materials is converted to less crystalline cellulose
(cellulose type II) at atmospheric pressure and 60 ◦ C using a cellulose
solvent (i.e., H3PO4 at a concentration more than 81%), a volatile
organic solvent (e.g., acetone), and a washing agent (i.e., water). The
advantages of this pretreatment are that the residual phosphoric acid has
no inhibitory effect on hydrolysis and fermentation. Moreover, the sol­
vents are easily recoverable, and the dissolution of cellulose in phos­
phoric acid occurs at low temperatures (Satari et al., 2018).
Using a proper microorganism for fermentation also affects ethanol
yield at high solids loading. Mucor indicus is capable of producing
ethanol from various substrates (Karimi and Zamani, 2013). Research on
this species began more than 200 years ago. Mucor species can be found
in dairy products, meats, fruits, and vegetables because of their ability to
break down different carbohydrates and proteins. This fungus is used to
produce renin, amylase, organic acids, alcohol, ethyl alcohol, cheese,
and tempeh (a type of traditional food in southeast Asia) (Krishna,
2005). The natural physiological abilities of fungi make them suitable
options for high solid fermentation (Mitchell et al., 1992). Therefore, M.
indicus, with dimorphic characteristics, is proper to produce high-yield
ethanol. This fungus is an atmospheric species that can withstand tem­
peratures above 40 ◦ C. This fungus has several advantages over indus­
trial microorganisms, e.g., Saccharomyces cerevisiae, including the
capability of fermenting a wide range of sugars such as pentoses, the
valuable biomass rich in chitin and chitosan, the ability of growing at
upper temperatures than conventional yeasts, and more exceptional
ability to tolerate inhibition (Aghbashlo et al., 2017). Besides, the
biomass of M. indicus has high amounts of proteins and lipids, making it
possible to be used as animal feeds (Molaverdi et al., 2019b). All of these
advantages have led to the study of the fungus as a suitable microor­
ganism to produce ethanol from industrial substrates in recent years
(Asachi et al., 2011). To the best of our knowledge, no research has been
published on the effects of NMMO and phosphoric acid pretreatments on
simultaneous saccharification and fermentation (SSF) of rice straw at
solid-state using M. indicus.
This study aimed to efficiently convert rice straw into ethanol using a
high solid loading (solid-state) process without mixing, prehydrolysis,
neutralization, and detoxification by applying solvents pretreatment
and M. indicus. After pretreatments, hydrolysis and SSF were performed
on the substrate at high solids loadings. Also, the impacts of different
enzyme loadings on hydrolysis and SSF yields at high solids loadings
were studied.
2. Materials and methods
In this section, all materials and methods used in this study for the
production of high titer ethanol by high solid SSF using solvent pre­
treatments are reported in detail.
2.1. Materials and chemicals
Rice straw was provided from a field located in Pir Bakran, (Isfahan,
Iran, 32◦ 28′ 8′′ N, 51 33′ 28′′ E). The straw was milled into small par­
ticles by a coffee mill (Krups, Germany). Then, the particles within the
range of 0.178–0.853 mm were screened using 20 and 80 meshes and
dried at ambient temperature. The moisture content after drying was
about 5.5%.
The blend of cellulase and hemicellulase by 1:9 ratio from Cellic®
HTec2 (VHN00002) with the enzyme activity of 23 FPU/mL and Cellic®
CTec2 (VCNI0013) with the enzyme activity of 100 FPU/mL was pre­
pared and applied (samples were provided by Novozymes, Denmark,
M. Molaverdi et al.
free of charge). The enzyme activity of the blend (95 FPU/mL) was
measured according to the standard method developed by National
Renewable Energy Library (NREL) (Adney and Baker, 1996).
The NMMO used in this study was industrial grade (50% w/w, BASF,
Ludwigshafen, Germany). At 100 mbar (absolute) and >130 ◦ C, a rotary
evaporator was used to increase the NMMO concentration to 85%.
M. indicus (No. 22424) was obtained from the microbial culture
collection of the University of Gothenburg (CCUG), Sweden. It was
cultured on a solid medium containing glucose, 40 g/L, agar, 20 g/L, and
peptone, 10 g/L. After obtaining a homogenous solution using a mag­
netic stirrer, the medium was sterilized using an autoclave (120 ◦ C for
15 min), cooled down, poured into Petri dishes under a microbial hood,
and incubated at 32 ◦ C for 24 h, to be sure that no germs were grown on
them. Then, the fungal spores were inoculated and incubated for 48 h at
32 ◦ C, where the cotton-like fungal hypha covered all the surface of Petri
dishes.
2.2. Pretreatments
In this section, NMMO and phosphoric acid solvent pretreatments
performed to open up the structure of untreated rice straw are explained.
2.2.1. N-methylmorpholine N-oxide pretreatment
According to Shafiei et al. (2010), rice straw and the NMMO solution
(85%) were mixed with a ratio of 1:19 g/g, respectively, heated up to
120 ◦ C and kept for 3 h at this temperature. After finishing the desired
time, 60 mL of deionized water (the antisolvent) was rapidly poured into
the suspension to regenerate the dissolved cellulose. Then, the precipi­
tated solids washed with boiling water to remove the NMMO remained
in the precipitated materials, i.e., NMMO pretreated straw.
2.2.2. Phosphoric acid pretreatment
To perform this pretreatment, phosphoric acid (8 mL, 85%) was
added to 1 g of the straw in a centrifuge tube (50 mL), and it was placed
in a water bath at 60 ◦ C for 45 min. The reaction of cellulose dissolution
was stopped by adding 20 mL acetone as an anti-solvent. The solid
fraction was separated from the supernatant by centrifugation (Uni­
versal 320 R, Tuttlingen, Germany) at 4,000×g for 15 min and dis­
charging the liquid fraction. Then, 40 mL of acetone was poured into the
tubes containing the solid fraction and mixed, and the solid fraction was
separated by centrifugation (at 4,000×g for 15 min). Afterward, the
solid fraction was mixed with distilled water and separated by centri­
fugation and supernatant decanting. The pretreated straw was dried by a
freeze drier (Martin Christ, Alpha 1–2 LD plus) at − 48 ◦ C for 48 h
(Mirmohamadsadeghi et al., 2016).
2.3. Enzymatic hydrolysis
The untreated and pretreated lignocellulosic samples were enzy­
matically hydrolyzed at 15, 20, and 30 wt % substrate loading using 2.5,
5, 10, and 20 FPU/g substrate enzyme loadings for 72 h at 45 ◦ C. In
addition to the untreated and pretreated straw samples, sodium citrate
buffer solution (pH 4.8, 0.05 M) was also added into 118 mL glass
bottles, and then the mixture was sterilized by autoclaving. Finally, the
enzyme mixture was added to the bottles after reaching room temper­
ature. All analyses and experiments were performed in duplicates, and
their average amounts were reported.
2.4. Biomass production
The M. indicus biomass was produced in a medium including 50 g/L
glucose, 5 g/L yeast extract, 7.5 g/L (NH4)2SO4, 3.5 g/L K2HPO4, 1 g/L
CaCl2⋅2H2O, and 0.75 g/L MgSO4⋅7H2O, at the pH of 5.5. After auto­
claving the media at 121 ◦ C for 20 min, they were inoculated with the
fungal spores. For this purpose, sterile distilled water (10 mL) was
poured into the agar plates, and the spores were released using a sterile
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Journal of Cleaner Production 339 (2022) 130702
Table 1
Recoveries and compositions of untreated rice straw as well as straw pretreated
with NMMO and phosphoric acid on the dry basis.
Composition (%) Untreated straw pretreated straw
Phosphoric acid NMMO
Glucan 40.2 ± 0.8 46.0 ± 1.1 43.5 ± 0.7
Xylan 12.5 ± 1.4 7.3 ± 0.7 11.2 ± 0.1
Other carbohydrates 4.2 ± 0.4 5.4 ± 0.1 5.0 ± 0.2
Lignin 16.7 ± 0.6 16.4 ± 0.1 13.2 ± 0.3
Ash 6.1 ± 0.1 7.4 ± 0.9 8.2 ± 0.5
Solid recovery – 55 68
loop. This spore suspension was applied to prepare 50 mL inoculum
anaerobically fermented for 24 h (at 32 ◦ C and 120×g). Then, the pro­
duced fungus was centrifuged at 3,000×g for 10 min (Hettich-Uni­
versa320R, Tuttlingen, Germany) and applied for the main fermentation
experiments (Molaverdi et al., 2019b).
2.5. Simultaneous saccharification and fermentation
SSF of untreated and pretreated rice straw was conducted at 15, 20,
and 30% (w/w) solid loadings and 2.5, 5, 10, and 20 FPU/g substrate
enzyme loadings (Karimi et al., 2006). The prepared straw samples with
buffer solution (sodium citrate, 0.05 M) were autoclaved in 118 mL glass
bottles at 121 ◦ C for 20 min and left to reach ambient temperature. Then,
5 g/L of the fungal biomass and certain volumes of enzymes were added
to the glass bottles and incubated at 37 ◦ C under anaerobic conditions.
The ethanol yield was calculated according to Equation (1) (Poor­
nejad et al., 2013):
Produced ethanol (g/L)
Ethanol yield (%) = × 100 (1)
Cellulose content (g/L) × 0.51 × 1.111
where 0.51 (g/g) represents the theoretical ethanol yield from hexoses,
and 1.111 is the glucan hydration factor.
2.6. Analytical methods
The compositional analyses of untreated and treated straw samples
were performed based on the NREL LAP procedure (Sluiter et al., 2008).
The liquid samples, taken from compositional analysis, SSF, and hy­
drolysis, were injected into a high-performance liquid chromatograph
(HPLC). The HPLC was equipped with a refractive index detector (Agi­
lent Technologies 1260 Infinity, Germany). The remaining glucose and
ethanol were detected by separation in an Aminex HPX-87H column
eluted with the mobile phase of 0.6 mL/min H2SO4 (5 mM) at 60 ◦ C. An
Aminex HPX-87P column (Bio-Rad, CA, USA) eluted with HPLC grade
water at 80 ◦ C was used for sugar analysis. Total reducing sugars con­
centrations were analyzed using Miller’s technique (Miller, 1959). To
investigate the effect of pretreatments on the crystallinity of the straw, a
Fourier transform infrared spectrophotometer (FTIR, Rayleigh
WQF-510A) was used. The final spectra with a resolution of 4 cm− 1 were
obtained from the average of 32 scans from 4400 to 400 cm− 1.
3. Results and discussion
High titer ethanol production using high solid SSF at different solids
(15, 20, and 30%) and enzyme loadings (20, 10, 5, and 2.5 FPU/g
substrate) was investigated. The impacts of two solvent pretreatments
and using Mucor indicus in SSF on the efficiency of ethanol production
were studied. In this section, the obtained results are reported, dis­
cussed, and compared with those of the other studies.
3.1. Pretreatment impacts on the straw composition
The composition of the straw before and after NMMO and
M. Molaverdi et al.
Fig. 1. FTIR spectra of untreated, NMMO-pretreated, and phosphoric acid-pretreated rice straw.
Table 2
The crystallinity index of untreated straw and the straw pretreated with NMMO
and phosphoric acid.
Substrate Pretreatment interval CI TCI
Untreated rice straw – 1.45 1.55
Pretreated rice straw with:
NMMO 3h 1.31 1.29
Phosphoric acid 45 min 1.29 1.17
phosphoric acid pretreatments are presented in Table 1. The untreated
straw contained 40.2% glucan, 12.5% xylan, 16.7% lignin and 23%
extractive. The impacts of pretreatments on glucan and lignin contents
of straw were not considerable. However, phosphoric acid pretreatment
significantly decreased the xylose content (about 40%) because of the
acid hydrolysis of hemicellulose. These results are consistent with other
studies (Poornejad et al., 2013; Shafiei et al., 2010). In another study,
Poornejad et al. (2013) investigated the NMMO pretreatment impact on
ethanol production from rice straw. The compositional analysis showed
that the glucan content was enhanced from 40.7% to 44.9%, while the
lignin content was reduced from 19.8% to 16.4%. The mechanism of
solvent pretreatments is the dissolution of cellulose only by the break­
down of intermolecular forces without considerable alteration in the
chemical composition. Then, the reaction is stopped by adding
anti-solvent, and the dissolved materials, with an open-up structure,
precipitate on a solid surface (Shafiei et al., 2010).
3.2. Pretreatment impacts on straw crystallinity
The structural changes by pretreatments on rice straw were investi­
gated using FTIR. The FTIR spectra of all straw samples are shown in
Fig. 1. The absorbance ratios of A1375:A2900 and A1430:A896 represent the
total crystallinity index (TCI) and crystallinity index (CI) values,
respectively (Poornejad et al., 2013). The TCI and CI values are reported
in Table 2. Both pretreatments notably decreased the crystallinity
indices by completely dissolving type I cellulose with high crystallinity,
with the anti-solvents re-sedimenting it in the form of type II cellulose
with less crystallinity. Similar results have been observed for the cellu­
lose solvent pretreatments (Amiri and Karimi, 2013). In another study,
Poornejad et al. (2013) investigated the effect of NMMO pretreatment
on the crystallinity of rice straw. The FTIR of samples indicated that the
TCI and CI were decreased from 1.69 to 0.46 for the untreated straw to
1.62 and 0.40 for NMMO treated straw, respectively.
4
Journal of Cleaner Production 339 (2022) 130702
The effect of phosphoric acid and NMMO pretreatment was also
studied on the hydrolysis of cotton fiber with a cellulose content of over
99 wt % (Amiri and Karimi, 2013). After these pretreatments, the FTIR
absorption band showed a major reduction in CI and TCI. Therefore, it
could be concluded that cellulose I, untreated (native) cellulose, was
regenerated to cellulose II and amorphous cellulose. The NMMO pre­
treatment of pinewood similarly decreased biomass crystallinity in a
study by Shafiei et al. (2014).
3.3. Enzyme loading impact on enzymatic hydrolysis
The main challenge of enzymatic hydrolysis or SSF at high solids
loadings is the extra need for enzymes to have high ethanol production
yields. Therefore, studying the impacts of enzyme and solid loadings on
solid-state hydrolysis is essential. To determine the effect of enzyme
loading, enzymatic hydrolysis was conducted at 2.5 to 20 FPU/g straw at
high solid loading (15% w/w). Typically, higher enzyme loadings are
expected to result in higher hydrolysis yields. However, this cannot
happen due to the phenomenon referred to as jamming or overcrowding.
The cellulase enzyme molecules are meaningly larger than their ultimate
substrate. The binding/activation of an enzyme molecule by a substrate
molecule would typically not prevent the other binding/activation of
enzyme and substrate molecules in a homogeneous solution. However,
when the substrate molecules are limited in a small space by their
immobility or insolubility, this leads to a jamming among the enzyme
molecules. It should be noted that cellulose fibers are structured in
parallel to each other by ~ 4–6 Å. However, cellobiohydrolases (CBH)
are prolate ellipsoids with dimensions of ~ 67 Å × 45 Å × 45 Å. The
movement of CBH enzymes on the surface of the crystalline cellulose
fibers is one dimensional along the fiber. At high concentrations of the
enzyme, several molecules of CBH bind to each other, locating on a small
surface of cellulose and because of the different rate of the bound en­
zymes, they tend to interfere with each other. This effect is called jam­
ming or overcrowding, leading to a reduced overall reaction rate. Also,
active enzymes can be trapped between bound but inactive enzymes.
Therefore, this effect seems to be of particular importance for the hy­
drolysis of cellulose by CBH because of the regular packing of cellulose
chains, the difficulty of separating anchor enzymes, and the size dif­
ference between the enzyme and the inter-chain distance (Igarashi et al.,
2006; Xu and Ding, 2007). Bommarius et al. (2008) proposed the
overcrowding of enzyme molecules as the corresponding mechanism for
decreasing the hydrolysis yield at very high concentrations of enzymes.
The glucose and total reducing sugars concentrations gained from
M. Molaverdi et al.
Fig. 2. Impacts of enzyme load on the concentrations of (a) total reducing sugars and (b) glucose from enzymatic hydrolysis at 15% solid loading.
enzymatic hydrolysis of untreated and pretreated straw are presented in
Fig. 2. As can be seen, the highest total reducing sugars produced from
72-h enzymatic hydrolysis of raw, the NMMO-pretreated, and the
phosphoric acid-pretreated straw were 60.0, 120.8, and 118.3 g/L,
respectively, obtained with 20 FPU/g substrate enzyme load. Therefore,
NMMO and phosphoric acid pretreatments could increase total sugar
concentrations by about two folds. The interesting point about NMMO
pretreatment was that small changes in total sugar concentrations were
observed with changing the enzyme loading. In other words, a sharp
Fig. 3. Impact of different enzyme loads on ethanol concentrations from SSF at
15% solid loads by Mucor indicus.
5
Journal of Cleaner Production 339 (2022) 130702
decrease in enzyme load from 20 to 2.5 FPU/g straw led to a slight
decline in total sugar concentrations from 121 to 92 g/L. However, the
same reduction in enzyme loading after phosphoric acid pretreatment
sharply decreased total sugar concentrations from 118 to 46 g/L. These
results indicate that NMMO pretreatment has considerably altered the
structure and improved the enzyme’s accessibility even at low enzyme
loadings. However, phosphoric acid pretreatment could not significantly
improve the enzymatic hydrolysis of the straw at high solids and low
enzyme loadings.
Glucose concentrations (Fig. 2b) also showed the same trend as total
sugars concentration (Fig. 2a). Similarly, phosphoric acid pretreatment
did not considerably improve the glucose concentration obtained from
enzymatic hydrolysis at high solids (15% w/w) and low enzyme load­
ings (less than 5 FPU/g straw) in comparison with the untreated straw. It
can be said that the enzyme concentration was the limiting factor at low
enzyme loads. However, the effect of this pretreatment at higher enzyme
loadings is evident, with the glucose concentration increasing from
about 50 g/L for raw rice straw to approximately 100 g/L at 20 FPU/g
straw. The positive impact of NMMO pretreatment on the resulting
glucose concentration is considerable, even at low enzyme loadings. As
can be seen, 87%, 75%, and 50% decrease in enzyme loadings reduced
the glucose content only by 27%, 10%, and 6%, respectively. The impact
of enzyme load on enzymatic hydrolysis of unbleached hardwood pulp
and organosolv pretreated poplar was investigated by Zhang et al.
(2009). They reported a decrease of only 21% in glucose concentration
by a 50% reduction in enzyme load. In our previous study (Molaverdi
et al., 2019b), the effect of sodium carbonate pretreatment was inves­
tigated on ethanol production from rice straw using different enzyme
loadings (10, 5, and 2.5 FPU/g substrate). The maximum glucose and
M. Molaverdi et al. Journal of Cleaner Production 339 (2022) 130702

Fig. 4. Effect of enzyme and solid loads on ethanol concentrations from 72 h-SSF of rice straw at (a) 20, (b) 10, (c) 5, and (d) 2.5 FPU/g straw and 15, 20, and 30%
solid loading using cellulose solvents (phosphoric acid and NMMO) pretreatments.

total sugar concentrations were 52.8 g/L and 89.22 g/L, respectively,
using 15% solids loading and 10 FPU/g enzyme. That study showed the
effect of pretreatment and enzyme loading on the hydrolysis process. In
another study conducted by Molaverdi et al. (2021), the impact of
modified mild alkaline pretreatment (steam explosion combined with
sodium carbonate pretreatment) was studied to enhance ethanol pro­
duction at high solids loading. The maximum glucose concentration was
92.3 g/L from solid-state hydrolysis of corn stover at 30% solids and 15
FPU/g enzyme loading. The mentioned studies revealed the efficiency of
solvent pretreatment on lignocelluloses to improve the hydrolysis and

6
M. Molaverdi et al. Journal of Cleaner Production 339 (2022) 130702

Table 3
Comparison of ethanol yield and concentration from high-solid SSF of several pretreated feedstocks.
Feedstock Pretreatment Microorganism Solid loading (%) Ethanol yield (%) Ethanol concentration (g/L) Ref.

Rice straw Sodium carbonate M. indicus 15 89.5 53.6 Molaverdi et al. (2019b)
Bermudagrass Phosphoric acid S. cerevisiae 20 65.1 56.1 Li et al. (2009)
Rapeseed Phosphoric acid S. cerevisiae 20 84.7 36.6 Li et al. (2009)
Wheat straw Dilute acid S. cerevisiae 24.4 80.0 57 Mohagheghi et al. (1992)
Rice straw Acid-alkali combined pretreatment Kluyveromyces sp. 10 84.6 23.2 Narra et al. (2015)
Corncob Acid–alkali combined pretreatment S. cerevisiae 19 81.2 69.2 (M. M. Zhang et al., 2010)
Reed Phosphoric acid S. cerevisiae 20 59.9 55.0 Li et al. (2009)
Rice straw NMMO pretreatment M. indicus 15 100 44.2 The present study
Rice straw Phosphoric acid Mucor indicus 15 73.7 34.2 The present study

glucose yield.
Table 4
Ethanol yields from untreated and pretreated rice straw with phosphoric acid
3.4. Simultaneous saccharification and fermentation at high solid loads and NMMO at different conditions.
Substrate Solid Enzyme SSF Ethanol Total ethanol
SSF experiments of pretreated and untreated rice straw with NNMO loading loading time yield (%) produced (g
and phosphoric acid were conducted at 15% solid loading by M. indicus (%) (FPU/g (h) per kg of
substrate) initial straw)
at different enzyme loads (2.5, 5, 10, and 20 FPU/g straw) (Fig. 3). The
highest ethanol concentrations were 44.2 g/L and 34.2 g/L, obtained Phosphoric 15 20 72 73.7±0.0 194
from the SSF of rice straw pretreated with NMMO and phosphoric acid, acid- 10 72 67.2±0.1 177
pretreated 5 120 65.7±0.2 174
respectively, using the enzyme load of 20 FPU/g substrate. In addition, straw 2.5 120 45.0±0.3 119
the results indicate that only at the high enzyme level of 20 FPU/g Phosphoric 20 20 72 66.1±0.3 172
substrate, there was a considerable difference between ethanol con­ acid- 10 72 45.8±0.5 116
centration from straw pretreated with phosphoric acid and NMMO. pretreated 5 120 59.8±0.2 156
straw 2.5 120 47.6±0.4 124
However, at the lower enzyme levels, the improvement in ethanol
Phosphoric 30 20 72 49.0±0.1 128
concentration by NMMO and phosphoric acid pretreatments was acid- 10 72 60.8±0.5 159
approximately the same. Therefore, only the positive effects of these two pretreated 5 72 53.8±0.3 140
pretreatments on ethanol concentrations were considerable compared straw 2.5 120 44.5±0.4 115
with the untreated straw. NMMO- 15 20 72 100.0±0.7 247
In the continuation of this study, the effects of changing solid load­ pretreated 10 72 75.3±0.2 185
ings (20, 15, and 30%) at different enzyme loads (2.5, 5, 10, and 20 straw 5 72 56.5±0.1 140
2.5 120 61.8±0.4 135
FPU/g straw) were investigated on ethanol production (Fig. 4). The
NMMO- 20 20 72 75.0±0.1 184
maximum ethanol concentration using 20 FPU/g enzyme (Fig. 4a) was pretreated 10 72 57.0±0.2 140
63.4 g/L obtained from the straw pretreated with NMMO at 30% solids straw 5 72 56.2±0.0 120
loading. However, the ethanol concentration produced from phosphoric 2.5 120 49.2±0.3 116
acid pretreated straw was 69.0 g/L at 10 FPU/g substrate, Fig. 4b. NMMO- 30 20 72 59.1±0.6 145
pretreated 10 72 56.9±0.3 140
Although the standard deviation of ethanol concentration is high for this straw 5 72 48.8±0.0 120
observation, the decrease in ethanol concentration by enhancing the 2.5 120 35.3±0.1 85
enzyme loads from 10 to 20 FPU/g is meaningful. This observation can
Untreated 15 20 72 52.6±0.3 129
be due to the enzyme overcrowding phenomena that cause competition straw 10 72 50.4±0.6 124
on free sites of cellulose and prevent obtaining the highest performance 5 120 35.1±0.0 84
of the enzyme (Bommarius et al., 2008). As seen in Fig. 4b–d, the 2.5 120 34.7±0.6 84
improvement of ethanol concentration by phosphoric acid pretreatment Untreated 20 20 72 41.4±0. 5 92
straw 10 72 33.3±0.0 80
was almost similar to that of NMMO pretreatment at 15 and 20% solids 5 120 28.2±0.3 68
loadings, even better than NMMO at 30% solids loading. 2.5 120 33.8±0.1 56
An important achievement that can decrease ethanol distillation Untreated 30 20 72 24.2±0.4 60
costs is reaching ethanol concentrations of more than 40 g/L (Galbe straw 10 72 24.0±0.3 60
5 120 22.2±0.0 51
et al., 2007; Zhao et al., 2011), which was gained in this research even at
2.5 120 22.7±0.2 51
low enzyme loadings. A 50% decrease in the enzyme loads from 10 to 5
FPU/g substrate only decreased the ethanol concentration from 69 g/L
to 61 g/L and from 61 g/L to 52 g/L using phosphoric acid and NMMO cerevisiae (Li et al., 2009). The present study achieved promising ethanol
pretreatments, respectively. An ethanol concentration of 25 g/L was concentration and yield by choosing appropriate pretreatment and
stated by (J. J. Zhang et al., 2010) through the SSF of steam-exploded microorganism for solid-state fermentation (Table 4) compared to
corn stover at 200 ◦ C at 20% solid loading. Furthermore, Jørgensen similar studies.
et al. (2007) used wheat straw pretreated with hot water at 205 ◦ C,
followed by hydrolysis and fermentation at 20% solids load and 11.8 3.4.1. Impact of SSF time at low enzyme loads
FPU/g glucan cellulase load. They obtained ethanol yields of less than To increase the ethanol production yield at low enzyme levels (5 and
50%. The ethanol production results in the current study were compared 2.5 FPU/g straw), the effect of increasing the time of SSF was analyzed,
with other studies on SSF (Table 3). As can be seen, NMMO and phos­ and the results are shown in Fig. 5. Comparing the results presented in
phoric acid pretreatment showed an acceptable ethanol yield. NMMO Fig. 5a with Fig. 4c shows that prolonging SSF from 72 to 120 h was
pretreatment and using M. indicus showed the highest ethanol yield by inefficient at 5 FPU/g substrate since the ethanol concentrations did not
SSF at high solid loading without mixing, fed-batch process, or prehy­ change or decreased slightly. This finding indicates that 72 h was suf­
drolysis. While the ethanol yields for reed, rapeseed, and bermudagrass ficient for SSF of pretreated straw at 5 FPU/g enzyme, and the minor
were 59.9, 84.7 and 65.1% by phosphoric acid pretreatment and S.

7
M. Molaverdi et al.
Fig. 5. Effect of solids and enzyme loads on ethanol concentrations from prolonged SSF for 120 h using the enzyme loads of (a) 5 and (b) 2.5 FPU/g straw at 15, 20,
and 30% solid loadings.
reduction in ethanol concentration over time could be due to ethanol
evaporation. In contrast, for the lower level of enzyme loading, i.e., 2.5
FPU/g substrate, prolonging the process to 120 h improved the ethanol
concentration. The highest ethanol concentration was enhanced from
42.8 g/L to 50.5 g/L by increasing process time from 72 to 120 h using
phosphoric acid pretreatment. However, this trend was not observed for
NMMO pretreated and untreated rice straw, as the ethanol concentra­
tions did not change with increasing the process duration.
The maximum ethanol production obtained from untreated and
pretreated rice straw with phosphoric acid and NMMO is reported in
Table 4. The maximum ethanol yield was 100% (247 g/kg rice straw)
and 73.7% (194 g/kg rice straw) of the theoretical yield, obtained from
SSF of rice straw pretreated with NMMO and phosphoric acid at lowest
solid loading (15% w/w) and highest enzyme loading (20 FPU/g sub­
strate). An important achievement of using high solids loading is
reaching ethanol concentrations of more than 40 g/L that considerably
declines ethanol distillation costs and energy consumption (Galbe et al.,
2007; Zhao et al., 2011). In this study, the highest ethanol concentration
for untreated rice straw was 25 g/L. Therefore, in this study, this goal
was not achieved from untreated rice straw. Also, heat and mass transfer
are two main problems in SSF using high solids loading, and these
problems were observed especially at the higher solids loading (at 30%)
8
Journal of Cleaner Production 339 (2022) 130702
even with mixing. These problems led to lower ethanol yields at 30%
solids loading than that at 15% solids loading (Table 4).
3.5. Mass balance
The high-solids SSF process yielding the highest ethanol yield (close
to 100% theoretical efficiency for NMMO pretreatment and 74% for
phosphoric acid pretreatment) was studied from the total mass balance
perspective and based on one kg of raw rice straw (Fig. 6). According to
data tabulated in Table 1, NMMO and phosphoric acid pretreatments
followed by a wash and then a neutralizing process resulted in approx­
imately 32% and 45% solids loss, respectively. Then, the residual solids
were applied for SSF at high solids for ethanol production. Finally, 168 g
ethanol with a concentration of 44.2 g/L and 106 g of ethanol with a
concentration of 34.2 g/L were obtained using solid-state SSF at 15%
solids and 20 FPU/g substrate loadings of pretreated straw with NMMO
and phosphoric acid, respectively.
As mentioned earlier, ethanol production from lignocellulosic
biomass, i.e., the second-generation bioethanol, is highly advantageous
because of the abundance of these resources, lack of interference with
food sources, and the environmental problems associated with cellulosic
waste disposal (Aghbashlo et al., 2018). However, despite many pilot
M. Molaverdi et al.
Fig. 6. Total mass balance of high-solids loading SSF processing (15% solids and 20 FPU/g substrate enzyme loading) of rice straw pretreated with (A) NMMO and
(B) phosphoric acid.
units developed for the biological conversion of lignocellulosic materials
into ethanol, none has been industrialized yet due to low-profit margin.
The findings of the present study, through boosting the efficiency of
ethanol production from lignocellulosic materials using high solids
processes, could contribute to overcoming these challenges. However, it
should also be noted that increasing solid content to values higher than
20% could also increase inhibitor generation, cause enzyme penetration
problems, mixing restrictions, and mass transfer limitations (Kristensen
et al., 2009). Hence, future studies should be focused on solving these
challenges to maximize the benefits of the high-solid loading strategy. In
addition, comprehensive diagnostic assessment tools like exergy
(Khounani et al., 2021), exergoeconomic (Aghbashlo et al., 2019), and
exergoenvironmental (Aghbashlo et al., 2021) approaches need to be
applied to find the most thermodynamically efficient, economically
viable, and environmentally sustainable process conditions.
4. Conclusions
Combining cellulose solvent pretreatments and M. indicus species
showed promising results for high titer ethanol production at low
cellulase loadings and without mixing and prehydrolysis. The maximum
ethanol concentrations were 69 g/L and 63 g/L (at 30% solids loadings),
and maximum ethanol yields were 74% and 100% (at 15% solids
loadings) from phosphoric acid- and NMMO-pretreated straw samples,
respectively. Therefore, phosphoric acid and NMMO pretreatments are
promising processes for high solid fermentation to produce bioethanol
cost-effectively. This is ascribed to the fact that these processes could
facilitate the fractionation of lignocelluloses, leading to high ethanol
concentrations (more than 40 g/L) from the cellulose-rich streams. Such
features are well aligned with the requirements to develop the
9
Journal of Cleaner Production 339 (2022) 130702
biorefinery concept.
CRediT authorship contribution statement
Maryam Molaverdi: Writing – original draft, Formal analysis,
Software, Data curation, Methodology. Safoora Mirmohamadsadeghi:
Data curation, Methodology. Keikhosro Karimi: Conceptualization,
Resources, Funding acquisition, Supervision, Writing – review & editing.
Mortaza Aghbashlo: Formal analysis, Writing – review & editing.
Meisam Tabatabaei: Conceptualization, Supervision, Writing – review
& editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was financially supported by the Research Institute for
Biotechnology and Bioengineering, Isfahan University of Technology.
The authors appreciate Novozymes, Denmark, for providing the en­
zymes used in this study. This research did not receive any specific grant
from funding agencies in the public, commercial, or not-for-profit
sectors.
M. Molaverdi et al. Journal of Cleaner Production 339 (2022) 130702

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