Industrial Crops & Products 155 (2020) 112837

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Industrial Crops & Products

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Enabling butanol production from crude sugarcane bagasse hemicellulose T

hydrolysate by batch-feeding it into molasses fermentation
Suranny Jiménez Chacóna, Gabriela Matiasa, Carla Ferreira dos Santos Vieiraa,
Thaddeus Chukwuemeka Ezejib, Rubens Maciel Filhoa, Adriano Pinto Marianoa,*
a
Laboratory of Optimization, Design, and Advanced Control – Fermentation Division (LOPCA-Ferm), School of Chemical Engineering, University of Campinas
(UNICAMP), Campinas, SP, 13083-852, Brazil
b
The Ohio State University, Department of Animal Sciences, Ohio State Agricultural Research and Development Center, Wooster, OH, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Butanol is a chemical and advanced biofuel whose fermentative production from hemicellulose hydrolysates is
Sugarcane hindered by lignocellulose-derived microbial inhibitory compounds. In this work, Clostridium sacchar-
Hemicellulose hydrolysate operbutylacetonicum DSM 14923 was not able to ferment a sugarcane hemicellulose hydrolysate supplemented
Molasses with either laboratory-grade nutrients or sugarcane molasses. To circumvent this problem, the crude hemi-
Fed-batch fermentation
cellulose hydrolysate was fed, after 24 h, to a molasses fermentation containing 45 g/L total reducing sugars.
Butanol
Without the need of supplementing nutrients, the culture was able to co-ferment sucrose, glucose, fructose, and
xylose (conversion of sucrose and xylose were 81 and 90 %, respectively) in a fermentation containing a diluted
molasses-to-hydrolysate volume ratio of 3:1. Butanol yield (0.31 g/g) was remarkable, and butanol titer was
10.0 g/L after 72 h of fermentation. Therefore, this research demonstrated that sugarcane molasses ‒ a by-
product of the sugar industry rich in sucrose and nutrients ‒ can be an efficient feedstock for enabling the
production of butanol from sugarcane bagasse hemicellulose hydrolysate.

1. Introduction
Sugarcane bagasse is a promising source of second-generation su-
gars to produce fuels and chemicals such as butanol in sugarcane pro-
ducing countries such as Brazil, India, Colombia, China, and the United
States (Mariano et al., 2013). However, the sugar obtained from the
hydrolysis of the hemicellulose fraction of the bagasse, xylose, is not
metabolized by several natural-occurring microorganisms, including
the Saccharomyces cerevisiae strains currently used in ethanol mills.
Furthermore, the hemicellulose hydrolysate generally contains bio-
mass-derived toxic compounds (furans, organic acids, and phenolic
compounds), which affect the performance of xylose-fermenting mi-
croorganisms such as butanol-producing clostridia (Baral and Shah,
2014; Amiri and Karimi, 2018). The poor fermentability of hemi-
cellulose hydrolysates is a barrier to the economic feasibility of second-
generation sugarcane biorefineries (Vasconcelos et al., 2020), and
consequently, this problem has been tackled using different approaches.
One approach is the detoxification of the hemicellulose hydrolysate.
Different techniques have been applied to allow the fermentative pro-
duction of butanol, including overliming (Qureshi et al., 2008 and
2010; Liu et al., 2010; Ranjan and Moholkar, 2013; Plaza et al., 2017),
adsorption using ion-exchange resins (Ezeji et al., 2007) and activated
carbon (Zhang et al., 2018; Pratto et al., 2020;Mirfakhar et al., 2020),
nanofiltration (Sun and Liu, 2012), pervaporation (Cai et al., 2013), and
microbial treatment (Agu et al., 2016). Furthermore, increased toler-
ance towards toxic compounds has been achieved through genetic and
metabolic engineering (Liu et al., 2018; Okonkwo et al., 2019) and
supplementation of the fermentation medium with up to 10 g/L calcium
carbonate (Zhang and Ezeji, 2014; Wang et al., 2019; Wu et al., 2019).
However, detoxification techniques can result in sugar loss, generate
solid waste such as gypsum and lime sludge, and overload wastewater
with inorganic material (Humbird et al., 2011). Also, engineered in-
hibitor-tolerant microorganisms may not perform as expected on the
industrial scale. These negative aspects can, thus, narrow even more the
tight profit margins of a commodity product such as butanol.
Alternatively, recent studies have proposed an operation strategy in
which crude (non-detoxified) hemicellulose hydrolysate is batch-fed to
an inhibitors-free broth such as cellulose hydrolysates. The dilution of
the toxic compounds allowed, for example, the conversion of crude
sorghum bagasse hemicellulose hydrolysate (with toxic levels of acetic
acid) into butanol. More specifically, the bioreactor was loaded with
sorghum bagasse cellulose hydrolysate (supplemented with laboratory-

⁎
Corresponding author:
E-mail address: adpm@unicamp.br (A.P. Mariano).

https://doi.org/10.1016/j.indcrop.2020.112837
Received 27 April 2020; Received in revised form 22 July 2020; Accepted 27 July 2020
0926-6690/ © 2020 Elsevier B.V. All rights reserved.
S.J. Chacón, et al.
grade nutrients) and the hemicellulose hydrolysate was fed after 24 h
(Qureshi et al., 2018). In another instance, sugarcane bagasse hemi-
cellulose hydrolysate was added, after 8 h, to a mineral medium sup-
plemented with glucose, peptone, and yeast extract (Gomes et al.,
2019). However, nutrient supplementation, including industrial-grade
nutrients such as diammonium phosphate (DAP) and urea, adds to
production cost and carbon emission. Moreover, the cellulose hydro-
lysate of sugarcane bagasse may not be available for butanol production
because this glucose stream is a promising feedstock for expanding
ethanol production by S. cerevisiae in sugarcane biorefineries
(Junqueira et al., 2017; Vasconcelos et al., 2020).
In the face of these issues, we propose the use of molasses fermen-
tation as the initial stage of the fed-batch fermentation. Sugarcane
molasses ‒ a by-product of the sugar industry ‒ is rich in sucrose and
nutrients and could serve as a low-cost medium where the culture can
grow before the feeding of hydrolysates containing microbial inhibitory
compounds. We tested our hypothesis by conducting fed-batch fer-
mentation experiments where the ability of Clostridium sacchar-
operbutylacetonicum DSM 14923 to convert xylose into butanol was
assessed considering different volume ratios of diluted molasses and
crude hemicellulose hydrolysate.
2. Materials and methods
2.1. Feedstock
Sugarcane molasses (hereafter referred to as molasses) and bagasse
were purchased from a sugarcane mill located in Cosmópolis, SP, Brazil
(22.658951 °S, 47.211819 °W). The molasses contained 608.5 g/L su-
gars (g/L, 449.9 sucrose, 89.9 glucose, and 68.7 fructose) and was used
in the fermentation experiments without hydrolysis treatment. To
produce the hemicellulose hydrolysate, the bagasse (20 dry kg) was
treated (145 °C, 12 min) with 200 L dilute sulfuric acid (0.5 % v/v) in a
350-L Hastelloy C-276 reactor (POPE Scientific Inc., Saukville, USA) at
the Brazilian Biorenewables National Laboratory (LNBR) (CNPEM,
Campinas, Brazil). The experimental protocol developed by the LNBR
was aimed at producing hydrolysates rich in xylose and poor in in-
hibitory compounds (Oliveira et al., 2018). The hemicellulose hydro-
lysate contained 29.8 g/L sugars (23.3 xylose and 6.5 glucose), and its
pH was adjusted from 1.5 to 6.5 using NaOH pellets before fermenta-
tion.
2.2. Microorganism and inoculum preparation
Clostridium saccharoperbutylacetonicum DSM 14923 (N1–4) procured
from the German Collection of Microorganisms and Cell Culture,
Braunschweig, Germany (DSMZ- Deutsche Sammlung von
Mikroorganismen und Zellkulturen) was used for butanol production.
Culture stocks were maintained as cell suspensions in glycerol solution
(40 % v/v) at −80 °C. To prepare the fermentation inoculum, the cell
suspension was inoculated (400 μL) into 10 mL anoxic pre-sterilized
TGY medium (g/L, 30 tryptone, 20 glucose, 10 yeast extract, 1 L-cy-
steine) and incubated in an anaerobic chamber (COY Type A vinyl
chamber) for 20 h at 30 °C under the atmosphere of 96 % nitrogen and
4% hydrogen. The actively growing cells were transferred to 90 mL
anoxic pre-sterilized TGY medium and cultivated under the same con-
ditions for 6 h, during which the optical density of cells at 600 nm
(G6860A Agilent Cary 60 UV–Vis spectrophotometer) attained 1.3–1.5
(fermentation inoculum).
2.3. Batch fermentation
Batch culture (200 mL) of C. saccharoperbutylacetonicum DSM 14923
was conducted in 250-mL screw-capped bottles (triplicate) incubated
without shaking for 72 h at 30 °C in an anaerobic chamber (COY Type A
vinyl chamber). The fermentation medium was sterilized in an
2
Industrial Crops & Products 155 (2020) 112837
autoclave (121 °C; 20 min) followed by cooling and overnight storage in
the anaerobic chamber, and inoculation with C. sacchar-
operbutylacetonicum DSM 14923 (10% v/v). These experiments were
designed to test the fermentability of glucose (60 g/L + nutrients), xy-
lose (60 g/L + nutrients), molasses (40 and 60 g/L with and without
nutrients supplementation), and the hemicellulose hydrolysate [to
which we added nutrients or molasses (increasing the concentration of
total reducing sugars, TRS, to 40 g/L)]. Laboratory nutrients were yeast
extract (1 g/L) and filter-sterilized (0.22 μm) P2 stock solutions (2 mL of
each buffer, mineral, and vitamin solutions) (Vieira et al., 2020).
2.4. Fed-batch fermentation
The two components (diluted molasses and hemicellulose hydro-
lysate) of the fermentation medium were sterilized in an autoclave
(121 °C; 20 min) followed by storage in the anaerobic chamber for
anaerobiosis. The fermentation was initiated with diluted molasses
(45 g/L TRS) and inoculum (10 % v/v of the final fermentation volume)
of actively growing C. saccharoperbutylacetonicum DSM 14923 in the
anaerobic chamber as described in Section 2.3. The hemicellulose hy-
drolysate was added after 24 h when the fermentation was vigorous
(intense bubbling) and approximately 40 % of the sugar content of the
fermentation medium had been utilized by C. sacchar-
operbutylacetonicum DSM 14923. The final fermentation volume was
200 mL, and in five experimental conditions the diluted molasses-to-
hydrolysate volume ratio (M:H ratio) varied as follows: 3:1; 3:1 and
nutrients (P2 stock solutions +1 g/L yeast extract added to the diluted
molasses); 1.5:1; 0.75:1; and 1:3. The fed-batch fermentation was
conducted in 250-mL screw-capped bottles (triplicate) incubated
without shaking for 72 h at 30 °C in an anaerobic chamber.
2.5. Analytical methods and calculations
Concentrations of butanol, ethanol, acetic acid, and sugars (sucrose,
glucose, xylose, and fructose) were measured by high-performance li-
quid chromatography (HPLC) (Agilent 1260 Infinity) using a Bio-Rad
Aminex® HPX-87H column (at 15 °C; 3 mM H2SO4 as mobile phase at a
flow rate of 0.5 mL/min) and a refractive index detector (RID). Furfural
and 5-hydroxymethylfurfural (HMF) were separated using a Nova-Pak
C18 HPLC column (30 °C; 88:11:1 v/v water:acetonitrile:acetic acid
solution as mobile phase at a flow rate of 0.8 mL/min) and detected by
UV–Vis at 280 nm. Total phenolics were measured by a colorimetric
assay (Ainsworth and Gillespie, 2007).
Substrate conversion (%) was calculated as the amount of substrate
consumed (g) divided by the total substrate loading (g). Butanol pro-
ductivity was calculated as the amount of butanol produced (g) divided
by the culture volume (L) and fermentation time (h). Butanol yield (g/
g) was defined as the amount of butanol produced (g) divided by the
amount of substrate consumed (g). The difference between means was
statistically assessed by Tukey’s test (p < 0.05) using the web-based
open-access tool Astatsa Online Web Statistical Calculators (Navendu
Vasavada, astatsa.com).
3. Results and discussion
3.1. Batch fermentation
C. saccharoperbutylacetonicum DSM 14923 was not able to produce
butanol from the crude hemicellulose hydrolysate supplemented with
either laboratory-grade nutrients or molasses. The fermentation was
inhibited by microbial inhibitors contained in the hydrolysate (g/L, 0.5
furfural, 0.6 HMF, 3.7 acetic acid, and 1.3 phenolic compounds), al-
though most of them were presumably not at toxic levels. For example,
in the case of the furans, C. saccharoperbutylacetonicum can even be
stimulated if their concentration is lower than 2 g/L (Yao et al., 2017).
This strain was also able to produce butanol from a sugarcane
S.J. Chacón, et al. Industrial Crops & Products 155 (2020) 112837

hemicellulose hydrolysate containing 3.4 g/L acetic acid (Zetty-Arenas obtained in the Glu (1.22 g/L) and Xyl fermentations (1.21 g/L), re-
et al., 2019). However, in the referred study, the sugarcane hemi- sulting in increased B:E ratio from 12:1 (Glu and Xyl) to 18:1 (M-40). As
cellulose hydrolysate was produced using a milder pretreatment tech- for the acetone, no information was obtained because of the poor HPLC
nique (hydrothermal pretreatment followed by dilute sulfuric acid hy- separation of acetone and butyric acid, a fermentation intermediate.
drolysis). Consequently, lignin was probably degraded to a lesser But we estimate that the acetone concentration was lower than 1 g/L
extent, and the resulting concentration of phenolics (g/L, 0.06 syr- based on other studies on C. saccharoperbutylacetonicum DSM 14923
ingaldehyde, 0.18 p-coumaric acid) was lower than that of the current (Zetty-Arenas et al., 2019). Given the relatively low yield of the co-
study and those (g/L, 0.8 syringaldehyde, 0.4 p-coumaric acid) con- products [i.e. below the typical ABE ratio of 3:6:1 (Jones and Woods,
sidered toxic to C. saccharoperbutylacetonicum (Yao et al., 2017). 1986)], variations in their output may have had a limited effect on the
Therefore, in our studies, the culture was probably most affected by the butanol yield. Thus, a third factor that may have contributed to im-
phenolic compounds produced during the dilute acid pretreatment. proving the butanol yield was the consumption of other carbon sources
Additionally, the cumulative and synergistic effects of acids (including (e.g., organic acids and amino acids) inherently present in sugarcane
the possible presence of formic acid, which derives from the degrada- molasses (Lino et al., 2018).
tion of HMF and furfural; not measured), furans, and phenolic com- In the fermentation experiments of sugarcane molasses containing
pounds contained in the crude hemicellulose hydrolysate probably ex- 60 g/L TRS (M-60 and M-60-P2), the addition of nutrients significantly
acerbated its toxicity (Ezeji et al., 2007), and consequently, negatively improved the conversion of sugars, especially sucrose, to butanol
affected the growth of C. saccharoperbutylacetonicum and butanol fer- (Table 1). Interestingly, the culture consumed all sugars at the same
mentation. time regardless of the supplementation of the fermentation medium
In contrast, in the fermentation of molasses containing 40 g/L TRS with nutrients (Fig. 1). Supplementation of the fermentation medium
(fermentation M-40), the sugars were almost exhausted, and the bu- with nutrients also stimulated butanol productivity, which increased by
tanol yield was remarkable (Table 1). Moreover, the conversion of su- 28 %. Note that the productivity of the fermentation medium con-
gars (97 %) and the butanol yield (0.31) did not vary regardless of taining 40 g/L TRS likewise increased due to supplementation of the
nutrients supplementation (fermentation M-40-P2). Interestingly, the fermentation medium with nutrients (M-40-P2 compared to M-40);
butanol yield from molasses exceeded those on synthetic medium however, the initial sugar concentration in M-40-P2 was higher than
containing either glucose (Glu) or xylose (Xyl) (0.23 and 0.27, respec- that of the M-40. Therefore, given that we aimed to eliminate the need
tively). We attribute this improved yield to three factors. First, given for nutrients supplementation, the condition with the initial TRS con-
that the sugar content of sugarcane molasses is predominantly sucrose, centration of 40 g/L (M-40) was chosen for the fed-batch fermentation.
the higher molecular weight of sucrose compared with glucose may
have influenced the increased butanol yield from sugarcane molasses. 3.2. Fed-batch fermentation
Indeed, solventogenic Clostridium species possess sucrose phosphoe-
nolpyruvate (PEP)-dependent phosphotransferase system (PTS) for The fed-batch operation enabled C. saccharoperbutylacetonicum DSM
transporting and metabolizing sucrose without first hydrolyzing the 14923 to convert the crude hemicellulose hydrolysate into butanol;
disaccharide into the two individual sugar components, glucose and however, the conversion decreased with increasing amounts of hydro-
fructose (Tangney et al., 1998). lysate in the medium (Table 2). In the fermentation containing the least
Second, the superior butanol yield from molasses, when compared amount of hydrolysate (M:H ratio of 3:1), most of the xylose (89.6 %)
to glucose substrate, may have also resulted from a shift in the meta- and the other sugars were consumed (conversion of TRS was 84.0 %).
bolism of the cells that increased carbon flux to butanol production And the butanol yield (0.31) was as high as that observed in the batch
while decreasing flux to the production of co-products (ethanol and fermentation of the molasses (M-40). Furthermore, the fermentation
acetone). As evidence of this fact, ethanol concentration was statisti- with supplementary nutrients (P2 stock solutions + yeast extract)
cally lower in the M-40 fermentation (0.70 g/L) when compared to that showed that all sugars can almost be exhausted in 72 h (fermentation
3:1 + nutrients). Interestingly, C. saccharoperbutylacetonicum, which
was once used in commercial butanol plants run on molasses (Poehlein
Table 1 et al., 2017), can co-ferment sucrose, glucose, fructose, and xylose
Performance of the batch cultivation of C. saccharoperbutylacetonicum DSM (Fig. 2). Nevertheless, the conversion of sucrose and xylose was as low
14923 considering different carbon sources. The fermentation time was 72 h. as 30 % in the fermentation containing the most amount of hydrolysate
Glu Xyl M-40 M-40-P2 M-60 M-60-P2 (Table 2).
Butanol yield, productivity, and concentration were also negatively
Initial TRS (g/L) 61.9 60.6 39.3 42.8 61.9 63.0
affected by increasing amounts of hydrolysate (Table 2). On the one
sucrose (g/L) - - 29.6 32.1 44.8 46.6
glucose (g/L) 61.9 - 4.8 5.2 8.2 8.0 hand, the loss in butanol production was slightly reduced by the fact
fructose (g/L) - - 3.3 3.7 6.6 6.0 that C. saccharoperbutylacetonicum was able to consume part of the
xylose (g/L) - 60.6 - - - - acetic acid present in the hemicellulose hydrolysate (Fig. 2). Notably,
Residual TRS (g/L) 0.3 15.1 1.2 1.4 36.9 26.6 acetic acid is a metabolic intermediate in the acetone-butanol-ethanol
sucrose (g/L) - - 1.1 1.2 33.8 23.7
glucose (g/L) - 0.0 0.1 0.7 0.7
production pathway, which can also be assimilated by the solvento-
fructose (g/L) - - 0.1 0.0 0.7 0.8 genic Clostridium species and converted into acetone and butanol (Ezeji
xylose (g/L) - 15.1 - - - - et al., 2010). But on the other hand, we estimate that the losses in
TRS conversion (%) 99.5a 76.6c 97.0ab 96.9ab 44.3e 60.8d fermentation performance make the process economically infeasible.
sucrose (%) - - 96.5 96.5 29.5 52.6
For instance, any loss in butanol yield and sugar conversion increases
glucose (%) 99.5 - 99.3 98.4 92.6 91.5
fructose (%) - - 98.5 98.9 89.8 86.9 the quantity of feedstock needed and increases the cost of butanol
xylose (%) - 75.0 - - - - production because feedstock is invariably the major cost component of
Butanol (g/L) 15.0a 13.5b 12.5c 14.4a 9.7d 12.8bc fuels and chemical commodities. Also, the dilution of the product
Butanol yield (g/g) 0.23c 0.27b 0.31a 0.32a 0.33a 0.31a stream [butanol concentration decreased from 10.0 (3:1) to 3.2 (1:3) g/
Butanol productivity (g/L h) 0.21a 0.19b 0.17c 0.20a 0.14d 0.18bc
L] would significantly increase the operating costs of downstream se-
Glu: glucose fermentation; Xyl: xylose fermentation; M: molasses fermentation; paration and disposal of wastewater (Mariano and Maciel Filho, 2012).
P2: nutrients supplementation (P2 stock solutions + yeast extract); TRS: total Moreover, the observed loss in butanol productivity would increase
reducing sugars. Means followed by different letters are statistically different the investment cost of the fermentation. For example, for a plant that
from each other (Tukey’s test; p < 0.05). produces 10 kton/a butanol, we estimated the number of fermentation

3
S.J. Chacón, et al. Industrial Crops & Products 155 (2020) 112837

Fig. 1. Concentration of sugars and butanol during the fermentation of the molasses (M-60 stands for 60 g/L TRS).

Table 2 since 16 MMUSD is worth 188 USD/t butanol in terms of production

Performance of the fed-batch cultivation of C. saccharoperbutylacetonicum DSM cost (purchase cost was annualized over 20 years considering a discount
14923 considering different diluted molasses-to-hydrolysate volume ratios. The rate of 10 %). Thus, due to economic and technical factors, the volume
fermentation time was 72 h. of hemicellulose hydrolysate fed to the fermentation should not exceed
3:1 3:1+nutrients 1.5:1 0.75:1 1:3 the M:H ratio of 3:1.
However, the recommended ratio may limit the capacity of the
Initial TRS in the molasses (g/ 44.7 43.7 47.6 46.8 46.5 butanol plant. In the fermentation containing the M:H ratio of 3:1,
L)
TRS loading (molasses + C5) 7.3 6.9 7.3 6.8 5.7
xylose accounts for only 10 % of the total amount of sugars (Table 2). If
(g) this value is put into the context of a sugarcane mill that converts
Xylose fraction (%) 10.0 7.5 24.0 43.1 61.8 500 tons of sugarcane per hour into ethanol and table sugar (each
Residual sugars (g/L) 6.2 0.8 15.4 14.6 19.5 process consuming half of the sugarcane input), the amount of sugar
Residual sugars (g) 1.2 0.2 2.9 2.7 3.6
from molasses available in the mill would not match the amount re-
Xylose fraction (%) 6.4 14.9 19.7 55.6 68.6
TRS conversion (%) 84.0b 97.8a 62.6c 63.2c 44.1d quired by the butanol plant. In that mill, the production of molasses is
sucrose (%) 80.9 97.9 45.2 59.7 32.1 10 t/h (total reducing sugars) and lower compared to xylose (11 t/h) if
glucose (%) 91.3 97.7 88.9 91.9 92.0 75 % of the bagasse is used to produce lignocellulosic sugars (Mariano
fructose (%) 97.1 98.9 100.0 100.0 100.0 et al., 2013). Thus, either the amount of bagasse used to produce sugars
xylose (%) 89.6 95.8 67.7 48.1 29.1
Butanol (g/L) 10.0b 10.8a 7.0c 5.2d 3.2e
would have to be adjusted accordingly (to 8% of the total amount in
Butanol (g) 1.9 2.0 1.3 1.0 0.6 this case) or, probably more promising, additional molasses would have
Butanol yield (g/g) 0.31ab 0.31ab 0.27b 0.21c 0.21c to be purchased from other mills. Note that the recommended M:H ratio
Butanol productivity (g/L h) 0.14b 0.15a 0.10c 0.07d 0.04e of 3:1 may have to be adjusted depending on the composition of the
hemicellulose hydrolysate, which may vary according to pretreatment
TRS: total reducing sugars; C5: hemicellulose hydrolysate. Means followed by
conditions, variety of sugarcane (Benjamin et al., 2013), and other
different letters are statistically different from each other (Tukey’s test;
p < 0.05).
factors such as edaphoclimatic conditions and maturity stage at har-
vesting (Andrade et al., 2017).
tanks required by the plant as a function of butanol productivity
(Fig. 3). The number of fermentation tanks varies significantly de- 4. Conclusions
pending on the M:H ratio and can range from 15 (3:1) to staggering 52
(1:3) tanks. Consequently, the respective purchase cost of the tanks Fed-batch feeding of bagasse hemicellulose hydrolysate to molasses
varies from 6 to 22 MMUSD. Yet, the lowest number of tanks required fermentation was an efficient strategy to allow the conversion of the
by the butanol plant is still impressive. For the sake of comparison, the hydrolysate into butanol without prior detoxification treatment and
production of 250 kton/a ethanol in a sugarcane ethanol mill would supplementation of nutrients. The operational strategy exploited the
require 12 tanks assuming ethanol productivity is 4.5 g/L∙h (J. Fin- ability of C. saccharoperbutylacetonicum DSM 14923 to co-ferment su-
guerut, Institute of Technology for Sugarcane, personal communica- crose and xylose; however, the amount of hemicellulose hydrolysate fed
tion). Nevertheless, the operation considering the least amount of hy- to the fermentation is limited by the tolerance of the microorganism to
drolysate in the medium can have a significant economic advantage bagasse-derived microbial inhibitors. For a hemicellulose hydrolysate

4
S.J. Chacón, et al.
Fig. 2. Concentration of sugars, acetic acid, and butanol during the fed-batch fermentation considering different diluted molasses-to-hydrolysate volume ratios. The
red arrow indicates the time the crude hemicellulose hydrolysate was fed to the fermentation.
produced by dilute acid pretreatment, the volume of hydrolysate in the
fermentation should be at least three times lower than that of molasses.
Regardless of this restriction, the combination of first (molasses)- and
second (sugarcane bagasse)-generation feedstock is a promising
strategy to improve the economics of sugarcane biorefineries designed
to produce butanol from the hemicellulose fraction of the sugarcane
bagasse.
CRediT authorship contribution statement
Suranny Jiménez Chacón: Investigation, Conceptualization,
Writing - oiginal daft. Gabriela Matias: Investigation. Carla Ferreira
dos Santos Vieira: Investigation. Thaddeus Chukwuemeka Ezeji:
Conceptualization, Writing- rview & editing. Rubens Maciel Filho:
Conceptualization, Funding acquisition. Adriano Pinto Mariano:
Conceptualization, Supervision, Funding acquisition, Writing - review &
editing.
5
Industrial Crops & Products 155 (2020) 112837
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
We thank the National Council for Scientific and Technological
Development (CNPq) for providing a scholarship to S. J. Chacón, the
São Paulo Research Foundation (FAPESP) (Grant numbers 2015/
20630-4, 2018/23983-3, and 2018/25925-0) and the United States
Department of Agriculture – NIFA Hatch grant for the financial support,
and the Brazilian Biorenewables National Laboratory (LNBR) (CNPEM/
MCTIC) for producing the hemicellulose hydrolysate at no cost.
S.J. Chacón, et al.
Fig. 3. Fermentation tanks in a butanol plant as a function of butanol pro-
ductivity. The plant operates 200 days per year during the crop season. The
purchase cost of the fermentation tanks was calculated based on the quotation
available in Humbird et al. (2011), which was adjusted considering (i) a scaling
factor equal to 0.6, and (ii) the Chemical Engineering Plant Cost Index.
References
Agu, C.V., Ujor, V., Gopalan, V., Ezeji, T.C., 2016. Use of Cupriavidus basilensis-aided
bioabatement to enhance fermentation of acid-pretreated biomass hydrolysates by
Clostridium beijerinckii. J. Ind. Microbiol. Biotechnol. 43, 1215–1226.
Ainsworth, E.A., Gillespie, K.M., 2007. Estimation of total phenolic content and other
oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat. Protoc. 2,
875–877.
Amiri, H., Karimi, K., 2018. Pretreatment and hydrolysis of lignocellulosic wastes for
butanol production: challenges and perspectives. Bioresour. Technol. 270, 702–721.
Andrade, L.P., Crespim, E., Oliveira, N., Campos, R.C., Teodoro, J.C., Galvão, C.M.A.,
Maciel Filho, R., 2017. Influence of sugarcane bagasse variability on sugar recovery
for cellulosic ethanol production. Bioresour. Technol. 241, 75–81.
Baral, N.R., Shah, A., 2014. Microbial inhibitors: formation and effects on acetone-bu-
tanol-ethanol fermentation of lignocellulosic biomass. Appl. Microbiol. Biotechnol.
98, 9151–9172.
Benjamin, Y., Cheng, H., Görgens, J.F., 2013. Evaluation of bagasse from different vari-
eties of sugarcane by dilute acid pretreatment and enzymatic hydrolysis. Industr.
Crops Prod. 51, 7–18.
Cai, D., Zhang, T., Zheng, J., Chang, Z., Wang, Z., Qin, P., Tan, T., 2013. Biobutanol from
sweet sorghum bagasse hydrolysate by a hybrid pervaporation process. Bioresour.
Technol. 145, 97–102.
Ezeji, T., Qureshi, N., Blaschek, H.P., 2007. Butanol production from agricultural re-
sidues: impact of degradation products on Clostridium beijerinckii growth and butanol
fermentation. Biotechnol. Bioeng. 97, 1460–1469.
Ezeji, T., Milne, C., Price, N.D., Blaschek, H.P., 2010. Achievements and perspectives to
overcome the poor solvent resistance in acetone and butanol-producing micro-
organisms. Appl. Microbiol. Biotechnol. 85, 1697–1712.
Gomes, A.C., Rodrigues, M.I., Passos, D.F., Castro, A.M., Santa Anna, L.M.M., Pereira Jr.,
N., 2019. Acetone–butanol–ethanol fermentation from sugarcane bagasse hydro-
lysates: Utilization of C5 and C6 sugars. Electronic J. Biotechnol. 42, 16–22.
Humbird, D., Davis, R., Tao, L., Kinchin, C., Hsu, D., Aden, A., 2011. Process design and
economics for biochemical conversion of lignocellulosic biomass to ethanol–dilute-
Industrial Crops & Products 155 (2020) 112837
acid pretreatment and enzymatic hydrolysis of corn stover. Technical report, NREL/
TP-5100-47764. .
Jones, D.T., Woods, D.R., 1986. Acetone–butanol fermentation revisited. Microbiol.
Reviews 50, 484–524.
Junqueira, T.L., Chagas, M.F., Gouveia, V.L.R., Rezende, M.C.A.F., Watanabe, M.D.B.,
Jesus, C.D.F., Cavalett, O., Milanez, A.Y., Bonomi, A., 2017. Techno‑economic ana-
lysis and climate change impacts of sugarcane biorefineries considering different time
horizons. Biotechnol. Biof. 10, 50.
Lino, F.S.O., Basso, T.O., Sommer, M.O.A., 2018. A synthetic medium to simulate su-
garcane molasses. Biotechnol. Biof. 11, 221.
Liu, Z., Ying, Y., Li, F., Ma, C., Xu, P., 2010. Butanol production by Clostridium beijerinckii
ATCC 55025 from wheat bran. J. Ind. Microbiol. Biotechnol. 37, 495–501.
Liu, J., Lin, Q., Chai, X., Luo, Y., Guo, T., 2018. Enhanced phenolic compounds tolerance
response of Clostridium beijerinckii NCIMB 8052 by inactivation of Cbei_3304. Microb.
Cell Fact. 17, 35.
Mariano, A.P., Maciel Filho, R., 2012. Improvements in biobutanol fermentation and their
impacts on distillation energy consumption and wastewater generation. Bioen. Res. 5,
504–514.
Mariano, A.P., Dias, M.O.S., Junqueira, T.L., Cunha, M.P., Bonomi, A., Maciel Filho, R.,
2013. Utilization of pentoses from sugarcane biomass: techno-economics of biogas vs.
butanol production. Bioresour. Technol. 142, 390–399.
Mirfakhar, M., Asadollahi, M.A., Amiri, H., Karimi, K., 2020. Co-fermentation of hemi-
cellulosic hydrolysates and starch from sweet sorghum by Clostridium acetobutylicum:
A synergistic effect for butanol production. Indust. Crops Prod. 151, 112459.
Okonkwo, C.C., Ujor, V., Ezeji, T.C., 2019. Chromosomal integration of aldo-keto-re-
ductase and short-chain dehydrogenase/reductase genes in Clostridium beijerinckii
NCMB 8052 enhanced tolerance to lignocellulose-derived microbial inhibitory com-
pounds. Sci. Rep. 9, 7634.
Oliveira, R.A., Rossell, C.E.V., Venus, J., Rabelo, S.C., Maciel Filho, R., 2018.
Detoxification of sugarcane-derived hemicellulosic hydrolysate using a lactic acid
producing strain. J. Biotechnol. 278, 56–63.
Plaza, P.E., Gallego-Morales, L.J., Peñuela-Vásquez, M., Lucas, S., García-Cubero, M.T.,
Coca, M., 2017. Biobutanol production from brewer’s spent grain hydrolysates by
Clostridium beijerinckii. Bioresour. Technol. 244, 166–174.
Poehlein, A., Solano, J.D.M., Flitsch, S.K., Krabben, P., Winzer, K., Reid, S.J., Jones, D.T.,
Green, E., Minton, N.P., Daniel, R., Dürre, P., 2017. Microbial solvent formation re-
visited by comparative genome analysis. Biotechnol. Biof. 10, 58.
Pratto, B., Chandgude, V., Sousa Junior, R., Cruz, A.J.G., Bankar, S., 2020. Biobutanol
production from sugarcane straw: Defining optimal biomass loading for improved
ABE fermentation. Indust. Crops Prod. 148, 112265.
Qureshi, N., Ezeji, T.C., Ebener, J., Dien, B.S., Cotta, M.A., Blaschek, H.P., 2008. Butanol
production by Clostridium beijerinckii. Part I: use of acid and enzyme hydrolyzed corn
fiber. Bioresour. Technol 99, 5915–5922.
Qureshi, N., Saha, B.C., Dien, B., Hector, R.E., Cotta, M.A., 2010. Production of butanol (a
biofuel) from agricultural residues: Part I-Use of barley straw hydrolysate. Biom.
Bioen. 34, 559–565.
Qureshi, N., Saha, B.C., Klasson, K.T., Liu, S., 2018. High solid fed-batch butanol fer-
mentation with simultaneous product recovery: Part II-Process integration.
Biotechnol. Prog. 34, 967–972.
Ranjan, A., Moholkar, V.S., 2013. Comparative study of various pretreatment techniques
for rice straw saccharification for the production of alcoholic biofuels. Fuel 112,
567–571.
Sun, Z., Liu, S., 2012. Production of n-butanol from concentrated sugar maple hemi-
cellulosic hydrolysate by Clostridia acetobutylicum ATCC824. Biom. Bioen. 39, 39–47.
Tangney, M., Rousse, C., Yazdanian, M., Mitchell, W.J., 1998. Note: sucrose transport and
metabolism in Clostridium beijerinckii NCIMB 8052. J. Appl. Microbiol. 84, 914–919.
Vasconcelos, M.H., Mendes, F.M., Ramos, L., Dias, M.O.S., Bonomi, A., Jesus, C.D.F.,
Watanabe, M.D.B., Junqueira, T.L., Milagres, A.M.F., Ferraz, A., dos Santos, J.C.,
2020. Techno-economic assessment of bioenergy and biofuel production in integrated
sugarcane biorefinery: Identification of technological bottlenecks and economic
feasibility of dilute acid pretreatment. Energy 199, 117422.
Wang, J., Yang, H., Qi, G., Liu, X., Gao, X., Shen, Y., 2019. Effect of lignocellulose-derived
weak acids on butanol production by Clostridium acetobutylicum under different pH
adjustment conditions. RSC Adv. 9, 1967–1975.
Wu, Y., Bai, Y., Zhang, D., Cheng, C., Chen, L., Bai, F., Xue, C., 2019. Pleiotropic reg-
ulation of a glucose‑specific PTS in Clostridium acetobutylicum for high‑efficient bu-
tanol production from corn stover without detoxification. Biotechnol. Biof. 12, 264.
Yao, D., Dong, S., Wang, P., Chen, T., Wang, J., Yue, Z.B., Wang, Y., 2017. Robustness of
Clostridium saccharoperbutylacetonicum for acetone-butanol-ethanol production: ef-
fects of lignocellulosic sugars and inhibitors. Fuel 208, 549–557.
Zetty-Arenas, A.M., Alves, R.F., Portela, C.A.F., Mariano, A.P., Basso, T.O., Tovar, L.P.,
Maciel Filho, R., Freitas, S., 2019. Towards enhanced n-butanol production from
sugarcane bagasse hemicellulosic hydrolysate: Strain screening, and the effects of
sugar concentration and butanol tolerance. Biom. Bioen. 126, 190–198.
Zhang, Y., Ezeji, T.C., 2014. Elucidating and alleviating impacts of lignocellulose-derived
microbial inhibitors on Clostridium beijerinckii during fermentation of Miscanthus gi-
ganteus to butanol. J. Ind. Microbiol. Biotech. 41, 1505–1516.
Zhang, Y., Xia, C., Lu, M., Tu, M., 2018. Effect of overliming and activated carbon de-
toxification on inhibitors removal and butanol fermentation of poplar pre-
hydrolysates. Biotechnol. Biof. 11, 178.