Fungal pretreatment: An alternative in second-generation ethanol from wheat straw

Davinia Salvacha
a
, Alicia Prieto
a
, Mara Lpez-Abelairas
b
, Thelmo Lu-Chau
b
, ngel T. Martnez
a
,
Mara Jess Martnez
a,
a
Centro de Investigaciones Biolgicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
b
Institute of Technology, University of Santiago de Compostela, E-15782 Santiago de Compostela, Spain
a r t i c l e i n f o
Article history:
Received 17 March 2011
Received in revised form 10 May 2011
Accepted 13 May 2011
Available online 19 May 2011
Keywords:
Wheat straw
Pretreatment
Basidiomycetes
Enzymatic hydrolysis
Bioethanol
a b s t r a c t
The potential of a fungal pretreatment combined with a mild alkali treatment to replace or complement
current physico-chemical methods for ethanol production from wheat straw has been investigated.
Changes in substrate composition, secretion of ligninolytic enzymes, enzymatic hydrolysis efciency
and ethanol yield after 7, 14 and 21 days of solid-state fermentation were evaluated. Most fungi degraded
lignin with variable selectivity degrees, although only eight of them improved sugar recovery compared
to untreated samples. Glucose yield after 21 days of pretreatment with Poria subvermispora and Irpex lac-
teus reached 69% and 66% of cellulose available in the wheat straw, respectively, with an ethanol yield of
62% in both cases. Conversions from glucose to ethanol reached around 90%, showing that no inhibitors
were generated during this pretreatment. No close correlations were found between ligninolytic enzymes
production and sugar yields.
2011 Elsevier Ltd. All rights reserved.
1. Introduction
Lignocellulosic materials are the major component of biomass
and represent the most abundant renewable energy resource avail-
able on earth (Lin and Tanaka, 2006). Among them, agricultural
wastes are the most extended and the cheapest, especially wheat
straw which is the most plentiful in Europe and the second one
worldwide after rice straw (Kim and Dale, 2004). Moreover, wheat
straw is a residue that does not compete with human food re-
sources constituting an auspicious alternative to generate renew-
able biofuels.
Second-generation bioethanol production from wheat straw in-
cludes three main steps: (i) pretreatment, (ii) enzymatic hydrolysis
of cellulose and hemicellulose, and (iii) ethanol fermentation
(Talebnia et al., 2010). The aim of the pretreatment is the disrup-
tion of the lignocellulose structure, improving cellulose and hemi-
cellulose accessibility. Nowadays, steam explosion, which requires
high pressures and temperatures, is the most common and effec-
tive pretreatment for this purpose, although the severity of this
process generates by-products that affect adversely subsequent
steps (Alvira et al., 2010; Jurado et al., 2009). An alternative to
avoid these problems is the use of biological pretreatments, which
present additional advantages as being cheaper, safer, less energy-
consuming and more environmentally friendly.
Biopretreatment is based on the capacity of some organisms of
degrading lignin to gain access to cellulose and hemicellulose. Nev-
ertheless, the literature shows a big controversial about this topic,
since the largest lignin degradations not always correspond with
the best sugar recoveries (Capelari and Toms-Pej, 1997; Shi
et al., 2008). Biological degradation of lignocellulose is a complex
process where many factors, as fungal strain, culture conditions,
fungal enzymatic secretion and oxidative mechanisms, are impli-
cated (Guilln et al., 2000; Wan and Li, 2010). Therefore, analysis
of the whole process is relevant to understand the mechanisms
of fungal degradation and to get the best fungi and optimum
growth conditions for obtaining the maximum amount of ferment-
able sugars.
The pretreatment of wheat straw by using basidiomycetes to
produce ethanol has been barely studied. Moreover, the rates of
this type of pretreatment are still far from industrial purposes be-
sides of presenting disadvantages, as long storage times or ex-
tended cellulose and hemicellulose consumptions (Galbe and
Zacchi, 2007). Nevertheless, it could be used alone or combined
with other pretreatments to result more effective. Alkali pretreat-
ments are well known to improve sugar recovery because they
cause lignin solubilisation (Kumar et al., 2009; Talebnia et al.,
2010) but only a few investigations have complemented it with a
biological pretreatment (Hatakka, 1983; Yu et al., 2010). In the
present study, a fungal screening using 21 basidiomycetes has
been carried out, combined with a very mild alkali washing, to se-
lect the best fungal strains to be included in the pretreatment step
for second generation bioethanol production from wheat straw.
The relationship among different variables, such as lignin degrada-
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.05.027

Corresponding author. Address: Centro de Investigaciones Biolgicas, Depart-

ment of Environmental Biology, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Tel.: +34 918373112; fax: +34 915360432.
E-mail address: mjmartinez@cib.csic.es (M.J. Martnez).
Bioresource Technology 102 (2011) 75007506
Contents lists available at ScienceDirect
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j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
tion, cellulose and hemicellulose digestibility, and ethanol produc-
tion was analysed, and the role of the ligninolytic enzymes in the
process is discussed.
2. Methods
2.1. Fungal strains and culture media
The strains of basidiomycetes used in the present study were
obtained from different fungal collections: Centraalbureau voor
Schimmelcultures (CBS; Baarn, The Netherlands), Instituto Jaime
Ferrn de Microbiologa (IJFM; Centro Investigaciones Biolgicas,
Madrid, Spain) and Coleccin Espaola de Cultivos Tipo (CECT;
Burjassot, Valencia, Spain). Most of the fungi used in this study
were white-rot fungi: Bjerkandera anamorph IJFM A757, Bjerkan-
dera adusta CBS 595.78, Coriolopsis rigida CECT 20449, Fomes fasci-
atus IJFM A772, Fomes fomentarius IJFM A166, Ganoderma australe
IJFM A130, Irpex lacteus IJFM A792, Lentinus tigrinus IJFM A790, Pa-
nus tigrinus (a synonym of L. tigrinus) IJFM A768, Phanerochaete
chrysosporium CBS 481.73, Phellinus robustus IJFM A788 , Phlebia
radiata CBS 184.83, Phlebiopsis gigantea CBS 935.7, Pleurotus eryngii
CBS 613.91, Pleurotus ostreatus CBS 411.71, Polyporus alveolaris
IJFM A794, Poria subvermispora (a synonym of Ceriporiopsis subver-
mispora) IJFM A718, Pycnoporus coccineus IJFM A780, Stereum hirsu-
tum IJFM A793, and Trametes versicolor IJFM A136. In addition one
brown-rot fungus was used, Postia placenta IJFM A781. Strains
were maintained on 2% malt extract agar (w/v) and preserved at
4 C.
Fungal strains were individually cultured at 28 C for 7 days on
MEA plates. Four agar plugs of about 1 cm
2
were cut from actively
growing mycelium and inoculated into 250 mL Erlenmeyer asks
with 30 mL of growth medium (pH 5.6) and incubated at 28 C,
and 180 rpm for 7 days. The growth medium contained (L
1
): glu-
cose, 40 g; FeSO
4
7H
2
O, 0.4 g; (NH
4
)
2
SO
4
, 9 g; KH
2
PO
4
, 4 g; corn
steep solids, 26.3 g; CaCO
3
, 7 g; soybean oil, 2.8 mL. Each culture
was aseptically homogenised (Omnimixer, Sorvall), and 2.5 mL
were used to inoculate second generation cultures, which were
incubated for 5 days as described above. These cultures will be
used as inocula for solid state fermentation (SSF) experiments.
2.2. Pretreatment of wheat straw
2.2.1. Fungal screening
Wheat (Triticum aestivum) straw was harvested from Galicia
elds (Spain) and chopped (<1 cm). Two grams of dry grinded
wheat straw plus 6 mL of distilled water were autoclaved at
121 C for 15 min into 100 mL Erlenmeyer asks capped with
hydrophobic cotton. These asks were inoculated with 5-day-old
mycelia (2 mL) and incubated at 28 C. Triplicate asks of each fun-
gal culture were sampled after 7, 14 and 21 days. Samples were
washed with distilled water (15 mL) at 28 C and 180 rpm for
1 h, and ltered under vacuum to remove most of the water-solu-
ble components, which were stored at 4 C. The solid fractions
from biopretreated wheat straw were dried in an aeration oven
at 65 C and weighted. This value was used to calculate weight loss
of the samples. Non-inoculated samples were incubated and trea-
ted under the same conditions, being used as control.
2.2.2. Mild alkali treatment
Solid fractions (300 mg, dry weight) were subjected to a mild al-
kali treatment with a nal concentration of 0.1% sodium hydroxide
(5% w/v), at 50 C and 165 rpm for 1 h. The alkali-treated material
was ltered and washed, until neutrality, with distilled water at
50 C. Total reducing sugars were measured (Somogyi, 1945) in
the ltrates, and the solid residue was dried at 60 C and weighted.
The effect of the alkali treatment on subsequent enzymatic hydro-
lysis was investigated by comparison of different samples with and
without alkali washing.
2.3. Enzymatic hydrolysis and sugar yield estimation
Solid residues or solid fractions, with or without alkali treat-
ment respectively, were hydrolysed in duplicate at 5% (w/v) by en-
zyme complexes (Novozymes Bagsvaerd, Denmark) as 15 FPU g
1
of cellulases (Celluclast and NS50010) and 30 U g
1
of xylanases
(NS50013 and NS50030) in 100 mM sodium citrate buffer (pH
4.8) at 50 C, and 165 rpm for 60 h. Tetracycline (200 lg mL
1
)
was also added to avoid bacterial growth during enzymatic treat-
ments. After hydrolysis, 0.5 mL of treated material were centri-
fuged at 5000 rpm for 5 min, and glucose and xylose content
were measured in the supernatants. The Glucose-TR kit (Spinre-
act) was used to quantify glucose. Xylose content was calculated as
the difference between total reducing sugars (Somogyi, 1945) and
glucose. A set of samples was chosen to quantify glucose and xy-
lose also by gas chromatography (GC) (Prieto et al., 2008), to com-
pare both methodologies.
To probe enzymatic hydrolysis efciency, cellulose (D
c
) and
hemicellulose (D
h
) digestibilities were evaluated and expressed,
according to Eq. (1), as the quotient between the percentage of glu-
cose (G
r
) and xylose (X
r
) released from either the solid fraction or
the solid residue from alkali washing and the theoretical maximum
amount of glucose (G
s
) and xylose (X
s
) in the solid fraction,
respectively.
D
c
orD
h
% g G
r
orX
r
=g G
s
orX
s
100 1
To calculate sugar yields, glucose (G
i
) and xylose (X
i
) per gramof
dry wheat straw, glucose (G
f
) and xylose (X
f
) remaining in pre-
treated wheat straw, and cellulose (D
g
) and hemicellulose (D
h
)
digestibilities were taken into account as shown in Eq. (2). Thus,
this yield considers both, the sugar digestibility (D
c
or D
h
) and
the percentage of sugar loss during the biological pretreatment
(G
f
/G
i
or X
f
/X
i
).
Sugaryield% G
f
D
g
X
f
D
h
=G
i
X
i
2
Glucose and xylose recovery yields were separately estimated
applying Equations (3) and (4), respectively.
Glucoseyield% G
f
D
c
=G
i
3
Xyloseyield% X
f
D
h
=X
i
4
2.4. Fermentation to ethanol
To evaluate the potential inhibitory effect of fungal pretreat-
ment and alkali washing on yeast growth, solid fractions or solid
residues, hydrolysed with cellulases and xylanases as previously
indicated, were subsequently fermented with Saccharomyces cere-
visiae (Fermentis LPA 3035). The yeast (0.5 g L
1
inoculum) was
grown at 32 C and 200 rpm for 24 h in 250 mL Erlenmeyer asks
containing 50 mL of a liquid medium. The medium was composed
of (L
1
) glucose (20 g), yeast extract (3 g), peptone (5 g), and tetra-
cycline (200 mg). Seven millilitres of hydrolysed wheat straw sam-
ples were inoculated with 350 lL of a yeast suspension (OD
625 nm = 2) in 10 mL glass tubes. Tubes were sealed with rubber
plugs and incubated at 32 C and 200 rpm for 72 h. Then, tubes
were centrifuged for 2 min at 7000 rpm and 5 mL of the superna-
tant were extracted with ethanol-free chloroform (0.5 mL) to
determine the ethanol content in the organic phase by gas chroma-
tography (GC). Methanol (1%) was added in the samples before
chloroform extraction, as internal standard. GC analyses were per-
formed on an Agilent 7890A instrument equipped with a ame
D. Salvacha et al. / Bioresource Technology 102 (2011) 75007506 7501
ionisation detector (FID), using a HP5-MS capillary column
(30 m 0.25 mm, 0.25 lm lm thickness) and helium (25 psi) as
the carrier gas. Separation was carried out isothermically at
28 C, and injector and detector were maintained at 100 C. Peaks
were identied on the basis of sample coincidence with retention
times of commercial standards, and quantied using peak areas
and the corresponding response factors. All the experiments were
carried out by triplicate. Finally, the ethanol conversion yield was
calculated as shown in Eq. (5) taking into account the glucose
recovery after enzymatic hydrolysis. The value 0.511 corresponds
to the conversion factor from glucose to ethanol (Maiorella et al.,
1981).
Glucose conversion %

gL
1
ethanol produced in fermentation broth
gL
1
initial glucose in fermentation broth 0:511
100
5
2.5. Substrate characterisation and analysis methods
Weight loss was calculated as the percentage of total solids lost
after each biopretreatment. Klason lignin content and polysaccha-
ride composition of untreated and biopretreated wheat straw were
determined on hydrolysates according to standard Tappi methods
(Tappi, 1974, 1975). Glucose and xylose were also measured as de-
scribed in Section 2.3. The content of cellulose was calculated from
glucose while hemicellulose was calculated from xylose, using an
anhydro correction of 0.90 and 0.88 for both sugars, respectively.
Total reducing sugars in the water-soluble extracts were deter-
mined using the Somogyi method (Somogyi, 1945).
2.6. Estimation of ligninolytic activities
Enzymatic activities were evaluated in water-soluble extracts of
biopretreated wheat straw (Section 2.2.1). Laccase activity was as-
sayed using 5 mM 2,6-dimethoxyphenol (DMP) in 100 mM sodium
citrate buffer (pH 5.0; e
469
= 27,500 M
1
cm
1
, referred to DMP
concentration). Mn
2+
-oxidising peroxidase was estimated by mea-
suring the formation of Mn
+3
-tartrate complex (e
238
=
6500 M
1
cm
1
) during the oxidation of 0.1 mM MnSO
4
in
100 mM sodium tartrate buffer (pH 5.0) in the presence of
0.1 mM H
2
O
2
. Lignin peroxidase was assayed by veratraldehyde
formation from 2 mM veratryl alcohol (3,4-dimethoxybenzyl alco-
hol) in 100 mM sodium tartrate buffer (pH 3), in the presence of
0.4 mM H
2
O
2
. International enzymatic units (lmoles per minute)
were used.
3. Results and discussion
3.1. Fungal pretreatment of wheat straw
3.1.1. Cell wall components degradation
The objective of this study was to select fungal species, in a
wide screening, to produce second generation bioethanol from bio-
pretreated wheat straw. The selected strain should be the one giv-
ing the highest amount of fermentable sugars from wheat straw in
the shortest period of time. A screening of fungi was carried out to
evaluate lignin and polysaccharides degradation from wheat straw
after 7, 14 and 21 days of SSF. Most fungi colonised the substrate
appropriately, except F. fasciatus which presented a poor evolution.
Polysaccharide content was rst estimated from GC analysis.
Untreated wheat straw used as control (0 and 21 days of incuba-
tion), was composed of 36.9% cellulose and 23% hemicellulose
(18% xylan, 3.4% arabinan, 1.1% mannan, and 0.5% galactan). Cellu-
lose and hemicellulose content were also evaluated by colorimetric
methods. Glucose, assayed by the Glucose-TR kit (Spinreact), and
xylose, determined by difference between total sugars and glucose,
gave values not signicantly different to those detected by GC. In
the case of xylose this result could be explained because it is the
major hemicellulose component in wheat straw (as shown by
GC). In addition, this lignocellulosic material contained 24% lignin
(22.8% acid-insoluble lignin and 1.2% acid-soluble lignin).
Weight loss of the cultures gives an estimation of the extent of
substrate degradation. The greatest weight losses, after 21 days of
SSF, were caused by B. adusta, F. fomentarius and P. coccineus (up to
35%), and the lowest by P. eryngii, P. gigantea and P. placenta (down
to 6%). Composition of wheat straw after fungal treatment was
analysed (Table 1) and different degradation patterns were appre-
ciated among the studied basidiomycetes. Some fungi, as P. chry-
sosporium and P. gigantea, as well as the brown-rot P. placenta,
were not able to degrade lignin under the assayed culture condi-
tions, showing a polysaccharidic preferential degradation. Other
fungi, as P. tigrinus and P. radiata, degraded lignin and sugars
simultaneously. Finally, basidiomycetes as P. eryngii and P. robustus
were able to remove lignin selectively and faster than the carbohy-
drate components in wheat straw.
On the other hand, sugar degradation can be balanced or prefer-
ential from cellulose or hemicellulose. B. adusta degraded both
polymers equitably (54% and 43% respectively). S. hirsutum only al-
tered cellulose (43%) and P. coccineus degraded almost all hemicel-
lulose (98%) but less cellulose (31%). These data give information
about the amount of glucose and xylose (from cellulose and hemi-
cellulose, respectively) available for alcohol fermentation, since
largest degradations should imply a performance decrease.
3.1.2. Water-soluble fraction analysis
To evaluate the total sugar recovery, water-soluble sugars from
the hydrosoluble fraction were also analysed, since they could also
be potentially fermented. The greatest recovery reached only 6%
after 21 days of pretreatment with P. radiata. Signicantly less
water-soluble sugars were quantied at 7 or 14 days with this fun-
gus, and even at 21 days with the remaining fungi screened (data
not shown). These results are in agreement with previous studies
which showed that the two rst weeks of incubation correspond
to an early delignication stage with lowwater-soluble sugars con-
tent because fungi consume monosaccharides and disaccharides to
grow (Valmaseda et al., 1991). Because of its low amount of sugars,
the water-soluble fraction was not enzymatically hydrolysed for
further ethanol production by yeasts. Consequently, this fraction
was not taken into account in the nal process yield.
3.2. Enzymatic hydrolysis
3.2.1. Digestibility
Digestibility represents the yield of conversion of the raw mate-
rial into fermentable sugars. To increase this value in biopretreated
wheat straw, samples are subjected to a very mild alkali treatment
before enzymatic hydrolysis with cellulases and xylanases (Kumar
et al., 2009; Yu et al., 2010, 2009). Preliminary studies in our labo-
ratory showed that alkali treatment with only 0.1% NaOH at 50 C
during 1 h does not affect xylose recovery, probably because hemi-
cellulose does not form packed crystalline structures like cellulose,
becoming a substrate more accessible to fungi and enzymatic
hydrolysis (Xu et al., 2009). However, this step is crucial to improve
glucose release from cellulose, since digestibility at 21 days in-
creased more than twice in several biopretreated samples (data
not shown). Recently the use of a combined biological and mild
chemical pretreatment of cornstalks has been reported (Yu et al.,
2010). They obtained values of glucan digestibility comparable to
those found in the present study, using similar temperatures and
7502 D. Salvacha et al. / Bioresource Technology 102 (2011) 75007506
incubation times but with a NaOH concentration tenfold higher.
There are several advantages of using low amounts of alkali for
the chemical treatment. First of all, the effect of the biological pre-
treatment can be clearly observed, since it is not masked as a result
of more aggressive alkali pretreatments. In addition, the process is
cheaper and the generation of inhibitors for downstream steps of
the process is diminished or even avoided.
Cellulose and hemicellulose conversion to fermentable sugars
from biopretreated wheat straw is depicted in Fig. 1. No differences
were found between controls analysed at the beginning (0 days)
and at the end of the incubation time (21 days). In both cases,
the conversion of cell wall polysaccharides to glucose and xylose
was around 36% and 35%, respectively. Only eight of the fungal
strains studied increased digestibilities at 14 and 21 days of bio-
pretreatment with respect to the controls, and only one of them,
P. tigrinus, was able to improve them after 7 days of SSF. The great-
est values for glucose and xylose recovery were 82% and 78% in
samples pretreated for 21 days with I. lacteus and P. tigrinus,
respectively. Our results show higher increases in wheat straw
digestibility in shorter incubation times than those reported in pre-
vious studies (Capelari and Toms-Pej, 1997; Dias et al., 2010;
Valmaseda et al., 1991).
Lignin polymers are the main obstacle to the efcient utilisation
of lignocellulosic materials. Consequently, a preferential deligni-
Table 1
Degradation of wheat straw components (% of the initial content) produced by 21 basidiomycetes after incubation periods of 7, 14, and 21 days. Data are means of triplicates
(SD). CEL, cellulose; HEM, hemicellulose; LIG, lignin. Bjerkandera

= Bjerkandera anamorph.
Fungi Loss (%)
7 days 14 days 21 days
CEL HEM LIG CEL HEM LIG CEL HEM LIG
Bjerkandera

14 0 7 1 4 3 16 0 14 3 1 0 21 1 12 1 6 0
B. adusta 27 0 0 0 9 0 42 2 16 2 28 1 54 6 43 5 37 0
C. rigida 18 0 28 6 15 0 22 1 52 10 24 1 36 2 42 12 34 1
F. fasciatus 0 0 0 1 0 0 0 0 0 3 0 0 0 0 0 1 0 0
F. fomentarius 17 1 2 0 0 0 30 1 28 4 17 0 45 1 51 27 35 1
G. australe 4 0 13 0 0 0 4 0 16 1 9 0 15 0 23 3 25 1
I. lacteus 9 0 13 7 11 0 17 1 13 4 27 1 17 0 26 7 34 2
L. tigrinus 24 1 46 10 0 0 28 1 68 12 15 1 40 1 58 9 23 1
P. tigrinus 12 0 24 7 17 1 20 1 41 7 32 1 24 1 60 26 47 4
P. chrysosporium 23 0 36 8 0 0 31 2 22 2 0 0 35 0 70 24 0 0
P. robustus 4 0 0 1 0 0 6 0 0 0 21 1 8 1 3 0 25 2
P. radiata 13 1 36 11 8 0 20 2 40 10 29 1 24 3 41 5 40 2
P. gigantea 8 0 0 0 0 0 9 1 0 0 0 0 7 0 9 1 0 0
P. eryngii 0 0 0 0 2 0 0 0 4 0 14 0 0 0 8 1 17 1
P. ostreatus 10 1 14 1 2 0 14 1 38 9 18 0 22 1 52 13 27 1
P. alveolaris 14 1 8 1 18 1 18 0 28 9 34 1 28 2 42 8 43 2
P. subvermispora 1 0 33 10 8 0 4 0 35 6 25 1 13 0 36 6 30 1
P. placenta 3 0 11 2 0 0 0 0 15 3 0 0 2 0 9 3 1 0
P. coccineus 12 0 74 43 11 0 26 2 77 20 26 1 31 1 98 1 36 1
S. hirsutum 24 0 0 1 15 1 37 4 1 0 30 1 43 2 2 0 37 2
T. versicolor 12 0 5 1 24 1 18 1 23 3 33 0 23 1 21 6 46 1
Cellulose digestibility (%)
Untreated
*
Bjerkandera*
B. adusta
C. rigida
F. fasciatus
F. fomentarius
G. australe
I. lacteus
L. tigrinus
P. tigrinus
P. chrysosporium
P. robustus
P. radiata
P. gigantea
P. eryngii
P. ostreatus
P. alveolaris
P. subvermispora
P. placenta
P. coccineus
S. hirsutum
T. versicolor
Hemicellulose digestibility (%)
100 90 80 70 60 50 40 30 20 10 0
0 10 20 30 40 50 60 70 80 90 100
Fig. 1. Cellulose and hemicellulose digestibility (%) from pretreated wheat straw. White, dotted and black bars correspond to 7, 14, and 21 day cultures respectively. Control
corresponds to non-inoculated wheat straw. Data are means of triplicates. Bjerkandera

= Bjerkandera anamorph.
D. Salvacha et al. / Bioresource Technology 102 (2011) 75007506 7503
cation would improve the process performance because it would
facilitate the access of hydrolytic enzymes to polysaccharides
(Camarero et al., 1994; Kuhar et al., 2008; Valmaseda et al.,
1991) maintaining at the same time a good level of fermentable
sugars, which would be only slightly consumed for fungal growth.
With the aim to correlate both variables, lignin degradation and
digestibility were compared. The two fungi that generated the
highest lignin degradation, T. versicolor (46%) and P. tigrinus
(47%), gave pretreated wheat straw with signicant differences in
cellulose and hemicelluloses digestibilities. After treatment with
T. versicolor, cellulose digestibility was 25% less than after pretreat-
ing with P. tigrinus. Otherwise, hemicellulose digestibility was not
improved by T. versicolor treatment as compared with untreated
wheat straw while, after growing of P. tigrinus, a 78% of conversion
was reached. In contrast, fungi as P. eryngii and P. robustus, which
did not produce high lignin losses, were able to raise cellulose
and hemicellulose digestibility of the substrate. According to these
data, although lignin attack is essential to the efciency of the
enzymatic hydrolysis of cell wall polysaccharides, the highest lig-
nin degradation is not always positively correlated with the high-
est levels of cellulose and hemicellulose digestibility. These results
agree with previous reports (Capelari and Toms-Pej, 1997; Ml-
ler and Trsch, 1986), which remark that the level of delignication
cannot be considered as the only parameter to assess if a microor-
ganism is a valid candidate for biological pretreatment.
3.2.2. Fermentable sugar yields
Digestibility values and carbohydrate losses during biopretreat-
ment were essential to quantify the amount of potentially ferment-
able sugars. Sugars were not found in the liquid fraction after alkali
washing what indicates that only lignin was removed in this step.
The treatments which improved the recovery of fermentable
sugars, compared to untreated wheat straw, are presented in
Fig. 2. Glucose yields increased in most cases after 21 days of fun-
gal treatment, especially with P. subvermispora (69%) and I. lacteus
(66%). In addition, only these two fungi led to a signicant increase
in glucose yield in samples pretreated for 14 days. During the rst
2 weeks of incubation, fungi consume a huge amount of glucose
and energy for their own growth. After this time, they continue
on wheat straw degradation consuming less sugars, which in-
creases the nal sugar recovery (Valmaseda et al., 1991). In the
present work we have obtained the highest yields reported so far
from wheat straw combining a 21 days-biological pretreatment
with a very mild chemical reagent. Recent studies described simi-
lar glucose recoveries by using corn stover treated with C. subver-
mispora during 35 days (Wan and Li, 2010) and rice straw treated
with P. chrysosporium for 15 days (Bak et al., 2009) although in this
case the raw material was autoclaved twice, before and after
biopretreatment.
Concerning xylose yields, the differences between untreated
and pretreated samples were not very signicant, excluding I. lac-
teus pretreatment which reached 62% and 47% at 14 and 21 days,
respectively. This nding set out that, among the studied basidio-
mycetes, this fungus would be the best candidate to recover xylose
from biological pretreatment. At the present time, xylose cannot be
fermented at industrial scale; however it is important to know the
extent of hemicellulose conversion to xylose to be taken into ac-
count to improve process yields in the future.
Considering the total sugar yield (data not shown), the best re-
sults were obtained after 21 days of incubation with I. lacteus (62%)
and P. subvermispora (61%). On the contrary, treatment with P.
ostreatus did not increase signicantly this yield and was not in-
cluded in further ethanol fermentation experiments.
3.2.3. Relationship between fungal enzymes and sugar yield
Differences in wheat straw degradation have been related to
variations in the pattern and levels of ligninolytic enzymes (Cam-
arero et al., 1996; Manubens et al., 2007; Pedersen and Meyer,
2009). Consequently, those enzymes could be used as markers of
the process yield. In this study, Mn
2+
-oxidising peroxidases, laccase
and LiP activities, as models of enzymes closely related to lignin
degradation, were analysed in the water-soluble extracts from
the 21 fungal strains during the 3 weeks of incubation (Fig. 3).
LiP was not detected in any case.
The largest Mn
2+
-oxidising peroxidase activities (per gram of
dry wheat straw) were detected in the seven cultures that gave
an improved sugar recovery, with the maximum values in 14-days
cultures of P. radiata (6.9 U g
1
) and P. robustus (6.7 U g
1
). In any
case, these high activities were not correlated with the best sugar
recoveries. On the other hand, the highest laccase activities were
found in P. robustus (3.3 U g
1
) and P. eryngii (2.4 U g
1
) at 7 and
14 days, respectively. Both fungi displayed a preferential degrada-
tion of lignin, which could be related to the high laccase secretion
detected in these fungi. Fungi as I. lacteus and P. subvermispora,
which showed low laccase activity (<0.25 U g
1
) and not very high
Mn
2+
-oxidising peroxidase activity (<3.6 U g
1
), gave the best su-
gar yields after wheat straw biopretreatment. These species
showed a simultaneous degradation of all lignocellulosic compo-
nents. Alternatively, other fungi which produced high lignin degra-
dation presented very low ligninolytic activities (as B. adusta and C.
rigida).
These results corroborate again that it is not easy to nd a direct
correlation among enzyme production, lignin degradation, and su-
gar yield in biopretreated wheat straw. As previously stated, lignin
degradation is an oxidative and rather nonspecic process where
extracellular ligninolytic enzymes participate, but also low molec-
ular-weight extracellular oxidant compounds (e.g. Mn
3+
and oxy-
gen free radicals), which can be generated during the process,
having a very important role (Guilln et al., 2000; Hammel et al.,
2002).
3.3. Ethanol production
Evaluation of ethanol production is necessary to quantify the
process nal performance. At industrial level, only glucose is being
fermented with high ethanol production yields while xylose fer-
mentation, which is also essential for the economical success of lig-
nocellulosic ethanol, continues being investigated to raise the low
yields obtained so far (Grio et al., 2010; Lee, 1997).
Glucose fermentations by S. cerevisiae were carried out on the
seven enzymatic hydrolysates which gave signicantly improved
G
l
u
c
o
s
e

a
n
d

x
y
l
o
s
e

y
i
e
l
d

(
%
)
0
10
20
30
40
50
60
70
80
Glucose (14d) Glucose (21d)
Xylose (14d) Xylose (21d)
Fig. 2. Glucose and xylose yield (%) from 14 and 21 days pretreated wheat straw.
Fungi are listed in order of the best sugar yields (left to right). Control corresponds
to non-inoculated wheat straw. Data are means of triplicates.
7504 D. Salvacha et al. / Bioresource Technology 102 (2011) 75007506
sugar recoveries as compared to control samples. Most conversion
yields from available glucose to ethanol were superior to 90% ex-
cept those coming from pretreatments with P. tigrinus, P. eryngii
and P. robustus, which showed conversions of 84%, 81% and 79%,
respectively. These results indicate that the fungal plus alkali
washing pretreatment of wheat straw does not generate signicant
inhibitors of yeast growth. Based on the dry weight of wheat straw
(1 g), the glucose availability (0.41 g per gram of dry wheat straw)
and the stoichiometry of the reaction (1 glucose ?2 ethanol + 2
CO
2
), and estimating that 5% of glucose is used for yeast metabo-
lism, the theoretical maximum ethanol production is approxi-
mately 0.2 g per gram of dry wheat straw (Eq. (5)). In this study,
after checking total glucose consumption for yeast growth, the
largest ethanol production found, which corresponds to the highest
process yield, was around 62% of the theoretical maximum in sam-
ples pretreated with I. lacteus and P. subvermispora for 21 days (Ta-
ble 2). These results were slightly higher than those reported for
35 day-pretreated corn stover with P. subvermispora (Wan and Li,
2010) and similar to those obtained from rice straw, autoclaved
twice and pretreated for 15 days with P. chrysosporium (62.7%)
(Bak et al., 2009). In contrast, much lower ethanol production has
been obtained from 14 days pretreated cotton stalks with P. chry-
sosporium (13%) (Shi et al., 2009).
The complete process from wheat straw to ethanol with the
fungi which improved sugar recoveries, as compared to biologi-
cally untreated wheat straw is summarised in Table 2. It has been
previously stated that to increase the nal sugar yields it would be
desirable to have a low consumption of sugars during biopretreat-
ment. However, it can be highlighted that this is not the most
important variable. It can be observed that although P. eryngii
and P. robustus (fungi which degraded selectively the lignin) con-
sumed less glucose for their growth than the other fungi, enzy-
matic hydrolysis after biopretreatments released the lowest
amounts of glucose. On the other hand P. alveolaris, which con-
sumed the highest amount of glucose from wheat straw, gave a
glucose recovery after enzymatic hydrolysis slightly higher than
those found in the above mentioned species. Finallly, I. lacteus
and P. subvermispora, showed intermediate levels of glucose con-
sumption but gave the best glucose recoveries after enzymatic
hydrolysis and also the best yields of ethanol production. Then,
they can be considered as the best species to be potentially used
for wheat straw biopretreatment. Since conversions from glucose
to ethanol were high in all cases, it can be guessed that the main
differences in the whole process should arise from biopretreat-
ment. Lignocellulose degradation mechanisms are very difcult
to predict because of their complexity and variety, and inuence
Fig. 3. Ligninolytic enzymes secreted by fungi during wheat straw biopretreatment: (a) Mn
2+
-oxidising peroxidase and (b) laccase. White, dotted and black bars correspond
to 7, 14, and 21 day cultures, respectively. Data are means of triplicates. Bjerkandera

= Bjerkandera anamorph.
Table 2
Monitoring of glucose content (GLC) and ethanol production per gram of dry wheat straw (g WS). Wheat straw samples were biopretreated (B) during 21 days. A mild alkali
washing (AW) was done after biopretreatment. Data are means of triplicates (SD). EH = enzymatic hydrolysis.
Process step Initial WS Pretreatment (B + AW) EH GLC fermentation Process yield (%)
GLC (mg/g WS) GLC (mg/g WS) GLC (mg/g WS) Ethanol (mg/g WS)
Theoretical maximum 410 410 410 199 100
Control
a
410 9 410 9 133 7 68 2 35
I. lacteus 410 9 340 2 254 8 123 5 62
P. subvermispora 410 9 357 1 260 14 122 8 62
P. radiata 410 9 311 13 202 13 97 9 49
P. tigrinus 410 9 311 3 227 18 97 4 49
P. alveolaris 410 9 295 10 207 7 94 8 47
P. robustus 410 9 377 5 193 21 78 0 39
P. eryngii 410 9 410 0 151 22 62 9 31
a
Control is a biologically untreated sample, only subjected to alkali washing.
D. Salvacha et al. / Bioresource Technology 102 (2011) 75007506 7505
the subsequent enzymatic hydrolysis step. Then, a complete study
of the process is required for each fungal treatment, in order to
analyse the efciency of the biological pretreatment on ethanol
production.
Chen et al. (2007) reported on a chemical pretreatment of
wheat straw using acid and alkaline reagents, which allowed the
recovery of more glucose (>10%) but with an ethanol production
only 3% higher than the maximum reached in this study. Probably
this is because, as stated above, biopretreatment does not generate
toxic by-products affecting yeast growth, while steam explosion
does (Alvira et al., 2010). Our results are still far from the high
yields obtained using combined steam explosion and alkaline per-
oxide pretreatments (Chen et al., 2008), but suggest that the bio-
logical pretreatment with I. lacteus or P. subvermispora,
complemented with a very mild alkali washing, could be an alter-
native to replace certain current chemical pretreatments without
generating inhibitors of the fermentation step.
4. Conclusions
Our data showed that very few fungi are suitable to increase su-
gar recoveries from wheat straw. The combination of a biological
pretreatment by I. lacteus or P. subvermispora with a mild alkali
pretreatment did not produce inhibitors for downstream pro-
cesses, improving signicantly ethanol production. These results
turn both methods into possible and environmentally friendly
alternatives in the production of second-generation ethanol. At
the present time, pretreatments of wheat straw with the selected
fungi are being carried out to scale up the process and check its via-
bility at industrial level.
Acknowledgements
This work was supported mainly by the CENIT I+DEA project
(funded by CDTI, Spain) and carried out in collaboration with
Abengoa Bionerga Nuevas Tecnologas. Authors thank also the
Galician government (I. Barreto program), DEMO-2 and Lignodeco
EU projects for additional supports, and Novozymes for providing
commercial enzymes. D.S. thanks a FPU fellowship from the
MICINN.
References
Alvira, P., Toms-Pej, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment
technologies for an efcient bioethanol production process based on
enzymatic hydrolysis: a review. Bioresour. Technol. 101, 48514861.
Bak, J.S., Ko, J.K., Choi, I.G., Park, Y.C., Seo, J.H., Kim, K.H., 2009. Fungal pretreatment
of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice
straw. Biotechnol. Bioeng. 104, 471482.
Camarero, S., Galletti, G.C., Martnez, A.T., 1994. Preferential degradation of phenolic
lignin units by two white rot fungi. Appl. Environ. Microbiol. 60, 45094516.
Camarero, S., Bockle, B., Martnez, M.J., Martnez, A.T., 1996. Manganese-mediated
lignin degradation by Pleurotus pulmonarius. Appl. Environ. Microbiol. 62, 1070
1072.
Capelari, M., Toms-Pej, E., 1997. Lignin degradation and in vitro digestibility of
wheat straw treated with Brazilian Tropical species of white-rot fungi. Folia
Microbiol. 42, 481487.
Chen, Y., Sharma-Shivappa, R., Keshwani, D., Chen, C., 2007. Potential of agricultural
residues and hay for bioethanol production. Appl. Biochem. Biotechnol. 142,
276290.
Chen, H., Han, Y., Xu, J., 2008. Simultaneous saccharication and fermentation of
steam exploded wheat straw pretreated with alkaline peroxide. Process
Biochem. 43, 14621466.
Dias, A.A., Freitas, G.S., Marques, G.S.M., Sampaio, A., Fraga, I.S., Rodrigues, M.A.M.,
Evtuguin, D.V., Bezerra, R.M.F., 2010. Enzymatic saccharication of biologically
pre-treated wheat straw with white-rot fungi. Bioresour. Technol. 101, 6045
6050.
Galbe, M., Zacchi, G., 2007. Pretreatment of lignocellulosic materials for efcient
bioethanol production. Adv. Biochem. Eng./Biotechnol. 108, 4165.
Grio, F.M., Fonseca, C., Carvalheiro, F., Duarte, L.C., Marques, S., Bogel-Lukasik, R.,
2010. Hemicelluloses for fuel ethanol: a review. Bioresour. Technol. 101, 4775
4800.
Guilln, F., Muoz, C., Gomez-Toribio, V., Martnez, A.T., Martnez, M.J., 2000.
Oxygen activation during oxidation of methoxyhydroquinones by laccase from
Pleurotus eryngii. Appl. Environ. Microbiol. 66, 170175.
Hammel, K.E., Kapich, A.N., Jensen Jr., K.A., Ryan, Z.C., 2002. Reactive oxygen species
as agents of wood decay by fungi. Enzyme Microb. Technol. 30, 445453.
Hatakka, A.I., 1983. Pretreatment of wheat straw by white-rot fungi for enzymic
saccharication of cellulose. Appl. Microbiol. Biotechnol. 18, 350357.
Jurado, M., Prieto, A., Martnez-Alcal, A., Martnez, A.T., Martnez, M.J., 2009.
Laccase detoxication of steam-exploded wheat straw for second generation
bioethanol. Bioresour. Technol. 100, 63786384.
Kim, S., Dale, B.E., 2004. Global potential bioethanol production from wasted crops
and crop residues. Biomass Bioenerg. 26, 361375.
Kuhar, S., Nair, L.M., Kuhad, R.C., 2008. Pretreatment of lignocellulosic material with
fungi capable of higher lignin degradation and lower carbohydrate degradation
improves substrate acid hydrolysis and the eventual conversion to ethanol. Can.
J. Microbiol. 54, 305313.
Kumar, P., Barrett, D., Delwiche, M., Stroeve, P., 2009. Methods for pretreatment of
lignocellulosic biomass for efcient hydrolysis and biofuel production. Ind. Eng.
Chem. Res. 48, 37133729.
Lee, J., 1997. Biological conversion of lignocellulosic biomass to ethanol. J.
Biotechnol. 56, 124.
Lin, Y., Tanaka, S., 2006. Ethanol fermentation from biomass resources: current state
and prospects. Appl. Microbiol. Biotechnol. 69, 627642.
Maiorella, B., Wilke, Ch.R., Blanch, H.W., 1981. Alcohol production and recovery.
Adv. Biochem. Eng./Biotechnol. 20, 4392.
Manubens, A., Canessa, P., Folch, C., Avila, M., Salas, L., Vicua, R., 2007. Manganese
affects the production of laccase in the basidiomycete Ceriporiopsis
subvermispora. FEMS Microbiol. Lett. 275, 139145.
Mller, H.W., Trsch, W., 1986. Screening of white-rot fungi for biological
pretreatment of wheat straw for biogas production. Appl. Microbiol.
Biotechnol. 24, 180185.
Pedersen, M., Meyer, A.S., 2009. Inuence of substrate particle size and wet
oxidation on physical surface structures and enzymatic hydrolysis of wheat
straw. Biotechnol. Prog. 25, 399408.
Prieto, A., Leal, J.A., Bernab, M., Hawksworth, D.L., 2008. A polysaccharide from
Lichina pygmaea and L. Connis supports the recognition of Lichinomycetes.
Mycol. Res. 112, 381388.
Shi, J., Chinn, M.S., Sharma-Shivappa, R.R., 2008. Microbial pretreatment of cotton
stalks by solid state cultivation of Phanerochaete chrysosporium. Bioresour.
Technol. 99, 65566564.
Shi, J., Sharma-Shivappa, R.R., Chinn, M., Howell, N., 2009. Effect of microbial
pretreatment on enzymatic hydrolysis and fermentation of cotton stalks for
ethanol production. Biomass Bioenerg. 33, 8896.
Somogyi, M., 1945. A new reagent for the determination of sugars. J. Biol. Chem.
160, 6173.
Talebnia, F., Karakashev, D., Angelidaki, I., 2010. Production of bioethanol from
wheat straw: an overview on pretreatment, hydrolysis and fermentation.
Bioresour. Technol. 101, 47444753.
Tappi, 1974. Acid-insoluble lignin in wood and pulp. Tappi Rule. T 222-os, -74.
Tappi, 1975. Carbohydrate composition of extractive-free wood and wood pulp by
gasliquid chromatography. Tappi Rule. T 249-pm, -75.
Valmaseda, M., Martnez, M.J., Martnez, A.T., 1991. Kinetics of wheat straw solid-
state fermentation with Trametes versicolor and Pleurotus ostreatus: lignin and
polysaccharide alteration and production of related enzymatic activities. Appl.
Microbiol. Biotechnol. 35, 817823.
Wan, C., Li, Y., 2010. Microbial pretreatment of corn stover with Ceriporiopsis
subvermispora for enzymatic hydrolysis and ethanol production. Bioresour.
Technol. 101, 63986403.
Xu, C., Ma, F., Zhang, X., 2009. Lignocellulose degradation and enzyme production
by Irpex lacteus CD2 during solid-state fermentation of corn stover. J. Biosci.
Bioeng. 108, 372375.
Yu, J., Zhang, J., He, J., Liu, Z., Yu, Z., 2009. Combinations of mild physical or chemical
pretreatment with biological pretreatment for enzymatic hydrolysis of rice hull.
Bioresour. Technol. 100, 903908.
Yu, H., Du, W., Zhang, J., Ma, F., Zhang, X., Zhong, W., 2010. Fungal treatment of
cornstalks enhances the delignication and xylan loss during mild alkaline
pretreatment and enzymatic digestibility of glucan. Bioresour. Technol. 101,
67286734.
7506 D. Salvacha et al. / Bioresource Technology 102 (2011) 75007506