fermentation

Article
Effect of Various Pretreatment Methods on Bioethanol
Production from Cotton Stalks
Konstantinos Dimos 1,† , Thomas Paschos 1,† , Argiro Louloudi 2 , Konstantinos G. Kalogiannis 3 ,
Angelos A. Lappas 3 , Nikolaos Papayannakos 2 , Dimitris Kekos 1 and Diomi Mamma 1, *
1 Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens,
Zografou Campus, 15780 Athens, Greece; [email protected] (K.D.);
[email protected] (T.P.); [email protected] (D.K.)
2 Chemical Process Engineering Laboratory, School of Chemical Engineering, National Technical University
of Athens, Zografou Campus, 15780 Athens, Greece; [email protected] (A.L.);
[email protected] (N.P.)
3 Laboratory of Environmental Fuels and Hydrocarbons, Chemical Process and Energy Resources
Institute (CPERI), Centre for Research and Technology Hellas (CERTH), 6th km Harilaou-Thermi Rd,
57001 Thessaloniki, Greece; [email protected] (K.G.K.); [email protected] (A.A.L.)
* Correspondence: [email protected]; Tel.: +30-210-7723158
† These authors contributed equally to this paper.




Received: 3 December 2018; Accepted: 20 December 2018; Published: 1 January 2019


Abstract: Cotton stalks (CS) are considered a good candidate for fuel-ethanol production due to its
abundance and high carbohydrate content, but the direct conversion without pretreatment always
results in extremely low yields due to the recalcitrant nature of lignocelluloses. The present study was
undertaken to investigate the effect of various chemical and physicochemical pretreatment methods,
i.e., alkali, microwave-assisted acid, organosolv, hydrothermal treatment, and sequentially organosolv
and hydrothermal pretreatment, on chemical composition of CS and subsequent ethanol production
applying pre-hydrolysis and simultaneous saccharification and fermentation (PSSF) at high solid
loading. The best results in terms of ethanol production were achieved by the sequential combination
of organosolv and hydrothermal pretreatment (32.3 g/L, using 15% w/v substrate concentration
and 6 h pre-hydrolysis) with an improvement of 32% to 50% in ethanol production compared to the
other pretreatments. Extending pre-hydrolysis time to 14 h and increasing substrate concentration to
20% w/v, ethanol production reached 47.0 g/L (corresponding to an ethanol yield of 52%) after 30 h
of fermentation.

Keywords: cotton stalks; alkali pretreatment; hydrothermal treatment; microwave-assisted acid

pretreatment; organosolv; bioethanol; high solids pre-hydrolysis and simultaneous saccharification
and fermentation

1. Introduction
The global economy can no longer depend on fossil fuels and non-renewable carbon and thus
in the last two decades, global research programs on alternative energy have been directed towards
discovering new and sustainable energy sources [1].
Depletion of fossil fuels, accumulation of atmospheric CO2 , increasing energy demand, national
energy security and rural economic development are the main reasons bringing about a transitional
change from a fossil fuel based economy to a more sustainable carbon-neutral bio-economy, where
agriculture not only will continue to provide food security but also biomass as a renewable raw
material for industry [1,2].

Fermentation 2019, 5, 5; doi:10.3390/fermentation5010005 www.mdpi.com/journal/fermentation

Fermentation 2019, 5, 5 2 of 12

Biomass, like agricultural crop residues such as wheat straw, corn stover, rice straw, and cotton
stalks, forestry residues, grasses, sawdust, wood chips offers a promising source for biofuels because
it is abundant, inexpensive and does not compete with food and feed applications. Ethanol derived
from biomass has the potential to be renewable transportation fuel due to several advantageous
properties [3].
Cotton plant is an important crop playing a key role in economic, political and social affairs of the
world [4]. According to Food and Agricultural Organization of the United Nations cotton production
in 2014 was approximately 26 million tones worldwide [5]. Cotton stalk (CS), a by-product of cotton
production, generated in huge volumes annually, approximately 2 metric tons of dry matter/ha, is a
major low-cost source of sugars that can be converted into bio-based products such as fuel ethanol [6].
The typical scheme of bioethanol production from lignocellulosic biomass involves pre-treatment,
hydrolysis, fermentation, and product separation/purification [3]. The main components of lignocelllulosic
biomass i.e., cellulose, hemicellulose and lignin, are strongly intermeshed and bonded through covalent
or non-covalent bonds forming the lignocellulosic matrix. Therefore, the degradation of any of the
above class of ingredients is subject to the constraints caused by other ingredients, resulting in relatively
low digestibility of raw lignocellulosic biomass [7,8]. Pretreatment is thus, a crucial step for bioethanol
production from lignocellulosic materials causing changes in microstructure, macrostructure and
chemical composition of lignocellulose in order to obtain a solid phase enriched in cellulose and more
susceptible to enzymatic attack [7]. To be efficient any selected pretreatment method should improve
the availability of monomeric sugars, prevent their degradation, avoid inhibitor formation and be low
cost [9]. Pretreatment methods can be classified as physical, chemical, physico-chemical, biological
methods and their combinations [7,8]. Chemical and physico-chemical pretreatment technologies were
extensively used for lignocellulosic biomass, although both technologies demonstrate distinct action
mechanisms for cell wall destruction. For instance, the outcome of the dilute acid or hydrothermal
pretreatment is the hydrolysis of hemicellulose components while alkali or organosolv pretreatment
targets mainly lignin [10–13]. A single pretreatment method alone is not always effective and therefore
combined pretreatment methods have been recently considered as a promising approach for higher
ethanol yield [7].
The processes generally used in fermentation of lignocellulosic materials are simultaneous
saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF). The SHF
process has been traditionally used except SSF is superior for ethanol production since it can get
better ethanol yields by removing end product inhibition and eliminates the need for separate reactors.
This process is also cost effective although difference in optimum temperature conditions of enzyme
for hydrolysis and fermentation poses some restrictions [14]. Ethanol concentration in the fermentation
broth superior to 4% (w/v) is considered a prerequisite to diminish the energy demand in the distillation
stage [15]. In order to achieve ethanol concentration of 4% (w/v), the amount of sugars released in the
hydrolysis step should be at least 80 g/L, hence high-solids loadings (>15% solids) are required during
enzymatic hydrolysis [16]. Operating hydrolysis at high solid loading creates technical difficulties
due to high initial viscosity of the material, which makes mixing difficult resulting in mass and heat
transfer problems [17]. Pre-hydrolysis and the Simultaneous Saccharification and Fermentation (PSSF)
process, is a variation of the SSF process, where the substrate is subjected to a pre-hydrolysis step at
optimum temperature for cellulolytic enzymes (50 ◦ C) followed by fermentation of the resulted slurry.
By PSSF reduction of viscosity of the solid-liquid mixture can be achieved prior to the addition of the
microorganism [17].
The aim of this work was to evaluate the potential of CS as a feedstock for high titer ethanol
production. In this context, different chemical (alkali, organoslov), physico-chemical (hydrothermal,
microwave-assisted acid pretreatment) and combined pretreatment methods (organosolv followed
by hydrothermal treatment) were examined. Compositional analysis of the pretreated materials was
conducted. The process configuration for ethanol production applied was PSSF at high solids loading.
Fermentation 2019, 5, 5 3 of 12

Parameters that influenced the performance of PSSF such as substrate concentration and length of
pre-hydrolysis time were also examined.

2. Materials and Methods

2.1. Raw Material and Reagents

Cotton stalks (CS) were obtained from the Laboratory of Farm Mechanization, Department of
Agriculture, Crop Production and Rural Environment, University of Thessaly. Prior to compositional
analysis, the biomass which consisted primarily of stalks, leaves and cotton residue, was ground to
a particle size of 3 mm. Commercial cellulase Cellic® CTec2 was kindly provided by Novozymes
Corporation (Bagsværd, Denmark). All chemicals were analytical grade, provided by Sigma S.A.
(St. Louis, MO, USA).

2.2. Pretreatment of Cotton Stalks

2.2.1. Hydrothermal Treatment

Hydrothermal treatment of biomass and organosolv treated CS was carried out in a Hastelloy
C-276 Parr autoclave with a volume of 975 cm3 . A total of 30 g of solid feedstock were fed into
the reactor and 300 g of distilled water were then poured so as to achieve a liquid to solid ratio of
10:1 w/w. The reactor was tightly sealed and pressurized up to 12 bars with N2 . Temperature was set
at 175 ◦ C and reaction time was 2 h in all cases. Heat up and cool down times were around 15 min
each. The reaction mixture was filtered to separate the solid residues from the liquid phase. Distilled
water was used to wash the solid residue and the washing water was mixed with the liquid sample
received from the hydrolysis. The solid residue was dried overnight in an oven at a low temperature of
50 ◦ C and mass constancy was checked the following day to ensure total removal of residual moisture.
More details can be found elsewhere [18].

2.2.2. Microwave-Assisted Acid Pretreatment

Microwave-assisted acid pretreatment was conducted as described elsewhere [19]. Briefly, a
microwave digestion equipment (speed waveTM MWS-2, Berghof Instruments GmBH, Eningen,
Germany) was employed for substrate pretreatment, operating at microwave power of 700 W. The charge
of solids, measured by the liquor to solid ratio (LSR), was kept at 6.7 g·g−1 . The experimental settings
were 210 ◦ C, 10 min in the presence of 2 g sulfuric acid/100 g of dry CS. The pretreated solid was filtered,
washed thoroughly with deionized water, dried in an air-circulated oven overnight at 50 ◦ C to remove all
residual moisture, and used for the subsequent fermentation experiments.

2.2.3. Alkali Treatment

Conditions for alkali treatment of CS were adopted from a previous study [20]. Briefly, aqueous
solution of NaOH at concentration 1.5% (w/v) was used to pretreat CS sample at a solid loading of
15% (w/v) (corresponding to 10 g NaOH/100 g of CS). Treatments were performed in an autoclave at
121 ◦ C with 15 psi (103.4 kPa) pressure for residence time of 30 min. The pretreated solid was filtered,
washed thoroughly with deionized water, dried in an air-circulated oven overnight at 50 ◦ C to remove
all residual moisture and used for the subsequent fermentation experiments.

2.2.4. Organosolv Treatment

Single stage Milox cooking were carried out in 500 cm3 conical flasks at atmospheric pressures.
Formic acid (FA) was used at a 10:1 solvent to biomass weight ratio. Hydrogen peroxide (HP) was
added at a concentrations of 2 wt.% on solvent, and the reaction temperature was 80 ◦ C. Typically, 30 g
of biomass were treated in each run for reaction time of 1 h. The biomass was suspended in the FA and
heated up to the desired temperature prior to the H2 O2 addition. The delignified biomass samples were
Fermentation 2019, 5, 5 4 of 12

filtered and washed with six volumes of the corresponding solvent. Finally, the resulting pulp was
washed with distilled water and dried in an oven overnight at 50 ◦ C to remove all residual moisture
and used for the subsequent fermentation experiments. Further details can be found elsewhere [18].

2.3. Bioconversion of Pretreated CS to Ethanol

Pretreated CS at concentration of 15% (w/v) was subjected to pre-hydrolysis using 80 FPU/g
cellulose of Cellic® CTec2 at 50 ± 0.5 ◦ C and pH 5.0 in an orbital shaker (200 ± 2 rpm) for 6 h
unless otherwise stated. Following pre-hydrolysis, compressed baker’s yeast (Yiotis, Athens, Greece)
corresponding to 15 mg yeast per gram of dry pretreated CS was added in all cases, while no nutrients
supplemented the fermentation broth. Fermentation was carried out in an orbital shaker at 30 ± 0.5 ◦ C,
initial pH 5.0 and 80 ± 2 rpm. At certain time intervals, samples were withdrawn and analyzed for
ethanol. All experiments were conducted in duplicate.

2.4. Analytical Methods

Total cellulase activity (FPU), glucose and ethanol concentration were determined as described
elsewhere [20]. Structural carbohydrate content (cellulose and hemicellulose) and lignin of native
CS as well as of the pretreated CS samples was determined according to the laboratory analytical
procedure (LAP) of National Renewable Energy Laboratory [21]. For each sample, the carbohydrate
content analysis was carried out in triplicate.

2.5. Calculations
The following equations were used for the calculation of glucose potential (Equation (1)), maximum
theoretical ethanol (Equation (2)), hydrolysis yield (Equation (3)) and ethanol yield (Equtaion (4)).
h i
G potential = f ·[S0 ]·1.111, (1)

[ EtOHtheoretical ] = 0.511· f ·[S0 ]·1.111, (2)

[ Glucoset − Glucose0 ]
HY (%) = , (3)
f ·[S0 ]·1.111
[ EtOHt − EtOH0 ]
TEY (%) = , (4)
0.511 · f · [S0 ]·1.111
h i
where, G potential is the maximum (theoretical) amount of glucose that could be released if all cellulose
in the biomass converts to glucose (g/L), [ EtOHtheoretical ] the maximum (theoretical) ethanol that could
be produced from the cellulose present in the biomass (g/L), HY hydrolysis yield (%), TEY theoretical
ethanol yield (%), [S0 ] is the initial biomass concentration (g/L), [ Glucoset − Glucose0 ] is the glucose
produced during pre-hydrolysis (g/L), [ EtOHt − EtOH0 ] is the ethanol produced during fermentation
(g/L), f is the cellulose fraction of the biomass, 0.511 is the conversion factor for glucose to ethanol and
1.111 is the conversion factor for cellulose to equivalent glucose.

3. Results and Discussion

3.1. Composition of Native and Pretreated CS

Native CS used in the present study, contained 37.9% (w/w dry basis) cellulose and 14.7% (w/w
dry basis) hemicellulose [20]. Lignin, the major barrier in enzymatic hydrolysis of cellulose, was found
to be 25.8% (w/w). In the literature, cellulose content of native CS ranged from 30% to 39.8% (w/w)
while hemicellulose from 10.7% to 16.7% (w/w). The total lignin content of CS is comparable to that
reported elsewhere (approximately 27% to 30%, w/w) [4,22,23]. According to Agblevor et al. [24]
composition of CS varies depending on the growing location, season, harvesting methods, as well
Fermentation 2019, 5, 5 5 of 12

as analysis procedures. The total holocellulose content (sum of cellulose and hemicellulose) of CS
is 52.7% (w/w), making CS a potential feedstock for ethanol production. The total polysaccharide
content of cotton stalk can be fairly compared with other extensively explored lignocellulosic materials,
such as sugarcane bagasse, 67.1%; corn stover, 58.3%; wheat straw, 54.0%; and sorghum straw, 61.0%;
and widely accepted switch grass, 52.9%, for ethanol production [6].
In the present study, chemical and physico-chemical pretreatments were applied to CS.
More specifically, CS was subjected to alkali, microwave-assisted acid, organosolv, hydrothermal
treatment and combination of the organosolv and hydrothermal treatment. Compositional analysis of
the solid residue after each pretreatment revealed that varying amounts of hemicellulose and lignin
remained in the biomass, which might affect the fermentable sugar release (Table 1).
Hydrothermal pretreatment is an environmentally friendly pretreatment method, maintains water
in the liquid state at elevated temperatures (160–240 ◦ C) and high pressures in order to promote
disintegration and separation of the lignocellulosic matrix. During hydrothermal treatment, most of
hemicellulose and part of lignin could be degraded [11]. In the present study, CS was subjected to
hydrothermal pretreatment at 175 ◦ C for 2 h and led to a solid recovery of 67% (w/w) on dry basis.
Cellulose content in the solid residue after pretreatment was found to be 47.3% (w/w) (corresponding
to a cellulose recovery of 83.5%), while approximately 82% of hemicellulose was removed. When the
heating source of hydrothermal pretreatment is changed to microwave irradiation (MWI) in the
presence or absence of an acid catalyst (usually sulfuric acid, acetic acid or phosphoric acid), it could
be considered as microwave or microwave-assisted acid pretreatment [25]. Approximately 58.5% of
hemicellulose was removed after MWI pretreatment (210 ◦ C for 10 min) in the presence of 2% w/w
H2 SO4 while lignin remained in the solid residue. Approximately 81.8% of cellulose was recovered
after treatment. However, lignin content increased in the solid residues after both hydrothermal and
microwave-assisted acid treatment compared to native CS. These pretreatments as mentioned mainly
remove hemicelluloses from the raw biomass, which in turn increased the relative lignin content.
The latter is consistent with the reports of Silvertein et al. [26] and Wang et al. [27].
Alkaline pretreatment is one of the extensively studied chemical pretreatment methods, which
aims to remove lignin and part of hemicellulose, by employing various alkali compounds [12].
Hemicellulose and lignin removal during the alkali pretreatment (10%, w/w NaOH, 121 ◦ C, 30 min)
led to an enrichment of cellulose which constituted 55.3% (w/w) of the pretreated biomass (Table 1).
Approximately 93% of CS cellulose was recovered after alkali treatment, while 42.2% and 9.2% of the
total lignin and hemicellulose respectively were removed.

Table 1. Compositional analysis of pretreated cotton stalks (CS).

Content (g/100 g of Pretreated CS)

Component Microwave- Organosolv-
Hydrothermal b Alkali c Organosolv d
Assisted Acid a Hydrothermal e
Cellulose 48.6 ± 0.3 47.3 ± 2.1 55.3 ± 1.5 77.0 ± 0.7 79.6 ± 0.2
Hemicellulose 9.6 ± 0.8 3.9 ± 0.1 21.2 ± 0.7 1.4 ± 0.3 0.4 ± 0.1
Lignin Klason 38.3 ± 1.5 39.1 ±1.3 22.2 ± 1.0 9.7 ± 0.6 10.4 ± 0.5
Acid Soluble Lignin 2.3 ± 0.0 1.7 ± 0.0 1.1 ± 0.0 1.1 ± 0.1 0.8 ± 0.0
Total lignin 40.6 ± 1.6 40.8 ± 1.3 23.3 ± 1.0 10.8 ± 0.7 11.2 ± 0.5
Solid recovery: a 64% ± 0.8%, b 67% ± 0.7%, c 64% ± 0.5 %, d 45% ± 0.5%, e 34% ± 0.8%.

Organic solvent pretreatment (organosolv) can be used to provide treated cellulose suitable for
enzymatic hydrolysis, using an organic or aqueous organic solvent to remove or decompose the
network of lignin and possibly a part of the hemicellulose. The advantage of this process is the
option to recover lignin as a value added product [13]. Solid recovery after organosolv treatment was
45.0% on dry basis. The cellulose content of organosolv treated CS samples was found to be 77.0%
(w/w), corresponding to a 91% of cellulose recovery (Table 1). The hemicellulose and lignin contents
dramatically decreased (96% and 81% removal, respectively).
Fermentation 2019, 5, 5 6 of 12

Organosolv pretreated CS was subjected to hydrothermal treatment and resulted in an almost

hemicellulose free solid residue with low lignin content. Combined pretreatment led to a cellulose
recovery of2018,
Fermentation 71%. 4, x FOR PEER REVIEW 6 of 12
The results of Table 1 showed that the order for cellulose content was organosolv-hydrothermal
treatment > organosolv
The results of Table > alkali treatment
1 showed > microwave-assisted
that the order for cellulose content acid
waspretreatment > hydrothermal
organosolv-hydrothermal
treatmentand
treatment > organosolv > alkali ittreatment
for lignin content > microwave-assisted
was hydrothermal treatment > acid pretreatment > hydrothermal
microwave-assisted acid pretreatment
>treatment and for
alkali treatment lignin content it was hydrothermal
> organosolv-hydrothermal treatment >respectively.
treatment > organosolv, microwave-assisted acid
pretreatment > alkali treatment
Various pretreatments of >CS organosolv-hydrothermal
have been reported in treatment
literature >i.e.,organosolv, respectively.[4,6,26],
alkali pretreatment
Various pretreatments of CS have been reported in literature i.e.,
high pressure-assisted alkali pretreatment and ultrasound-assisted alkali pretreatment [27,28],alkali pretreatment [4,6,26],
steam
high pressure-assisted alkali pretreatment and ultrasound-assisted alkali pretreatment
explosion [29], liquid hot water pretreatment [23], dilute acid pretreatment [27,30], pretreatment [27,28], steamwith
explosion
ILs [29], treatment
[22], ozone liquid hot[4]. water
Thepretreatment
outcome of each[23], pretreatment
dilute acid pretreatment [27,30],and
in terms of lignin pretreatment
hemicellulose
with ILs [22], ozone treatment [4]. The outcome of each pretreatment in terms of lignin and
solubilization depends on the pretreatment method and the conditions applied.
hemicellulose solubilization depends on the pretreatment method and the conditions applied.
Pretreatment of lignocellulosic biomass represents one of the main economic costs in the process
Pretreatment of lignocellulosic biomass represents one of the main economic costs in the
of bioethanol production. The operating cost of the combined pretreatment appears to be relatively
process of bioethanol production. The operating cost of the combined pretreatment appears to be
high compared to a single step pretreatment, but the usage of biomass appears to be more efficient.
relatively high compared to a single step pretreatment, but the usage of biomass appears to be more
The increased amount of ethanol produced translates into a higher ethanol concentration in the
efficient. The increased amount of ethanol produced translates into a higher ethanol concentration in
products
the productsstream,
stream, meaning
meaning more
moresavings
savings potential
potential in in the
thedownstream
downstream purification
purification process
process [31]. [31].
Moreover, lignin, the largest non-carbohydrate phenolic polymer not
Moreover, lignin, the largest non-carbohydrate phenolic polymer not involved in the fermentationinvolved in the fermentation
process
processto to produce
produce ethanol, couldbe
ethanol, could beobtained
obtainedasasco-product
co-product of of
thethe pretreatment
pretreatment process.
process. Its unique
Its unique
material properties allow lignin utilization in value-added markets that
material properties allow lignin utilization in value-added markets that can more likely afford can more likely afford
the the
extra
extracosts
costsfor
forpre-recovery
pre-recovery by by pretreatments,
pretreatments, such
such asas organosolv
organosolv and ionic liquid, contributing
contributing to to the
competitiveness
the competitiveness of bioethanol
of bioethanol [32].[32].

3.2.
3.2.Conversion
Conversion of Pre-Treated CS
of Pre-Treated CS to
toBioethanol
BioethanolApplying
ApplyingPSSF
PSSF
Pretreated
Pretreated CS samples wereconverted
samples were convertedtotobioethanol
bioethanol applying
applying PSSF
PSSF at 15%
at 15% (w/v)
(w/v) solids
solids loading,
loading,
ininorder
ordertotoevaluate
evaluatethe thepretreatment
pretreatmentprocesses.
processes.The
Thelength
lengthofofpre-hydrolysis
pre-hydrolysisstepstepwaswasset
setatat6 h6 while
h
anwhile an enzyme
enzyme load ofload of 80 FPU/g
80 FPU/g cellulose
cellulose was was
usedused
[20].[20]. In general,
In general, 7 to7 33
to FPU/g
33 FPU/g substrateare
substrate areused
used
for for cellulose
cellulose hydrolysis
hydrolysis in lignocellulosic
in lignocellulosic material,
material, butbutin in some
some caseshigher
cases higherenzyme
enzyme loadings
loadings are
are necessary
necessary to efficiently
to efficiently hydrolyze
hydrolyze the substrate
the substrate [27,33].
[27,33]. GlucoseGlucose release
release duringduring pre-hydrolysis
pre-hydrolysis step was
step was
found to befound to beondependent
dependent on the applied
the pretreatment pretreatment applied
and ranged and18.77
from ranged from 18.77 g/L to a
g/L (corresponding
(corresponding
hydrolysis yield to
of a23.81%)
hydrolysis yield of 23.81%)treated
for hydrothermally for hydrothermally
CS (Figure 1a) treated CSg/L
to 35.81 (Figure 1a) to 35.81 to a
(corresponding
g/L (corresponding to a hydrolysis yield of 27.00 %) for organosolv-hydrothemally
hydrolysis yield of 27.00 %) for organosolv-hydrothemally treated CS (Figure 1b). treated CS
(Figure 1b).
50 50

(α) (b)
Prehydrolysis
Prehydrolysis
Ethanol and glucose concentration (g/L)

Ethanol and glucose concentration (g/L)

40 40

30 30

20 20

10 10

0 0
-10 0 10 20 30 40 50 60 70 -10 0 10 20 30 40 50 60 70

Time (h) Time (h)

Figure 1.
Figure 1. Bioethanol
Bioethanol production
productionfrom
frompretreated CSCS
pretreated at at
15% (w/v)
15% substrate
(w/v) concentration
substrate afterafter
concentration 6h 6h
pre-hydrolysis. (a) Microwave-assisted acid pretreatment () ethanol and () glucose; alkali
pre-hydrolysis. (a) Microwave-assisted acid pretreatment ( ) ethanol and (#) glucose; alkali treatment
treatment
(N ) ethanol ()
and (ethanol and hydrothermolysis
4) glucose; () glucose; hydrothermolysis
(H) ethanol and() ethanol (b)
(5) glucose; andorganosolv
() glucose; (b)
() ethanol
organosolv () ethanol and () glucose; combined organosolv-hydrothermolysis () ethanol and
and () glucose; combined organosolv-hydrothermolysis () ethanol and (3) glucose.
() glucose.

The order of glucose concentration after 6 h pre-hydrolysis is organosolv-hydrothermal

treatment > organosolv > alkali treatment> microwave-assisted acid pretreatment > hydrothermal
Fermentation 2019, 5, 5 7 of 12

The order of glucose concentration after 6 h pre-hydrolysis is organosolv-hydrothermal treatment

> organosolv > alkali treatment> microwave-assisted acid pretreatment > hydrothermal treatment.
Glucose release during the pre-hydrolysis step seemed nearly inversely proportional to the lignin
content in the pretreated CS substrates, indicating that lignin removal facilitated cellulose hydrolysis.
Lignin acts as a physical barrier and can affect enzymatic hydrolysis by limiting access of the cellulase to
the cellulose in cell wall, as well as through the non-productive adsorption of cellulase. Non-productive
absorption of cellulases to lignin is unavoidable and leads to reduced cellulase activity, requiring
higher enzyme loadings or longer hydrolysis times to reach economic conversion levels [34]. Berlin et
al. [35] reported that almost 70% of all enzymes added for hydrolysis can become unproductive due to
the nonspecific adsorption by lignin.
Ethanol fermentation was initiated by the addition of dry yeast in the pre-hydrolyzed slurries
(Figure 1). The high ethanol production rate due to glucose availability was observed in the first 24 h
of fermentation, where yeast rapidly consumes glucose, followed by a decline in the production rate.
Enzymes, which are present in the fermentation medium, continued to hydrolyze the substrate, at a
slower rate due to suboptimal working temperatures (30 ◦ C), and was the reason for the increase in
bioethanol production after almost complete exhaustion of the initial glucose (Figure 1).
It is obvious that the pretreatment method affects severely the outcome of the process (Table 2).
Ethanol production increased as the lignin content of the solid residues decreased. Fermentation
of microwave-assisted acid and alkali treated samples resulted in the lowest ethanol production
(approximately 15.9 g/L) (Table 2).

Table 2. Ethanol production from pretreated CS in relation to pretreatment method.

Pretreatment Method Ethanol Yield Theoretical Ethanol Ethanol

Ethanol (g/L)
(Pretreatment Conditions) (g/100 g Pretreated CS) Yield (%) 1 Productivity (g/L/h)
Alkali treatment (10% w/w
15.9 ± 0.7 10.6 ± 0.5 33.9 ± 1.5 0.33 ± 0.02
NaOH, 121 ◦ C, 30 min)
Microwave-assisted acid
pretreatment (210 ◦ C, 10 min, 15.9 ± 1.8 10.6 ± 1.2 38.5 ± 1.3 0.33 ± 0.02
2%, w/w sulfuric acid)
Hydrothermolysis (175 ◦ C,
18.8 ± 0.3 12.5 ± 0.3 54.6 ± 1.3 0.45 ± 0.04
2 h, N2 )
Organosolv 22.1 ± 0.2 14.8 ± 0.1 33.8 ± 0.4 0.52 ± 0.01
Organosolv and hydrothermal
32.3 ± 2.2 21.5 ± 1.5 47.6 ± 2.7 0.49 ± 0.05
treatment (175 ◦ C, 2 h, N2 )
1 Calculated by Equation (4).

The individual effect of the organosolv and hydrothermal pretreatments acted synergistically,
producing significant improvement in ethanol production. The maximum attainable ethanol
production was 32.3 g/L (corresponding to an ethanol yield of 47.6%) after fermentation of the
solid residue resulted from the combined pretreatment. Productivities ranged from 0.33 to 0.52 g/L/h
(Table 2). The organosolv-hydrothermally treated CS was used in further experimentations.

3.3. Effect of Organosolv-Hydrothermally Treated CS Concentration and Pre-Hydrolysis Time on

Bioethanol Production
The effect of pre-hydrolysis time and substrate concentration on bioethanol production was
evaluated. At 15% (w/v) substrate concentration, glucose concentration after 6 h pre-hydrolysis (PSSF
(6 h pre-hydrolysis)) was 35.8 g/L (corresponding to 27.0% cellulose conversion), while extending
pre-hydrolysis time to 14 h glucose increased by approximately 49% (69.6 g/L, corresponding to 52.5%
cellulose conversion) (Figure 2). The 14 h pre-hydrolyzed slurry was fermented to ethanol by the
yeast and resulted in 39.7 g/L bioethanol, corresponding to an ethanol yield of 58.5%, after 30 h of
fermentation (Figure 2).
Fermentation 2019, 5, 5 8 of 12
Fermentation 2018, 4, x FOR PEER REVIEW 8 of 12

100

Prehydrolysis
90

Ethanol and glucose concentration (g/L)

80

70

60

50

40

30

20

10

0
-20 -10 0 10 20 30 40 50 60 70 80

Time (h)

Figure 2. Bioethanol production from organosolv-hydrothermally treated CS at 15% and 20% (w/v)
Figure 2. Bioethanol production from organosolv-hydrothermally treated CS at 15% and 20% (w/v)
substrate concentration applying different pre-hydrolysis times. Symbols: ( ) ethanol and (#) glucose 6
substrate concentration applying different pre-hydrolysis times. Symbols: () ethanol and ()
h pre-hydrolysis at 15% (w/v) substrate concentration; (N) ethanol and (4) glucose 14 h pre-hydrolysis
glucose 6 h pre-hydrolysis at 15% (w/v) substrate concentration; () ethanol and () glucose 14 h
at 15% (w/v) substrate concentration; (H) ethanol and (5) glucose 14 h pre-hydrolysis at 20% (w/v)
pre-hydrolysis at 15% (w/v) substrate concentration; () ethanol and () glucose 14 h pre-hydrolysis
substrate concentration.
at 20% (w/v) substrate concentration.
The increase in bioethanol production compared to 6 h pre-hydrolysis prior to fermentation was
Thehigher
18%. The increase in bioethanol
availability production
of glucose comparedoftothe
at the beginning 6h pre-hydrolysis
fermentation priorthe
affected tofermentation
fermentation
was 18%. The higher availability of glucose at the beginning of the fermentation affected the
time and as a consequence the productivity of the process. According to Ma et al. [36] sugar
fermentation time and as a consequence the productivity of the process. According to Ma et al., [36]
transportation to yeast cell is the rate limiting step of glycolysis pathway in Saccharomyces cerevisiae; a
sugar transportation to yeast cell is the rate limiting step of glycolysis pathway in Saccharomyces
sugar concentration gradient across the plasma membrane is needed to activate this process, thus the
cerevisiae; a sugar concentration gradient across the plasma membrane is needed to activate this
presence of fermentable sugars at the beginning of the fermentation could have enhanced this activation
process, thus the presence of fermentable sugars at the beginning of the fermentation could have
resulting in reduced fermentation time. The productivity of the PSSF (6 h pre-hydrolysis) process was
enhanced this activation resulting in reduced fermentation time. The productivity of the PSSF (6 h
found to be 0.49 g/L/h, while that of the PSSF (14 h pre-hydrolysis) was 1.32 g/L/h. The ethanol
pre-hydrolysis) process was found to be 0.49 g/L/h, while that of the PSSF (14 h pre-hydrolysis) was
produced by the PSSF (14 h pre-hydrolysis) process is close to the limit for economical distillation [15].
1.32 g/L/h. The ethanol produced by the PSSF (14 h pre-hydrolysis) process is close to the limit for
It is evident that the increase in pre-hydrolysis time had a positive effect on bioethanol production.
economical distillation [15]. It is evident that the increase in pre-hydrolysis time had a positive effect
In some previous studies no effect, or a small negative effect, has been reported after pre-hydrolysis
on bioethanol production. In some previous studies no effect, or a small negative effect, has been
prior to fermentation [37], while a clear improvement in ethanol yield has been found in other studies
reported after pre-hydrolysis prior to fermentation [37], while a clear improvement in ethanol yield
when using pre-hydrolysis [17,38].
has been found in other studies when using pre-hydrolysis [17,38].
In an attempt to increase ethanol titer and meet the demands for economical distillation, higher
In an attempt to increase ethanol titer and meet the demands for economical distillation, higher
substrate concentration was used (20% w/v), while the pre-hydrolysis time was set at 14 h. The choice
substrate concentration was used (20% w/v), while the pre-hydrolysis time was set at 14 h. The
of pre-hydrolysis time for the 20% (w/v) substrate concentration was based on previous results [20].
choice of pre-hydrolysis time for the 20% (w/v) substrate concentration was based on previous
Glucose in the pre-hydrolyzed slurry was found to be 90.9 g/L (corresponding to 51.5% cellulose
results [20]. Glucose in the pre-hydrolyzed slurry was found to be 90.9 g/L (corresponding to 51.5%
conversion), which was rapidly converted to ethanol by the yeast (Figure 2). Ethanol production
cellulose conversion), which was rapidly converted to ethanol by the yeast (Figure 2). Ethanol
reached 47.0 g/L (corresponding to an ethanol yield of 52%) after 30 h of fermentation leading to a
production reached 47.0 g/L (corresponding to an ethanol yield of 52%) after 30 h of fermentation
productivity of 1.57 g/L/h. Even though high ethanol titer was achieved, ethanol yield ranged at
leading to a productivity of 1.57 g/L/h. Even though high ethanol titer was achieved, ethanol yield
a moderate level. In general, for high efficiency bioethanol production the balance between ethanol
ranged at a moderate level. In general, for high efficiency bioethanol production the balance between
concentration and yield is very important. One way to improve the overall performance of PSSF could
ethanol concentration and yield is very important. One way to improve the overall performance of
be the recycling the solid residue contained the unhydrolyzed carbohydrates, adsorbed enzymes,
PSSF could be the recycling the solid residue contained the unhydrolyzed carbohydrates, adsorbed
and yeast cells, as demonstrated by Qin et al. [39].
enzymes, and yeast cells, as demonstrated by Qin et al. [39].
A comparison of different processes reported in literature for bioethanol production from
A comparison of different processes reported in literature for bioethanol production from
pretreated CS is summarized on Table 3. Compared to the reported values, bioethanol production
pretreated CS is summarized on Table 3. Compared to the reported values, bioethanol production
obtained in this work is excellent.
obtained in this work is excellent.

Table 3. Comparison of different processes for ethanol production from pretreated CS.
Fermentation 2019, 5, 5 9 of 12

Table 3. Comparison of different processes for ethanol production from pretreated CS.

Fermenting Solids Loading Type of Ethanol Productivity

Type of Pretreatment Reference
Microorganism (%, w/v) Process Conc. (g/L) (g/L/h)
Alkaline (4% w/v NaOH, Pichia kudriavzevii
10.0 PSSF (12 h) 1 19.48 0.41 [4]
121 ◦ C/15psi for 60 min) HOP-1
Ozone treatment (ozone
concentration of 45 mg/L P. kudriavzevii
10.0 SHF 10.96 0.46 [4]
and flow rate of 0.37 L/min HOP-1
for 150 min)
Dilute acid (0.8% v/v (1.5%
S. cerevisiae
w/v), H2 SO4 at 180 ◦ C for 10.0 SSF 12.88 0.13 [33]
Thermosacc® Dry
12 min)
Liquid Hot Water (severity
Dry yeast 8.0 PSSF (24 h) 1 14.0 0.12 [23]
factor: 4.34)
Alkaline (3% NaOH at
S. cerevisiae VS3
room temperature for 24 h).
and Pichia stipitis 10.0 3 - 11.64 0.24 [6]
Two-stage acid hydrolysis
NCIM-3498
of cellulose
High pressure-assisted
alkali pretreatment (HPAP)
S. cerevisiae 2.0 2 SHF 3.32 0.07 [27]
(3.0% NaOH, 121 ◦ C,
130 kPa for 40 min)
Ionic Liquids (EMIMAc) S. cerevisiae NRRL
15.0 2 SHF 22.9 1.92 [22]
(150 ◦ C for 30 min) Y-132
Ultrasonication and hot
S. cerevisiae
water and ligninolytic 10.0 2 SHF 5.5 0.08 [40]
Ethanol Red
enzymes pretreatment
Organosolv and
Dry yeast 20.0 PSSF (14 h) 1 47.0 1.57 Present study
hydrothermal treatment
1 Pre-hydrolysis time, 2 in the case of SHF solids loading is referred to enzymatic hydrolysis step preceded the
fermentation, 3 solid loading of alkali delignified cotton stalk subjected to two-stage dilute acid hydrolysis.

It is evident that ethanol production from CS depends on a number of different parameters that
should be considered for the optimal outcome, i.e., type of pretreatment and the changes these cause
in the composition and structure of the solid residue, type of enzymes, solids and enzyme loading,
fermenting microorganism, process configuration etc.
Despite the high ethanol titer achieved in the present study, further efforts will be devoted to
better exploitation of cellulose content of pretreated CS through recycling of PSFF or implementation
of fed-batch process.

4. Conclusions
Cotton stalk can be a promising feedstock for bio-ethanol production and preferably suited to
the biorefinery approach due to its worldwide abundance, its high carbohydrate content and the
fact that it does not compete with the land available for food and feed production. In the present
study alkali pretreatment, microwave-assisted acid pretreatment, organosolv, hydrothermal treatment
and combination of the organosolv and hydrothermal treatment were comparatively investigated to
produce ethanol from CS applying PSSF at high solids loading. Cellulose, hemicellulose and lignin
content of the pretreated materials depended on the type of pretreatment applied. Fermentation of the
pretreated CS samples carried out by the PSSF process at 15% (w/v) substrate concentration revealed
that ethanol production increased as the lignin content of the solid residues decreased. Among the
different pretreatments, sequentially organosolv and hydrothermal pretreatment of CS generated the
highest ethanol production. The extent of pre-hydrolysis and substrate concentration were important
factors that affected ethanol production as well as the productivity of the process. The highest ethanol
concentration reached in the present study was 47.0 g/L at 20% (w/v) substrate concentration, applying
14 h pre-hydrolysis prior to SSF. Under the conditions described in the present study 235 g of ethanol
from 1 kg organosolv-hydrothermally treated CS could be obtained.
Fermentation 2019, 5, 5 10 of 12

Author Contributions: Conceptualization, K.G.K., A.A.L., N.P., D.K. and D.M.; Methodology, K.G.K. and D.M.;
Investigation, K.D., T.P., A.L.; Writing-Original Draft Preparation, D.M.; Writing-Review & Editing, all authors.
Acknowledgments: The authors would like to thank Theofanis Gemtos of the Laboratory of Farm Mechanization,
Department of Agriculture, Crop Production and Rural Environmnent, University of Thessaly for providing
the cotton stalks. The authors would also like to thank Novozymes Corporation for generously providing the
cellulose enzyme samples.
Conflicts of Interest: The authors declare no conflict of interest.

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