Chemical Engineering Research and Design 196 (2023) 568–576

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Chemical Engineering Research and Design

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

Recovery and characterization of cellulosic ethanol

from fermentation of sugarcane bagasse
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⁎
Celina K. Yamakawa a, , Sebastian T. Rojas b, William E. Herrera c,
Carlos E.V. Rossell d, Maria Regina Wolf Maciel c, Rubens Maciel Filho c
a
Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 223,
2800 Kongens Lyngby, Denmark
b
Institute for Molecular Bio Science, Goethe University Frankfurt, Frankfurt, Germany
c
School of Chemical Engineering, State University of Campinas, Campinas, SP, Brazil
d
Interdisciplinary Center on Energy Planning, State University of Campinas, Campinas, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Industrial production of ethanol by fermentation using renewable feedstock such as su­
Received 19 December 2022 garcane stalks has been demonstrated as a sustainable fuel chain in Brazil. This work
Received in revised form 29 May focused on the production of cellulosic ethanol from sugarcane bagasse in a pilot scale
2023 unit by applying the current bioprocessing strategies with the aim of recovering and
Accepted 26 June 2023 characterizing the end products. The feedstock was pretreated at 190 °C and a residence
Available online 28 June 2023 time of 10 min. Enzymatic hydrolysis was performed with commercial cellulolytic en­
zymes. Fermentation’s substrates were formulated with hydrolysate and supplemented
Keywords: with 8%wt sugarcane molasses. The fermentations were set up to mimic the conventional
Sugarcane bagasse industrial fermentation in Brazil’s ethanol distilleries at high cell density with cell re­
Lignocellulosic biomass cycling. The fermentation resulted in a reproducible performance by the yield of 0.49 g/g,
Cellulosic ethanol productivity of 6.96 g/(L∙h), and cell viability of 95.3%. Ethanol was recovered in a lab-scale
Fermentation distillation batch system. Distilled fractions showed higher content of higher alcohols and
Cell recycling sulfur content than the standard specification of ANP (National Agency of Petroleum -
Distillation Brazilian Agency) for ethanol fuel. The distillation bottom product (vinasse) presented
Vinasse most characteristics suitable for fertilizer or biogas applications, except for sodium and
Ethanol fuel standard sulfate content. Therefore, for a successful technology, transference processing adjust­
ments should be made to make the product commercially suitable and the side stream
compatible for disposal as fertilizer or digestion for biogas production.
© 2023 Institution of Chemical Engineers. Published by Elsevier Ltd. All rights reserved.

produced during the fermentation of sugars by Saccharomyces

1. Introduction
cerevisiae yeast. Currently, the most used sources of sugars
are sugarcane, corn, and sugar beet (Zabed et al., 2017). Par­
Biofuels play a crucial role in the advancement of the dec­
ticularly, Brazilian bioethanol distilleries are energy self-
arbonization of the economy, mitigating climate change by
sufficient, where the steam and electricity are generated
using renewable resources for the production of fuels (Letti
from bagasse and straw in the thermoelectric plant installed
et al., 2022). A classic example of biofuel is ethanol, which is
together with the production site (de Souza Dias et al., 2015).
Ethanol has been used intensely as an additive, fuel or
blended with conventional gasoline (Mizik, 2021). Beyond
⁎
Corresponding author. fuels, ethanol is widely used in food, pharmaceuticals, cos­
E-mail addresses: celinayamakawa@gmail.com, metics, detergents, and medical care areas. Also, it is a raw
cikiya@dtu.dk (C.K. Yamakawa).
https://doi.org/10.1016/j.cherd.2023.06.053
0263-8762/© 2023 Institution of Chemical Engineers. Published by Elsevier Ltd. All rights reserved.
 Chemical Engineering Research and Design 196 (2023) 568–576
material to generate several industrial products of interest
such as ethylene, propylene, and butadiene (Rossi et al.,
2021). Brazil and USA are the largest producers responsible
for 75% of ethanol production worldwide (Bergmann et al.,
2018). Nowadays, lignocellulosic biomass such as sugarcane
bagasse, wheat straw, and cultivated wood have been
exploited for the production of ethanol (Guigou et al., 2019;
Soccol et al., 2010; Townsend et al., 2017) being called as
second-generation ethanol. Sugarcane bagasse is generated
after extracting sucrose by crushing the sugarcane stalks.
This fiber residue contains 50% of moisture (Dias et al., 2012).
Today there is a surplus of bagasse due to the advances in
process efficiency like energy optimization and modern and
more efficient equipment. Therefore, there is room to utilize
the lignocellulosic sugarcane residues as a feedstock to in­
crease the productivity of ethanol without land-use compe­
tition with food crops. Integration of the current ethanol
process and the second-generation processes is a realistic
approach and strategic principle to minimize the capital in­
vestment, optimize the feedstock logistic, and promote the
advancement of biorefineries by producing added-value
chemicals (Dias et al., 2013; Klein et al., 2017).
The polymeric structure of cellulose of bagasse can be
hydrolysate by the application of heat, chemicals, and cel­
lulolytic enzymes to obtain a stream rich in glucose together
with pentoses, organic acids, furan derivatives, and phenolic
compounds. In the integration concept, this new stream of
sugars is combined with the current stream of fermentation
substrate formulated with sugarcane juice and molasses.
The configuration of the conventional bioprocess in Brazil
comprises high cell density and cell recycling (Lopes et al.,
2016; Walker and Basso, 2020). Most of the installations apply
fed-batch mode with 4 h of feeding followed by 2 h or more to
complete sugars exhaustion. At the end of fermentation, the
cells are recovered by centrifugation, washed at acidic con­
dition of pH 2–3 adjusted with sulfuric acid, and reused in the
following new batch. The amount of cell suspension corre­
sponds to 1/3 of the final volume, and the rest is dedicated for
substrate feeding. The current ethanol titer at the end of
fermentation is between 8% and 12% (v/v), reaching product
yield of 92%. Ethanol is recovered and concentrated by dis­
tillation. Conventional distillation consists of a set of 3 dis­
tillation columns and 2 rectification columns (Dias et al.,
2011; Rossell et al., 2016). In the distillation columns is ob­
tained ethanol-rich stream containing approximately 40% (v/
v) ethanol, and the bottom product stream is named vinasse.
Vinasse is practically free of ethanol, but rich in salts and
organic matter. Currently, vinasse can be converted into
biogas (Volpi et al., 2022), but it is mostly used in the fertili­
zation of sugarcane crops at a limited amount to avoid soil
salinization (Rossell et al., 2016). The ethanol-rich stream in a
vapor phase goes to the rectification columns to concentrate
near the binary ethanol-water azeotrope. Either a heater
exchange or direct steam injection is used in the distillation
column. Moreover, during the distillation, impurities are re­
moved. The distillation columns are also designed to remove
separately secondary alcohols and incondensable compo­
nents (CO2 and SO2). The rectification columns are con­
figurated with condensers to remove the compounds named
fusel oil and higher oil streams (Rossell et al., 2016). Fusel oil
is composed mainly of 3-Methyl-1-butanol (amyl alcohol)
and 2-Methyl-1-propanol (isobutyl alcohol) (Mayer et al.,
2015). Higher oil is composed of n-propanol, isobutanol, and
n-butanol. The current distillation yield is 99.5%, and the
569
generation of the bottom product is between 10 and 15 L of
vinasse/L of ethanol (Bergmann et al., 2018). Therefore,
ethanol production from sugarcane in Brazil has a noticeably
high efficiency and well-established technology. But in the
light of using lignocellulosic biomass, introducing a new
sugar stream could cause impacts during fermentation, dis­
tillation, effluent disposal, and the quality of the end product.
For instance, lignocellulosic hydrolysates are generated
using different approaches, such as diluted acid hydrolysis
and saccharification, that bring a new sugar stream with the
presence of inhibitors, salts, and proteins. This work ad­
dressed the application of sugarcane bagasse hydrolysate for
ethanol production using the current methodology to ana­
lyze the characteristics of the final product (ethanol) and the
effluent (vinasse) in comparison to the Brazilian fuel stan­
dard regulation of ANP (National Agency of Petroleum -
Brazilian Agency).
2. Material and methods
2.1. Hydrolysate preparation
Sugarcane bagasse was obtained from the Granelli sugar mill
(Charqueada, São Paulo, Brazil). First, mineral impurities
were removed from the feedstock using a vibratory sieve
Multideck® (MultiVibro, BR). Three pretreatment campaigns
were performed to generate cellulosic hydrolysate for fer­
mentation. The pretreatment was performed in a 350-L
stirred reactor (Pope Scientific Inc., USA) at 190 °C and 10 min
of residence time using 10% (w/v) of solid loading, and 0.5%
(v/v) of sulfuric acid. The reactor’s vessel was made of cor­
rosion-resistant nickel-chromium-molybdenum wrought
alloy (Hastelloy C276). Pretreated bagasse was recovered in a
140-L Nutsche filter (Pope Scientific Inc., USA) made in
Hastelloy C22. The solid fraction, rich in cellulose and lignin
(cellulignin), was transferred to a basket centrifuge VTC 400/
200 ha (Ferrum, CH) with capacity of 12.8-L. Water was in­
jected in the basket for washing the cellulignin until the
outlet water reached stable value of pH and %brix. The cel­
lulignin generated by three pretreatment campaigns was
transferred to the 350-L stirred reactor (Pope Scientific Inc.,
USA) for the enzymatic hydrolysis procedure. That was per­
formed at 50 °C, pH of 4.8, adjusted with 2 M H2SO4 and 2 M
NaOH, during 72 h, 10% (w/v) of solid loading, and 10 FPU/mL
of cellulolytic enzymes Cellic® Ctec2 (Novozymes, DK). After
completing the hydrolysis, the liquid fraction (cellulosic hy­
drolysate) was recovered in a 140-L Nutsche filter (Pope
Scientific Inc., USA) and concentrated in a wiped film eva­
porator (Pope Scientific Inc., USA) at 80 °C and 475 mbar. The
hydrolysate concentration was done until it reached a sugars
concentration of 212 g/L that corresponded to the equivalent
of total sugars, in term of reducing sugars, to obtain a final
ethanol concentration of 7.5°GL (59.2 g/L) in a configuration
of an industrial fed batch at 90% of product yield (Basso
et al., 2010).
2.2. Substrate preparation
The obtained concentrated hydrolysate was conditioned as a
substrate for fermentation. The conditioning steps and the
substrate formulation were done in the same manner as
previous work (Yamakawa et al., 2016) to meet the nutrient
requirement for the yeast’s maintenance. The conditioning
step, called clarification, removes particles and proteins. This
570 Chemical Engineering Research and Design 196 (2023) 568–576

industrial procedure benefits yeast cell recycling by avoiding
obstruction of centrifuges nozzles and the fermentation itself
by minimizing the foaming formation due to the presence of
protein, and minimizing the cell yield. Clarification treat­
ment relies on the pH drop with phosphoric acid, followed by
pH arising with lime and particle aggregation by adding non-
ionic polymer. The clarification treatment of concentrated
hydrolysate was performed in a stainless steel 19.5 L-Bior­
eactor (New Brunswick Scientific, USA) with the addition of
0.4 mL/L of phosphoric acid (85%) followed by titration with
78 g/L calcium oxide solution until pH of 6.4 and heating up
to 95 °C. Afterwards, the temperature was immediately de­
creased to 25 °C, and 4 ppm of non-ionic polyacrylamide was
added. After 10 min, the hydrolysate was centrifuged in a
basket centrifuge VTC 400/200 ha (Ferrum, CH). The hydro­
lysate was distributed in 2-L Erlenmeyer flasks for steriliza­
tion in an autoclave AV-100 Plus (Phoenix, BR) at 121 °C for
15 min. The fermentation substrate was formulated with
sterile hydrolysate and 8%wt of sugarcane molasses. Su­
garcane molasses support the nutrient demand for the
yeast’s maintenance during fermentation with cell recycling
(Yamakawa et al., 2016).
2.3. Microorganism, inoculum, and yeast propagation
An isolated strain of yeast Saccharomyces cerevisiae originally
obtained from Santa Adélia sugarcane mill (Jaboticabal, São
Paulo, Brazil) was used in this work. The stock culture was
maintained in YPD (10 g/L yeast extract, 20 g/L peptone and
20 g/L dextrose) at - 80 °C with 30% (v/v) glycerol. A pre-cul­
ture was prepared by transferring the stock culture to a 500-
mL Erlenmeyer flask containing 100 mL of YPD broth fol­
lowed by incubation at 33 °C and 250 rpm. After 24 h, an
aliquot of pre-culture was transferred to 100 mL of inoculum
medium in a 500-mL Erlenmeyer flask. Inoculum medium
was prepared with 5.0 g/L of (NH4)2HPO4 and 80 g/L of sugars.
The source of sugars corresponded to a mixture of sugarcane
juice and molasses in a proportion of 79%wt of sugarcane
juice and 21%wt in terms of reducing sugars (Rivera et al.,
2017). The inoculum was incubated in the same conditions as
the pre-culture. After 12 h, cells were recovered in the high-
performance centrifuge Avanti J-26 XP (Beckman Coulter,
USA) with rotor JLA-16.250 at 5509 x g, 10 °C for 15 min. The
pellet was diluted with sterile tap water and transferred to
the 7.5-L bioreactor (New Brunswick, USA) for yeast propa­
gation to obtain the biomass needed for fermentation at high
cell density. The yeast propagation was conducted in the
same manner as previous work (Rivera et al., 2017) but using
a carbon source of a mixture of sugarcane juice and molasses
as cited previously.
2.4. Ethanol fermentation
Ten repeated fermentations in fed-batch were performed in
a 2-L Bioflo 115 bioreactor (New Brunswick, USA) at 33 °C,
200 rpm, without any gas sparging. The first fed-batch was
done by adjusting the cell concentration at 70 g/L (dry basis)
in sterile tap water. The final weight corresponded to 600 g
and was transferred to the bioreactor. The substrate for
feeding corresponded to 1400 g at a concentration of 195 g/L
of sugars. After completing each fermentation, yeast cells
were recovered by centrifugation in a centrifuge Avanti J-26
XP (Beckman Coulter, EUA) at 13,225 x g, 15 min, and 20 °C.
The supernatant was kept refrigerated for further distilla­
tion. The cells pellet was resuspended in sterile tap water,
and its concentration was adjusted to 70 g/L of cells in a total
of 600 g. Then, the cell suspension was transferred back to
the bioreactor’s vessel to perform acidic yeast treatment
before new fed-batch fermentation. The yeast treatment was
performed at 33 °C, 800 rpm, and 0.2 L/min of air, followed by
pH adjustment at 2.5 with 2 M sulfuric acid. After 30 min of
acidic treatment, the air supplier was cut off, the stirrer was
adjusted at 200 rpm, and the substrate feeding started. These
3 stages of fermentation are shown in Fig. 1.
2.5. Distillation
All supernatants recovered after centrifugation of the fer­
mented broth were combined for ethanol recovery in a batch
distillation system Autodest® 800AC (iFISCHER, DE) con­
trolled by Indusoft Web studioTM v6.216. The distillation
column system comprehended a column packed with Pro-
Pak® of 6 mm in 316 SS. The column was equipped with a
silvered vacuum mantle and a thermometer for temperature
control. The temperature set-point was adjusted to 78 °C. The
reboiler was equipped with an external mantle and two
temperature sensors. Temperature set-points were adjusted
at 100 °C (top jacket) and 170 °C (bottom jacket). A cryostat
with a capacity of 360 Kcal at 0 °C was used in the condenser.
The reflux ratio of 3 was adopted. This batch distillation
system is equivalent to a column of 15 theoretical plates
(ASTM D 2892). The distillate streams were collected at 5 time
points between 38 °C and 80 °C.

Fig. 1 – Photos of cellulosic ethanol fermentation with cell recycling in 2-L bioreactor in fed-batch with cell recycling during
different steps a) Step of yeast cell treatment for 0.5 h, followed immediately by b) step of substrate feeding distributed in 6 h,
and followed by final c) step of sugars depletion for 2 h.
 Chemical Engineering Research and Design 196 (2023) 568–576
2.6. Analytical methods
2.6.1. Feedstock composition, fermentation substrate and
metabolites
The feedstock was characterized according to NREL’s stan­
dard procedure (Hames et al., 2008; Sluiter et al., 2012, 2008).
The composition of hydrolysate and the fermentation me­
tabolites were analyzed by high-performance liquid chro­
matography (HPLC). Sucrose, glucose, fructose, and xylose
were determined in an Agilent Infinity 1260 with IR detector
at 50 °C, Aminex column HPX-87 P 300 mm × 7.8 mm at 60 °C
and 0. 5 mL/min of ultrapure Milli-Q water as eluent phase.
Acetic acid, ethanol, formic acid, and glycerol were de­
termined in a Dionex Ultimate 3000 with IR detector Shodex
RI-101, Aminex column HPX-87 H 300 mm × 7.8 mm at 50 °C
and 0.5 mL/min of 5 mM sulfuric acid as eluent phase. Fur­
fural and 5-HMF were determined in an Agilent Infinity 1260
with UV detector at 274 nm, Acclaim 120 - C18 150 × 4.8 mm
at 25 °C and 0.8 mL/ min of acetonitrile with water (1:8) with
8% of acetic acid as eluent phase.
2.6.2. Fermentation volatile metabolites and characterization
of ethanol
The composition of distillate products was quantified by gas
chromatography (GC) in a GC-2010 Shimadzu equipment and
following the European Standard EN 15721. The distilled
streams were characterized by color (ASTM D 1209); acidic
(ABNT NBR 16047) using a potentiometric titration KEM
(Kyoto Electronics); electrical conductivity at 25 °C (ABNT
NBR 10547) using Orion Star conductivity meter; sodium
content (ABNT NBR 10422); permanganate test (ASTM D
1363); sulfur content (ASTM D 5453).
2.6.3. Vinasse characterization
The bottom product (vinasse) was characterized according to
the Standard Method for the Examination of Water and
Wastewater (SMEWW) 22nd edition.
2.6.4. Yeast concentration, viability, and budding
The cell concentration in dry basis of the initial and final
samples of each fermentation was determined gravime­
trically in triplicate after centrifuging 1 mL of samples,
washing the cell pellets twice with deionized water, and
drying it at 80 °C until a constant weight was obtained in an
analytical scale. Yeast cell viability and budding were quan­
tified by classical staining technique with methylene blue
(Lee et al., 1981) and counted using a Neubauer chamber in
an optical microscope Eclipse CI-S (Nikon, JP).
2.6.5. Fermentation monitoring
During the course of fermentation, sugars and ethanol were
monitored in real-time, soon after the sampling, in a mid-
infrared spectrometer (Bruker Optics Inc., USA) that had been
calibrated accordingly (Rivera et al., 2017). Cell concentration
was monitored by optical density at 600 nm in a spectro­
photometer using water as a blank. Also, total soluble solids
in %brix were measured using an optical refractometer
(Atago, JP).
2.6.6. Fermentation performance parameters
The parameters of fermentation performance were calcu­
lated based on the weight of the transferred mass of the cell
suspension and substrate to the bioreactor, and the mass of
fermented broth at the end of each fed-batch. Substrate to
571
cell conversion yield (YX/S) was obtained as the ratio between
produced cells and total sugars consumed. Sugars corre­
sponded to the sum of glucose and equivalent reducing su­
gars (glucose and fructose) from sucrose. Substrate to
product conversion yield (YP/S) was obtained as the ratio be­
tween the produced ethanol and total sugars consumed. The
volumetric productivity (Qp) was calculated by dividing the
ethanol produced per final volume of the fermented medium
and the total fermentation time.
3. Results and discussion
3.1. Feedstock characterization and hydrolysate
composition
The composition of the lignocellulosic biomass corresponded
to in wt%: cellulose, 38.57 ± 6.23; hemicellulose, 25.10 ± 4.09;
lignin, 28.30 ± 0.97; acetic acid, 2.84 ± 0.36; extractives,
3.13 ± 0.08; ashes, 5.42 ± 0.11. This composition agrees with
the reported values (Canilha et al., 2012; Silva et al., 2011), but
the ash content found in this work was much higher and
justified the need for impurities removal by vibratory sieves.
These mineral impurities can damage equipment such as
pretreatment bioreactor and nozzle centrifuges used for
yeast cell recovery. The moisture content of biomass corre­
sponded to 19.42 ± 0.46%. The composition of the con­
centrated cellulosic hydrolysate corresponded to in g/L:
glucose, 212.29; xylose, 11.69; glycerol, 0.10; acetic acid, 0.72;
HMF, 0.092; furfural; 0.002.
3.2. Ethanol fermentation
The substrate used for ethanol fermentation was composed
in g/L: sucrose, 27.80; glucose, 121.19; fructose, 10.85; xylose,
8.46; acetic acid, 0.53; lactic acid, 4.76; glycerol, 0.15, HMF,
0.10; furfural, 0.05. Sucrose and lactic acid came from su­
garcane molasses; lactic acid bacteria are the most common
contaminants found in sugar and ethanol production site
(Lino et al., 2020). Industrial yeast strain of Saccharomyces
cerevisiae can well ferment sucrose, glucose, and fructose into
ethanol under anaerobic environment. Xylose will end as
residual sugar contributing to the organic matter in the dis­
tillation bottom product. The organic acids and furan deri­
vatives compounds inhibit yeast growth and affect cell
physiology significantly, such as decreasing intracellular pH
and viability (Palmqvist and Hahn-Hägerdal, 2000). However,
the inhibitory effects can be minimized in a fed-batch fer­
mentation where the microorganism will face a low con­
centration of inhibitors. Thus, this primary characterization
of the substrate formulated with lignocellulosic biomass
hydrolysate can give an essential overview to follow the
coming results of ethanol fermentation with cell recycling.
The fermentation profiles of all cycles were combined in a
single graphic as presented in Fig. 2. Cell concentration de­
creased over time due to substrate feeding, whereas ethanol
accumulated resulting in a final ethanol titer of
59.09 ± 2.39 g/L. The initial ethanol value of 17.89 ± 4.07 g/L
corresponded to the ethanol with the cells; ethanol was re­
leased after acidic cell washing. After this step, the substrate
was fed immediately over the washing water. Decreasing
external pH plays an important role in the yeast cell per­
meability (Jones and Greenfield, 1987) that changes the in­
tracellular pH (Charoenbhakdi et al., 2016). Regarding the
sugars, they accumulated up to 12 g/L until 6 h, which was
572 Chemical Engineering Research and Design 196 (2023) 568–576

Table 2 – Fractions of distilled product at different

column heating temperatures and sampling time-point
at 72–80 °C with corresponding volume and ethanol titer.
Fraction Temp. Volume Ethanol titer
(°C) (mL) (%v/v)

1 25–38 110 33.45

2 38–72 524 81.78
3 72–80 440 79.50
4 72–80 500 43.90
5 72–80 120 13.06

lost as residual sugars. Nowadays, on-line measurement

such as mid-infrared spectroscopy combined with real-time
analysis has been applied in several ethanol plants to elevate
the production management for making more accurate de­
cisions (Ethanol Producer Magazine, 2021).
Fig. 2 – Ethanol fermentation profile in fed-batch mode with
Therefore, the downscaling process of ethanol fermenta­
cell recycling: sugars accumulation (black square symbol
tion showed that the operation of ethanol plant using lig­
and line), cells dilution (orange circle symbol and line), and
nocellulosic biomass hydrolysate will deliver similar key
accumulation of produced ethanol (green triangle symbol
parameter indicators. In addition, 8%wt of sugarcane mo­
and line).
lasses were sufficient for cell maintenance and low growth.

kept constant during the last hour to complete the fermen­

tation. The residual sugars corresponded to xylose. More­
over, Table 1 shows the fermentation performance in terms
of cell and ethanol yields, ethanol titer, volumetric product
productivity, and cells viability and budding. The fermenta­
tion conditions benefited the product yield by reaching
0.49 ± 0.02 g/g in contrast to the cell yield of 0.04 ± 0.01 g/g.
Ethanol titer corresponded to 59.09 ± 2.39 g/L or approxi­
mately to 7.5°GL as expected. Industrial yields have been
reported to be 90% of ethanol efficiency and cell yield be­
tween 0.04 and 0.05 g of cell/g of ethanol (Della-Bianca et al.,
2013; Yamakawa et al., 2017). In this work, yields were higher
than typical industrial values, which could be attributed to
the better homogeneous system in a small-scale bioreactor.
In consequence, the volumetric productivity of ethanol
reached a value of 6.96 ± 0.21 g/(L·h), which is higher than
the typical industrial value of 2.6 g/(L·h) (Yamakawa et al.,
2019) for fed-batch operation. In addition, on-line measure­
ment of sugars and ethanol led to more accurate data to
decide the end of the fermentation where all sugars were
exhausted (Rivera et al., 2013; Yamakawa et al., 2019). It
shortens the fermentation time and minimizes the sugars
3.3. Ethanol recovery and characterization
The fermented media free of cells from all performed fer­
mentations totalized 15.3 L that was submitted to distillation
in a batch system. Five fractions were collected from the
initial heating until stabilized at 80 °C. The volume of each
fraction and its ethanol titer are shown in Table 2. The
equivalent amount in absolute ethanol corresponded to
0.94 L. Dividing it by the lignocellulosic biomass gives 12.5 L
ethanol/tonne of bagasse (dry basis), which is far from the
expected value of 150 L ethanol/tonne of bagasse in the sce­
nario of 90% efficiency for enzymatic hydrolysis (Yamakawa,
2016). In this work, very low enzymatic hydrolysis conversion
was obtained due to the ineffective mixture of solids and
enzymes in a conventional stirrer reactor. Customized re­
actors and operation strategies have been developed to
maximize enzymatic hydrolysis of lignocellulosic biomass,
for instance, membrane reactors and novel continuous mode
operation (Andrić et al., 2010; Lischeske and Stickel, 2019;
Narendranath, 2015; Olivieri et al., 2021). About the fractions
collected during the distillation, Fig. 3 shows the appearance

Table 1 – Fermentation performance parameters of 10 repeated fed-batch cycles with cell recycling in term of cell yield
(YX/S), product yield (YP/S), final ethanol titer, volumetric productivity (QP), percentage of cell viability, and percentage of
cell budding.
Cycle YX/S YP/S Ethanol titer QP Cell viability Cell budding
(g/g) (g/g) (g/L) (g/ (L·h)) (%) (%)

1 0.09 0.47 53.55 6.74 96.61 8.27

2 0.06 0.43 56.74 6.63 98.84 5.66
3 0.07 0.48 59.37 6.88 98.16 2.40
4 0.05 0.51 60.33 6.97 98.82 6.42
5 0.01 0.51 60.04 7.03 93.83 7.56
6 0.04 0.51 61.93 7.36 90.58 8.31
7 0.05 0.50 60.00 7.10 96.00 8.85
8 0.02 0.49 60.89 7.15 90.62 11.38
9 0.01 0.49 58.65 6.89 94.17 6.46
10 0.01 0.47 59.37 6.91 94.20 6.28
Mean 0.04 0.49 59.09 6.96 95.18 7.16
Deviation 0.01 0.02 2.38 0.21 3.06 2.36
 Chemical Engineering Research and Design 196 (2023) 568–576 573

alcohols can be removed from the rectifier column to prevent

operational difficulties and an inability to produce high
quality ethanol (Rossell et al., 2016).
Characterization of the distilled fractions was done for
Fraction 3 and Fraction 4, as shown in Table 4, following the
standard specification of ANP (National Agency of Petroleum
- Brazilian Agency) for ethanol fuel. Fraction 3 presented
most of the desired characteristics such as total acidic,
electrical conductivity, and pH. The high color index can be
attributed to the hydrolysate itself that contained furfural, 5-
Fig. 3 – Photos of distilled fractions obtained at different HMF, and phenolic compounds, molasses, and byproducts
heating column temperatures and time-point when formed during the pretreatment step, also the sterilization in
reached 72–80 °C, left to right, fraction 1 (25 – 38 °C), 2 autoclave contribute to the formation of color due to de­
(38–72 °C), 3 (72–80 °C), 4 (72–80 °C), and 5 (72–80 °C). gradation of xylose at 121 °C. Clarification treatment applied
on the hydrolysate was not so effective for hydrolysate,
meaning that is necessary to adjust the current method for
Table 3 – Content of volatile compounds in the distilled hydrolysate-based substrate. Clarification process is com­
fractions.
monly applied in the sugar and ethanol processing for re­
Compound Fraction 2 Fraction Fraction Fraction 5 moving colloids particles and soluble proteins. Moreover, the
(mg/kg) 3 (mg/kg) 4 (mg/kg) (mg/kg)
composition of the hydroalcoholic fraction obtained in this
Butanol 65 34.4 3.8 < 10 work was slightly different from previous work that applied
3-Methyl-1- 1086 41.3 5.0 21 sugarcane juice fermentation using the same distillation
butanol system. The reported values (Alvarez et al., 2013) were:
Propanol 772 500 95 28
ethanol, 80% (w/w); 3-Methyl-1-butanol, 1.5% (w/w); acet­
2-Methyl-1- 1960 602 36.6 38
aldehyde, 22.5 mg/L; acetone, 7 mg/L; ethyl acetate, 30 mg/L;
propanol
Acetaldehyde 425 60 31 < 50 propanol, 70 mg/L; 2-Methyl-1-propanol, 325 mg/L; butanol,
Acetal 42 < 10 12.2 < 16 6 mg/L. The content of butanol and propanol of Fraction 3
Acetone 26 < 10 < 10 < 10 and Fraction 4 were pronounced higher compared to this
Ethyl acetate 207 10.5 < 10 < 10 work. Concerning other parameters to assess the quality of
ethanol, sulfur content did not meet the standard specifica­
tion of ANP. Sulfur content is not permitted for ethanol fuel.
of all fractions. Fraction 1 exhibited a whitish appearance Sulfur content originates in fermentation at the stage of
while Fraction 2 presented a yellowish appearance. These acidic cell washing with sulfuric acid. However, in this work,
colors can be attributed to the presence of impurities. an extra addition of sulfuric acid occurred during the titra­
Whereas Fractions 3, 4, and 5 were colorless. tion of enzymatic hydrolysis, and the sulfuric acid added
Regarding the volatile compounds besides ethanol, apart during the pretreatment was removed from the solids after
from Fraction 1, the superior alcohols were quantified intense washing with water. Therefore, adjustments on the
(Table 3). Fraction 2 presented the highest amount of all vo­ distillation system at industrial scale will be expected in
latiles analyzed (butanol, isoamyl alcohol, propanol, isobutyl order to obtain high quality cellulosic ethanol in accordance
alcohol, acetaldehyde, acetal, acetone, and ethyl acetate). with standard specifications. In this work, ethanol recovered
Such by-products mixtures (isoamyl alcohol (3-Methyl-1- by using batch equipment was important to analyze the
butanol), propanol, and isobutyl alcohol (2-Methyl-1-pro­ impact of the new substrate on the final product quality.
panol)) are called higher alcohols or fusel oils that can be
converted into valuable compound such as organic carbo­ 3.4. Vinasse characterization
nates (Bergmann et al., 2018). The excess of amino acids
during fermentation leads to amino acid deamination and Approximately 13.5 L of vinasse was generated as distillation
decarboxylation (Ehrlich Pathway) forming these higher al­ bottom product. Vinasse is an important side stream product
cohols (Walker and Stewart, 2016). Molasses is a source of that is currently used as fertilizer (Rossell et al., 2016), but it
amino acids (Alcantara et al., 2020); also, the cellulolytic en­ can be applied for the production of biogas (Fuess et al.,
zymes broth can contribute to the nitrogen content. Higher 2018). Table 5 presents the vinasse characterization in

Table 4 – Characterization of distilled fractions in comparison to standard specification of ANP

(National Agency of Petroleum - Brazilian Agency) needed to meet the fuel requirements sale.
Parameter Fraction 3 Fraction 4 ANP standard

Color < 20 < 20 5

Total acidic (mg/L) 29.3 51.1 30 (max.)
Electrical conductivity (µS/m) 312 980 300 (max.)
pH 5.6 4.9 6.0–8.0
Permanganate test (min) <1 <1 Not detected
Sodium content (mg/kg) 2.7 0.9 2.0 (max.)
Sulfur content (mg/kg) < 3.5 < 3.5 Not detected
Ethanol (% v/v) 79.5 43.9 92.5 – 94.6
Methanol (%v/v) < 0.03 < 0.03 0.5 (max.)
574 Chemical Engineering Research and Design 196 (2023) 568–576

Table 5 – Distillation bottom product (vinasse) characterization in comparison to vinasse obtained from ethanol
fermentation of cellulosic hydrolysate and sugarcane juice.
Compound/ parameter Value (this work) Value (Petrobras, 2012) Value (Petrobras, 2012)
Cellulosic hydrolysate Cellulosic hydrolysate Sugarcane juice

Calcium (mg/L) 404 ± 2 8–12 130–1540

Cupper (mg/L) 0.024 ± 0.001 - -
Iron (mg/L) 26 ± 1 - -
Magnesium (mg/L) 211 ± 1 16–24 200–490
Manganese (mg/L) 7.315 ± 0.004 - -
Molybdenum (mg/L) 0.230 ± 0.001 - -
Nickel (mg/L) 6.11 ± 0.08 - -
Potassium (mg/L) 1194 ± 1 40–80 1200–2100
Selenium (mg/L) < 0.005 - -
Sodium (mg/L) 10,752 ± 18 - -
Sulfate (mg/L) 24,870 ± 100 44–366 600–760
Zinc (mg/L) 0.500 ± 0.003 - -
Total nitrogen (mg NH3/L) 28.1 ± 0.4 - -
Total nitrogen (mg N/L) 108.60 ± 0.35 205 – 462 150–700
Total phenolic (mg/L) < 0.010 n.d. 0.4 – 12.4
Total phosphorous (mg/L) 128 ± 4 100.5 10–210
Total solids (mg/L) 62,490 ± 86 454 – 5805 23,700
Biochemical oxygen demand (mg/L) 25,049 ± 4 31,500–87,700 6000–16,500
Chemical oxygen demand (mg O2/L) 54,062 ± 26 75,800 – 109,700 15,000–33,000

Note: (-) means not determined and (n.d.) means not detected.

comparison to reported values (Petrobras, 2012) for the dis­
tillation of fermented sugarcane juice and cellulosic hydro­
lysate. The vinasse originated from cellulosic ethanol can be
applied as fertilizer, because the content of phosphorous
(128 ± 4 mg/L) and magnesium (211 ± 1 g/L) was similar to
the current values. Also, it can be applied for biogas pro­
duction, since the quantity of biochemical oxygen demand
was high (25,049 ± 4 mg/L), also higher compared to the
current values (6000 - 16,500 mg/L). However, sodium and
sulfate contents were higher than expected.
CRediT authorship contribution statement
Conceptualization, C.K.Y., R.M.F. and C.E.V.R.; methodology,
C.K.Y., S.T.R. and W.E.H.; investigation, C.K.Y.; data curation,
C.K.Y and S.T.R.; writing-reviewing and editing, C.K.Y.;
R.W.M. and R. M. F.; visualization, C.K.Y.; supervision, R.M.F.
“All authors have read and agreed to the published version of
the manuscript.”
Conflicts of interest

The authors declare no competing interests.

4. Conclusions

The fermentation of cellulosic hydrolysate obtained from

sugarcane bagasse by applying the current technologies was
well succeed with similar industrial performance. The char­
acterization of the distilled products showed discrepancies
compared to the standard values required for fuel, which can
be solved by adjusting the operating parameter to remove all
impurities (volatile compounds). The bottom product pre­
sented similar characteristics of the current vinasse from the
conventional process, except for sodium and sulfate con­
tents. Suitable equipment and operation of enzymatic hy­
drolysis can minimize the use of buffers; also other
substances can be used for pH control. Ideally, generating the
correspondent salt that will be metabolized by the micro­
organism (e.g., ammonium sulfate). However, for ethanol
production, yeast growth must be minimized. In conclusion,
efforts should focus on improving enzymatic hydrolysis,
such as reactors design and process strategies combined
with more online measurements to maximize the solid-li­
quid transference and accurate processing control.
Funding
This work was supported by the State of São Paulo Research
Foundation, FAPESP, Brazil, (grant numbers 2011/51902-9,
2015/20630-4 and 2017/23335-9).
Acknowledgements
This work used facilities at the National Laboratory of
Biorenewables (LNBR), at the National Research Center for
Energy and Materials (CNPEM), a Social Organization su­
pervised by the Ministry of Science, Technology, and
Innovation (MCTI). We thank LNBR/CNPEM and UNICAMP for
providing the infrastructure and sharing expertise.
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