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Gorse (Ulex europæus) as a possible source of xylans by hydrothermal treatment
Industrial Crops and Products 33 (2011) 205–210
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Gorse (Ulex europæus) as a possible source of xylans by hydrothermal treatment Pablo Ligero a , Alberto de Vega a,∗ , Johannes C. van der Kolk b , Jan E.G. van Dam b a b
Department of Physical Chemistry and Chemical Engineering, Faculty of Sciences, University of A Coru˜ na, Alejandro de la Sota, 1, 15004 A Coru˜ na, Spain Food and Biobased Research, Wageningen University and Research Centre, P.O. Box 7 17, 6700 AA Wageningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 28 May 2010 Received in revised form 9 October 2010 Accepted 11 October 2010
Keywords: Autohydrolysis Ulex europæus Hemicelluloses Xylans
a b s t r a c t Autocatalytic hydrothermal process conditions were used to study Ulex europæus (Gorse) as a source of xylan compounds. The aim was to study the possibilities for using this unutilised biomass material to produce xylans. Ulex is an evergreen shrub that grows in the northwest of Spain and has no economic value. Therefore, Ulex is considered a promising candidate as a biomass source. Ulex showed a total xylose content of 12%, thus qualifying it as a suitable material to extract xylan-derived compounds. Autohydrolysis was applied to extract xylans from Ulex. To find the best conditions for xylan extraction, samples of Ulex were subjected to different temperatures and time conditions. Results indicate that autohydrolysis is a suitable method to selectively extract xylans at temperatures between 160 and 190 ◦ C for 5–30 min, reaching a maximum xylan recovery of almost 63% of the initial xylan at 180 ◦ C for 30 min, with only small effects on cellulose and lignin contents. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Because of the increasing demands from society and governments for cleaner and CO2 neutral energy production, novel biorefinery production systems are of interest for conversion of alternative biomass sources for energy or chemicals. In this context, the exploitation of largely unexplored biomass species, such as Ulex, is of interest. Considering the advantages of the abundant distribution of this renewable and cheap biomass source, it is an interesting crop for study. Currently, lignocellulose feedstock (LCF) biorefinery concepts are introduced as a possible method to valorise biomass. LCF biorefinery can be understood as a set of processes through which the main biomass components can be transformed into chemicals, fuels and energy, according to its natural constitution. In particular, LCF organosolv biorefineries could contribute to large economic benefits from the valorisation of fractionated products (Bachmann and Riese, 2006). These biorefineries fractionate the biomass complex into its main components (i.e., cellulose, hemicelluloses, lignin and extractives) using organic solvents. Subsequently, each fraction is further processed to marketable products. The aim of such biorefineries is to maximise the value of each biomass fraction by chemical or physical transformations. In this sense, compatible techniques should be integrated to obtain the optimum use of all wood cell wall components to increase their value. A pretreatment of the raw material to selectively remove the main hemicellulose fraction prior to any
∗ Corresponding author. Tel.: +34 981 167000; fax: +34 981167065. 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.10.011
organosolv pulping process can be considered as a valorisation preprocessing technique. Furthermore, the harvesting of Ulex, which is very abundant in some areas, will help to reduce the serious threat of forest fires, as Ulex is an important substrate for its propagation. In this way, a system of sustainable forest management can be combined with a strategy for economic development. Gorse (Ulex europæus) is a spiny, perennial, evergreen shrub belonging to the Fabaceæ family, a very common wild species in Galicia (N.W. Spain) that is present in 74% of woodland. Gorse can be found in many parts of the world, but it is native to western and central Europe. Due to its great reproductive capacity, prolonged latency of the seed, long vegetative period, fast growth and absence of natural enemies, gorse plays an important role in the spread of forest fires, as mentioned previously (Matthei, 1995). For this reason, it is sometimes removed preventively from the forests before the start of the dry season. In the past, this material was successfully used as a source of protein in animal food, (Bao et al., 1998), but its most common use was as source of lectin (Gomez et al., 1995; Ezpeleta et al., 1999). In addition to these uses, Ulex is also a suitable candidate material to produce pentosans or xylan compounds. The hemicellulose fraction is the most abundant non-cellulose polysaccharide part of the biomass. Xylose is the main sugar compound within the hemicellulose fraction, and its potential use is of high importance. Many different biomass materials are used for the extraction of xylan, including cereals (Garrote et al., 2002; Carvalheiro et al., 2004, Nabarlatz et al., 2007), nuts (Nabarlatz et al., 2004), hardwood (Springer and Harris, 1982; Garrote et al., 2001) and grass (Aoyama and Seki, 1999). Its potential applications
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in the food and pharmaceutical industries (Imaizumi et al., 1991; Ebringerová, 2006) are the main support for studying the extraction of xylan. The autohydrolysis process takes advantage of the presence of acetyl groups in the biomass, which are liberated as acetic acid into the reaction media and catalyse xylan depolymerisation. Moreover, this process causes a selective solubilisation of hemicelluloses (Garrote et al., 2002), leaving cellulose slightly affected and lignin almost unaltered. Therefore, this pretreatment of biomass can be considered as a preparation step for a number of treatments as organosolv pulping, cellulose hydrolysis, etc. Furthermore, autohydrolysis can remove the extractable compounds, avoiding pitch deposits in the machinery (Caparrós et al., 2007). Autohydrolysis has been widely used to successfully cause xylan depolymerisation (Garrote et al., 2001; Parajó et al., 2004; Nabarlatz et al., 2007). The aim of our study was to use Ulex as a biomass source for xylo-oligosaccharides by means of autohydrolysis. 2. Materials and methods 2.1. Raw material Ulex was manually collected in Galicia in the autumn of 2008. The sample was taken from specimens of about 1–1.5 m in height. The sample obtained was transferred to the laboratory and left to dry at room temperature for a long period. Later, pricks and flowers were removed, and stems were cut in a garden shredder and milled in a hammer mill equipped with a 0.3-mm sieve at the outlet. A final moisture equilibration period was allowed, after which a value of 9.6% humidity was measured. The milled samples were stored in polypropylene bags. 2.2. Analytical methods Tappi standards were used to determine ash (T211 om-02), cold water solubility (T207 om-99), 1% NaOH solubility (T212 om-02), acid insoluble and acid soluble lignin (Tappi T222 om-88) and ␣-, - and ␥-cellulose (T203 om-93). Ethanol–toluene extractives were determined in an accelerated solvent extractor [DIONEX ASE 200] where milled samples were subjected to extraction with boiling ethanol–toluene (65:35 by volume) by fully automated methods. A second extraction step was carried out semi-automatically by putting the organic extractivefree samples in a glass flask in a heating block for extraction with boiling water for 1 h. In both cases, extractive contents were calculated from the weight difference between the starting raw material and the extractive-free material. Quantitative acid hydrolysis was performed by a two-step procedure following standard methods (Browning, 1967). Namely, treatment with 72% sulphuric acid (3.75 mL) was added to 0.375 g of sample for 2 h at 30 ◦ C. Next, water was added to obtain 4% sulphuric acid solution, and the mixture was kept at 100 ◦ C for 3 h. From this acid hydrolysis, two fractions were obtained: a solid fraction and a liquid fraction. The solid was the acid insoluble fraction that was considered as Klason lignin, after burning the solid to 575 ◦ C to correct for the acid insoluble ash content. The liquid fraction was a solution from the acid treatments. This was neutralised before determination of monosaccharides, furfural, hydroxymethylfurfural (HMF) and acetic acid, by HPLC. Holocellulose determination was performed by the chlorite method through successive extractions with sodium chlorite in acidic medium according to a previously described procedure (Browning, 1967). Quantitative determination of individual sugars was performed using HPLC. The samples were diluted with water until a suit-
able concentration of sugars was reached. Fucose was added as an internal standard, neutralised with barium carbonate, and filtered through a 0.45 m membrane. A Dionex HPLC equipped with an anion exchange column (Carbopack PA1) thermostated to 35 ◦ C and a pulsed amperometric detector (PAD) was used. HPLC chromatography also enabled the determination of other significant components as acetic acid, furfural and hydroxymethylfurfural. Uronic acids concentrations were determined spectrophotometrically using galacturonic acid as a standard for quantification (Blumenkrantz and Asboe-Hansen, 1973). 2.3. Hydrothermal processing Autohydrolysis experiments were carried out simultaneously in four 100-mL stainless steel reactors submerged in an oil bath at the desired temperature. Samples (2 g) were treated in duplicate for 5, 15, 30 and 60 min at different temperatures between 160 and 200 ◦ C. A liquid/solid ratio of 20 was used in all experiments. After the reaction, the mixture was cooled. Undissolved materials were removed by filtration, and the solids were washed three times with water. 2.4. Chemical analysis of hydrolysate fractions An aliquot of the combined liquids from hydrolysis (liquor and washing water) was used to determine the solid residue, which was considered as non-volatile dissolved matter. The difference between this value and the total mass loss after treatment was considered as volatile dissolved matter. The combined liquid autohydrolysis fraction was split into two aliquots. One was submitted directly to HPLC to determine the percentages of monosaccharides, acetic acid, furfural and hydroxymethylfurfural. The second aliquot was subjected to quantitative acid hydrolysis with 4% sulphuric acid at 121 ◦ C for 1 h in an autoclave. After that, the samples were neutralised to pH 7 prior to HPLC determination of monosaccharides, furfural, hydroxymethylfurfural (HMF) and acetic acid. The difference between the xylose amounts before and after hydrolysis was considered as recovered xylan. Solid residues were analysed to determine the yield of dissolved carbohydrates, acid insoluble lignin and ␣-cellulose. 3. Results and discussion 3.1. Characterisation of raw material The results of the chemical composition analysis of U. europæus are presented in Table 1. The resulting ␣-cellulose content in Ulex was quite high (50% ␣-cellulose), while the corresponding value found for glucose content was rather low (32%). These values are less than those found in hardwoods such as Eucalyptus globulus, which contains about 46.3% of glucose (Garrote et al., 2001), similar to those of cereal straws (Nabarlatz et al., 2007), and higher than the levels in almond shells, olive stone, rice husks, wheat straw and barley straw (Nabarlatz et al., 2007). In addition, previously reported values for the same material (Vega and Bao, 1993) are also higher (39%), probably due to the use of plants of different ages in previous studies. The monosaccharide composition was dominated by glucose and xylose, followed by minor quantities of arabinose, galactose, mannose and rhamnose. Uronic acids were also detected in significant quantity (Table 1). The acid insoluble lignin content showed a similar level to the values found previously (Vega and Bao, 1993). In summary, the Ulex substrate is ranked among non-wood species with relatively high contents of lignin and carbohydrates (Han, 1998).
P. Ligero et al. / Industrial Crops and Products 33 (2011) 205–210 Table 1 Chemical analysis of unextracted Ulex europæus stems.
3.2. Dissolved matter, volatile and non-volatile matter % On dry basis
Ash Solubility Extractives
Lignin Polysaccharides
Monosaccharides
Cold water 1% NaOH Ethanol/toluene Ethanol Hot water Acid insoluble Acid soluble Holocellulose ␣-cellulose -celullose ␥-cellulose Arabinose Xylose Mannose Galactose Glucose Rhamnose Uronic acids Acetic acid
207
1.2 19.2 30.8 5.1 0.7 3.8 21.2 1.6 74.2 50.0 10.2 9.2 1.5 12.4 0.5 1.1 31.9 0.3 3.8 8.4
The pH profiles at different temperatures (Fig. 1a) showed a kinetic profile that follows the hydrothermal autocatalysed release of the acetate groups. The release of acetate was important throughout the heating period and during the first 5 min after reaching the reaction temperature (pH between 3.4 and 3.8). This liberation continued until 30 min of reaction time was reached, after which the liberation of acidic groups decreased substantially. The amount of dissolved material increased strongly with temperature and progressed, especially in the first 15 min. Beyond this time, the curve rose slowly until the maximum value was reached in the range of 25–40%, depending on the applied temperature (Fig. 1b). The dissolved material during autohydrolysis can be divided into the two categories of volatile and non-volatile matter. Fig. 1c and d shows the solubilisation rates of non-volatile and volatile matter, respectively, as a function of time and temperature. The volatile matter percentage increased progressively with temperature and time. This is in accordance with the concurrent formation of the main compounds of this group, including acetic acid, furfural and hydroxymethylfurfural, which showed individual concentrations in liquors that followed similar trends.
Fig. 1. Evolution of (a) pH and formation of (b) dissolved, (c) volatile and (d) non-volatile components during autohydrolysis of Ulex.
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reactions of the non-volatile fraction. This can be verified by the acid catalysed formation of dehydration products (i.e., furural and HMF) from released monosaccharides. 3.3. Xylose, xylan and arabinose The yield of xylan indicated that its recovery was strongly dependent on temperature and reaction time (Fig. 2a). Xylans were removed quickly in the first minutes of the reaction and then underwent decomposition reactions that were also temperature dependent. Thus, at the lowest reaction temperature (160 ◦ C), the xylan production profile was always increasing, while at higher temperatures the degradation reactions became more important. The maximum recovery of xylan (62% of initial) was obtained at 180 ◦ C after 30 min. This xylan recovery rate was similar to other reports for cereal grain (Carvalheiro et al., 2004) and eucalyptus wood (Garrote et al., 2001). Fig. 2b shows the curves for recovered xylose in extraction liquors as a function of temperature and reaction time. Xylose yields increased with time and temperature, proving that acid catalysed hydrolysis of the extracted xylan occurred. At the highest reaction temperature (200 ◦ C), xylose degradation (dehydration) reactions became predominant after 15 min. Nevertheless, the proportion of hydrolysed xylose in the extracts remained at relatively low levels, which is indicative of an acceptable selectivity. Concerning the liberation of arabinose, a qualitative explanation can be given similar to that described for xylose (Fig. 2c), with the only difference being that arabinose was present in lower amounts in the liquor, and degradation reactions became predominant earlier and at lower temperatures. 3.4. Minor sugars and others compounds Glucose was also removed from the solid matrix, but in small quantities. Values ranging from 0.20% to 0.45% were measured in dry initial weight (Table 2), probably resulting from minor hemicellulose glycans comprising glucose units (galactoglucomannans, xyloglucans) and cellulose degradation. Nevertheless, the percentages of liberated glucose were sufficiently low, indicating that cellulose was only slightly affected. This fact was seen even in the most severe conditions assayed, reinforcing the idea of the selectivTable 2 Solubilised components (% untreated gorse) from the autohydrolysis process at different temperature and reaction times. Temperature (◦ C)
Fig. 2. Evolution of (a) recovered xylan, (b) xylose and (c) arabinose for Ulex with reaction time.
Time profiles for the formation of non-volatile dissolved solids showed the typical shapes of a process consisting of two consecutive reactions involving formation and consequent degradation of intermediate products. Thus, production maxima of non-volatiles appeared at different times depending on the process temperature. At the most severe conditions, the volatiles were the main component of dissolved material due to their fast formation from the raw material and the contribution of the abovementioned degradation
Time (min)
Acetic acid
Furfural
HMF
Galactose released
Glucose released
160
5 15 30 60
0.79 0.93 0.81 0.78
0.002 0.014 0.039 0.098
0.004 0.005 0.019 0.036
0.000 0.024 0.000 0.091
0.206 0.204 0.267 0.301
170
5 15 30 60
0.95 0.51 0.84 1.42
0.008 0.043 0.003 0.296
0.004 0.019 0.003 0.074
0.000 0.055 0.090 0.150
0.233 0.257 0.267 0.284
180
5 15 30 60
0.33 0.82 1.28 2.19
0.027 0.134 0.313 0.952
0.011 0.038 0.073 0.154
0.049 0.080 0.158 0.253
0.256 0.230 0.283 0.312
190
5 15 30 60 5 15 30 60
0.69 1.17 2.81 3.15 1.56 3.28 4.56 4.91
0.080 0.351 1.162 2.540 0.385 1.341 2.707 4.533
0.028 0.075 0.176 0.348 0.067 0.194 0.367 0.879
0.081 0.157 0.260 0.325 0.135 0.261 0.302 0.211
0.249 0.244 0.292 0.384 0.219 0.275 0.371 0.448
200
P. Ligero et al. / Industrial Crops and Products 33 (2011) 205–210
209
Fig. 3. ␣-cellulose and acid insoluble lignin behaviour at different temperatures, for 60 min treatment time. Values for log R0 are: 3.54, 3.84, 4.13, 4.43, and 4.72, for 160, 170, 180, 190, and 200 ◦ C, respectively.
ity of the process. Moreover, the influences of treatment time and temperature proved to be much lower in the liberation of glucose. The liquors showed that the galactose concentrations were even lower than those of glucose (Table 2). Only two differences were found. First, the time and temperature effects were quantitatively higher, although in a restricted range (0.0–0.3%). Second, a degradation reaction was observed at 200 ◦ C. The formation of acetic acid increased with treatment time and temperature (Table 2). Acetic acid production was especially important at 200 ◦ C and long reaction times, where 60% of the initial content of acetate groups were liberated. The pattern of acetic acid formation showed that the deacetylation rate of xylan was slower than xylan depolymerisation. This result agrees with results previously reported using corncob (Nabarlatz et al., 2004). Furfural and hydroxymethylfurfural are the main degradation products of pentoses and hexoses, respectively. As pentoses are more easily dehydrated than hexoses, the concentration of furfural was always higher. As expected, the concentration of furfural in the liquors increased exponentially with temperature and time. The highest furfural concentration found reached a percentage close to 5% (Table 2). The profiles of hydroxymethylfurfural formation in the liquors were similar to those of furfural, although evidently at lower concentration values. Because glucose suffered no apparent degradation, galactose was the more likely source of hydroxymethylfurfural.
Fig. 4. Effect of severity factor on (a) the recovery of xylan and on (b) release of acetic acid.
3.6. Fractionation as a function of the severity factor The severity factor or reaction ordinate (RO ) was developed by Abatzoglou to give a simplified kinetic equation for reactions where several processes take place in a complex substrate, such as the lignocellulose matrix (Abatzoglou et al., 1992). The use of the severity factor allows the observation of the combined effect of temperature and time in a single variable. RO is defined as
RO =
exp 0
3.5. Cellulose and lignin Samples extracted at the most prolonged times (60 min) at different temperatures were subjected to analysis for the determination of lignin and ␣-cellulose to understand how the autohydrolytic conditions affected these compounds (Fig. 3). As expected, ␣-cellulose suffered greater alteration with increasing temperature. Roughly 3% (initial dry basis) of ␣-cellulose decomposed for every 10 ◦ C increase. At the most severe conditions, the ␣-cellulose remained at levels close to 50%. Moreover, the proportion of lignin in the solid remained at a value of 17.5% (initial dry weight), showing no variations with temperature. Therefore, within the ranges studied herein, it seemed that only a limited proportion of lignin could be solubilised regardless of the process temperature.
t
T − T r
ω
dt,
where T is the reaction temperature, Tr is a reference temperature (100 ◦ C), ω is an empirical factor depending on activation energy (in our case ω = 14.75 ◦ C according to bibliography) and t is reaction time. Fig. 4 shows the system behaviour as a function of log RO . By combining time and temperature in a single variable, the curves for different temperatures almost overlap. This is particularly interesting when observing the production of xylans in solution (Fig. 4a). As shown, the maxima of xylan released from Ulex at each temperature was around log RO = 3.65 min. This is in accordance with an important effect of liberation of acetic acid at the same RO (Fig. 4b). In addition, the behaviour of other compounds (e.g., degradation of arabinose and formation of furfural and hydroxymethylfurfural) is closely linked to this value of RO . A similar behaviour was reported in the non-isothermal, autohydrolysis of sugar beet pulp (Martínez
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et al., 2009). The adoption of this value allows the calculation of the optimal treatment time required for each temperature to obtain the maximum xylan recovery (i.e., 84.8, 47.3, 28.7, 17.9 and 12.15 min for 160, 170, 180, 190 and 200 ◦ C, respectively). Because the amount of xylan released is virtually the same for each one of these combinations, selecting the best time/temperature combination will depend on other factors besides the hydrolytic process itself. 4. Conclusions Samples of gorse (U. europæus) were used to test the potential use of this material as a source of xylan. Hydrothermal process conditions were applied to selectively extract xylan. Different reaction temperatures and times were tested to determine the best process conditions. Extraction of xylan showed a strong dependence on temperature and time. Increasing temperature yielded an increased amount of recovered xylan, reaching a maximum value of 62% (wt.% on initial xylan) at 180 ◦ C for 30 min, which is in agreement with other studies using non-wood material. The analysis of the kinetic behaviour as a function of the logarithm of the severity factor, log RO , allowed the identification of a set of time/temperature combinations that were the most favourable to produce the maximum amount of xylan in solution, corresponding to the value of log RO = 3.6. Due to its characteristics and its behaviour in the extraction process, U. europæus is a suitable material to extract xylan compounds. Moreover, the autohydrolysis conditions described herein showed a selective method to obtain xylan while only slightly affecting other fractions in the biomass such as cellulose and lignin. References Abatzoglou, N., Chornet, E., Belkacemi, K., 1992. Phenomenological kinetics of complex systems: the development of a generalised severity parameter and its application to lignocellulosics fractionation. Chem. Eng. Sci. 47, 1109–1122. Aoyama, M., Seki, K., 1999. Acid catalysed steaming for solubilization of bamboo grass xylan. Bioresour. Technol. 69, 91–94. Bachmann, R., Riese, J., 2006. Industrial biotech – setting conditions to capitalize on the economic potential. In: Kamm, B., Gruber, P.R., Kamm, M. (Eds.), Biorefineries – Industrial Processes and Products, vol. 2. Wiley, pp. 445–462. Bao, M., Rodríguez, J.L., Domínguez, M.J., Crespo, I., Vega, A., 1998. Ulex europæus as a protein source for the agrifood industry in Galicia, Spain. In: El Bassam, N., Behl,
R.K., Prochnow, B. (Eds.), Intl. Conf. on Sustainable Agriculture for Food, Energy and Industry: Strategies Towards Achievement. Braunschweig (Germany), pp. 716–721. Blumenkrantz, N., Asboe-Hansen, G., 1973. New method for quantitative determination of uronic acids. Anal. Chem. 54, 484–489. Browning, B.L., 1967. Methods of Wood Chemistry. Wiley & Sons, New York, pp. 726–727. Caparrós, S., Ariza, J., Garrote, G., Lopez, F., Diaz, M.J., 2007. Optimization of Paulownia Fortunei L. Autohydrolysis-organosolv pulping as a source of xylooligomers and cellulose pulp. Ind. Eng. Chem. Res. 46, 623–631. Carvalheiro, F., Esteves, M.P., Parajó, J.C., Pereira, H., Gírio, F.M., 2004. Production of oligosaccharides by hydrolysis of brewery’s spent grain. Bioresour. Technol. 91, 93–100. Ebringerová, A., 2006. Structural diversity and application potential of hemicellulose. Macromol. Symp. 232, 1–12. Ezpeleta, I., Arangoa, M.A., Irache, J.M., Stainmesse, S., Chabenat, C., Popineau, Y., Orecchioni, A., 1999. Preparation of Ulex europæus lectin–gliadin nanoparticle conjugates and their interaction with gastrointestinal mucus. Int. J. Pharm. 191, 25–32. Garrote, G., Dominguez, H., Parajó, J.C., 2001. Study on the deacetylation of hemicellulose during the hydrothermal processing of eucalyptus wood. Holz Roh Werkst. 59, 53–59. Garrote, G., Domínguez, H., Parajó, J.C., 2002. Autohydrolysis of corncob: study of non-isothermal operation for xylooligosaccharide production. J. Food Eng. 52, 211–218. Gomez, D.E., Yoshiji, H., Kim, J.C., Thorgeirsson, U.P., 1995. Ulex europæus I lectin induces activation of matrix-metalloproteinase-2 in endothelial cells. Biochem. Biophys. Res. Commun. 216, 177–182. Han, J.S., 1998. Properties of nonwood fibers. In: Proceedings of the Korean Society of Wood Science and Technology Annual Meeting, pp. 3–12. Imaizumi, K., Nakatsu, Y., Sato, M., Sedarnawati, Y., Sugano, M., 1991. Effects of xylooligosaccharides on blood glucose, serum and liver lipids and cecum shortchain fatty acids in diabetic rats. Agric. Biol. Chem. 55, 199–205. Martínez, M., Gullón, B., Schols, H., Alonso, J.L., Parajó, J.C., 2009. Assessment of the production of oligomeric compounds from sugar beet pulp. Ind. Eng. Chem. Res. 48, 4681–4687. Matthei, O., 1995. Manual de las malezas que crecen en Chile. Editorial Alfabeta Impresores, Santiago. Nabarlatz, D., Farriol, X., Montané, D., 2004. Kinetic modelling of the autohydrolysis of lignocellulosic biomass for production of hemicellulose derived oligosaccharides. Ind. Eng. Chem. Res. 43, 4124–4131. Nabarlatz, D., Ebringerová, A., Montané, D., 2007. Autohydrolysis of agricultural by-products for the production of xylo-oligosaccharides. Carbohydr. Polym. 69, 20–28. Parajó, J.C., Garrote, G., Cruz, J.M., Domínguez, H., 2004. Production of xylooligosaccharides by autohydrolysis of lignocellulosic materials. Trends Food Sci. Technol. 15, 115–120. Springer, E., Harris, J.F., 1982. Prehydrolysis of aspen wood with water and with dilute aqueous sulfuric acid. Svensk Papperstidning 82, R152–R154. Vega, A., Bao, M., 1993. Organosolv fractionation of Ulex europæus with dilute hydrochloric and phenol. Two simple kinetic models for pre-hydrolysis and delignification. Wood Sci. Technol. 27, 61–68.
Contents lists available at ScienceDirect
Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop
Gorse (Ulex europæus) as a possible source of xylans by hydrothermal treatment Pablo Ligero a , Alberto de Vega a,∗ , Johannes C. van der Kolk b , Jan E.G. van Dam b a b
Department of Physical Chemistry and Chemical Engineering, Faculty of Sciences, University of A Coru˜ na, Alejandro de la Sota, 1, 15004 A Coru˜ na, Spain Food and Biobased Research, Wageningen University and Research Centre, P.O. Box 7 17, 6700 AA Wageningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 28 May 2010 Received in revised form 9 October 2010 Accepted 11 October 2010
Keywords: Autohydrolysis Ulex europæus Hemicelluloses Xylans
a b s t r a c t Autocatalytic hydrothermal process conditions were used to study Ulex europæus (Gorse) as a source of xylan compounds. The aim was to study the possibilities for using this unutilised biomass material to produce xylans. Ulex is an evergreen shrub that grows in the northwest of Spain and has no economic value. Therefore, Ulex is considered a promising candidate as a biomass source. Ulex showed a total xylose content of 12%, thus qualifying it as a suitable material to extract xylan-derived compounds. Autohydrolysis was applied to extract xylans from Ulex. To find the best conditions for xylan extraction, samples of Ulex were subjected to different temperatures and time conditions. Results indicate that autohydrolysis is a suitable method to selectively extract xylans at temperatures between 160 and 190 ◦ C for 5–30 min, reaching a maximum xylan recovery of almost 63% of the initial xylan at 180 ◦ C for 30 min, with only small effects on cellulose and lignin contents. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Because of the increasing demands from society and governments for cleaner and CO2 neutral energy production, novel biorefinery production systems are of interest for conversion of alternative biomass sources for energy or chemicals. In this context, the exploitation of largely unexplored biomass species, such as Ulex, is of interest. Considering the advantages of the abundant distribution of this renewable and cheap biomass source, it is an interesting crop for study. Currently, lignocellulose feedstock (LCF) biorefinery concepts are introduced as a possible method to valorise biomass. LCF biorefinery can be understood as a set of processes through which the main biomass components can be transformed into chemicals, fuels and energy, according to its natural constitution. In particular, LCF organosolv biorefineries could contribute to large economic benefits from the valorisation of fractionated products (Bachmann and Riese, 2006). These biorefineries fractionate the biomass complex into its main components (i.e., cellulose, hemicelluloses, lignin and extractives) using organic solvents. Subsequently, each fraction is further processed to marketable products. The aim of such biorefineries is to maximise the value of each biomass fraction by chemical or physical transformations. In this sense, compatible techniques should be integrated to obtain the optimum use of all wood cell wall components to increase their value. A pretreatment of the raw material to selectively remove the main hemicellulose fraction prior to any
∗ Corresponding author. Tel.: +34 981 167000; fax: +34 981167065. 0926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2010.10.011
organosolv pulping process can be considered as a valorisation preprocessing technique. Furthermore, the harvesting of Ulex, which is very abundant in some areas, will help to reduce the serious threat of forest fires, as Ulex is an important substrate for its propagation. In this way, a system of sustainable forest management can be combined with a strategy for economic development. Gorse (Ulex europæus) is a spiny, perennial, evergreen shrub belonging to the Fabaceæ family, a very common wild species in Galicia (N.W. Spain) that is present in 74% of woodland. Gorse can be found in many parts of the world, but it is native to western and central Europe. Due to its great reproductive capacity, prolonged latency of the seed, long vegetative period, fast growth and absence of natural enemies, gorse plays an important role in the spread of forest fires, as mentioned previously (Matthei, 1995). For this reason, it is sometimes removed preventively from the forests before the start of the dry season. In the past, this material was successfully used as a source of protein in animal food, (Bao et al., 1998), but its most common use was as source of lectin (Gomez et al., 1995; Ezpeleta et al., 1999). In addition to these uses, Ulex is also a suitable candidate material to produce pentosans or xylan compounds. The hemicellulose fraction is the most abundant non-cellulose polysaccharide part of the biomass. Xylose is the main sugar compound within the hemicellulose fraction, and its potential use is of high importance. Many different biomass materials are used for the extraction of xylan, including cereals (Garrote et al., 2002; Carvalheiro et al., 2004, Nabarlatz et al., 2007), nuts (Nabarlatz et al., 2004), hardwood (Springer and Harris, 1982; Garrote et al., 2001) and grass (Aoyama and Seki, 1999). Its potential applications
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in the food and pharmaceutical industries (Imaizumi et al., 1991; Ebringerová, 2006) are the main support for studying the extraction of xylan. The autohydrolysis process takes advantage of the presence of acetyl groups in the biomass, which are liberated as acetic acid into the reaction media and catalyse xylan depolymerisation. Moreover, this process causes a selective solubilisation of hemicelluloses (Garrote et al., 2002), leaving cellulose slightly affected and lignin almost unaltered. Therefore, this pretreatment of biomass can be considered as a preparation step for a number of treatments as organosolv pulping, cellulose hydrolysis, etc. Furthermore, autohydrolysis can remove the extractable compounds, avoiding pitch deposits in the machinery (Caparrós et al., 2007). Autohydrolysis has been widely used to successfully cause xylan depolymerisation (Garrote et al., 2001; Parajó et al., 2004; Nabarlatz et al., 2007). The aim of our study was to use Ulex as a biomass source for xylo-oligosaccharides by means of autohydrolysis. 2. Materials and methods 2.1. Raw material Ulex was manually collected in Galicia in the autumn of 2008. The sample was taken from specimens of about 1–1.5 m in height. The sample obtained was transferred to the laboratory and left to dry at room temperature for a long period. Later, pricks and flowers were removed, and stems were cut in a garden shredder and milled in a hammer mill equipped with a 0.3-mm sieve at the outlet. A final moisture equilibration period was allowed, after which a value of 9.6% humidity was measured. The milled samples were stored in polypropylene bags. 2.2. Analytical methods Tappi standards were used to determine ash (T211 om-02), cold water solubility (T207 om-99), 1% NaOH solubility (T212 om-02), acid insoluble and acid soluble lignin (Tappi T222 om-88) and ␣-, - and ␥-cellulose (T203 om-93). Ethanol–toluene extractives were determined in an accelerated solvent extractor [DIONEX ASE 200] where milled samples were subjected to extraction with boiling ethanol–toluene (65:35 by volume) by fully automated methods. A second extraction step was carried out semi-automatically by putting the organic extractivefree samples in a glass flask in a heating block for extraction with boiling water for 1 h. In both cases, extractive contents were calculated from the weight difference between the starting raw material and the extractive-free material. Quantitative acid hydrolysis was performed by a two-step procedure following standard methods (Browning, 1967). Namely, treatment with 72% sulphuric acid (3.75 mL) was added to 0.375 g of sample for 2 h at 30 ◦ C. Next, water was added to obtain 4% sulphuric acid solution, and the mixture was kept at 100 ◦ C for 3 h. From this acid hydrolysis, two fractions were obtained: a solid fraction and a liquid fraction. The solid was the acid insoluble fraction that was considered as Klason lignin, after burning the solid to 575 ◦ C to correct for the acid insoluble ash content. The liquid fraction was a solution from the acid treatments. This was neutralised before determination of monosaccharides, furfural, hydroxymethylfurfural (HMF) and acetic acid, by HPLC. Holocellulose determination was performed by the chlorite method through successive extractions with sodium chlorite in acidic medium according to a previously described procedure (Browning, 1967). Quantitative determination of individual sugars was performed using HPLC. The samples were diluted with water until a suit-
able concentration of sugars was reached. Fucose was added as an internal standard, neutralised with barium carbonate, and filtered through a 0.45 m membrane. A Dionex HPLC equipped with an anion exchange column (Carbopack PA1) thermostated to 35 ◦ C and a pulsed amperometric detector (PAD) was used. HPLC chromatography also enabled the determination of other significant components as acetic acid, furfural and hydroxymethylfurfural. Uronic acids concentrations were determined spectrophotometrically using galacturonic acid as a standard for quantification (Blumenkrantz and Asboe-Hansen, 1973). 2.3. Hydrothermal processing Autohydrolysis experiments were carried out simultaneously in four 100-mL stainless steel reactors submerged in an oil bath at the desired temperature. Samples (2 g) were treated in duplicate for 5, 15, 30 and 60 min at different temperatures between 160 and 200 ◦ C. A liquid/solid ratio of 20 was used in all experiments. After the reaction, the mixture was cooled. Undissolved materials were removed by filtration, and the solids were washed three times with water. 2.4. Chemical analysis of hydrolysate fractions An aliquot of the combined liquids from hydrolysis (liquor and washing water) was used to determine the solid residue, which was considered as non-volatile dissolved matter. The difference between this value and the total mass loss after treatment was considered as volatile dissolved matter. The combined liquid autohydrolysis fraction was split into two aliquots. One was submitted directly to HPLC to determine the percentages of monosaccharides, acetic acid, furfural and hydroxymethylfurfural. The second aliquot was subjected to quantitative acid hydrolysis with 4% sulphuric acid at 121 ◦ C for 1 h in an autoclave. After that, the samples were neutralised to pH 7 prior to HPLC determination of monosaccharides, furfural, hydroxymethylfurfural (HMF) and acetic acid. The difference between the xylose amounts before and after hydrolysis was considered as recovered xylan. Solid residues were analysed to determine the yield of dissolved carbohydrates, acid insoluble lignin and ␣-cellulose. 3. Results and discussion 3.1. Characterisation of raw material The results of the chemical composition analysis of U. europæus are presented in Table 1. The resulting ␣-cellulose content in Ulex was quite high (50% ␣-cellulose), while the corresponding value found for glucose content was rather low (32%). These values are less than those found in hardwoods such as Eucalyptus globulus, which contains about 46.3% of glucose (Garrote et al., 2001), similar to those of cereal straws (Nabarlatz et al., 2007), and higher than the levels in almond shells, olive stone, rice husks, wheat straw and barley straw (Nabarlatz et al., 2007). In addition, previously reported values for the same material (Vega and Bao, 1993) are also higher (39%), probably due to the use of plants of different ages in previous studies. The monosaccharide composition was dominated by glucose and xylose, followed by minor quantities of arabinose, galactose, mannose and rhamnose. Uronic acids were also detected in significant quantity (Table 1). The acid insoluble lignin content showed a similar level to the values found previously (Vega and Bao, 1993). In summary, the Ulex substrate is ranked among non-wood species with relatively high contents of lignin and carbohydrates (Han, 1998).
P. Ligero et al. / Industrial Crops and Products 33 (2011) 205–210 Table 1 Chemical analysis of unextracted Ulex europæus stems.
3.2. Dissolved matter, volatile and non-volatile matter % On dry basis
Ash Solubility Extractives
Lignin Polysaccharides
Monosaccharides
Cold water 1% NaOH Ethanol/toluene Ethanol Hot water Acid insoluble Acid soluble Holocellulose ␣-cellulose -celullose ␥-cellulose Arabinose Xylose Mannose Galactose Glucose Rhamnose Uronic acids Acetic acid
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1.2 19.2 30.8 5.1 0.7 3.8 21.2 1.6 74.2 50.0 10.2 9.2 1.5 12.4 0.5 1.1 31.9 0.3 3.8 8.4
The pH profiles at different temperatures (Fig. 1a) showed a kinetic profile that follows the hydrothermal autocatalysed release of the acetate groups. The release of acetate was important throughout the heating period and during the first 5 min after reaching the reaction temperature (pH between 3.4 and 3.8). This liberation continued until 30 min of reaction time was reached, after which the liberation of acidic groups decreased substantially. The amount of dissolved material increased strongly with temperature and progressed, especially in the first 15 min. Beyond this time, the curve rose slowly until the maximum value was reached in the range of 25–40%, depending on the applied temperature (Fig. 1b). The dissolved material during autohydrolysis can be divided into the two categories of volatile and non-volatile matter. Fig. 1c and d shows the solubilisation rates of non-volatile and volatile matter, respectively, as a function of time and temperature. The volatile matter percentage increased progressively with temperature and time. This is in accordance with the concurrent formation of the main compounds of this group, including acetic acid, furfural and hydroxymethylfurfural, which showed individual concentrations in liquors that followed similar trends.
Fig. 1. Evolution of (a) pH and formation of (b) dissolved, (c) volatile and (d) non-volatile components during autohydrolysis of Ulex.
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reactions of the non-volatile fraction. This can be verified by the acid catalysed formation of dehydration products (i.e., furural and HMF) from released monosaccharides. 3.3. Xylose, xylan and arabinose The yield of xylan indicated that its recovery was strongly dependent on temperature and reaction time (Fig. 2a). Xylans were removed quickly in the first minutes of the reaction and then underwent decomposition reactions that were also temperature dependent. Thus, at the lowest reaction temperature (160 ◦ C), the xylan production profile was always increasing, while at higher temperatures the degradation reactions became more important. The maximum recovery of xylan (62% of initial) was obtained at 180 ◦ C after 30 min. This xylan recovery rate was similar to other reports for cereal grain (Carvalheiro et al., 2004) and eucalyptus wood (Garrote et al., 2001). Fig. 2b shows the curves for recovered xylose in extraction liquors as a function of temperature and reaction time. Xylose yields increased with time and temperature, proving that acid catalysed hydrolysis of the extracted xylan occurred. At the highest reaction temperature (200 ◦ C), xylose degradation (dehydration) reactions became predominant after 15 min. Nevertheless, the proportion of hydrolysed xylose in the extracts remained at relatively low levels, which is indicative of an acceptable selectivity. Concerning the liberation of arabinose, a qualitative explanation can be given similar to that described for xylose (Fig. 2c), with the only difference being that arabinose was present in lower amounts in the liquor, and degradation reactions became predominant earlier and at lower temperatures. 3.4. Minor sugars and others compounds Glucose was also removed from the solid matrix, but in small quantities. Values ranging from 0.20% to 0.45% were measured in dry initial weight (Table 2), probably resulting from minor hemicellulose glycans comprising glucose units (galactoglucomannans, xyloglucans) and cellulose degradation. Nevertheless, the percentages of liberated glucose were sufficiently low, indicating that cellulose was only slightly affected. This fact was seen even in the most severe conditions assayed, reinforcing the idea of the selectivTable 2 Solubilised components (% untreated gorse) from the autohydrolysis process at different temperature and reaction times. Temperature (◦ C)
Fig. 2. Evolution of (a) recovered xylan, (b) xylose and (c) arabinose for Ulex with reaction time.
Time profiles for the formation of non-volatile dissolved solids showed the typical shapes of a process consisting of two consecutive reactions involving formation and consequent degradation of intermediate products. Thus, production maxima of non-volatiles appeared at different times depending on the process temperature. At the most severe conditions, the volatiles were the main component of dissolved material due to their fast formation from the raw material and the contribution of the abovementioned degradation
Time (min)
Acetic acid
Furfural
HMF
Galactose released
Glucose released
160
5 15 30 60
0.79 0.93 0.81 0.78
0.002 0.014 0.039 0.098
0.004 0.005 0.019 0.036
0.000 0.024 0.000 0.091
0.206 0.204 0.267 0.301
170
5 15 30 60
0.95 0.51 0.84 1.42
0.008 0.043 0.003 0.296
0.004 0.019 0.003 0.074
0.000 0.055 0.090 0.150
0.233 0.257 0.267 0.284
180
5 15 30 60
0.33 0.82 1.28 2.19
0.027 0.134 0.313 0.952
0.011 0.038 0.073 0.154
0.049 0.080 0.158 0.253
0.256 0.230 0.283 0.312
190
5 15 30 60 5 15 30 60
0.69 1.17 2.81 3.15 1.56 3.28 4.56 4.91
0.080 0.351 1.162 2.540 0.385 1.341 2.707 4.533
0.028 0.075 0.176 0.348 0.067 0.194 0.367 0.879
0.081 0.157 0.260 0.325 0.135 0.261 0.302 0.211
0.249 0.244 0.292 0.384 0.219 0.275 0.371 0.448
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209
Fig. 3. ␣-cellulose and acid insoluble lignin behaviour at different temperatures, for 60 min treatment time. Values for log R0 are: 3.54, 3.84, 4.13, 4.43, and 4.72, for 160, 170, 180, 190, and 200 ◦ C, respectively.
ity of the process. Moreover, the influences of treatment time and temperature proved to be much lower in the liberation of glucose. The liquors showed that the galactose concentrations were even lower than those of glucose (Table 2). Only two differences were found. First, the time and temperature effects were quantitatively higher, although in a restricted range (0.0–0.3%). Second, a degradation reaction was observed at 200 ◦ C. The formation of acetic acid increased with treatment time and temperature (Table 2). Acetic acid production was especially important at 200 ◦ C and long reaction times, where 60% of the initial content of acetate groups were liberated. The pattern of acetic acid formation showed that the deacetylation rate of xylan was slower than xylan depolymerisation. This result agrees with results previously reported using corncob (Nabarlatz et al., 2004). Furfural and hydroxymethylfurfural are the main degradation products of pentoses and hexoses, respectively. As pentoses are more easily dehydrated than hexoses, the concentration of furfural was always higher. As expected, the concentration of furfural in the liquors increased exponentially with temperature and time. The highest furfural concentration found reached a percentage close to 5% (Table 2). The profiles of hydroxymethylfurfural formation in the liquors were similar to those of furfural, although evidently at lower concentration values. Because glucose suffered no apparent degradation, galactose was the more likely source of hydroxymethylfurfural.
Fig. 4. Effect of severity factor on (a) the recovery of xylan and on (b) release of acetic acid.
3.6. Fractionation as a function of the severity factor The severity factor or reaction ordinate (RO ) was developed by Abatzoglou to give a simplified kinetic equation for reactions where several processes take place in a complex substrate, such as the lignocellulose matrix (Abatzoglou et al., 1992). The use of the severity factor allows the observation of the combined effect of temperature and time in a single variable. RO is defined as
RO =
exp 0
3.5. Cellulose and lignin Samples extracted at the most prolonged times (60 min) at different temperatures were subjected to analysis for the determination of lignin and ␣-cellulose to understand how the autohydrolytic conditions affected these compounds (Fig. 3). As expected, ␣-cellulose suffered greater alteration with increasing temperature. Roughly 3% (initial dry basis) of ␣-cellulose decomposed for every 10 ◦ C increase. At the most severe conditions, the ␣-cellulose remained at levels close to 50%. Moreover, the proportion of lignin in the solid remained at a value of 17.5% (initial dry weight), showing no variations with temperature. Therefore, within the ranges studied herein, it seemed that only a limited proportion of lignin could be solubilised regardless of the process temperature.
t
T − T r
ω
dt,
where T is the reaction temperature, Tr is a reference temperature (100 ◦ C), ω is an empirical factor depending on activation energy (in our case ω = 14.75 ◦ C according to bibliography) and t is reaction time. Fig. 4 shows the system behaviour as a function of log RO . By combining time and temperature in a single variable, the curves for different temperatures almost overlap. This is particularly interesting when observing the production of xylans in solution (Fig. 4a). As shown, the maxima of xylan released from Ulex at each temperature was around log RO = 3.65 min. This is in accordance with an important effect of liberation of acetic acid at the same RO (Fig. 4b). In addition, the behaviour of other compounds (e.g., degradation of arabinose and formation of furfural and hydroxymethylfurfural) is closely linked to this value of RO . A similar behaviour was reported in the non-isothermal, autohydrolysis of sugar beet pulp (Martínez
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et al., 2009). The adoption of this value allows the calculation of the optimal treatment time required for each temperature to obtain the maximum xylan recovery (i.e., 84.8, 47.3, 28.7, 17.9 and 12.15 min for 160, 170, 180, 190 and 200 ◦ C, respectively). Because the amount of xylan released is virtually the same for each one of these combinations, selecting the best time/temperature combination will depend on other factors besides the hydrolytic process itself. 4. Conclusions Samples of gorse (U. europæus) were used to test the potential use of this material as a source of xylan. Hydrothermal process conditions were applied to selectively extract xylan. Different reaction temperatures and times were tested to determine the best process conditions. Extraction of xylan showed a strong dependence on temperature and time. Increasing temperature yielded an increased amount of recovered xylan, reaching a maximum value of 62% (wt.% on initial xylan) at 180 ◦ C for 30 min, which is in agreement with other studies using non-wood material. The analysis of the kinetic behaviour as a function of the logarithm of the severity factor, log RO , allowed the identification of a set of time/temperature combinations that were the most favourable to produce the maximum amount of xylan in solution, corresponding to the value of log RO = 3.6. Due to its characteristics and its behaviour in the extraction process, U. europæus is a suitable material to extract xylan compounds. Moreover, the autohydrolysis conditions described herein showed a selective method to obtain xylan while only slightly affecting other fractions in the biomass such as cellulose and lignin. References Abatzoglou, N., Chornet, E., Belkacemi, K., 1992. Phenomenological kinetics of complex systems: the development of a generalised severity parameter and its application to lignocellulosics fractionation. Chem. Eng. Sci. 47, 1109–1122. Aoyama, M., Seki, K., 1999. Acid catalysed steaming for solubilization of bamboo grass xylan. Bioresour. Technol. 69, 91–94. Bachmann, R., Riese, J., 2006. Industrial biotech – setting conditions to capitalize on the economic potential. In: Kamm, B., Gruber, P.R., Kamm, M. (Eds.), Biorefineries – Industrial Processes and Products, vol. 2. Wiley, pp. 445–462. Bao, M., Rodríguez, J.L., Domínguez, M.J., Crespo, I., Vega, A., 1998. Ulex europæus as a protein source for the agrifood industry in Galicia, Spain. In: El Bassam, N., Behl,
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