Industrial Crops & Products 169 (2021) 113642

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Industrial Crops & Products

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The water consumption of sugarcane bagasse post-washing after protic

ionic liquid pretreatment and its impact on 2G ethanol production
P.Y.S. Nakasu a, J.L. Ienczak b, S.C. Rabelo c, A.C. Costa a, *
a
Process and Products Development Department, School of Chemical Engineering, State University of Campinas (UNICAMP), Campinas, SP, Brazil
b
Department of Chemical Engineering and Food Engineering, Federal University of Santa Catarina (UFSC), Florianópolis, Santa Catarina, Brazil
c
Department of Bioprocess and Biotechnology, College of Agricultural Sciences, São Paulo State University (UNESP), Botucatu, SP, Brazil

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

Keywords: Despite the water consumption of the first-generation ethanol (1G) production from sugarcane being widely
Water consumption studied, there are only a few experimental studies on water consumption — especially in the pulp washing — in
Ionic liquid second generation (2G) ethanol production. A quantitative evaluation of water wash consumption in the pre­
Pretreatment
treatment with the protic ionic liquid (PIL) monoethanolammonium acetate [MEA][OAc] has shown that a water
Sugarcane bagasse
Washing
consumption between 600− 1,000 wt% for two temperatures, 25 and 80 ◦ C, decreased overall enzyme perfor­
mance in saccharification compared to the full wash sample. The best condition in terms of high yield and low
water consumption was found to be 800 wt%/80 ◦ C, with up to 83 and 61.8 % of glucose and xylose yields
against 97.9 and 85 % for the full wash sample. However, despite the full wash still presenting a superior ethanol
yield, 87.4 %, the best condition with limited amount of water, 800 wt%/80 ◦ C, also had a high ethanol yield of
86.1 %. Mass balance calculations confirmed that optimized [MEA][OAc] pretreatment with 800 wt%/80 ◦ C and
full wash conditions provided the highest ethanol yield per ton of biomass amongst several types of PIL and non-
PIL pretreatments, 299 and 379 L 2G ethanol per ton of biomass, with an estimated water consumption of 56 and
647 ton, paving the way for an effective and more water-sustainable pretreatment technology for 2G ethanol
production.

1. Introduction enzyme’s accessibility. Phenolic compounds from lignin degradation

Water consumption must be considered when it comes to designing a
sustainable ethanol production process. The sugar-ethanol industry de­
mands high quantities of water; for instance, an annexed mill that pro­
duces 50 % ethanol and 50 % sugar consumes 22 m3 of water per ton of
cane on average, and approximately 10 % of this amount is used to wash
cane (UNICA, 2013). In the second-generation process, there is an even
higher consumption of wash water, since a post-washing step of the
pretreated material is required after most pretreatments to recover sol­
ubilized sugars or to remove potential inhibitors of the enzymatic
saccharification and/or alcoholic fermentation.
The enzymatic saccharification consists of the biochemical decon­
struction of the lignocellulosic biomass. Cellulolytic cocktails have been
engineered and upgraded to maximize sugar monomer production with
increased robustness. However, some chemicals may interfere with the
decrease enzyme activity in the medium, therefore, acting as cellulase
inhibitors (Qin et al., 2016; Ximenes et al., 2010; Zhai et al., 2018). A
careful understanding of the dynamics of the chemical transformations
that occur during pretreatment is needed to avoid lignin/carbohydrate
degradation into potential inhibitors (Jönsson and Martín, 2016).
Alcoholic fermentation is in an important step in the production of
2G ethanol, and it validates the efficiency of the enzymatic saccharifi­
cation and pretreatment steps. In this stage, any chemicals such as
furfural or phenolic compounds that might have been generated during
pretreatment or released during enzymatic saccharification may impact
negatively on the yeast metabolism (Chandel et al., 2007; Clark and
Mackie, 1984; Jönsson and Martín, 2016; Larsson et al., 1999; Palmqvist
and Hahn Hägerdal, 2000). There are, however, several ways to over­
come the presence of fermentation inhibitors, such as chemical, or
biological detoxification methods, and adaptation of yeasts to the

Abbreviations: ABR, acid-base ratio; APIL, aprotic ionic liquids; C5, pentose sugar; C6, hexose sugar; DCM, dry cell mass; DI, deionized; 1G, first generation; FPU,
Filter paper unit; LMW, low molecular weight; PIL, protic ionic liquid; 2G, second generation; TRS, total reducing sugars.
* Corresponding author.
E-mail address: accosta@feq.unicamp.br (A.C. Costa).

https://doi.org/10.1016/j.indcrop.2021.113642
Received 22 January 2021; Received in revised form 12 May 2021; Accepted 15 May 2021
Available online 26 May 2021
0926-6690/© 2021 Elsevier B.V. All rights reserved.
P.Y.S. Nakasu et al.
presence of inhibitors (Jönsson and Martín, 2016; Soares et al., 2020).
Despite generating an inhibitor-free hydrolysate, detoxification means
an additional step before fermentation that can ultimately impact the
final ethanol selling price.
The water consumption of a biofuel is an interesting parameter that
examines the full life cycle, starting with the water required to grow the
crop, including water from rainfall (Hoekstra and Mekonnen, 2012;
Mekonnen and Hoekstra, 2011). Despite some studies on simulation of
the water use on 2G ethanol production (Argo et al., 2013; Chiu and Wu,
2012), there are no experimental studies on water consumption (or use)
post PIL pretreatment. However, some works on water use after dilute
acid (Lee et al., 2015), steam explosion (Söderström et al., 2004) and
alkaline pretreatments (Wang et al., 2016) have been discussed in the
literature. Establishing quantitative correlations between water con­
sumption and the yields in saccharification and fermentation is not only
important for more accurate input data in process simulations, but also
for a deeper understanding of the importance of water in the overall
process.
Protic ionic liquid (PIL) pretreatment has shown a potential to reduce
or produce practically no saccharification/fermentation inhibitors
(Gschwend et al., 2018; Malaret et al., 2020; Nakasu et al., 2020;
Semerci and Güler, 2018). It is also known that PIL synthesis is simpler
and cheaper than the more commonly used aprotic ionic liquids (APILs)
(Mäki-Arvela et al., 2010; Rashid et al., 2016; Wei et al., 2012). How­
ever, incomplete pulp washing results in the presence of residual PIL
that might interfere with the subsequent steps in two ways: 1) by
altering the ionic strength of the medium and decreasing the enzyme
activity and/or yeast performance (Turner et al., 2003); 2) by inhibiting
the yeast growth due to the presence of acetate (Felipe et al., 1995;
Ibraheem and Ndimba, 2013). This work aims to evaluate the fermen­
tation of the hydrolysates obtained with [MEA][OAc] pretreatment;
also, the impact of wash water will be investigated on both enzymatic
hydrolysis and fermentation once the enzymes and yeast are affected by
the presence of PIL and/or degradation products.
2. Experimental
The experimental setup of this work was summarized in Fig. 1. Once
we aim to calculate a material balance, all the steps in Fig. 1 were
gravimetrically measured.
Fig. 1. Scheme of the study of the impact of washing on enzymatic saccharification and alcoholic fermentation.
2
Industrial Crops & Products 169 (2021) 113642
2.1. Feedstock and pretreatment
Sugarcane bagasse was provided by São João Mill (Araras-SP,
Brazil). PIL monoethanolammonium acetate, [MEA][OAc], was syn­
thesized by the equimolar addition of acetic acid in a stirring jacketed
glass reactor at 10 ◦ C to monoethanolamine according to Pin et al.
(2019).
For the pretreatment assays, approximately 8.3 g of unground
bagasse (7 wt% moisture content), 34.6 g of [MEA][OAc] and 10.7 g of
water were added to custom-built stainless-steel reactors of 500 mL with
the following conditions: 150 ◦ C, 2 h, 15 wt% solids loading, 20 wt%
water content. After the reaction time, the reactors were cooled in an ice
bath; the slurries were then subjected to the washing experiments at
different temperatures (20 and 80 ◦ C) and water amounts (200 to 1,000
wt%). The washed pulps were air-dried and then used in the enzymatic
saccharification.
2.2. Washing experiments
Pretreatment slurry post-washing was carried out considering a
sequential washing step, the amount of water fed in the process was
based in the initial mass of the system “biomass + ionic liquid + water
(from the pretreatment)”, which corresponded to 53.6 g (100 %).
Therefore, in the washing assays of 200, 400, 600, 800, and 1,000 wt%
the amount of wash water corresponded to multiples of 53.7 g, which
are 107, 214, 322, 429, and 536 g, that were split into 1, 2, 3, 4, and 5
washing batches, respectively. At the end of each batch, the material was
kept under agitation for 5 min, in an orbital shaker at 150 RPM (MA-
832, Marconi, Brazil), the system was then filtered in a press filter
(Maxwell and Williams, JD0004, Brazil) and fresh water was added to
the system to continue the washing process. A full wash sample was also
generated by thoroughly washing the pulp in cloth bags; it was
considered fully washed when the water pH reached that of tap water
(near 6.0). Washing experiments were performed in duplicates. More
information about the experimental setup was provided in the ESI.
The liquid fractions were centrifuged at 3,663 × g for 10 min to
precipitate the lignin and the supernatant was evaporated by rotary
evaporation (Nova Técnica, NT810, Brazil) to yield a concentrated
recovered [MEA][OAc] which had its moisture content determined by
Karl Fischer (Mettler Toledo, DL31, Switzerland) titration in triplicate.
P.Y.S. Nakasu et al.
2.3. Enzymatic saccharification
The air-dried washed pulps were subjected to enzymatic saccharifi­
cation. Saccharification conditions were 10 wt% solids loading, 15 FPU
of cellulases g biomass− 1 (Cellic Ctec2, Novozymes), 72 h in 0.05 M
sodium citrate buffer (Selig et al., 2008). Prior to saccharification,
although the medium was buffered, the pH of the samples was adjusted
to the range between 4.5–5.0 with H2SO4 6 mol⋅L− 1 due to the presence
of residual PIL. After saccharification, the hydrolysates were centrifuged
at 3,663 × g for 10 min to precipitate the solid residue and then filtered
under vacuum through 0.45 μm cellulose membranes to 125 mL Erlen­
meyer flasks and subjected to the alcoholic fermentation.
2.4. Alcoholic fermentation
2.4.1. Microorganism and inoculum preparation
In this work, the C5 and C6 fermenting microorganism Spathaspora
passalidarum NRRL Y-27907 was employed. The yeast was stored at -80
◦
C and it was activated in XYMP media whose composition was xylose,
10 g⋅L− 1; yeast extract, 5 g⋅L− 1; malt extract, 20 g⋅L− 1; and K2HPO4, 2
g⋅L-1 during 24 h in an orbital shaker (Marconi, MA 830, Brazil) at 28 ◦ C
and 200 RPM (Santos et al., 2015). After, 10 % (v⋅v− 1) were transferred
to the inoculum with the same composition previously described, and
incubated during 24 h in an orbital shaker at 28 ◦ C and 200 RPM (Farias
et al., 2014). Inoculum was used to inoculate propagation that was
performed according to Biazi et al. (2020) and Santos et al. (2015) in
500 mL Erlenmeyer flasks. At the end of propagation, the culture was
harvested by centrifugation at 3,663 × g for 20 min at 4 ◦ C and resus­
pended in a minimum volume of water. The cell suspension was then
stored at 4 ◦ C for the fermentation assays.
2.4.2. Fermentation assays
S. passalidarum cells obtained by propagation were added (repre­
senting around 1.5 g L− 1 of DCM) to the hydrolysates (obtained by 200,
400, 600, 800,1,000 wt % and full wash of the slurries) with KH2PO4,
(NH4)2HPO4, MgSO4.7H2O, yeast extract, malt extract, and trace ele­
ments solution with the following concentrations: 4.0 g⋅L− 1, 2.0 g⋅L− 1,
0.5 g⋅L− 1, 3.0 g⋅L− 1, 3.0 g⋅L-1 and 1 mL⋅L− 1, respectively (Farias et al.,
2014). The trace elements solution composition was H3BO3, 0.05 g⋅L-1;
CaCl2.2H2O, 1.25 g⋅L-1; ZnSO4.7H2O, 0.30 g⋅L-1; MnSO4.H2O, 0.19 g⋅L-1;
CoCl2.6H2O, 0.025 g⋅L-1; CuSO4.5H2O, 0.025 g⋅L-1; NaMoO4.2H2O,
0.035 g⋅L-1; FeSO4.7H2O, 0.9 g⋅L-1. Initial total reducing sugars (TRS)
concentrations varied from 27 to 65 g⋅L-1 for the washings performed at
25 ◦ C, and from 29 to 88 g⋅L-1 for the washings performed at 80 ◦ C. A
fermentation control consisting of glucose (60 g⋅L− 1) and xylose (30
g⋅L− 1) was also prepared. Fermentations were performed in 150 mL
Erlenmeyer flasks with 50 mL of work volume at 110 RPM and 28 ◦ C in
shaker. The fermentation profiles were obtained by sampling 1.5 mL of
each sample in the following intervals: 0, 6, 12, 24, 36, 48, and 72 h.
2.5. Analytical methods
The untreated and pretreated materials were submitted to compo­
sitional analysis according to Sluiter et al. (2016). Dry cell mass (DCM)
concentrations were determined gravimetrically after centrifuging the
samples for 5 min at 11,970 × g, washing with DI water, and
oven-drying the cells at 50 ◦ C for 24 h. The supernatants from the
fermentation samples were used for the determination of sugars
(glucose, xylose, and arabinose), polyols (glycerol and xylitol), acetic
acid, and ethanol by using an HPLC system 1260 Infinity (Agilent, Santa
Clara, USA) equipped with a refractive index (RI) detector. Columns and
mobile phase conditions were employed according to Nakasu et al.
(2016).
3
Industrial Crops & Products 169 (2021) 113642
2.6. Fermentation parameters calculation
1
Ethanol yield factor — YP/S (gEthanol⋅ g−TRS) — was calculated as the
ratio between the produced ethanol concentration (g⋅L− 1) and the initial
TRS concentration (g⋅L− 1). The percentage ethanol yield (%) was
calculated as the ratio between the ethanol produced (g⋅L− 1) and the
initial TRS concentration (g⋅L− 1), divided by the theoretical yield, 0.511
g g− 1. The conversion factor of TRS into cell mass — YX/S (gcells g−TRS
1
)—
was calculated as the ratio between cell growth concentration (g⋅L− 1)
and TRS consumed (g⋅L− 1). The ethanol volumetric productivity — QP
(gEthanol⋅L− 1 h− 1) — was defined as the ratio between the maximum
ethanol concentration (g⋅L− 1) and its respective fermentation time (h).
3. Results and discussion
3.1. [MEA][OAc] pretreatment
Recovery of biomass components after pretreatment is shown in
Fig. 2. Pretreatment with [MEA][OAc], which is an alkaline PIL, and
whose alkalinity was boosted by using an acid-base ratio (ABR, the
molar ratio between the acid and base used in the PIL synthesis) of 0.5
(Nakasu et al., 2020), resulted mainly in lignin solubilisation. Nearly 80
wt% of the lignin, 4 wt% of the hemicellulose, and no cellulose present
in the untreated bagasse was solubilized by the PIL. Such feature of PIL
pretreatment (Rashid et al., 2016) shows how selective it can be towards
lignin solubilization, leaving a carbohydrate-rich pulp that can be then
subjected to enzymatic saccharification and be effectively deconstructed
into glucose and pentose (xylose and arabinose) monomers.
3.2. Interplay of PIL and enzyme performance in saccharification
PIL recovery as a function of the amount of wash water together with
glucose and pentose yields in saccharification for the washing experi­
ments are shown in Fig. 3a-f. Recovery of [MEA][OAc] increases with
higher amounts of wash water, as expected, and the effect of tempera­
ture was not pronounced as it is shown in the comparison between
lowercase letters (Fig. 3a, b). A Tukey test done on the PIL recovery for
the washing at 25 ◦ C showed that there were no significant differences
amongst samples with 600 wt% wash water or higher. While for PIL
recovery from washing at 80 ◦ C, the Tukey test detected significant
differences amongst all samples, except between 800− 1,000 wt%. It is
interesting to note that PIL recovery was high for 800 and 1,000 wt%
wash water for both temperatures studied. The full wash sample did not
present quantitative recovery due to PIL incorporation into the biomass
(Nakasu et al., 2020) which results in inexorable PIL loss. Therefore,
recovery values much higher than the full wash would not be expected
due to PIL-biomass interactions.
It is worth mentioning that the washing protocol in this study
Fig. 2. Recovered solids of untreated and pretreated sugarcane bagasse.
P.Y.S. Nakasu et al. Industrial Crops & Products 169 (2021) 113642

Fig. 3. PIL recovery (orange bars) as a function of wash water at 25 ◦ C (a) and 80 ◦ C (b); Glucose (c)-(d) and pentose (e)-(f) yields in saccharification (blue bars)
along with different wash water contents at 25 ◦ C and 80 ◦ C. A full wash sample (green bar) was also plotted as a matter of comparison. Uppercase letters: comparison
of conditions at the same temperature. Lowercase letters: comparison of conditions at different temperatures. Means that do not share a letter are signifi­
cantly different.

provides information and evidence regarding the relationship between
the impact of LIP residue on subsequent process steps. However, it is
known that in a large-scale operation, the countercurrent washing
process would be more recommended, due to its higher efficiency and
lower water demand (Svarovsky, 2001).
A study by Ninomiya et al. (2015) on the washing of bagasse pre­
treated with cholinium acetate (a PIL), [Ch][OAc], and [EMIM][OAc]
(APIL) showed that even after almost 4,000 wt% of wash water there
was still nearly 10 and 5 wt% of residual [Ch][OAc] and [EMIM][OAc]
in the pulps, showing that these ILs might interact by chemo-sorption or
chemically bonding to the biomass, especially [EMIM][OAc], whose
residual values were higher than [Ch][OAc].
There seems to be a sudden increase in enzyme activity between 200
and 400 wt% wash water tests (Fig. 3c-f), which indicates there is an
upper threshold that ensures cellulase activity. Pentose yields (Fig. 3e, f)
were lower than glucose with only 20 % of yield when 200 wt% wash
water was tested, which shows that enzyme inhibition by the PIL
impacts more on the breakage of hemicellulose bonds. Besides the
lowest value at 200 wt%, the yields were significantly different every
two levels apart, i.e., 400–800, 600− 1,000 wt%, etc. For the glucose
yields (Fig. 3c, d), a Tukey test showed that from 400 to 1,000 wt% of
wash water there were no significant differences between the water
quantities for both temperatures, which also did not differ, as indicated
by the lowercase letters. Despite almost all [MEA][OAc] being extracted
from the pulps at 1,000 wt% (Fig. 2), saccharification yields were still
lower than the full wash sample. The presence of unremoved low mo­
lecular weight lignin (LMW) particles in the biomass might have
contributed to a lower enzyme performance (Fig. 2). Ninomiya et al.
(2015) showed that the enzymes present higher tolerance to [Ch][OAc]
than [EMIM][OAc], with the latter presenting almost twice the cEC50
value, i.e., a minimum IL concentration (in wt%) to decrease the enzyme
activity by 50 %, which shows that the imidazolium cation interacts
more strongly with the enzymes than cholinium.
PIL recovery values were used to estimate the amount of residual PIL

4
P.Y.S. Nakasu et al.
in the hydrolysates, which were plotted against the yields in sacchari­
fication (Fig. 4). Strong negative correlations were found — with R2
values ranging from 0.91 to 0.99 — showing that the presence of re­
sidual [MEA][OAc] is one of the causes of the decrease in enzyme ac­
tivity. By analyzing the graphs, it appears the aforementioned upper
threshold is in between 10− 15 wt% of the PIL.
Ninomiya et al. (2015) also correlated the amount of residual [Ch]
[OAc] and [EMIM][OAc] with saccharification yields. However, they
obtained lower R2 values (values were not calculated) which could be
inferred by data points distribution in the graphs. In a study by Sun et al.
(2017) on the effect of increasing loadings of [MEA][OAc] on sacchar­
ification yields of pretreated switchgrass, there was no yield drop be­
tween the fully washed pulp and the ones with 5, 10, and 20 wt% PIL.
However, different methodology was employed. After pretreatment,
water was added to dilute the slurry to the desired PIL loading, the solids
loading of the saccharification was not 10 wt%. By diluting the medium,
the mass transfer properties of the system change and therefore enzyme
inhibition by LMW lignin particles is less probable. On the other side, the
threshold between 10− 15 wt% of PIL to ensure a reasonable enzyme
activity also seemed to appear, a property that does not seem to depend
on the solids loading of the system.
Cellulase inhibition by the presence of ILs has been the subject of
study in the literature (Latiffah and Zahari, 2019; Turner et al., 2003).
Although the mechanism of enzyme denaturation is not fully under­
stood, it is known that they become irreversibly denatured under high
ionic strength media. A decrease of the enzyme activity may be due to a
negatively induced conformational change within the enzyme upon
refolding. The high ionic strength of the media forces protein aggrega­
tion during refolding (Summers and Flowers, 2000; Turner et al., 2003).
These aggregates serve to “lock” proteins in inactive conformations and
may be the culprit behind the drop of activity of the refolded cellulase in
the [MEA][OAc] solutions (Turner et al., 2003). The development of IL
Fig. 4. Linear regression between (a) glucose, (b) pentose (xylose and arabi­
nose) yields in saccharification (washing at 25 ◦ C in blue and 80 ◦ C in orange)
and the amount of residual PIL in the medium. Condition marks: – 1000 wt
%, – 800 wt%, – 600 wt%, – 400 wt%, – 200 wt%.
5
Industrial Crops & Products 169 (2021) 113642
biocompatible (and thermo-tolerant) cellulases (Madeira Lau et al.,
2004) is an interesting approach to overcome enzyme inhibition and
may eventually allow enzyme recyclability, which would then ensure
the economic feasibility of the process.
3.3. Interplay of PIL and alcoholic fermentation performance
The fermentation profiles of the hydrolysates obtained from the
washing experiments are shown in Fig. 5. Fermentation parameters are
shown in Table 1. The washing temperature did not impact on the
ethanol yield factor, as can be noticed from Fig. 5a-h. However, YX/S
values were higher for the samples at 25 ◦ C (Table 1); which means that,
despite producing practically the same amount of ethanol, the TRS in
such samples were more easily converted into cell mass than at 80 ◦ C;
which is not desired, since cell mass production implies less carbon
utilization for ethanol fermentation. 200 and 400 wt% samples did not
ferment, which is directly linked to the amount of residual PIL (Fig. 4).
Acetic acid concentration was higher for the samples at 25 ◦ C,
especially for the 600 wt%, which started with 4.5 g⋅L− 1. However,
overall, initial acetic acid concentrations were below 2.0 g⋅L− 1. Acetic
acid is known to be a potent yeast inhibitor (Felipe et al., 1995; van Zyl
et al., 1991). In its undissociated form, which is liposoluble, it can
diffuse across the plasma membrane. In the cytosol, it dissociates due to
the neutral intracellular pH, therefore decreasing the cytosolic pH. The
cell then diverts its metabolism from growth and ethanol production in
order to restore intracellular pH neutrality (Palmqvist and Hahn
Hägerdal, 2000).
Wash water contents of 200 and 400 wt% did not ferment due to the
very high acetic acid concentrations from the residual PIL, which varied
from around 20 g⋅L− 1 for the 400 wt% and 40 g⋅L− 1 for the 200 wt%.
Soares et al. (2020) studied the effect of inhibitory compounds such as
acetic acid, HMF, furfural, and vanillin, on the fermentation perfor­
mance of S. passalidarum, and found an upper threshold of 2.5 g⋅L− 1 of
acetic acid to ensure satisfactory yeast performance, which might seem
the case for this work, in which acetic acid assimilation from 600 wt% at
25 ◦ C sample decreased its concentration below the threshold.
The YP/S values for the 25 ◦ C wash — ranging between 0.418− 0.441
— however, were not substantially different from the 80 ◦ C wash —
ranging between 0.428− 0.444, which consequently reflected on similar
theoretical yield ranges, 82–86 % and 83–87 %, respectively, which
could drive us to think there was no overall difference between
fermentation yields for both temperatures. Ninomiya et al. (2015) ob­
tained 60 % and 24 % of the theoretical ethanol yield for the fermen­
tation of bagasse hydrolysates from [Ch][OAc] and [EMIM][OAc]
pretreatment by using approximatelly 1,600 wt% of wash water. In the
present work, higher yields (84 %) were obtained by using half the
amount of wash water with both temperatures.
YX/S values for the 25 ◦ C wash samples — 0.19− 0.16 g⋅L− 1 h− 1 —
were higher than the 80 ◦ C wash —0.14− 0.13 g⋅L− 1 h− 1, which may be
related to the high acetic acid concentration in the lowest temperature.
The yeast responds to the presence of inhibitors by trying to assimilate/
metabolize them; by growing, chances to accomplish that are higher.
Polyol accumulation in 72 h (ESI) was proportional to the ethanol yields;
80 ◦ C wash samples had higher polyol levels — glycerol, 0.18− 0.34
g⋅L− 1 and xylitol 0.13− 0.17 g⋅L− 1 from 600 to 1,000 wt% wash,
respectively.
Glycerol content was higher for the control, 0.41 g⋅L− 1 against 0.32
-1
g⋅L for the full wash sample. On the other hand, the full wash sample
had the highest xylitol content, 0.55 g⋅L-1, which was the same as the
control (ESI). Xylitol accumulation, however, was lower than Nakanishi
et al. (2017) in a fed-batch fermentation of sugarcane bagasse hydro­
lysate from alkaline pretreatment (NaOH with anthraquinone as an
additive); xylitol levels varied from 2.20 g⋅L-1 in the first batch to 0.67
g⋅L-1 in the fourth one. Su et al. (2015) found even higher xylitol con­
tents — 2.9–3.8 g⋅L− 1 — on the fermentation of a synthetic media with
12 wt% xylose and 3 wt% glucose. Xylitol is the first intermediary in the
P.Y.S. Nakasu et al. Industrial Crops & Products 169 (2021) 113642

Fig. 5. Fermentation profiles of the samples from the washing experiments. (a) 600 wt% at 25 ◦ C; (b) 600 wt% at 80 ◦ C; (c) 800 wt% at 25 ◦ C; (d) 800 wt% at 80 ◦ C;
(e) 1000 wt% at 25 ◦ C; (f) 1000 wt% at 80 ◦ C; (g) Full wash; (h) Fermentation control. Ethanol, Glucose, Xylose, DCM,
Acetic acid.

6
P.Y.S. Nakasu et al.
Table 1
Fermentation parameters for the washing experiments with S. passalidarum.
Temperature Sample Initial sugars (g⋅L− 1) Ethanol (g⋅L− 1)
600 61.9 ± 3.03 25.9 ± 0.27
25 ◦ C 800 64.8 ± 0.62 28.5 ± 0.27
1,000 65.2 ± 0.29 28.8 ± 0.13
600 63.7 ± 1.42 27.3 ± 1.35
80 ◦ C 800 65.6 ± 0.02 28.9 ± 1.51
1,000 67.1 ± 0.64 29.8 ± 0.05
Full wash 81.4 ± 3.03 36.3 ± 0.03
Control 87.9 ± 3.03 39.4 ± 0.17
xylose metabolism by the yeast, its accumulation implies the lack of
NAD+ (nicotinamide adenine dinucleotide) or NAD(P)+ (nicotinamide
adenine dinucleotide phosphate), two cofactors that are essential for
xylitol oxidation into xylulose by xylitol dehydrogenase (Agbogbo and
Coward Kelly, 2008; Nakanishi et al., 2017). The absence of NAD+ or
NADP+ indicates a lack of oxidizing agents, i.e., molecular oxygen, with
the microaerophily being the main cause behind it.
The full wash sample had a better overall performance than the
samples washed with a limited amount of water, showing YP/S of 0.446 g
g− 1; such values were comparable to the fermentation control, 0.448 g
g− 1, showing that, once inhibitors are removed by fully washing the
pulp, pretreatment with [MEA][OAc] generates a highly digestible and
fermentable mixture. Despite quite satisfactory, fermentation results led
us to think of strategies to increase sugar intake and ethanol productivity
whilst dealing with the presence of acetic acid in the medium.
Some fermentation studies with S. passalidarum for glucose and
xylose consumption have been performed by many research groups
(Bonan et al., 2020; Hou, 2012; Long et al., 2012; Nakanishi et al., 2017;
Ninomiya et al., 2018, 2015; Soares et al., 2020). Different initial xylose
and glucose concentrations were used in each work, which ranged from
7 to 140 g⋅L− 1 for xylose and from 0 to 67 g⋅L− 1 for glucose. Most works
were performed in flasks, except for Nakanishi et al. (2017), who
employed a fed-batch system in a bioreactor and were able to obtain a
high ethanol titer, 58 g⋅L− 1, and Bonan et al. (2020), who used a batch
system. It is noteworthy mentioning that, amongst all works (0.32 to
0.43 YP/S range), we were able to obtain the highest YP/S value, 0.446,
with the full wash sample without a previous optimization of fermen­
tation parameters, i.e., the yeast was able to efficiently consume
monosaccharides and produce ethanol. However, the productivity ob­
tained in this study, 0.51 g⋅L− 1 h− 1, still falls behind most studies
(0.08–1.04 g⋅L− 1 h− 1 QP/S range), confirming the need for a more
in-depth look into aeration rates, which is related to the headspace
volume of the flask and the agitation rate. Ninomiya et al. (2018) ob­
tained considerable productivity on the fermentation of a bagasse hy­
drolysate from [Ch][OAc] pretreatment and by using a genetically
modified S. cerevisiae strain able to ferment pentoses. [Ch]+ cation
structure is quite similar to that of [MEA]+ with a quaternary ammo­
nium ion and a 2-hydroxyethyl group. Negative Crabtree yeasts divert
their metabolic pathway in face of oxygen availability, favoring cell
growth in the presence of higher dissolved oxygen content in the me­
dium, consequently affecting ethanol production (Agbogbo and Coward
Kelly, 2008; du Preez, 1994; Soares et al., 2020). For this group of mi­
croorganisms, ethanol production is favored under OD limiting
conditions.
Ninomiya et al.(2018), by using S. cerevisiae YPH499XU able to
metabolize xylose, were able to deplete both glucose and xylose in 24 h;
this strain co-expresses xylitol dehydrogenase and xylose reductase from
P. stipitis. However, they provided no further information about acetic
acid or polyol accumulation. These authors obtained a YP/S value of
0.43, which was quite high considering that nearly 25 wt% of residual
[Ch][OAc] was still present in the pulp as they did not wash the pulps
after pretreatment once they employed a very low solids loading, 2 wt%.
Sun et al. (2017), on a simultaneous saccharification and
Industrial Crops & Products 169 (2021) 113642
YP/S Yield (%) QP (gEtOH⋅L− 1. h− 1) 1
YX/S (gDCM g−substrate )
0.418 ± 0.017 81.8 ± 3.78 0.37 ± 0.001 0.19 ± 0.010
0.439 ± 0.000 85.9 ± 0.07 0.40 ± 0.088 0.17 ± 0.002
0.441 ± 0.006 86.4 ± 0.37 0.40 ± 0.137 0.16 ± 0.005
0.428 ± 0.003 83.7 ± 4.33 0.38 ± 0.016 0.14 ± 0.015
0.440 ± 0.013 86.1 ± 2.60 0.40 ± 0.030 0.14 ± 0.031
0.444 ± 0.005 86.9 ± 0.37 0.41 ± 0.099 0.13 ± 0.014
0.446 ± 0.009 87.4 ± 1.69 0.51 ± 0.000 0.09 ± 0.001
0.448 ± 0.008 87.8 ± 1.50 0.55 ± 0.001 0.09 ± 0.021
fermentation (SSF) of a switchgrass hydrolysate from [MEA][OAc]
pretreatment, obtained a YP/S value of 0.36, a quite lower value even
though the hydrolysates presented 5 wt% of PIL. They also employed
lower enzyme and yeast loadings, 2 and 0.1 wt%, which ultimately
impacts on equally low productivities. Additionally, an SSF strategy
with such diluted media would require a considerable energetic input
for water evaporation to yield feasible ethanol titers.
3.4. Mass balances for overall 2G ethanol production
Mass balances for the whole 2G ethanol process with [MEA][OAc]
were depicted in Fig. 6 for 800 wt% wash (Fig. 6a) and full wash of the
pulp (Fig. 6b). Both conditions, 1,000 wt% and 800 wt% at 80 ◦ C,
provided the highest ethanol yields. However, we decided to choose 800
wt%/80 ◦ C due to the lower water consumption for such condition, that
produced nearly 299 L of ethanol ton− 1 bagasse; while for the full wash
approximately 379 L of ethanol ton− 1 bagasse was achieved, a difference
of 80 L ton− 1 bagasse. The water consumption was much higher for the
full wash sample, 647 ton of wash water ton-1 pulp against only 56 ton of
wash water ton-1 pulp. Despite producing nearly 20 % less ethanol,
partial wash of the biomass decreased the water consumption by 90 %. It
is important to notice the PIL will be diluted with the wash water and
eventually recovered by water evaporation, so the energetic demand for
PIL recovery with the full wash sample is higher. An in-depth techno-
economic analysis is necessary in order to quantitatively estimate
important 2G ethanol parameters such as the minimum ethanol selling
price — MESP.
An assessment of some PIL pretreatments, subsequent saccharifica­
tion and fermentation and their overall ethanol yield is shown in
Table 2. Although a direct comparison amongst the ethanol yields
cannot be made (due to different saccharification/fermentation condi­
tions), it is a good indication of the overall productivity of the process.
We were able to achieve the highest ethanol yields for almost both
conditions, the full wash of the pulp and 800 wt%/80 ◦ C with 299 and
379 L ton− 1 bagasse.
Sun et al. (2017); Ninomiya et al. (2018), and Rochat et al. (2017)
obtained up to 266, 218, and 148 L of ethanol ton− 1 bagasse, which are
also below the yields obtained in this work. Ninomiya’s pretreatment
time is longer than ours, 21 h compared to 2 h, and they also employed
lower solids loading 2 wt%, which require a higher water consumption
for dilution. Also, they did not wash the pulps after pretreatment, which
end up in PIL losses along with the cycles. Although Sun et al. (2017)
also employed [MEA][OAc] with an SSF strategy, the lower ethanol
yield might be explained by their lower enzyme and yeast loadings,
which ultimately decreased overall ethanol productivity. Rocha et al.
(2017) employed similar pretreatment conditions; however, they
employed a 1.0 acid-base ratio, while we used a lower one, 0.5, which
ultimately impacts on PIL performance due to PIL degradation. We
aimed to ensure the pretreatment parameters we employed (water and
unground biomass loading) would be feasible for a future scaleup and
impact less on PIL degradation (low ABR values).
7
P.Y.S. Nakasu et al. Industrial Crops & Products 169 (2021) 113642

Fig. 6. Mass balance for 2G ethanol production from [MEA][OAc] pretreatment of sugarcane bagasse with (a) 800 wt% wash water and (b) full wash of the biomass.

Table 2
PIL pretreatment, enzymatic saccharification and alcoholic fermentation conditions with their overall ethanol yield.
Pretreatment conditions Saccharification conditions Fermentation conditions Overall ethanol yield Reference
ton− 1⋅biomass

[MEA][OAc], 150 ◦ C, 2 h, 15 wt% 10 wt% solids, 15 FPU of cellulases g 0.15 wt% cell loading, 81 g⋅L− 1 379 This study (full wash)
solids, 20 wt% H2O, 0.5:1 ABR biomass− 1, 72 h initial sugar concentration, 72 h a
[MEA][OAc], 150 ◦ C, 2 h, 15 wt% 10 wt% solids, 15 FPU of cellulases g 0.15 wt% cell loading, 66 g⋅L− 1 299 This study (800 wt%
solids, 20 wt% H2O, 0.5:1 ABR biomass− 1, 72 h initial sugar concentration, 72 h a wash water at 80 ◦ C)
[MEA][OAc], 150 ◦ C, 2 h, 10 wt% 5 (w/v)% solids, 15 FPU of cellulases g Estimated fermentation data b 218 Rochat et al. (2017)a
solids, 20 wt% H2O, 1:1 ABR biomass− 1, 20 IU of β-glucosidase g biomass− 1,
72 h
[MEA][OAc], 160 ◦ C, 0.5 h, 10 wt 10 wt% solids, 20 mg of cellulases/ 0.1 wt% cell loading, 72 h c 148 Sun et al. (2017)a
% solids, 1:1 ABR hemicellulases g biomass− 1, 72 h
[Ch][OAc], 110 ◦ C, 21 h, 2 wt% 10 wt% solids loading, 10 FPU of cellulases g 100 g cell⋅L− 1, 33 g⋅L− 1
initial sugar 266 Ninomiya et al. (2018)a
solids, 1:1 ABR biomass− 1, 72 h concentration, 48 h d
a
Spathaspora passalidarum NRRLY-7124, 28 ◦ C, 200 RPM.
b
Estimated ethanol yield based on 95 % of the theoretical from glucose and 60 % from xylose.
c
Saccharomyces cerevisiae BY4741, SSF temperature not disclosed, agitation rate not disclosed.
d
Saccharomyces cerevisiae YPH499XU, 30 ◦ C, 150 RPM.

3.5. Water management
The washing experiments have shown that it is possible to recover
the PIL almost quantitatively from the wash waters. A wastewater
management route can be thought out based on a scaled-up counter-
current washing configuration, to minimize water consumption and
improve the efficiency of extraction of phenolic compounds, hydrolyzed
carbohydrate, and unrecovered PIL. The process could be similar to the
one used in the extraction of sucrose in the sugar cane plants (Tzia and
Liadakis, 2003). Then, the water can be separated from the PIL in a
distillation process, and it can be reused in the pulp washing processes.
Another wastewater from the process, 2G vinasse, obtained at the end of
the ethanol distillation process, can be used in the biogas production
process, since such residue is known to have a high chem­
ical/biochemical oxygen demand (Silverio et al., 2019). In addition to
the issue of recycling the PIL in the process, which brings benefits in
terms of the costs of the final product, solvent recovery is also important
because its presence in biodigesters can decrease the biogas yield
(Czarny et al., 2019; Li et al., 2017). A thorough evaluation of waste­
water treatment and reuse is necessary but, as it is not in the scope of this
work, it was not further explored.
4. Conclusions
A quantitative evaluation of the wash water consumption in the
pretreatment with [MEA][OAc] has shown that water contents between
600− 1,000 wt% (for both temperatures, 25 and 80 ◦ C) decreased overall
enzyme performance in saccharification compared to the full wash
sample with up to 83.6 % and 61.8 % of glucose and xylose yields with
the 800 wt% at 80 ◦ C sample against 97.9 and 84.3 % for the full wash.
This was mainly due to the presence of residual PIL on the pretreated
materials. However, despite the full wash still presenting a superior
ethanol yield, 87.4 %, the best water wash condition, 800 wt% at 80 ◦ C,
had an equally high ethanol yield of 86.1 %. Mass balance calculations
confirmed that optimized [MEA][OAc] pretreatment with 800 wt% at
80 ◦ C and full wash conditions provided the highest ethanol yield per

8
P.Y.S. Nakasu et al.
ton of biomass amongst several types of PIL and non-PIL pretreatments,
299 and 379 L 2G ethanol per ton of biomass. However, the scaled-up
water consumption for the process must also be considered; even
though 800 wt%/80 ◦ C provided 299 L 2G ethanol per ton of biomass, its
water consumption was 12.5 times lower than the full wash, 56 ton
against 647 tons of wash water, which would take a toll on the energetic
requirements of the plant for [MEA][OAc] recovery through distillation.
CRediT authorship contribution statement
P.Y.S. Nakasu: Conceptualization, Methodology, Data curation,
Visualization, Investigation, Writing - original draft. J.L. Ienczak:
Methodology, Data curation. S.C. Rabelo: Project administration,
Conceptualization, Methodology, Formal analysis, Supervision. A.C.
Costa: Project administration, Conceptualization, Formal analysis,
Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
The authors would like to thank the financial support from FAPESP
— São Paulo Research Foundation [grant numbers 2015/14042-2,
2015/20630-4, and 2016/06142-0], and from the National Council for
Scientific and Technological Development, CNPQ [grant number
304944/2018-1].
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2021.113642.
References
Agbogbo, F.K., Coward Kelly, G., 2008. Cellulosic ethanol production using the naturally
occurring xylose-fermenting yeast, Pichia stipitis. Biotechnol. Lett. 30, 1515–1524.
https://doi.org/10.1007/s10529-008-9728-z.
Argo, A.M., Tan, E.C.D., Inman, D., Langholtz, M.H., Eaton, L.M., Jacobson, J.J.,
Wright, C.T., Muth Jr, D.J., Wu, M.M., Chiu, Y.-W., Graham, R.L., 2013.
Investigation of biochemical biorefinery sizing and environmental sustainability
impacts for conventional bale system and advanced uniform biomass logistics
designs. Biofuels, Bioprod. Biorefining 7, 282–302. https://doi.org/10.1002/
bbb.1391.
Biazi, L.E., Martínez-Jimenez, F.D., Bonan, C.I.D.G., Soares, L.B., Morais, E.R., Ienczak, J.
L., Costa, A.C., 2020. A differential evolution approach to estimate parameters in a
temperature-dependent kinetic model for second generation ethanol production
under high cell density with Spathaspora passalidarum. Biochem. Eng. J. 161,
107586 https://doi.org/10.1016/j.bej.2020.107586.
Bonan, C.I.D.G., Biazi, L.E., Dionísio, S.R., Soares, L.B., Tramontina, R., Sousa, A.S., de
Oliveira Filho, C.A., Costa, A.C., Ienczak, J.L., 2020. Redox potential as a key
parameter for monitoring and optimization of xylose fermentation with yeast
Spathaspora passalidarum under limited-oxygen conditions. Bioprocess Biosyst. Eng.
43, 1509–1519. https://doi.org/10.1007/s00449-020-02344-2.
Chandel, A.K., Kapoor, R.K., Singh, A.K., Kuhad, R.C., 2007. Detoxification of sugarcane
bagasse hydrolysate improves ethanol production by Candida shehatae NCIM 3501.
Biores. Technol. 98, 1947–1950.
Chiu, Y.-W., Wu, M., 2012. Assessing county-level water footprints of different cellulosic-
biofuel feedstock pathways. Environ. Sci. Technol. 46, 9155–9162. https://doi.org/
10.1021/es3002162.
Clark, T.A., Mackie, K.L., 1984. Fermentation inhibitors in wood hydrolysates derived
from the softwood Pinus radiata. J. Chem. Technol. Biotechnol. Biotechnol. 34,
101–110. https://doi.org/10.1002/jctb.280340206.
Czarny, J., Piotrowska-Cyplik, A., Lewicki, A., Zgoła-Grześkowiak, A., Wolko, Ł.,
Galant, N., Syguda, A., Cyplik, P., 2019. The toxic effect of herbicidal ionic liquids on
biogas-producing microbial community. Int. J. Environ. Res. Public Health 16, 916.
https://doi.org/10.3390/ijerph16060916.
du Preez, J.C., 1994. Process parameters and environmental factos affecting D-xylose
fermentation by yeasts. Enzym. Microb Technol 16, 944–956.
Farias, D., Andrade, R.R., Maugeri-Filho, F., 2014. Kinetic modeling of ethanol
production by scheffersomyces stipitis from xylose. Appl. Biochem. Biotechnol. 172,
361. https://doi.org/10.1007/s12010-013-0546-y.
9
Industrial Crops & Products 169 (2021) 113642
Felipe, M.G., Vieira, D.C., Vitolo, M., Silva, S.S., Roberto, I.C., Manchilha, I.M., 1995.
Effect of acetic acid on xylose fermentation to xylitol by Candida guilliermondii.
J. Basic Microbiol. 35, 171–177.
Gschwend, F.J.V., Malaret, F., Shinde, S., Brandt-Talbot, A., Hallett, J.P., 2018. Rapid
pretreatment of miscanthus using the low-cost ionic liquid triethylammonium
hydrogen sulfate at elevated temperatures. Green Chem. 20, 3486–3498. https://doi.
org/10.1039/c8gc00837j.
Hoekstra, A.Y., Mekonnen, M.M., 2012. The water footprint of humanity. Proc. Natl.
Acad. Sci. 109, 3232–3237. https://doi.org/10.1073/pnas.1109936109.
Hou, X., 2012. Anaerobic xylose fermentation by Spathaspora passalidarum. Appl.
Microbiol. Biotechnol. 94, 205–214. https://doi.org/10.1007/s00253-011-3694-4.
Ibraheem, O., Ndimba, B.K., 2013. Molecular adaptation mechanisms employed by
ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds.
Int. J. Biol. Sci. 9, 598–612. https://doi.org/10.7150/ijbs.6091.
Jönsson, L.J., Martín, C., 2016. Pretreatment of lignocellulose: formation of inhibitory
by-products and strategies for minimizing their effects. Bioresour. Technol. 199,
103–112. https://doi.org/10.1016/j.biortech.2015.10.009.
Larsson, S., Reimann, A., Nilvebrant, N., 1999. Comparison of different methods for the
detoxification of lignocellulose hydrolysates of spruce. Appl. Biochem. Biotechnol.
77–79, 91–103.
Latiffah, K., Zahari, S.M.S.N.S., 2019. Stability of cellulases in ionic liquids. Malaysian J.
Fundam. Appl. Sci. 15, 432–435.
Lee, C., Zheng, Y., VanderGheynst, J.S., 2015. Effects of pretreatment conditions and
post–pretreatment washing on ethanol production from dilute acid pretreated rice
straw. Biosyst. Eng. 137, 36–42. https://doi.org/10.1016/j.
biosystemseng.2015.07.001.
Li, X., Schwede, S., Li, H., Yu, X., Yu, Z., Zhu, K., 2017. Toxicity of ionic liquid on
anaerobic digestion. Energy Procedia 142, 938–942. https://doi.org/10.1016/j.
egypro.2017.12.150.
Long, T.M., Su, Y.K., Headman, J., Higbee, A., Willis, L.B., Jeffries, T.W., 2012.
Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast
Spathaspora passalidarum. Appl. Environ. Microbiol. 78, 5492–5500. https://doi.
org/10.1128/AEM.00374-12.
Madeira Lau, R., Sorgedrager, M.J., Carrea, G., van Rantwijk, F., Secundo, F., Sheldon, R.
A., 2004. Dissolution of Candida antarctica lipase B in ionic liquids: effects on
structure and activity. Green Chem. 6, 483–487. https://doi.org/10.1039/
B405693K.
Mäki-Arvela, P., Anugwom, I., Virtanen, P., Sjöholm, R., Mikkola, J.P., 2010. Dissolution
of lignocellulosic materials and its constituents using ionic liquids-A review. Ind.
Crops Prod. 32, 175–201. https://doi.org/10.1016/j.indcrop.2010.04.005.
Malaret, F., Gschwend, F.J.V., Lopes, J.M., Tu, W.C., Hallett, J.P., 2020. Eucalyptus red
grandis pretreatment with protic ionic liquids: effect of severity and influence of
sub/super-critical CO2 atmosphere on pretreatment performance. RSC Adv. 10,
16050–16060. https://doi.org/10.1039/d0ra02040k.
Mekonnen, M.M., Hoekstra, A.Y., 2011. The green, blue and grey water footprint of crops
and derived crop products. Hydrol. Earth Syst. Sci. 15, 1577–1600. https://doi.org/
10.5194/hess-15-1577-2011.
Nakanishi, S.C., Soares, L.B., Biazi, L.E., Nascimento, V.M., Costa, A.C., Rocha, G.J.M.,
Ienczak, J.L., 2017. Fermentation strategy for second generation ethanol production
from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and
Scheffersomyces stipitis. Biotechnol. Bioeng. 114, 2211–2221. https://doi.org/
10.1002/bit.26357.
Nakasu, P.Y.S., Ienczak, L.J., Costa, A.C., Rabelo, S.C., 2016. Acid post-hydrolysis of
xylooligosaccharides from hydrothermal pretreatment for pentose ethanol
production. Fuel. https://doi.org/10.1016/j.fuel.2016.07.069.
Nakasu, P.Y.S., Clarke, C.J., Rabelo, S.C., Costa, A.C., Brandt-Talbot, A., Hallett, J.P.,
2020. Interplay of acid–base ratio and recycling on the pretreatment performance of
the protic ionic liquid monoethanolammonium acetate. ACS Sustain. Chem. Eng. 8,
7952–7961. https://doi.org/10.1021/acssuschemeng.0c01311.
Ninomiya, K., Omote, S., Ogino, C., Kuroda, K., Noguchi, M., Endo, T., Kakuchi, R.,
Shimizu, N., Takahashi, K., 2015. Saccharification and ethanol fermentation from
cholinium ionic liquid-pretreated bagasse with a different number of post-
pretreatment washings. Bioresour. Technol. 189, 203–209. https://doi.org/
10.1016/j.biortech.2015.04.022.
Ninomiya, K., Utami, A.R.I., Tsuge, Y., Kuroda, K., Ogino, C., Taima, T., Saito, J.,
Kimizu, M., Takahashi, K., 2018. Pretreatment of bagasse with a minimum amount
of cholinium ionic liquid for subsequent saccharification at high loading and co-
fermentation for ethanol production. Chem. Eng. J. 334, 657–663. https://doi.org/
10.1016/j.cej.2017.10.113.
Palmqvist, E., Hahn Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysates. II:
inhibitors and mechanisms of inhibition. Bioresour. Technol. 74, 25–33. https://doi.
org/10.1016/S0960-8524(99)00161-3.
Pin, T.C., Nakasu, P.Y.S., Mattedi, S., Rabelo, S.C., Costa, A.C., 2019. Screening of protic
ionic liquids for sugarcane bagasse pretreatment. Fuel. https://doi.org/10.1016/j.
fuel.2018.08.122.
Qin, L., Li, W.-C., Liu, L., Zhu, J.-Q., Li, X., Li, B.-Z., Yuan, Y.-J., 2016. Inhibition of
lignin-derived phenolic compounds to cellulase. Biotechnol. Biofuels 9, 70. https://
doi.org/10.1186/s13068-016-0485-2.
Rashid, T., Kait, C.F., Regupathi, I., Murugesan, T., 2016. Dissolution of kraft lignin using
Protic Ionic Liquids and characterization. Ind. Crops Prod. 84, 284–293. https://doi.
org/10.1016/j.indcrop.2016.02.017.
Rochat, E.C.A., Pin, T.C., Rabelo, S.C., Aline, C.C., 2017. Evaluation of the use of protic
ionic liquids on biomass fractionation. Fuel 206, 145–154. https://doi.org/10.1016/
j.fuel.2017.06.014. In press.
Santos, S.C., Dionísio, S.R., De Andrade, A.L.D., Roque, L.R., Da Costa, A.C., Ienczak, J.L.,
2015. Fermentation of xylose and glucose mixture in intensified reactors by
P.Y.S. Nakasu et al.
scheffersomyces stipitis to produce ethanol. Int. J. Biol. Biomol. Agric. Food
Biotechnol. Eng. 9, 482–487.
Selig, M., Weiss, N., Ji, Y., 2008. Enzymatic Saccharification of Lignocellulosic Biomass.
Laboratory Analytical Procedure (LAP).
Semerci, I., Güler, F., 2018. Protic ionic liquids as effective agents for pretreatment of
cotton stalks at high biomass loading. Ind. Crop. Prod. 125, 588–595. https://doi.
org/10.1016/j.indcrop.2018.09.046.
Silverio, M.S., Calegari, R.P., Leite, G.M.F.L., Baptista, A.S., 2019. Biogas production
from second generation ethanol vinasse. In: de Oliveira, A.C. (Ed.), Agronomy: Link
in the Production Chain. Atena Editora.
Sluiter, J.B., Chum, H., Gomes, A.C., Tavares, R.P.A., Azevedo, V., Pimenta, M.T.B.,
Rabelo, S.C., Marabezi, K., Curvelo, A.A.S., Alves, A.R., Garcia, W.T., Carvalho, W.,
Esteves, P.J., Mendonça, S., Oliveira, P.A., Ribeiro, J.A.A., Mendes, T.D.,
Vicentin, M.P., Duarte, C.L., Mori, M.N., 2016. Evaluation of Brazilian sugarcane
bagasse characterization: an interlaboratory comparison study. J. AOAC Int. 99,
579–585. https://doi.org/10.5740/jaoacint.15-0063.
Soares, L.B., Bonan, C.I.D.G., Biazi, L.E., Dionísio, S.R., Bonatelli, M.L., Andrade, A.L.D.,
Renzano, E.C., Costa, A.C., Ienczak, J.L., 2020. Investigation of hemicellulosic
hydrolysate inhibitor resistance and fermentation strategies to overcome inhibition
in non-saccharomyces species. Biomass & Bioenergy 137, 105549. https://doi.org/
10.1016/j.biombioe.2020.105549.
Söderström, J., Galbe, M., Zacchi, G., 2004. Effect of washing on yield in one- and two-
step steam pretreatment of softwood for production of ethanol. Biotechnol. Prog. 20,
744–749. https://doi.org/10.1021/bp034353o.
Su, Y.K., Willis, L.B., Jeffries, T.W., 2015. Effects of aeration on growth, ethanol and
polyol accumulation by Spathaspora passalidarum NRRL Y-27907 and
Scheffersomyces stipitis NRRL Y-7124. Biotechnol. Bioeng. 112, 457–469. https://
doi.org/10.1002/bit.25445.
Summers, C.A., Flowers II, R.A., 2000. Protein renaturation by the liquid organic salt
ethylammonium nitrate. Protein Sci. 9, 2001–2008. https://doi.org/10.1110/
ps.9.10.2001.
10
Industrial Crops & Products 169 (2021) 113642
Sun, J., Konda, N.V.S.N.M., Parthasarathi, R., Dutta, T., Valiev, M., Xu, F., Simmons, B.
A., Singh, S., 2017. One-pot integrated biofuel production using low-cost
biocompatible protic ionic liquids. Green Chem. 19, 3152–3163. https://doi.org/
10.1039/c7gc01179b.
Svarovsky, L., 2001. Countercurrent washing of solids, 15. In: Svarovsky, Ladislav (Ed.),
Solid-Liquid Separation (Fourth Edition). Butterworth-Heinemann, Oxford,
pp. 442–475. https://doi.org/10.1016/B978-075064568-3/50041-9.
Turner, M.B., Spear, S.K., Huddleston, J.G., Holbrey, J.D., Rogers, R.D., 2003. Ionic
liquid salt-induced inactivation and unfolding of cellulase from Trichoderma reesei.
Green Chem. 5, 443–447. https://doi.org/10.1039/B302570E.
Tzia, C., Liadakis, G. (Eds.), 2003. Extraction Optimization in Food Engineering, 1st
editio. CRC Press.
UNICA, 2013. FNS - Gestão de recursos hídricos na agroindústria canavieira. Água,
Indústria e Sustentabilidade.
van Zyl, C., Prior, B.A., du Preez, J.C., 1991. Acetic acid inhibition of d-xylose
fermentation by Pichia stipitis. Enzyme Microb. Technol. 13, 82–86.
Wang, W., Wang, Q., Tan, X., Qi, W., Yu, Q., Zhou, G., Zhuang, X., Yuan, Z., 2016. High
conversion of sugarcane bagasse into monosaccharides based on sodium hydroxide
pretreatment at low water consumption and wastewater generation. Bioresour.
Technol. 218, 1230–1236. https://doi.org/10.1016/j.biortech.2016.07.074.
Wei, L., Li, K., Ma, Y., Hou, X., 2012. Dissolving lignocellulosic biomass in a 1-butyl-3-
methylimidazolium chloride-water mixture. Ind. Crops Prod. 37, 227–234. https://
doi.org/10.1016/j.indcrop.2011.12.012.
Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M., 2010. Inhibition of cellulases by
phenols. Enzyme Microb. Technol. 46, 170–176. https://doi.org/10.1016/j.
enzmictec.2009.11.001.
Zhai, R., Hu, J., Saddler, J.J.N., 2018. Extent of enzyme inhibition by phenolics derived
from pretreated biomass is significantly influenced by the size and carbonyl group
content of the phenolics. ACS Sustain. Chem. Eng. 6, 3823–3829. https://doi.org/
10.1021/acssuschemeng.7b04178.