Fuel 89 (2010) 28912896

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Fuel
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Determination of optimal pre-treatment conditions for ethanol production

from olive-pruning debris by simultaneous saccharication and fermentation
Manuel Cuevas a, Sebastin Snchez a,*, Vicente Bravo b, Juan Francisco Garca b, Jaime Baeza c,
Carolina Parra c, Juanita Freer c
a
Department of Chemical, Environmental and Materials Engineering, University of Jan, Campus Las Lagunillas, 23071 Jan, Spain
b
Department of Chemical Engineering, University of Granada, Campus Fuentenueva, 18071 Granada, Spain
c
Renewable Resources Laboratory, Biotechnology Center, University of Concepcin, Casilla 160-C Concepcin, Chile

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

Article history: Ethanol generation from lignocellulose materials provides an alternative energy-production system. This
Received 21 October 2009 study investigates the effect of pre-treatment conditions: maximum temperature (range 423.15
Received in revised form 29 January 2010 483.15 K) and sulfuric-acid concentration (interval 0.0020.059 kmol/m3) on fuel-ethanol production
Accepted 2 February 2010
from simultaneous saccharication and fermentation (SSF) of olive-pruning debris by Saccharomyces
Available online 13 February 2010
cerevisiae IR2-9a (a thermal acclimatized microorganism, 313.15 K). The inuence of these two variables
was determined by using a response-surface methodology. Cellulose percentage in pre-treated solids
Keywords:
reached a maximum of 71.6% of the content in raw material at 483.15 K and 0.010 kmol/m3 of acid con-
Bioethanol
Olive-pruning debris
centration. The conversion of hemicellulose into monosaccharides and oligosaccharides also was ana-
Diluted-acid pre-treatment lyzed. After the wash and ltration of solids, a signicant quantity of D-glucose was obtained in the
SSF liquid fraction. For ethanol generation, the bio-fuel yield (maximum of 9.6 kg from 100 kg olive-pruning
Saccharomyces cerevisiae debris), and volumetric ethanol productivity (maximum of 0.27 kg/(m3 h)), strongly depended on pre-
treatments conditions. According to statistical optimization, the highest ethanol yield (9.9 kg ethanol
from 100 kg olive-pruning debris) is achieved at 480.15 K using a catalyst concentration of 0.016 kmol/
m3. A maximum overall process yield of 15.3 kg ethanol/100 kg olive-pruning debris may result when
taking into account ethanol from SSF and D-glucose present in the pre-hydrolysate, assuming its theoret-
ical conversion (22.8 kg ethanol/100 kg raw material, also considering the total conversion of D-xylose in
the ltrate).
2010 Elsevier Ltd. All rights reserved.

1. Introduction
The production of fuel-ethanol from lignocellulose biomass is of
growing interest around the world because it can provide a num-
ber of environmental advantages over conventional fossil fuels,
most notably a reduction in greenhouse-gas emissions. However,
the higher production cost for bioethanol (1.3 dollars/dm3, from
lignocellulose) as compared to gasoline, and its lower heating va-
lue (26,700 kJ/kg at ambient temperature) are some problems of
this alternative fuel [1].
Traditionally, olive-tree is cultivated in Mediterranean coun-
tries (especially in Spain, Italy, Greece, Morocco and Tunisia) but
in recent times it has been cultivated in regions of ve continents
(West Coast of USA, Mexico, Australia, Argentina, Chile, etc.) with a
total area of more than 7  104 km2 [2]. In olive-tree cultivation,
pruning is a necessary biennial operation to eliminate old branches
* Corresponding author. Tel.: +34 953 212219; fax: +34 953 212141.
E-mail address: ssanchez@ujaen.es (S. Snchez).
and to regenerate the tree. Debris from olive-tree pruning repre-
sents a great volume of renewable biomass in Spain (between
4.3  109 and 7.5  109 kg/year) and a great potential resource
for ethanol production (maximum of 1.3  106 m3/year).
The conversion of biomass to ethanol generally includes four
steps: pre-treatment, hydrolysis of polysaccharides and oligosac-
charides into monomer sugars, fermentation of sugars to ethanol
and, nally, ethanol concentration to absolute alcohol (for use as
motor fuel, ethanol must be concentrated to >99.8%).
Extensive literature is available on different pre-treatment
methods to enhance the digestibility of lignocellulose materials
[3]. Diluted-acid pre-treatment, at temperatures of 423.15
493.15 K, is one of the most important procedures and has been re-
viewed for different materials [46], working with sulfuric-acid or
other acids (e.g. nitric acid, hydrochloric acid). During this thermal
process, hemicellulose is depolymerized into a mixture of sugar
oligomers and monomers, whereas less alteration is caused in lig-
nin and cellulose [7]. Removal of hemicellulose increases porosity
and therefore improves enzymatic digestibility of cellulose.

0016-2361/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fuel.2010.02.005
2892 M. Cuevas et al. / Fuel 89 (2010) 28912896
Depending on the hydrolysis conditions, carbohydrates degrada-
tion products can form, and interfere with microbial activities.
SSF processes combine enzymatic hydrolysis of cellulose with
simultaneous fermentation of the D-glucose obtained to ethanol
[8]. The presence of yeast together with cellulases reduces the
accumulation of D-glucose, thereby increasing the saccharication
rate and ethanol yield. SSF also lowers capital costs and reduces the
risk of microbial contamination [9].
This study evaluates the inuence of pre-treatment conditions
(temperature and acid concentration) on the simultaneous sac-
charication and fermentation of olive-pruning debris (OPD) using
response-surface methodology and the best-known ethanol-fer-
menting yeast, Saccharomyces cerevisiae. The generation of sugars
and ethanol from this raw material has been studied by using dif-
ferent pre-treatments: concentrated sulfuric-acid [10], concen-
trated phosphoric-acid [11] and steam explosion. This research
can complete the available information on the utilization of ol-
ive-pruning debris for ethanol production.
2. Experimental
2.1. Raw material
Olive-pruning debris (thin branches and leaves) were obtained
from a local farm in the province of Jan, Spain, air-dried at room
temperature until reaching 810% moisture, milled, using a blade
mill (Retsch, mod. SM1, Germany), to a particle size between
0.425 and 0.600 mm, homogenized and stored until used. The sam-
ples were characterized following ASTM methods [12,13].
Cellulose and hemicellulose content was determined based on
monomer content measured after an acid hydrolysis in two steps.
In the rst, 300 mg of free-extractive material was hydrolyzed with
3 cm3 72% (v/v) H2SO4 at 303.15 K for 60 min. In the second step,
the reaction mixture was diluted to 4% H2SO4 and autoclaved at
394.15 K for 1 h. The resultant product was ltered on a Gosh lter
No. 4. The solid fraction, acid-insoluble lignin (AIL), was dried and
weighed. The sugars in the hydrolysates were determined by nor-
mal phase HPLC (Merck Hitachi), equipped with refractive index
detector by using a Aminex HPX-87H column (Bio-Rad Labs, Hercu-
les, CA) at 318.15 K, mobile phase H2SO4 5 mol/m3 and a ow rate of
0.6 cm3/min [14]. All the analyses were carried out in triplicate.
2.2. Liquid hot-water pre-treatment
Autohydrolysis was conducted in a 1-dm3 stainless steel Parr
reactor, Series 4521 (Moline, IL), loaded with 50 g of dry solid
and 300 cm3 of dilute sulfuric-acid solution. In all experiments,
the reactor was heated to the selected temperature by an external
heating jacket (heating rate was between 6.1 and 6.6 K/min). After
5 min at maximum temperature, the reactor was removed from
the heating system and cooled in a water bath to about 303.15 K
in less than 10 min.
After pre-treatment, the material was separated into solid resi-
due and liquid hydrolysate by ltration. The solid was washed with
water (30 times the mass of the obtained pre-treated material),
and then dried at room temperature to equilibrium moisture of
about 6070% and stored in sealed plastic bags at 281.15 K. The
chemical composition of the pre-treated material was analyzed
as described above. The water-soluble extract was analyzed for su-
gar and acid-soluble lignin (ASL).
2.3. Simultaneous saccharication and fermentation (SSF)
The enzymatic hydrolysis was carried out using a cellulolytic
complex, Celluclast 1.5 L (Novo Nordisk, Denmark), supplemented
with a b-glucosidase preparation (Novozyme 188, Novo Ltd.). The
enzymatic activity in all the experiments was 20 Filter Paper Units
(FPU)/gsubstrate (cellulase activity) and 40 International Unit (IU)/
gsubstrate (b-glucosidase activity). Cellulase activity was determined
following the standard procedure recommended by the Commis-
sion on Biotechnology, IUPAC [15].
The yeast strain used was S. cerevisiae IR2-9a, a thermal accli-
matized microorganism at 313.15 K [16]. The inoculum was grown
in 100 cm3 of liquid culture made of D-glucose, 50 g/dm3; yeast ex-
tract, 5.0 g/dm3; peptone, 5.0 g/dm3; KH2PO4, 1.0 g/dm3;
MgSO47H2O, 0.5 g/dm3; NH4Cl, 2.0 g/dm3 at pH 4.8 (buffer sodium
citrate, 0.05 kmol/m3) in a 250 cm3 Erlenmeyer ask. The culture
was incubated for 48 h at 313.15 K in an orbital shaker at
150 rpm. The cells were collected by centrifugation at 1500 rpm
for 5 min by a Beckman centrifuge, model GS-GR (Fullerton, CA),
washed with a diluted NaCl solution and used for the inoculum.
The biomass concentration at the beginning of each experiment
was approximately 6 g/dm3.
The SSF experiments were performed in 125 cm3 Erlenmeyer
asks with a weight of 3 g (pre-treated material on a dry basis,
6070% moisture) suspended in 30 cm3 of 0.05 kmol/m3 citrate buf-
fer solution (pH 4.8). Nutrients consisting in KH2PO4, 1.0 g/dm3;
MgSO47H2O, 0.50 g/dm3; peptone, 5.0 g/dm3; yeast extract, 5.0 g/
dm3, were added. After enzyme addition, the yeast inoculum was
added. SSF was performed at 313.15 K for 72 h. Samples were with-
drawn after 12, 24, 48 and 72 h, and analyzed regarding ethanol by
gas chromatography (GC) on a PerkinElmer Autosystem XL using a
FID detector, and a column HPSMS of 30 m. The GC program:
323.15 K (3 min), 10 K/min, 373.15 K (1 min), 25 K/min and
398.15 K (1 min). The temperature of the injector and detector were
473.15 K and 573.15 K, respectively. The theoretical SSF yield is cal-
culated by assuming that all the potential D-glucose in pre-treated
material is available for fermentation, and a yield of 0.51 kg etha-
nol/kg D-glucose. All the experiments were performed in duplicate.
2.4. Experimental design
The conventional methodology of varying one factor at a time,
maintaining the other variables inuencing the process at a con-
stant level, does not, in fact, point out the combined effect of all
the process variables. The autohydrolysis variables studied to
determine the optimal conditions to achieve maximum ethanol
yield, from the pre-treated material, by SSF process were temper-
ature and acid concentration. The inuences of these two variables
were determined through a response-surface methodology (RSM)
as published elsewhere [17,18]. The methodology was a 22 facto-
rial design, augmenting the central composite (CCD) design with
a total of 11 experiments (Fig. 1). Four star points distributed at
a distance of 1.4 from the central point were carried out, whereas
three central points were included to give important information
on the reproducibility of the experiments and on the suitability
of the proposed model [19]. The experimental runs were carried
out in random order, and the results were summarized and ana-
lyzed with the software MODDE 7.0 (Umetrics AB, Ume, Sweden).
A second-order function to describe the system behaviour was
determined by a Multiple Lineal Regression (MLR) and the statisti-
cal validation was performed by the Fishers test for analysis of var-
iance (ANOVA) with a 95% condence.
3. Results and discussion
3.1. Raw material
Table 1 shows the composition of raw material (mean values
and standard deviation of three determinations). Cellulose (as
 M. Cuevas et al. / Fuel 89 (2010) 28912896
Fig. 1. Factorial experimental design (square domain) and star points (circular
domain). Each black dot represents a pre-treatment conducted at the variable
values indicated. The coded values are bracketed (1 is dened as the lowest value
of a variable and +1 is dened as its highest value, whereas the star points are
distributed at a distance of 1.4 from central point).
Table 1
Percentage (%) of the main components in olive-pruning debris (dry basis)a.
Cellulose Hemicellulose AIL ASL Extractives
30.3 0.7 17.9 0.3 21.0 0.3 3.1 0.2 14.5 0.1
AIL: acid-insoluble lignin.
ASL: acid-soluble lignin.
a
Mean values and standard deviations of three determinations.
D-glucose) and hemicellulose (as sum of D-xylose, D-arabinose, D-
galactose, and D-mannose) contents agree with those reported in
previous studies [20] and make this biomass an adequate substrate
for ethanol production.
3.2. Pre-treated materials characterization
The behaviour of OPD, during high-temperature diluted-acid
hydrolysis, was similar to that of other lignocellulose materials.
Regarding the percentage of non-soluble solid, the values ranged
between 40.7% and 67.3% (Fig. 2). These results are consistent with
the literature. Some authors [21] reported a percentage of non-sol-
uble solid from 44% to 83% in dilute H2SO4 pre-treatment of car-
doon (temperature, 433.15473.15 K; acid loading, 00.2%, w/w).
The non-soluble solid was very sensitive to increases in both tem-
perature and acid concentration. Thus, the parameter raised up
from values of 49.0% and 40.7%, when pre-treatment was carried
out at 483.15 K (with a sulfuric-acid concentration of 0.010 and
0.050 kmol/m3, respectively), to values of 67.351.5% at 433.15 K
(using the same catalyst concentrations). Fig. 2 also shows the
behaviour of acid-soluble and acid-insoluble lignins. The percent-
age of acid-insoluble lignin in pre-treated solids increased from
28.8% to 47.5%, due to the depolymerization of hemicellulose,
while acid-soluble lignin remained relatively constant at values
of around 2.3%.
The major weight loss in olive-pruning debris, after pre-treat-
ment, was attributed mainly to the hydrolysis of the hemicellulose
fraction. Table 2 illustrates the weight of hemicellulose in the pre-
2893
Fig. 2. Total gravimetric residue ( ) and lignin composition (j, acid-insoluble and
h, acid-soluble) of water insoluble fraction resulting from dilute-acid pre-
treatment.
treated solid and hydrolysate, in relation to weight of the copoly-
mer (D-xylose, D-galactose, D-mannose, and D-arabinose) in the ori-
ginal material. The results reveal a strong increase of the
hemicellulose hydrolysis with increasing temperature. From solids
pre-treated at 423.15 K and 0.030 kmol/m3, 433.15 K and
0.010 kmol/m3, 433.15 K and 0.050 kmol/m3, and 458.15 K and
0.002 kmol/m3 a signicant quantity of solid hemicellulose was
obtained (34%, 49.1%, 10.6%, and 18.9%, respectively). Under these
mild conditions, a substantial part of the polymer is not trans-
formed to monomers but to oligomers. For instance, at 458.15 K
and 0.002 kmol/m3 about 60% of the total hemicellulose-sugars
were in an oligomeric form. This result is in the order of that re-
ported by Nabarlatz et al. [22], where an oligosaccharides yield
close to 58% was achieved after corncob autohydrolysis at
463.15 K and 15 min.
Nevertheless, oligosaccharides cannot be directly metabolized
by yeasts. The highest total hemicellulose-sugar yield in the liquid
(sum of carbohydrates released in monomer and oligomer form)
resulted when the raw material was pre-treated at 458.15 K and
0.030 kmol/m3. Under such condition, 76.0 0.9 kg of sugars (as
monomers) and 21.8 6.2 kg of sugars (as oligomers) were pro-
duced from 100 kg of hemicellulose in OPD, while only
2.4 0.1 kg of original hemicellulose remained in solid. The highest
yield of monomeric sugars (78.9% of initial hemicellulose-sugars)
along with a low yield of oligosaccharides (2.3%) was found for a
pre-treatment temperature of 458.15 K and 0.059 kmol/m3 of acid
concentration. These results concur with those reported by Karimi
et al. [23] in the sulfuric-acid pre-treatment of rice straw, render-
ing 80.8% of the theoretical yield of D-xylose in the transformation
of xylan to D-xylose.
Finally, at higher pre-treatment temperatures considerable
hemicellulose-derived sugars were lost. Thus, the yield in these
carbohydrates decreased progressively to 47.6%, 15.2% and 14.9%
for pre-treatments at 483.15 K and 0.010 kmol/m3, 483.15 K and
0.050 kmol/m3, and 493.15 K and 0.030 kmol/m3, respectively. This
may be attributed to the thermal-degradation process.
Concerning the cellulose bers, Table 2 shows the cellulose per-
centage in the pre-treated solid (expressed in relation to the initial
content of cellulose) and the D-glucose yield in the hydrolysate li-
quor. The relative content of cellulose in pre-treated materials in-
creased 1.11.5 times compared to the raw material, but the
cellulose percentage (in relation to its initial content) in the solid
fraction was low for all the conditions, and ranged between
2894 M. Cuevas et al. / Fuel 89 (2010) 28912896

Table 2
Composition of ltrate and water insoluble solid resulting from diluted-acid pre-treatment of olive-pruning debris.

Pre-treatment conditions Cellulose fraction Hemicellulose fraction

3
T (K) H2SO4 (kmol/m ) A B C A B C
423.15 0.03 68.5 13.5 17.7 34.0 19.3 25.7
433.15 0.01 71.6 8.6 23.6 49.1 2.9 27.2
0.05 67.5 30.2 2.9 10.6 69.1 22.5
458.15 0.002 69.4 7.7 24.0 18.9 14.7 60.6
0.03 65.7 30.4 4.3 2.3 74.9 18.6
0.03 66.8 29.7 9.3 2.6 76.0 30.5
0.03 69.7 30.4 4.1 2.3 77.2 16.2
0.059 67.4 36.4 1.5 0.0 78.9 2.3
483.15 0.01 72.1 15.7 7.5 2.0 45.6 0.0
0.05 55.6 19.1 3.5 0.0 15.2 0.0
493.15 0.03 61.6 18.5 2.1 0.0 14.9 0.0

Percentage of sugars in the pre-treated solid (A), and monomers (B) and oligomers yields (C) in the hydrolysate, expressed in relation to the initial content of cellulose, or
hemicellulose, from the olive-pruning debris.

55.6% and 71.6%. This result can be explained assuming the exis-
tence of a high fraction of amorphous cellulose that is transferred
to liquid after a mild pre-treatment. The great capacity of acid
hydrolysis to produce D-glucose at mild conditions from olive-tree
pruning debris had been reported by some authors [24].
The D-glucose content of the ltrates rose progressively with
the pre-treatment temperature and acid concentration, reaching
a maximum yield of 36.4 kg D-glucose from 100 kg of original cel-
lulose (at 458.15 K and 0.059 kmol/m3), and then values lowered.
As reected in Table 2, the oligosaccharides yield declined at a con-
stant temperature when the acid concentration was augmented.
The most severe pre-treatment conditions (483.15 K and
0.050 kmol/m3; 493.15 K and 0.030 kmol/m3) were needed in or-
der to increase the hydrolysis of the crystalline cellulose fraction.
Under those conditions, a signicant degradation of monosaccha-
rides and oligosaccharides was observed. Nevertheless, maximum
D-glucose loss (21.8% and 36.3%, for pre-treatment at 483.15 K with
0.050 kmol/m3 and 493.15 K with 0.030 kmol/m3, respectively)
was produced to a lesser extent than hemicellulose-sugar losses.
Fig. 3. Ethanol concentration from SSF of pre-treated olive-pruning debris under
different conditions: () 458.15 K and 0.030 kmol/m3; (D) 483.15 K and
3.3. Simultaneous saccharication and fermentation 0.010 kmol/m3; () 433.15 K and 0.050 kmol/m3; (h) 433.15 K and 0.010 kmol/
m3. Data are the mean of two independent samples.
The solids resulting from pre-treatment were subjected to the
simultaneous saccharication and fermentation (performed in
Table 3
duplicate) using S. cerevisiae. The time courses of SSF with four dif- Experimental values of YE, EPTC and QE calculated for SSF.
ferent pre-treatments are shown in Fig. 3. In general, it can be seen
that the bio-fuel production increased fast during rst 24 h of fer- Pre-treatment conditionsa YE (%)b EPTC (%)c QE (kg/(m3 h))d

mentation. After 48 h, no signicant differences stood out by com- 423.150.030 3.0 0.0 25.7 0.2 0.073 0.000
paring the nal ethanol concentration. The ethanol concentration 433.150.010 2.6 0.1 21.1 0.7 0.055 0.000
433.150.050 7.3 0.1 62.8 0.4 0.23 0.00
strongly depended on pre-treatments conditions and it increased
458.150.002 6.8 0.2 55.4 1.8 0.27 0.02
from 3.9 kg/m3, after a pre-treatment at 433.15 K and 458.150.030 9.8 0.1 83.6 1.2 0.25 0.01
0.010 kmol/m3 to 20.3 kg/m3, after pre-treatments at 458.15 K 458.150.030 9.5 0.2 76.6 1.2 n.d.
and 0.030 kmol/m3 or at 458.15 K and 0.059 kmol/m3. 458.150.030 8.5 0.1 79.1 0.6 0.24 0.00
458.150.059 8.9 0.1 76.0 1.1 0.24 0.01
Table 3 lists the experimental results (ethanol yield, YE, and vol-
483.150.010 9.6 0.0 75.1 0.1 0.17 0.01
umetric ethanol productivity, QE) collected by tests planned 483.150.050 6.6 0.0 67.0 0.0 0.13 0.01
according to the two-level factorial design. Also, indicated are the 493.150.030 8.7 0.0 82.9 0.1 0.17 0.01
percentages of ethanol obtained in relation to the theoretical max-
n.d. = Not determined.
imum quantities of ethanol that could be produced from the cellu- a
Temperature (K) sulfuric-acid concentration (kmol/m3).
lose of the pre-treated material (EPTC). b
Ethanol yield (kg ethanol per 100 kg dry olive-pruning debris).
c
The volumetric ethanol productivity (QE) was evaluated by the Percentage of ethanol produced in relation to the theoretical maximum quan-
differential method for analysis of kinetic data, following proce- tity of ethanol that could be produced from the cellulose of the pre-treated material.
d
Volumetric ethanol productivity at 12 h of fermentation.
dures established in previous works [25,26]. It was considered that
the ethanol concentration changed during SSF according to,
  
ET B ET
At 1 ln ln lnlnA B lnt 2
ET  E ET  E
where ET represents the maximum concentration of obtainable
product (on the basis of GayLussac yield) while A and B are two and its parameters calculated by least-squares t. In this way, QE
empirical parameters. The above equation was linearized, was determined using the expression,
 M. Cuevas et al. / Fuel 89 (2010) 28912896 2895

B
Q E ET BlnAtB1 At 3
The ethanol concentration followed the typical batch-fermentation
prole, with a fast product release (during the rst 12 h of culture),
and then the bio-fuel-production rate slowed down. The poor volu-
metric ethanol productivity from SSF could be explained by the fact
that the QE values were calculated at 12 h of fermentation (Table 3).
However, QE was affected notably by the pre-treatment tempera-
ture with an initial rise (0.073 kg/(m3 h)), a middle peak (0.27 kg/
(m3 h)), and a nal fall (0.17 kg/(m3 h)) when the temperature rose
from 423.15 to 493.15 K. This was probably due to the fact that mild
pre-treatments showed partial hemicellulose conversion (hemicel-
lulose is a physical barrier that can protect the cellulose from enzy-
matic attack) while under harsh conditions a minimum cellulose/
lignin ratio appeared in the pre-treated solid (Fig. 2 and Table 2).
With regard to the EPTC, the values ranged between 21.1% (with
a pre-treatment at 433.15 K and 0.010 kmol/m3 acid concentra-
tion) and 83.6% (at 458.15 K and 0.030 kmol/m3 acid concentra-
tion), Table 3. The values agree with those reported by Martn
et al. [27], who found a maximum cellulose conversion to ethanol
of 87.5% during SSF of wet-oxidized cloverryegrass using S. cere-
visiae, or by Karimi et al. [28], who reported an EPTC of 4074% dur-
ing SSF of dilute-acid pre-treated rice straw using the same yeast.
Fig. 5. Response-surface: ethanol yield (kg per 100 kg of olive-pruning debris) as a
As shown in Fig. 4, the ethanol production rst increased lineally
simultaneous function of pre-treatment temperature (423.15483.15 K) and sulfu-
with increasing percentages of cellulose in the solid residue and ric-acid concentration (0.0100.050 kmol/m3).
then remained nearly constant. Thus, when the pre-treated solids
reached a cellulose percentage above 42%, no increase on EPTC
was detected. This limitation may be attributed to a low cellulase where YE is the response and X1 and X2 are the coded values of tem-
load (all experiments have been performed using the same enzyme perature and sulfuric-acid concentration, respectively (Fig. 1). In the
dosage per gram of pre-treated solid), to a high lignin content model, r2 (the goodness of t) and Q2 (the goodness of prediction)
(above 39% for the most severe pre-treatments, Fig. 2) or to the were, respectively, 0.973 and 0.878. The F-value (35.82) and p-value
presence of a recalcitrant cellulose fraction that is not possible to (0.001) did demonstrate that the regression was statistically signif-
break down under the assayed operational conditions. icant. The equation shows that the effect of pre-treatment temper-
For the analysis of simultaneous saccharication and fermenta- ature, on YE, was higher than the sulfuric-acid concentration.
tion process, YE is more suitable than EPTC, because a fraction of cel- Three-dimensional representation of the response-surface (YE)
lulose in OPD is transferred to liquid phase after the pre-treatment. is given in Fig. 5. Maximum production of bio-fuel occurred with
The SSF experiments performed with the solids pre-treated at pre-treatment conditions that coincided with high temperatures
458.15 K and 0.030 kmol/m3, and 483.15 K and 0.010 kmol/m3, pro- and low acid concentrations. The optimum values for both vari-
duced 9.20.2 kg ethanol and 9.6 0.0 kg ethanol from 100 kg of raw ables were determined by the SIMPLEX method from the maxi-
material, respectively. The quadratic model calculated for YE was, mum values of the response-surface. The predicted values were
480.15 K and 0.016 kmol/m3 of acid concentration, with a response
Y E 9:27  0:34 1:80  0:21X 1 0:58  0:21X 2
of 9.9 kg ethanol per 100 kg olive-pruning debris.
 1:79  0:25X 21  0:79  0:25X 22  1:93  0:29X 1 X 2 4 Economically, for energy-production from lignocellulose mate-
rials by biochemical pathways, it is necessary to consume not only
the cellulose fraction but also hemicellulose-sugars, employing
yeasts capable of fermenting these fractions to ethanol (e.g. Pachys-
olen tannophilus). To optimize the overall process, two aims must
be achieved: high cellulose content in the pre-treated solid (the
cellulose must be susceptible to hydrolysis by cellulases) and high
concentration of hemicellulose-sugars (in monomeric form) in
hydrolysate, free of fermentation inhibitors. The highest total eth-
anol yield (sum of fuel generated from pre-treated solid, by SSF,
and ethanol obtained, from D-glucose present in the ltrate) would
be obtained when olive-pruning debris is pre-treated at 458.15 K
and 0.059 kmol/m3 or at 458.15 K and 0.030 kmol/m3; under such
conditions 15.3 kg or 14.5 kg of ethanol could be produced, respec-
tively, from 100 kg olive-pruning debris (22.8 kg ethanol per
100 kg raw material, also considering the total conversion of hemi-
cellulose-sugars in the ltrate).

4. Conclusions

In this study of the conversion of olive-pruning debris to bio-

Fig. 4. Ethanol produced (calculated as percentage of bioproduct in the SSF divided
fuel ethanol, via diluted-acid pre-treatment and the SSF process,
by potential ethanol from the cellulose in the pre-treated material) related to the was found that as the process temperature and sulfuric-acid con-
cellulose percentage in the pre-treated solid. centration increased, hemicellulose was degraded to water-soluble
2896 M. Cuevas et al. / Fuel 89 (2010) 28912896
compounds (in monomeric and oligomeric form). Pre-treatment
step solubilized all the hemicellulose and increased the cellulose
content to 46.3% (1.5 times more compared to the initial raw mate-
rial). Simultaneous saccharication and fermentation of washed
pre-treated solids gave ethanol percentages higher than 80%
(based on the cellulose content in the pre-treated solids, EPTC)
but low SSF ethanol yields (based on the weight of the raw mate-
rial, YE), because of the existence of a cellulose fraction apt for mild
pre-treatments that is transferred to hydrolysate.
Therefore, it is necessary to increase the cellulose content of the
solids through other pre-treatments, and to ferment the pentoses
in the hydrolysate, in order to make the process economically
feasible.
Acknowledgement
This work was nancially supported by the Project 01272/2005
and by a mobility grant of the Consejera de Innovacin, Ciencia y
Empresa, Junta de Andaluca (Spain).
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