Valorization of Vine Shoots Based on the Autohydrolysis Fractionation

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Valorization of vine shoots based on the autohydrolysis fractionation optimized by a kinetic approach Beatriz Gullón, Gemma Eibes, Izaskun Dávila, Carlos Vila, Jalel Labidi, and Patricia Gullón Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b02833 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 11, 2017

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Industrial & Engineering Chemistry Research

Valorization of vine shoots based on the autohydrolysis fractionation optimized by

a kinetic approach Beatriz Gullón1, Gemma Eibes*1, Izaskun Dávila2, Carlos Vila3, Jalel Labidi2, Patricia Gullón2 1

Department of Chemical Engineering, Institute of Technology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain

2

BioRP Group, Department of Chemical and Environmental Engineering, University of Basque Country, UPV/EHU, 20018 San Sebastián, Spain 3

Department of Chemical Engineering, Faculty of Science, University of Vigo (Campus Ourense), As Lagoas, 32004 Ourense, Spain

*Corresponding author: E-mail address: [email protected] (G. Eibes)

Abstract Non-isothermal autohydrolysis treatments of vine shoots were used as a method for hemicellulose solubilization and xylooligosaccharides (XOS) production. The proposed kinetic model allowed the selection of the operational conditions that maximize the oligosaccharides content and minimize the generation of undesired compounds such as monosaccharides and sugar-decomposition products. In this sense, the maximum value of XOS (9.9 g/100 g raw material) was obtained at 200 ºC. The liquid fraction containing oligosaccharides was highly stable against heat and pH (degradation below 10%), demonstrating its potential as functional food ingredients. On the other hand, the cellulose-enriched solid fraction was subjected to enzymatic digestibility, obtaining 49.5% of total glucan hydrolysis after 96 h. In this work, the holistic use of wine shoots following a biorefinery concept has been proposed. However, a delignification step of the cellulose-enriched solid fraction should be included in order to increase the enzymatic digestibility, and hence allowing the integral use of this by-product.

Keywords: Hydrothermal treatment, Biorefinery, Xylooligosaccharides, Reaction kinetics, Heat stability, Enzymatic hydrolysis

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1.

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Introduction Winemaking industry, one of the main economic agro-food activities in the

Mediterranean countries, involves the generation of significant amounts of residues with more than 60 million tons produced worldwide annually.1 Among the grape processing residues, one of the main sub-product of vineyards are the vine shoots, which annual pruning results in the generation of a fluctuating amount of these lignocellulosic leftovers, approximately between 1.4 and 2.0 tons per hectare.2 Although these residues are usually left in the ground as organic material for fertilization purposes, vine shoots constitute an abundant, cheap, renewable and easily available raw-material with a great economic potential for a sustainable development.3 In this context, an efficient, profitable and ecofriendly alternative to their conventional uses could be their transformation in high-added-value products with applications in food, pharmaceutical and cosmetic industries.1 Literature regarding the holistic use of vine shoots within a biorefinery approach is quite limited (Dávila et al., 2017).4 Among the various approaches described in the literature for the integral valorization of lignocellulosic biomass, the aqueous treatment (namely autohydrolysis) was considered the most appropriate choice5. This technology allows the solubilization of hemicelluloses (mainly oligosaccharides), remaining both cellulose and lignin in solid phase with scarce chemical alteration; these fractions are suitable for further specific applications.6 In this sense, the holistic use of lignocellulosic materials following the biorefinery concept has been proposed as a key tool to develop the so-called biobased economy.7 When lignocellulosic biomass rich in xylan is subjected to an autohydrolysis process, an extensive solubilization of hemicelluloses takes place by their conversion into substituted xylooligosaccharides (XOS).6 One of the main advantages of this


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treatment is that, when performed under suitable conditions, a XOS mixture with a wide range of degree of polymerization and rich substitution patterns can be obtained.5 Moreover, under certain autohydrolysis conditions the formation of degradation compounds is limited, allowing the use of the solubilized hemicelluloses solutions for food applications without further refining treatment. In this regard, autohydrolysis has been successfully applied to several feedstocks, including wheat bran8, rice husks9, sugar beet pulp10, pine wood11, orange peel wastes12, birch wood13 inter alia in order to obtain different types of oligosaccharides. In the last two decades, there has been a growing interest in these compounds due to their potential application in a variety of fields such as biomaterials, biofuel, pharmaceuticals, cosmetic and functional food.14,15 Nowadays, XOS are regarded as food ingredients which cause prebiotic effects due to their ability to modulate the bowel function by conferring beneficial health effects.16 From a technological point of view, XOS, which are important for their use in processed and functional foods, present advantages with respect to other non-metabolizable oligosaccharides, such as their stability in acidic media, resistance to heat, low available energy and low daily intakes to achieve biological effects.16,17 Moreover, further biological actions such as antioxidant, antiallergy, antimicrobial, anti-infection, anti-inflammatory, antiadhesive, immunomodulatory, selective cytotoxicity, antihyperlipidemic inter alia, have been described for XOS.16 Taking into account the multiple properties reported for XOS, particularly their application as prebiotic ingredients, and considering their high selling price, the manufacture of XOS using xylan-containing lignocellulosic materials such as agricultural residues has gained interest in the last years. In order to produce food-grade XOS from hydrothermal treatments, operational conditions that maximize the


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oligosaccharide content and minimize the generation of undesired compounds such as monosaccharides and sugar-decomposition products must be selected. Thus, understanding the hydrolytic processing of biomass is crucial for the optimization, design and scale-up of the process. Consequently, the development of accurate kinetic models for the comprehension of the autohydrolysis process is essential in order to provide the optimal conditions that allow the maximum solubilization of hemicelluloses and the minimum generation of degradation products.18,19 In this context, our research group has carried out the production of hemicellulosic oligosaccharides from vine shoots by autohydrolysis processing20; however, the integral valorization of the different fractions obtained from the autohydrolysis of the residue was not evaluated in detail. Here, we expanded that work in two important directions. First, a kinetic model was developed to accurately predict the behaviour of the hemicellulosic fraction present in the vine shoots subjected to nonisothermal autohydrolysis treatments. To our knowledge, the kinetic modeling of the autohydrolysis of hemicelluloses from vine shoots is still unexplored in the scientific literature. Secondly, with the aim of performing an integral valorization of vine shoots following a biorefinery concept, and the zero waste goal strategy, the two fractions obtained after the hydrothermal processing were evaluated in order to explore their possible applications. The thermal and pH stability of the liquid fraction containing XOS was assessed, in order to incorporate these solubilized products in food as functional ingredients. The cellulose-enriched solid fraction, obtained under selected conditions, was subjected to enzymatic hydrolysis to determine its suitability for glucose production. The advantages and limitations of autohydrolysis as a first stage of a biorefinery scheme for vine-shoot holistic valorization were discussed.


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2. Materials and methods 2.1. Raw material The vine shoots (variety Hondarribi Zuri) collected from a local winery (Aldako Bodega S.L. in Oiartzun, Basque Country, Spain) were milled and sieved to pass through a 0.4 mm screen. They were homogenized in a single lot to avoid compositional variations among aliquots, and then they were air-dried up to constant weight and stored until use. Aliquots from the above lot were subjected to analytical determinations following the analytical procedures described by Dávila et al.20 The chemical composition of the vine shoots lot, determined by Dávila et al. 20 was: 33.2 ± 0.9% of glucan, 27 ± 1.2% of hemicelluloses (including xylan, arabinose substituents, galactosyl substituents, manosyl substituents, acetyl groups and galacturonic acid) and 26.7 ± 1.3% of Klason lignin; minor components such as ashes (2.6 ± 0.1%) and extractives (3.1 ± 0.3%) were determined for a better knowledge of the feedstock composition. 2.2. Autohydrolysis conditions The experimental procedure of hydrothermal treatments of vine shoots was described by Dávila et al. 20 Briefly, these authors carried out the experiments with a liquor to solid ratio of 8 kg/kg oven-dried solid and reacted in a 1.5 L stainless steel 5100 Parr reactor under non-isothermal conditions. The heat treatment was performed in the temperature range of 180-215 ºC (corresponding to severity factors, So, in the range 3.29-4.65) based on related works.6,11,13 At the end of the treatments, the reactor was cooled down to 60 ºC and the resultant suspension was filtered to obtain a liquid phase and a solid phase. The autohydrolysis experiments were performed in duplicate.


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2.3. Analysis of the fractions from the autohydrolysis treatments Spent solids from the autohydrolysis treatment recovered by filtration were processed as follows: in a set of experiments these solids were washed with water, airdried and quantified to calculate the solubility of the raw material; then, an aliquot was milled to a particle size below 0.25 mm and analyzed following the same methodology used for raw material characterization. 20 Other set of experiments were carried out to recover the spent solids which were washed with water, filtered and stored to 4 ºC until they were used in the enzymatic digestibility experiments. Samples from each autohydrolysis liquors were filtered through 0.22 µm membranes and analyzed by HPLC to determine the content of monosacharides, acetic acid, galacturonic acid and degradation products (furfural and hydroxymethylfurfural). Another aliquot from each sample was subjected to quantitative acid posthydrolysis (treatment with 4% H2SO4 at 121 ºC for 30 min) and analyzed by HPLC for the determination of monosaccharides and attached substituents. The increase in the concentrations of monosaccharides, galacturonic acid and acetic acid caused by the acid posthydrolysis provided a measure of the oligomer concentration and their degree of substitution by acetyl and galacturonyl groups. More detailed information about the analytical procedure and the HPLC methodology can be found in the work published by Dávila et al. 20 The analyses described above were carried out in triplicate. In this work, the autohydrolysis reaction was carried out under the optimal conditions predicted by the model to obtain the oligosaccharides required for the stability tests and to recover the remaining solid for the enzymatic saccharification.


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2.4. Fitting of data The results obtained by Dávila et al.

20

were used in this study to develop a

kinetic model that can describe the autohydrolysis process for vine shoots. In order to numerically solve the differential equations involved in the kinetic model, empirical equations were used to fit the temperature profiles. Fourth order Runge-Kutta method was employed to solve the kinetic model equations using the optimization routine based on Newton-Raphson method of the Microsoft Excel software. The preexponential factors and the activation energies were calculated by minimizing the sum of the deviation squares between experimental and calculated data. 2.5. Stability assessment of oligosaccharides The oligosaccharides obtained at the optimal temperature of non-isothermal autohydrolysis treatment were previously freeze-dried to evaluate their heat and pH stability at different conditions by simulating the cooking process and the human digestion, respectively, according to Rumpagaporn et al.21 Briefly, the freeze-dried oligosaccharides were dissolved to obtain a concentration of 2% (w/v) in citratephosphate buffers with pH values of 3 and 7. The solutions were incubated at 37 ºC for 3 h (simulating in vitro human digestion) and at 100 ºC for 1 h (simulating cooking procedures). To monitor the stability of oligosaccharides, an aliquot of the treated solutions of oligosaccharides was filtered through a 0.22 µm membrane filter and analyzed by HPLC; another aliquot was subjected to quantitative acid posthydrolysis using the methodology described above and reported elsewhere.20


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2.6. Enzymatic hydrolysis of the spent solid from the optimal hydrothermal treatment The enzymatic hydrolysis of the wet spent solid obtained from the autohydrolysis treatment under the optimal conditions was performed at 48.5 ºC in Erlenmeyer flasks with orbital agitation (150 rpm). The pH of the samples was kept at 4.85 using 0.05N citric acid-sodium citrate buffer. The liquid to solid ratio was fixed at 20 g/g. The enzyme concentrates, “Celluclast 1.5L” cellulases from Trichoderma reesei and “Novozym 188” β-glucosidase from Aspergillus niger, were kindly supplied by Novozymes (Copenhagen, Denmark). A cellulase to solid ratio of 25 FPU/g and a βglucosidase to cellulase ratio of 5 IU/FPU were used in the experiments. The enzymatic hydrolysis experiments lasted up to 96 h. At the desired reaction times, samples were withdrawn from the reaction media, centrifuged, filtered and analyzed by HPLC for monosaccharides and acetic acid composition. At the end of the experiments, the medium was filtered and the solid was recovered, washed several times with water and dried at room temperature. This solid was weighed, its moisture was determined and the percentage of solubilization was gravimetrically calculated. Aliquots of the recovered spent solids were milled and characterized by quantitative acid hydrolysis and analyzed by HPLC. 3. Results and discussion 3.1. Composition of the autohydrolysis liquors Based on data collected from Dávila et al.20 (2016), during the hydrothermal process of the vine shoots the xylan percentage on the remaining solids decreased with the increase of the temperature until it reached a minimum value near 8% (Table 1). The main components of the liquid phase were the XOS generated by the hydrolysis of


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the xylan fraction, reaching the highest yield at 200 ºC (83.1%), which supposes a concentration of 12.2 g/L. Under the most severe conditions assayed (215 ºC) this yield progressively decreased down to 18.7% (2.7 g/L XOS) which concurs with the maximum yield of xylose (7%, corresponding to 1.02 g/L). This behavior has been reported by other authors using similar hemicellulosic heteropolysaccharides.6,22 Regarding the arabinosyl groups linked to oligomers (ArOS), an analogous behaviour to the xylan fraction was observed: at 200 ºC the ArOS yield achieved a maximum conversion percentage of 42.9% (0.74 g/L in the liquid phase) with respect to the initial amount of this fraction in the raw material. Higher temperatures resulted in a marked decrease of the yield of ArOS down to 19.7% (0.34 g/L). This decrease can be explained by the partial hydrolysis of the ArOS to give arabinose (Ara), which subsequently is degraded to furfural. Ara achieves a concentration of 1.81 g/L at the highest temperature assayed. On the basis of the data presented in Table 1, the acetyl group content of the spent solids (Acn) diminished with the severity of treatments until it reached a 10.8% with respect to the acetyl groups contained in raw material at the highest temperature tested. The acetyl groups linked to oligosaccharides (AcO) accomplished a maximum conversion of 30.9% when the autohydrolysis treatment was carried out at 190 ºC which supposes a concentration of 3.12 g/L in the autohydrolysis liquor. This percentage decreased until 15% (1.56 g/L) at the highest temperature evaluated, whereas a continuous increase in the acetic acid (AcH) present in the autoydrolysis liquors was observed when the temperature increased, achieving a concentration of 5.41 g/L at the severest conditions studied. This behavior was in agreement with the reported by other authors.23


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Finally, it is important to remark that the concentration of sugar-degradation compounds (mainly furfural and HMF) reached 0.35 g /L at 200 ºC.20 3.2. Kinetic modeling Among the advantages of kinetic models are the better understanding and the prediction of the behavior of the autohydrolysis process, and the formulation of mathematical equations that can be used for techno-economic optimization studies.24 Indeed, the process optimization using kinetic models is a key procedure for scaling-up the concept level of assay and its later trade step.25 In order to elucidate the main mechanisms involved in the hemicelluloses solubilization of vine shoots by autohydrolysis treatment,20 a kinetic model considering a pseudo-homogeneous behaviour by sequential first-order reactions with an Arrhenius type temperature difference was developed. Kinetic models supply a more complete description of the processes that take place in the hydrothermal treatment, such as the solubilization of hemicelluloses, the formation and breaking of oligomers, the release of monomers and the generation of degradation products.22 Although the hydronium catalyzed degradation of hemicelluloses has been studied by several authors,6,22,24,26,27 kinetic parameters strongly depend on the pretreatment method and raw materials. The reactions considered in the kinetic model of the depolymerization of hemicelluloses from vine shoots can be seen in Scheme 1 and was based on previous studies carried out by Caparrós et al.24 on the autohydrolysis of Arundo donax L., and on the work with rye straw carried out by Gullón et al.5


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Xns Arns

k1

XOSH k5

Acns

k2

XOSL

AcO

k9

Xyl

k4 F

k6

ArOS

k8

k3

Ara

k7

AcH

Scheme 1. Reaction pathway for the depolymerization of hemicelluloses from vine shoots A sequential first-order reactions kinetic scheme was proposed to model the time course of these compounds. The proposed mechanism for xylan degradation assumed the existence of two xylan fractions with different susceptibility towards hydrolysis; Xns is the susceptible xylan fraction that hydrolyzed to give high molecular weight xylooligomers (XOSH), which further decompose to low molecular weight xylooligomers (XOSL). Simplifications by considering only one of the xylan fractions28 lead to a worse fit of experimental results. In the late reaction stages, the xylooligomers with low molecular weight are hydrolyzed to xylose. Arabinosyl groups which are substituents (ArOS) of the xylan chain are easily hydrolyzed to give arabinose. Both, xylose and arabinose, decompose generating furfural. The acetyl group content followed a similar trend to the cleavage of the arabinosyl groups. For calculation purposes, the experimental data employed for constructing the model were expressed in grams of component per 100 g of dry raw material. On the basis of the considerations mentioned above and according to the scheme proposed in Table 2, the equations describing the hydrothermolysis of the hemicellulosic fraction can be expressed by Eqs. (1)-(15) dXn s = − k 1 ⋅ Xn s dt

(eq. 1)


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Xn= Xns + (1−αXn) ⋅ XnRM Xn s Xn

α Xn = dXOS dt dXOS dt

L

(eq. 2)

(eq. 3)

RM

= k 1 ⋅ Xn s − k 2 ⋅ XOS

H

= k 2 ⋅ XOS

XOS = XOS

H

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H

− k 3 ⋅ XOS

+ XOS

(eq. 4)

H

L

(eq. 5) (eq. 6)

L

dXyl = k 3 ⋅ XOS L − k 4 ⋅ Xyl dt

(eq. 7)

dArn = − k 5 ⋅ Arn dt

(eq. 8)

dArOS = k 5 ⋅ Arn − k 6 ⋅ ArOS dt

(eq. 9)

dAra = k 6 ⋅ ArOS − k 7 ⋅ Ara dt

(eq. 10)

dAcn s = − k 8 ⋅ Acn s dt

(eq. 11)

Acn= Acns + (1−αAcn) ⋅ AcnRM

(eq. 12)

α Acn =

Acn s Acn

(eq. 13) RM

dAcO = k 8 ⋅ Acn s − k 9 ⋅ AcO dt

(eq. 14)

AcH = Acn RM − Acn − AcO

(eq. 15)

where subscript “s” corresponds to the susceptible fraction of each component, subscript “RM” is the feedstock; αj (j = Xn or Acn) are the susceptible fractions (g susceptible fraction/g total fraction); ki (h-1) with i= 1 to i =9 represent the kinetic


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coefficients. It is also considered that the temperature dependence of these kinetic coefficients follows the Arrhenius equation:  Ea i   − R ⋅T (t )  

k i = k 0i ⋅ e 

(eq. 17)

where k0i (h-1) are the pre-exponential factors, Eai (kJ/mol) the activation energies and T(t) is the temperature reached at time t. The calculated values of the pre-exponential factors, susceptible fractions, activation energies and R2 are collected in Table 2. Figures 1 and 2 represent experimental data and the fitted model predictions for the hydrolysis of xylan and acetyl groups, respectively. In general, the good agreement between the calculated and experimental data for the fractions studied observed in Figures 1 and 2, was also reflected in the R2 values, hence the kinetic model can be used for the optimization of the hydrothermal process. The results presented in Figure 1 show that the hydrothermal treatment favors the removal of the xylan contained in the raw material, reaching a minimum value of about 1.7 g xylan/100 g oven-dried raw material at the highest temperature (215 ºC). The value calculated for the reactive xylan (measured by the “susceptible fraction”, α), was 0.881 g susceptible xylan/g xylan. This value agrees fairly well with the results reported by Gullón et al.6 for rye straw (0.865), by Rivas et al.13 for Betula alba (0.880) and by Parajó et al.29 for corn cobs (0.865). The maximum value of XOS was 9.9 g XOS/100 g raw material at 200 ºC, and the amount of xylose, under the same conditions, achieved just 0.23 g/100 g raw material, supporting that substantial amounts of XOS with limited decomposition can be obtained under these conditions. It is important to highlight that the kinetic model predicts that 200 ºC would be the optimal


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temperature for the hydrothermal treatment of the vine shoots, which coincides with one of the evaluated experimental temperatures. The activation energy value obtained in the xylan hydrolysis (156.9 KJ/mol) is close to the value found for the hydrolysis of Arundo donax L. (149 KJ/mol)24 and it was slightly lower than for rye straw (225.2 KJ/mol).6 The highest activation energy in the xylan hydrolysis corresponded to the high molecular weight xylooligomers conversion to low molecular weight XOS, in accordance with the results reported by Gullón et al.6 This effect suggests that, under certain temperatures, an accumulation of xylooligomers in the pretreatment liquor can occur and that the decomposition to the corresponding monomers and to furfural is faster at higher temperatures.22,30 The activation energy for the hydrolysis of the XOSL to xylose (94.2 KJ/mol) and the activation energy for its dehydration to furfural (125.8 KJ/mol) are significantly lower than those obtained for almond shells.31 Recently, dos Santos Rocha et al.22 reported a value of activation energy for the conversion of the XOS to monomers 2.3 times higher than that found in our work. In contrast, Branco et al.25 reported much lower activation energy values for the hemicellulose degradation of Annona cherimola Mill. seeds than those found for the aforementioned feedstocks. These results can be explained in basis to the different compositions of the raw materials evaluated. With regard to the residual arabinosyl groups in solid phase, the maximum removal was obtained at 200 ºC (see Table 1). These results indicate the high susceptibility of this fraction to the hydrothermal treatment. Similar behaviour was also described by Yáñez et al.32 for the hydrothermal processing of Acacia dealbata. The activation energy for the hydrolysis of the arabinosyl moieties was higher than the one observed by Gullón et al.6 using rye straw as feedstock (48.6 KJ/mol).


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For the hydrolysis of acetyl groups into oligomers, the activation energy value obtained (274.4 KJ/mol) was substantially higher than the one observed for rye straw, Betula alba, A. donax and A. dealbata.6,13,24,32 However, the activation energy value obtained for the transformation of the AcO into acetic acid is in the order of magnitude to those obtained from the previous feedstocks. Finally, the value obtained for the susceptible fraction of the acetyl groups (αAcn= 0.884 g susceptible acetyl groups/g acetyl groups) is close to the results reported by Yañéz et al.32 for A. dealbata. 3.3. Thermal and pH stability assessment Taking into account that the main industrial application of the oligosaccharides is their use as prebiotic food ingredients, their heat stability and their capacity to resist the acidic gastric conditions were evaluated. In order to assess this possibility, and based on the experimental data of autohydrolysis processing, the liquors from the treatment performed at 200 ºC in non-isothermal regime were used, as these conditions allowed the highest concentration of substituted xylooligosaccharides. A few research papers have analyzed the stability of the xylooligosaccharides during the thermal processing in acid media, being this the most common condition used for the incorporation of these compounds in food matrices.25 Moreover, the studies reported in the literature about the thermal and pH stability have been carried out using oligosaccharides obtained by enzymatic or alkali methods.21,34 Based on this information, the in vitro assessment of both thermal and pH stability of XOS obtained by hydrothermal treatments is important and therefore, it was an aspect studied in this work. In the current study, the oligosaccharide (OS) solution obtained at optimal conditions was kept at two pH values (3.0 and 7.0) at 100 ºC during 1 h to simulate cooking procedures. Figure 3A shows the recovery profile of the total oligosaccharides


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under the above conditions. As it can be observed, the oligosaccharides derived from vine shoots are very stable under the conditions tested and only a small decomposition of 4 and 8% for pH 3 and pH 7, respectively, were detected. It should be noted that this lower recovery at pH 7 is due to the degradation of the acetyl groups linked to the xylooligosaccharides (results not shown). In a related study, Chapla et al. (2012)33 studied the stability of XOS obtained from corncob xylan, and they reported low decomposition levels at high temperature and low pH. Courtin et al.34 also evaluated the stability of XOS, fructooligosaccharides (FOS) and arabinoxylooligosaccharides (AXOS) at acid pH and 100 ºC and they reported that XOS and AXOS were more stable than FOS. Similar findings were also observed by Wang et al. (2009)35 using XOS from wheat bran. More recently, Branco et al.25 reported that XOS obtained from Annona cherimola Mill. seeds were very stable at 100 ºC for 1 h and acid pH. Another important requirement of these compounds in order to be considered as prebiotics, is their capacity to resist the conditions of the acidic gastric environment.15 To this end, the oligosaccharides solution was kept at 37 ºC for 3 h and pH 3.0, simulating human digestion. The oligosaccharides tested were resistant to the digestive conditions, and only 5% of them were degraded after the treatment (Figure 3B). Sánchez et al.36 reported the same conclusions when they studied the susceptibility of arabinoxylan-oligosaccharides to simulated gastric conditions. In general, it can be concluded that the cooking processing conditions as well as the capacity to resist the digestive process do not affect these functional components. Therefore, they are suitable for food applications, although additional studies are necessary to confirm these in vitro results.34 3.4 Enzymatic digestibility of the hydrothermally processed solids


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The current trend in the revalorization of feedstocks is focused on their integral use following the biorefinery approach with a zero waste generation. This holistic approximation involves obtaining each of the constituent fractions of a feedstock separately, for their use to manufacture marketable final products, seeking the total exploitation of the starting residue. Therefore, within this framework, to achieve a whole exploitation of the vine shoots after the autohydrolysis processing, the remaining solid also must be valorized. The spent solids, enriched in cellulose and lignin, are susceptible to be enzymatically treated to saccharify the cellulose yielding a sugar solution, suitable for several uses (e.g. fermentation to ethanol), and a residual solid enriched in lignin with a low percentage of cellulose. This fraction enriched in lignin could be used as substrate for the endophytic fungi growth for producing ligninolytic enzymes.37,38 In order to evaluate this alternative, in a preliminary assessment, the spent solid from the pretreatment carried out at 200 ºC in non-isothermal regime was selected, as it provided the maximal oligosaccharides recovery and the minimal undesirable compounds formation. The material balance of the enzymatic hydrolysis of the spent solid from the autohydrolysis at 200 ºC of the vine shoots is collected in Figure 4. After 96 h, 49.5% of the total glucan of the spent solid was hydrolyzed, leading to solutions containing up to 11.0 g glucose/L (yield of 21.9 kg/100 kg of autohydrolyzed vine shoots) corresponding to a glucose/cellulase ratio of 0.0088 g glucose/FPU and to a volumetric productivity of 0.114 g glucose/L·h. The reported digestibility in this work is in agreement with the percentage reported for other byproducts such as the Eucalyptus residues (54%) using autohydrolysis as a pretreatment and similar enzymatic hydrolysis conditions.39


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As it can be seen in the Figure 4, the spent solid after the enzymatic hydrolysis contained a high percentage of glucan (25.8%) confirming that the autohydrolyzed vine shoots exhibited a low susceptibility for the enzymatic digestibility. Cellulose is intrinsically resistant to enzymatic degradation due to its high crystallinity and because is preserved by the surrounding matrix of lignin and hemicellulose. Although the autohydrolysis pretreatment provokes the breakdown of lignocelluloses removing most of the hemicellulose and hence, improving the enzymatic susceptibility,40 a scarce conversion of cellulose into glucose was observed. Autohydrolysis pretreatments do not cause the delignification of the raw material but instead increases the percentage of lignin due to the removal of hemicelluloses (from 26.7% in untreated vine shoots to 46.7% in autohydrolyzed vine shoots). It is well known that lignin has an unfavorable effect on the enzymatic hydrolysis of cellulose, hindering the hydrolysis by two mechanisms: by making a physical barrier that prevents the accessibility of enzymes to cellulose and by forming unproductive linkages with cellulolytic enzymes.37 Therefore, additional treatments will be necessary to reduce the effect of the residual lignin allowing modifications in the structure of cellulosic substrates by shattering the lignin matrix and by altering the crystalline structure of cellulose, improving the enzymatic hydrolysis. Several authors have carried out different processes of delignification that enable a greater digestibility of the pretreated solid as for instance, Yáñez et al.41 obtained 47.3 g glucose per 100 g autohydrolyzed Acacia dealbata after an alkaline treatment. Zhu et al.42 proposed the integrated process combining autohydrolysis and organosolv delignification for the value-added utilization of Eucommia ulmoides Oliver wood. In this case, the cellulose-rich residue achieved a glucose yield of 89.3%. According to the results shown here, in order to obtain an


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improved enzymatic susceptibility that allows the integral use of this by-product, a delignification step (suitable for other applications) would be desirable.

Conclusions In this study, the experimental data concerning the solubilization of xylan, arabinosyl and acetyl groups obtained in the hydrothermal processing of vine shoot were interpreted by means of kinetic models based on the Arrhenius equation. High temperature processing conditions as well as acidic gastric environment did not substantially affect the oligosaccharide content, being them considered suitable for food applications. The cellulose-enriched solid fraction obtained after the autohydrolysis treatment of the vine shoots at the optimum conditions was subjected to enzymatic hydrolysis and showed a limited digestibility, suggesting the necessity of a previous delignification step to improve the glucose yield. Nevertheless, in this study we propose the integral use of vine shoots within a multipurpose-multiproduct biorefinery scheme.

ACKNOWLEDGEMENTS This work was funded by the Spanish Ministry of Economy and Competitiveness (projects PCIN-2015-031 and CTQ2016-78689-R). The authors B. Gullón and G. Eibes belong to the Galician Competitive Research Group GRC 2013-032 and to the CRETUS Strategic Partnership (AGRUP2015/02). All these programs are co-funded by FEDER (EU). Beatriz Gullón and Patricia Gullón thank the Spanish Ministry of Economy and Competitiveness for their postdoctoral grants (Grant references IJCI2015-25305 and IJCI-2015-25304, respectively). Izaskun Dávila thanks the scholarship of young researchers training of the Basque Government.


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(21) Rumpagaporn, P.; Kaur, A.; Campanella, O. H.; Patterson, J. A.; Hamaker, B. R. Heat and pH Stability of alkali-extractable corn arabinoxylan and its xylanasehydrolyzate and their viscosity behavior. J. Food Sci. 2012, 77, H23. (22) dos Santos Rocha a, Bruna Pratto, M. S. R.; de Sousa Júnior, R.; Garcia Almeida, R. M. R.; da Cruz, A. J. G. A kinetic model for hydrothermal pretreatment of sugarcane Straw. Bioresour. Technol. 2017, 228, 176. (23) Garrote, G.; Domínguez, H.; Parajó, J. C. Mild autohydrolysis: an environmentally friendly technology for xylooligosaccharide production from Wood. J Chem. Technol. Biotechnol. 1999, 74, 1101. (24) Caparrós, S.; Garrote, G.; Ariza, J.; López, F. Autohydrolysis of Arundo donax L., a kinetic assessment. Ind. Eng. Chem. Res. 2006, 45, 8909. (25) Branco, P.C.; Dionísio, A.M.; Torrado, I.; Carvalheiro, F.; Castilho, P. C.; Duarte, L. C. Autohydrolysis of Annona cherimola Mill. seeds: optimization, modeling and products characterization. Biochem. Eng. J. 2015, 104, 2. (26) Garrote, G.; Parajó, J. C. Non-isothermal autohydrolysis of eucalyptus wood. Wood Sci. Technol. 2002, 36, 111. (27) González, D.; Santos, V.; Parajó, J. C. Manufacture of fibrous reinforcements for biocomposites and hemicellulosic oligomers from bamboo. Chem. Eng. J. 2011, 167, 278. (28) Yat, S. C.; Berger, A.; Shonnard, D. R. Kinetic characterization for dilute sulfuric acid hydrolysis of timber varieties and switchgrass. Bioresour. Technol. 2008, 99, 3855. (29) Parajó, J. C.; Garrote, G.; Cruz, J. M.; Dominguez, H. Production of xylooligosaccharides by autohydrolysis of lignocellulosic materials. Trends Food Sci. Tech. 2004, 15, 115. (30) Zhuang, X.; Yuan, Z.; Ma, L.; Wu, C.; Xu, M.; Xu, J.; Zhu, S.; Qi, W. Kinetic study of hydrolysis of xylan and agricultural wastes with hot liquid water. Biotechnol. Adv. 2009, 27, 578. (31) Nabarlatz, D.; Farriol, X.; Montane, D. Autohydrolysis of almond shells for the production of xylo-oligosaccharides: product characteristics and reaction kinetics. Ind. Eng. Chem. Res. 2005, 44, 7746.


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digestibility of autohydrolysed Acacia dealbata. J. Chem Technol Biotechnol. 2009b, 84, 1070. (42) Zhu, M. Q.; Wen, J. L.; Su, Y. Q.; Wei, Q.; Sun, R. C. Effect of structural changes of lignin during the autohydrolysis and organosolv pretreatment on Eucommia ulmoides Oliver for an effective enzymatic hydrolysis. Bioresour. Technol. 2015, 185, 378.

Abbreviations: Ac: acetyl groups linked to oligosaccharides AcH: acetic acid Acn: acetyl groups in solids AcnRM: acetyl groups in raw material Acns: acetyl groups susceptible to hydrolysis AcO: acetyl groups linked to oligosaccharides ArOS: arabinooligosaccharides Ara: arabinose Arn: arabinosyl substituents in solids ArnRM: arabinosyl substituents in raw material F: furfural Xn: xylan in solids XnRM: xylan in raw material Xns: xylan susceptible to hydrolysis XOS: xylooligosaccharides XOSH: high molecular weight xylooligomers XOSL: low molecular weight xylooligomers Xyl: xylose NVC: non-volatile compounds


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Figure captions Figure 1. Experimental and calculated values of variables measuring xylan hydrolysis. Figure 2. Experimental and calculated values of variables measuring acetyl groups

hydrolysis. Figure 3. Recovery of oligosaccharides simulating cooking procedures at 100 ºC for 1 h (A) and simulating human digestion at 37 ºC for 3 h (B). Figure 4. Material balance for the enzymatic hydrolysis of the solid fraction from autohydrolyzed vine shoots at 200 ºC.


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Figure 1


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Figure 2


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A

B

Figure 3


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Autohydrolyzed solid at 200 ºC (100 kg db) Glucan: 39.9 kg Hemicellulose: 5.8 kg Lignin: 46.7 kg Others: 7.5 kg

Water, Buffer, Enzymes

Enzymatic Hydrolysis LSR: 20 g/g, ESR: 25 FPU/g, 5 IU/FPU, 96 h

Spent solid Glucan: 19.6 kg Hemicellulose: 2.7 kg Lignin: 44.9 kg

Liquid phase containing glucose

Glucose: 21.9 kg

Figure 4


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Table 1. Conversion yields for hemicellulose (expressed as g of monomer equivalents/100 g of monomer equivalents in raw material) during the autohydrolysis treatments. Experimental data collected by Dávila et al. (2016)19. Monomer/oligomer/polymer

Temperature (ºC) 180

185

190

195

200

205

210

215

Xylose

0.4

0.5

0.6

1.7

1.9

4.2

6.8

7.0

Xylooligosaccharides

29.0

39.4

46.9

74.5

83.1

79.0

39.2

18.7

Xylan (solid)

77.5

49.7

33.7

22.6

16.4

12.4

8.5

8.0

Arabinose

25.0

26.3

31.4

19.0

21.2

18.3

11.2

10.4

Arabinosyl groups linked to oligomers 37.8

40.1

47.7

38.5

42.9

19.7

16.8

19.7

Arabinan (solid)

48.0

40.0

34.1

16.8

0.0

0.0

0.0

0.0

Acetic acid

12.7

14.2

16.9

34.8

38.8

57.5

75.2

105.8

24.3

25.9

30.9

18.5

20.6

17.8

15.5

15.0

47.7

35.8

29.1

22.3

17.4

15.4

13.0

10.8

Acetyl groups oligomers

linked

Acetyl groups (solid)

to


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Table 2. Values of susceptible fractions (αi), pre-exponetial factors (k0i), activation energies (Eai), and R2 determined in the model.

Reaction

Coefficient

LnK0i (K0i in h-1)

Eai (KJ/mol)

R2

Xylan and arabinan autohydrolysis (αXn=0.881 g susceptible xylan/g xylan) XnS→XOSH

k1

43.5

156.9

0.990

XOSH→XOSL

k2

58.7

225.5

0.995

XOSL→Xyl

k3

26.0

94.2

0.977

Xyl→F

k4

34.5

125.8

0.996

Arn→ArOS

k5

30.4

108.6

0.939

ArO→Ara

k6

4.9

17.9

Valorization of Vine Shoots Based on the Autohydrolysis Fractionation

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