Wood Sci Technol (2017) 51:669–684 DOI 10.1007/s00226-017-0899-4 ORIGINAL

Potential of different poplar clones for sugar production David Ibarra1 • Marı´a E. Eugenio1 • Isabel Can˜ellas2 Hortensia Sixto2 • Raquel Martı´n-Sampedro1

•

Received: 23 May 2016 / Published online: 27 February 2017 Ó Springer-Verlag Berlin Heidelberg 2017

Abstract In a poplar clonal plantation with different species and hybrids, established at 13,333 cuttings ha-1, biomass production was assessed at the end of the first rotation (3 years) and the potential of the different clones for sugar production was evaluated. The highest biomass production was observed for clones ‘AF2’ and ‘Viriato’ (46.5 and 42 t dm ha-1, respectively). After a mild acid pretreatment and an enzymatic saccharification, the highest sugar digestibility was found for ‘Viriato’ and ‘Unal’. A clear relation between sugar digestibility and xylan removal during pretreatment was observed, while no relation with lignin content was found. Taking into account the results, the highest production of sugars per hectare was estimated for the poplar clone ‘Viriato’, being 18–48% higher than that achieved with the other clones. Therefore, this clone is a promising candidate to be used as feedstock for sugar production in a forest biorefinery.

Introduction Lignocellulosic biomass is the most promising feedstock to be used in biorefineries for the production of biofuels, chemicals and other biomass-derived products. At the same time, obtaining lignocellulosic biomass from dedicated crops is especially interesting from a logistical perspective since it tends to be localized, both in time and space, and provides an opportunity for rural development. One of the most effective ways to produce it involves using short-rotation coppice (SRC). In Europe, & Raquel Martı´n-Sampedro [email protected] 1

Forestry Products Department, INIA – CIFOR, Ctra. de La Corun˜a km 7.5, 28040 Madrid, Spain

2

Silviculture and Forest Management Department, INIA – CIFOR, Ctra. de La Corun˜a km 7.5, 28040 Madrid, Spain

123

670

Wood Sci Technol (2017) 51:669–684

the suitability of the Salicaceae family (willows and poplars) for this purpose is well known (Weih 2004), being very fast growing species, widely adapted to different scenarios and easily to regrow. Poplar is one of the forest species with the greatest potential as bioenergy crop in the short term in the Mediterranean due to its fast growth and high yields, pests and diseases tolerance, among others (El Bassam 1996; Sannigrahi et al. 2010; Sixto et al. 2014). Its species and hybrids generally show coppicing capacity and facility for plantation establishment from cuttings. In general, poplar has high cellulose content (42–49%), low amounts of ash and extractives (0.6–2.7 and 1.4–3.6%, respectively), and moderate lignin and hemicellulose contents (21–29 and 16–23%, respectively), which make it a desirable feedstock for the production of biofuels and other value-added bio-based products (Sannigrahi et al. 2010). Obtaining fermentable sugars from lignocellulosic biomass is usually achieved by enzymatic hydrolysis. However, this process requires a previous pretreatment step to disrupt the complex and recalcitrant plant cell wall structure and thus render the cellulose more accessible to hydrolytic enzymes (Ma et al. 2014). An effective pretreatment should be cost-effective and provide more accessible and celluloserich biomass with high enzymatic digestibility, preventing degradation or loss of carbohydrates and formation of inhibitory products (Alvira et al. 2010). These pretreatments can be classified into biological, physical, chemical and physicochemical pretreatments, according to the different forces or energy consumed in the pretreatment process (Alvira et al. 2010). One of the most promising options because of cost-effectiveness and ease of scalability employs dilute acid (generally 0.5–1.0% sulfuric acid) at moderate temperatures (130–190 °C) to hydrolyze hemicelluloses, disrupt lignin and enhance sugar yield in subsequent enzymatic deconstruction of cellulose (Singh et al. 2015; Sun et al. 2014b). Another very common pretreatment is autohydrolysis or hydrothermal treatment (without adding acid or base), which is associated with lower capital and production costs and reduced formation of degradation products (Sun et al. 2014b). The effectiveness of the different pretreatments would depend not only on the operational conditions, but also on the characteristic of the lignocellulosic material used. Therefore, the aim of this work is to compare the sugar production from different poplar clones which are currently tested in SRC plantations for biomass production. To this end, both autohydrolysis and acid pretreatments were evaluated to enhance enzymatic saccharification of five poplar clones. These data together with the biomass production and composition will allow selecting the most highly promising poplar clone to be used as sugar platform for the production of biofuels and/or other value-added products.

Materials and methods Chemicals All chemicals used were reagent grade and were obtained from Merck (Barcelona, Spain), Panreac (Barcelona, Spain) or Sigma-Aldrich (Madrid, Spain). Hydrolytic

123

Wood Sci Technol (2017) 51:669–684

671

enzymes used in this work were Celluclast 1.5L and Novozyme 188, both supplied as donation by Novozymes (Bagsvaerd, Denmark). Raw material The biomass used comes from a short-rotation plantation established in the Autonomous Community of Aragon (Spain). The poplar clones included in the study were as follows: ‘AF2’, ‘I-214’, ‘Guardi’ (Populus x canadensis Moensch.), ‘Monviso’ (P.x interamericana 9 P. nigra L.), ‘Unal’ (P.x generosa Henry.) and ‘Viriato’ (P. deltoides Bartr.). The plantation (2 ha) was established at a density of 13,333 cuttings ha-1 and included 3 replications in a completely randomized design. Twenty-five central trees not affected by the border effect were evaluated on each clone and block. The plantation was cut in the third year since its installation (first rotation), in order to assess the biomass production of the different clones. Yield was evaluated by recording total aboveground (stem and branches) biomass. The fresh weight of the trees was determined. A subsample of the most representative diameter frequency distribution trees (3 trees per clone) was selected both for the chemical analysis and the estimation of dry weight. The yield data were expressed as dry weight after estimating the humidity content of the subsample which was oven-dried to constant weight at 100 °C. Analytical methods The composition of the raw material and pretreated samples was determined by standard analytical methods (National Renewable Energy Laboratory NREL/TP510-42618). The extractives were calculated as the soluble material after extensive Soxhlet extraction with ethanol. The free-extractive samples were subjected to quantitative acid hydrolysis in two steps to determine the carbohydrate composition. The hydrolyzed liquid obtained was then analyzed for carbohydrate content by HPLC in an Agilent Technologies 1260 chromatograph fitted with a refractive index detector (Agilent, Waldbronn, Germany), using an Agilent Hi-PlexPb column (Agilent, Waldbronn, Germany) operated at 70 °C with Milli-Q water as mobile phase pumped at a rate of 0.6 mL min-1. The solid residue remaining after the acid hydrolysis is considered acid-insoluble lignin (Klason lignin). Acid-soluble lignin was quantified at 205 nm using a Jasco V-500 spectrophotometer (Jasco, Japan). Glucose and xylose concentrations were determined in the hydrolyzates obtained during enzymatic hydrolysis. Hydrolyzate samples were directly used for determination of monosaccharides, using an Agilent Technologies 1260 HPLC fitted with a refractive index detector (Agilent, Waldbronn, Germany), using an Agilent HiPlexH column (Agilent, Waldbronn, Germany) operated at 65 °C with a mobile phase containing 5 mmol L-1 sulfuric acid pumped at a rate of 0.6 mL min-1. The compositions of the liquid fractions or prehydrolyzates obtained during the acid pretreatments were also determined. An aliquot (1 mL) was filtered through 0.45 lm nylon syringe membranes and used for direct HPLC determination of monosaccharides, acetic acid, furfural and 5-hydromethylfurfural (5-HMF), using

123

672

Wood Sci Technol (2017) 51:669–684

an Agilent Hi-PlexH column (Agilent, Waldbronn, Germany). A second aliquot of 25 ml was subjected to quantitative posthydrolysis with 4% H2SO4, at 120 °C, for 60 min, before HPLC analysis (according to NREL/TP-510-42623). Increments in the concentrations of monosaccharides and acetic acid caused by posthydrolysis were used to measure the concentrations of oligomers and acetyl groups bound to oligosaccharides, respectively. Autohydrolysis and acid pretreatments A representative sample of each poplar clone (stems and branches) was cut into chips and ground in a Willey mill. Sawdust samples were sieved to select a size of 0.71–0.40 mm prior to autohydrolysis or acid pretreatment. Both pretreatments were performed in an autoclave (Trade Raypa S.L., Spain) at 130 °C during 60 min, with a liquid to solid ratio of 6:1. When acid pretreatment was carried out, sulfuric acid was added in a final concentration of 3% (w/w over dry wood), while no chemicals were added for autohydrolysis pretreatment. Pre-soaking of the materials in the water or diluted acid was done at room temperature for 17 h. Pretreatments were performed by duplicate. After pretreatments, the whole slurries obtained were vacuum-filtered and separated into a solid fraction and a liquid fraction or prehydrolyzate. The solid fraction was used for enzymatic hydrolysis experiments without washing. Enzymatic hydrolysis Pretreated samples were subjected to enzymatic hydrolysis. All experiments were carried out by triplicate. A cellulolytic complex (Celluclast 1.5L), supplemented with b-glucosidase (Novozyme 188) was added to a 5% w/w wood suspension in 50 mM sodium citrate buffer (pH 4.8). Celluclast 1.5L is a cellulase preparation (92.5 FPU mL-1) with some xylanase activity, but practically no b-glucosidase activity; therefore, supplementation with Novozyme 188, which mainly presents bglucosidase activity (1274 UI mL-1), is typically applied in saccharification processes. The enzyme doses were 15 FPU of Celluclast 1.5L and 15 UI of bglucosidase per gram of dry sample. Enzymatic hydrolysis was carried out in a thermostatic rotary shaker at 50 °C and 120 rpm during 72 or 168 h for autohydrolyzed or acid-pretreated samples, respectively. Samples of 1.5 mL were taken after 24, 48, 72, 120 and 168 h of hydrolysis to evaluate the glucose and xylose concentrations by high-pressure liquid chromatography (HPLC). Prior to the determination of sugars, these liquid samples were heated in boiling water for 10 min to stop the enzymatic reaction and, after cooling, filtered through a 0.45-lm nylon syringe filter. Glucose (DG) and xylose (DX) digestibility were calculated according to Eq. 1, while glucose (YG), xylose (YX) and total sugar (YT) yields were calculated as indicated in Eq. 2. Thus, digestibility evaluates the percentage of sugars that were converted during the enzymatic hydrolysis per grams of sugars in the pretreated material (material subjected to enzymatic hydrolysis) and sugar yield takes also into account the loss of sugars during the pretreatment, calculating the yield per gram of

123

Wood Sci Technol (2017) 51:669–684

673

dry initial material (material subjected to pretreatment).

DG or DX ð%Þ ¼

g of sugar in liquid phase C h  Vh
 100 ð1Þ  100 ¼ g of sugar in pretreated material mp  Cp

YG or YX or YT ð%Þ ¼

g of sugar in liquid phase C h  Vh
 100 ¼  100 g of dry initial material mp =Yp

ð2Þ

where Ch is the concentration of sugars (glucose, xylose or total sugars) in the hydrolyzate at the end of the enzymatic hydrolysis, expressed in g L-1; Vh is the volume of hydrolyzate in L; mp is the g of dry pretreated material subjected to enzymatic hydrolysis; Cp is the concentration of sugars (glucose, xylose or total sugars) in the pretreated material, expressed as a percentage; and Yp is the solid yield of the pretreatment, expressed as a percentage. Statistical analysis One-way analysis of variance (ANOVA) was performed to evaluate the effect of clone on biomass yield. Newman–Keuls test was used to determine differences in pairwise comparisons between the clones for the variable. The level of statistical significance was set as p = 0.05.

Results and discussion Biomass production The lignocellulosic dry matter (dm) biomass production (stem and branches) of the different clones tested at the end of the first rotation ranged between 27.1 and 46.6 Mg dm ha-1 (Fig. 1). Highly variable productions for different genotypes and sites have been referred to previously in the Mediterranean. Productivities between 3.0 and 15.1 Mg dm ha-1 year-1 have been reported in Italy (Facciotto et al. 2005; Paris et al. 2011). In Spain, the potential productivity estimated from a wide range of trials has been considered between 10.9 and 15.3 Mg dm ha-1 year-1 (Pe´rezCruzado et al. 2014), which agrees with the productivities reported for the best clones in Greece (Aravanopoulos 2010). The clones ‘AF2’,‘Viriato’ and ‘Monviso’ have previously shown significantly higher biomass production under different soil and climate conditions (Sixto et al. 2013).

123

674

Wood Sci Technol (2017) 51:669–684 60 50

a

ab

Mg dm ha-1

b 40

c

30

cd

c

Guardi

Unal

20 10 0 AF2

Viriato Monviso

I214

Fig. 1 Lignocellulosic biomass production (stem and branches) per hectare in poplar clones at the end of the 1st rotation. Means followed by the same letter are not significantly different according to NK test (p \ 0.005). Error bars represent standard deviation

Poplar clones composition Table 1 shows the chemical composition of the different poplar clones. Ash content includes the inorganic elements present in biomass which act as a waste stream during its conversion to biofuels and are the source of biochar and slagging during thermochemical conversion. Therefore, low ash content would be desirable. All studied clones presented ash content between 2.1 and 2.7%, except ‘Viriato’ which showed a lower ash content (1.6%). These values are in the range of those reported by Sannigrahi et al. (2010) for 17 hybrid poplar species (ash content between 0.6 and 2.7%) and San Martin-Davison et al. (2015) for 4 hybrid poplars and Populus nigra (1.7–2.2%). In general, the ash content of hybrid poplar clones is similar to that in hardwood and slightly higher than softwood biomass, but substantially lower than other non-wood materials such as switchgrass, corn stover, wheat straw, giant cane, oat straw and tobacco stalk (Ates et al. 2008; Khiari et al. 2010; Sannigrahi et al. 2010). Ethanol extractives include waxes, chlorophyll and other minor components which can interfere in chemical and biological treatments. However, for large-scale lignocellulosic biorefinery operations, extractives can be a potential source of valueadded coproducts (Sannigrahi et al. 2010). The lowest ethanol extractive content was found for the poplar clone ‘I-214’ (1.6%), followed by ‘Monviso’ and ‘AF2’, while ‘Unal’ presented the highest ethanol extractive content (4.0%). Sannigrahi et al. (2010) reported values in the same range for different species of poplar and hybrid poplars, while San Martin-Davison et al. (2015) reported values slightly higher for different hybrid poplars (6.8–7.1%). Compared to other raw materials, the studied poplar clones show similar ethanol extractive content to corn stover, pine, eucalyptus or false yucca, but much lower than switchgrass (15.5%), olive wood (10.4%), giant cane (9.2%), wheat straw (4.6–9.2%) or Sorghum stalks (8.0%) (Khiari et al. 2010; Martı´n-Sampedro et al. 2012, 2014; Sannigrahi et al. 2010).

123

2.2 ± 0.2

2.1 ± 0.1

Guardi

Unal

1.6 ± 0.2

2.6 ± 0.1

1.6 ± 0.1

I-214

Viriato

4.0 ± 0.1

3.4 ± 0.2

3.5 ± 0.3

2.4 ± 0.2

2.5 ± 0.1

2.7 ± 0.1

2.4 ± 0.2

Monviso

AF2

Ethanol extractives

Ash content

Sample

21.4 ± 0.2

19.4 ± 0.1

22.8 ± 0.0

21.1 ± 0.3

23.6 ± 0.0

23.0 ± 0.1

Klason lignin

2.8 ± 0.1

2.5 ± 0.1

2.2 ± 0.0

2.8 ± 0.0

2.2 ± 0.0

2.9 ± 0.1

Ac. Sol. lignin

24.2 ± 0.3

21.9 ± 0.2

25.1 ± 0.0

23.9 ± 0.3

25.8 ± 0.0

25.9 ± 0.2

Total lignin

41.9 ± 0.4

41.4 ± 0.1

42.0 ± 0.4

46.3 ± 0.1

43.5 ± 0.4

41.5 ± 0.2

Glucan

Table 1 Chemical composition of the poplar clones (expressed as percentages of each compound per dry matter)

18.6 ± 0.5

19.9 ± 0.1

21.0 ± 0.2

19.7 ± 0.1

19.6 ± 0.2

18.6 ± 0.1

Xylan

2.3 ± 0.2

2.0 ± 0.0

1.7 ± 0.1

0.9 ± 0.0

0.7 ± 0.0

1.9 ± 0.0

Arabinan

1.9 ± 0.2

1.8 ± 0.1

1.1 ± 0.2

1.4 ± 0.3

1.3 ± 0.3

1.4 ± 0.2

Mannan

Wood Sci Technol (2017) 51:669–684 675

123

676

Wood Sci Technol (2017) 51:669–684

The proportion of cellulose, hemicelluloses and lignin in a biomass feedstock is a very important criterion in determining its suitability as an economically viable feedstock and also in deciding on the optimum pathway for its conversion. From a saccharification point of view, high cellulose (glucan) and low lignin contents are desirable. Thus, poplar clone ‘I-214’ seems to be a promising candidate for sugar production, since it showed the highest glucan content (46.3%) and the highest glucan to total lignin ratio. Nevertheless, all the poplars clones presented a glucan and total lignin content of 41.4–46.3 and 21.9–25.9%, respectively, similar to those reported for different poplar species and hybrids, and also for other hardwood and softwood (Garrote et al. 1999; Sannigrahi et al. 2010). Other non-wood materials such as switchgrass, corn stover, wheat straw and oat straw present a lower lignin content (between 9 and 18%), but also lower cellulose content (33–43%) (Ates et al. 2008; Garrote et al. 1999; Sannigrahi et al. 2010). Hemicelluloses content usually influences negatively the glucose hydrolysis yield, so a low content would be desirable to increase glucan conversion to bioethanol. However, they are also a source of valuable sugars (such as xylose), so a total sugar yield should be taken into account for many applications, especially when yeast able to ferment xylose is used. The six poplar clones showed a xylan content ranging from 18.6 to 21.0%, similar to that reported for different poplar hybrids (San Martı´n-Davison et al. 2015; Sannigrahi et al. 2010) and other hardwoods such as Eucalyptus globulus, higher than those reported for softwood and lower than other alternative non-wood raw materials such as switchgrass, corn stover or wheat straw (Garrote et al. 1999; Martı´n-Sampedro et al. 2014; Sannigrahi et al. 2010). Selection of pretreatment In order to select the most adequate pretreatment, both autohydrolysis and acid hydrolysis were evaluated prior to enzymatic hydrolysis of the six poplar clones. The objective of these pretreatments is to increase the saccharification yields without masking the influence of the composition and structure of the different clones, as a result of more aggressive hydrothermal pretreatment. Then, the selected severity of both pretreatments was low [S0 = 2.66, according to equation defined by Overend and Chornet (1987)]. Due to this selection, the sugar concentrations obtained after the enzymatic hydrolysis were very low (Table 2). Nevertheless, once the most adequate poplar clone is selected, a more intensive pretreatment could be carried out, in order to maximize the sugar recovery (taking into account both the sugar loss on the pretreatment and the sugar yield on the enzymatic hydrolysis). Comparing the sugar content of the enzymatic hydrolyzates, it was clear that the addition of 3% H2SO4 in the acid pretreatment increased the glucose production for the six poplar samples, while xylose production remained almost constant (Table 2). The highest glucose production increments were observed for poplar clones ‘I-214’ and ‘AF2’, followed by ‘Viriato’, ‘Guardi’ and ‘Unal’. Contrary, ‘Monviso’ showed the lowest increase. Both autohydrolysis and acid pretreatments enhance enzymatic digestibility not only through dissolution of the hemicelluloses (which increases porosity and improves enzymatic digestibility) but also through lignin redistribution

123

Wood Sci Technol (2017) 51:669–684

677

Table 2 Glucose and xylose concentrations (g L-1) in the hydrolyzates obtained after 72 h of enzymatic hydrolysis of the pretreated solid materials Sample

Autohydrolysis -1

Glucose (g L )

Acid pretreatment -1

Xylose (g L )

Glucose (g L-1)

Xylose (g L-1)

Monviso

2.0 ± 0.1

0.6 ± 0.0

2.9 ± 0.1

0.6 ± 0.0

AF2

0.5 ± 0.1

0.4 ± 0.0

3.6 ± 0.1

0.7 ± 0.0

I-214

1.0 ± 0.1

0.8 ± 0.0

4.2 ± 0.0

0.9 ± 0.0

Viriato

1.8 ± 0.0

0.6 ± 0.0

4.6 ± 0.1

0.5 ± 0.0

Guardi

1.4 ± 0.1

0.7 ± 0.0

3.7 ± 0.1

0.8 ± 0.0

Unal

2.4 ± 0.1

0.8 ± 0.0

4.4 ± 0.0

0.7 ± 0.0

as distinct aggregates rather than forming a coating on cellulose fibrils (Singh et al. 2015). According to Meng et al. (2015) and Sun et al. (2014b), when an acid is added, higher removal of hemicelluloses happens, increasing the accessible surface area of cellulose which would explain the higher glucose concentration obtained in the enzymatic hydrolyzates. Furthermore, the solid fractions subjected to acid pretreatment content lower amount of hemicellulose (mainly xylans) compared to those subjected to autohydrolysis, which limits the xylose concentration on the hydrolyzate, though the increase in accessibility caused by the hemicellulose removal also improves the enzymatic saccharification of the remained xylans. Selection of poplar clones for sugar production After selecting the acid pretreatment, the digestibility of the six acid-pretreated clones was evaluated during 168 h of enzymatic hydrolysis (Fig. 2) and compared with the composition of the acid-pretreated clones (Table 3). The highest glucose digestibility after 168 h was found for ‘Viriato’ (23.7%), in spite of showing a slower increase during the first 48 h than that of the other clones. This clone had the highest glucose content after the acid pretreatment (50.3%), while pretreated ‘Monviso’ and ‘AF2’ showed the lowest glucose contents (42.1 and 47.2%, respectively) and also the lowest glucose digestibility values (16.6 and 17.1%, respectively). According to these results and those of the other clones, a direct relation between the glucose content after the acid pretreatment and the glucose digestibility was found (coefficient of determination, R2, of 0.84, excluding data for the clone ‘Monviso’, whose glucose content is lower than expected). Furthermore, acid-pretreated ‘Monviso’ and ‘AF2’ presented also the highest lignin contents (27.4 and 27.6%, respectively). Similar to other authors, the presence and surface distribution of lignin have a detrimental effect on the enzymatic hydrolysis of lignocellulosic biomass, not only by physically limiting the cellulose accessibility but also by reversible/irreversible adsorption of cellulases onto lignin through functional groups such as lignin phenolic hydroxyl groups (Berlin et al. 2006; Chandra et al. 2008; Hoeger et al. 2012; Martı´n-Sampedro et al. 2013; Rahikainen et al. 2013). However, no relation was found between lignin content and glucose digestibility for the other clones, which is in agreement with several other authors (Cianchetta et al. 2014; Martı´n-Sampedro et al. 2015; Salvachu´a et al. 2011).

123

678

Wood Sci Technol (2017) 51:669–684

(b) 30%

Xylose Digestibility

Glucose Digestibility

(a) 25% 20% 15% 10% 5% 0% 0

50

100

150

90% 75% 60% 45% 30% 15% 0%

200

-5%

-15%

Time (hours) Monviso

105%

AF2

I-214

0

50

100

150

200

Time (hours) Viriato

Guardi

Unal

Fig. 2 Time course of glucose (a) and xylose (b) digestibility obtained during the enzymatic hydrolysis of poplar solid fractions resulting from acid pretreatment. Error bars represent standard deviation

Meng et al. (2015) reported that when severity of diluted acid pretreatment increased, the accessibility increased to a certain level that it became the dominating factor, causing higher sugar release despite retaining a large lignin fraction. Similarly, Sun et al. (2014a) suggested that partial delignification with hemicelluloses removal during diluted acid pretreatment instead of complete lignin removal could better benefit the sugar yield because not only it reduces irreversible adsorption of lignin to the enzyme, but it also increases specific surface area and, only to a limited extent, causes the cellulose fibril coalescence to provide an optimal pretreated biomass for subsequent enzymatic deconstruction. Leu and Zhu (2013), after a revision of recently published works about enzymatic saccharification of lignocellulosic materials, concluded that hemicelluloses removal is more important than lignin removal for creating cellulose accessible pores, and therefore to enhance saccharification. Apart from published experimental data, they based this conclusion on the structure of lignocellulose, which is represented by elemental cellulose fibrils being cross-linked by hemicelluloses and embedded in a non-cellulosic polysaccharide matrix of hemicelluloses and lignin; therefore, lignin is not directly cross-linked with cellulose chains on the surface of the elemental fibrils and its removal would be less significant in increasing accessibility, although partial delignification is needed to achieve satisfactory saccharification of lignocelluloses with high lignin content. The current results also corroborate this conclusion, finding an indirect correlation between xylan content in acid-pretreated samples and glucose digestibility, except for the clone ‘AF2’ (coefficient of determination, R2, of 0.86). When the removal of hemicelluloses during the acid pretreatment is taken into account (Fig. 3), similar correlation was found. In this sense, the higher the removal of hemicelluloses, the higher the glucose digestibility. However, no relation with lignin removal (nor lignin content) was found (coefficient of determination, R2, lower than 0.3). Thus, for the poplar clone ‘Viriato’, which showed the highest glucose digestibility, the acid pretreatment removed 91.6 and 14.5% of the xylose and lignin contained in the untreated material, respectively, while only 0.6% of the glucose was removed. The lowest percentage of xylose

123

82.4 ± 0.2

80.3 ± 0.3

Guardi

Unal

4.8 ± 0.1

87.5 ± 0.3

83.0 ± 0.4

I-214

Viriato

5.8 ± 0.3

3.9 ± 0.1

3.5 ± 0.2

4.7 ± 0.2

4.6 ± 0.4

86.6 ± 0.2

83.2 ± 0.4

Monviso

AF2

Ethanol extractives

Yield

Sample

25.1 ± 0.0

23.9 ± 0.3

23.9 ± 0.5

22.6 ± 0.2

25.7 ± 0.6

24.9 ± 0.4

Klason lignin

2.1 ± 0.2

2.4 ± 0.1

1.9 ± 0.2

2.8 ± 0.1

1.9 ± 0.1

2.5 ± 0.0

Ac. Sol. lignin

48.6 ± 0.9

47.6 ± 0.2

50.3 ± 0.2

48.9 ± 0.7

47.2 ± 0.9

42.1 ± 1.0

Glucan

2.3 ± 0.1

3.3 ± 0.7

2.1 ± 0.0

3.8 ± 0.0

2.1 ± 0.3

4.3 ± 0.3

Xylan

Table 3 Solid fraction yields (%) of acid pretreatment and chemical composition of the resulting pretreated solid fractions

1.4 ± 0.1

1.1 ± 0.2

1.0 ± 0.1

1.8 ± 0.2

1.1 ± 0.2

1.0 ± 0.1

Arabinan

1.7 ± 0.1

1.9 ± 0.3

1.1 ± 0.2

2.2 ± 0.4

1.5 ± 0.3

1.8 ± 0.1

Mannan

Wood Sci Technol (2017) 51:669–684 679

123

680

Wood Sci Technol (2017) 51:669–684 Xylan

Glucan

Lignin

Unal Guardi Viriato I-214 AF2 Monviso 0%

20%

40%

60%

80%

100%

% Extracted from untreated material

Fig. 3 Percentages of xylan, glucan and lignin extracted from the untreated samples during acid pretreatment. Error bars represent standard deviation

removed during the pretreatment was found for ‘Monviso’ (79.9%) which also corresponds to the clone showing the lowest glucose digestibility jointly with ‘AF2’. However, this last clone was the only one that did not follow the general tendency, since xylose removal during acid pretreatment was higher than expected (91.3%). On the other hand, both poplar clones ‘Monviso’ and ‘AF2’ showed the highest glucose removal during pretreatment (12.1 and 10%, respectively), which could indicate a lignocellulose structure with higher cross-linking between cellulose and hemicelluloses. Regarding xylose digestibility (Fig. 2b), higher values than for glucose digestibility were found (except for Monviso H2). Nevertheless, it should be taken into account that the xylose content in acid-pretreated samples was low (2.1–4.6%), so the xylose hydrolysis meant only an increase in total sugar digestibility of 1.4–2.7%. As it happens with glucose digestibility, a correlation between xylose digestibility and xylan removal during acid pretreatment (and also with xylan content in acid-pretreated samples; coefficients of determination, R2, of 0.79 and 0.81, respectively) was found, whereas no correlation was found with lignin removal (nor lignin content; coefficients of determination, R2, lower than of 0.1). Table 4 shows the composition of the liquid fractions obtained after the acid pretreatments. These data corroborate the removal of hemicelluloses from the solid fraction. However, xylooligosaccharides recovered in the liquid fraction were found lower than those extracted according to solid fraction composition (Fig. 3). This loss of hemicelluloses could be due to the degradation of the dissolved xylooligosaccharides to furfural. However, according to the amount of furfural detected in the liquid, only 1.4–2.9% of the original xylan was degraded to furfural. Nevertheless, it is possible that these volatile products were not quantitatively recovered in the liquid fraction because they were partially lost to the atmosphere after pretreatment (Emmel et al. 2003).

123

2.39 ± 0.03

0.11 ± 0.00

0.09 ± 0.00

Arabino-oligomers

Acetil groups

1.18 ± 0.02

1.51 ± 0.02

0.43 ± 0.00

0.03 ± 0.00

Acetic acid

Furfural

5-HMF

Degradation products

0.04 ± 0.00

0.96 ± 0.00

4.86 ± 0.09

1.01 ± 0.06

2.82 ± 0.03

Arabinose

13.1 ± 0.30

0.43 ± 0.01

Glucose

0.84 ± 0.01

0.51 ± 0.02

0.29 ± 0.00

4.55 ± 0.12

1.01 ± 0.10

AF2

Xylose

Monomeric form

0.51 ± 0.03

Gluco-oligomers

Xylo-oligomers

Oligomeric form

Monviso

0.03 ± 0.00

0.61 ± 0.01

2.13 ± 0.02

1.16 ± 0.01

4.53 ± 0.18

0.25 ± 0.01

0.9 ± 0.01

0.04 ± 0.00

4.78 ± 0.03

0.74 ± 0.01

I-214

0.06 ± 0.00

1.01 ± 0.02

4.3 ± 0.06

1.63 ± 0.02

11.51 ± 0.39

1.10 ± 0.02

0.15 ± 0.01

0.06 ± 0.00

5.88 ± 0.12

0.18 ± 0.01

Viriato

0.05 ± 0.00

0.74 ± 0.01

3.25 ± 0.01

1.35 ± 0.05

7.88 ± 0.30

0.66 ± 0.01

0.68 ± 0.00

0.25 ± 0.00

5.45 ± 0.08

1.01 ± 0.05

Guardi

0.06 ± 0.00

0.46 ± 0.00

4.35 ± 0.04

1.74 ± 0.02

10.63 ± 0.06

1.19 ± 0.01

0.33 ± 0.00

0.05 ± 0.00

8.18 ± 0.04

0.42 ± 0.01

Unal

Table 4 Composition of liquid fractions (g L-1) obtained after acid pretreatment of the different poplar clones expressed as grams per 100 g of raw material treated in each pretreatment

Wood Sci Technol (2017) 51:669–684 681

123

682

Wood Sci Technol (2017) 51:669–684

Table 5 Biomass production, glucose content, sugar yield per gram of biomass and sugar yield per hectare of the different poplar clones studied Sample

Production (Mg dm ha-1)

Glucose content (%)

Sugar yield (%)

Sugar yield per hectare (Mg ha-1)

Monviso

39.4

41.5

7.3

2.89

AF2

46.6

43.5

8.1

3.78

I-214

30.9

46.3

10.0

3.09

Viriato

42

42.0

11.0

4.62

Guardi

27.1

41.4

8.9

2.42

Unal

27.7

41.9

9.7

2.68

Taking into account sugar digestibility, the most promising clones to be used as sugar feedstock for the production of biofuels and/or other value-added products were the poplar clone ‘Viriato’, followed by ‘Unal’. These clones showed a total sugar digestibility of 25.2 and 23.7%, respectively. However, when the sugar yield is calculated per gram of dry initial material (which takes into account also the loss of sugar during the pretreatment), the clone ‘I-214’ became also very interesting: total sugar yield of 11.0, 9.7 and 10.0% for poplar clones ‘Viriato’, ‘Unal’ and ‘I214’, respectively. On the other hand, the lowest total sugar yields were found for ‘Monviso’ (7.3%), followed by ‘AF2’ (8.1%) and ‘Guardi’ (8.9%). Similar enzymatic hydrolysis yields (4–10%) were reported by Zhang et al. (2015) after acid pretreatment of poplar using different particle sizes between 0.5 and 10 mm. However, most authors reported higher saccharification yields for different poplar species and hybrids after hydrothermal or acid pretreatments. Thus, Meng et al. (2015) reported a glucose yield of approximately 50% when poplar was subjected to an acid pretreatment with sulfuric acid at 160 °C and 60 min. In the same way, San Martin-Davison et al. (2015) carried out enzymatic hydrolysis of different hybrid poplars after steam explosion, obtaining hydrolysis yields of 20.6–28 and 25.9–35.7% for pretreatments at 200 °C (S0 = 3.42) and 220 °C (S0 = 4.01), respectively. Finally, Negro et al. (2003) obtained 26–40 g of glucose per 100 g of pretreated poplar after steam explosion pretreatments with severity factors between 3.25 and 4.14. In all these cases, severity of pretreatment (taking into account treatment time and temperature and/or acid concentration) was higher than in the current experiments. As aforementioned, a mild pretreatment was selected because the objective of the pretreatment in the present work was to increase the hydrolysis yield without masking the influence of the composition and structure of the different clones, as a result of more aggressive hydrothermal pretreatment. Nevertheless, once ‘Viriato’ has been selected as the most promising poplar clone, an optimization of the acid pretreatment could be carried out to maximize the sugar yield. Not only the sugar yield, but also the biomass production of the different clones should be taken into account in order to select the most promising clone for being used as feedstock in a forest biorefinery. As mentioned above, the higher biomass production per hectare in the first rotation was found for ‘AF2’ followed by ‘Viriato’. Combining these results with sugar yield, the higher sugar production per

123

Wood Sci Technol (2017) 51:669–684

683

hectare was found for the clone ‘Viriato’, being 18 and 33% higher than that found for clones ‘AF2’ and ‘I-214’, respectively (Table 5). The lowest sugar production was observed for ‘Guardi’ (being 48% lower than for ‘Viriato’) which showed the lowest biomass production.

Conclusion Six poplar clones cultivated in a short-rotation plantation were compared for sugar production. Biomass production after the first rotation, wood composition and sugar yields after an acid pretreatment followed by enzymatic saccharification were studied, calculating the sugar production per hectare. ‘Viriato’ showed a sugar production per hectare between 18 and 48% higher than the other clones. Therefore, this poplar clone seems to be a promising raw material to be used as sugar feedstock for the production of biofuels and/or other value-added products. Acknowledgements This research was funded by MINECO (Spain) throughout the Projects CTQ201128503-C02-01, CTQ2013-47158-R, PSE-On Cultivos and RTA2011-00006-00-00 and Program PTA 2011-4857-I. We would like to thank Fernando Sebastia´n and Maider Go´mez (CIRCE-UZ) and M.J. Herna´ndez and E. Viscasillas (INIA) for their technical support.

References Alvira P, Toma´s-Pejo´ E, Ballesteros M, Negro M (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 101:4851–4861 Aravanopoulos F (2010) Breeding of fast growing forest tree species for biomass production in Greece. Biomass Bioenergy 34:1531–1537 Ates S, Ni Y, Akgul M, Tozluoglu A (2008) Characterization and evaluation of Paulownia elongota as a raw material for paper production. Afr J Biotechnol 7:4153–4158 Berlin A, Balakshin M, Gilkes N, Kadla J, Maximenko V, Kubo S, Saddler J (2006) Inhibition of cellulase, xylanase and b-glucosidase activities by softwood lignin preparations. J Biotechnol 125:198–209 Chandra RP, Esteghlalian AR, Saddler JN (2008) Assessing substrate accessibility to enzymatic hydrolysis by cellulases. In: Hu TQ (ed) Characterization of lignocellulosic materials. Wiley Online Library, New York, pp 60–80 Cianchetta S, Di Maggio B, Burzi PL, Galletti S (2014) Evaluation of selected white-rot fungal isolates for improving the sugar yield from wheat straw. Appl Biochem Biotechnol 173:609–623 El Bassam N (1996) Renewable energy: potential energy crops for Europe and the Mediterranean region. In REU Technical Series (FAO) no 46. FAO/FAL, Rome, p 200 Emmel A, Mathias AL, Wypych F, Ramos LP (2003) Fractionation of Eucalyptus grandis chips by dilute acid-catalysed steam explosion. Bioresour Technol 86:105–115 Facciotto G, Bergante S, Lioia C, Mughini G, Zenone T (2005) Produttivita` di cloni di pioppo e salice in piantagioni a turno breve (Productivity of poplar and willow clones in short rotation plantations) (In Italian). In: V National Congress SISEF—’’Forest and Society: changes, conflicts, synergies’’,Grugliasco, pp 27–30 Garrote G, Dominguez H, Parajo J (1999) Hydrothermal processing of lignocellulosic materials. Holz Roh- Werkst 57:191–202 Hoeger I, Filpponen I, Martin-Sampedro R, Johansson LS, Osterberg M, Laine J, Kelley S, Rojas OJ (2012) Bicomponent lignocellulose thin films to study the role of surface lignin in cellulolytic reactions. Biomacromolecules 13:3240–3328

123

684

Wood Sci Technol (2017) 51:669–684

Khiari R, Mhenni M, Belgacem M, Mauret E (2010) Chemical composition and pulping of date palm rachis and Posidonia oceanica—a comparison with other wood and non-wood fibre sources. Bioresour Technol 101:775–780 Leu S-Y, Zhu JY (2013) Substrate related factors affecting enzymatic saccharification of lignocelluloses: a review and analysis of recent literature. BioEnergy Res 6:405–415 Ma J, Zhang X, Zhou X, Xu F (2014) Revealing the changes in topochemical characteristics of Poplar cell wall during hydrothermal pretreatment. BioEnergy Res 7:1358–1368 Martı´n-Sampedro R, Eugenio M, Villar J (2012) Effect of steam explosion and enzymatic pre-treatments on pulping and bleaching of Hesperaloe funifera. Bioresour Technol 111:460–467 Martı´n-Sampedro R, Rahikainen JL, Johansson L-S, Marjamaa K, Laine J, Kruus K, Rojas OJ (2013) Preferential adsorption and activity of monocomponent cellulases on lignocellulose thin films with varying lignin content. Biomacromolecules 14:1231–1239 Martı´n-Sampedro R, Revilla E, Villar JC, Eugenio ME (2014) Enhancement of enzymatic saccharification of Eucalyptus globulus: steam explosion versus steam treatment. Bioresour Technol 167:186–191 ´ , Ibarra D, Eugenio ME (2015) Use of new endophytic fungi as pretreatment Martı´n-Sampedro R, Fillat U to enhance enzymatic saccharification of Eucalyptus globulus. Bioresour Technol 196:383–390 Meng X, Wells T, Sun Q, Huang F, Ragauskas A (2015) Insights into the effect of dilute acid, hot water or alkaline pretreatment on the cellulose accessible surface area and the overall porosity of Populus. Green Chem 17:4239–4246 Negro MJ, Manzanares P, Ballesteros I, Oliva JM, Caban˜as A, Ballesteros M (2003) Hydrothermal pretreatment conditions to enhance ethanol production from poplar biomass. Appl Biochem Biotechnol 105:87–100 Overend R, Chornet E (1987) Fractionation of lignocellulosics by steam-aqueous pretreatments. Philos Trans R Soc Lond Ser A 321:523–536 Paris P, Mareschi L, Sabatti M, Pisanelli A, Ecosse A, Nardin F, Scarascia-Mugnozza G (2011) Comparing hybrid Populus clones for SRF across northern Italy after two biennial rotations: survival, growth and yield. Biomass Bioenergy 35:1524–1532 Pe´rez-Cruzado C, Sanchez-Ron D, Rodrı´guez-Soalleiro R, Herna´ndez MJ, Mario Sa´nchez-Martı´n M, Can˜ellas I, Sixto H (2014) Biomass production assessment from Populus spp. short-rotation irrigated crops in Spain. GCB Bioenergy 6:312–326 Rahikainen JL, Martin-Sampedro R, Heikkinen H, Rovio S, Marjamaa K, Tamminen T, Rojas OJ, Kruus K (2013) Inhibitory effect of lignin during cellulose bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption. Bioresour Technol 133:270–278 ´ T, Martı´nez MJ (2011) Fungal Salvachu´a D, Prieto A, Lo´pez-Abelairas M, Lu-Chau T, Martı´nez A pretreatment: an alternative in second-generation ethanol from wheat straw. Bioresour Technol 102:7500–7506 San Martı´n-Davison J, Ballesteros M, Manzanares P, Sepu´lveda XP-B, Vergara-Ferna´ndez A (2015) Effects of temperature on steam explosion pretreatment of poplar hybrids with different lignin contents in bioethanol production. Int J Green Energy 12:832–842 Sannigrahi P, Ragauskas AJ, Tuskan GA (2010) Poplar as a feedstock for biofuels: a review of compositional characteristics. Biofuels Bioprod Bior 4:209–226 Singh J, Suhag M, Dhaka A (2015) Augmented digestion of lignocellulose by steam explosion, acid and alkaline pretreatment methods: a review. Carbohyd Polym 117:624–631 Sixto H, Herna´ndez MJ, de Miguel J, Can˜ellas I (2013) Red de parcelas de cultivos len˜osos en alta densidad y turno corto (Network of plots of woody crops in high density and short rotation). Monogr INIA: Serie For 25:188 (in Spanish) Sixto H, Gil P, Ciria P, Camps F, Sa´nchez M, Can˜ellas I, Voltas J (2014) Performance of hybrid poplar clones in short rotation coppice in Mediterranean environments: analysis of genotypic stability. GCB Bioenergy 6:661–671 Sun Q et al (2014a) Effect of lignin content on changes occurring in poplar cellulose ultrastructure during dilute acid pretreatment. Biotechnol Biofuels 7:150 Sun Q et al (2014b) Comparison of changes in cellulose ultrastructure during different pretreatments of poplar. Cellulose 21:2419–2431 Weih M (2004) Intensive short rotation forestry in boreal climates: present and future perspectives. Can J For Res 34:1369–1378 Zhang M, Ju X, Song X, Zhang X, Pei Z, Wang D (2015) Effects of cutting orientation in poplar wood biomass size reduction on enzymatic hydrolysis sugar yield. Bioresour Technol 194:407–410

123