Bioresource Technology 192 (2015) 228–237

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Bioresource Technology
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Ethanol production from sugars obtained during enzymatic hydrolysis

of elephant grass (Pennisetum purpureum, Schum.) pretreated by steam
explosion
Angélica Luisi Scholl a, Daiane Menegol a, Ana Paula Pitarelo b,c, Roselei Claudete Fontana a,
Arion Zandoná Filho b, Luiz Pereira Ramos b,d,1, Aldo José Pinheiro Dillon a, Marli Camassola a,⇑
a
University of Caxias do Sul, Enzyme and Biomass Laboratory, 1130 Francisco Vargas Street, Caxias do Sul, RS 95070-560, Brazil
b
Federal University of Paraná, Department of Chemistry, Research Center in Applied Chemistry (CEPESQ), P.O. Box 19032, Curitiba, PR 81531-980, Brazil
c
Cane Technology Center (CTC), Fazenda Santo Antônio, Piracicaba, SP 13400-907, Brazil
d
INCT in Energy and Environment (INCT E&A), Federal University of Paraná, Department of Chemistry, Curitiba, PR 81531-980, Brazil

h i g h l i g h t s

 Higher pretreatment temperature resulted in the highest yields of sugars and ethanol.
 The highest yield of RS was obtained to the pretreatment at 200 °C for 10 min.
 Pretreatment at 190 °C for 10 min enabled the highest release of glucose.
 The highest ethanol production was obtained to the pretreatment at 200 °C for 6 min.
 The washing of sample after pretreatment was favourable to ethanol production.

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

Article history: In this work, steam explosion was used a pretreatment method to improve the conversion of elephant
Received 15 March 2015 grass (Pennisetum purpureum) to cellulosic ethanol. This way, enzymatic hydrolysis of vaccum-drained
Received in revised form 18 May 2015 and water-washed steam-treated substrates was carried out with Penicillium echinulatum enzymes while
Accepted 19 May 2015
Saccharomyces cerevisiae CAT-1 was used for fermentation. After 48 h of hydrolysis, the highest yield of
Available online 27 May 2015
reducing sugars was obtained from vaccum-drained steam-treated substrates that were produced
after 10 min at 200 °C (863.42 ± 62.52 mg/g). However, the highest glucose yield was derived
Keywords:
from water-washed steam-treated substrates that were produced after 10 min at 190 °C
Elephant grass
Steam explosion
(248.34 ± 6.27 mg/g) and 200 °C (246.00 ± 9.60 mg/g). Nevertheless, the highest ethanol production
Enzymatic hydrolysis was obtained from water-washed steam-treated substrates that were produced after 6 min at 200 °C.
Fermentation These data revealed that water washing is a critical step for ethanol production from steam-treated
Cellulosic ethanol elephant grass and that pretreatment generates a great deal of water soluble inhibitory compounds for
hydrolysis and fermentation, which were partly characterized as part of this study.
Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction However, lignocellulose requires a pretreatment method to induce

Lignocellulosic materials have been considered a prominent
source for the production of fuels and chemicals and the primary
option for a gradual replacement of fossil fuels in the transporta-
tion sector (Menegol et al., 2014a,b; Ramos et al., 2015).
⇑ Corresponding author. Tel./fax: +55 54 3218 2149.
E-mail addresses: ana.pitarelo@gmail.com (A.P. Pitarelo), luiz.ramos@ufpr.br
(L.P. Ramos), mcamassola@gmail.com (M. Camassola).
1
Tel.: +55 41 3361 3175, +55 41 3361 3198.
changes in its chemical structure and composition, making plant
polysaccharides such as cellulose and hemicelluloses accessible
to the enzymes that are able to convert them into fermentable sug-
ars (Mohanty et al., 2009; Mosier et al., 2005; Rocha et al., 2012;
Shafiei et al., 2013).
Elephant grass (Pennisetum purpureum) is a tropical species
with very high productivity (45 t of dry matter/ha/year) compared
to other species (Somerville et al., 2010). This grass is planted sim-
ilarly to sugarcane, requiring little additional nutrients for growth.
Therefore, together with sugarcane bagasse, elephant grass is a

http://dx.doi.org/10.1016/j.biortech.2015.05.065
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
 A.L. Scholl et al. / Bioresource Technology 192 (2015) 228–237
good option for cellulosic ethanol and for several other high
value-added biotechnological applications (Menegol et al.,
2014a). The increased photosynthetic efficiency of elephant grass
is attributed to its C4 metabolism (Euclides et al., 2008).
Steam explosion is recognised as one of the most effective and
widely used pretreatment methods developed to date (Mosier
et al., 2005; Ramos, 2003; Ramos et al., 1992, 2000). Steam explo-
sion allows a relatively easy fractionation and recovery in high
yields of the three main components of the plant cell wall:
cellulose, hemicellulose, and lignin (Brugnago et al., 2011). As a
result, steam-exploded materials can be converted to a range of
value-added products such as fuels, chemicals, materials, and
enzymes (Liew et al., 2014).
Many microorganisms are able to produce enzyme complexes
with a good potential for the enzymatic hydrolysis of lignocellu-
losic materials. Among them, mutant strains of Penicillium echinu-
latum are known to secrete high levels of filter paper activities
(FPA) (Dillon et al., 2011; Reis et al., 2014) and a good ratio
between b-glucosidases and FPA when compared to Trichoderma
reesei (Martins et al., 2008). Also, these enzymes are very stable
at temperatures around 50 °C (Camassola et al., 2004).
In this work, elephant grass was steam-exploded to improve its
susceptibility to enzymatic hydrolysis using cellulases and xyla-
nases produced by P. echinulatum S1M29, and the fermentability
of the corresponding substrate hydrolysates was carried out using
Saccharomyces cerevisiae CAT-1.
2. Methods
2.1. Microorganism
For the production of enzymes, i.e., cellulases and xylanases, the
strain S1M29 strain of P. echinulatum was used. This strain was
derived from the 9A02S1 strain (Dillon et al., 2011) and belongs
to the culture collection of the Enzymes and Biomass Laboratory
of the University of Caxias do Sul (Caxias do Sul, RS, Brazil).
Hexose fermentation was carried out with the S. cerevisiae
CAT-1 strain, which was kindly donated by Professor Luis
Humberto Gomes from the Luiz de Queiroz School of Agriculture
(ESALQ) of the University of São Paulo (Piracicaba, SP, Brazil).
2.2. Growth and maintenance of strains
P. echinulatum S1M29 was transferred to test tubes containing
the growth media proposed by (Dillon et al., 2006) and incubated
for seven days at 28 °C until the formation of conidia. The tubes
were stored at 4 °C or used directly for the preparation of the coni-
dial suspensions that were subsequently used for enzyme
production.
The yeast S. cerevisiae CAT-1 was maintained in YEPD (yeast
extract peptone dextrose) media containing 5 g/L yeast extract,
10 g/L peptone, 10 g/L glucose, and 20 g/L agar.
2.3. Biomass pretreatment
The elephant grass was collected in Nova Petrópolis, RS, Brazil
(29°210 39.1800 S and 51°020 53.8800 W, 619-m above sea level) and
ground in a 70 TrappÒ forage chopper to obtain a particle size
range between 0.075 and 4.75 mm. The samples were then dried
at 60 °C for three days and stored at ambient temperature for fur-
ther use (Menegol et al., 2014b).
The steam explosion was performed at the Federal University of
Paraná (Curitiba, PR, Brazil) using a 10-L stainless steel reactor. A
600-g sample of untreated elephant grass containing 10% (w/w)
moisture was pretreated at various times and temperatures as
229
summarised in Table 1. The pretreatment severity was calculated
according to (Overend et al., 1987). After pretreatment, a mass bal-
ance was performed to determine the efficiency of each pretreat-
ment condition. The mass balance was expressed as the
difference in the sample mass before and after pretreatment, con-
sidering the moisture content of each sample.
The steam-exploded material was drained in a Buchner funnel
and the pretreatment solids were used for enzymatic hydrolysis
and fermentation. The liquid fraction of this filtration procedure
was named primary water solubles, whereas the solid retentate
was designated as unwashed (UW) steam-exploded substrate. A
portion of the solid retentate was washed with water to remove
any possible substances that would still inhibit both hydrolysis
and fermentation. For the washing procedure, 5% (w/v) of the pre-
treated biomass was dispersed in distilled water and stirred at
600 rpm for 1 h at ambient temperature. Then, the suspension
was filtered through a polyester fabrics and the excess of liquid
was removed via vacuum filtration. In this case, the liquid fraction
and solid retentate of this filtration procedure were named sec-
ondary water solubles and water-washed (W) steam-exploded
substrate, respectively. A portion of the solid retentate (approxi-
mately 2 g) was removed for determining its moisture content
while the rest was accommodated in plastic bags and stored at
4 °C for further use.
2.4. Production of enzymes for the hydrolysis of lignocellulosic biomass
P. echinulatum enzymes were produced via submerged cultiva-
tion. The production medium was formulated in a 10% (v/v) aque-
ous salt solution (Mandels and Reese, 1957) containing 0.5% (w/v)
wheat bran, 0.2% (w/v) soybean meal, 0.05% (w/v) ProdexÒ, 0.1%
(w/v) Tween 80Ò, and 1% (w/v) steam-exploded elephant grass that
was produced at 190 °C for 8 min. The salt solution mentioned
above contained 20 g/L KH2PO4, 3 g/L (NH4)2SO4, 3 g/L CO(NH2)2,
3 g/L MgSO47H20, 3 g/L CaCl2, 0.050 g/L FeSO47H2O, 0.0156 g/L
MnSO4H2O, 0.014 g/L ZnSO47H2O, and 0.0020 g/L CoCl2.
Enzyme production was performed in 500 mL Erlenmeyer flasks
containing 100 mL of medium. The medium was inoculated with
the P. echinulatum S1M29 spore suspension to obtain a final con-
centration of 1  105 conidia/mL. The flasks were kept under recip-
rocal agitation at 180 rpm and 28 °C for 5 days. Afterwards, the
content was centrifuged and the enzyme broth was evaluated for
its filter paper activity (FPA) as described by (Ghose, 1987) with
the modifications proposed by (Camassola and Dillon, 2012). The
FPA units were defined as the amount of enzyme capable of releas-
ing 1 lmol/mL of total reducing sugars per minute. The best sub-
strate for enzyme production was identified as the one that
produced the highest total FPA values at the end of fermentation
since this activity represents the set of enzymes that is directly
involved in enzymatic hydrolysis of cellulose (Scholl et al., 2015).
2.5. Characterization of elephant grass before and after pretreatment
Moisture was determined in cellulosic materials by mass loss at
105 °C until constant weight. Inorganic components (ash) were
determined by weight (dry basis) after calcination of the biomass
samples at 575 °C for 6 h.
The chemical composition of elephant grass was characterized
according to the National Renewable Energy Laboratory methods
(NREL-TP-510-42618, NREL-TP-510-42619, NREL-TP-510-42621)
with the adaptations described by (Menegol et al., 2014a). For this,
elephant grass samples were extracted with water and ethanol to
remove low molar mass extractable components prior to sulphuric
acid hydrolysis to quantify glucans (mostly cellulose), hemicellu-
loses (mostly acetylated heteroxylans) and both acid soluble and
acid insoluble lignins. Carbohydrate analysis was carried out in
230 A.L. Scholl et al. / Bioresource Technology 192 (2015) 228–237

Table 1 The concentration of fermentation inhibitors (furfural and

Pretreatment conditions for the steam explosion for elephant grass. hydroxymethylfurfural) that were produced during pretreatment
Test Temperature (°C) Time (min) Pressure (kgf/cm3) Severity factor were analysed in a Shimadzu HPLC using a C18 column and a
1 1 (180) 1 (6) 13 3.13 UV/Vis detector SPD-20A (Shimadzu) at 280 nm and 30 °C. The
2 +1 (200) 1 (6) 13 3.36 mobile phase was prepared by mixing 9 volumes of 0.1% aqueous
3 1 (180) +1 (10) 17 3.43 formic acid with 1 volume with 1% formic acid in methanol. The
4 +1 (200) +1 (10) 17 3.55 flow rate was 0.5 mL/min while quantification was performed by
5 0 (190) 1 (6) 17 3.65
6 0 (190) +1 (10) 21 3.72
external calibration.
7 0 (190) 0 (8) 21 3.94

3. Results and discussion

sulphuric acid hydrolysates by high performance liquid chro-
matography (HPLC) using an Aminex HPX-87H column at 60 °C
with 5 mmol/L H2SO4 as the mobile phase and a flow rate of
0.6 mL/min. Detection of soluble sugars was carried out by refrac-
tive index and quantification was performed by external calibra-
tion. The determination of both total nitrogen and protein
contents were carried out using the Kjeldahl’s method (Lynch
and Barbano, 1999).
2.6. Scanning electron microscopy
Scanning electron microscopy (50 and 1000) of elephant
grass samples was carried out at the Laboratory of Materials
Characterization (LCMAT), University of Caxias do Sul, Brazil. The
method employed was based on magnetron-sputtering plasma
deposition, commonly known as physical vapour gold or physical
vapour deposition (PVD), using a Superscan Shimadzu SSX-550
scanning electron microscope.
2.7. Enzymatic hydrolysis
Enzymatic hydrolysis was carried out according to (Menegol
et al., 2014a) using various steam-exploded elephant grass samples
(unwashed and water-washed). Untreated elephant grass,
CeluflockÒ cellulose (Cotia, São Paulo, Brazil) and Avicel PH101
(Fluka, Buchs, Ireland) were used as controls. All experiments were
carried out in three replicates at 50 °C using an enzyme loading of
10 FPU/g total solids and a substrate concentration of 4% (w/v) in
acetate buffer 50 mmol/L pH 4.8. Reaction aliquots were with-
drawn at 24 and 48 h of hydrolysis and the release of reducing sug-
ars (RS) was measured according to (Miller, 1959) using the
3,5-dinitrosalicylic acid (DNS) reagent. Also, these reaction aliquots
were analysed by HPLC using an Aminex HPX-87H as described
above.
2.8. Fermentation
Fermentation of samples after 48 h of enzymatic hydrolysis was
carried out in 2 mL reaction tubes for 24 h at 28 °C. These samples
were supplemented with 4 g/L of ProdexÒ and 1 g/L of (NH4)2SO4
and inoculated with S. cerevisiae CAT-1 to reach a total of 1  108
viable cells per mL.
Samples were taken at different intervals to determine the
kinetics of sugar consumption and ethanol production. Sugar and
ethanol concentrations were analysed by HPLC using an Aminex
HPX-87H column as described above. The ethanol yield was
expressed as Y(P/S) and calculated as follows (Eq. (1)),
x1
Y ðP=SÞ ¼
x2
where x1 is the amount of ethanol produced by fermentation of sub-
strate hydrolysates and x2 is the amount of biomass used for
fermentation.
3.1. Chemical composition of elephant grass before and after
pretreatment
The chemical composition of elephant grass before and after
pretreatment has been already reported elsewhere (Scholl et al.,
2015). In general, steam explosion modified the chemical composi-
tion of elephant grass primarily by decreasing its hemicellulose
content as previously observed for other lignocellulosic materials
such as cane bagasse (Pitarelo et al., 2012) and Eucalyptus grandis
(Emmel et al., 2003). Most of the hemicellulose sugars that were
released by acid hydrolysis during steam explosion were recovered
in the primary and secondary water soluble fractions, which were
obtained from the steam-treated materials by filtration and exten-
sive water washing, respectively. Hemicellulose removal reflected
in an increase in both glucan (mostly cellulose) and lignin contents
in the steam-treated cellulosic substrate. Hence, hemicelluloses
were the most severely affected biomass component during steam
explosion and this explains the high concentration of pentose sug-
ars in the water solubles, even at relatively mild pretreatment con-
ditions (Pitarelo et al., 2012).
Largest percentages of ash were recorded in elephant grass
before and after pretreatment. Experiments at 190 °C for 10 min
yielded the highest ash concentration (9.44%), whereas the lowest
was obtained after pretreatment at 180 °C for 10 min (2.77%). On
the other hand, untreated elephant grass contained 8.53% of ash
in its composition. Hence, it seems that lignocellulosic materials
with an increased capacity to retain inorganic materials were gen-
erated under increased pretreatment severities and that these
inorganic materials were not easy to remove even by extensive
water washing.
Untreated elephant grass exhibited a protein content of 5.55%
but, after pretreatment, unwashed substrates had higher protein
contents than water-washed samples. The highest protein content
(8.74%) was derived from pretreatment at 200 °C for 6 min
(unwashed sample), whereas the lowest (1.63%) was produced at
190 °C for 8 min (water-washed sample). The presence of higher
protein contents in substrates produced at higher pretreatment
temperatures is probably due to protein denaturation, which
causes their precipitation and/or adsorption onto the lignocellu-
losic fibers (Jaenicke, 1991).
3.2. Mass recovery of pretreatment fractions
Pretreatment usually results in a considerable mass loss of plant
biomass components, depending on the type of pretreatment
method, the applied experimental conditions, and the type of bio-
mass used for conversion (Brugnago et al., 2011; Emmel et al.,
2003; Iroba et al., 2014; Menardo et al., 2013). As shown in
ð1Þ Tables 1 and 2, the pretreatment severity influenced the percent-
age of mass loss. Likewise, higher losses were observed when dif-
ferent pretreatment residence times were compared at the same
temperature. As a result, the mildest pretreatment condition of
180 °C for 6 min had the lowest mass loss and the results at more
 A.L. Scholl et al. / Bioresource Technology 192 (2015) 228–237
Table 2
Percent of mass loss of the elephant grass biomass under different steam explosion
pretreatment conditions.
Pretreatment (°C/min) Unwashed (UW) Washed (W)
Untreated – –
180/6 1.64 1.66
180/10 7.50 7.62
190/6 14.81 15.01
190/8 18.91 19.00
190/10 28.93 28.97
200/6 20.73 20.78
200/10 23.16 23.20
drastic conditions agreed relatively well with their corresponding
severity factor.
Other authors have observed similar mass losses for the steam
explosion of other lignocellulosic materials such as barley straw
and sugarcane trash (Iroba et al., 2014; Oliveira et al., 2013) and
these have been primarily attributed to the dehydration of pentose
sugars such as arabinose and xylose. According to Pitarelo et al.
(2012), low pretreatment recovery yields are partially due to the
loss of volatiles in the flash steam and these would contain consid-
erable amounts of furfural, hydroxymethylfurfural, and acetic acid,
among others.
3.3. Scanning electron microscopy
In addition to the influence of pretreatment severity on the
recovery yield and chemical composition of elephant grass,
changes were also observed in the physical structure of
steam-treated biomass. Fig. 1S (Supplementary material) presents
the morphological analysis of both untreated and
steam-exploded elephant grass by scanning electron microscopy.
Samples that were pretreated at higher temperatures suffered
greater fragmentation and these changes increased the accessibil-
ity of cellulose to the subsequent enzymatic hydrolysis. Also, these
morphological changes agreed well with the observed mass loss
(Table 2). With a decrease in biomass particle size at increasing
temperature and extraction time, higher concentrations of low
molar mass compounds were observed in the water solubles, such
as in the case of xylose and arabinose (Table 3A).
By comparing the images obtained for steam-exploded elephant
grass with those presented by (Aver et al., 2014), it seems that
steam explosion promotes an extensive fragmentation of the bio-
mass fibers while ionic liquids restrict their pretreatment action
on the dissolution of biomass components such as plant
polysaccharides.
3.4. Characterization of the pretreatment water solubles
Pretreatment water solubles were characterized to reveal the
presence of inhibitory compounds to hydrolysis and fermentation.
For reactions carried out at 180 °C for 6 min, lower concentrations
of xylose and arabinose were observed in these liquid fractions.
Hence, these conditions were not strong enough to promote
detectable modifications in the chemical composition of elephant
grass, which remained almost unchanged after pretreatment
(Table 2). The concentration of xylose and arabinose increased in
the water solubles when higher severities were used for pretreat-
ment (Table 3A). As a result, cellulosic substrates pretreated at high
severities had lower concentrations of these carbohydrate compo-
nents. Water washing of steam-exploded samples that were
drained after pretreatment removed much lower concentrations
of water soluble components (Table 3B). On the other hand, most
of the elephant grass glucans remained in the solid fraction after
pretreatment.
231
Several compounds that are released and/or produced during
steam explosion of plant biomass, such as formic acid, acetic acid,
phenolic compounds, furfural, and 5-hydroxymethylfurfural, can
be highly inhibitory to microorganisms and cause adverse effects
on the subsequent hydrolysis and fermentation processes (Lu
et al., 2010). Lower concentrations of these inhibitory chemicals
were observed in samples pretreated at 180 °C but when the tem-
perature was increased to 190 and 200 °C, there was a considerable
accumulation of acetic acid, furfural and hydroxymethylfurfural in
the pretreatment water solubles (Table 3A).
According to (Palmqvist and Hahn-Hägerdal, 2000), when sev-
ere pretreatment conditions are used, xylose and glucose can be
degraded to furfural and hydroxymethylfurfural, respectively.
Most of these dehydration by-products are recovered in the
pretreatment water solubles (Table 3) but some were also lost in
the pretreatment volatiles or even converted to other chemicals
such as formic acid and levulinic acid.
3.5. Enzymatic hydrolysis and fermentation
Fig. 1 shows the reducing sugar concentrations that were
obtained after enzymatic hydrolysis of untreated and
steam-exploded elephant grass. Fig. 1A–C are related to
water-washed steam-treated materials whereas Fig. 1D–F provide
the enzymatic hydrolysis profile of unwashed steam-exploded
substrates. Controls were also performed using untreated elephant
grass, CeluflockÒ and AvicelÒ using the same hydrolysis conditions.
Hydrolysis of water-washed (Fig. 1A–C) and unwashed
(Fig. 1D–F) steam-exploded substrates yielded higher reducing
sugar concentrations when compared to that of untreated elephant
grass. However, these yields were lower than those obtained from
the enzymatic hydrolysis of delignified cellulose fibers (CeluflockÒ)
or microcrystalline cellulose (AvicelÒ). Hydrolysis of CeluflockÒ
exhibited a peak in the release of reducing sugars at 36 h, which
corresponded to 1054.82 ± 16.11 mg/g (dry basis), whereas the
reducing sugars released from AvicelÒ peaked at
935.96 ± 46.33 mg/g at the end of hydrolysis (48 h). As expected,
untreated elephant grass exhibited the lowest release of reducing
sugars, reaching only 310.10 ± 3.12 mg/g after 36 h of hydrolysis.
Considering the hydrolysis of substrates that were produced
under different pretreatment conditions, water-washed
(Fig. 1A–C) and unwashed (Fig. 1D–F) steam-exploded samples
exhibited similar profiles of reducing sugar release. The hydrolysis
of unwashed samples pretreated at 200 °C for 10 and 6 min
released 863.42 ± 62.52 mg/g and 785.73 ± 31.81 mg/g of reducing
sugars in 36 h, respectively (Fig. 1F). These values are comparable
to those obtained from the enzymatic hydrolysis of CeluflockÒ
and AvicelÒ. Hence, pretreatment made the biomass a more
accessible target to the concerted action of the enzymes.
In Fig. 1, the release of reducing sugars appears to decrease in
some situations and this was probably due to interferences of
non-carbohydrate components in the response of the DNS method.
By contrast, when the concentration of sugars was determined by
HPLC (Table 4), such trend was not observed for both glucose and
xylose release. This indicates that, although widely used for the
determination of hydrolysis efficiency, the DNS method was
not reliable for monitoring the enzymatic hydrolysis of
steam-exploded elephant grass, particularly when unwashed
substrates were used for hydrolysis.
In Table 4 and Fig 2, it is clear that both CeluflockÒ and AvicelÒ
were better substrates for hydrolysis than any of the
steam-exploded materials derived from elephant grass. Menegol
et al. (2014b) observed a maximum release of 127.80 ± 2.17 and
504.52 ± 26.66 mg/g of glucose after hydrolysis of untreated and
alkali-extracted elephant grass for 48 h with an enzyme loading
of 15 FPU/g. In the present investigation, the largest release of
232 A.L. Scholl et al. / Bioresource Technology 192 (2015) 228–237

Table 3
Substances that were quantified by HPLC in the (A) primary and (B) secondary water solubles that were obtained after steam explosion of elephant grass at different temperatures
and for residence times in the steam reactor.

Pretreatment Detected substances (g/L)

(°C/min)
Cellobiose Glucose Xylose Arabinose Acetic acid HMF Furfural
A 180/6 n.d. 0.43 ± 0.00 1.04 ± 0.00 0.29 ± 0.00 0.57 ± 0.00 0.05 ± 0.01 0.03 ± 0.00
180/10 0.86 ± 0.00 1.49 ± 0.00 4.54 ± 0.00 0.83 ± 0.00 3.90 ± 0.02 0.22 ± 0.03 0.18 ± 0.01
190/6 0.93 ± 0.13 1.70 ± 0.15 5.99 ± 0.66 0.80 ± 0.10 7.58 ± 0.38 0.37 ± 0.05 0.44 ± 0.05
190/8 0.62 ± 0.11 1.36 ± 0.23 5.07 ± 0.76 0.94 ± 0.06 5.40 ± 1.92 0.32 ± 0.12 0.44 ± 0.12
190/10 0.68 ± 0.19 1.40 ± 0.36 5.14 ± 0.92 0.61 ± 0.29 5.59 ± 3.21 0.21 ± 0.04 0.40 ± 0.06
200/6 n.d. 1.62 ± 0.06 5.68 ± 0.18 0.81 ± 0.03 7.75 ± 0.46 0.47 ± 0.02 0.45 ± 0.01
200/10 n.d. 1.27 ± 0.00 4.40 ± 0.04 0.5 ± 0.02 5.67 ± 0.13 0.34 ± 0.01 0.38 ± 0.01
B 180/6 n.d. 0.11 ± 0.00 0.22 ± 0.01 0.15 ± 0.02 0.00 ± 0.00 0.01 ± 0.00 0.01 ± 0.00
180/10 n.d. 0.07 ± 0.00 0.07 ± 0.03 0.07 ± 0.02 0.01 ± 0.01 0.01 ± 0.00 0.02 ± 0.00
190/6 n.d. 0.08 ± 0.00 0.28 ± 0.01 0.06 ± 0.00 0.14 ± 0.01 0.02 ± 0.00 0.03 ± 0.01
190/8 n.d. 0.08 ± 0.00 0.24 ± 0.01 0.05 ± 0.00 0.09 ± 0.01 0.02 ± 0.00 0.05 ± 0.01
190/10 n.d. 0.07 ± 0.00 0.05 ± 0.02 0.05 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.04 ± 0.00
200/6 n.d. 0.08 ± 0.00 0.24 ± 0.00 0.05 ± 0.00 0.11 ± 0.00 0.02 ± 0.00 0.05 ± 0.00
200/10 0.03 ± 0.02 0.07 ± 0.00 0.04 ± 0.01 0.04 ± 0.00 0.02 ± 0.01 0.01 ± 0.00 0.02 ± 0.00

A, primary pretreatment water-solubles; B, secondary pretreatment water-solubles.

n.d.: not detected.

1200 1200 1200

C
Reducing sugars (mg/g)

1000
A 1000
B 1000

800 800 800

600 600 600

400 400 400

200 200 200

0 0 0
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
Time (hours) Time (hours) Time (hours)

1200 1200 1200

Reducing sugars (mg/g)

1000
D 1000
E F
1000

800 800 800

600 600 600

400 400 400

200 200 200

0 0 0
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
Time (hours) Time (hours) Time (hours)

6 min 8 min 10 min Untreated EG Celuflock ® Avicel®

Fig. 1. Reducing sugars release during the enzymatic hydrolysis of CeluflockÒ(Cel), AvicelÒ, untreated elephant grass, and steam-exploded elephant grass for both washed
(A–C) and unwashed (D–F) substrates using 4% (w/v) total solids and 10 FPU/g.

glucose was obtained from elephant grass after steam explosion at of xylose were detected in the enzymatic hydrolysates of
190 °C for 10 min, giving 248.34 ± 6.27 mg/g after 48 h of hydroly- unwashed substrates. This may be due to the retention of
sis using an enzyme loading of 10 FPU/g. xylooligomers in the unwashed substrates that were not quantified
Xylose was not detected in the hydrolysis of AvicelÒ (Fig. 2B) in Table 3 because both primary and secondary water solubles
but some was released from CeluflockÒ due to its relatively high derived from pretreatment were not subjected to a
hemicellulose content. Interestingly, AvicelÒ was a better substrate post-hydrolysis prior to HPLC analysis.
for the Penicillium enzymes than CeluflockÒ, regardless of its high For unwashed steam-exploded substrates, the best glucose
crystallinity index. Regarding pretreatment, water washing of yield after 48 h of hydrolysis was obtained after pretreatment at
steam-exploded materials was beneficial for enzymatic hydrolysis, 190 °C for 10 min (228.31 ± 3.32 mg/g), while water-washed
revealing that even the small amount of water solubles (also steam-exploded samples exhibited higher glucose yields when
referred to as C5 fraction) that is retained in the pretreated mate- pretreated at 190 °C for 8 or 10 min (243.29 ± 6.19 and ± 248.34
rial after filtration contains adverse factors and/or inhibitory com- 6.27 mg/g, respectively) and 200 °C for 10 min (246.00 ±
pounds that are enough to compromise the concerted action of 9.60 mg/g). For the release of glucose in the hydrolysis of corn
Penicillium cellulases (Fig. 2A). By contrast, higher concentrations stover pretreated via steam explosion, (Lu et al., 2010) found that
 A.L. Scholl et al. / Bioresource Technology 192 (2015) 228–237 233

Table 4
Substances detected by HPLC in the enzymatic hydrolysate of CeluflockÒ, AvicelÒ, untreated elephant grass (EG) and steam-exploded materials that were obtained from EG at
different pretreatment conditions.

Pretreatment (°C/min) Hydrolysis time (h) Type of substrate Detected substances (g/L)
Cellobiose Arabinose Xylitol Acetic acid HMF Furfural
Elephant grass 24 Untreated n.d. 0.24 ± 0.01 n.d. n.d. n.d. n.d.
48 0.05 ± 0.04 0.46 ± 0.02 0.11 ± 0.01 n.d. n.d. n.d.
CeluflockÒ 24 Untreated 0.94 ± 0.18 0.04 ± 0.00 n.d. n.d. n.d. n.d.
48 1.50 ± 0.35 0.07 ± 0.09 n.d. n.d. n.d. n.d.
AvicelÒ 24 Untreated 0.84 ± 0.04 n.d. 0.02 ± 0.00 n.d. n.d. n.d.
48 1.12 ± 0.07 n.d. 0.01 ± 0.01 n.d. n.d. n.d.
180/6 24 W n.d. 0.08 ± 0.01 n.d. 0.07 ± 0.01 n.d. n.d.
UW n.d. 0.41 ± 0.15 n.d. 0.46 ± 0.04 n.d. n.d.
48 W 0.14 ± 0.06 0.07 ± 0.00 n.d. 0.09 ± 0.01 n.d. n.d.
UW n.d. 0.51 ± 0.03 n.d. 0.33 ± 0.01 n.d. n.d.
180/10 24 W 0.23 ± 0.02 0.05 ± 0.00 n.d. n.d. n.d. n.d.
UW 0.31 ± 0.02 0.10 ± 0.00 n.d. 0.52 ± 0.01 n.d. n.d.
48 W n.d. 0.04 ± 0.00 n.d. n.d. n.d. n.d.
UW 0.26 ± 0.01 0.55 ± 0.01 n.d. 0.46 ± 0.02 0.04 ± 0.00 n.d.
190/6 24 W 0.55 ± 0.02 0.25 ± 0.02 n.d. n.d. n.d. n.d.
UW 0.25 ± 0.07 0.07 ± 0.00 n.d. 0.77 ± 0.0 n.d. n.d.
48 W 0.27 ± 0.09 n.d. 0.01 ± 0.01 n.d. n.d. n.d.
UW 0.47 ± 0.07 0.59 ± 0.12 n.d. 0.92 ± 0.3 0.05 ± 0.00 0.06 ± 0.01
190/8 24 W 0.55 ± 0.11 0.25 ± 0.02 n.d. n.d. n.d. n.d.
UW 0.43 ± 0.11 0.13 ± 0.06 n.d. 0.59 ± 0.06 n.d. n.d.
48 W 0.31 ± 0.16 n.d. 0.09 ± 0.16 n.d. n.d. 0.01 ± 0.00
UW 0.23 ± 0.01 0.52 ± 0.03 n.d. 0.76 ± 0.18 0.05 ± 0.00 0.08 ± 0.01
190/10 24 W 0.57 ± 0.02 0.30 ± 0.04 n.d. n.d. n.d. n.d.
UW 0.51 ± 0.03 0.07 ± 0.00 n.d. 0.35 ± 0.02 n.d. n.d.
48 W 0.34 ± 0.01 0.03 ± 0.00 0.02 ± 0.00 n.d. n.d. n.d.
UW 0.54 ± 0.02 0.48 ± 0.01 n.d. 0.51 ± 0.05 0.03 ± 0.00 0.07 ± 0.01
200/6 24 W 0.67 ± 0.06 0.26 ± 0.08 n.d. n.d. n.d. n.d.
UW 0.38 ± 0.03 0.14 ± 0.00 n.d. 0.66 ± 0.03 n.d. n.d.
48 W 0.41 ± 0.04 0.25 ± 0.15 0.02 ± 0.00 n.d. n.d. n.d.
UW 0.35 ± 0.05 0.54 ± 0.02 n.d. 0.79 ± 0.12 0.06 ± 0.00 0.08 ± 0.00
200/10 24 W 0.84 ± 0.17 0.38 ± 0.13 n.d. n.d. n.d. n.d.
UW 0.40 ± 0.05 0.11 ± 0.05 n.d. 0.47 ± 0.11 n.d. n.d.
48 W 0.49 ± 0.03 0.04 ± 0.00 0.02 ± 0.01 n.d. n.d. n.d.
UW 0.35 ± 0.03 0.40 ± 0.06 n.d. 0.59 ± 0.24 0.04 ± 0.01 0.07 ± 0.01

W: sample that was water-washed after the pretreatment.

UW: sample not subject to water washing after pretreatment.
n.d.: not detected.

most of the sugar release occurred after 96 h (103.3 g/L to wt% con-
centration of substrate) when washed and pretreated straw was
used, whereas unwashed samples exhibited only a 85.1 g/L glucose
release concentration.
The concentration of fermentation inhibitors in 48-h substrate
hydrolysates was determined by HPLC for both unwashed and
water-washed substrates as well as for AvicelÒ and CeluflockÒ,
which were used as unlignified substrate controls (Table 4).
Furfural and 5-hydroxymethylfurfural (HMF) were only detectable
in the hydrolysates of unwashed substrates that were produced
under higher pretreatment severities but, even so, their concentra-
tion was always below inhibitory conditions (less than 0.1 g/L).
(Ewanick and Bura, 2011) also reported non-inhibitory concentra-
tions of HMF and furfural in the steam explosion liquid streams
derived from switchgrass and sugarcane bagasse. Since unwashed
substrates were not as good for hydrolysis as water-washed sub-
strates, it seems that inhibition came from the water-soluble car-
bohydrate moiety (mostly oligomers) of these steam exploded
materials and this would be consistent with the findings of (Qing
et al., 2010), who showed that the presence of xylooligomers dra-
matically decreases the conversion rates and yields of cellulose
hydrolysis.
Acetic acid was undetectable by HPLC in water-washed samples
that underwent hydrolysis for 48 h (Table 4), except for the
substrate that was produced under the lowest pretreatment sever-
ity. This indicates that elephant grass hemicelluloses were almost
completely deacetylated above a severity factor of 3.13, which cor-
responds to 180 °C for 6 min, and this is consistent with the low
acetyl group content of these substrates as reported elsewhere
(Scholl et al., 2015). Hence, the amount of acetic acid that was
detected in all enzymatic hydrolysates derived from unwashed
samples corresponds to the amount of this analyte in the fraction
of the pretreatment liquid stream (C5 fraction) that was retained
in the pretreated substrate after filtration. Not surprisingly, acetic
acid was not found in the enzymatic hydrolysates of AvicelÒ and
CeluflockÒ because the former is a hemicellulose-free commercial
form of microcrystalline cellulose while the latter is originated
from a pulping process in which acetyl groups are known to be
completely hydrolysed and washed away from the cellulose fibers.
Fig. 3 shows the concentrations of sugars (glucose and xylose)
and ethanol after fermentation of 48-h substrate hydrolysates by
S. cereviseae CAT-1. By comparing the performance of substrates
derived from different pretreatment conditions, the highest etha-
nol yields were obtained from samples that were water-washed
after steam explosion at 200 °C for 6 or 10 min
(4.27 ± 0.15 mg/mL and 4.25 ± 0.20 mg/mL, respectively) when a
total fermentation time of 12 h was used. Other conditions, such
as pretreatment at 180 °C for 6 min and 190 °C for 8 min or
234 A.L. Scholl et al. / Bioresource Technology 192 (2015) 228–237

500
A
Glucose (mg/g)

400

300

200 Washed (W)

Unwashed (UW)
100

0
24 h 48 h 24 h 48 h 24 hin 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

in

in
in

in

el

l
in

in

ce
tE

C
m

m
m
m
m

vi
U
8

10

10
6
10
6

A
C

C
C

C
C

0°
0°

0°

0°
0°

0°

0°
20
18

19

19
18

19

20
Pretreatment (°C and min) and hydrolysis time (h)
500
B
Xylose (mg/g)

400

300
Washed (W)
200 Unwashed (UW)

100

0
24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h
in

in

G
in

el

l
in
in

in
in

ce
tE

C
m

m
m

m
m

vi
U
6

10
6

10
10

A
°C

°C
°C

°C
°C
°C

°C
0

0
0

0
18

19

19

0
0

20
18

19

20

Pretreatment (°C and min) and hydrolysis time (h)

Fig. 2. Concentration of glucose (A) and xylose (B) in the enzymatic hydrolysate of CeluflockÒ (Cel), AvicelÒ, and unwashed (UW) and water-washed (W) steam-exploded
elephant grass after 24 and 48 h of hydrolysis.

10 min, resulted in a significant decrease in ethanol concentration, controls. Hence, this demonstrates the efficiency and importance
whereas for other conditions, ethanol concentration remained con- of steam explosion for the production of ethanol from elephant
stant. Lu et al. (2010) reported that the concentrations of ethanol grass. Furthermore, both substrate controls had fermentation pro-
derived from the fermentation of corn stover pretreated via steam files that were very similar to those of steam-exploded substrates.
explosion and subjected to washing increased at the beginning of AvicelÒ hydrolysates containing 20.12 ± 0.75 mg/mL of glucose
the process and remained constant thereafter. Regarding ethanol resulted in 4.82 mg/mL ± 0.03 mg/mL of ethanol after 24 h of fer-
yields obtained using samples that were not washed after the pre- mentation, while the 18.24 ± 0.44 mg/mL of CeluflockÒ hydroly-
treatment process were similar for the various fermentation times sates were converted 4.42 ± 1.52 mg/mL of ethanol in the same
profiles when compared the yields obtained using washed sam- reaction time. Notably, hydrolysates from untreated elephant grass
ples. However, the unwashed sample after pretreatment at had a much lower glucose concentration at the beginning of fer-
190 °C for 10 min exhibited the highest yield of ethanol after mentation (4.06 ± 0.18 mg/mL) and this affected the ethanol pro-
24 h of fermentation compared with the washed sample for the duction from this substrate.
same condition. Table 1S presents the analytes that were detected and quanti-
In general, the highest consumption of glucose occurred during fied using HPLC in the substrate hydrolysates after fermentation.
the first 6 h of fermentation and the decrease in glucose concentra- Arabinose was detected in most samples and was generally more
tion coincided with an increase in ethanol production. Regardless of concentrated in experiments carried out with unwashed sub-
the conditions used for pretreatment, the amount of glucose avail- strated. The highest acetic acid concentration was detected when
able for fermentation was higher in water-washed substrates and unwashed steam-exploded substrates were used for hydrolysis
this explains why their ethanol yields was higher than that of and fermentation. Under some conditions of pretreatment, the
unwashed substrates. Also, after 6 h of fermentation, the consump- analyte was not detected in the pretreated, washed samples at
tion of glucose remained stable until it was exhausted from the 0 h of fermentation. Lu et al. (2010) reported that concentrations
medium and, at this point, the ethanol yield stabilized as well. of 3.3 g/L acetic and 145 mh/L of furfural were considered inhibi-
The concentrations of xylose (Fig. 3) remained constant during tory to the fermentation of corn stover pretreated via explosion
the entire fermentation process for both unwashed and steam, whereas in the present study, minimal concentrations of
water-washed substrates because the yeast was not able to ferment these compounds were detected.
it. And as mentioned before, unwashed substrates generally exhib- Substrate hydrolysates were also analysed by HPLC after fer-
ited higher amounts of xylose in the fermentation medium, com- mentation by S. cereviseae CAT-1 using the same method described
pared to experiments carried out with water-washed substrates. above for characterizing the pretreatment water solubles (data not
After most of the available glucose was depleted and the high- shown). Apart from non-fermentable sugars (e.g., xylose and arabi-
est ethanol concentration was reached, which corresponded to nose), acetic acid and other organic components that were not uti-
12 h of fermentation, a decrease in ethanol concentration was lized by the yeast metabolism, glycerol and xylitol were also
observed and this was probably due to the consumption of ethanol detected after fermentation of substrate hydrolysates derived from
by the yeasts. The use of untreated elephant grass gave much lower unwashed and water-washed steam-exploded samples as well as
yields compared steam-exploded substrates and the substrate substrate controls (CeluflockÒ and AvicelÒ). In most cases, the
 A.L. Scholl et al. / Bioresource Technology 192 (2015) 228–237 235

25 7 25 7
180°C - 6 min 180°C - 10 min
6 6
20 Glucose (W)

Ethanol (mg/mL)
20

Ethanol (mg/mL)

Sugars (mg/mL)
Sugars (mg/mL)

5 5
Glucose (UW)
15 15 4
4 Ethanol (W)
3 3 Ethanol (UW)
10 10
2
Xylose (W)
2
5 5 Xylose (UW)
1 1

0 0 0 0
0 6 12 18 24 0 6 12 18 24
Time (hours) Time (hours)
25 7 25 7
190°C - 6 min 190°C - 8 min
6 6
20 Glucose (W)

Ethanol (mg/mL)
20

Ethanol (mg/mL)

Sugars (mg/mL)
Sugars (mg/mL)

5 5
Glucose (UW)
15 15 4
4 Ethanol (W)
3 3 Ethanol (UW)
10 10
2
Xylose (W)
2
5 5 Xylose (UW)
1 1

0 0 0 0
0 6 12 18 24 0 6 12 18 24
Time (hours) Time (hours)
25 7 25 7
190°C - 10 min 200°C - 6 min
6 6
20 20 Glucose (W)
Ethanol (mg/mL)

Ethanol (mg/mL)
Sugars (mg/mL)

Sugars (mg/mL)

5 5
Glucose (UW)
15 4 15 4 Ethanol (W)
3 3 Ethanol (UW)
10 10
2 2
Xylose (W)
5 5 Xylose (UW)
1 1
0 0 0 0
0 6 12 18 24 0 6 12 18 24
Time (hours) Time (hours)
25 7 25 7
200°C - 10 min Untreated
6 6
20
Ethanol (mg/mL)

20
Ethanol (mg/mL)
Sugars (mg/mL)

Sugars (mg/mL)

5 5 Glucose
15 4 15 4 Ethanol
3 3 Xylose
10 10
2 2
5 5
1 1
0 0 0 0
0 6 12 18 24 0 6 12 18 24
Time (hours) Time (hours)
25 7 25 7
® ®
Celuflock Avicel
6 6
20
Ethanol (mg/mL)

20
Ethanol (mg/mL)

Sugars (mg/mL)
Sugars (mg/mL)

5 5 Glucose
15 15 4
4 Ethanol
3 3 Xylose
10 10
2 2
5 5
1 1
0 0 0 0
0 6 12 18 24 0 6 12 18 24
Time (hours) Time (hours)

Fig. 3. Concentration of sugars (glucose and xylose) and ethanol after 48 h of fermentation of substrate hydrolysates derived from CeluflockÒ, AvicelÒ and different samples of
unwashed (UW) and water-washed (W) steam-exploded elephant grass.

highest concentrations of these analytes were observed in experi- concentrations higher than xylitol, which was not even detected
ments carried out with unwashed samples. Glycerol and xylitol in many fermentation experiments.
are known yeast metabolites and differences in their concentration Table 5 demonstrates the positive influence of pretreatment on
can be attributed to differences in the chemical composition of the the ethanol production from elephant grass glucans. Water wash-
fermentation medium. However, glycerol was usually found in ing of the pretreated biomass was also beneficial for this same
236 A.L. Scholl et al. / Bioresource Technology 192 (2015) 228–237

Table 5 Dillon, A.J., Bettio, M., Pozzan, F.G., Andrighetti, T., Camassola, M., 2011. A new
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hydrogen peroxide mutagenesis and screening with 2-deoxyglucose. J. Appl.
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