Dp

Sa

b

ARRA

KDOHA

1

wAcralpada(

0h

Industrial Crops and Products 49 (2013) 196– 203

Contents lists available at SciVerse ScienceDirect

Industrial Crops and Products

journa l h om epa ge: www.elsev ier .com/ locate / indcrop

etoxification of hemicellulosic hydrolyzate from olive treeruning residue

oledad Mateoa, Inês Conceic ão Robertob, Sebastián Sáncheza, Alberto J. Moyaa,∗

Department of Chemical, Environmental and Material Engineering, University of Jaén, 23071 Jaén, SpainDepartment of Biotechnology, College of Chemical Engineering of Lorena, P.O. Box 116, Lorena, São Paulo, Brazil

a r t i c l e i n f o

rticle history:eceived 17 January 2013eceived in revised form 24 April 2013ccepted 30 April 2013

eywords:etoxificationlive tree pruningemicellulosic hydrolyzatectivated charcoal

a b s t r a c t

One of the major problems in commercial production of lignocellulosic ethanol and xylitol, are the toxiccompounds generated during the hydrolytic process. A concentrated hemicellulosic hydrolyzate fromolive tree pruning residue has been detoxified using several methods in order to minimize the presenceof inhibitory compounds, improving subsequent fermentation stage with yeasts. Thus, in a first stage,activated charcoal has been used to study the influence of pH, agitation, temperature and the propor-tion of this type of adsorbent, using a 24 experimental design. Further, liming and overliming processeswere performed with three different alkalis, CaO, Ca(OH)2 and NaOH. Finally, the use of three organicsolvents, 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 havechecked the reduction in acetic acid, total phenolic compounds, furfural, hydroxymethylfurfural and totalsugars concentrations. The volume loss of hydrolyzate has also been determined. In the adsorption pro-cesses with activated charcoal, the variables that have proved most influential on the responses testedwere the percentage of this compound and pH. It could be an excellent alternative to decrease inhibitorsfrom olive tree pruning residue hydrolyzates (removing 46% of acetic acid, 81% of phenolic compoundsand 98% of total furans). The detoxification treatments with solvents revealed that the experimental

conditions which combined the best inhibitor removal and lower alteration of sugar yields could beachieved using ethyl acetate because this extractor agent was able to remove near 50% of total phenoliccompounds and 57% of total furans. Less favorable results were obtained when n-hexane was employed.The liming and overliming processes allowed a significant reduction of acetic acid (in some case upto 53%), lignin-derived compounds (close to 72%) and total furans (near 83%) mainly furfural, but alsohydroxymethylfurfural.

. Introduction

Olive tree pruning residue (OTPR) is a renewable, cheap andidely available resource of polysaccharides, primarily at thendalusian region (Spain). The acid hydrolysis of this residue can beonceived as the first stage of an integrated global strategy for thisesidue utilization to generate interesting products as bioethanolnd xylitol. Among the treatments tried with other lignocellu-osic materials, dilute acid hydrolysis appears to be in the bestosition in economic terms (Wyman, 1994). Through this mech-nism it is possible to obtain some sugars such as d-xylose and

-glucose as the major reaction products, but one problem associ-ted with this hydrolytic process is the toxic compounds generationfurans, 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).

926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2013.04.046

© 2013 Elsevier B.V. All rights reserved.

great limiting factor in bioconversion processes using acid hydrol-ysis and complicating the hydrolyzates fermentability (Purwadiet al., 2004; Larsson et al., 1999).

Hemicellulose is the second major polysaccharide in woodybiomass, typically comprising 15–35% of the dry wood mass. Itconsists 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 detoxifica-tion process for the subsequent fermentation of the concentratedhydrolyzate, several treatments have been tested attending tothe literature (Palmqvist and Hahn-Hägerdal, 2000; Convertiet al., 2000; Martínez et al., 2001; Mussatto and Roberto,

2004).

Since the level of detoxification would be conditioned bythe fermenting microorganisms tolerance to certain inhibitorycompounds, and to minimize the costs of different treatments,

ps and Products 49 (2013) 196– 203 197

soh

dpriiaclae

fd(ti1

lCvoiictirp

2

2

rmag(

ew(pa4wbb

aA

riparrp

Table 1Olive tree pruning residue composition.

Composition % of dry mattera

Glucosans 28.6 ± 0.4Pentosans 13.9 ± 0.2d-xylose 10.1 ± 0.3l-arabinose 3.5 ± 0.2

Acid-insoluble lignin 21.4 ± 0.5Acid-soluble lignin 2.3 ± 0.6Acetyl groups 1.5 ± 0.1Ash 3.8 ± 0.3Extractives 21.4 ± 0.2

In water 18.8 ± 0.1d-xylose 2.7 ± 0.1d-glucose 5.8 ± 0.1

S. Mateo et al. / Industrial Cro

imple detoxification methods are proposed instead of combinednes, usually posed as a system of inhibitors reduction in theydrolyzate.

Furfural and 5-hydroxymethylfurfural (HMF) are two furanserivatives which are formed by the further hydrolysis of sugars,entoses and hexoses, respectively. These furans are available inelatively high concentration in the hydrolyzates and are seriousnhibitors for microorganisms. The furans are probably the mostmportant group of inhibitors, since the fermentability of dilute-cid hydrolyzates is inversely related to the concentration of thisompounds (Taherzadeh et al., 1997). Phenolic compounds causeoss of integrity of biological membranes, thereby affecting theirbility to serve as selective barriers and enzyme matrices (Kangt al., 2012).

Acetic acid, the major aliphatic compound present, is releasedrom the hemicellulosic acetyl groups and formic acid is a degra-ation product of HMF (Ulbricht et al., 1984). When these acidsin the undissociated form) enter in the cell, become dissociated inhe protoplasm because its pH. This fact leads to a decrease in thentracellular pH and can generate the cellular death (Verduyn et al.,990).

This study describes several detoxification methods of hemicel-ulosic hydrolyzates from OTPR (liming and overliming with NaOH,aO and Ca(OH)2, activated charcoal adsorption and organic sol-ents use) in order to find a treatment that allows efficient removalf major inhibitory compounds of fermentation processes presentn these hemicellulosic hydrolyzates. Although the needs for detox-fication must be evaluated in each case, yeast tolerance to theseomponents depends on the types of microorganism considered sohat, a decrease of the microbial inhibitors concentrations presentn the medium could be particularly beneficial by decreasing theeaction times and increasing the efficient sugars utilization andrincipal products formation.

. Materials and methods

.1. Raw material characterization

OTPR was collected locally after the fruit harvest, air-dried atoom temperature to equilibrium moisture content (8–12%) andilled using a laboratory hammer mill (Retsch); fraction graded to

particle size between 0.60 mm and 0.425 mm, according to ASTMuidelines, was stored until used. This fraction consists of leaves30%) and wood chips (70%).

For chemical characterization (of original residue withoutxtracts), the modified methodology proposed by Browning (1967)as used. It consisted of the hydrolysis of 2 g of sample with H2SO4

10 cm3) at 72% (w/w) and maintained at 50 ◦C for 7 min. After thisre-treatment, distilled water (275 cm3) was added to dilute thebove acid and the solution was autoclaved at 1 atm and 121 ◦C for5 min. After filtration of the hydrolyzate, a solution is obtainedhich will allow the determination of the acid soluble lignin, car-

ohydrates, acetic acid, furfural and hydroxymethylfurfural (HMF)y HPLC.

Ash contents were determined by weight difference before andfter incineration of the OTPR sample in a furnace at 550 ◦C for 4 h.ll determinations were carried out in triplicate.

The correct characterization of OTPR is not an easy issue toesolve. Using traditional methods we can mistake in allocat-ng the d-glucose into the cellulosic fraction if this monomer isart of the hemicellulosic fraction or whether there is d-glucose

nd d-xylose in the parenchyma surrounding the cell wall aseserve material in form of insoluble �-1,3 glucans or like oleu-opein that show a higher concentration in leaves extracts. Thisolyphenolic glycoside, formed by the union of three molecules:

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 (Silvaet al., 2006). We think this would be the situation in the OTPR, sowe have made some determinations in order to confirm the cor-rect composition, Table 1. This biomass can provide high amountsof sugar for every unit mass of dry residue, considering that fer-mentable sugars may represent almost 50% and can be processedinto bioproducts of interest by a subsequent fermentation.

2.2. Preparation of hemicellulosic hydrolyzate

Acid hydrolysis was conducted in a stainless steel reactor of50 dm3 capacity, with a useful volume of 40 dm3, provided witha system of electrical resistance heating and agitation by rotationabout its own axis. The reactions were carried out with H2SO4 (2%w/v) at 120 ◦C, for 90 min, using 1:10 solid/liquid ratio. Treatmentconditions were chosen by preliminary tests with the aim of obtain-ing the highest concentration of total sugars in the liquid. The solidand liquid phases were separated by filtration. The hydrolyzate wasconcentrated three to four-fold under vacuum in a 4 dm3 evap-orator at 70 ± 5 ◦C. The concentrated hydrolyzate was chemicallycharacterized.

2.3. Detoxification procedures

Treatments of lignocellulosic hydrolyzate could removal somecompounds which would improve their fermentability in asubsequent step. We study different detoxification methods fun-damentally based on chemical treatment to diminish inhibitorspresent in this one.

2.3.1. Activated charcoal adsorptionThe concentrated hydrolyzate was treated, at room tem-

perature, with activated charcoal (Labsynth in powder form,granulometry of 32% retained on 325 mesh, specific surface areaof 860 m2 g−1 and an average particle size of 0.43 cm). Charcoalwas mixed with 50 g of concentrated hydrolyzate for 30 min. Sub-sequently, the charcoal was separated from the hydrolyzate byfiltration.

The temperature was at the range 25–35 ◦C, the agitation inter-val in 100–200 rpm, the concentration of adsorbent between 2%and 8% and the pH (adjusted with NaOH or H2SO4) between 2 and6.

2.3.2. Liming and overliming processes

We used three different alkalis: CaO, Ca(OH)2 and NaOH at room

temperature. The liming process consisted on neutralization to pH5.5 and centrifugation (10,000 rpm for 10 min) of the precipitateobtained. On the other hand, the overliming experiments involved

198 S. Mateo et al. / Industrial Crops and Products 49 (2013) 196– 203

Table 2Chemical composition of the original and concentrated hydrolyzate of OTPR.

Group of compounds Compounds Concentration (g dm−3) % Reduction

Original Concentrated

Sugars d-xylose 9.71 ± 0.27 37.93 ± 2.13 2.3d-glucose 12.73 ± 0.59 49.50 ± 2.50 2.8l-arabinose 2.79 ± 0.11 10.55 ± 0.62 5.5

Furan derivates HMF 0.22 ± 0.02 0.84 ± 0.02 4.5Furfural 0.04 ± 0.01 0.14 ± 0.03 12.5Totals 0.51 ± 0.02 1.70 ± 0.04 16.7

1.67

0.45

3.76

tAgc

2

n1

2

atltc0u

idaatuaTi

cu7dp

tdpa

uFm6a

3

s

Aliphatic acids Acetic acid

Formic acid

Phenolic compounds

he pH adjust to 10 by the addition of these different compounds.fter that, the solid and liquid fraction was separated by centrifu-ation acidifying, the last one, to pH 5.5 with 72% (w/w) H2SO4 andentrifuged again for precipitate removal.

.3.3. Organic solvents treatmentsFinally, we studied the use of three organic solvents: chloroform,

-hexane and ethyl acetate in four different proportions, 2:1, 1:1,:2 and 1:3 (hydrolyzate: solvent, v/v).

.4. Analytical methods

The quantification of carbohydrates (d-glucose, d-xylose and l-rabinose) as well as acetic acid concentrations (in order to estimatehe acetyl groups content) were determined by high-performanceiquid chromatography (HPLC) using a WATERS equipment, inhe conditions: a BIO-RAD Aminex HPX-87H (300 mm × 7.8 mm)olumn at 45 ◦C, 0.005 M sulfuric acid as eluant, flow rate of.6 cm3 min−1, refraction index (RI) detector and 20 �L sample vol-me.

Furfural and HMF were analyzed by HPLC using a WATERSnstrument with a UV detector (at 276 nm), in the following con-itions: a Waters Resolve C18 5 �m (300 mm × 3.9 mm) columnt ambient temperature, acetonitrile/water (1/8 with 1% of aceticcid) degassed, with addition of phosphoric acid for pH correctiono 2.5 as eluant, flow rate of 0.8 cm3 min−1 and 20 �L sample vol-me. The samples were previously diluted with ultrapure waternd filtered through membranes HAWP 04700 with 0.45 �m pores.he concentrations of these compounds were calculated from cal-bration curves obtained from standard solutions.

To calculate the cellulose, hemicellulose and Klason lignin per-entages, the methodology proposed by Irick et al. (1988) wastilized and moisture composition by the TAPPI norm T12 os-5. Furthermore, the concentration of acid-soluble lignin wasetermined by the method proposed by Rocha (2000) and ash com-osition using the procedure established by Browning (1967).

Extractives (nonstructural components such as pectins, fats,erpenes, phenolic compounds, tannins, uronic acids, etc.) wereetermined gravimetrically using a two-step sequential extractionrocess by Soxhlet to remove water and ethanol soluble materialccording to a procedure adapted from Sluiter et al. (2008).

The total phenol content, expressed as concentration of fer-lic acid, was determined colorimetrically at 760 nm, usingolin–Ciocalteu modified method (Singleton et al., 1999). To esti-ate the total furan content (Martínez et al., 2000) a Beckman DU

40B spectrophotometer model was employed. The samples werenalyzed using two different wavelengths, 284 nm and 320 nm.

. Results and discussion

Dilute sulfuric acid has achieved good yields of monomericugars from many cellulosic materials. OTPR was submitted to

± 0.08 2.77 ± 0.35 58.5± 0.02 1.34 ± 0.02 25.6± 0.13 12.65 ± 0.52 15.9

acid hydrolysis in conditions that promoted selective genera-tion of simple carbohydrates and, subsequently, the hydrolyzatewas concentrated to increase sugars content. The fermentablemonosaccharides concentration and the toxic compounds presentin original and concentrated hydrolyzate are shown in Table 2.

All dilution steps during the treatments have been consideredin the calculations of the final concentrations of those analyzedcompounds. Although the amounts of principal carbohydrates andinhibitory compounds were measured before and after the treat-ments, to take account possible volume variations, the eliminationpercentage of both sugars and fermentation process inhibitors werecalculated as reduction in weight of the concerned substancespresent in the detoxified hydrolyzates in relation to global contentof them in the initial concentrated.

The main purpose of this study was to investigate the effects ofdifferent detoxification processes using some chemicals agents.

3.1. Hemicellulosic hydrolyzate of olive tree residue

The liquid obtained after hydrolysis from OTPR with dilutedsulfuric acid contained 25.2 g dm−3 of total fermentable sugars.After the concentration process (4.0 times), the simple sugarscontent was 90.45 g dm−3 (density: 1087 g dm−3), being the totalamount of hemicellulosic carbohydrates approximately the sameas d-glucose. There was no sugar degradation during the vacuumevaporation process according the concentration factor employed,fact also evidenced by Rivas et al. (2002).

Concentration step by evaporation could act as a detoxificationsystem for several compounds, as it is shown in Table 2 at the %of reduction or loss column. The significant presence of phenoliccompounds after evaporation treatment, to a greater extent thanacetic acid must be noted. Moreover, the increase of acetic acidconcentration may be a consequence of the system evaporationconducted under vacuum, obtaining about a 58% of elimination rel-ative to the amount of aliphatic acid that could be present. The factthat this treatment has been performed at low pH (0.97) may havefavored this compounds removal which are volatile only in its pro-tonated form (Larsson et al., 1999). Furthermore, the evaporationat 70 ◦C was able to produce a partial removal of furfural (12.5%),fact also evidenced in Canilha et al. (2005), but the evaporation hada negligible effect in HMF removing (4.5%), as also noted Dehkhodaet al. (2009), because of this lower volatility specially under acidicconditions.

3.2. Activated charcoal

In order to research the effectiveness of this kind of treatment,if this detoxification method for OTPR concentrated hydrolyzate

was able to reduce some inhibitor concentrations, we have con-sidered the main parameters which affect the adsorption processwith activated charcoal: pH (X1), agitation (X2), temperature (X3)and % of activated charcoal (X4) (Palmqvist and Hahn-Hägerdal,

S. Mateo et al. / Industrial Crops and Products 49 (2013) 196– 203 199

Table 3Matrix of the factorial design for activated charcoal detoxification study.

Assay Variables: real values (coded values)

X1 X2 X3 X4

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)

XX

2tatcca

ltttvs

avoirtm

Y

Y

Y

hc

a

b

c

1: pH. X2: agitation (rpm).3: T (◦C). X4: % act. char. (w/w).

000; Mussatto and Roberto, 2004). The study of the different fac-ors influence was carried out, during a fixed time of 30 min, using

24 experimental design with three central points. Table 3 showshe matrix of the experimental design done, including the real andoded values of the studied variables. The experimental runs werearried out in random order and the results were summarized andnalyzed with the software STATISTICA 6.0 (Statsoft, USA).

The Pareto charts, Fig. 1, describes calculated “student t” abso-ute values, also called standardized effects, providing lengths ofhe bars which in turn are arranged in decreasing order. All fac-ors or interactions for which the length of the bars, representinghe corresponding statistical significance, is positioned beyond theertical line drawn to a confidence level of 95%, p = 0.05, will beignificant.

The mathematical models (by elimination of those terms thatre not statistically significant for the treatment, those which p-alor are above the significance level = 0.05) used to predict theptimal value, for each response variable, were expressed accord-ng to Eqs. (1)–(3). Response values of some dependent variables (%eduction of volume, Y1; % reduction of total sugars, Y2 and % reduc-ion of furfural, Y3) as well as the error in parameter estimation by

odel, are shown in Table 4.

1 = 22.62188 + 3.7625X1 − 2.275X2 − 1.5X3 + 4.6X4 + 1.05X1X3

+ 1.6125X2X3 + 1.7375X2X4 + 1.1875X3X4 (1)

2 = 28.9875 + 3.15562X1+3.39313X2 − 3.19063X3 + 5.62312X4

+ 2.35688X2X3 + 1.94813X2X4 + 2.56563X3X4 (2)

3 = 94.35 + 0.0875X1 + 0.3375X2 − 0.66255X3 + 0.55X4

− 0.67505X1X2 + 0.625X1X3 + 1.8625X1X4 + 0.5875X2X4

(3)

The results reveal that in almost all cases, the variables thatave proved most influential on the responses tested were the per-entage of activated charcoal and pH. As their effects are positive,

Fig. 1. Pareto charts of standardized effects for reduction of (a) volume; (b) totalsugars; and (c) furfural.

an increase of both independent variables values, would imply anincrease in the value of the response. Fig. 2 shows the responsesurfaces at the central point of the rest of variables for reduction ofvolume, Y1; total sugars, Y2 and furfural, Y3.

Results of analysis of variance (ANOVA) with estimation of thecurvature for the dependent variables tested is used to determinethe degree of fit between the experimentally observed values andthose obtained by the model.

2

For all the analyzed parameters, correlation coefficients (R ) areacceptable (R2> 0.943); thus, for Y1, R2 = 0.978 pointing out thatonly 2.2% of the total variations in the response were not explainedby the model (is explained by the residue), or what is the same,

200 S. Mateo et al. / Industrial Crops and Products 49 (2013) 196– 203

Table 4Analysis of variance (ANOVA) of the models describing the % reduction of volume (Y1), total sugars (Y2) and furfural (Y3).

DFa SSb MSc

Y1 Y2 Y3 Y1 Y2 Y3 Y1 Y2 Y3

Model 8 7 7 813.98 1267.25 88.25 101.75 181.04 12.61Curvature 1 1 1 0.36 4.92 0.02 0.36 4.92 0.02Residual 9 10 10 18.02 75.99 3.98 2.00 7.60 0.40Lack of fit 7 8 8 16.30 69.04 3.89 2.33 8.63 0.49Pure error 2 2 2 1.73 6.95 0.09 0.86 3.48 0.04Total 18 18 18 832.37 1348.17 92.24

F-value p-value

Y1 Y2 Y3 Y1 Y2 Y3

Model 50.81 23.82 31.70 0.0001 0.0001 0.0001Curvature 0.18 0.65 0.04 0.6802 0.4399 0.8379ResidualLack of fit 2.70 2.48 11.22 0.2970 0.3187 0.0844Pure errorTotal

R2 for the Y1 model: 0.978; R2 for the Y2 model: 0.943; R2 for the Y3 model: 0.957.

ei

iXY

stio

bi3osawpdm

bd

(6fodmae

weaetp

a Degrees of freedom.b Sum of squares.c Mean square.

xplains 97.8% of variation in the response, showing good accuracyn predicting the response value.

Considering Eqs. (1)–(3), and to minimize Y1 and Y2 while Y3s maximized, the best solution is achieved when X1 = −1, X2 = 1,3 = 0.12 and X4 = −1 with a desirability of 0.901 being Y1 = 16.3%,2 = 8.1% and Y3 = 95.9%.

The reduction of volume, Y1, is strongly influenced by X4 and X1,o that the lower volume losses were obtained using the lower pH,he lower charcoal concentration and higher temperature tested,ndependently of agitation. Lower reductions in the concentrationf total sugars were also obtained under these conditions.

Although losses of carbohydrates in many researches have noteen very significant, the reduction of fermentable sugars reported

n literature data may be 20% (Wang and Chen, 2011) or up to0%, (Lee et al., 2011), coinciding with the highest removal ratesbtained in the established experimental design. The amount ofugars eliminated depend on the hydrolyzate composition anddsorption conditions. It seems that reduction of sugars was higherhen a greater proportion of activated charcoal is used and atH = 6, perhaps by precipitation or interaction of these carbohy-rates with other components present in the hydrolyzate whenedium became more basic.In relation to the reduction of acetic acid, it is greatly influenced

y the concentration of activated charcoal and the pH, althoughirectly and inversely proportional respectively.

It is noteworthy that very high furfural reductions are obtained87–97%) and total elimination of furan is situated in the range0–98%. Lee et al. (2011) found a nearly total adsorption of fur-ural and HMF when 2.5 wt % charge of activated carbon and 1 hf contact time was used. Furfural and HMF concentrations wereramatically reduced (92% and 68%, respectively) by the treat-ent with this adsorbent (Carvalheiro et al., 2005), although other

uthors indicate below removal of HMF percentages, 8.8% (Nápolest al., 2006).

In general, elimination percentages of total phenolic compoundsere significant (42–81%); higher values corresponded to the

xperiments done with a larger amount (8%) of activated charcoal

nd the best results, except in one of the assays, are agree with thosexperiments at pH 2. Mussatto and Roberto (2004) established thathe adsorption of weak organic acids is favored when the mediumH 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 detoxification methodfor sago trunk hydrolyzates, enabled a reduction of 78% of phenoliccompounds in the best conditions.

While some authors point out that, generally, the adsorption offuran 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 nosignificant effect on this kind of inhibitors elimination or in sug-ars reduction, at least at tested low values. Use of relatively lowtemperatures in the study would be motivated by an attempt tominimize heating costs which would entail an adsorption processat high temperature.

Temperature is statistically insignificant, so that the responsesurfaces of % furans versus % charcoal and temperature for a givenagitation values resulted to be planes. It indicates that reductionpercentage of furans was not affected by temperature.

The effect caused by the use of activated carbon on phenoliccompounds was greater than the reduction of acetic acid; greatereliminations of this aliphatic acid are obtained when activated char-coal load was 8% (removal rates up to 45%), using a 4% intermediateconcentration (25–28%) and slightly lower for activated charcoalrates 2% (<15%). In general, better adsorptions are performed whenhydrolyzate pH was 2 vs. pH 6, independently of the temperatureand agitation degree employed.

Finally, notice that for none of the experiments carried out, therewas a significant decrease in hydrolyzate color. The largest reduc-tions in this parameter were achieved with 8% of activated charcoalat pH 2 and 35 ◦C.

3.3. Liming and overliming

The liming and overliming terms have been used in this studyto make reference to alkalinization processes, carried out at pH 5.5or 10, respectively, in which the bases employed have been NaOH,Ca(OH)2 and CaO. All experiments have been performed at roomtemperature to minimize the costs involved in hydrolyzate heating.

Both methods entailed a loss of hydrolyzate volume when CaOis used, between 16% and 20%; with Ca(OH)2 there were only lostof concentrated liquid when overliming was employed while thevolume was maintained constant using NaOH; one reason that can

S. Mateo et al. / Industrial Crops and Products 49 (2013) 196– 203 201

Fo

ada

h

10 with calcium hydroxide like detoxificant 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 decreasein the fermentable sugars of 20% (Horvath et al., 2005; Nigam, 2001)

ig. 2. Response surfaces at the central point of the rest of variables for reductionf: (a) volume; (b) total sugars; and (c) furfural.

ffect to the volume change is related to the dilution experiencedue to the use of neutralizing agents and acidifying involved in the

bove process.

The mechanism behind the technique is not fully specified but itas 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 detoxification.

this operation (Baek and Kwon, 2007) or transformed into othertypes of chemical substances (Persson et al., 2002). When over-liming technique were carried out, a great formation of precipitatewere obtained with CaO and Ca(OH)2, possibly due to calcium sul-fate precipitation (gypsum) (Alriksson et al., 2006), by increasingthe amount of base added while the solid amount from NaOH waslower. Furthermore, because these detoxification processes led toa partial removal of furfural and HMF, furans aldehydes may beinvolved in alkali-assisted aldol condensation reactions (Horvathet al., 2005). In this regard, and in relation to the formation of a largeamount of precipitate from insoluble salts (calcium sulfate, calciumoxalate among other compounds), a significant decrease of the ini-tial conductivity of the concentrated hydrolyzate (138.2 mS/cm)was observed to values of 8.5–8.8 mS/cm with calcium salts and51.1–51.4 mS/cm for NaOH. These conductivity values are similarto those obtained for treated hydrolyzates with bases, Martínezet al. (2001) .

In relation to the removal of both, free sugars and inhibitorspresent in the hydrolyzates, Figs. 3 and 4, while NaOH detoxifica-tion treatments did not involve a significant loss of sugars (<5%)or acetic acid (<7%), if calcium salts were used, sugar reductionswere higher, presenting the greatest losses by adjusting the pH to

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 detoxification.

202 S. Mateo et al. / Industrial Crops and

Table 5Reduction percentages in detoxification process with organic solvents.

Solvent a h:sb % Reduction

volc tsd AAe phef furg HMF

EA 2:1 0 19.6 − 34.9 88.4 0.0EA 1:1 0 4.1 − 40.1 91.3 21.0EA 1:2 4 9.2 − 48.3 94.4 39.6EA 1:3 8 10.2 − 49.5 74.1 40.0Hex 2:1 0 0.0 0.0 14.7 0.0 0.0Hex 1:1 0 0.0 0.0 18.9 13.3 0.0Hex 1:2 4 0.0 0.0 20.4 68.7 0.0Hex 1:3 4 5.5 0.1 24.1 66.3 0.0Chl 2:1 0 0.0 6.6 30.6 94.8 0.0Chl 1:1 0 5.6 9.9 29.3 97.2 0.0Chl 1:2 0 7.2 9.9 28.0 98.5 0.0Chl 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.

btp(cwbt

lf(hfsicelc

fbr

3

te(

phacicmoc

pw

e Acetic acid.f Phenolic compounds.g Furfural.

ut, in any case, although the magnitude of these losses depend onhe composition and nature of the hydrolyzate the shift to highH during the overliming process must be kept to a minimumAmartey and Jeffries, 1996); these authors suggested that the cal-ium ions catalyze the monosaccharides degradation by interactionith their intermediate enolate. With CaO and Ca(OH)2 was possi-

le to reduce the amount of above aliphatic acid in 32–52%, similaro the results reported by Ge et al. (2011) by overliming process.

The furans and phenolic compounds elimination occurred in aarge extension; the liming process with CaO and Ca(OH)2 allowedurans elimination (56–79%) and phenolic compounds reduction62–69%), whereas overliming removal percentages were slightlyigher, Fig. 4. Martínez et al. (2001) reported 51% removal of total

urans after optimal overliming of diluted acid hydrolyzates fromugarcane bagasse. Reduction of phenolic compounds, when NaOHs used, was quite low (28–31%) for both alkaline detoxification pro-esses, while calcium bases had a marked effect allowing a greaterlimination of these lignin derived constituents (56–71%). Over-iming with CaO followed by activated charcoal reduced phenolicompounds by 82.1 ± 5.3% (Dubey et al., 2012).

Treated hydrolyzates were obtained with acetic acid reductionsor overliming between 50–53% or 32–50% for liming process whenases such as CaO or Ca(OH)2 were employed; similar data wereeflected by Ge et al. (2011).

.4. Organic solvents treatments

Finally, we studied the use of three organic solvents to achievehe elimination of toxic compounds, chloroform, n-hexane andthyl acetate in four different proportions, 2:1, 1:1, 1:2 and 1:3hydrolyzate/solvent, v/v), Table 5.

In general, it was observed how the quantity of inhibitorsresent in the medium was increased by using a smaller ratioydrolyzate:solvent to carry out the extraction process. Ethylcetate was the most effective solvent for furans removal, espe-ially furfural in a percentage ranging between 74% and 94%, ast was also evidenced by Frazer and McCaskey (1989), but HMFould only be removed in significant quantities (40%) by using asuch of this solvent. n-Hexane was unable to remove HMF and

nly reduced total furans content in a maximum of 7% while for

hloroform, reductions are ranged from 19% to 34%.

The use of n-hexane as an extracting agent of inhibiting com-ounds of olive tree pruning hydrolyzates is not a suitable methodhen it seeks to eliminate furfural, acetic acid and HMF although

Products 49 (2013) 196– 203

it may moderately reduce (24%) the amount of total phenolic com-pounds. Chloroform slightly reduces the content of furans andphenolic inhibitors (34|acetic acid concentration (17%), while theethyl acetate allowed moderate levels of lignin-derived compoundsand furans total elimination (49% and 57%, respectively), but a sol-vent removal should be performed if the yeast is not tolerant toacetate ions, which often contribute positively in the fermentationprocess.

The losses of sugars produced due to the extraction process, evenfor higher relations hydrolyzate:solvent, were null using n-hexane,irrelevant if chloroform was employed (<4%), and not too high whenthe detoxification was carried out with ethyl acetate (<8%).

The acetic acid cannot be extracted using n-hexane but removalrates were obtained for this aliphatic compound close to 17% if theextractor solvent ratio of chloroform was 1:3 while, if the extractorsolvent ratios are lower, the reduction of acid would be between 7%and 10%. The acidic pH of the concentrated hydrolyzate might havefavored the mentioned acid dissolution in this solvent extractorbecause the protonated form is probably more soluble in an organicmedium than the non-protonated.

The ethyl acetate was the solvent that shown a higher totalphenolic compounds extracting capacity, allowing reductions of34–49%, followed by chloroform (30%), and finally the n-hexane(14–24%). In fact, hydroxytyrosol is one of the o-catechols withgreater presence in the olive tree, either in free form or as deriva-tives such as oleuropein, and various solvent of this nature areemployed for their recuperation. As a result of ethyl acetate extrac-tion, Stoutenburg et al. (2011) got to remove a 25% of this phenolderivates in sugar maple hydrolyzate.

4. Conclusions

The most suitable detoxification procedure to improve the sub-sequent fermentation of the concentrated acid liquid obtained, stilldepending on the nature of the hydrolyzate, would be one thatwould 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 theprocess might be sufficient partial and not complete elimination ofthose compounds with greater negative repercussions on yeast cellmetabolism, as long as they are situated at levels considered nontoxic to the microorganism.

In view of the experiments carried out with different detoxifyingagents and considering techniques of neutralization, adsorption,precipitation and extraction. It must be pointed out that, in general,the best results for solvent detoxification were achieved with 1:3ratio which is the greatest of those studied in the present research.As the results for this proportion hydrolyzate:solvent, are very closeto those obtained with a smaller relationship (1:2) the latest oneshould be used in order to minimize the process costs.

When NaOH was used, the decrease in phenolic compoundswas minimal (28–31%), for both alkaline detoxification processes,and slight acetic acid reduction (<7%) was achieved although it didnot involve a significant loss of sugars, while calcium bases had amarked effect allowing a greater removal of these lignin derivedconstituents (56–71%), acetic acid (similar for both bases treat-ments, next to 50% except liming with CaO, 32%) and total furanscontent (62–69% for liming and 80–83% for overliming). The draw-back is that reduction of sugar amounts, for liming (15–34%) and foroverliming (26–46%), are very high; this method, therefore, couldnot be advisable for olive tree pruning concentrated hydrolyzates.

In the adsorption processes with activated charcoal, the vari-ables that have proved most influential on the responses testedwere the percentage of this compound and pH. It could be anexcellent alternative to decrease inhibitors from olive tree pruning

ps and

rper

hmtp

A

ftt

R

A

A

B

B

C

C

C

C

D

D

F

G

H

I

S. Mateo et al. / Industrial Cro

esidue hydrolyzates (46% of acetic acid, 81% of phenolic com-ounds and 98% of total furans), with possibility to reuse themployed activated charcoal through regeneration methods toeduce the costs of these processes.

In summary, among the detoxification processes studied for theemicellulosic hydrolyzate from olive tree pruning residue, theost promising one for the reduction of phenolic compounds and

otal furans is the adsorption with 2% of activated charcoal at 30 ◦C,H = 2 and 200 rpm.

cknowledgements

The authors are grateful to Andalusia Regional Governmentor financial support (Project Ref. AGR-6509). On the other hand,hanks given to Professor M.J. Olmo for her assistance in the statis-ical treatment of the data.

eferences

lriksson, B., Sjöde, A., Nilvebrant, N., Jönsson, L.J., 2006. Optimal conditions foralkaline detoxification of dilute-acid lignocellulose hydrolysates. Appl. Biochem.Biotechnol. 130, 599–611.

martey, S., Jeffries, T., 1996. An improvement in Pichia stipitis fermentationof acid-hydrolyses hemicellulose achieved by overliming (calcium hydroxidetreatment) and strain adaptation. World J. Microbiol. Biotechnol. 12, 281–283.

aek, S.C., Kwon, Y.J., 2007. Optimization of the pretreatment of rice straw hemi-cellulosic hydrolyzates for microbial production of xylitol. Biotechnol. Bioproc.Eng. 12 (4), 404–409.

rowning, B.L., 1967. Determination of lignin. In: Methods of Wood Chemistry.Interscience Publishers, New York, pp. 785–823.

anilha, L., Carvalho, W., Almeida-Silva, J.B., 2005. Influence of medium compositionon xylitol bioproduction from wheat straw hemicellulosic hydrolysate. World J.Microbiol. Biotechnol. 21, 1087–1093.

arvalheiro, F., Duarte, L.C., Lopes, S., Parajó, J.C., Pereira, H., Gírio, F.M., 2005.Evaluation of the detoxification of brewerys spent grain hydrolysate for xyli-tol production by Debaryomyces hansenii CCMI 941. Process Biochem. 40 (3–4),1215–1223.

heng, K.K., Ge, J.P., Zhang, J.A., Ling, H.A., Zhou, Y.Z., Yang, M.D., Xu, J.M., 2007.Fermentation of pretreated sugarcane bagasse hemicellulose hydrolysate to

ethanol by Pachysolen tannophilus. Biotechnol. Lett. 29 (7), 1051–1055.onverti, A., Domínguez, J.M., Perego, P., Silva, S.S., Zilli, M., 2000. Wood hydrolysis

and hydrolysate detoxification for subsequent xylitol production. Chem. Eng.Technol. 23, 1013–1020.

ehkhoda, A., Brandberg, T., Taherzadeh, M.J., 2009. Comparison of vacuum andhigh pressure evaporated wood hydrolyzate for ethanol production by repeatedfed-batch using flocculating Saccharomyces cerevisiae. Bioresources 4 (1),309–320.

ubey, A.K., Gupta, P.K., Garg, N., Naithani, S., 2012. Bioethanol production fromwaste paper acid pretreated hydrolyzate with xylose fermenting Pichia stipitis.Carbohydr. Polym. 88, 825–829.

razer, F.R., McCaskey, T.A., 1989. Wood hydrolyzate treatments for improved fer-mentation of wood sugars to 2,3-butanediol. Biomass 18 (1), 31–42.

e, J.P., Cai, B.Y., Liu, G.M., Ling, H.Z., Fang, B.Z., Song, G., Yang, X.F., Ping, W.X.,2011. Comparison of different detoxification methods for corn cob hemicellu-ose hydrolysate to improve ethanol production by Candida shehatae ACCC 20335.Afr. J. Microbiol. Res. 5 (10), 1163–1168.

orvath, I.S., Sjöde, A., Alriksson, B., Jönsson, L.J., Nilvebrant, N.O., 2005. Criticalconditions for improved fermentability during overliming of acid hydrolysates

from spruce. Appl. Biochem. Biotechnol. 121–124, 1031–1044.

rick, T.J., West, K., Brownell, H.H., Schwald, W., Saddler, J.N., 1988. Compari-son of colorimetric and HPLC techniques for quantitating the carbohydratecomponents of steam-treated wood. Appl. Biochem. Biotechnol. 17 (1–3),137–149.

Products 49 (2013) 196– 203 203

Kamal, S.M.M., Mohamad, N.L., Abdullah, A.G.L., Abdullahb, N., 2011. Detoxifica-tion of sago trunk hydrolysate using activated charcoal for xylitol production.Procedia Food Sci. 1, 908–913.

Kang, L., Lee, Y.Y., Yoon, S.H., Smith, A.J., Krishnagopalan, G.A., 2012. Ethanol pro-duction from the mixture of hemicellulose prehydrolysate and paper sludge.Bioresurces 7 (3), 3607–3626.

Larsson, S., Reimann, A., Nilvebrant, N., Jönsson, L.J., 1999. Comparison of differentmethods for the detoxification of lignocellulose hydrolyzates of spruce. Appl.Biochem. Biotechnol. 77-79, 91–103.

Lee, J.M., Venditti, R.A., Jameel, H., Kenealy, W.R., 2011. Detoxification ofwoody hydrolyzates with activated carbon for bioconversion to ethanol bythe thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum.Biomass Bioenergy 35 (1), 626–636.

Martínez, A., Rodríguez, M.E., Wells, M.L., York, S.W., Preston, J.F., Ingram, L.O., 2001.Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnol.Progr. 17, 287–293.

Martínez, A., Rodríguez, M.E., York, S.W., Preston, J.F., Ingram, L.O., 2000. Use of UVabsorbance to monitor furans in dilute acid hydrolyzates of biomass. Biotechnol.Progr. 16 (4), 637–641.

Miyafuji, H., Danner, H., Neureiter, M., Thomasser, C., Braun, R., 2003. Effect of woodash treatment on improving the fermentability of wood hydrolysate. Biotechnol.Bioeng. 84 (3), 390–393.

Mussatto, S.I., Roberto, I.C., 2004. Alternatives for detoxification of diluted-acid lig-nocellulosic hydrolyzates for use in fermentative processes: a review. Bioresour.Technol. 93 (1), 1–10.

Nápoles, A.I., Ortiz, Y., Vinals-Verde, M., Manganelly, E., Acosta, E., 2006. Purificationof sugarcane bagasse hydrolysates using activated charcoal and ion-exchangeresins. Ciencia y Tecnología Alimentaria 5 (2), 124–128.

Nigam, J.N., 2001. Ethanol production from wheat straw hemicellulose hydrolysateby Pichia stipitis. J. Biotechnol. 87 (1), 17–27.

Palmqvist, E., Hahn-Hägerdal, B., 2000. Fermentation of lignocellulosic hydrolysatesI: inhibition and detoxification. Bioresour. Technol. 74 (1), 17–24.

Persson, P., Andersson, J., Gorton, L., Larsson, S., Nilvebrant, N.O., Jönsson, L.J., 2002.Effect of different forms of alkali treatment on specific fermentation inhibitorsand on the fermentability of lignocellulose hydrolysates for production of fuelethanol. J. Agr. Food Chem. 50 (19), 5318–5325.

Purwadi, R., Niklasson, C., Taherzadeh, M.J., 2004. Kinetic study of detoxification ofdilute-acid hydrolyzates by Ca(OH)2. J. Biotechnol. 114 (1–2), 187–198.

Rivas, B., Domínguez, J.M., Domínguez, H., Parajó, J.C., 2002. Bioconversion of posthy-drolysed autohydrolysis liquors: an alternative for xylitol production from corncobs. Enzyme Microb. Technol. 31, 431–438.

Rocha, G.J.M., 2000. Deslignificac ao de bagac o de cana de ac úcar assistida poroxigênio. IQSC, USP, São Carlos, Brazil, Ph.D. (doutorado em química).

Silva, S., Gomes, L., Leitão, F., Coelho, A.V., Vilas-Boas, L., 2006. Phenolic compoundsand antioxidant activity of Olea europaea L. fruits and leaves. Food Sci. Technol.Int. 12 (5), 385–395.

Singleton, V.L., Orthofer, R., Lamuela-Raventós, R.M., 1999. Analysis of total phenolsand other oxidation substrates and antioxidants by means of Filon–Ciocalteureagent. Methods Enzymol. 299, 152–178.

Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008. Determination ofextractives in biomass, Laboratory Analytical Procedure NREL/TP-510-42619.National Renewable Energy Laboratory, Colorado (USA). 1617 Cole Boulevard,Golden, Colorado 80401-3393.

Stoutenburg, R.M., Perrotta, J.A., Nakas, J.P., 2011. Overcoming inhibitors in a hemi-cellulosic hydrolysate: improving fermentability by feedstock detoxificationand adaptation of Pichia stipitis. J. Ind. Microbiol. Biotechnol. 38 (12), 1939–1945.

Taherzadeh, M.J., Eklund, R., Gustafsson, L., Niklasson, C., Gunniar, L., 1997. Char-acterization and fermentation of dilute-acid hydrolyzates from wood. Ind. Eng.Chem. Res. 36 (11), 4659–4665.

Ulbricht, R.J., Northup, S.J., Thomas, J.A., 1984. A review of 5-hydroxymethylfurfuralHMF in parenteral solutions. Fundam. Appl. Toxicol. 4 (5), 843–853.

Verduyn, C., Postma, E., Scheffers, W.A., van Dijken, J.P., 1990. Physiology of Sac-charomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J. Gen.Microbiol. 136 (3), 395–403.

Wang, L., Chen, H., 2011. Increased fermentability of enzymatically hydrolyzedsteam-exploded corn stover for butanol production by removal of fermentationinhibitors. Process Biochem. 46 (2), 604–607.

Wyman, C.E., 1994. Ethanol from lignocellulosic biomass: technology economicsand opportunities. Bioresour. Technol. 50, 3–16.