Bioresource Technology 282 (2019) 103–109

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Improving ethanol yields with deacetylated and two-stage pretreated corn T

stover and sugarcane bagasse by blending commercial xylose-fermenting
and wild type Saccharomyces yeast
⁎
Zhaoqin Wanga, Bruce S. Dienb, Kent D. Rauscha, M.E. Tumblesona, Vijay Singha,
a
Department of Agricultural and Biological Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA
b
National Center for Agricultural Utilization Research, USDA, Peoria, IL, USA

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

Keywords: Corn stover and sugarcane bagasse are the most widely available agriculture processing biomass and could serve
Corn stover as feedstocks for production of biofuel. In this study, three different technologies are combined to develop a
Sugarcane bagasse more efficient conversion process for each of these feedstocks. The three technologies are diluted alkaline
Deacetylation deacetylation process, combined thermochemical and mechanical shear pretreatment, and fermentation using a
Bioethanol
combined inoculum of two commercial Saccharomyces yeast strains. The two yeast strains used were a non-GMO
Mixed yeast culture
Two-stage pretreatment
and GMO strain engineered for xylose fermentation. The final ethanol concentrations obtained were 35.7 g/L
from deacetylated corn stover and 32.9 g/L from sugarcane bagasse. Blending the two yeast reduced residual
xylose content from 1.24 g/L to 0.48 g/L and increased ethanol production by 6.5% compared to solely using the
C5/C6 yeast. The optimized yeast blend also lowered the amount of C5/C6 yeast required for inoculation by
80%.

1. Introduction
Cellulosic ethanol has long been sought as a strategy to expand
biofuel production beyond first-generation crops. Presently, most
bioethanol is produced using sugarcane and small grain crops.
Sustainable use of sugarcane bagasse and corn stover (e.g. stalks,
leaves, and cobs) for ethanol production has great promise because
their use would not require re-purposing arable land. Based on esti-
mates from USDA/DOE, 1.3 billion tons of sustainable cellulosic bio-
mass can be grown and collected for biofuel conversion (2016 Billion-
Ton Report: Volume 1 Fact Sheets, Department of Energy). Lig-
nocellulose is primarily composed of cellulose, hemicellulose and
lignin. The complex and recalcitrant nature of the plant cell wall
structure make it challenging to deconstruct and extract fermentable
sugars, which poses a major obstacle to cellulosic ethanol commercia-
lization (Yang and Wyman, 2008). Reducing this recalcitrance requires
processing the biomass under harsh pretreatment conditions prior to
hydrolysis and fermentation (Menon and Rao, 2012).
To reduce recalcitrance, biomass is typically pretreated either singly
or in combination using physical, thermal, and/or chemical steps.
Physical pretreatment methods, which include chopping, grinding and
milling, can effectively reduce the biomass particle size but have
limited effect on improving sugar and ethanol yields (Kim et al., 2016).
Chemical methods such as pretreating with dilute acids or alkalis have
been widely used to produce cellulosic ethanol (Yang et al., 2017), but
their use requires detoxification steps to remove enzyme and microbial
inhibitors generated by the harsh chemical catalysts as well as the use
of expensive acid or base resistant reactors. Dealing with waste acid or
base can also be a concern. Mild pretreatment methods have been
proposed that reduce production of enzyme and microbial inhibitors
and, thereby, eliminate the need for removing inhibitors. Liquid hot
water (LHW) pretreatment is a promising alternative method, where
biomass is pretreated using water alone (200 ± 20 °C) because it re-
duces recalcitrance and incurs minor sugar degradation (Jönsson and
Martín, 2016; Wang et al., 2018). However, compared with the che-
mical pretreatment methods, these mild pretreatment methods result in
lower sugar and ethanol yields (Zhuang et al., 2016; Yang et al., 2017).
Combined pretreatments methods have been widely studied to mi-
tigate the drawbacks of individual pretreatments. One promising ex-
ample is the two-stage pretreatment consisting of LHW pretreatment
followed by mechanical milling, which has been shown to improve
glucose yield from biomass by 34 to 41% compared to LWH pretreat-
ment alone (Hideno et al., 2012; Kim et al., 2016; Wang et al., 2018). In
addition, Chen et al. (2016) reported favorable results for a

⁎
Corresponding author.
E-mail address: vsingh@illinois.edu (V. Singh).

https://doi.org/10.1016/j.biortech.2019.02.123
Received 15 January 2019; Received in revised form 27 February 2019; Accepted 28 February 2019
Available online 01 March 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Z. Wang, et al.
Fig. 1. Process diagram indicating mass loss of overall process.
combination pretreatment method using alkaline deacetylation fol-
lowed by high temperature pretreatment and subsequent two-stage
milling before sugar and ethanol production. The final sugar yield and
ethanol production reached more than 10% v/v when fermented at
28% solids loading (ibid). Deacetylation is a bio/chemical treatment
that uses dilute alkaline or acetyl xylan esterase to free acetyl groups
from the xylan backbone, which hinder the glycosidic bonds accessi-
bility by cellulases. Deacetylation with a mild NaOH solution frees
acetyl groups from the xylan backbone before pretreatment, thus re-
sulting in increase in biomass specific surface area (SSA) and xylan
hydrolysis yield (Chen et al., 2012a; Chen et al., 2012b; Jiang et al.,
2016).
Native Saccharomyces cerevisiae yeast strains only ferment hexose
(C6) sugars (e.g. glucose), but cellulosic materials are rich in both C6
and pentose (C5) sugars. Native yeast species that can ferment xylose
have shortcomings that have limited their commercialization
(Schneider et al., 1981; Jeffries, 1982; Jeffries and Jin, 2004). There-
fore, researchers have sought to engineer Saccharomyces and other
yeasts to ferment hexose and pentose sugars into bioethanol. Strategies
have included expressing a xylose metabolic pathway (Jeffries and Jin,
2004, Wang et al., 2016) and xylose cell membrane transporters (Apel
et al., 2016). This research resulted in engineered yeast strains for
bioethanol production that have been successfully evaluated in small
pilot scale fermentations (Lian et al., 2018; Lane et al., 2018). However,
xylose fermentation appears to be partially inhibited in the presence of
glucose even though the former is not a natural substrate for Sacchar-
omyces (Azhar et al., 2017). Combining conventional and engineered
yeast might circumvent glucose uptake repression and glucose/xylose
metabolic pathway competition in the C5/C6 yeast. To date, however,
no previous studies have addressed the possibility of this combination
strategy in cellulosic ethanol production.
In this study, corn stover and sugarcane bagasse were used to study
the impact of adding a step for removal of acetyl groups prior to two-
stage pretreatment (LHW followed by disk milling) on the sugar pro-
duction yield. Sugarcane bagasse and corn stover were chosen because
they are readily available to first generation ethanol production facil-
ities. We also evaluated and compared ethanol production yields for
blends of commercial GMO xylose-fermenting and traditional
Saccharomyces yeast.
2. Material and methods
2.1. Feedstock
Corn stover and sugarcane bagasse were used for this study. Corn
104
Bioresource Technology 282 (2019) 103–109
stover was harvested in 2012 from the University of Illinois South Farm
(Urbana, IL) and dried sugarcane bagasse (harvested in 2013,
Patoutville, LA) was obtained from the Energy Bioscience Institute at
University of Illinois (Urbana, IL). Corn stover samples were oven dried
to a moisture content less than 5% and hammer milled (model W-8-H,
Schutte-Buffalo Hammermill, Buffalo, NY) with a 2.38 mm sieve.
Bagasse samples were milled (model MHM4, Glen Mills, Clifton, NJ)
with a 2.0 mm sieve. Ground stover and bagasse samples were stored in
Ziploc bags at 4 °C.
2.2. Deacetylation and washing
Ground biomass samples were first deacetylated. Fifty grams of dry
biomass was mixed with 0.1 M NaOH at 1:9 solids:liquid ratio (at a 10%
w/w solids loading), and the mixture was heated in a water bath to
80 °C and incubated for 3 h with continuous agitation at 70 rpm. Three
replicates were performed for both corn stover and sugarcane bagasse.
After deacetylation, biomass samples were washed with DI water and
filtered through Whatman glass microfiber filters for acetyl group re-
moval and slurry neutralization. Washed biomass samples were col-
lected and dried at 49 °C overnight and stored in plastic bags at 4 °C.
Process flow and mass balances are reported in Fig. 1.
2.3. Liquid hot water (LHW) pretreatment and wet disk milling
Pretreatment and wet disk milling were performed as described
previously (Wang et al., 2018). Briefly, biomass samples were mixed
with DI water at a ratio of 1:4 and loaded into stainless steel reactors
(316 stainless with 4.125″ (10.478 cm) length × 0.75″ (1.905 cm) outer
diameter × 0.065″ (0.165 cm)) (SS-T12-S-065-20, Swagelok, Chicago
Fluid system Technologies, Chicago, IL). Capped reactors were im-
mersed in a fluidized sand bath (IFB-51 Industrial Fluidized Bath,
Techne Inc., Burlington, NJ) at a preset reaction temperature of 180 °C.
The in situ reaction temperature was recorded once per second using a
thermocouple (Penetration/Immersion Thermocouple Probe Mini Conn
(−418 to 1652F), McMaster-Carr, Robbinsville, NJ.) inserted into the
reactor and connected to a data logger (HH306/306A, Datalogger
Thermometer, Omega, Stamford, CT). Reactors were heated to the
target temperature within 5 min. After 10 min at reaction temperature,
reactors were transferred to a water-bath to quench the reaction.
Disk milling processes were performed immediately after pretreat-
ment on the whole slurry. LHW pretreated wet samples were disk
milled using an electrical powered disc mill (model 4E, Quaker city
grinding mill, Straub Co., Philadelphia, PA) with an output speed of
89 rpm. The setting for the distance between the stationary and rotating
Z. Wang, et al. Bioresource Technology 282 (2019) 103–109

plates was set to zero and biomass samples were sequentially milled 3
times. For each pretreatment three replicates were performed.
2.4. Enzymatic hydrolysis
The enzymatic hydrolysis was performed to analyze the sugar re-
covery from processed biomass following NREL/TP-5100-63351 pro-
cedures. Raw and processed (deacetylated only or deacetylated, pre-
treated and disk milled) substrates were transferred to pre-sterilized
polypropylene tubes (Corning® CLS430829). The pH was adjusted by
adding sodium citrate buffer (1 M citrate buffer, pH 4.5) at 50 mM and
solids to 10% w/w by adding dH2O. Finally, cellulases and hemi-
cellulases were added to initiate hydrolysis. The cellulases and hemi-
cellulases formulations used were cellulase Cellic® Ctec2 (protein con-
tent: 76.3 mg/mL by BSA analysis) (Novozymes North America, Inc.,
Franklinton, NC, USA) at 0.215 ml/g dry substrate (30 FPU/g biomass)
and hemicellulase NS 22,244 (protein content: 72.80 mg/mL by BSA
analysis) (Novozymes North America, Inc., Franklinton, NC, USA) at a
quarter of the cellulase dose (0.054 ml/g dry substrate). Enzymatic
hydrolysis was conducted at 50 °C with 120 rpm constant shaking for
72 h using a shaker/incubator. Background sugars were accounted for
using enzyme controls (e.g. reactions without added biomass). Sugar
content after 72 h hydrolysis were measured by HPLC and recovery
yield were calculated as reported by Wyman et al. (2005). Enzymatic
digestions were conducted in triplicate.
2.5. Simultaneously saccharification and co-fermentation (SScF)
Two Saccharomyces cerevisiae yeast strains were used to ferment
biomass in this study: Ethanol Red™ and a genetically modified C5/C6
yeast. Ethanol Red (ER) yeast (Lesaffre yeast corporation, Milwaukee,
WI) is an industrial yeast used for corn ethanol fermentations (Chen
et al., 2013). The C5/C6 yeast M11205 (Lallemand Inc., Milwaukee,
WI) is genetically modified to simultaneously ferment glucose and xy-
lose. The ER yeast culture were prepared by mixing 5 g dry yeast with
25 ml deionized water and incubating in water bath set to 32 °C while
agitating at 110 rpm for 25 min (Chen et al., 2013). The OD600nm was
measured (Evolution 60S spectrophotometer, Thermo Scientific, Wal-
tham MA) and diluted to a OD600nm value of 50 by adding sterile DI
water. The C5/C6 yeast seed culture was prepared as described by
Wang et al. (2018). The C5/C6 yeast culture was adjusted to an
OD600nm value of 50 as well before biomass SScF. The prepared ER and
C5/C6 culture were mixed at target ratios of (1:0 (ER only), 5:1, 1:1 and
0:1(C5/C6 only)) before inoculating the SScF cultures.
The co-fermentation of deacetylated, LHW pretreated and disk
milled biomass samples were all carried under sterile conditions. The
biomass samples were adjusted to pH 4.5 by adding sodium citrate
buffer (50 mM, pH 4.5), supplemented with YP (2% bactopeptone and
1% yeast extract) and diluted to 10% w/w solids with dH2O. SScF was
initiated by adding the same enzyme doses used for the enzymatic
hydrolysis experiments and yeast to a beginning OD of 1.5. Cultures
were fermented for 96 h in a shaker/incubator set to 35 °C and 160 rpm
shaking. Fermentations were sampled at 3, 6, 9, 24, 48, 72 and 96 h and
sugars and ethanol measured using HPLC (Bio-Rad Aminex HPX-87H,
Biorad, Hercules, CA). Fermentation controls were conducted without
added biomass to account for background ethanol levels. Each experi-
ment was run in triplicate.
2.6. Analytical methods
Similar as previous study (Wang et al., 2018), chemical components
of raw and deacetylated biomass were determined via two-stage acid
hydrolysis procedure according to laboratory analytical procedure for
biomass analysis from National Renewable Energy Laboratory (NREL).
Extractives were removed and measured by both deionized water and
ethanol extraction using Soxhelt approach based on NREL/TP-510-
42619 (2008). Two-step acid hydrolysis was performed following
NREL/TP-510-42618 (2012). Extracted biomass was hydrolyzed with
72% sulfuric acid for 1 h at 30 °C. The mixture was diluted to a 4% acid
concentration by adding deionized water and autoclaved at 121 °C for
1 h. After two-step acid hydrolysis, hydrolysates were vacuum filtered
by filter crucible. Filtrates were used to measure sugar concentration
and acetic acid by HPLC (Bio-Rad Aminex HPX-87H, Biorad, Hercules,
CA). Acid soluble lignin (ASL) content were determined by measuring
filtrate absorbance at 205 nm using spectrophotometer. Remaining so-
lids in crucibles were further analyzed for acid insoluble lignin (AIL)
and ash contents by being dried at 105 °C for 24 h and followed by
575 °C ± 25 °C for 4 h according to NREL/TP-510-42622 (2008).
Deacetylated and washed biomass samples were analyzed for carbo-
hydrates, acetyl group, lignin and ash contents using the protocol de-
scribed above without water/ethanol extractions.
Liquid samples were collected immediately after pretreatment and
used to determine concentrations of organic acid (acetate) and other
inhibitors (furans). Liquid samples were obtained by centrifugation of
pretreated biomass at 15,000×g for 45 min and analyzed by HPLC (Bio-
Rad Aminex HPX-87H, Biorad, Hercules, CA).
HPLC samples used to measure sugar and ethanol analysis were
centrifuged at 9000×g for 5 min and supernatants filtered through
0.2 μm PTFE filters before HPLC analysis. HPLC (Bio-Rad Aminex HPX-
87H, Biorad, Hercules, CA) was used to determine monosaccharide and
ethanol concentrations. Replicate analyses were performed on every
HPLC sample and the detection limit was 0.001% w/v.
3. Results and discussion
3.1. Effect of deacetylation on compositional changes in biomass material
Chemical compositions of corn stover and sugarcane bagasse before
and after deacetylation treatment are listed in Table 1. Sugarcane ba-
gasse contained less extractives than corn stover but higher glucan and
xylan contents; however, corn stover had higher acetyl groups than
bagasse. The initial acetyl contents were 2.16% in bagasse and 2.75% in
stover. Similarly, Chen et al. (2011) reported 2.87% acetyl content
presented in raw corn stover and Rabelo et al. (2009) reported 2.4 to

Table 1
Compositions (% w/w) of raw, deacetylated biomass.a
Sample Mass recoveryb (%) Extractives (%) Glucan (%) Xylan (%) AILc (%) ASLd (%) Ashe (%) Acetyl group (%)

Raw bagasse NA 7.82 ± 0.88 37.31 ± 1.72 20.40 ± 2.61 18.18 ± 0.48 2.22 ± 0.04 3.65 ± 0.47 2.16 ± 0.18
Raw corn stover NA 11.84 ± 0.62 31.33 ± 0.91 16.53 ± 0.14 14.10 ± 0.35 2.48 ± 0.02 3.04 ± 0.05 2.75 ± 0.01
Deacetylated bagasse 87.63 ± 0.48 NA 34.40 ± 0.88 15.88 ± 1.32 17.60 ± 1.91 1.92 ± 0.08 3.02 ± 0.36 0.74 ± 0.02
Deacetylated stover 84.03 ± 0.89 NA 33.46 ± 1.49 17.93 ± 1.73 13.62 ± 0.25 2.28 ± 0.37 3.63 ± 0.55 1.11 ± 0.02

a
Mean ± standard deviation; biomass composition is on a dry basis.
b
Mass recovery rate following DEA step.
c
AIL: Acid insoluble lignin (Klason lignin).
d
ASL: Acid soluble lignin.
e
Ash: Acid insoluble ash measured after combusting AIL samples in a muffle furnace.

105
Z. Wang, et al.
2.5% acetyl content in bagasse.
Compositions of bagasse and stover were measured following dea-
cetylation. Biomass extracted during this pre-process were also tracked
by using a mass balance. For corn stover and bagasse, mass losses were
16.9% and 12.5%, respectively, following deacetylation. Deacetylation
was effective for removal of acetyl side groups: acetyl content decreased
70.0 ± 0.8% and 66.1 ± 2.7% in bagasse and corn stover, respec-
tively. Chen et al. (2011) reported removal of 65.9% of the acetyl
groups in corn stover using the same experimental conditions. Lignin
content decreased in both biomass samples including acid insoluble
lignin (AIS) and acid soluble lignin (ASL). Zhu et al. (2008) reported
decrease lignin contents with deacetylation by dilute alkali, pre-
sumably, lignin groups disrupted during deacetylation are removed
during the washing step. In deacetylated bagasse samples, glucan and
xylan contents decreased: 19.2% of the glucan and 31.9% of the xylan
present in the initial bagasse sample was removed during deacetylation
and washing. These results indicate that sugar loss occurs during dea-
cetylation of bagasse and would affect overall conversion efficiency. In
contrast, relative glucose and xylose contents increased in deacetylated
corn stover compared with raw material. Chen et al. (2012a,b) ob-
served a slight increase in carbohydrate contents (3.6% in glucose and
1.6% in xylose) after deacetylation. While glucan and xylan con-
centrations were enriched through deacetylation, glucan and xylan
contents relative to the beginning sample decreased 7.6% and 8.8%
respectively. Deacetylation with 0.1 M NaOH at 80 °C for 3 h success-
fully removed two thirds of the acetyl group from either biomass and
co-extraction of glucan and xylan was worse for bagasse versus stover.
3.2. Effect of deacetylation on inhibitor formations during LHW
pretreatment
Inhibitor concentrations (i.e. acetic acid, formic acid, levulinic acid,
hydroxymethylfurfural (HMF) and furfural) measured for pretreated
samples w/o deacetylation treatment are summarized in Table 2. The
most common inhibitors produced during LHW pretreatment are acetic
acid, levulinic acid, and furans (Hu and Ragauskas, 2012; Jönsson and
Martín, 2016). For both treatments, neither HMF or levulinic acid was
detected, indicating that glucose degradation was minimal during LHW
pretreatment of raw and deacetylated biomass. Deacetylation was ef-
fective at reducing acetic acid concentrations for biomass hydrolysates.
For corn stover hydrolysates including a deacetylation step reduced
acetic acid concentration from 1.94 g/L to 0.539 g/L and for bagasse
from 1.15 g/l to 0.291 g/L. This was expected because the deacetylation
step removed two-thirds of the acetyl groups (Table 1). Lower acetic
acid concentrations in the hydrolysate might potentially relieve acetate
inhibition of yeast fermentations (Larsson et al., 1999). Furfural, and to
lesser extent formic acid, is formed from degradation of released ara-
binose and xylose (Kim et al., 2016). Amounts of furfural and formic
acid are indicators of the extent of xylose loss. Inhibitor concentrations
show that raw biomass produced more furfural and formic acid than
deacetylated samples for both sugarcane bagasse and corn stover,
which is indicative that xylan degradation during LHW pretreatment
were reduced when deacetylation was performed on biomass materials
prior to pretreatment.
Table 2
Inhibitor concentrationsa of liquid hot water pretreated hydrolysates.
Pretreatment Substrate Formic Acid (g/L) Acetic Acid (g/L)
Raw bagasse 0.234 ± 0.013 1.151
Raw corn stover 0.543 ± 0.109 1.942
Deacetylated bagasse 0.286 ± 0.122 0.291
Deacetylated stover 0.273 ± 0.078 0.539
a
Mean ± standard deviation.
b
HMF: 5-Hydroxymethylfurfural.
c
BDL: Below HPLC detection limit (0.001% w/w).
Bioresource Technology 282 (2019) 103–109
Fig. 2. Overall sugar recovery yields (A: glucose; B: xylose) from 72 h enzy-
matic hydrolysis. Data label represents the final sugar content in hydrolyzate.
DEA: deacetylated only biomass; DEA + P + DM: deacetylated, LHW pre-
treated (P) and disk milled (DM) biomass sample.
3.3. Sugar recovery from enzymatic hydrolysis
Sugar yields from enzymatic hydrolysis were compared as follows:
no treatment (e.g. raw biomass); deacetylated (DEA); and deacetylated,
LHW (180 °C) pretreated, and disk milled (DEA + P + DM). Glucose
and xylose recovery yields (Fig. 2) are based on raw biomass values
(Table 2) and final sugar concentrations (after 72 h hydrolysis) and are
reported on top of plotted bars (Fig. 2).
Glucose recoveries for pretreated biomass (DEA + P + DM) were
very high: 92.2% for corn stover and 80.3% for bagasse and glucose
concentrations were 44.4 and 40.4 g/l, respectively. Kim et al. (2016)
reported a glucose recovery of 79.1% from corn stover after LHW
pretreatment followed by disk milling. Chen et al. (2011) reported si-
milar findings. Therefore, the glucose yield for corn stover was in-
creased by 16.5% by deacetylating the biomass prior to pretreatment.
Glucose recovery from sugarcane bagasse was reported to be only
78–83% for a two-stage chemical and milling type pretreatment (de
Barros et al., 2013; Wang et al., 2018). The reduced overall yield was
mainly due to sugar lost during washing, however, including a DEA step
did increase the final glucose concentration following enzymatic hy-
drolysis from 37.8 g/L (Wang et al., 2018) to 40.01 g/L (Fig. 2A).
However, for unpretreated biomass, DEA reduced glucose yields for
bagasse and stover compared to raw biomass samples (Fig. 2A), which
most likely reflects sugar losses incurred from the DEA and washing
steps.
Including a DEA step improved overall xylan hydrolysis recoveries
(Fig. 2B) for corn stover and sugarcane bagasse. Xylose recovery yields
were 12.6% from raw bagasse and 18.7% from raw stover and in-
creased to 20.1% for deacetylated bagasse and 23.1% for deacetylated
stover. Xylose yields from deacetylated plus LHW pretreated and disk
milled samples were 75.3% for corn stover and 63.5% for bagasse.
Levulinic Acid (g/L) HMFb (g/L) Furfural (g/L)
c
± 0.004 BDL BDL 0.215 ± 0.006
± 0.036 BDL BDL 0.411 ± 0.020
± 0.020 BDL BDL BDL
± 0.067 BDL BDL BDL
106
Z. Wang, et al. Bioresource Technology 282 (2019) 103–109

Ethanol
A B G
Glucose
4 3 4 % (3) 3
Xylose

3 3

2 2

Ethanol (% w/v)
Ethanol (% w/v)

Sugar (% w/v)

Sugar (% w/v)
2 2

1 1

1 1

0 0 0 0
0 24 48 72 96 0 24 48 72 96
Fermentation Time (hr) Fermentation Time (hr)
Fig. 3. Ethanol and glucose/xylose profiles of deacetylated and two-stage pretreated corn stover (A) and bagasse (B) during simultaneously saccharification and
cofermentation. Standard deviations are included in graphs.

Deacetylation is reported to improve xylan hydrolysis by breaking the
hemicellulose backbone into simpler xylo-oligomers with fewer side-
chains, which reduces recalcitrance and increases enzyme accessibility
(Maloney et al., 1985; Conner, 2007; Li and Xu, 2013; Jiang et al.,
2016). Chen et al. (2011) reported a xylose yield of 58% from deace-
tylated and steam explosion pretreated corn stover. Final xylose con-
centrations for low solids (10% w/w) hydrolyzed biomass achieved
20.4 g/L and 16.4 g/L in corn stover and bagasse, respectively and these
are higher than the 12.8 g/L and 15.2 g/L values reported earlier (Kim
et al., 2016; Wang et al., 2018) for two-stage pretreated (P + DM only)
corn stover and bagasse, respectively. It is also higher than the xylose
concentration (14.5 g/L) reported for corn stover that was deacetylated
and pretreated with dilute-acid but not disc milled (Chen et al., 2011).
Therefore, incorporating deacetylation with a two-stage pretreatment
led to increased glucose and xylose production.
3.4. Co-fermentation of deacetylated biomass by C5/C6 yeast strain
stage pretreated sugarcane bagasse with around 3 g/L xylose un-
fermented in the slurry (Wang et al., 2018). Combining deacetylation
with LHW pretreatment and disk milling decreased unfermented xylose
by 58% for bagasse; however, the residual sugar observed for both
stover and bagasse cultures warrants further experimentation.
Maximum ethanol production rates occurred during the first 24 h
for both stover and bagasse. After 24 h, rates of fermentation were
higher in stover than bagasse culture. The ethanol yields increased
13.2% after 24 h in stover fermentations, while it increased only 4.9%
in bagasse fermentations. Final ethanol titers (96 h) were 3.57% (w/v)
(0.348 g/g of corn stover) and 3.29% (w/v) (0.343 g/g of bagasse).
Ethanol yields from two-stage pretreated corn stover and bagasse
without DEA were reported to be 2.91% and 3.34%, respectively (Kim
et al., 2016; Wang et al., 2018). Incorporating deacetylation improved
ethanol yield for stover and (slightly) worsened yield for bagasse. The
decrease in ethanol yield for bagasse is probably due to losses of glucan
and xylan during deacetylation and washing as discussed above.

Pretreated bagasse and stover (DEA + P + DM) were fermented
using a commercial C5/C6 yeast strain to evaluate ethanol production
(Fig. 3). Glucose concentrations were maximum at the beginning of
fermentation (3 h) and declined to nearly zero by 9 h. Maximum glu-
cose concentrations were 12.1 g/L in stover and 16.6 g/L in bagasse.
However, high glucose concentrations inhibit cellulase (Himmel et al.,
2007; Kristensen et al., 2009; Teugjas and Väljamäe, 2013), which is
undesired for efficient enzyme usage and bioethanol production in
SScF. Ideally glucose should be fermented to ethanol as readily as it is
released by the cellulase enzymes. After 9 h, glucose concentrations
remained low but increased slightly (from 0.5% to 0.7% in stover and
0.3–0.4% in bagasse) near the end of fermentations.
Xylose was consumed more slowly than glucose. Maximum xylose
concentrations were observed at 6 h and were 9.29 g/L for stover and
9.42 g/L for bagasse and by 9 h xylose concentration had fallen near
zero for both fermentations. These differences in consumption rates
suggest that there might be a time lag between fermentation of glucose
and xylose because of xylose transport limitations (Sedlak and Ho,
2004; Apel et al., 2016). Nevertheless, similar to glucose concentration
profiles, xylose concentration begins to increase towards the end of
SScF and rose to 1.91 g/L in corn stover and 1.24 g/L in bagasse after
96 h. A previous study observed this increase in sugar content for two-
3.5. Bagasse co-fermentation by binary yeast culture
Traditional industrial Saccharomyces yeast used for production of
ethanol only ferment hexoses (Azhar et al., 2017). The availability of
commercial Saccharomyces strains for fermentation of xylose is a recent
phenomenon, albeit achieved through decades of concentrated research
effort. It is possible that combining a traditional Saccharomyces with
one engineered specifically for xylose fermentation might lead to a
more efficient fermentation than using a pure culture. To test for this
possibility, fermentations using two different blends of traditional (e.g.
C6 yeast) and C5/C6 Saccharomyces were compared to fermentations
solely inoculated with either C6 or C6/C5 yeast strains (Fig. 4). The
blends of C6:C5/C6 tested were 5:1 (low ratio) and 1:1 (high ratio).
Expectedly, when using C6 yeast alone, there was negligible re-
sidual glucose and xylose was not consumed; and xylose final con-
centration was 17 g/L. Final ethanol concentration was only 2.28% (w/
v). However, by mixing in C5/C6 yeast, final ethanol titers improved by
53.9% and 53.1% for both low ratio (5:1) and high ratio (1:1), re-
spectively. Comparing the two C6:C5/C6 blends, final ethanol pro-
ductions were comparable (3.51% in 5:1 mixture and 3.49% in 1:1
mixture) but initial sugar consumption rates were higher in the high
ratio. For example, 98% of glucose was fermented within 9 h using the

107
Z. Wang, et al.
Fig. 4. Ethanol and glucose/xylose profiles of deacetylated and two-stage pretreated bagasse during simultaneously saccharification and cofermentation by C6 only
(A), low mixing ratio with C6: C5/C6 = 5:1 (B) and high mixing ratio with C6: C5/C6 = 1:1 (C) culture. Standard deviations are included in graphs.
Fig. 5. Unfermented residual glucose/xylose content after 96 h co-fermenta-
tion. Standard deviations are included in graphs.
high ratio, but with low ratio fermentation it took 24 h to consume the
glucose. In addition, at 24 h, 96.1% of the xylose was fermented in the
high ratio culture but only 87.5% with low ratio culture. These results
indicate that blending in less C5/C6 yeast does not decrease final
ethanol yield but does slow the initial rate of fermentation at the be-
ginning. Using a low blend led to less residual sugar compared to the
pure C5/C6 and high blend fermentations.
The unfermented sugars remaining in final fermentation broth are
reported in Fig. 5. Final glucose content was 0.35 g/L by C6 yeast,
0.31 g/L by C6: C5/C6 mixed in a ratio of 5:1, 0.35 g/L by C6: C5/C6
mixed in a ratio of 5:1 and 0.40 g/L by C5/C6 yeast culture. Blending in
the C6 yeast reduced the amount of residual xylose. Residual xylose for
unblended C5/C6 fermentation was 1.24 g/l and for the low and high
blends it was 0.48 and 0.67 g/l, respectively. One explanation is that
competition between glucose and xylose consumption in C5/C6 yeast
was reduced when glucose was removed by including C6 yeast, hence
promoting xylose utilization.
4. Conclusion
Deacetylation effectively removed 66% of acetyl groups present in
corn stover and sugarcane bagasse. The combination of deacetylation
with two-stage pretreatment decreased acetic acid formation during
pretreatment, resulting in increased final glucose/xylose content from
108
Bioresource Technology 282 (2019) 103–109
enzymatic hydrolysis. However, sugar recovery yields decreased in
bagasse due to the mass loss during deacetylation. Glucose/xylose co-
fermentation of biomass by engineered Saccharomyces cerevisiae effec-
tively fermented the glucose but not all of the xylose. Residual xylose
was eliminated (< 0.05%) by blending a traditional C6 yeast with the
C5/C6 yeast. Using a blend also lowers by 80% the C5/C6 inoculum
needed for fermentation.
Acknowledgments
This work was funded by the DOE Center for Advanced Bioenergy
and Bioproducts Innovation (U.S. Department of Energy, Office of
Science, Office of Biological and Environmental Research under Award
Number DE-SC0018420). Any opinions, findings, and conclusions or
recommendations expressed in this publication are those of the authors
and do not necessarily reflect the views of the U.S. Department of
Energy.
References
Apel, A.R., Ouellet, M., Szmidt-Middleton, H., Keasling, J.D., Mukhopadhyay, A., 2016.
Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization
in Saccharomyces cerevisiae. Sci. Rep. 6, 19512.
Azhar, S.H.M., Abdulla, R., Jambo, S.A., Marbawi, H., Gansau, J.A., Faik, A.A.M.,
Rodrigues, K.F., 2017. Yeasts in sustainable bioethanol production: a review.
Biochem. Biophys. Rep. 10, 52–61.
Chen, X., Shekiro, J., Elander, R., Tucker, M., 2011. Improved xylan hydrolysis of corn
stover by deacetylation with high solids dilute acid pretreatment. Ind. Eng. Chem.
Res. 51 (1), 70–76.
Chen, X., Shekiro, J., Franden, M.A., Wang, W., Zhang, M., Kuhn, E., Johnson, D.K.,
Tucker, M.P., 2012a. The impacts of deacetylation prior to dilute acid pretreatment
on the bioethanol process. Biotechnol. Biofuels 5, 8.
Chen, X., Tao, L., Shekiro, J., Mohaghaghi, A., Decker, S., Wang, W., Smith, H., Park, S.,
Himmel, M.E., Tucker, M., 2012b. Improved ethanol yield and reduced Minimum
Ethanol Selling Price (MESP) by modifying low severity dilute acid pretreatment with
deacetylation and mechanical refining: 1) Experimental. Biotechnol. Biofuels 5, 60.
Chen, M.H., Kaur, P., Dien, B., Below, F., Vincent, M.L., Singh, V., 2013. Use of tropical
maize for bioethanol production. World J. Microbiol. Biotechnol. 29, 1509–1515.
Chen, X., Kuhn, E., Jennings, E.W., Nelson, R., Tao, L., Zhang, M., Tucker, M.P., 2016.
DMR (deacetylation and mechanical refining) processing of corn stover achieves high
monomeric sugar concentrations (230 g L− 1) during enzymatic hydrolysis and high
ethanol concentrations (> 10% v/v) during fermentation without hydrolysate pur-
ification or concentration. Energy Environ. Sci. 9, 1237–1245.
Conner, A.H., 2007. Kinetic modeling of hardwood prehydrolysis. Part I. Xylan removal
by water prehydrolysis. Wood Fiber Sci. 16, 268–277.
de Barros, R.D.R.O., de Sousa Paredes, R., Endo, T., da Silva Bon, E.P., Lee, S.H., 2013.
Association of wet disk milling and ozonolysis as pretreatment for enzymatic sac-
charification of sugarcane bagasse and straw. Bioresour. Technol. 136, 288–294.
Department of Energy, 2016 Billion-Ton Report: Volume 1 Fact Sheets. URL: https://
www.energy.gov/eere/bioenergy/downloads/2016-billion-ton-report-volume-1-fact-
sheets (accessed 11.18.2018).
Hideno, A., Inoue, H., Yanagida, T., Tsukahara, K., Endo, T., Sawayama, S., 2012.
Combination of hot compressed water treatment and wet disk milling for high sugar
recovery yield in enzymatic hydrolysis of rice straw. Bioresour. Technol. 104,
743–748.
Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Foust,
Z. Wang, et al.
T.D., 2007. Biomass recalcitrance: engineering plants and enzymes for biofuels pro-
duction. Science 315, 804–807.
Hu, F., Ragauskas, A., 2012. Pretreatment and lignocellulosic chemistry. Bioenergy Res.
5, 1043–1066.
Jeffries, T.W., 1982. A comparison of Candida tropicalis and Pachysolen tannophilus for
conversion of xylose to ethanol. Biotechnol. Bioeng. Symp. 12, 103–110.
Jeffries, T.W., Jin, Y.S., 2004. Metabolic engineering for improved fermentation of pen-
toses by yeasts. Appl. Microbiol. Biotechnol. 63, 495–509.
Jiang, W., Chang, S., Qu, Y., Zhang, Z., Xu, J., 2016. Changes on structural properties of
biomass pretreated by combined deacetylation with liquid hot water and its effect on
enzymatic hydrolysis. Bioresour. Technol. 220, 448–456.
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.
Kim, S.M., Dien, B.S., Tumbleson, M.E., Rausch, K.D., Singh, V., 2016. Improvement of
sugar yields from corn stover using sequential hot water pretreatment and disk
milling. Bioresour. Technol. 216, 706–713.
Kristensen, J.B., Felby, C., Jørgensen, H., 2009. Yield-determining factors in high-solids
enzymatic hydrolysis of lignocellulose. Biotechnol. Biofuels 2, 11.
Lane, S., Dong, J., Jin, Y.S., 2018. Value-added biotransformation of cellulosic sugars by
engineered Saccharomyces cerevisiae. Bioresour. Technol In press.
Larsson, S., Palmqvist, E., Hahn-Hägerdal, B., Tengborg, C., Stenberg, K., Zacchi, G.,
Nilvebrant, N.O., 1999. The generation of fermentation inhibitors during dilute acid
hydrolysis of softwood. Enzyme Microb. Technol. 24, 151–159.
Li, H., Xu, J., 2013. Optimization of microwave-assisted calcium chloride pretreatment of
corn stover. Bioresour. Technol. 127, 112–118.
Lian, J., Mishra, S., Zhao, H., 2018. Recent advances in metabolic engineering of
Saccharomyces cerevisiae: new tools and their applications. Metab. Eng In press.
Maloney, M.T., Chapman, T.W., Baker, A.J., 1985. Dilute acid hydrolysis of paper birch:
kinetics studies of xylan and acetyl-group hydrolysis. Biotechnol. Bioeng. 27,
355–361.
109
Bioresource Technology 282 (2019) 103–109
Menon, V., Rao, M., 2012. Trends in bioconversion of lignocellulose: biofuels, platform
chemicals & biorefinery concept. Prog. Energy Combust. Sci. 38 (4), 522–550.
Sedlak, M., Ho, N.W., 2004. Characterization of the effectiveness of hexose transporters
for transporting xylose during glucose and xylose co-fermentation by a recombinant
Saccharomyces yeast. Yeast 21, 671–684.
Schneider, H., Wang, P.Y., Chan, Y.K., Maleszka, R., 1981. Conversion of D-xylose into
ethanol by the yeast Pachysolen tannophilus. Biotechnol. Lett. 3 (2), 89–92.
Rabelo, S.C., Maciel Filho, R., Costa, A.C., 2009. Lime pretreatment of sugarcane bagasse
for bioethanol production. Appl. Biochem. Biotechnol. 153, 139–150.
Teugjas, H., Väljamäe, P., 2013. Product inhibition of cellulases studied with 14 C-labeled
cellulose substrates. Biotechnol. Biofuels 6, 104.
Wang, M., Yu, C., Zhao, H., 2016. Directed evolution of xylose specific transporters to
facilitate glucose-xylose co-utilization. Biotechnol. Bioeng. 113 (3), 484–491.
Wang, Z., Dien, B.S., Rausch, K.D., Tumbleson, M.E., Singh, V., 2018. Fermentation of
undetoxified sugarcane bagasse hydrolyzates using a two stage hydrothermal and
mechanical refining pretreatment. Bioresour. Technol. 261, 313–321.
Wyman, C.E., Dale, B.E., Elander, R.T., Holtzapple, M., Ladisch, M.R., Lee, Y.Y., 2005.
Comparative sugar recovery data from laboratory scale application of leading pre-
treatment technologies to corn stover. Bioresour. Technol. 96, 2026–2032.
Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic
ethanol. Biofuels. Bioprod. Biorefin. 2, 26–40.
Yang, B., Tao, L., Wyman, C.E., 2017. Strengths, challenges, and opportunities for hy-
drothermal pretreatment in lignocellulosic biorefineries. Biofuels. Bioprod. Biorefin.
12, 125–138.
Zhu, L., O’Dwyer, J.P., Chang, V.S., Granda, C.B., Holtzapple, M.T., 2008. Structural
features affecting biomass enzymatic digestibility. Bioresour. Technol. 99,
3817–3828.
Zhuang, X., Wang, W., Yu, Q., Qi, W., Wang, Q., Tan, X., Zhou, G., Yuan, Z., 2016. Liquid
hot water pretreatment of lignocellulosic biomass for bioethanol production ac-
companying with high valuable products. Bioresour. Technol. 199, 68–75.