Optimization of Hydrothermal Pretreatment of Hardwood and Softwood

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Optimization of hydrothermal pretreatment of hardwood and softwood lignocellulosic residues for selective hemicellulose recovery and improved cellulose enzymatic hydrolysis Christos K. Nitsos, Theodora Choli-Papadopoulou, Konstantinos A. Matis, and Kostas S. Triantafyllidis ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00535 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 6, 2016

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ACS Sustainable Chemistry & Engineering

Optimization of hydrothermal pretreatment of hardwood and softwood lignocellulosic residues for selective hemicellulose recovery and improved cellulose enzymatic hydrolysis Christos K. Nitsos1, Theodora Choli-Papadopoulou2, Konstantinos A. Matis1 and Kostas S. Triantafyllidis1,3,* 1

Laboratory of General and Inorganic Chemical Technology, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

2

Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece 3

Chemical Process & Energy Resources Institute, Centre for Research and TechnologyHellas, 57001 Thermi, Thessaloniki, Greece

*Corresponding author e-mail: [email protected]

Abstract The sustainable utilization of lignocellulosic biomass as a renewable and abundant source lies at the core of the emerging bio-based economy for the production of fuels, materials and platform chemicals. The first step in the implementation of many biomass valorization technologies is the “pretreatment” that aims at biomass fractionation and recovery of its main structural components, i.e. cellulose, hemicellulose and lignin, which can be then converted by down-stream (bio)catalytic processes to targeted high added value intermediate chemicals or final products. In this respect, hydrothermal pretreatment in pure water (also called as Liquid Hot Water or Autohydrolysis) offers a method with low operational costs, free of organic solvents and corrosive acids or bases, and with no use of “external” liquid or solid catalysts. In the present work, the hydrothermal pretreatment of three types of lignocellulosic forestry and agricultural residues/byproducts was studied. They are representative of hardwood (residual poplar branches from logging operations and grapevine pruning) and softwood (pine sawdust) biomass. The pretreatment experiments were conducted in a batch-mode, high-pressure reactor under autogeneous pressure at varying temperature (170-220oC) and time (15-180 min) regimes, and at liquid-to-solid (LSR) of 15. The intensification of the process was expressed by the severity factor, log Ro. The process was optimized for increasing the recovery of 1 ACS Paragon Plus Environment


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hemicellulose in the form of monomeric sugars (xylose, mannose, galactose) or the respective oligo-saccharides as well as for improving the production of glucose in the subsequent enzymatic hydrolysis of the pretreated biomass. Maximum hemicellulose recovery for poplar, grapevine and pine in the liquid products was around 60 % at ~70-85 % hemicellulose removal, based on initial hemicellulose content of each biomass type, and was achieved at relatively moderate treatment severities (log Ro = 3.8 - 4.1). Formation of major degradation products, such as acids (i.e. formic and levulinic acid) and furans (i.e. furfural, HMF) was relatively low and below ca. 1 mg/ml for the whole range of pretreatment severities. Enzymatic hydrolysis of the parent lignocellulosic materials towards glucose was very low (i.e. 10%) and remained low for the pretreated pine biomass (16%) but was substantially improved for poplar (49%) and especially for grapevine (77%) as a result of hydrothermal pretreatment at the highest severity (log Ro = 4.7). The significant improvement of enzymatic hydrolysis of grapevine was attributed to the nearly complete removal of hemicellulose and to the changes in the morphological and textural characteristics of biomass particles, with the most pronounced one being the 9-fold increase of surface and pore volume.

Keywords: Hydrothermal pretreatment, poplar branches, grapevine pruning, pine sawdust, cellulose hydrolysis, enzymes, hemicellulose recovery

Introduction The vision for a new bio-based economy is slowly but constantly emerging with the aim to supplement and partially replace the current fossil-based economy, for the production of fuels and platform chemicals. With regard to the availability of sources, it is based on the utilization of biodegradable feedstocks from a variety of sources, such as industrial, agricultural, forestry, and municipal wastes and byproducts1-3. Among these, lignocellulosic biomass which includes all plant dry matter have attracted increased attention due to the fact that it is the most abundant renewable source on earth. The main structural components of biomass are the carbohydrates cellulose (crystalline polysaccharide consisting of linear chains of several hundred to thousands of D-glucose primary units) and hemicellulose (amorphous polysaccharide consisting of C5 and/or C6 sugar primary units), and the aromatic polymer lignin (comprised of phenylpropane 2 ACS Paragon Plus Environment

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units). These three bio-polymers are arranged in the form of fibrils where the crystalline cellulose microfibrils are surrounded by a hemicellulose and lignin matrix4. Depending on the source type, a number of other components such as protein, pectins, ash and extractives may also be present at varying but usually small concentrations. This complex structure together with cellulose crystallinity and lignin hydrophobic aromatic polymer nature, gives lignocellulose its high resistance to chemical and biological treatment and limits the availability of its components for conversion into fuels or platform chemicals5. To overcome this natural recalcitrance of lignocellulose, an effective pretreatment strategy must be applied before any microbial, enzymatic or chemical conversion process. Main goals of the pretreatment methods are to disrupt the lignocellulosic structure, separate its components and expose cellulose in the core for subsequent processing, mainly via enzymatic or chemical hydrolysis to its glucose monomers 6-10. Among the various methods developed, the hydrothermal (also referred to as Liquid Hot Water, Hot Water Extraction or Autohydrolysis) pretreatment is one of the mildest since it utilizes hot water as the sole reagent and relatively low treatment temperatures (up to ca. 230oC) and low autogeneous pressures.

11-13 14

. Thus, the avoidance of expensive and hazardous

chemicals, such as strong inorganic acids/bases or various organic solvents, minimizes corrosion of equipment and eliminates the need for post-process solvent separation, recycling or neutralization, making it simple, cost effective, and environmentally friendly. Although the main target of the hydrothermal pretreatment is to selectively dissolve and hydrolyse the hemicellulose fraction to its primary sugar units (i.e. xylose, arabinose, glucose, etc.), the formation of sugar transformation products, such as furfural, HMF, formic acid, and levulinic acid, is also taking place, especially at relatively more intense reaction temperature and time conditions. In our previous related study on the hydrothermal pretreatment of beech wood 11, it was shown that the optimum process conditions (mainly temperature and time) can be selected 3 ACS Paragon Plus Environment


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in order to obtain a process liquid enriched either in xylan/xylose or in furfural and acetic acid (derived from the acetyl units of hemicellulose in beech wood, playing also the role of the in situ formed “acidic catalyst” which enhanced hydrolytic and dehydration reactions). At the same time, the degree of enzymatic hydrolysis of the remaining solid biomass (cellulose plus lignin) was significantly improved, reaching as high as 70 % conversion of cellulose without any additional processing. The hydrothermal method has been used for the pretreatment of a variety of raw materials including agricultural biomass such as wheat straw15, 16, barley straw17, sugarcane baggase18, 19, shorghum baggase20, corn stover21 and olive tree pruning16, energy crops such as switchgrass21, 22

, and forestry biomass such as eucalyptus 16, 23, poplar24, beech wood11. Apart from the main

target of promoting the enzymatic or chemocatalytic hydrolysis of the remaining cellulose-rich biomass towards ethanol production, the hydrothermal pretreatment of biomass has been also used for the enhancement of biomethane production25. The aim of the current work was to evaluate the hydrothermal pre-treatment of three biomass types, namely pine wood sawdust, residual poplar branches from logging operations and grapevine pruning. The first one is a representative softwood species while the latter two are hardwoods. Grapevine pruning is an agricultural waste/by-product without much attention up to date. Unmerchantable poplar branches and grapevine pruning are relatively untapped biomass resources and a low cost – less demanding valorization process, including the hydrothermal pretreatment in neat water would be highly desirable. In this direction, the potential of using such a mild and simple method for pine pretreatment is rather scarce in the literature. A systematic investigation of the time and temperature regimes of the hydrothermal pretreatment of the three biomass types was performed. The effect of these pretreatment parameters on the chemical and physicochemical characteristics of the pretreated biomass as well as the chemical composition of the liquid fractions is reported. Efficient removal of 4 ACS Paragon Plus Environment

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hemicellulose from all biomass species is possible under the reported conditions with good recovery yields of hemicellulosic oligosaccharides in the liquid products. Formation of degradation products is limited, while the effects of pretreatment on enzymatic hydrolysis, range from poor to very good depending on biomass source type. Results and Discussion Solubilization of biomass The biomass samples from poplar branches, grapevine pruning and pine sawdust were analyzed for the determination of their structural components, i.e., cellulose (determined as glucan), hemicellulose (determined as xylan, galactan, manan, arabinan and acetyl groups) and lignin (determined as acid-insoluble and -soluble lignin), as well as their non-structural constituents (i.e., ash and extractives). The respective results can be seen in Table 1. Table 1 The composition varies depending on the type of biomass with cellulose concentration ranging from 34 to 40 wt.%, hemicellulose from 18 to 23 wt.% and lignin from 15 to 25 wt.%. The content of ash is low (ca. 0.1 - 3 wt.%) in contrast to the high amount of extractives which reaches 17-19 wt.% for all three biomass types. It is also worth mentioning the relatively low acetyl content of pine biomass (at only 1 wt.%) which is typical for all softwoods. The hydrothermal pretreatment experiments were organized in two sets, aiming to investigate separately the effect of process temperature and time. The experimental conditions and the respective samples prepared are shown in Table 2. The first set was conducted at constant temperature of 170oC by varying systematically the treatment time (15 – 180 min), while in the second set, the time was kept constant (15 min) and the temperature was progressively increased (170 – 220oC). The severity factor, log Ro, which is an expression of the combined effect of temperature and time, was used to quantify the intensity of the process (see the Experimental Section for more details). 5 ACS Paragon Plus Environment


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Table 2 The degree of solubilisation at the above experimental conditions (expressed as log Ro) for the three types of biomass can be seen in Figure 1. Figure 1 A good correlation between solubilization and log Ro is observed for both sets of experiments, with the experiments performed at 15 min reaction time (at temperatures of 170 to 220oC) showing slightly higher solubilization compared to the ones performed at 170 oC (for 15 to 180 min) at similar log Ro values. This means that for the same hydrothermal treatment severity, the use of higher temperature (and short time) favors biomass dissolution slightly more compared to when longer time (and low temperature) is used. Solubilization was higher for grapevine pruning ranging from 28% at log Ro 3.3 (170 oC, 15 min), up to 48% for log Ro > 4 (15 min & ≥200 oC or 170oC & ≥ 90 min). Comparable, but at lower values, was the behavior of poplar pruning, with solubilization beginning from 23.5 % at log Ro 3.3 and reaching almost 40% at log Ro 4.1 and above. Pine wood sawdust proved to be the most resistant to hydrothermal pretreatment, between the three types of biomass, with solubilisation ranging from 19% at log Ro 3.3 up to 34% at log Ro 4.7 (220oC, 15 min), the harshest conditions applied in our work. It should be noted, that the solubilisation of the biomass samples, especially at the more intense hydrothermal conditions, is attributed to the dissolution of hemicellulose, extractives and ash, as well as of small portions of cellulose and lignin, as is discussed below. The effect of hydrothermal treatment severity (log Ro) on the composition of biomass with regard to its three main structural components (cellulose, hemicellulose, and lignin) can be seen in Figure 2. These results are presented as “normalized wt.% content” (i.e., the content of each component in the treated biomass samples expressed as percentage of its content in the parent biomass). Cellulose and lignin are more or less unaffected from the hydrothermal pretreatment exhibiting less than ca. 6% decrease, while the hemicellulose content is reduced with the 6 ACS Paragon Plus Environment

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increase of log Ro and at 220 oC/15 min (log Ro, 4.7) almost no hemicellulose remained in all three types of biomass. Figure 2

Physicochemical characteristics of hydrothermally treated biomass The X-ray diffraction (XRD) patterns of the parent and hydrothermally treated biomass samples (not shown for brevity) were used to determine the respective crystallinity index values, which are listed in Table 3. As the severity of the treatment increases, leading to almost complete removal of hemicellulose, the crystallinity index also increases due to higher concentration of the crystalline cellulose in the treated solids. For poplar and grapevine pruning the more intense pretreatment (220 οC, 15 min) leads to an increase in crystallinity from 72 and 70% (parent biomass) to 83 and 86%, respectively, while for pine the increase is from 77% to 85%. One of the main targets of pretreatment, especially when a down-stream enzymatic hydrolysis is foreseen, is to increase the available surface and pore volume of biomass, thus increasing the accessibility of cellulose to enzymes. As can be seen from the data in Table 3, both the specific surface area (SSA) and total pore volume increase progressively with increasing treatment severity for all three types of biomass. N2 sorption at -196oC is usually applied for the study of micro-/mesoporous catalysts and adsorbents with high surface areas and pore volumes, such as zeolites or activated carbons; however, it can still identify small but systematic changes in the surface area and pore volume induced in the hydrothermally treated biomass samples relative to the parent biomass 11. A gentle outgassing of the biomass samples is however required in order not to significantly affect their textural properties; i.e. in the present study outgassing prior to N2 sorption was performed at 90oC under vacuum. The SSA for poplar and grapevine raises from 0.38 to 2.06 m2g-1 and from 0.48 to 4.55 m2g-1, which account for a 7 ACS Paragon Plus Environment


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5.4- and 9.5-fold increase, respectively. On the other hand, the more severely treated pine sample exhibits a SSA of 0.96 m2g-1, which is lower compared to those of the corresponding poplar and grapevine samples, but still significantly higher compared to the SSA of the parent pine which is negligible. Similar trends can be also observed in the total pore volume of the biomass samples, showing a 3 to 9-fold increase for the more severely treated samples. Table 3 The effect of hydrothermal treatment on the morphology and physical characteristics of the biomass samples can be seen in the photographs and SEM images of Figure 3. A progressive darkening of the light brown colour of the biomass particles can be clearly observed, especially for the poplar and vine biomass. This is a typical effect of the hydrothermal treatment and has been also previously shown for beech wood11, etc. It can be attributed to the partial solubilization and depolymerization of lignin and its recondensation on the external surface of the biomass particles, as well as to caramelization of released sugars. Additionally, it can be related to the formation of aromatic polycondensates, named “pseudo-lignin”, which originate from the repolymerization of polysaccharides degradation products and/or polymerization with lignin26, 27. The deposition of the recondensed lignin or pseudo-lignin in the form of nearly spherical droplets on the surface of biomass particles can be clearly seen in the SEM images of Fig. 3, especially in the case of poplar and vine, in accordance with previous studies 11, 28, 29 . A slight change/destruction of the microstructure of biomass fibrils can be also identified by the SEM images. Figure 3 The deposition of these condensates is relatively heavy for poplar (Fig. 3A) and moderate in the case of vine (Fig. 3B) for the samples pretreated at the highest severity (220 οC, 15 min). In the case of pine, however, (Fig. 3C) it is evident that the biomass surface remains free of droplet formation even at the most severe pretreatment conditions. This is indicative of the more 8 ACS Paragon Plus Environment

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resistant nature of softwood lignin compared to the lignin of hardwood species due to the difference in its composition. Softwood lignins are mostly made up of guaiacyl units (one methoxyl group in the phenolic ring) whereas hardwood lignins contain both guaiacyl and syringyl groups (two methoxyl groups in the phenolic ring). Hardwood lignins, therefore, which contain more methoxyl groups substituted aromatic rings are less condensed and therefore more amenable to hydrothermal treatment 30. However, despite the absence of droplets on the surface of hydrothermally treated pine samples, the darker colour of the particles compared to the parent ones, may be attributed to the relocation of small portion of lignin on the outer surface of the particles as well as to the formation of sugar caramelization products or pseudo-lignin, as discussed above.

Chemical composition of hydrothermal pretreatment liquids - sugars As has already been shown in Figure 2 hemicellulose is the structural carbohydrate that is primarily removed from the biomass during hydrothermal pretreatment, while cellulose was affected to a much lesser extent. Thus, the main sugars expected to be found in hydrothermal pretreatment liquids from the solubilisation and partial hydrolysis of the two hardwoods (poplar and grapevine) were xylose and xylo-oligosaccahrides (with minor contribution of galactose, mannose and arabinose), whereas for softwood/pine they were galactose, mannose and galactomannan oligosaccharides (with minor contribution of arabinose). In the case of poplar, at low severities of log Ro 3.3 (170 oC, 15 min) the concentration of total xylose (i.e. xylose monomer plus xylo-oligomers) is relatively low at 1.73 mg/ml (Table 4). A maximum value of ~6.5 mg/ml is achieved at log Ro ~ 3.8 (170 oC, 60 min or 190 oC, 15 min) and is subsequently reduced down to 0.36 mg/ml at the highest severity of log Ro 4.69 (220 oC, 15 min). The concentration of monomeric xylose follows a similar trend, reaching a maximum value of 1.55 mg/ml at log Ro 4.31 (170oC, 180 min) followed by reduction at higher 9 ACS Paragon Plus Environment


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severities (i.e. 0.23 mg/ml, at log Ro 4.7, 220oC, 15min). A smooth exponential decrease of the oligomeric-to-monomeric xylose ratio versus the severity factor log Ro exists, as can be seen in Figure S2 in the Supporting Information. The significant decrease of total xylose, as well as of monomeric xylose, at the harshest conditions is attributed to the enhanced hydrolysis of xylooligomers towards momomeric xylose and the subsequent transformation/degradation of xylose to furfural and formic acid, as is described below and has been also previously shown for beech wood derived xylose 11. In the grapevine experiments total xylose is measured at 2.59 mg/ml at log Ro 3.3, with a maximum of 6.13 mg/ml achieved at log Ro of 4.01 (170 oC, 90 min), followed by reduction to a minimum value of 0.92 mg/ml at the highest log Ro of 4.69. Monomeric xylose concentration is also severity dependent as already seen with poplar; it is initially 0.56 mg/ml at log Ro 3.33 (170 oC, 15min), reaches a maximum of 2.99 mg/ml at log Ro 4.14 (170 oC, 120 min), and is subsequently reduced down to 0.67 mg/ml at the highest severity. Finally, for pine biomass a significant differentiation in the composition of the hydrothermal liquids is observed, compared to poplar and grapevine biomass, due to the different nature and composition of hemicellulose between hardwoods and softwoods, as discussed above (Table 1). For example the concentration of total mannose/galactose sugars (expressed as mannose in Table 4) is considerably high (5mg/ml) at low log Ro values of 3.3 (170oC, 15 min) and it continues to increase with log Ro and reaches its maximum values, 7.67 mg/ml and 7.77 mg/ml, at log Ro 4.01 (170oC, 90 min) and 4.14 (170oC, 120 min), respectively. Following that it starts to decrease, but its concentration remains relatively high at 2.21 mg/ml even at the maximum severity of log Ro 4.69. With regard to the monomeric mannose/galactose, they show a similar trend, reaching a maximum concentration of 5.0 mg/ml at log Ro 4.31 (170oC, 180 min), followed by reduction to a minimum value of 2.05 mg/ml at the highest severity but still higher than the respective values observed for poplar and grapevine. This differentiation between the 10 ACS Paragon Plus Environment

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hardwood and softwood biomass, may reflect the higher resistance of softwood hemicelluloses to hydrothermal treatment30, which can offer an additional pool of C6 monomeric sugars for down-stream upgrading to bioethanol, HMF, levulinic acid, etc. The smooth correlation between the oligomeric-to-monomeric xylose ratio and the severity factor log Ro observed for poplar, has been also evidenced for grapevine and pine biomass as well (Fig. SI-2). It is however, important to notice that relatively high amounts of glucose (monomeric plus oligomeric) are also present in these hydrothermal treatment liquids for all three biomass types, and at all severity (log Ro) values. Maximum glucose concentration for poplar (3.52 mg/ml), grapevine (7.68 mg/ml) and pine (2.71 mg/ml) were observed at severity values of 4.01, 4.01 and 4.31, respectively (Table 4). Usually the presence of glucose in biomass treatment products is due to the hydrolysis of cellulose, as it is the main glucose containing carbohydrate in the biomass. However, as can be seen in Fig 2, and in accordance with previous reports 11, 31, 32, the hydrothermal pretreatment in pure water is not severe enough to cause cellulose dissolution and hydrolysis, at least to that extent. Thus, in addition to the contribution of the limited cellulose hydrolysis, glucose can also originate from the extractives of the raw biomass. It is apparent from the data in Table 1 that all three types of biomass contain a significant amount, of nonstructural components (extractives) which amount to 18.9 % for poplar, 16.8 % for grapevine and 18.3% for pine. The analysis of the liquid samples from the Soxhlet extraction for the determination of the water extractives, showed glucose concentrations of 0.42 mg/ml for poplar, 1.35 mg/ml for grapevine and 0.35 mg/ml for pine. Analysis after acid hydrolysis of these samples (for converting gluco-oligomers to monomeric glucose) revealed even greater glucose concentrations of 1.34 mg/ml, 2.94 mg/ml, and 0.92 mg/ml, respectively. It is therefore apparent that glucose is present in the extractable fraction of the biomass both as free glucose as well as gluco-oligosaccharides, that can be present in the living plants

33

. The poplar and grapevine

biomass samples used in this work were collected immediately after forestry logging or 11 ACS Paragon Plus Environment


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grapevine pruning, were chopped in the knife mill as is (wet) without prior debarking, and were then dried under IR lamps. Hardwood bark may contain a large amount of water extractives34 which can account for the increase in total extractives content observed for these two samples. In addition, bark can also contain relatively high amounts of easily extractable sugars, either in easily hydrolysable or monomeric form34 , which can account for the increased glucose content observed in the pretreatment liquids. Therefore, it is most possible that all the aforementioned carbohydrates could have remained in the biomass, and were released during Soxhlet extraction as well as during the hydrothermal pretreatment experiments. In the case of pine wood, hemicellulose could also be considered as a source of glucose since glucomannans and galactoglucomannans, that contain glucose, are typical hemicellulose components in softwoods 35, 36

. Estimation of the percent of glucose originating from extractives and that derived from

cellulose in the hydrothermal treatment liquids is difficult, as a portion of glucose is simultaneously converted to degradation products (HMF, levulinic acid, etc.), depending on the severity of the HT treatment. Thus, at higher severities, glucose originating from extractives may have been more readily converted to HMF, while glucose originating from the slower cellulose hydrolysis may have predominantly remained intact in the process liquid.

Chemical composition of hydrothermal pretreatment liquids – degradation products As was briefly mentioned above, the relatively intense hydrothermal treatment conditions lead to the transformation of sugars to other chemicals, also referred to as degradation products. The main degradation products of the biomass components can be classified into two categories; acids and furans. The main acids detected in the hydrothermal treatment liquids were acetic acid, formic acid and levulinic acid, whereas the furans were furfural and hydroxymethylfurfural (HMF). The concentration of acetic acid depends on the acetylation level of hemicellulose and on the treatment severity, mainly related to the 12 ACS Paragon Plus Environment

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temperature of the process. As has been previously reported14 and discussed also in our previous work on hydrothermal treatment of beech wood

11

, the initial hydrolysis of hemicellulose is

attributed to the mild acidic conditions due to the formation of water oxonium ions (hydroniums, H3O+) that can be generated at the relatively mild subcritical water conditions applied (i.e. temperatures 170-220oC). Thus, the more intense conditions induce enhanced hydrolysis of hemicellulose and of the released acetyl units towards acetic acid in the aqueous medium. Therefore it is logical that the biomass with the highest level of hemicellulose acetylation should have the highest acetic acid concentration present in the hydrothermal pretreatment liquids. This is confirmed by the chemical analysis of the liquid products as the grapevine biomass with the highest acetyl content of 3% (Table 1) showed the highest concentration of acetic acid of 2.8 mg/ml at log Ro 4.69 (Figure 4). An analogously higher value of ca. 3.4 mg/ml has been determined in the hydrothermal treatment liquid of beech wood (Lignocel HBS) for the same log Ro value, considering that the acetyl content of Lignocel was 3.8 wt.% 11 . For poplar with 2.13% acetyl content, an acetic acid concentration of 2 mg/ml was reached in the liquid products, whereas, in pine with only 1% acetyl content, only 0.9 mg/ml of acetic acid was observed at the highest log Ro value. For all the types of biomass, an increase in the concentration of acetic acid with the increase of log Ro is observed, as explained above. At the highest pretreatment severity conditions (log Ro =4.69), around 90% of acetyl groups of grapevine biomass were recovered in the pretreatment liquid as acetic acid, while the corresponding percentages for poplar and pine were 83% and 84%, respectively. Figure 4 The other two acids detected in the hydrothermal pretreatment liquids are formic and levulinic acid (Figure 4). These are furan degradation products; formic acid is produced from both furfural and hydroxymethylfurfural (HMF), whereas levulinic acid is produced from HMF only. Furfural and HMF are formed via dehydration of xylose (or arabinose) and glucose (or 13 ACS Paragon Plus Environment


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galactose and mannose), respectively, under the relatively mild acidic conditions of the hydrothermal treatment induced by hydroniums and the progressively released acetic acid. The pH values of the liquid products at room temperature were measured between 4.5 (for log Ro = 3.3) and 2.5 (for log Ro = 4.69), accounting for all the above mentioned acids. The overall reactions of sugar transformation to the respective acids are schematically shown in Figure 5. The concentration of formic acid for all three types of biomass, increases from around 0.2 mg/ml to about 1 mg/ml for severity factor (log Ro) values of 3.3 and 4.69, respectively (Fig. 4). The concentration of levulinic acid in the poplar hydrothermal pretreatment liquids remains relatively low for the whole range of log Ro (0.05 to 0.3 mg/ml), while it is slightly higher for grapevine and pine, i.e. reaches 0.9 mg/ml and 1.4 mg/ml, respectively, at the highest severity (log Ro = 4.69). Figure 5 Furfural which is a dehydration product of the C5 sugars of hemicellulose (xylose, arabinose), has similar concentrations for all three biomass types of about 0.2 mg/ml at the mildest hydrothermal treatment conditions, i.e. log Ro of 3.3 (Figure 6). Its concentration increases with log Ro and then stabilizes at the higher log Ro values. This is attributed to the conversion of furfural to formic acid which is favoured at the more severe conditions via acid hydrolysis, as depicted in Fig. 5, and is in accordance with the observed steep increase of the concentration of formic acid at the highest log Ro values (Fig. 4). The formation of furfural in the liquid products of poplar and grapevine is attributed mainly to xylose since their hemicellulose comprises mainly of xylan, while in the case of pine, arabinose (from arabinan) could be the only source of furfural. Hydroxymethylfurfural (HMF), which is the dehydration product of the C6 sugars (glucose, mannose and galactose) remains relatively low in the case of poplar, with a concentration of ~ 0.2 mg/ml for all pretreatment severities (Figure 6). This result justifies the similarly low concentration of levulinic acid (Fig. 4), as this acid is produced 14 ACS Paragon Plus Environment

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from acid hydrolysis of HMF (Fig. 5). In the grapevine experiments a small increase of HMF can be observed from 0.3 mg/ml at log Ro 3.3 to 0.52 mg/ml at log Ro 4.69. The formation of HMF in the liquids of poplar and grapevine treatment is attributed to galactose and mannose existing in their hemicellulose as well as to the glucose originating from the extractives, as discussed above. Finally in the pine experiments the HMF concentration reaches a higher value of 0.71 mg/ml at log Ro 4.69, probably due to the high content of C6 sugars, i.e. mannose and galactose, in pine hemicellulose, as discussed earlier. The higher HMF concentration in the hydrothermal pretreatment liquids of pine can explain also the higher levulinic acid concentration observed with this type of wood (Fig. 4). Figure 6

Tuning the hydrothermal treatment severity for maximum recovery of hemicellulose sugars Adjustment of the pretreatment parameters (i.e., selection of the appropriate time and temperature combination, expressed by the severity factor log Ro) allows to control the composition of the obtained liquid and solid products. The effect of the log Ro value on the removal of hemicellulose (xylan) from the hardwood biomass (poplar and grapevine) and its recovery in the liquid products of the hydrothermal pretreatment as monomeric or oligomeric xylose can be seen in Figures 7A & B, respectively. Figure 7 For the poplar hydrothermal treatment experiments (Figure 7A) at the mild conditions of log Ro 3.3 about 60% of the total xylan remained in the solid biomass while 40% has been solubilized. For the 15 min series of experiments (with increasing temperature), the removal of xylan is increased with increased log Ro values and at the harshest conditions of log Ro 4.69 almost no xylan is present in the solid biomass. On the other hand, in the 170oC series of experiments (with increasing time), removal of xylan reaches a limit in the order of 80% (with 15 ACS Paragon Plus Environment


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around 20% remaining in the biomass) which is in accordance with our previous results regarding the hydrothermal treatment of another hardwood, i.e. beech wood (Lignocel) 11. This behaviour has been also previously identified and is attributed to the existence of a fraction of hemicellulose that is more difficult to remove. Kinetic studies in hydrothermal treatment experiments have shown the existence of a threshold of about 75–85% hemicellulose removal from different types of lignocellulosic biomass for temperatures up to ~170oC and relatively prolonged reaction times14, 37-41 . Similar behaviour with regard to the effect of hydrothermal treatment conditions on hemicellulose solubilisation is observed in the experiments with grapevine biomass (Figure 7B), where the hard to remove xylan was around 18%. For pine biomass (Figure 7C), such a differentiation in the removal of hemicellulose (which in this case refers to mannan and galactan) between constant time and constant temperature experiments is not observed. The removal of galactomannan reaches a maximum of 90% at higher log Ro values with ~ 10% remaining in the solid biomass. Xylan recovery for poplar and grapevine and mannan/galactan recovery for pine in the process liquids reaches a maximum of around 60% (Fig. 7). This is observed at log Ro 3.8 for poplar, between log Ro 3.8 and 4.01 for grapevine and between 3.8 and 4.14 for pine. The sum of xylan remaining in the biomass and of xylan recovered in the process liquids, at these log Ro values is around 90% for poplar and around 80-90% for grapevine and pine (in the case of pine these values refer to mannan and galactan). Increased treatment severities with log Ro > 4.1 (170oC, 180 min or 210-220oC, 15 min) leads to steep decrease of xylan and mannan/galactan in the liquid products due to their transformation to furans and acids, as described above (Fig. 5). However, the increase of all these compounds, i.e. formic and levulinic acid (Fig. 4) and furfural and HMF (Fig. 6) is not analogously high, possibly due to the formation of humins (carbonaceous solids formed upon polymerization of furans or of furans with the parent sugars) or formation of gaseous products (not measured) such as CO and CO2. 16 ACS Paragon Plus Environment

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Another parameter of the hydrothermal pretreatment that may have a considerable effect is the heat-up time. For example, in the case of pine biomass, when pretreated at 190oC for 0 min (experiment stopped when target temperature was reached) the concentration of free mannose plus galactose in the pretreatment liquid was ~0.6 mg/ml; the concentration of these hemicellulose sugars increased to 1.6 mg/ml after accounting for the 15 min isothermal treatment at 190oC, due to more extensive hydrolysis. Concentrations of other sugars such as glucose and arabinose remained relatively stable. A different behaviour was observed when the pine biomass was pretreated at 220oC. The mannose/galactose released at 0 min of pretreatment time was as high as 3.1 mg/ml and was reduced to 2 mg/ml after 15 min of isothermal pretreatment at 220oC. The more severe conditions (higher temperature and longer time to reach targeted temperature) allowed for a greater hemicellulose removal at time zero, while at the same time, the relatively high isothermal treatment temperature favoured reactions of sugars transformation into degradation products such as HMF, levulinic acid, etc. Similar effects were observed for the other types of biomass at similar conditions (not reported for brevity). Coolingtime effects in our study were minimal due to the immediate quenching of the reactor after the isothermal treatment. Poplar, among the three biomass types studied in this work, has been more systematically investigated up to date since it represents an important candidate tree species for energy cultures. In previously reported studies on hydrothermal pretreatment of poplar

42

, the

maximum total recovery of hemicellulose in the liquid was around 65% (with 25% remaining in the solid biomass) achieved at a log Ro value of 3.84 (210 oC, 4 min). In the steam explosion pretreatment of poplar

43

, the maximum yield of pentosans (monomeric and oligomeric C5

sugars) was achieved at log Ro 3.8 (220 oC, 2 min) and corresponded to 60% recovery in the liquid. These results are in good agreement with the results of the present study for poplar pretreatment where a total xylan recovery of 61.5% in the liquid has been achieved at log Ro 17 ACS Paragon Plus Environment


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3.82 (190 oC, 15 min). Similar literature reports are, to the best of our knowledge, scarce in the case of grapevine pruning. In one of the few available studies, the hydrothermal pretreatment has been used for the extraction of hemicellulosic sugars that were then hydrolysed by acid and used for the production of lactic acid with microbial fermentation

44

. Due to the more

recalcitrant nature of pine (and softwoods in general) its hydrothermal pretreatment in neat water has not been a subject of systematic research in the open literature. In order to improve the enzymatic saccharification of the more recalcitrant softwood derived biomass, more severe pretreatment methods are required. For this purpose the pretreatment of various softwood biomass samples, such as lodgepole pine and spruce has been studied with methods including dilute sulfuric acid hydrolysis45 and steam explosion without the use of additional catalysts 46 as well as the use of sulfuric acid or SO2

47-50

as acidic catalysts to enhance the hydrolytic

removal of hemicellulose from the biomass. The curves in Figure 8A & B present the correlation between the concentration of total xylose (including monomeric and oligomeric xylose) and that of its main degradation product, furfural, in the liquid products as a function of hemicellulose removal, for the two hardwoods (i.e. poplar and grapevine). For poplar it can be seen that a maximum xylose concentration of 6.5 mg/ml was achieved at around 70% removal of hemicellulose, and the concentration of furfural remained relatively low at ca. 0.4 mg/ml. The maximum concentration of xylose measured in grapevine was 6.1 mg/ml and was observed at 84% hemicellulose removal, corresponding also to a relatively low furfural concentration of 0.5 mg/ml. In the case of pine (Fig. 8C), the maximum concentration of total mannose and galactose (including monomeric and oligomeric mannose and galactose) concentration obtained was 7.8 mg/ml at about 80% hemicellulose removal. The concentration of HMF, being the main degradation product of C6 sugars such as glucose, mannose and galactose, was kept at relatively low levels of 0.65 mg/ml. Furfural (not shown in Fig. 8C) in the hydrothermal liquid products of pine was also at low 18 ACS Paragon Plus Environment

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levels (ca. 0.6 mg/ml) and its formation is attributed to the small amounts of arabinose included in the hemicellulose fraction of pine. Figure 8 This comparative presentation illustrates the capability of the hydrothermal pretreatment in neat water to induce high recoveries of xylose (for poplar and grapevine) and mannose/galactose (for pine) in the form of monomeric sugars and soluble oligo-saccharides, at high percentages of hemicellulose removal, of ca. 70-85%, keeping the concentration of their primary degradation products furfural and HMF at relatively low levels. This can be achieved by applying hydrothermal treatment conditions of moderate severity with log Ro values of ca. 3.8 – 4.1, which correspond to 170oC and 60-120 min, or 15 min and 190-200oC. At these conditions, the secondary degradation products of sugars, i.e. formic and levulinic acid, remain also at low levels of below 0.6 mg/ml. A hydrothermal treatment liquid with the above composition could be utilized in fermentation processes for the production of ethanol/butanol or other chemicals (i.e. lactic acid) or for the selective chemo-catalytic conversion of the C5 and C6 sugars to a series of platform chemicals (i.e. furfural, HMF, levulinic acid) and final products, such as fuel additives or plastics. Although the valorization of these liquid hemicellulose streams would increase the economic viability of the biochemical production of ethanol/butanol, still, the main target of the hydrothermal pretreatment is the improvement of the enzymatic hydrolysis of the treated biomass.

Enzymatic hydrolysis of hydrothermally pretreated biomass The effect of hydrothermal pretreatment of poplar biomass on the maximum enzymatic hydrolysis of its cellulose can be seen in Fig. 9A. Untreated poplar biomass gives an enzymatic hydrolysis of only 9%. This is improved at all pretreatment severities with the highest digestibility of 49% reached at the highest pretreatment severity of 220 oC, 15min (log Ro = 19 ACS Paragon Plus Environment


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4.69). The inset in Fig. 9A also shows that digestibility is well correlated with log Ro while the pretreatment protocol of short time (15 min) and increasing temperature (170-220oC) seems to slightly favor enzymatic hydrolysis compared to the protocol of low temperature (170oC) and increasing time (15-180 min). The relatively moderate enzymatic hydrolysis observed in our study can be attributed to the presence of significant amounts of microspheres on the surface of the

severely

pretreated

samples

(Fig.

3A),

which

could

be

associated

with

depolymerized/recondensed lignin deposits, as well as pseudo-lignin and sugar caramelization products, as discussed above. In our previous work, it was also shown that the enzymatic hydrolysis of hydrothermally pretreated beech wood decreased from 67% after relatively moderate pretreatment conditions (log Ro = 3.8) to 31% at the highest severity applied (log Ro = 4.69) 11. This behavior was attributed to the dense lignin layer deposited on the surface of the beechwood particles after being treated at this high severity. In addition to the recondensed lignin, pseudo-lignin and caramel deposits, the relatively moderate enzymatic hydrolysis of the treated poplar can be also related to the presence of bark in the parent biomass samples. Poplar bark contains phenolics, such as phenolic glycosides and tannins 51. The inhibition of enzymatic hydrolysis of biomass by phenolic glycosides 52 and tannins 53, among others, has been shown in previous investigations. Thus, it is possible that the presence of these phenolics in the treated poplar biomass due to insufficient removal of bark under the applied hydrothermal treatment, could lead to reduced enzymatic hydrolysis of cellulose. In the case of grapevine biomass, a more pronounced positive effect of hydrothermal pretreatment on enzymatic hydrolysis was observed, as can be seen in Fig 9B. Initially digestibility of untreated grapevine biomass was relatively low (12%) but it was substantially increased to 77% for the more severely treated sample (at 220 oC, 15min; log Ro = 4.69). There is a good correlation of enzymatic hydrolysis with the log Ro values (inset of Fig. 9B), showing

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also that the pretreatment protocol of short time (15 min) and increasing temperature (170220oC) induces slightly higher digestibility as in the case of poplar. Figure 9 In contrast to poplar, the presence of the lignin or sugar derived microspheres on the surface of hydrothermally pretreated grapevine particles do not seem to inhibit the enzymatic hydrolysis, possibly due to the less intense deposition compared to poplar, as can be seen in Fig. 3A & B. Although, the formation of condensation products on the outer surface of biomass and the presence of phenolics originating from the remaining bark, can influence the effectiveness of enzymes, other physicochemical properties of the treated biomass could play a more decisive role on cellulose digestibility. The hydrothermal pretreatment in neat water at temperatures below ca. 220oC does not affect/decrease the crystallinity of cellulose but has certainly an effect on the morphology and texture of the biomass particles, as can be realized from the SEM images of Fig. 3 and the porosity data in Table 3, respectively. As discussed above, the surface area of treated grapevine biomass exhibits a 9-fold increase compared to the parent biomass, while a 5-fold increase has been found for the similarly treated poplar samples. Such a more “open” biomass particle morphology would enhance the accessibility of enzymes and favor interaction with cellulose, leading to increased digestibility. For pine biomass, the hydrothermal pretreatment had almost no effect on enzymatic hydrolysis. A marginal increase of digestibility from 10 (untreated pine) to 16% for the sample treated at the highest severity of log Ro 4.69 was observed (Fig. 9C). Despite the extensive removal of hemicellulose at these intense conditions (Fig. 2), it can be suggested on the basis of the SEM images (Fig. 3C) and the porosity data (Table 3), that the morphology and textural characteristics of the parent pine biomass were not significantly affected, at least to the point of inducing a noticeable improvement in enzymatic hydrolysis of cellulose. In addition, it seems that lignin in pine is more resistant to hydrothermal pretreatment, at least for the conditions 21 ACS Paragon Plus Environment


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applied in this work. Both the hemicellulose and - mainly - the lignin of softwoods are more resistant compared to those of hardwoods 30. This is the reason why, in the literature more severe pretreatment approaches such as steam explosion, dilute acids and organosolv have been preferred for pine and other softwoods. Nevertheless, the hydrothermal pretreatment applied in this work was tuned to the point of recovering a relatively clean hemicellulose fraction enriched in the respective carbohydrates and with minimum formation of degradation products. In order to assess the effectiveness of the hydrothermal pretreatment regarding the fate of sugars, the content of glucose in the parent biomass samples (in extractives and in cellulose) and in the pretreatment and enzymatic hydrolysis liquid products has been calculated as grams per 100 gram of parent biomass (Table 5). Based on these data, the % recovery of glucose has been also determined. Two sets of conditions for the three biomass feeds have been selected, one being representative of the relatively moderate conditions favouring higher sugar yields in the pretreatment liquids and one of the more severe conditions favouring mainly enzymatic hydrolysis towards glucose as well as transformation of sugar in the pretreatment liquids towards their respective degradation products. As can be seen from Table 5, at the relatively moderate conditions ≥ 90 % of the glucose present in extractives can be recovered in the hydrothermal pretreamtent liquids for all three biomass samples. However, the effect on the enzymatic hydrolysis of cellulose was not that pronounced as only up to 40% of the glucose present in cellulose of the parent grapevine biomass could be recovered in the enzymatic hydrolysate. The reverse situation was observed at the more severe hydrothermal conditions, where about 74% of glucose present in cellulose of parent grapevine was recovered after enzymatic hydrolysis. The recovery of glucose in the pretreatment liquids at these conditions was lower, due to its transformation to HMF and levulinic acid, as it was discussed above. Table 5


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Conclusions The hydrothermal pretreatment was studied for two types of hardwood, i.e. poplar branches from logging operations and grapevine pruning, and one softwood, i.e. pine sawdust. The range of conditions applied was 170 to 220 oC for 15 min or 170 oC for 15 to 180 min, corresponding to severity factor log Ro values from 3.3 to 4.7. A good correlation of the studied parameters, i.e. biomass solubilisation, recovery of hemicellulose, enzymatic hydrolysis, with log Ro was observed for all three types of biomass and for both sets of experiments. Maximum biomass solubilization achieved was 40 wt.% for poplar branches, 48 wt.% for grapevine pruning, and 35% for pine sawdust, and was mainly attributed to removal of hemicellulose and extractives, and to a much smaller extent to cellulose, lignin and ash dissolution. Maximum and almost complete removal of hemicellulose was achieved for all biomass types at the harshest conditions applied (log Ro 4.7). However, maximum xylan recovery of about 60 % in the aqueous liquid product was achieved at moderate severity of log Ro 3.8 and 3.8-4.0 for poplar branches and grapevine pruning, respectively, For pine, due to the different hemicellulose composition, mannan and galactan were the carbohydrates mostly recovered in the liquids with maximum recovery of 60% at log Ro 3.8-4.14. The above recovery values corresponded to 7085% removal of hemicellulose. Further increase of pretreatment severity for all types of biomass, resulted in decreased hemicellulose recoveries in the liquid fractions with a simultaneous increase of the major degradation products such as acids (acetic, formic, levulinic) and furans (furfural and HMF). The effect of hydrothermal pretreatment on the morphological and porous characteristics of biomass was also profound. An increase of specific surface area and pore volume, as well as deposition of microspheres, possibly associated with depolymerized/recondensed lignin and sugar-derived pseudo-lignin and caramelization products, on the particles surface was observed for poplar and grapevine biomass. For pine, the changes in the physicochemical properties were 23 ACS Paragon Plus Environment


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modest and the surface condensation products could not be observed as clearly as for the other two biomass types. The effect of hydrothermal pretreatment on the enzymatic hydrolysis of the biomass samples was in accordance with all the physical and chemical characterization data. The least improvement was observed for pine experiments with only 16% enzymatic hydrolysis achieved at maximum severity (log Ro 4.7). This is more or less expected and can be attributed to the more recalcitrant nature of softwood, compared to hardwoods, even after substantial removal of hemicellulose. Poplar pretreated at the same highest severity provided a cellulose hydrolysis of 49%, while the best result achieved was with the grapevine pruning which reached about 77% enzymatic hydrolysis. The results obtained in this work provided evidence for the potential and limitations of hydrothermal pretreatment of representative hardwoods and softwood in neat water. Furthermore, the data presented can be utilized as reference case for hybrid (pre)treatment processes where the hydrothermal system could be combined with the use of an external solid or liquid catalyst. Experimental Section Biomass Feedstock Three types of lignocellulosic waste biomass were used in the current study. Residual poplar branches from logging operations, grapevine pruning and pine tree sawdust. Grapevine pruning originated from the Greek variety Vitis vinifera 'Xinomavro' that is cultivated in the Region of Central Macedonia in Northern Greece. Poplar trimmings were by-products of logging operations of 20 years old Populus deltoides trees from the area of Thessaloniki, also in the Region of Central Macedonia in Northern Greece. The pine sawdust was an industrial sample used for the manufacture of particleboards; it was Scots Pine (Pinus sylvestris) originated from Bulgaria. The poplar branches and grapevine pruning were chopped in a knife mill without any previous treatment, such as debarking. They were then dried under IR lamps for 24 hrs followed by equilibration at ambient conditions for another 24 hrs. The


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samples were then sieved and the 180-1000 μm fraction was used for the current study. The pine sawdust was already in the 180-1000 μm particle size range and was used as is.

Hydrothermal pretreatment of biomass The hydrothermal biomass pretreatment experiments were carried out in a laboratory scale autoclave batch reactor (Parr, Model 4563) with a total volume of 600 ml. The overall procedure (hydrothermal pretreatment experiments, preparation for analysis and characterization of solid and liquid samples) has been previously reported

11

and is schematically presented in Figure S1 in the

Supporting Information. In brief, biomass suspensions in deionized H2O (Liquid to Solids Ratio, LSR = 15) were heated (~7oC/min) at varying temperatures (170-220oC) under stirring (stirring speed of 150 rpm) and different reaction times (15-180 min). After the isothermal treatment, the reactor was immediately quenched in cold water. The detailed experimental conditions, as well as the Severity Factor (log Ro) which combines the effect of time and temperature of the hydrothermal pretreatment, are listed in Table 2. The severity factor log Ro is defined by the equation Ro= t*exp [(T-100)/14.75] where t is the time of the hydrothermal pretreatment in minutes and T is the temperature of the pretreatment in oC54, 55 . Only the isothermal time (at each targeted temperature) of the treatment was used for calculation of the log Ro values. The solid treated biomass was recovered by vacuum filtration, washed with deionized H2O until neutral pH of the filtrate, air dried and used for further analysis and for the enzymatic hydrolysis experiments. The liquid product was passed through 0.2μm filters and stored at -20oC for further analysis.

Chemical analysis Detailed description of all the analytical methods is provided in the Supporting Information, while a brief overview is given below. The chemical composition of the parent and the treated solid biomass samples, expressed as the content of the main structural carbohydrates and lignin, was determined according to the procedure described by NREL

56

, which is a modification of the ASTM E1758-01

“Standard method for the Determination of Carbohydrates by HPLC”. Non-structural components, i.e. ash and extractives, as well as moisture were also determined. The ash content of the parent and treated



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biomass samples was determined by a method similar to ASTM E1755-01 via gravimetric analysis of calcined biomass in air at 575oC for 12 hrs. The extractives of the parent biomass were determined by exhaustive (24 hrs) soxhlet extraction first with water and then with ethanol, followed by ethanol evaporation and gravimetric analysis of the remaining solids. The wt.% moisture of the biomass samples equilibrated at ambient conditions was determined by drying at 105oC for 6 hrs. The liquids recovered from the pretreatment experiments were analysed for monomeric sugars (glucose, xylose, galactose, mannose, and arabinose) as well as for the content of total sugars (oligomers and monomers) via hydrolysis with 4% H2SO4 for 1 h at 121oC, neutralization with CaCO3 and filtration with 0.2 μm filters. The determination of sugars was performed by high pressure liquid chromatography (HPLC, Shimadzu) using a Refractive Index (RI) detector (RID-6A) and a SP0810 Sugar Column (Shodex) with ultrapure H2O as the mobile phase. The soluble lignin in the above liquids was also determined by means of Ultra violet (UV) spectroscopy, measuring the absorbance at 205 nm and using a molecular extinction coefficient of 113 L.g-1.cm-1 as adopted from the literature57. Organic acids and furans, such as acetic acid, formic acid, levulinic acid, furfural and 5hydroxymethylfurfural (HMF) that can be also found in the liquid products of hydrothermally treated

biomass were also quantified by HPLC (Shimadzu) using a Refractive Index (RI) detector (RID-6A) and a Aminex HPX-87H organic acid column (Biorad) with 0.01N sulfuric acid as the mobile phase. The degree of biomass solubilization (expressed as wt.% on dry biomass) in the pretreatment experiments was determined by weight (W) difference, using the following equation: % solubilization = {(Winitial biomass – Wtreated biomass ) / Winitial biomass} x 100 The percent removal of carbohydrates (cellulose and hemicellulose) from the solid biomass was determined from the chemical composition analysis of solids (initial and treated biomass) and was calculated according to the equation: % removal = {(Wcarbohydrate initial biomass – Wcarbohydrate treated biomass) / Wcarbohydrate initial biomass } x 100 The percent recovery of carbohydrates (cellulose and hemicellulose) in the liquid product was determined by the content of the respective sugars (i.e. glucose for cellulose, and xylose, galactose,


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mannose, arabinose for hemicellulose) in the liquid products and was calculated according to the equation: % recovery = {Wcarbohydrate liquid samples of treated biomass / Wcarbohydrate initial biomass } x 100 Elemental analysis of the parent and treated biomass samples was also performed using a LECO800 CHN analyzer for C and H. Oxygen was calculated by difference and nitrogen was assumed negligible.

Physicochemical characterization of biomass The physicochemical characterization of the solid biomass samples was performed by Powder Xray Diffraction (XRD) for biomass crystallinity determination, N2 adsorption-desorption experiments performed at –196 oC for biomass textural properties; biomass samples have been previously outgassed at 90oC for 16 h under 5 x 10-9 Torr vacuum, and Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS) for biomass morphology investigation. Crystallinity indices (from XRD experiments) were calculated using the equation: % Crystallinity = {(I002 – Iam) / I002 } x 100 where, I002 is the intensity of the (002) peak at about 2θ = 22.5o and Iam is the intensity of the background at about 2θ = 18.3ο

11, 58

. Detailed description of all the physicochemical characterization methods is

provided in the Supporting Information.

Enzymatic hydrolysis tests The biomass enzymatic hydrolysis tests were performed following a procedure similar to that described by NREL59, using a commercial cellulase provided by Genencor (Accellerase 1500). The biomass samples containing 100mg of cellulose were added in 20ml scintillation vials together with 10ml of 50mM sodium citrate buffer (pH 4.8) and enzyme solution 60FPU/g of cellulase. The vials were incubated at 50 oC and 150 rpm in a water shaker-bath for 72 hours. The unconverted solid biomass in the samples was allowed to settle and the recovered supernatant liquid was incubated at 80 oC for 10 min to denature the suspended enzymes which were then removed via centrifugation (10 min at 10,000



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rpm). The final liquid sample was passed through 0.2 μm membranes and analyzed for sugars with HPLC as described above.

Supporting Information Detailed experimental methodology and procedures; chemical composition of liquid products – correlation of oligomeric-to-monomeric sugars ratio with the severity factor logRo.

Acknowledgements This research has been co-funded by the European Union (European Regional Development Fund) and Greek national funds through the operational program EPAN-II/ESPA 2007-2013/Action “Bilateral Cooperation Greece–Romania 2011–2012”. The authors wish to thank Genencor (Dr. Bart Koops) for kindly providing the Accelerase1500 Enzyme used in this work. The authors would also like to thank Chemist-Oenologist Mr. Antonios Kioseoglou of Kir-Yianni Estate for kindly providing the grapevine pruning samples and Mr. Christos Achelonoudis, Technical Representative and Pilot Production Manager of CHIMAR HELLAS S.A., for kindly providing the poplar branches and pine biomass.

Keywords Lignocellulosic biomass, residual poplar branches, grapevine pruning, pine sawdust, hydrothermal pretreatment, cellulose, hemicellulose, lignin, xylose, furfural, HMF, bioethanol, biofuels, enzymatic hydrolysis.

References 1. Klass, D. L. Biomass for renewable energy, fuels, and chemicals. http://www.knovel.com/knovel2/Toc.jsp?BookID=2245 2. Ellabban, O.; Abu-Rub, H.; Blaabjerg, F., Renewable energy resources: Current status, future prospects and their enabling technology. Renewable and Sustainable Energy Reviews 2014, 39, 748-764. 28 ACS Paragon Plus Environment

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3. McCormick, K., The bioeconomy and beyond: Visions and strategies. Biofuels 2014, 5, 191-193. 4. Gomez, L. D.; Steele-King, C. G.; McQueen-Mason, S. J., Sustainable liquid biofuels from biomass: the writing's on the walls. New Phytologist 2008, 178, 473-485. 5. Himmel, M. E.; Picataggio, S. K., Our Challenge is to Acquire Deeper Understanding of Biomass Recalcitrance and Conversion. In Biomass Recalcitrance, Blackwell Publishing Ltd.2009; pp 1-6. 6. Johnson, D. K.; Elander, R. T., Pretreatments for Enhanced Digestibility of Feedstocks. In Biomass Recalcitrance, Blackwell Publishing Ltd.2009; pp 436-453. 7. Kumar, P.; Barrett, D. M.; Delwiche, M. J.; Stroeve, P., Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Industrial & Engineering Chemistry Research 2009, 48, 3713-3729. 8. Bensah, E. C.; Mensah, M., Chemical pretreatment methods for the production of cellulosic ethanol: Technologies and innovations. International Journal of Chemical Engineering 2013. 9. Sathitsuksanoh, N.; George, A.; Zhang, Y. H. P., New lignocellulose pretreatments using cellulose solvents: A review. Journal of Chemical Technology and Biotechnology 2013, 88, 169-180. 10. Nitsos, C. K.; Mihailof, C. M.; Matis, K. A.; Lappas, A. A.; Triantafyllidis, K. S., Chapter 7 - The Role of Catalytic Pretreatment in Biomass Valorization Toward Fuels and Chemicals. In The Role of Catalysis for the Sustainable Production of Bio-fuels and Bio-chemicals, Michael Stöcker, K. S. T., Angelos A. Lappas Ed. Elsevier: Amsterdam, 2013; pp 217-260. 11. Nitsos, C. K.; Matis, K. A.; Triantafyllidis, K. S., Optimization of Hydrothermal Pretreatment of Lignocellulosic Biomass in the Bioethanol Production Process. ChemSusChem 2013, 6, 110-122. 12. Silva-Fernandes, T.; Duarte, L. C. C.; Carvalheiro, F.; Loureiro-Dias, M. C.; Fonseca, C.; Gírio, F., Hydrothermal pretreatment of several lignocellulosic mixtures containing wheat straw and two hardwood residues available in Southern Europe. Bioresource Technology 2015, 183, 213-220. 13. Merali, Z.; Collins, S. R.; Elliston, A.; Wilson, D. R.; Käsper, A.; Waldron, K. W., Characterization of cell wall components of wheat bran following hydrothermal pretreatment and fractionation. Biotechnology for Biofuels 2015, 8, 23. 14. Garrote, G.; Dominguez, H.; Parajo, J. C., Hydrothermal processing of lignocellulosic materials. Holz als Roh- und Werkstoff 1999, 57, 191-202. 15. Petersen, M. Ø., Optimization of hydrothermal pretreatment of wheat straw for production of bioethanol at low water consumption without addition of chemicals. Biomass & Bioenergy 2009, 33, 834-840. 16. Silva-Fernandes, T.; Duarte, L. C. C.; Carvalheiro, F.; Marques, S.; Loureiro-Dias, M. C.; Fonseca, C.; Gírio, F., Biorefining strategy for maximal monosaccharide recovery from three different feedstocks: eucalyptus residues, wheat straw and olive tree pruning. Bioresource Technology 2015, 183, 203-212. 17. Vargas, F.; Domínguez, E.; Vila, C.; Rodríguez, A.; Garrote, G., Agricultural residue valorization using a hydrothermal process for second generation bioethanol and oligosaccharides production. Bioresource Technology 2015, 191, 263-270. 18. Laser, M., A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol. Bioresource Technology 2002, 81, 33-44. 19. da Cruz, S. H.; Dien, B. S.; Nichols, N. N.; Saha, B. C.; Cotta, M. A., Hydrothermal pretreatment of sugarcane bagasse using response surface methodology improves digestibility and ethanol production by SSF. Journal of Industrial Microbiology & Biotechnology 2012, 39, 439-447. 29 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 48

20. Matsakas, L.; Christakopoulos, P., Fermentation of liquefacted hydrothermally pretreated sweet sorghum bagasse to ethanol at high-solids content. Bioresource Technology 2013, 127, 202-208. 21. Liggenstoffer, A. S.; Youssef, N. H.; Wilkins, M. R.; Elshahed, M. S., Evaluating the utility of hydrothermolysis pretreatment approaches in enhancing lignocellulosic biomass degradation by the anaerobic fungus Orpinomyces sp. strain C1A. Journal of Microbiological Methods 2014, 104, 43-48. 22. Hu, Z.; Ragauskas, A. J., Hydrothermal Pretreatment of Switchgrass. Industrial & Engineering Chemistry Research 2011, 50, 4225-4230. 23. Romaní, A., Bioethanol production from hydrothermally pretreated Eucalyptus globulus wood. Bioresource Technology 2010, 101, 8706-8712. 24. Trajano, H. L.; Pattathil, S.; Tomkins, B. A.; Tschaplinski, T. J.; Hahn, M. G.; Van Berkel, G. J.; Wyman, C. E., Xylan hydrolysis in Populus trichocarpa × P. deltoides and model substrates during hydrothermal pretreatment. Bioresource Technology 2015, 179, 202-210. 25. Qiao, W.; Yan, X.; Ye, J.; Sun, Y.; Wang, W.; Zhang, Z., Evaluation of biogas production from different biomass wastes with/without hydrothermal pretreatment. Renewable Energy 2011. 26. Pu, Y.; Hu, F.; Huang, F.; Davison, B. H.; Ragauskas, A. J., Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnology for Biofuels 2013, 6, 1-13. 27. Hu, F.; Ragauskas, A., Pretreatment and Lignocellulosic Chemistry. BioEnergy Research 2012, 5, 1043-1066. 28. Kristensen, J. B.; Thygesen, L. G.; Felby, C.; Jorgensen, H.; Elder, T., Cell-wall structural changes in wheat straw pretreated for bioethanol production. Biotechnology for Biofuels 2008, 1. 29. Selig, M. J.; Viamajala, S.; Decker, S. R.; Tucker, M. P.; Himmel, M. E.; Vinzant, T. B., Deposition of Lignin Droplets Produced During Dilute Acid Pretreatment of Maize Stems Retards Enzymatic Hydrolysis of Cellulose. Biotechnology Progress 2007, 23, 1333-1339. 30. Ramos, L. P., The chemistry involved in the steam treatment of lignocellulosic materials. Quimica Nova 2003, 26, 863-871. 31. Targonski, Z., Alkali process for enhancing susceptibility of autohydrolysed beech sawdust to enzymatic hydrolysis. Enzyme and Microbial Technology 1985, 7, 126-128. 32. Ballesteros, M.; Oliva, J. M.; Negro, M. J.; Manzanares, P.; Ballesteros, I., Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SFS) with Kluyveromyces marxianus CECT 10875. Process Biochemistry 2004, 39, 1843-1848. 33. Renaut, J.; Lutts, S.; Hoffmann, L.; Hausman, J. F., Responses of Poplar to Chilling Temperatures: Proteomic and Physiological Aspects. Plant Biology 2004, 6, 81-90. 34. Torget, R.; Himmel, M. E.; Grohmann, K., Dilute sulfuric acid pretreatment of hardwood bark. Bioresource Technology 1991, 35, 239-246. 35. Rättö, M.; Siika-aho, M.; Buchert, J.; Valkeajävi, A.; Viikari, L., Enzymatic hydrolosis of isolated and fibre-bound galactoglucomannans from pine-wood and pine kraft pulp. Applied Microbiology and Biotechnology 1993, 40, 449-454. 36. Bishop, C. T.; Cooper, F. P., CONSTITUTION OF A GLUCOMANNAN FROM JACK PINE (PINUS BANKSIANA, LAMB). Canadian Journal of Chemistry 1960, 38, 793-804. 37. Mittal, A.; Chatterjee, S. G.; Scott, G. M.; Amidon, T. E., Modeling xylan solubilization during autohydrolysis of sugar maple wood meal: Reaction kinetics. Holzforschung 2009, 63, 307-314. 38. Garrote, G.; Dominguez, H.; Parajo, J. C., Kinetic modelling of corncob autohydrolysis. Process Biochemistry 2001, 36, 571-578. 30 ACS Paragon Plus Environment

Page 31 of 48

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39. Conner, A. H., Kinetic Modeling of Hardwood Prehydrolysis .1. Xylan Removal by Water Prehydrolysis. Wood and Fiber Science 1984, 16, 268-277. 40. Conner, A. H.; Lorenz, L. F., Kinetic modeling of hardwood prehydrolysis. Part III. Water and dilute acetic acid prehydrolysis of southern red oak. Wood and Fiber Science 1986, 18, 248-263. 41. Mašura, M., Prehydrolysis of beechwood. Wood Science and Technology 1987, 21, 89100. 42. Negro, M. J.; Manzanares, P.; Ballesteros, I.; Oliva, J. M.; Cabanas, A.; Ballesteros, M., Hydrothermal pretreatment conditions to enhance ethanol production from poplar biomass. Applied Biochemistry and Biotechnology - Part A Enzyme Engineering and Biotechnology 2003, 108, 87-100. 43. Heitz, M.; Capek-Mιnard, E.; Koeberle, P. G.; Gagnι, J.; Chornet, E.; Overend, R. P.; Taylor, J. D.; Yu, E., Fractionation of Populus tremuloides at the pilot plant scale: Optimization of steam pretreatment conditions using the STAKE II technology. Bioresource Technology 1991, 35, 23-32. 44. Moldes, A. B.; Bustos, G.; Torrado, A.; Domínguez, J. M., Comparison between different hydrolysis processes of vine-trimming waste to obtain hemicellulosic sugars for further lactic acid conversion. Applied Biochemistry and Biotechnology 2007, 143, 244-256. 45. Larsson, S.; Palmqvist, E.; Hahn-Hagerdal, B.; Tengborg, C.; Stenberg, K.; Zacchi, G.; Nilvebrant, N. O., The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme and Microbial Technology 1999, 24, 151-159. 46. Cotana, F.; Cavalaglio, G.; Gelosia, M.; Coccia, V.; Petrozzi, A.; Nicolini, A., Effect of Double-Step Steam Explosion Pretreatment in Bioethanol Production from Softwood. Applied Biochemistry and Biotechnology 2014, 174, 156-167. 47. Tengborg, C.; Stenberg, K.; Galbe, M.; Zacchi, G.; Larsson, S.; Palmqvist, E.; HahnHägerdal, B., Comparison of SO2 and H2SO4 impregnation of softwood prior to steam pretreatment on ethanol production. Applied Biochemistry and Biotechnology 1998, 70-72, 315. 48. Kang, Y.; Bansal, P.; Realff, M. J.; Bommarius, A. S., SO2-catalyzed steam explosion: The effects of different severity on digestibility, accessibility, and crystallinity of lignocellulosic biomass. Biotechnology Progress 2013, 29, 909-916. 49. Stenberg, K.; Tengborg, C.; Galbe, M.; Zacchi, G., Optimisation of steam pretreatment of SO2-impregnated mixed softwoods for ethanol production. Journal of Chemical Technology and Biotechnology 1998, 71, 299-308. 50. Ewanick, S. M.; Bura, R.; Saddler, J. N., Acid-catalyzed steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol. Biotechnology and Bioengineering 2007, 98, 737-746. 51. Rowe, J. W. Extractives in eastern hardwoods : a review; WI: U.S. Department of Agriculture, Forest Service, Forest Products Laboratory1979; p 67 p. 52. Shibutani, S.; Igarashi, K.; Samejima, M.; Saburi, Y., Inhibition ofTrichoderma cellulase activity by a stilbene glucoside fromPicea glehnii bark. Journal of Wood Science 2001, 47, 135140. 53. Tejirian, A.; Xu, F., Inhibition of enzymatic cellulolysis by phenolic compounds. Enzyme and Microbial Technology 2011, 48, 239-247. 54. Hendriks, A. T. W. M.; Zeeman, G., Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresource Technology 2009, 100, 10-18. 55. Overend, R. P.; Chornet, E., Fractionation of Lignocellulosics by Steam-Aqueous Pretreatments. Philosophical Transactions of the Royal Society of London Series aMathematical Physical and Engineering Sciences 1987, 321, 523-536. 31 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 48

56. Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J., Determination of structural carbohydrates and lignin in biomass. Laboratory Analytical Procedures (LAP), National Renewable Energy Laboratory (NREL), Golden, CO. Revised version Jul 2011. There is no corresponding record for this reference 2012, 1-15. 57. Yasuda, S.; Fukushima, K.; Kakehi, A., Formation and chemical structures of acid-soluble lignin I: Sulfuric acid treatment time and acid-soluble lignin content of hardwood. Journal of Wood Science 2001, 47, 69-72. 58. Park, S.; Baker, J.; Himmel, M.; Parilla, P.; Johnson, D., Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance. Biotechnology for Biofuels 2010, 3, 10. 59. Brown, L.; Torget, R., Enzymatic Saccharification of Lignocellulosic Biomass. In Chemical and Testing Task: Laboratory Analytical Procedure 009 NREL: Golden, 1996.


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Table 1. Chemical composition of poplar branches, vineyard pruning and pine wood sawdust expressed as wt.% (dry basis) content of structural and non-structural components Component

Poplar

Vineyard

Pine wood

(wt.%)

(pruning)

(pruning)

(sawdust)

Glucan

40.5

33.8

37.9

Xylan

14.11

13.2

-

Galactan

0.97

1.63

7.18

Mannan

3.81

-

9.55

Arabinan

1.46

0.33

1.96

Acetyl groups

2.13

2.98

1.09

Lignin

14.7

25.3

23.9

Extractives

18.9

16.8

18.3

Ash

2.52

3.05

0.06

Total

99.0

97.8

99.9



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Table 2. Experimental conditions of hydrothermal (HT) pretreatment experiments Experiment

1

Temperature (oC)1

HT-1

Liquid to solids ratio (LSR) 15

170

Reaction time (min) 15

Severity factor (logRo) 3.33

HT-2

15

170

30

3.54

HT-3

15

170

60

3.84

HT-4

15

170

90

4.01

HT-5

15

170

120

4.14

HT-6

15

170

180

4.31

HT-7

15

180

15

3.53

HT-8

15

190

15

3.81

HT-9

15

200

15

4.12

HT-10

15

210

15

4.41

HT-11

15

220

15

4.69

The heating rate in all experiments was ~7 oC/min


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Table 3. Crystallinity and textural characteristics of parent and hydrothermally treated biomass solids Experimental Biomass type

conditions

Crystallinity index (%)

Poplar branches Poplar branches

parent o

190 C- 15min o

Specific Surface Area (m2g-1) [a]

Total Pore Volume (cm3g-1) [b]

72.2

0.38

0.007

79.9

1.15

0.018

Poplar branches

220 C - 15min

83.4

2.06

0.024

Vineyard pruning

Parent

70.3

0.48

0.007

80.7

2.31

0.021

Vineyard pruning

o

190 C - 15min o

Vineyard pruning

220 C - 15min

86.1

4.55

0.042

Pinewood sawdust

Parent

77.5

0.02

0.001

83.5

0.35

0.005

85.4

0.96

0.009

Pinewood sawdust Pinewood sawdust

o

190 C - 15min o

220 C - 15min

[a] From N2 adsorption at -196oC data, applying the multi-point Brunauer–Emmett–Teller method (ΒΕΤ). [b] At P/P0=0.99.



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Page 36 of 48

Table 4. Concentration of total (T) and monomeric (Μ) sugars measured in the hydrothermal pretreatment liquids of poplar, grapevine, and pine biomass at different conditions.

Poplar biomass

Hydrothermal

Grapevine biomass

Pine biomass

Glucose

Xylose

Glucose

Xylose

Glucose

Mannose &

(mg/ml)

(mg/ml)

(mg/ml)

(mg/ml)

(mg/ml)

Galactose

conditions

(mg/ml) (M)

(T)[a]

(M)

(T)[a]

(M)

(T)[a]

(M)

(T)[a]

(M)

(T)[a]

(M)

(T)[a]

170 oC - 15 min

1,05

3,28

0,15

1,73

1,12

2,35

0,56

2,59

0,79

2,07

0,4

5,00

170 oC - 30 min

1,09

3,39

0,63

4,88

1,51

7,04

1,29

4,21

0,96

2,30

0,77

6,94

170 oC - 60 min

1,13

3,39

1,02

6,53

1,56

7,65

2,05

5,45

1,03

2,41

1,87

7,63

170 oC - 90 min

1,16

3,52

1,18

5,53

1,61

7,68

2,74

6,13

1,07

2,45

2,91

7,67

170 oC - 120 min

1,19

3,24

1,26

4,18

1,79

7,16

2,99

5,77

1,16

2,53

3,66

7,77

170 oC - 180 min

1,23

2,98

1,55

3,21

2,72

6,68

2,67

4,75

1,38

2,71

5,04

7,00

180 oC - 15 min

1,11

3,40

0,56

4,47

1,37

4,37

1,29

5,19

1,01

2,43

0,68

6,03

190 oC - 15 min

1,13

2,50

1,03

6,30

1,48

5,80

2,09

6,08

1,11

2,55

1,53

6,98

200 oC - 15 min

1,15

3,06

1,23

5,221

1,64

6,68

2,83

5,74

1,20

2,64

3,52

7,65

210 oC - 15 min

1,30

2,45

1,27

2,72

2,59

5,01

2,03

3,22

1,45

2,61

3,88

5,21

220 oC - 15 min

1,09

1,86

0,23

0,36

1,96

2,51

0,67

0,92

1,63

2,36

2,05

2,21


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Table 5. Glucose mass balance and percent recovery in the hydrothermal pretreatment and enzymatic hydrolysates of poplar branches, vineyard pruning and pine wood sawdust Hydrothermal pretreatment conditions Biomass sample

Glucose in parent biomass sample a (g)

Glucose obtained in HT and enzymatic hydrolysis liquid products b (g)

Glucose recovery (% w/w)

c

Tempe rature (oC)

Time (min)

Extracti ves

Cellulo se

Total

HT pretreatm ent

Enzym atic hydroly sis

Total

HT pretreatme nt

Enzym atic hydroly sis

Total

Poplar

220

15

5.90

44.10

50.00

2.80

19.00

21.80

47.5

43.1

43.6

Grapevine

220

15

11.90

33.80

45.70

3.80

25.00

28.80

31.9

74.0

63.0

Pine

220

15

3.90

40.00

43.90

3.50

6.40

9.90

89.7

16.0

22.6

Poplar

170

90

5.90

44.10

50.00

5.30

10.30

15.60

89.8

23.4

31.2

Grapevine

170

90

11.90

33.80

45.70

11.50

13.20

24.70

96.6

39.1

54.0

Pine

170

90

3.90

40.00

43.90

3.70

5.50

9.20

94.9

13.8

21.0

a

Glucose contained in 100 g of untreated biomass in extractives and cellulose;

b

Glucose released from hydrothermal pretreatment and sequential enzymatic hydrolysis (based on

100g of parent biomass c

Glucose recovery in pretreatment was calculated from extractives and glucose recovery in enzymatic

hydrolysis was calculated from cellulose.



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Page 38 of 48

Figure captions Figure 1. Correlation of biomass solubilization (wt.%) with the severity factor (log Ro) in the hydrothermal pretreatment experiments. Filled symbols correspond to the set of experiments for 15 min at 170–220oC and empty symbols to the experiments at 170oC for 15–180 min. Figure 2. Main structural components (♦ cellulose, ■ lignin, and ▲ hemicellulose) of the hydrothermally treated solids expressed as percentage of their initial content in the parent biomass. Filled symbols correspond to the set of experiments for 15 min at 170–220oC, empty symbols to the experiments at 170oC for 15–180 min, and empty black symbols to the respective parent biomass. Figure 3. Photographs and SEM images of the parent (left column) and representative hydrothermally treated biomass samples of increasing severity (middle column: 190oC, 15 min; right column: 220oC, 15 min). (A) Poplar branches, (B) Grapevine pruning, and (C) Pine wood sawdust Figure 4. Correlation of the concentration of the three main acids (acetic, formic, and levulinic) that are detected in the hydrothermal pretreatment liquid products with log Ro Figure 5. Reaction pathways for the transformation of monomeric sugars to organic acids via furans formation under the mild acidic conditions of the biomass hydrothermal pretreatment Figure 6. Correlation of furfural and hydroxymethylfurfural/HMF concentration of the hydrothermal pretreatment liquid products with log Ro Figure 7. Fraction (percent) of xylan or galactomannan dissolved in the hydrothermal pretreatment liquids (blue symbols) and that remained in treated biomass (red symbols) versus the severity factor log Ro. (A) poplar biomass, (B) grapevine biomass, (C) pine biomass. Filled symbols correspond to the set of experiments for 15 min at 170–220oC and empty symbols to the experiments at 170oC for 15–180 min. Figure 8. Correlation of xylose, galactose & mannose, furfural and HMF concentration of the hydrothermal pretreatment liquids with % hemicellulose removal for (A) poplar biomass, (B) grapevine biomass, (C) pine biomass. Figure 9. Enzymatic hydrolysis of untreated and hydrothermally pretreated biomass samples. (A) poplar branches, (B) grapevine pruning, (C) pine sawdust. Bars show the fraction of cellulose (wt. %) that has been enzymatically hydrolyzed to glucose. The inset graph shows the correlation between hydrolysis (wt.% conversion of cellulose) of the samples and the severity factor log Ro.


Page 39 of 48

60

Vine 15 min

50

Solubilization (wt. %)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Vine 170 oC 40 Poplar 15 min Poplar 170 oC

30

Pine 15 min

20 Pine 170 oC

10 3

3.5

4

4.5

5

log Ro Figure 1. Correlation of biomass solubilization (wt.%) with the severity factor (log Ro) in the hydrothermal pretreatment experiments. Filled symbols correspond to the set of experiments for 15 min at 170–220oC and empty symbols to the experiments at 170oC for 15–180 min.


ACS Sustainable Chemistry & Engineering Poplar

45

Normalised content (wt. % )

40 35 30 25 20 15 10 5 0 3

3.5

4

4.5

5

4.5

5

4.5

5

log Ro Grapevine

45


40 35 30 25 20

15 10 5 0 3

3.5

4

log Ro Pine 45 40


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 48

35 30 25 20 15 10 5 0 3

3.5

4

log Ro

Figure 2. Main structural components (♦ cellulose, ■ lignin, and ▲ hemicellulose) of the hydrothermally treated solids expressed as percentage of their initial content in the parent biomass. Filled symbols correspond to the set of experiments for 15 min at 170–220oC, empty symbols to the experiments at 170oC for 15–180 min, and empty black symbols to the respective parent biomass.


Page 41 of 48

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(A)

(B)

(C)

Figure 3. Photographs and SEM images of the parent (left column) and representative hydrothermally treated biomass samples of increasing severity (middle column: 190oC, 15 min; right column: 220oC, 15 min). (A) Poplar branches, (B) Grapevine pruning, (C) Pine wood sawdust



C (mg/mL)

Acetic acid 3

Poplar 15 min - series

2.5

Poplar 170 oC - series

2

Grapevine 15 min - series

1.5

Grapevine 170 oC - series

1

Pine 15 min - series 0.5

Pine 170 oC - series

0 3

3.5

4

4.5

5

log Ro Formic acid 1.4


1.2


C (mg/mL)

1


0.8


0.6 0.4

Pine 15 min - series

0.2


0 3

3.5

4

4.5

5

log Ro Levulinic acid

1.6


1.4


1.2

C (mg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 48


1 0.8


0.6 0.4


0.2


0 3

3.5

4

4.5

5

log Ro Figure 4. Correlation of the concentration of the three main acids (acetic, formic and levulinic) that are detected in the hydrothermal pretreatment liquid products with log Ro 42 ACS Paragon Plus Environment

Page 43 of 48

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Reaction pathways for the transformation of monomeric sugars to organic acids via furans formation under the mild acidic conditions of the biomass hydrothermal pretreatment




Furfural

1.2


C (mg/mL)

1 0.8


0.6


0.4

Pine 15 min - series 0.2


0 3

3.5

4

4.5

5

log Ro 0.8


5-HMF

0.7


0.6

C (mg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 48


0.5 0.4


0.3 0.2


0.1


0

3

3.5

4

4.5

5

log Ro Figure 6. Correlation of furfural and hydroxymethylfurfural/HMF concentration of the hydrothermal pretreatment liquid products with log Ro


Page 45 of 48

ACS Sustainable Chemistry & Engineering 100

Xylan in liquid - 15 min series Xylan in liquid - 170 oC series Xylan in solid - 15 min series Xylan in solid 170 oC - series

(A)

90 80

Xylan (wt. %)

70 60 50

40 30 20 10 0 3

3.5

4

4.5

5

log Ro 100

Xylan (wt. %)

Xylan in liquid - 15 min series

(B)

90

Xylan in liquid - 170 oC series

80

Xylan in solid - 15 min series

70

Xylan in solid - 170 oC series

60 50 40 30 20 10 0 3

3.5

4

4.5

5

log Ro

100

Galactomannan in liquid - 15 min series

(C)

90

Galactomannan (wt. %)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Galactomannan in liquid - 170 oC series

80

Galactomannan in solid - 15 min series

70

Galactomannan in solid - 170 oC series

60

50 40 30 20 10 0

3

3.5

4

4.5

5

log Ro

Figure 7. Fraction (percent) of xylan or galactomannan dissolved in the hydrothermal pretreatment liquids (blue symbols) and that remained in treated biomass (red symbols) versus the severity factor log Ro. (A) poplar biomass, (B) grapevine biomass, (C) pine biomass. Filled symbols correspond to the set of experiments for 15 min at 170–220oC and empty symbols to the experiments at 170oC for 15–180 min



(A)

Xylose - 15 min series

Xylose - 170 oC series

Furfural - 15 min series

Furfural - 170 oC series

7

2 1.8

6

5

1.4 1.2

4

1 3

0.8

Furfural (mg/mL)

Xylose (mg/mL)

1.6

0.6

2

0.4 1

0.2

0

0 40

50

60

70

80

90

Hemicellulose removal (wt.%)

(Β)

Xylose - 15 min series

Xylose - 170 oC series

Furfural - 15 min series

Furfural - 170 oC series

7

2 1.8

6 5

1.4 1.2

4

1 3

0.8

Furfural (mg/mL)

Xylose (mg/mL)

1.6

0.6

2

0.4 1

0.2

0

0 40

50

60

70

80

90

Hemicellulose removal (wt.%) Galactose & mannose - 15 min series Galactose & mannose - 170 oC series HMF - 15 min series HMF - 170 oC series

(C) 2

8

1.8

7

1.6 1.4

6

1.2

5

1 4

0.8

3

HMF (mg/mL)

9

Galactose & Mannose (mg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 48

0.6

2

0.4

1

0.2

0

0 40

50

60

70

80

90

Hemicellulose removal (wt.%)

Figure 8. Correlation of xylose, galactose & mannose, furfural and HMF concentration of the hydrothermal pretreatment liquids with % hemicellulose removal for (A) poplar biomass, (B) grapevine biomass, and (C) pine biomass.


80

70

70

50

60

30

15 min series 170 oC series

60

(A)

40 20

50

49.1

10 3

3.5

4

4.5

41.8

5

log Ro

40

31.5 30.6

26.1 21.3 21.6 23.8

30

21.3

24.8

29.8

34.0

20 10

9.0

0

Biomass Sample 120

15 min series 170 oC series

Cellulose enzymatic hydrolysis (wt. % on cellulose)

90

100

(B)

70 50

80

77.0

30

68.0

10 3

3.5

4

4.5

62.0

5

log Ro

60

59.0

53.0

47.0

45.0 40 23.8 20

30.0

37.0

33.0 23.8

12.0

0

Biomass sample

30 Cellulose enzymatic hydrolysis (wt. % on cellulose)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cellulose enzymatic hydrolysis (wt. % on cellulose)

Page 47 of 48

15min series 170oC series

30

(C)

25

25

20 15

20

10 3

3.5

4

4.5

5

log Ro

15

12.8 11.8 11.6 12.0 12.6 11.0 11.4 11.5 10.4 11.0 11.0

16.0 14.0

10 5 0

Biomass Sample

Figure 9. Enzymatic hydrolysis of untreated and hydrothermally pretreated biomass samples. (A) poplar branches, (B) grapevine pruning, (C) pine sawdust. Bars show the fraction of cellulose (wt. %) that has been enzymatically hydrolyzed to glucose. The inset graph shows the correlation between hydrolysis (wt.% conversion of cellulose) of the samples and the severity factor log Ro.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Hydrothermal Pretreatment in neat H2O

Grapevine trimmings

Efficient Hemicellulose recovery

Improved Enzymatic hydrolysis

Title: Optimization of hydrothermal pretreatment of hardwood and softwood lignocellulosic residues for selective hemicellulose recovery and improved cellulose enzymatic hydrolysis Christos K. Nitsos, Theodora Choli-Papadopoulou, Konstantinos A. Matis and Kostas S. Triantafyllidis Synopsis: Tuning of the hydrothermal pretreatment conditions in neat water allows the selective recovery of hemicellulose sugars or the respective furans and acids, along with enhanced cellulose enzymatic hydrolysis of various hardwood biomass types. Grapevine pruning residuals represent a promising source of waste biomass which can be effectively used without prior debarking.

ACS Paragon Plus Environment

Optimization of Hydrothermal Pretreatment of Hardwood and Softwood

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