Industrial Crops and Products 49 (2013) 196203

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Industrial Crops and Products
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Detoxication of hemicellulosic hydrolyzate from olive tree
pruning residue
Soledad Mateo
a
, Ins Conceic o Roberto
b
, Sebastin Snchez
a
, Alberto J. Moya
a,
a
Department of Chemical, Environmental and Material Engineering, University of Jan, 23071 Jan, Spain
b
Department of Biotechnology, College of Chemical Engineering of Lorena, P.O. Box 116, Lorena, So Paulo, Brazil
a r t i c l e i n f o
Article history:
Received 17 January 2013
Received in revised form24 April 2013
Accepted 30 April 2013
Keywords:
Detoxication
Olive tree pruning
Hemicellulosic hydrolyzate
Activated charcoal
a b s t r a c t
One of the major problems in commercial production of lignocellulosic ethanol and xylitol, are the toxic
compounds generated during the hydrolytic process. A concentrated hemicellulosic hydrolyzate from
olive tree pruning residue has been detoxied using several methods in order to minimize the presence
of inhibitory compounds, improving subsequent fermentation stage with yeasts. Thus, in a rst stage,
activated charcoal has been used to study the inuence of pH, agitation, temperature and the propor-
tion of this type of adsorbent, using a 2
4
experimental design. Further, liming and overliming processes
were performed with three different alkalis, CaO, Ca(OH)
2
and NaOH. Finally, the use of three organic
solvents, chloroform, n-hexane and ethyl acetate, in four different proportions, 2:1, 1:1, 1:2 and 1:3
(hydrolyzate:solvent, v/v), was tested to achieve the toxic compounds elimination. In all assays we have
checked the reduction inacetic acid, total phenolic compounds, furfural, hydroxymethylfurfural and total
sugars concentrations. The volume loss of hydrolyzate has also been determined. In the adsorption pro-
cesses with activated charcoal, the variables that have proved most inuential on the responses tested
were the percentage of this compound and pH. It could be an excellent alternative to decrease inhibitors
from olive tree pruning residue hydrolyzates (removing 46% of acetic acid, 81% of phenolic compounds
and 98% of total furans). The detoxication treatments with solvents revealed that the experimental
conditions which combined the best inhibitor removal and lower alteration of sugar yields could be
achieved using ethyl acetate because this extractor agent was able to remove near 50% of total phenolic
compounds and 57% of total furans. Less favorable results were obtained when n-hexane was employed.
The liming and overliming processes allowed a signicant reduction of acetic acid (in some case up
to 53%), lignin-derived compounds (close to 72%) and total furans (near 83%) mainly furfural, but also
hydroxymethylfurfural.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Olive tree pruning residue (OTPR) is a renewable, cheap and
widely available resource of polysaccharides, primarily at the
Andalusianregion(Spain). The acidhydrolysis of this residue canbe
conceived as the rst stage of an integrated global strategy for this
residue utilization to generate interesting products as bioethanol
and xylitol. Among the treatments tried with other lignocellu-
losic materials, dilute acid hydrolysis appears to be in the best
position in economic terms (Wyman, 1994). Through this mech-
anism it is possible to obtain some sugars such as d-xylose and
d-glucose as the major reaction products, but one problemassoci-
atedwiththis hydrolytic process is thetoxic compounds generation
(furans, aliphatic acids and phenolic constituents), regarded as a

Corresponding author. Tel.: +34 953 212780; fax: +34 953 212140.
E-mail address: ajmoya@ujaen.es (A.J. Moya).
great limiting factor in bioconversion processes using acid hydrol-
ysis and complicating the hydrolyzates fermentability (Purwadi
et al., 2004; Larsson et al., 1999).
Hemicellulose is the second major polysaccharide in woody
biomass, typically comprising 1535% of the dry wood mass. It
consists of heterogeneous polymers of pentose (d-xylose and l-
arabinose) and hexoses (d-glucose, d-mannose and d-galactose),
which can be substituted with phenolic, uronic or acetyl groups
(Lee et al., 2011).
In order to investigate the effectiveness of the detoxica-
tion process for the subsequent fermentation of the concentrated
hydrolyzate, several treatments have been tested attending to
the literature (Palmqvist and Hahn-Hgerdal, 2000; Converti
et al., 2000; Martnez et al., 2001; Mussatto and Roberto,
2004).
Since the level of detoxication would be conditioned by
the fermenting microorganisms tolerance to certain inhibitory
compounds, and to minimize the costs of different treatments,
0926-6690/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.04.046
S. Mateo et al. / Industrial Crops and Products 49 (2013) 196203 197
simple detoxication methods are proposed instead of combined
ones, usually posed as a system of inhibitors reduction in the
hydrolyzate.
Furfural and 5-hydroxymethylfurfural (HMF) are two furans
derivatives which are formed by the further hydrolysis of sugars,
pentoses and hexoses, respectively. These furans are available in
relatively high concentration in the hydrolyzates and are serious
inhibitors for microorganisms. The furans are probably the most
important group of inhibitors, since the fermentability of dilute-
acid hydrolyzates is inversely related to the concentration of this
compounds (Taherzadeh et al., 1997). Phenolic compounds cause
loss of integrity of biological membranes, thereby affecting their
ability to serve as selective barriers and enzyme matrices (Kang
et al., 2012).
Acetic acid, the major aliphatic compound present, is released
from the hemicellulosic acetyl groups and formic acid is a degra-
dation product of HMF (Ulbricht et al., 1984). When these acids
(in the undissociated form) enter in the cell, become dissociated in
the protoplasmbecause its pH. This fact leads to a decrease in the
intracellular pHand can generate the cellular death (Verduyn et al.,
1990).
This study describes several detoxication methods of hemicel-
lulosic hydrolyzates fromOTPR (liming and overliming with NaOH,
CaO and Ca(OH)
2
, activated charcoal adsorption and organic sol-
vents use) in order to nd a treatment that allows efcient removal
of major inhibitory compounds of fermentation processes present
inthese hemicellulosic hydrolyzates. Althoughthe needs for detox-
ication must be evaluated in each case, yeast tolerance to these
components depends on the types of microorganismconsidered so
that, a decrease of the microbial inhibitors concentrations present
in the medium could be particularly benecial by decreasing the
reaction times and increasing the efcient sugars utilization and
principal products formation.
2. Materials and methods
2.1. Raw material characterization
OTPR was collected locally after the fruit harvest, air-dried at
room temperature to equilibrium moisture content (812%) and
milled using a laboratory hammer mill (Retsch); fraction graded to
a particle size between 0.60mmand 0.425mm, according to ASTM
guidelines, was stored until used. This fraction consists of leaves
(30%) and wood chips (70%).
For chemical characterization (of original residue without
extracts), the modied methodology proposed by Browning (1967)
was used. It consisted of the hydrolysis of 2g of sample with H
2
SO
4
(10cm
3
) at 72% (w/w) and maintained at 50

C for 7min. After this

pre-treatment, distilled water (275cm
3
) was added to dilute the
above acid and the solution was autoclaved at 1atmand 121

C for
45 min. After ltration of the hydrolyzate, a solution is obtained
which will allow the determination of the acid soluble lignin, car-
bohydrates, acetic acid, furfural and hydroxymethylfurfural (HMF)
by HPLC.
Ash contents were determined by weight difference before and
after incineration of the OTPR sample in a furnace at 550

C for 4h.
All determinations were carried out in triplicate.
The correct characterization of OTPR is not an easy issue to
resolve. Using traditional methods we can mistake in allocat-
ing the d-glucose into the cellulosic fraction if this monomer is
part of the hemicellulosic fraction or whether there is d-glucose
and d-xylose in the parenchyma surrounding the cell wall as
reserve material in form of insoluble -1,3 glucans or like oleu-
ropein that show a higher concentration in leaves extracts. This
polyphenolic glycoside, formed by the union of three molecules:
Table 1
Olive tree pruning residue composition.
Composition % of dry matter
a
Glucosans 28.6 0.4
Pentosans 13.9 0.2
d-xylose 10.1 0.3
l-arabinose 3.5 0.2
Acid-insoluble lignin 21.4 0.5
Acid-soluble lignin 2.3 0.6
Acetyl groups 1.5 0.1
Ash 3.8 0.3
Extractives 21.4 0.2
In water 18.8 0.1
d-xylose 2.7 0.1
d-glucose 5.8 0.1
In ethanol 2.6 0.1
a
Average of at least three determinations.
hydroxytyrosol, d-glucose and elenolic acid, could generate d-
glucose with hydrolytic conditions prone to their formation (Silva
et al., 2006). We think this would be the situation in the OTPR, so
we have made some determinations in order to conrm the cor-
rect composition, Table 1. This biomass can provide high amounts
of sugar for every unit mass of dry residue, considering that fer-
mentable sugars may represent almost 50% and can be processed
into bioproducts of interest by a subsequent fermentation.
2.2. Preparation of hemicellulosic hydrolyzate
Acid hydrolysis was conducted in a stainless steel reactor of
50dm
3
capacity, with a useful volume of 40dm
3
, provided with
a system of electrical resistance heating and agitation by rotation
about its own axis. The reactions were carried out with H
2
SO
4
(2%
w/v) at 120

C, for 90min, using 1:10 solid/liquid ratio. Treatment

conditions were chosenbypreliminarytests withthe aimof obtain-
ing the highest concentration of total sugars in the liquid. The solid
andliquidphases were separatedby ltration. The hydrolyzate was
concentrated three to four-fold under vacuum in a 4dm
3
evap-
orator at 705

C. The concentrated hydrolyzate was chemically

characterized.
2.3. Detoxication procedures
Treatments of lignocellulosic hydrolyzate could removal some
compounds which would improve their fermentability in a
subsequent step. We study different detoxication methods fun-
damentally based on chemical treatment to diminish inhibitors
present in this one.
2.3.1. Activated charcoal adsorption
The concentrated hydrolyzate was treated, at room tem-
perature, with activated charcoal (Labsynth in powder form,
granulometry of 32% retained on 325mesh, specic surface area
of 860m
2
g
1
and an average particle size of 0.43cm). Charcoal
was mixed with 50g of concentrated hydrolyzate for 30min. Sub-
sequently, the charcoal was separated from the hydrolyzate by
ltration.
The temperature was at the range 2535

C, the agitation inter-

val in 100200rpm, the concentration of adsorbent between 2%
and 8% and the pH (adjusted with NaOH or H
2
SO
4
) between 2 and
6.
2.3.2. Liming and overliming processes
We usedthree different alkalis: CaO, Ca(OH)
2
andNaOHat room
temperature. The liming process consisted on neutralization to pH
5.5 and centrifugation (10,000rpm for 10min) of the precipitate
obtained. On the other hand, the overliming experiments involved
198 S. Mateo et al. / Industrial Crops and Products 49 (2013) 196203
Table 2
Chemical composition of the original and concentrated hydrolyzate of OTPR.
Group of compounds Compounds Concentration (gdm
3
) % Reduction
Original Concentrated
Sugars d-xylose 9.71 0.27 37.93 2.13 2.3
d-glucose 12.73 0.59 49.50 2.50 2.8
l-arabinose 2.79 0.11 10.55 0.62 5.5
Furan derivates HMF 0.22 0.02 0.84 0.02 4.5
Furfural 0.04 0.01 0.14 0.03 12.5
Totals 0.51 0.02 1.70 0.04 16.7
Aliphatic acids Acetic acid 1.67 0.08 2.77 0.35 58.5
Formic acid 0.45 0.02 1.34 0.02 25.6
Phenolic compounds 3.76 0.13 12.65 0.52 15.9
the pH adjust to 10 by the addition of these different compounds.
After that, the solid and liquid fraction was separated by centrifu-
gation acidifying, the last one, to pH5.5 with 72% (w/w) H
2
SO
4
and
centrifuged again for precipitate removal.
2.3.3. Organic solvents treatments
Finally, westudiedtheuseof threeorganic solvents: chloroform,
n-hexane and ethyl acetate in four different proportions, 2:1, 1:1,
1:2 and 1:3 (hydrolyzate: solvent, v/v).
2.4. Analytical methods
The quantication of carbohydrates (d-glucose, d-xylose and l-
arabinose) as well as acetic acidconcentrations (inorder toestimate
the acetyl groups content) were determined by high-performance
liquid chromatography (HPLC) using a WATERS equipment, in
the conditions: a BIO-RAD Aminex HPX-87H (300mm 7.8mm)
column at 45

C, 0.005M sulfuric acid as eluant, ow rate of

0.6cm
3
min
1
, refraction index (RI) detector and 20L sample vol-
ume.
Furfural and HMF were analyzed by HPLC using a WATERS
instrument with a UV detector (at 276nm), in the following con-
ditions: a Waters Resolve C18 5m (300mm 3.9mm) column
at ambient temperature, acetonitrile/water (1/8 with 1% of acetic
acid) degassed, with addition of phosphoric acid for pH correction
to 2.5 as eluant, ow rate of 0.8cm
3
min
1
and 20L sample vol-
ume. The samples were previously diluted with ultrapure water
and ltered through membranes HAWP 04700 with 0.45mpores.
The concentrations of these compounds were calculated fromcal-
ibration curves obtained fromstandard solutions.
To calculate the cellulose, hemicellulose and Klason lignin per-
centages, the methodology proposed by Irick et al. (1988) was
utilized and moisture composition by the TAPPI norm T12 os-
75. Furthermore, the concentration of acid-soluble lignin was
determined by the method proposed by Rocha (2000) and ashcom-
position using the procedure established by Browning (1967).
Extractives (nonstructural components such as pectins, fats,
terpenes, phenolic compounds, tannins, uronic acids, etc.) were
determined gravimetrically using a two-step sequential extraction
process by Soxhlet to remove water and ethanol soluble material
according to a procedure adapted fromSluiter et al. (2008).
The total phenol content, expressed as concentration of fer-
ulic acid, was determined colorimetrically at 760nm, using
FolinCiocalteu modied method (Singleton et al., 1999). To esti-
mate the total furan content (Martnez et al., 2000) a Beckman DU
640B spectrophotometer model was employed. The samples were
analyzed using two different wavelengths, 284nmand 320nm.
3. Results and discussion
Dilute sulfuric acid has achieved good yields of monomeric
sugars from many cellulosic materials. OTPR was submitted to
acid hydrolysis in conditions that promoted selective genera-
tion of simple carbohydrates and, subsequently, the hydrolyzate
was concentrated to increase sugars content. The fermentable
monosaccharides concentration and the toxic compounds present
in original and concentrated hydrolyzate are shown in Table 2.
All dilution steps during the treatments have been considered
in the calculations of the nal concentrations of those analyzed
compounds. Although the amounts of principal carbohydrates and
inhibitory compounds were measured before and after the treat-
ments, to take account possible volume variations, the elimination
percentageof bothsugars andfermentationprocess inhibitors were
calculated as reduction in weight of the concerned substances
present in the detoxied hydrolyzates in relation to global content
of themin the initial concentrated.
The main purpose of this study was to investigate the effects of
different detoxication processes using some chemicals agents.
3.1. Hemicellulosic hydrolyzate of olive tree residue
The liquid obtained after hydrolysis from OTPR with diluted
sulfuric acid contained 25.2gdm
3
of total fermentable sugars.
After the concentration process (4.0 times), the simple sugars
content was 90.45gdm
3
(density: 1087gdm
3
), being the total
amount of hemicellulosic carbohydrates approximately the same
as d-glucose. There was no sugar degradation during the vacuum
evaporation process according the concentration factor employed,
fact also evidenced by Rivas et al. (2002).
Concentration step by evaporation could act as a detoxication
system for several compounds, as it is shown in Table 2 at the %
of reduction or loss column. The signicant presence of phenolic
compounds after evaporation treatment, to a greater extent than
acetic acid must be noted. Moreover, the increase of acetic acid
concentration may be a consequence of the system evaporation
conducted under vacuum, obtaining about a 58% of elimination rel-
ative to the amount of aliphatic acid that could be present. The fact
that this treatment has been performed at lowpH (0.97) may have
favored this compounds removal which are volatile only in its pro-
tonated form (Larsson et al., 1999). Furthermore, the evaporation
at 70

C was able to produce a partial removal of furfural (12.5%),

fact also evidenced in Canilha et al. (2005), but the evaporation had
a negligible effect in HMF removing (4.5%), as also noted Dehkhoda
et al. (2009), because of this lower volatility specially under acidic
conditions.
3.2. Activated charcoal
In order to research the effectiveness of this kind of treatment,
if this detoxication method for OTPR concentrated hydrolyzate
was able to reduce some inhibitor concentrations, we have con-
sidered the main parameters which affect the adsorption process
with activated charcoal: pH (X
1
), agitation (X
2
), temperature (X
3
)
and % of activated charcoal (X
4
) (Palmqvist and Hahn-Hgerdal,
S. Mateo et al. / Industrial Crops and Products 49 (2013) 196203 199
Table 3
Matrix of the factorial design for activated charcoal detoxication study.
Assay Variables: real values (coded values)
X
1
X
2
X
3
X
4
1 2 (1) 100 (1) 25 (1) 2 (1)
2 6 (+1) 100 (1) 25 (1) 2 (1)
3 2 (1) 200 (+1) 25 (1) 2 (1)
4 6 (+1) 200 (+1) 25 (1) 2 (1)
5 2 (1) 100 (1) 35 (+1) 2 (1)
6 6 (+1) 100 (1) 35 (+1) 2 (1)
7 2 (1) 200 (+1) 35 (+1) 2 (1)
8 6 (+1) 200 (+1) 35 (+1) 2 (1)
9 2 (1) 100 (1) 25 (1) 8 (+1)
10 6 (+1) 100 (1) 25 (1) 8 (+1)
11 2 (1) 200 (+1) 25 (1) 8 (+1)
12 6 (+1) 200 (+1) 25 (1) 8 (+1)
13 2 (1) 100 (1) 35 (+1) 8 (+1)
14 6 (+1) 100 (1) 35 (+1) 8 (+1)
15 2 (1) 200 (+1) 35 (+1) 8 (+1)
16 6 (+1) 200 (+1) 35 (+1) 8 (+1)
17 4 (0) 150 (0) 30 (0) 5 (0)
18 4 (0) 150 (0) 30 (0) 5 (0)
19 4 (0) 150 (0) 30 (0) 5 (0)
X
1
: pH. X
2
: agitation (rpm).
X
3
: T (

C). X
4
: % act. char. (w/w).
2000; Mussatto and Roberto, 2004). The study of the different fac-
tors inuence was carried out, during a xed time of 30min, using
a 2
4
experimental design with three central points. Table 3 shows
the matrix of the experimental design done, including the real and
coded values of the studied variables. The experimental runs were
carried out in randomorder and the results were summarized and
analyzed with the software STATISTICA 6.0 (Statsoft, USA).
The Pareto charts, Fig. 1, describes calculated student t abso-
lute values, also called standardized effects, providing lengths of
the bars which in turn are arranged in decreasing order. All fac-
tors or interactions for which the length of the bars, representing
the corresponding statistical signicance, is positioned beyond the
vertical line drawn to a condence level of 95%, p=0.05, will be
signicant.
The mathematical models (by elimination of those terms that
are not statistically signicant for the treatment, those which p-
valor are above the signicance level =0.05) used to predict the
optimal value, for each response variable, were expressed accord-
ing to Eqs. (1)(3). Response values of some dependent variables (%
reductionof volume, Y
1
; %reductionof total sugars, Y
2
and %reduc-
tion of furfural, Y
3
) as well as the error in parameter estimation by
model, are shown in Table 4.
Y
1
= 22.62188 +3.7625X
1
2.275X
2
1.5X
3
+4.6X
4
+1.05X
1
X
3
+1.6125X
2
X
3
+1.7375X
2
X
4
+1.1875X
3
X
4
(1)
Y
2
= 28.9875 +3.15562X
1
+3.39313X
2
3.19063X
3
+5.62312X
4
+2.35688X
2
X
3
+1.94813X
2
X
4
+2.56563X
3
X
4
(2)
Y
3
= 94.35 +0.0875X
1
+0.3375X
2
0.66255X
3
+0.55X
4
0.67505X
1
X
2
+0.625X
1
X
3
+1.8625X
1
X
4
+0.5875X
2
X
4
(3)
The results reveal that in almost all cases, the variables that
have proved most inuential on the responses tested were the per-
centage of activated charcoal and pH. As their effects are positive,
a
b
c
Fig. 1. Pareto charts of standardized effects for reduction of (a) volume; (b) total
sugars; and (c) furfural.
an increase of both independent variables values, would imply an
increase in the value of the response. Fig. 2 shows the response
surfaces at the central point of the rest of variables for reduction of
volume, Y
1
; total sugars, Y
2
and furfural, Y
3
.
Results of analysis of variance (ANOVA) with estimation of the
curvature for the dependent variables tested is used to determine
the degree of t between the experimentally observed values and
those obtained by the model.
For all the analyzed parameters, correlation coefcients (R
2
) are
acceptable (R
2
> 0.943); thus, for Y
1
, R
2
=0.978 pointing out that
only 2.2% of the total variations in the response were not explained
by the model (is explained by the residue), or what is the same,
200 S. Mateo et al. / Industrial Crops and Products 49 (2013) 196203
Table 4
Analysis of variance (ANOVA) of the models describing the % reduction of volume (Y
1
), total sugars (Y
2
) and furfural (Y
3
).
DF
a
SS
b
MS
c
Y
1
Y
2
Y
3
Y
1
Y
2
Y
3
Y
1
Y
2
Y
3
Model 8 7 7 813.98 1267.25 88.25 101.75 181.04 12.61
Curvature 1 1 1 0.36 4.92 0.02 0.36 4.92 0.02
Residual 9 10 10 18.02 75.99 3.98 2.00 7.60 0.40
Lack of t 7 8 8 16.30 69.04 3.89 2.33 8.63 0.49
Pure error 2 2 2 1.73 6.95 0.09 0.86 3.48 0.04
Total 18 18 18 832.37 1348.17 92.24
F-value p-value
Y
1
Y
2
Y
3
Y
1
Y
2
Y
3
Model 50.81 23.82 31.70 0.0001 0.0001 0.0001
Curvature 0.18 0.65 0.04 0.6802 0.4399 0.8379
Residual
Lack of t 2.70 2.48 11.22 0.2970 0.3187 0.0844
Pure error
Total
R
2
for the Y
1
model: 0.978; R
2
for the Y
2
model: 0.943; R
2
for the Y
3
model: 0.957.
a
Degrees of freedom.
b
Sumof squares.
c
Mean square.
explains 97.8% of variation in the response, showing good accuracy
in predicting the response value.
Considering Eqs. (1)(3), and to minimize Y
1
and Y
2
while Y
3
is maximized, the best solution is achieved when X
1
=1, X
2
=1,
X
3
=0.12 and X
4
=1 with a desirability of 0.901 being Y
1
=16.3%,
Y
2
=8.1% and Y
3
=95.9%.
The reduction of volume, Y
1
, is strongly inuenced by X
4
and X
1
,
so that the lower volume losses were obtained using the lower pH,
the lower charcoal concentration and higher temperature tested,
independently of agitation. Lower reductions in the concentration
of total sugars were also obtained under these conditions.
Although losses of carbohydrates in many researches have not
been very signicant, the reduction of fermentable sugars reported
in literature data may be 20% (Wang and Chen, 2011) or up to
30%, (Lee et al., 2011), coinciding with the highest removal rates
obtained in the established experimental design. The amount of
sugars eliminated depend on the hydrolyzate composition and
adsorption conditions. It seems that reduction of sugars was higher
when a greater proportion of activated charcoal is used and at
pH=6, perhaps by precipitation or interaction of these carbohy-
drates with other components present in the hydrolyzate when
mediumbecame more basic.
In relation to the reduction of acetic acid, it is greatly inuenced
by the concentration of activated charcoal and the pH, although
directly and inversely proportional respectively.
It is noteworthy that very high furfural reductions are obtained
(8797%) and total elimination of furan is situated in the range
6098%. Lee et al. (2011) found a nearly total adsorption of fur-
fural and HMF when 2.5wt % charge of activated carbon and 1h
of contact time was used. Furfural and HMF concentrations were
dramatically reduced (92% and 68%, respectively) by the treat-
ment with this adsorbent (Carvalheiro et al., 2005), although other
authors indicate belowremoval of HMF percentages, 8.8% (Npoles
et al., 2006).
Ingeneral, eliminationpercentages of total phenolic compounds
were signicant (4281%); higher values corresponded to the
experiments done with a larger amount (8%) of activated charcoal
andthebest results, except inoneof theassays, areagreewiththose
experiments at pH2. Mussatto and Roberto (2004) established that
the adsorption of weak organic acids is favored when the medium
pH is acidic because different products with inhibitory nature are
ionized and improves the adsorbability of this type of molecules.
Pursuant to Kamal et al. (2011), using this detoxication method
for sago trunk hydrolyzates, enabled a reduction of 78% of phenolic
compounds in the best conditions.
While some authors point out that, generally, the adsorption of
furan and phenolic compounds decrease with increasing the treat-
ment temperature for the wood charcoal (Miyafuji et al., 2003),
in this research was found that this parameter seems to have no
signicant effect on this kind of inhibitors elimination or in sug-
ars reduction, at least at tested low values. Use of relatively low
temperatures in the study would be motivated by an attempt to
minimize heating costs which would entail an adsorption process
at high temperature.
Temperature is statistically insignicant, so that the response
surfaces of % furans versus % charcoal and temperature for a given
agitation values resulted to be planes. It indicates that reduction
percentage of furans was not affected by temperature.
The effect caused by the use of activated carbon on phenolic
compounds was greater than the reduction of acetic acid; greater
eliminations of this aliphatic acidareobtainedwhenactivatedchar-
coal load was 8% (removal rates up to 45%), using a 4% intermediate
concentration (2528%) and slightly lower for activated charcoal
rates 2% (<15%). In general, better adsorptions are performed when
hydrolyzate pH was 2 vs. pH 6, independently of the temperature
and agitation degree employed.
Finally, notice that for none of the experiments carriedout, there
was a signicant decrease in hydrolyzate color. The largest reduc-
tions in this parameter were achieved with 8%of activated charcoal
at pH 2 and 35

C.
3.3. Liming and overliming
The liming and overliming terms have been used in this study
to make reference to alkalinization processes, carried out at pH 5.5
or 10, respectively, in which the bases employed have been NaOH,
Ca(OH)
2
and CaO. All experiments have been performed at room
temperature tominimize the costs involvedinhydrolyzate heating.
Both methods entailed a loss of hydrolyzate volume when CaO
is used, between 16% and 20%; with Ca(OH)
2
there were only lost
of concentrated liquid when overliming was employed while the
volume was maintained constant using NaOH; one reason that can
S. Mateo et al. / Industrial Crops and Products 49 (2013) 196203 201
Fig. 2. Response surfaces at the central point of the rest of variables for reduction
of: (a) volume; (b) total sugars; and (c) furfural.
affect to the volume change is related to the dilution experienced
due to the use of neutralizing agents and acidifying involved in the
above process.
The mechanismbehind the technique is not fully specied but it
has been suggested that toxic compounds are precipitated during
Fig. 3. Percentages of elimination of d-glucose, d-xylose, l-arabinose and total sug-
ars using CaO and Ca(OH)
2
for liming and overliming detoxication.
this operation (Baek and Kwon, 2007) or transformed into other
types of chemical substances (Persson et al., 2002). When over-
liming technique were carried out, a great formation of precipitate
were obtained with CaO and Ca(OH)
2
, possibly due to calciumsul-
fate precipitation (gypsum) (Alriksson et al., 2006), by increasing
the amount of base added while the solid amount fromNaOH was
lower. Furthermore, because these detoxication processes led to
a partial removal of furfural and HMF, furans aldehydes may be
involved in alkali-assisted aldol condensation reactions (Horvath
et al., 2005). Inthis regard, andinrelationto the formationof a large
amount of precipitate frominsoluble salts (calciumsulfate, calcium
oxalate among other compounds), a signicant decrease of the ini-
tial conductivity of the concentrated hydrolyzate (138.2mS/cm)
was observed to values of 8.58.8mS/cm with calcium salts and
51.151.4mS/cm for NaOH. These conductivity values are similar
to those obtained for treated hydrolyzates with bases, Martnez
et al. (2001) .
In relation to the removal of both, free sugars and inhibitors
present in the hydrolyzates, Figs. 3 and 4, while NaOH detoxica-
tion treatments did not involve a signicant loss of sugars (<5%)
or acetic acid (<7%), if calcium salts were used, sugar reductions
were higher, presenting the greatest losses by adjusting the pH to
10 with calcium hydroxide like detoxicant agent. Although sev-
eral authors point out lower losses of sugars with these processes
(Larsson et al., 1999; Cheng et al., 2007), others reported a decrease
inthe fermentable sugars of 20%(Horvathet al., 2005; Nigam, 2001)
Fig. 4. Percentages of elimination of acetic acid (AcA), total phenolic compounds
(Tph), total furans (Tfu), furfural (fur) and hydroxymethylfurfural (HMF) using CaO,
Ca(OH)
2
and NaOH for liming and overliming detoxication.
202 S. Mateo et al. / Industrial Crops and Products 49 (2013) 196203
Table 5
Reduction percentages in detoxication process with organic solvents.
Solvent
a
h:s
b
% Reduction
vol
c
ts
d
AA
e
phe
f
fur
g
HMF
EA 2:1 0 19.6 34.9 88.4 0.0
EA 1:1 0 4.1 40.1 91.3 21.0
EA 1:2 4 9.2 48.3 94.4 39.6
EA 1:3 8 10.2 49.5 74.1 40.0
Hex 2:1 0 0.0 0.0 14.7 0.0 0.0
Hex 1:1 0 0.0 0.0 18.9 13.3 0.0
Hex 1:2 4 0.0 0.0 20.4 68.7 0.0
Hex 1:3 4 5.5 0.1 24.1 66.3 0.0
Chl 2:1 0 0.0 6.6 30.6 94.8 0.0
Chl 1:1 0 5.6 9.9 29.3 97.2 0.0
Chl 1:2 0 7.2 9.9 28.0 98.5 0.0
Chl 1:3 0 0.1 17.4 29.7 85.2 11.0
a
EA: ethyl acetate; Hex: n-hexane; and Chl: chloroform.
b
Relation hydrolyzate:solvent (v/v).
c
Volume.
d
Total sugars.
e
Acetic acid.
f
Phenolic compounds.
g
Furfural.
but, in any case, although the magnitude of these losses depend on
the composition and nature of the hydrolyzate the shift to high
pH during the overliming process must be kept to a minimum
(Amartey and Jeffries, 1996); these authors suggested that the cal-
ciumions catalyze the monosaccharides degradationbyinteraction
with their intermediate enolate. With CaO and Ca(OH)
2
was possi-
ble to reduce the amount of above aliphatic acid in 3252%, similar
to the results reported by Ge et al. (2011) by overliming process.
The furans and phenolic compounds elimination occurred in a
large extension; the liming process with CaO and Ca(OH)
2
allowed
furans elimination (5679%) and phenolic compounds reduction
(6269%), whereas overliming removal percentages were slightly
higher, Fig. 4. Martnez et al. (2001) reported 51% removal of total
furans after optimal overliming of diluted acid hydrolyzates from
sugarcane bagasse. Reduction of phenolic compounds, when NaOH
is used, was quite low(2831%) for bothalkaline detoxicationpro-
cesses, while calciumbases had a marked effect allowing a greater
elimination of these lignin derived constituents (5671%). Over-
liming with CaO followed by activated charcoal reduced phenolic
compounds by 82.15.3% (Dubey et al., 2012).
Treated hydrolyzates were obtained with acetic acid reductions
for overliming between 5053%or 3250%for liming process when
bases such as CaO or Ca(OH)
2
were employed; similar data were
reected by Ge et al. (2011).
3.4. Organic solvents treatments
Finally, we studied the use of three organic solvents to achieve
the elimination of toxic compounds, chloroform, n-hexane and
ethyl acetate in four different proportions, 2:1, 1:1, 1:2 and 1:3
(hydrolyzate/solvent, v/v), Table 5.
In general, it was observed how the quantity of inhibitors
present in the medium was increased by using a smaller ratio
hydrolyzate:solvent to carry out the extraction process. Ethyl
acetate was the most effective solvent for furans removal, espe-
cially furfural in a percentage ranging between 74% and 94%, as
it was also evidenced by Frazer and McCaskey (1989), but HMF
could only be removed in signicant quantities (40%) by using as
much of this solvent. n-Hexane was unable to remove HMF and
only reduced total furans content in a maximum of 7% while for
chloroform, reductions are ranged from19% to 34%.
The use of n-hexane as an extracting agent of inhibiting com-
pounds of olive tree pruning hydrolyzates is not a suitable method
when it seeks to eliminate furfural, acetic acid and HMF although
it may moderately reduce (24%) the amount of total phenolic com-
pounds. Chloroform slightly reduces the content of furans and
phenolic inhibitors (34|acetic acid concentration (17%), while the
ethyl acetateallowedmoderatelevels of lignin-derivedcompounds
and furans total elimination (49% and 57%, respectively), but a sol-
vent removal should be performed if the yeast is not tolerant to
acetate ions, which often contribute positively in the fermentation
process.
Thelosses of sugars producedduetotheextractionprocess, even
for higher relations hydrolyzate:solvent, were null using n-hexane,
irrelevant if chloroformwas employed(<4%), andnot toohighwhen
the detoxication was carried out with ethyl acetate (<8%).
The acetic acid cannot be extracted using n-hexane but removal
rates were obtained for this aliphatic compound close to 17% if the
extractor solvent ratio of chloroformwas 1:3 while, if the extractor
solvent ratios are lower, the reduction of acid would be between 7%
and 10%. The acidic pHof the concentrated hydrolyzate might have
favored the mentioned acid dissolution in this solvent extractor
because the protonatedformis probably more soluble inanorganic
mediumthan the non-protonated.
The ethyl acetate was the solvent that shown a higher total
phenolic compounds extracting capacity, allowing reductions of
3449%, followed by chloroform (30%), and nally the n-hexane
(1424%). In fact, hydroxytyrosol is one of the o-catechols with
greater presence in the olive tree, either in free formor as deriva-
tives such as oleuropein, and various solvent of this nature are
employed for their recuperation. As a result of ethyl acetate extrac-
tion, Stoutenburg et al. (2011) got to remove a 25% of this phenol
derivates in sugar maple hydrolyzate.
4. Conclusions
The most suitable detoxication procedure to improve the sub-
sequent fermentation of the concentrated acid liquid obtained, still
depending on the nature of the hydrolyzate, would be one that
would allow greater removal of inhibitory compounds and pro-
duce a poor elimination of simple fermentable sugars. However,
for effective fermentation, once evaluated the parameters of the
process might be sufcient partial and not complete elimination of
those compounds with greater negative repercussions on yeast cell
metabolism, as long as they are situated at levels considered non
toxic to the microorganism.
Inviewof theexperiments carriedout withdifferent detoxifying
agents and considering techniques of neutralization, adsorption,
precipitation and extraction. It must be pointed out that, in general,
the best results for solvent detoxication were achieved with 1:3
ratio which is the greatest of those studied in the present research.
As theresults for this proportionhydrolyzate:solvent, areveryclose
to those obtained with a smaller relationship (1:2) the latest one
should be used in order to minimize the process costs.
When NaOH was used, the decrease in phenolic compounds
was minimal (2831%), for both alkaline detoxication processes,
and slight acetic acid reduction (<7%) was achieved although it did
not involve a signicant loss of sugars, while calcium bases had a
marked effect allowing a greater removal of these lignin derived
constituents (5671%), acetic acid (similar for both bases treat-
ments, next to 50% except liming with CaO, 32%) and total furans
content (6269% for liming and 8083% for overliming). The draw-
back is that reductionof sugar amounts, for liming (1534%) andfor
overliming (2646%), are very high; this method, therefore, could
not be advisable for olive tree pruning concentrated hydrolyzates.
In the adsorption processes with activated charcoal, the vari-
ables that have proved most inuential on the responses tested
were the percentage of this compound and pH. It could be an
excellent alternative to decrease inhibitors fromolive tree pruning
S. Mateo et al. / Industrial Crops and Products 49 (2013) 196203 203
residue hydrolyzates (46% of acetic acid, 81% of phenolic com-
pounds and 98% of total furans), with possibility to reuse the
employed activated charcoal through regeneration methods to
reduce the costs of these processes.
In summary, among the detoxication processes studied for the
hemicellulosic hydrolyzate from olive tree pruning residue, the
most promising one for the reduction of phenolic compounds and
total furans is the adsorption with 2% of activated charcoal at 30

C,
pH=2 and 200rpm.
Acknowledgements
The authors are grateful to Andalusia Regional Government
for nancial support (Project Ref. AGR-6509). On the other hand,
thanks given to Professor M.J. Olmo for her assistance in the statis-
tical treatment of the data.
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