Process Biochemistry 42 (2007) 1003–1009

www.elsevier.com/locate/procbio

Influence of solid loading on enzymatic hydrolysis of steam

exploded or liquid hot water pretreated olive tree biomass
Cristóbal Cara a, Manuel Moya a, Ignacio Ballesteros b, Ma José Negro b,
Alberto González b, Encarnación Ruiz a,*
a
Department of Chemical, Environmental and Materials Engineering, University of Jaén, Campus Las Lagunillas, 23071 Jaén, Spain
b
DER-CIEMAT, Avda. Complutense 22, 28040 Madrid, Spain
Received 22 December 2006; received in revised form 20 February 2007; accepted 23 March 2007

Abstract
Olive tree pruning biomass, pretreated by either liquid hot water or steam explosion under selected conditions, was used as a substrate for
enzymatic hydrolysis. The pretreated material was further submitted to alkaline delignification, the objective being to improve hydrolysis yields as
well as increasing cellulose content in the pretreated feedstock. The enzymatic hydrolysis of pretreated residues was performed using a commercial
cellulase mixture supplemented with b-glucosidase, using a solid loading range from 2 to 30% (w/v). The influence of substrate concentration on
the enzymatic hydrolysis yield and on glucose concentration was studied. Comparative results with and without a delignification step are presented.
Enzymatic hydrolysis at high substrate concentration (20%) is possible, yielding a concentrated glucose solution (>50 g/L). Nevertheless, a
cellulose fraction of the pretreated residue remains unaltered.
# 2007 Elsevier Ltd. All rights reserved.

Keywords: Olive tree biomass; Ethanol; Pretreatment; Delignification; Enzymatic hydrolysis; Substrate concentration

1. Introduction structure with no need of chemicals. Among them, liquid hot

Lignocellulosic resources such as agricultural residues
represent a renewable, low cost and largely available source
of energy [1]. Their utilization in an ethanol production process
solves, on the one side, environmental and disposal concerns;
on the other side, it is a promising way to decrease greenhouse
gas emissions and alleviate the shortage of fossil fuel energy
[2]. Ethanol is produced from lignocellulose by an integrated
process involving basically three steps: pretreatment, hydro-
lysis and fermentation. The final objective of both the
pretreatment and hydrolysis steps is to break down carbohy-
drate polymers present in plant cell walls into low-molecular-
weight sugars so that microorganisms can ferment them to
ethanol. Pretreatment is specially targeted to improve the
digestibility of the cellulose fraction in the hydrolysis step, by
removing lignin and hemicellulose. Hydrothermal pretreat-
ments result in substantial breakdown of lignocellulosic
* Corresponding author.
E-mail address: eruiz@ujaen.es (E. Ruiz).
water (LHW) and steam explosion (SE) pretreatments have
been applied to a wide range of agricultural residues [3–6]. To
improve the digestibility of the cellulosic residue, an alkaline
peroxide delignification step has been explored on several types
of biomass [7,8].
Hydrolysis is the key step in the bioconversion process
because (i) cellulose is the most abundant sugar polymer in
lignocellulosic residues and (ii) microorganisms performance on
glucose-to-ethanol conversion is easier than on any other simple
sugar. Hydrolysis may be performed either by acids or by enzyme
action. Enzymatic hydrolysis present several advantages when
comparing to acid hydrolysis, including lower equipment costs
(as hydrolysis is conducted at mild conditions) and higher
glucose yields without sugar-degradation products, which may
affect ulterior fermentation. On the other hand, enzyme purchase
is a costly part of ethanol production [9], moreover the presence
of residual lignin, not removed during pretreatment, has been
shown to play an important role in limiting the efficiency of
enzymatic hydrolysis of pretreated lignocellulosic materials
[10,11] as well as contributing to generation of degradation
products [12,13].

1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2007.03.012
1004 C. Cara et al. / Process Biochemistry 42 (2007) 1003–1009
Regarding the overall process economy, it is important to get a
glucose solution as concentrated as possible from the enzymatic
hydrolysis step, from which a concentrated ethanol solution may
be obtained as a result of fermentation, thus reducing separation
costs [14]. One way for obtaining concentrated glucose solutions
is to use as much substrate for the enzymatic hydrolysis as
possible, although problems related to material agitation and
inhibition by glucose may arise [15].
In olive tree cultivation, pruning is necessary to eliminate
old branches and to regenerate trees. A typical olive tree
pruning lot includes 70% thin branches (by weight, with
approximately one third of leaves) and 30% of wood (big
branches, diameter >5 cm approximately). Olive tree wood is
usually separated and put to domestic use as firewood or burnt
in small, local industries; it has been also considered as
feedstock for ethanol production [16,17]. The remaining
residue, left in the fields, is eliminated by burning, with
associated economical costs and environmental concerns. As an
alternative, this work deals with the use of this agricultural
residue as raw material for the production of fermentable sugars
in the frame of a biomass-to-ethanol conversion process.
In this work olive tree pruning biomass other than wood is
used as a raw material, the objective being to study the influence
of solid loading, or consistency, on the enzymatic hydrolysis of
the pretreated material in terms of both glucose concentration
and hydrolysis yields. As pretreatments, liquid hot water
(LHW) and steam explosion (SE) were used. We also
investigated how results are affected by a delignification step
performed on pretreated materials.
2. Materials and methods
2.1. Raw material and pretreatments
Olive tree pruning, consisting of leaves and thin branches (<5 cm diameter),
was collected locally after fruit-harvesting, air-dried at room temperature to
equilibrium moisture content of about 10%, milled using a laboratory hammer
mill (Retsch) to a particle size smaller than 10 mm, homogenised in a single lot
and stored until used.
Liquid hot water pretreatment was performed in a laboratory scale stirred
autoclave (model EZE-Seal, Autoclave Engineers, Erie, PA). The reactor has a
total volume of 2 L, with an electric heater and magnetic agitation. The
temperature/speed controller is a combination of furnace power control and
motor speed control with tachometer. Olive tree pruning was pretreated at
210 8C for 10 min. The amount of dry feedstock loaded was 200 g and water
was added at 1/5 (w/v) solid/liquid ratio. Overpressure in the reactor of 3 MPa
was kept to prevent formation of an aqueous vapour phase. The total heating
time required for reaching the operation temperature was approximately
50 min. Cooling water was circulated through a serpentine coil to cool the
reactor content at the end of each run. The content of the reactor cooled down to
100 8C in approximately 5 min.
Steam explosion of raw material was carried out in a custom-built batch
pilot unit based on Masonite technology and equipped with a 2-L reaction vessel
designed to reach a maximum operating pressure of 4.12 MPa. A more detailed
description of the plant is reported elsewhere [18]. The residue, 200 g (dry
matter) of feedstock per batch, was impregnated with 1 L water overnight and
then filtered. The reactor was charged with the filtered material (moisture
content approximately 50%) and heated to 240 8C with saturated steam for
5 min. The exploded material was recovered in a cyclone.
Both pretreatments were performed in triplicate batches, and the pretreated
materials from all batches were mixed together. After each pretreatment the wet
material was filtered for solid and liquid recovery. The water-insoluble fraction
was washed out with water and analyzed for cellulose, hemicellulose, and acid-
insoluble lignin content.
2.2. Alkaline peroxide delignification
The water-insoluble fiber from LHW and SE-pretreatments was delignified
using a hot alkaline peroxide treatment protocol adapted from Yang et al. [19] in
1% (w/v) H2O2 solution at 4% (w/v) solids concentration. The pH was adjusted
to 11.5 using 4 M NaOH. The treatment lasted for 45 min at 80 8C; then the
suspension was filtered and the solid fraction was water washed until neutral
pH. Delignified, dried solid was weighed and analyzed for sugars and lignin
composition. Triplicate experiments were performed. Mean values and standard
deviations are reported.
2.3. Enzymatic hydrolysis tests
The washed water-insoluble residue of pretreated olive tree pruning was
enzymatically hydrolyzed by a cellulase mixture (Celluclast 1.5 L) kindly
provided by Novozymes A/S (Denmark). Cellulase enzyme loading was 15
filter paper units (FPU)/g substrate. Fungal b-glucosidase (Novozym 188,
Novozymes A/S) was used to supplement the b-glucosidase activity with an
enzyme loading of 15 international unit (IU)/g substrate. Enzymatic hydrolysis
was performed in 0.05 M sodium citrate buffer (pH 4.8) at 50 8C on a rotary
shaker (Certomat-R, B-Braun, Germany) at 150 rpm for 72 h at 2, 5, 10, 20 and
30% (w/v) pretreated material concentration. Samples were taken after 4, 8, 12,
24, 48 and 72 h for glucose concentration determination. All enzymatic
hydrolysis experiments were performed in triplicate. Average results and
corresponding errors are given.
2.4. Analytical methods
Composition of raw material was determined according to the National
Renewable Energy Laboratory (NREL, Golden, CO) analytical methods for
biomass [20]. Prior to other determinations, raw material was extracted
consecutively with water and with ethanol (two-step extraction procedure).
After the first step, the sugar composition of the water-extract was determined
by high performance liquid chromatography (HPLC) in a waters 2695 liquid
chromatograph with refractive index detector. An AMINEX HPX-87P car-
bohydrate analysis column (Bio-Rad, Hercules, CA) operating at 85 8C with
ultrapure water as a mobile-phase (0.6 mL/min) was used. Free and oligo-
meric sugar compositions were determined before and after a posthydrolysis
process consisting in a treatment with sulphuric acid 3% (v/v) at 121 8C and
30 min. The cellulose and hemicellulose content of the extracted solid
residue was determined based on monomer content measured after a two-
step acid hydrolysis procedure to fractionate the fiber. A first step with 72%
(w/w) H2SO4 at 30 8C for 60 min was used. In a second step, the reaction
mixture was diluted to 4% (w/w) H2SO4 and autoclaved at 121 8C for 1 h.
This hydrolysis liquid was then analyzed for sugar content by HPLC as
described above. The remaining acid-insoluble residue is considered as acid-
insoluble lignin (AIL).
The composition of solid fraction after pretreatment was determined as
described for raw material except that no extraction was used. The sugar content
(glucose, xylose, arabinose, mannose and galactose) of the liquid fraction after
pretreatment (prehydrolysate) was determined by HPLC using the system
described above. Glucose concentration from enzymatic hydrolysis samples
was measured by an enzymatic determination glucose assay kit (Sigma GAHK-
20). All analytical determinations were performed in duplicate and average
results are shown. Relative standard deviations were in all cases below 5%.
3. Results and discussion
3.1. Raw material and pretreatments
Olive tree pruning has (oven dry weight) 25.0% of
cellulose (as glucose) and 15.8% of hemicellulosic sugars
 C. Cara et al. / Process Biochemistry 42 (2007) 1003–1009 1005

Table 1
Composition (% dry matter) of raw material and water-insoluble fibers after pretreatmenta
Total gravimetric recovery Cellulose as glucose Xylose Other sugarsb AIL
Raw material 25.0  1.2 11.1  0.6 4.7  0.6 16.6  0.5
LHW-pretreatedc 56.0  0.8 40.7  0.9 (22.8) 1.0  0.2 (0.6) nd 49.4  0.6 (27.7)
SE-pretreatedd 51.0  0.6 43.5  1.0 (22.2) 1.8  0.3 (0.9) nd 48.6  0.4 (24.8)
nd: not detected.
a
Data are expressed in parentheses as a percentage based on dry weight of raw material.
b
Sum of galactose, mannose and arabinose.
c
Liquid hot water at 210 8C for 10 min.
d
Steam explosion at 240 8C for 5 min.

with xylose as the main sugar (70%). Acid insoluble lignin
(AIL) accounts for 16.6%. Considering acid-soluble lignin
content, which refers to the small fraction of lignin that is
solubilized during the hydrolysis process used to determine
AIL, the total lignin value increases up to 18.8%. Acetyl
groups represent about 2.5% of raw material and ashes sum up
to 3.4%. It is worth noting that this lignocellulose residue has
an extractive content of 31.4%, which includes 7.9% of
glucose. This amount of glucose will enter the liquid fraction
after pretreatment.
Table 1 summarizes the main components of the raw
material as well as that of solids obtained after submitting it
to LHW and SE-pretreatments. Operation conditions (210 8C
for 10 min in LHW-pretreatment and 240 8C for 5 min and
water impregnation in steam explosion pretreatment) based
on the highest enzymatic hydrolysis yields at 5% solid
concentration and highest cellulose recovery of the insoluble
residues were selected among several experiments in which a
wide range of pretreatment conditions were explored [21,22].
As deduced from Table 1, solids from both pretreatments are
quite similar. Glucose content of pretreated solids increased
when compared to raw material because of almost complete
hemicellulose solubilization. Data in parentheses in Table 1
are expressed as a percentage based on dry weight of raw
material. Cellulose losses occurred in a relatively reduced
extent (around 10%). Regarding AIL content, values higher
than those of raw material were obtained, indicating that
acid-insoluble residue includes lignin-like structures from
recondensation reactions especially between sugars or sugar
degradation products and other components from the
extractive fraction [23,24].
As far as the liquid fractions issued from pretreatments are
concerned, a considerable proportion of solubilized sugars were
degraded under pretreatment conditions. It must be taken into
account that these liquors also contain the glucose present in the
extractive fraction, which accounts for up to 7.9%. So, there are
actually some glucose losses attributed to degradation
processes. 2.8 g glucose/100 g raw material and 1.3 g hemi-
cellulosic sugars/100 g were recovered in liquids from LHW-
pretreatment; SE-pretreatment resulted in sugar recoveries in
the liquid fraction of 5.4 g glucose and 5.4 g hemicellulosic
sugars (per 100 g raw material). Sugars were obtained mainly
as oligomers (76.5% of total sugars) when SE-pretreatment was
applied. In contrast, 45.5% of oligomers were released from
LHW-pretreatment.
3.2. Enzymatic hydrolysis on pretreated solids
Solids from both pretreatments were submitted to enzymatic
hydrolysis at varying substrate loadings, ranging from 2 to 30%
(w/v). Increasing substrate concentration leads to a highly
concentrated glucose solution, which in turn may yield high
ethanol fermentation levels and, consequently, lower separation
product costs. As a drawback, high hydrolysis consistency may
result in difficulties in stirring the material and end-product
enzyme inhibition.
Fig. 1 shows the time course of glucose concentrations
obtained as a function of substrate consistency from LHW-
pretreated solids submitted to enzymatic hydrolysis. Glucose
concentration follows the typical batch hydrolysis pattern, with
a rapid glucose release at the beginning of the process, and then
the glucose generation rate slowed down, as reported by other
researchers [2,25]; for example, 67% of the total glucose
released in the 72-h hydrolysis time was liberated within the
first 24 h when using a substrate concentration of 2%, with
increasing percentages as a function of solid loading, until
reach 78% glucose release within 24 h at 30% consistency. As
expected, the concentration of glucose increased as a function
of substrate concentration, and glucose solutions as concen-
trated as 61 g/L were obtained after 72 h of enzymatic attack
using 30% substrate consistency.
A similar behavior was found when hydrolyzing SE-
pretreated solids at varying substrate concentration (Fig. 2). As
Fig. 1. Hydrolysis profiles of LHW-pretreated olive tree pruning at different
solid loadings (%, w/v) at 15 FPU/g substrate and 50 8C. Pretreatment condi-
tions: 210 8C, 10 min.
1006 C. Cara et al. / Process Biochemistry 42 (2007) 1003–1009

between substrate concentration (20%) and hydrolysis yields

Fig. 2. Hydrolysis profiles of SE-pretreated olive tree pruning at different solid
loadings (%, w/v) at 15 FPU/g substrate and 50 8C. Pretreatment conditions:
240 8C, 5 min, water impregnated material.
an average, 83% of the total glucose concentration obtained
after 72 h hydrolysis was released within the first 24 h,
regardless the substrate concentration, although the final
glucose solutions were less concentrated (maximum of 52 g
glucose/L at 30% consistency) than those corresponding to
LHW-pretreated hydrolyzed solids. Another difference
between both pretreated materials is that SE-pretreatement
resulted in solids with an apparent lower density than LHW-
pretreated residues, making difficult for the enzymatic
hydrolyzing solution to access and to wet these materials
when using a high substrate concentration (30%). Disgregation
and solubilization of the residue was only detected after 72 h
(only one experimental point in Fig. 2). Nevertheless, 20%
consistency is considered a high concentrated biomass material
and no literature on enzymatic hydrolysis beyond this point has
been reported to our best knowledge. In fact, mixing problems
arise for some materials at consistencies 10% [26].
From glucose concentration data at different enzymatic
hydrolysis time, the hydrolysis yields have been evaluated
taking into account the potential glucose in pretreated material
(Table 1). Results are shown in Table 2. When enzymatic
hydrolysis was conducted on LHW-pretreated solids, hydro-
lysis yield after 72 h decreased as a function of substrate
concentration, thus making no use of a considerable proportion
of glucose in pretreated materials. By contrast, a high
concentrated glucose solution was obtained (Fig. 1). At lower
hydrolysis time, no definite influence may be established
and only at 30% consistency hydrolysis yields were smaller
than those obtained at any other conditions. This may be
attributable to a summative effect of both end-product
inhibition and physical problems related to a difficult agitation
of material in the enzymatic solution. These problems did not
appear at substrate concentration 20% when the pretreated
material was well-wetted.
Regarding SE-pretreated materials, enzymatic hydrolysis
yields were not affected by substrate consistency at any
hydrolysis time, except for 20% substrate concentration. In this
experiment (and of course at 30% consistency), the above
mentioned physical impediments were clearly evidenced from
the hydrolysis beginning.
Taking into account the operational conditions assayed and
comparing both pretreatments, a concentrated glucose solution
without a considerable losing of hydrolysis performance is
obtained at 20% consistency of LHW-pretreated materials, and
at 10% substrate concentration in the case of SE-pretreated
residue. For a 72-h hydrolysis and at a given substrate
concentration, enzymatic hydrolysis yields are greater when
using LHW-pretreatment. Nevertheless, SE-pretreatment leads
to better results within the first 24 h, for substrates at
consistencies 10%.
It is worth noting that, although high concentrated glucose
solutions are obtained, there is still a considerable proportion of
cellulose in the pretreated residue that is not hydrolyzed. In an
attempt to take advantage of cellulose content, a delignification
step was assayed, as lignin has been shown to limit the
efficiency of enzymatic hydrolysis of pretreated lignocellulosic
materials [11,27]. Moreover, since the pretreated residue has
still almost 50% of apparent lignin (Table 1), removal of lignin
may result in a higher cellulose content of the residue which, in
turn, would increase both glucose concentration and hydrolysis
yields.
3.3. Enzymatic hydrolysis on pretreated, delignified solids
Delignification conditions were selected based on previous
works using olive tree wood [16] where up to 80% of lignin
removal was reached and, at the same time, enzymatic
hydrolysis yields of steam pretreated solids were improved
compared to not delignified material. The composition of
pretreated solids after delignification is shown in Table 3.
Delignification recovery, also shown in Table 3, is defined as

Table 2
Enzymatic hydrolysis yields (%) of pretreated materials at varying hydrolysis time and solid loading
Hydrolysis time (h) LHW-pretreated solid concentration (%) SE-pretreated solid concentration (%)
2 5 10 20 30 2 5 10 20 30a
4 17.6 19.0 18.9 20.5 18.3 28.7 29.6 30.2 18.9 –
8 25.6 28.0 32.2 33.4 25.7 41.8 38.3 45.5 34.3 –
12 33.5 32.4 35.9 36.0 29.9 45.4 41.3 48.1 37.2 –
24 51.6 48.3 48.5 48.3 39.1 52.8 50.9 53.3 45.1 –
48 66.0 63.1 66.2 63.0 46.6 57.6 55.6 59.6 51.1 –
72 76.2 70.3 66.0 64.0 49.9 62.0 60.2 62.9 55.4 39.6
a
The enzymatic hydrolysis was not sampled due to very high consistency.
 C. Cara et al. / Process Biochemistry 42 (2007) 1003–1009 1007

Table 3
Composition (% dry matter) of water-insoluble fibers after pretreatment and delignificationa
Material Delignification recovery Cellulose as glucose Xylose Other sugarsb AIL
No pretreated, delignified 70.8  3.3 38.3  0.5 (27.1) 15.8  0.3 (11.2) 2.9  0.2 (2.1) 24.5  0.4 (17.3)
LHW-pretreatedc, delignified 58.8  2.5 56.7  2.0 (18.7) 1.8  0.2 (0.6) nd 37.1  0.3 (12.2)
SE-pretreatedd, delignified 65.3  1.4 60.2  1.6 (20.0) 2.1  0.1 (0.7) nd 33.5  0.5 (11.2)
nd: not detected.
a
Data are expressed in parentheses as a percentage based on dry weight of raw material.
b
Sum of galactose, mannose and arabinose.
c
Liquid hot water at 210 8C for 10 min.
d
Steam explosion at 240 8C for 5 min.

the weight of solid after delignification divided by weight of Enzymatic hydrolysis yields were evaluated by means of
pretreated solid. Data in parentheses are calculated taking into glucose concentrations and potential glucose in pretreated,
account both delignification recovery and total gravimetric delignified materials (Table 3). Results are shown in Table 4. As
recovery (pretreatment step, Table 1) and enable direct can be deduced by comparison between data in Tables 2 and 4,
comparison with raw material. Solubilization of lignin reached delignification did not improve enzymatic hydrolysis yields on
56 and 54.8% referred to AIL content in LHW and SE- LHW-pretreated solids and even some yield decrease was
pretreated materials, respectively (Table 1). Nevertheless, observed. This may be attributable to higher end-product
comparing with AIL content of the raw material, delignification inhibition derived from higher glucose concentrations. Another
causes an AIL reduction of 26.5 and 32.5%. Considering results fact that must be considered is that all experiments have been
shown in Table 3, it must be pointed out that a relatively high
AIL content remains after a delignification procedure. As
reported by Shevchenko et al., pretreatments may result in
dramatic changes in the chemical structure of lignin in the
residual material leading to a more condensed lignin that is
more difficult to remove [28]. The removal of lignin on
pretreated olive tree wood was greater [16] than on pretreated
olive tree pruning and lignin content after pretreatment
(referred to raw material) did not increase, in contrast to
present results (Table 1). Since olive tree pruning contains
approximately 30% by weight of leaves, extractive content is
higher than that of olive tree wood; this may produce
interferences in the lignin analysis method that could partially
explain these results, as reported by other researchers [29].
As a consequence of lignin solubilization, cellulose content
increased although glucose losses (accounting for 18% at LHW- Fig. 3. Hydrolysis profiles of LHW-pretreated, delignified olive tree pruning at
pretreated solids and for 10% in the case of SE-pretreated different solid loadings (%, w/v) at 15 FPU/g substrate and 50 8C. Pretreatment
materials) were detected. Hemicellulosic-derived sugars were conditions: 210 8C, 10 min.
not affected by delignification. For comparison purposes, raw
material without pretreatment was submitted to the same
delignification procedure as pretreated materials. As deduced
from Table 3, no lignin removal is detected, while the solubilized
material (near 30%) may correspond to extractive content.
After delignification, olive tree pruning residues were
submitted to enzymatic hydrolysis at the same operational
conditions as pretreated material. Glucose concentrations
obtained at different hydrolysis consistency are depicted in
Fig. 3 (LHW-pretreated, delignified materials) and Fig. 4 (SE-
pretreated, delignified residues). In all cases, glucose con-
centrations obtained from pretreated and delignified solids were
higher than those resulting from only pretreated materials,
reaching a maximum value of 73 g glucose/L (LHW-pretreated,
delignified solid at 30% hydrolysis consistency). Again, only
one hydrolysis sample was taken at 30% substrate concentra- Fig. 4. Hydrolysis profiles of SE-pretreated, delignified olive tree pruning at
tion in SE-pretreated material because solids remained different solid loadings (%, w/v) at 15 FPU/g substrate and 50 8C. Pretreatment
insoluble until 72 h hydrolysis time. conditions: 240 8C, 5 min, water impregnated material.
1008 C. Cara et al. / Process Biochemistry 42 (2007) 1003–1009

Table 4
Enzymatic hydrolysis yields (%) of pretreated and delignified materials at varying hydrolysis time and solid loading
Hydrolysis time (h) LHW-pretreated, delignified solid concentration (%) SE-pretreated, delignified solid concentration (%)
2 5 10 20 30 2 5 10 20 30 a
4 15.0 17.6 18.8 15.5 12.2 26.2 23.0 25.1 13.3 –
8 27.3 26.4 26.8 22.8 17.9 41.4 35.9 30.3 20.5 –
12 34.5 33.5 33.2 26.8 20.9 48.4 43.6 39.9 27.0 –
24 51.8 47.4 43.2 40.3 30.5 67.8 61.6 54.1 38.6 –
48 64.8 61.0 50.3 47.9 38.2 76.0 70.1 63.1 44.7 –
72 72.6 70.1 60.4 56.8 43.0 83.1 74.1 67.6 50.0 37.7
a
The enzymatic hydrolysis was not sampled due to very high consistency.

performed using the same enzyme dosage per gram of total
substrate; since LHW-pretreated, delignified solids have a
higher cellulose content (compared to only pretreated
materials), in fact there is a lesser amount of enzyme per
gram of cellulose, contributing perhaps to the yield decrease
observed. Nevertheless, delignification did improve hydrolysis
using an LHW-pretreated material at relatively soft conditions
(180 8C, 10 min) and hydrolysis yield increased from 16.7 up to
48.7%, although the lignin removal was of the same order as
that reported here (data not shown). It seems that there is always
a recalcitrant cellulose fraction, even at the harsher pretreat-
ment conditions, that remains inaccessible to enzymes even
after delignification. By contrast, enzymatic hydrolysis was
enhanced by delignification on SE-pretreated solids, except for
high consistency hydrolysis (20 and 30%) where glucose
inhibition is clearly evidenced. In our previous work on
enzymatic hydrolysis of SE-pretreated olive tree wood,
delignification also resulted in a yield improvement compared
to just pretreated material [16].
Considering results obtained after 72 h enzyme action on
LHW-pretreated materials, either or not delignified, enzymatic
hydrolysis yields decreased as a function of substrate
concentration whereas glucose concentration increased con-
comitantly with consistency in both types of materials. It seems
that using a 20% consistency of pretreated solids may be an
interesting option because enzymatic hydrolysis yield decrease
is not very important compared with other substrate concen-
trations and, in turn, high concentrated glucose solutions are
obtained (52.1 and 64.5 g/L, without and with delignification,
respectively). In the case of SE-pretreated residues, delignifica-
tion improved both yields and concentrations but only until a
certain consistency level (10%); beyond this point, enzymatic
hydrolysis yields of delignified materials are worse than those
of just steamed ones. This is now clearly attributable to glucose
inhibition, as physical concerns are independent of whether or
not the material was delignified (for the same substrate
concentration). These results are in agreement with those
reported by Zhu et al. [26], who suggest that, among several
factors affecting sugar yield decreases at high substrate
concentrations, end-product inhibition is the main one.
4. Conclusions
From the results reported in this study, the following
conclusions may be outlined:
1. Pretreated olive tree pruning biomass may be used as raw
material for enzymatic hydrolysis at high substrate
concentration (20%).
2. Enzyme saccharification of LHW-pretreated solids at 20%
consistency results in a glucose solution of 52 g/L with an
enzymatic hydrolysis yield of 64%.
3. A delignification step performed on pretreated materials
increases glucose concentration but, in turn, reduces
enzymatic yields at high substrate concentrations, due to
end-product inhibition. A techno-economic assessment is
needed to determine the convenience of this step to be
conducted.
4. Research on process configuration, for example conversion
of glucose as it is formed by a simultaneous saccharification
and fermentation scheme (to avoid end-product inhibition)
or enzymatic hydrolysis conditions (to reduce the fraction of
recalcitrant cellulose) should be investigated.
Acknowledgements
This work was partially financed by Ministerio de
Educación y Ciencia (Project ENE2005-08822) and FEDER
funds. Financial support from Azucareras Reunidas de Jaén,
S.A. is also gratefully acknowledged.
References
[1] Kim S, Dale BE. Global potential bioethanol production from wasted
crops and crop residues. Biomass Bioenergy 2004;26:361–75.
[2] Xiao Z, Zhang X, Gregg DJ, Saddler JN. Effects of sugar inhibition on
cellulases and b-glucosidase during enzymatic hydrolysis of softwood
substrates. Appl Biochem Biotechnol 2004;113–116:1103–14.
[3] Ballesteros I, Oliva JM, Negro MJ, Manzanares P, Ballesteros M. Enzymic
hydrolysis of steam exploded herbaceous agricultural waste (Brassica
carinata) at different particule sizes. Process Biochem 2002;38:
187–92.
[4] Belkacemi K, Turcotte G, Savoie P. Aqueous/steam-fractionated agricul-
tural residues as substrates for ethanol production. Ind Eng Chem Res
2002;41:173–9.
[5] Laser M, Schulman D, Allen SG, Lichwa J, Antal MJ, Lynd LR. A
comparison of liquid hot water and steam pretreatments of sugar
cane bagasse for bioconversion to ethanol. Biores Technol 2002;81:
33–44.
[6] Fernández-Bolaños J, Felizón B, Heredia A, Rodrı́guez R, Guillén R,
Jiménez A. Steam explosion of olive stones: hemicellulose solubilization
and enhancement of enzymatic hydrolysis of cellulose. Biores Technol
2001;79:53–61.
 C. Cara et al. / Process Biochemistry 42 (2007) 1003–1009
[7] Curreli N, Agelli A, Oisu B, Rescigno A, Sanjust E, Rinaldi A. Complete
and efficient enzymic hydrolysis of pretrated wheat straw. Process Bio-
chem 2002;37:937–41.
[8] Yáñez R, Alonso JL, Parajó JC. Enzymatic saccharification of hydrogen
peroxide-treated solids from hydrothermal processing of rice husks.
Process Biochem 2006;41:1244–52.
[9] Hamenlick C, van Hooijdonk G, Faaij A. Ethanol from lignocellulosic
biomass: techno-economic performance in short-, middle- and long-term.
Biomass Bioenergy 2005;28:384–410.
[10] Pan X, Zhang X, Gregg DJ, Saddler JN. Enhanced enzymatic hydrolysis of
steam-exploded douglas fir wood by alkali-oxygen post-treatment. Appl
Biochem Biotechnol 2004;113–116:1103–14.
[11] Adsul MG, Ghule JE, Shaikh H, Singh R, Bastawde KB, Gokhale DV,
et al. Enzymatic hydrolysis of delignified bagasse polysaccharides. Car-
bohydr Polym 2005;62:6–10.
[12] Cantarella M, Cantarella L, Gallifuoco A, Spera A, Alfani F. Comparison
of different detoxification methods for steam-exploded poplar wood as a
substrate for the bioproduction of ethanol in SHF and SSF. Process
Biochem 2004;39:1533–42.
[13] Tengborg C, Galbe M, Zacchi G. Reduced inhibition of enzymatic
hydrolysis of steam-pretreated softwood. Enzyme Microb Technol
2001;28:835–44.
[14] Ohgren K, Rudolf A, Galbe M, Zacchi G. Fuel ethanol production from
steam-pretreated corn stover using SSF at higher dry matter content.
Biomass Bioenergy 2006;30:863–9.
[15] Kadam KL, Rydholm EC, McMillan JD. Development and validation of a
kinetic model for enzymatic saccharification of lignocellulosic biomass.
Biotechnol Prog 2004;20:698–705.
[16] Cara C, Ruiz E, Ballesteros I, Negro MJ, Castro E. Enhanced enzymatic
hydrolysis of olive tree wood by steam explosion and alkaline peroxide
delignification. Process Biochem 2006;41:423–9.
[17] Ruiz E, Cara C, Ballesteros M, Manzanares P, Ballesteros I, Castro E.
Ethanol production from pretreated olive tree wood and sunflower stalks
by an SSF process. Appl Biochem Biotechnol 2006;130:631–43.
[18] Ballesteros M, Oliva JM, Negro MJ, Manzanares P, Ballesteros I. Ethanol
from lignocellulosic materials by a simultaneous saccharification and
fermentation process (SFS) with Kluyveromyces marxianus CECT 10875.
Process Biochem 2004;39:1843–8.
1009
[19] Yang B, Boussaid A, Mansfield SD, Gregg DJ, Saddler JN. Fast and
efficient alkaline peroxide treatment to enhance the enzymatic digest-
ibility of steam-exploded softwood substrates. Biotechnol Bioeng
2002;77:678–84.
[20] National Renewable Energy Laboratory (NREL). Chemical analysis and
testing laboratory analytical procedures. LAP-002 (1996). LAP-003
(1995). LAP-004 (1996). LAP-005 (1994). LAP-010 (1994) and LAP-
017 (1998). NREL, Golden, CO, USA. http://www.eere.energy. gov/
biomass/analytical_procedures.html.
[21] Cara C, Romero I, Oliva JM, Sáez F, Castro E. Liquid hot water
pretreatment of olive tree pruning residues. Appl Biochem Biotechnol;
in press.
[22] Cara C, Ruiz E, Ballesteros M, Manzanares P, Negro MJ, Castro E.
Production of fuel ethanol from steam-explosion pretreated olive tree
pruning. Fuel; submitted for publication.
[23] Nguyen QA, Tucker MP, Keller FA, Beaty DA, Connors KM, Eddy FP.
Dilute acid hydrolysis of softwood. Appl Biochem Biotechnol 1999;77–
79:133–42.
[24] Calvalheiro F, Esteves MP, Parajó JC, Pereira H, Gı́rio FM. Production of
oligosaccharides by autohydrolysis of brewery’s spent grain. Bioresour
Technol 2004;91:93–100.
[25] Lu Y, Yang B, Gregg D, Saddler JN, Mansfield SD. Cellulase adsorption
and an evaluation of enzyme recycle during hydrolysis of steam-exploded
softwood residues. Appl Biochem Biotechnol 2002;98–100:641–54.
[26] Zhu S, Wu Y, Yu Z, Liao J, Zhang Y. Pretreatment by microwave/alkali of
rice straw and its enzymic hydrolysis. Process Biochem 2005;40:
3082–6.
[27] Laureano-Perez L, Teymouri F, Alizadeh H, Dale BE. Understanding
factors that limit enzymatic hydrolysis of biomass. Appl Biochem Bio-
technol 2005;121–124:1081–99.
[28] Shevchenko SM, Beatson RP, Saddler JN. The nature of lignin from steam
explosion/enzymatic hydrolysis of softwood. Appl Biochem Biotechnol
1999;77–79:867–76.
[29] Zimbardi F, Ricci E, Viola E, Cuna D, Cardinale G, Cardinale M. Process
optimization in bioconversion of lignocellulosics into ethanol. In: Kyritsis
S, Beenackers AAC, Helm P, Grassi A, Chiaramonti D, editors. Proceed-
ings of the first world conference on biomass for energy and industry.
London: James & James; 2001. p. 1525–8.