Study on Conditioning of SO2âEthanolâWater Spent Liquor from

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Study on Conditioning of SO2−Ethanol−Water Spent Liquor from Spruce Chips/Softwood Biomass for ABE Fermentation Evangelos Sklavounos,*,† Mikhail Iakovlev,† and Adriaan van Heiningen†,‡ †

Department of Forest Products Technology, School of Chemical Technology, Aalto University, POB 16400, 00076 Aalto, Finland Department of Chemical and Biological Engineering, University of Maine, 5737 Jenness Hall, Orono, Maine 04469-5737, United States

‡

S Supporting Information *

ABSTRACT: The focus of this study is to develop a process for conditioning spent liquor produced by SO2−ethanol−water (SEW) fractionation of spruce chips/softwood biomass for fermentation to butanol, ethanol, and acetone/2-propanol (so-called ABE process) by Clostridia bacteria. This study is an integral part of a project which aims at creating an economic process that can utilize cheap forestry residues such as twigs, cones, treetops, branches, and bark to produce renewable chemicals and liquid fuels. The results of this study suggest that the proposed scheme for conditioning of SEW spent liquor from spruce/softwood biomass can be successfully applied to produce chemicals and biofuels by ABE fermentation. Butanol, acetone, and ethanol are produced at a total yield of around 0.25 g/g sugars.

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INTRODUCTION Rising oil prices, declining energy security, and imminent climate change have recently stimulated much research into the production of transportation fuels and chemicals from lignocellulosic feedstocks. Forest biomass remains a relatively cheap and sustainable energy source that provides a suitable feedstock for the production of renewable liquid fuels and chemicals. However, for economically feasible biofuel/biochemical production, it is important to have efficient fractionation of biomass. For instance, when the target is to produce biofuels by fermentation, hemicellulose sugars must be dissolved in high yield as monomers, while the cellulosic residue should be easily hydrolyzed by enzymes. The combined sugar solution must not contain compounds at a level which is inhibitory to fermentation. One fractionation method that potentially meets these requirements is the SO2−ethanol− water (SEW) fractionation process originally utilized by Schorning1 as a pulping method. The fractionation mechanisms and kinetics were recently revisited and reported by Iakovlev and van Heiningen.2,3 SEW fractionation is currently used to hydrolyze cellulosics to sugar monomers in a patent pending biorefinery process termed AVAP by American Process Inc.4 The SEW process has distinct advantages: it does not suffer from hemicellulose sugar degradation,2,5 it produces cellulose readily digestible by enzymes,6 it has a relatively low energy requirement because of the low temperature (130−150 °C) and liquid-to-wood ratio of 2−3 L kg−1, it does not create sticky lignin precipitates,7 it can simultaneously fractionate softwood and hardwood biomass,8 and it only requires evaporation of ethanol and SO2 for recovery of the fractionation chemicals due to the absence of a base (Mg or Na) in the cooking liquor. These characteristics make the SEW process uniquely suitable to dissolve wood sugars for microbial fermentation.9 The latter may be performed by utilizing Clostridia bacteria.10 An important feature of these microorganisms for acetone, butanol, © 2013 American Chemical Society

and ethanol (ABE) fermentation is that they allow utilization of all pentose and hexose sugars derived from wood. The requirement to ferment dissolved wood sugars to ABE solvents by Clostridia bacteria dictates that further processing and conditioning of the SEW spent liquor is needed. For example, the pH of the SEW spent liquor is 1.0 and adjustment to a neutral level is necessary to avoid harming the bacteria. Moreover, fermentation inhibitorssuch as SO2, formic acid, furanic compounds, and ligninmust be brought below critical levels.11,12 It was demonstrated that SO2 is inhibitory to Clostridia bacteria at concentrations as low as 10−50 ppm, while formic acid, furfural, and hydroxymethylfurfural are not tolerable at levels above 0.5, 1.0, and 1.5 g L−1, respectively. Soluble lignin compounds are not tolerable by Clostridia bacteria at levels above 1.0 g L−1. In addition to fermentability of the final liquor, an efficient recovery of the fractionation chemicals, i.e., SO2 and ethanol, is required to obtain an economical process with minimal environmental impact. The present paper describes a novel industry optimized scheme for conditioning of the spent liquor produced by SEW fractionation of spruce chips/softwood biomass. The conditioning scheme is a modified version of the original protocol for conditioning of SEW spent liquor from spruce developed by Sklavounos et al.13

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MATERIALS AND METHODS Air-dried spruce chips were screened to 2−4 mm thickness, and a mixture of the accept fractions were used for the experiments. Air-dried mixed softwood biomass was screened according to SCAN-CM 40:01 in order to remove humus and needles (7 and 13 mm hole screen accept fractions used). Fractionation Received: Revised: Accepted: Published: 4351

November 14, 2012 February 25, 2013 February 26, 2013 February 26, 2013 dx.doi.org/10.1021/ie303126x | Ind. Eng. Chem. Res. 2013, 52, 4351−4359

Industrial & Engineering Chemistry Research

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liquor from spruce chips/softwood biomass was exposed to a sequence of “conditioning” steps as shown in Figure 2. At first, the MSEW liquor was evaporated under vacuum for 120 min to remove most of the SO2 and ethanol. The water bath temperature was 95 °C and the vacuum was 300 mbar. Under these conditions, a reasonable evaporation rate and time to reduce the ethanol concentration to less than 10 g L−1 were achieved. About 72/67% of the original weight was evaporated for spruce wood chips/biomass, respectively, to reach SO2 levels of 4.9/5.6 g L−1 and ethanol levels of approximately 1 g L−1. The residual liquor, called EVAP, was then centrifuged (11 000 rpm for 20 min), whereby a light-colored precipitate was obtained (lignin−carbohydrate complex or LCC). The LCC was removed from the liquor before steam stripping. Steam stripping was performed for 120 min (steam temperature of 102 °C, flow of 0.7 L h−1) to further remove SO2 to a level of around 100 ppm. The steam-stripped liquor (STR) from spruce chips/softwood biomass was neutralized to pH 8.9 by addition of 6.1/6.4 g L−1 (about 0.7/1.1% based on ovendried feedstock (bof)) Ca(OH)2. The precipitate was removed by centrifugation (11 000 rpm for 15 min) and washed three times with alkaline water (Ca(OH)2 to pH 10). The wash waters collected as supernatants after the centrifugations were added to the neutralized LIME liquor. The remaining sulfite anions in the liquor were oxidized to sulfate anions by air bubbling through the solution for 1 h at 60 °C and in the presence of FeSO4·7H2O (about 20 mg L−1) as catalyst. This also brought the iron micronutrient level to that required for the fermentation microorganisms. The conditioned liquor called CATOX was treated with ASS 01G Cl− FINEX anionic resins at a resin-to-liquor ratio of 1:1.5 to further remove lignin. Finally, the treated liquor was 4-fold diluted and pH controlled to pH 6.5 with sulfuric acid. It was then fed continuously to a patent pending fermentation column.31 The column contained immobilized Clostridium acetobutylicum DSM 792 cells supplemented with production medium components. The supplement contained the following (in g/L): magnesium sulfate, 0.2; sodium chloride, 0.01; manganese sulfate, 0.01; iron sulfate, 0.01; potassium dihydrogen phosphate, 0.5; potassium hydrogen phosphate, 0.5; ammonium acetate, 2.2; biotin, 0.01; thiamin, 0.1; and p-aminobenzoic acid, 0.1. Glucose at a concentration of 35 g/L was added. The pH was adjusted to 6.5 with HCl. After preparation, the medium was purged with oxygen-free nitrogen and autoclaved at 105 Pa (121 °C) for 20 min and cooled. Reinforced clostridial medium (RCM) was used for the inoculum preparation. The RCM contained 10 g/L meat extract, 5 g/L peptone, 3 g/L yeast extract, 30 g/L D(+)glucose, 1 g/L starch, 5 g/L sodium chloride, 3 g/L sodium

was done in a HAATO 43427 silicon oil bath using six 220 mL bombs filled with 25 g of oven-dried spruce chips/softwood biomass. The fresh fractionation liquor was prepared by injecting gaseous sulfur dioxide into a 55% (by volume) ethanol−water solution cooled in an ice bath. The concentration of SO2 in the liquor was set to 12% by weight, and the liquor-to-wood ratio used was 3:1 L kg−1. Pulping was carried out at 150 °C (±1 °C) and 30 min, including the heating-up period of 8−9 min.14 After fractionation, the bombs were rapidly removed from the oil bath and cooled in cold water. SEW spent liquor was collected by squeezing the pulp suspension contained in a washing sock. The pulp was then washed twice with 40% ethanol−water at 60 °C and three times with deionized water at room temperature as shown in Figure 1.

Figure 1. SEW fractionation and pulp washings.

The liquor-to-wood ratio used for each washing step was 1.4:1 L kg−1. The pulp washings were added to the SEW liquor forming a dilute mixture called the MSEW liquor. The MSEW

Figure 2. SEW spent liquor conditioning scheme. 4352

dx.doi.org/10.1021/ie303126x | Ind. Eng. Chem. Res. 2013, 52, 4351−4359


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acetate, and 0.5 g/L L-cysteine hydrochloride (final pH 6.8 ± 0.2). The column temperature was maintained at 37 °C by continuously circulating water through the jacket. Fermentation was performed for 48 h until steady state was reached. Samples were taken from the top of the column and centrifuged at 13860g for 5 min. Supernatants were used for the substrate and product analysis. Pulp yields as well as dissolved components yields for all process liquors (the latter by evaporation to dryness at 105 °C) were determined (SCAN-C3:78 and SCAN-N1:61). Ash content was measured according to NREL/TP-510-42622. Extractives were removed from the pulps with acetone and analyzed gravimetrically (SCAN 49:03), while the spent liquor samples were evaporated to dryness in a vacuum evaporator prior to the determination of total carbohydrates, lignin, and (in pulps) acetyl groups within the same procedure (NREL/TP510-42618). The first step of hydrolysis consisted of 72% H2SO4, acid-to-material ratio of about 2−5 mL g−1 (for the liquors) and 10 mL g−1 (for pulps), 30 ± 3 °C, and 60 ± 5 min. The second step of hydrolysis consisted of 4% H2SO4, acid-tomaterial ratio of 50−130 mL g−1 (for the liquors) and 300 mL g−1 (for pulps), 121 ± 1 °C, and 60 min. The monosaccharides and acetic acid in the hydrolysate were analyzed by high performance anion exchange chromatography (HPAEC; Dionex ICS-3000; CarboPac PA20 column; pulsed amperometric detection (PAD)) and high performance liquid chromatography (HPLC; Dionex UltiMate 3000; Acclaim OA column; diode array detector), respectively. Acid insoluble lignin was determined gravimetrically, whereas acid soluble lignin was determined by measuring absorbance at 205 nm (Shimadzu UV-2550 spectrophotometer). An extinction coefficient of 128 L g−1 cm−1 was the basis for quantification.15 The pulps were also analyzed for kappa number (SCAN-C 1:00) and intrinsic viscosity in cupriethylenediamine (CED) solution using SCAN-CM 15:99. All pulps were exposed to chlorite delignification prior to viscosity measurement according to T230 om-66 since they had a kappa number higher than 35. The cellulose content of the feedstocks and the pulps was calculated based on the mannose-to-glucose ratio of 4.15 reported for softwood (galacto)glucomannan.16 Monosugar concentrations in the process liquors after each conditioning step were analyzed by HPAEC-PAD (Dionex ICS 3000, Sunnyvale, CA, USA) according to NREL/TP-51042623. Further instruments were used for various analyses: furfural and hydroxymethylfurfural by HPLC (Dionex UltiMate 3000 equipped with diode array detector); acetic acid, formic acid, and ethanol by an HP 1100 HPLC; aldonic acids by HPAEC equipped with a Dionex CarboPac PA10 column; uronic acids by methanolysis/GC-FID (Shimadzu GC-2010 Plus with NB-30 capillary column of 30 m length and 0.32 mm interior diameter) according to Sundberg et al.17 and Iakovlev and van Heiningen;2 concentrations of sulfate and sulfite anions (after adding NaOH) by ion chromatography (Dionex ICS 1500, Sunnyvale, CA, USA); inorganic analysis of the feedstocks by a Varian Liberty ICP-AAS; CHN/S analysis of the feedstocks by a 2400 Series II CHN/S PerkinElmer elemental analyzer (Jones factor of 6.25 used to convert nitrogen content to protein content according to Mariotti et al.18); sulfur content in the LCC by combustion in oxygen in a Schö niger flask, followed by absorption of SO2 in H2O2 solution, and the formed sulfate anions were determined by ion chromatography (SCAN-CM 57:99).

The produced ABE solvents were quantified by using gas chromatography as reported by Survase et al.31 Consumption of sugars during ABE fermentation was determined by measuring glucose, mannose, arabinose, galactose, and xylose concentrations by HPLC (Bio-Rad Laboratories, Hercules, CA, USA), equipped with an Inores S 259-H column (Inovex, Vienna, Austria) packed with an Inores cation exchanger (particle size, 9 mm). The column was heated at 70 °C, and the eluent (0.01 M H2SO4) was circulated with a flow rate of 0.60 mL min−1. A cellobiose (Roth, Karlsruhe, Germany) solution was added to the samples as an internal standard. A refractive index detector (Model 1755, Bio-Rad) was used for quantitation.

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RESULTS AND DISCUSSION In the following discussion “%” means “percent by weight”, and “% (bof)” means “weight percent based on feedstock”, if not otherwise indicated. Numbers separated by slashes denote respective values for spruce chips/softwood biomass liquors, respectively. Chemical Composition of the Feedstocks. Mixed softwood biomass has a significantly different chemical composition from that of spruce chips. This is due to the very heterogeneous nature of the former; i.e., softwood biomass contains forest residues such as needles, twigs, bark, etc., that lead to variations in chemical composition. Pine is likely to be the predominant constituent of the present softwood biomass as it is the most abundant tree species in Finland. In addition, contamination with nonsoftwood species such as hardwood residues is likely to occur. Spruce chips have a much higher carbohydrate content compared to softwood biomass: 62.2% vs 51.6%. This is in agreement with previous findings by Rakkolainen et al.19 Moreover, there are important differences in terms of extractives (1.5% for spruce vs 4.8% for softwood biomass), ash (0.3% vs 2.6%), and protein (0.3% vs 2.6%) contents. The significantly higher contents of these compounds in softwood biomass are explained by the presence of twigs and bark.20,21 Table 1 shows that ash of softwood biomass is rich in calcium cations in particular. On the other hand, the lignin content of the feedstocks is similar: 28.9% vs 29.8%. Table 1. Elemental Composition of the Ashes of Feedstocks As Determined by ICP-AES/AAS % (bof) element

spruce chips

K Na Al Mg Ca Si total

0.041 0.003 0.001 0.012 0.113 0.007 0.177

8

softwood biomass 0.141 0.009 0.038 0.050 0.577 0.123 0.938

SEW Fractionation Results. The current experiments were carried out at fractionation conditions of 12% SO2 in 55 v/v % ethanol−water, L:W = 3:1, 150 °C, and 30 min. These conditions form part of the modifications to the original scheme for conditioning of SEW spent liquor from spruce developed by Sklavounos et al.13 The applied SEW cooking conditions in that scheme were 3% SO2 in 55 v/v% ethanol− water, L:W = 6:1, 150 °C, and 120 min. 4353



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likely related to a lower extent of hydrolysis, rather than to different original DP values in spruce and softwood biomass. Hemicelluloses are hydrolyzed to produce short-chain oligomers and monomers. It must be noted that the relative amount of oligomers is substantially higher for softwood biomass (about 75−80%) compared to spruce (35−50%). The dehydration of sugars is not pronounced as inferred from the low concentrations of furanic compounds in the MSEW liquors. Their amount is, however, somewhat higher in the case of spruce. Several phenomena observed during fractionation of softwood biomass (compared to fractionation of spruce) including less delignification, slower depolymerization, and dehydration of polysaccharides are explained by the lower acidity of the cooking liquor in the case of softwood biomass. This is also confirmed by the measurement of spent SEW liquors pH (at room temperature): 1.4 vs 1.0. The acidity of SEW fractionation systems is governed by the amount of formed lignosulfonic acid groups and the amount of cations neutralizing them. Interestingly, lignin originating from fractionation of spruce and softwood biomass has similar sulfonic acid group content: S/C9 of 0.10/0.083 for residual lignin in the pulp fibers and 0.25/0.27 for dissolved lignin (Table 3). Since the concentration of hydrosulfite anions has

In the present set of experiments a liquid-to-wood ratio of L:W = 3:1 was used to investigate SEW fractionation at conditions more likely to be employed by the industry. A cooking time of only 30 min was selected because previous research by our group5,22 has demonstrated that it is possible to achieve efficient fractionation of lignocellulosics in only 30 min at 150 °C due to extremely fast impregnation and delignification of lignocellulosics by SO2−ethanol−water solutions. Moreover, from a process economics point of view it is generally accepted that a short fractionation time implies low equipment capital costs. The present spruce fractionation experiments resulted in pulp with kappa number 60 (lignin content 5% (bof)). This is in good agreement with the predicted value based on kinetic studies at L/W 6:1.22 However, the extent of delignification for softwood biomass was lower: the pulp had a kappa number of 104 (lignin content 9.7% (bof)). The latter kappa number value is comparable to the value of 97 obtained for softwood biomass at L/W 6:1.6 From these results it can be concluded that the liquor-to-wood ratio has little effect on bulk delignification (see also ref 7). In order to follow the fate of carbohydrates, it is advantageous to distinguish between cellulosehigh molecular weight, highly ordered, and therefore resistant polymerand short-chain amorphous hemicelluloses. Since anhydroglucose is present in softwoods in both cellulose and galactoglucomannan (GGM), the cellulose amount can be calculated by subtracting the GGM anhydroglucose from the total amount of anhydroglucose. We used the published ratio of mannose-toglucose in softwood GGM of 4.15.16 From the detailed information concerning carbohydrate dissolution of spruce chips/softwood biomass (Table 2), we observe that hemi-

Table 3. Lignin Sulfur Content feedstock

Table 2. Sugar Dissolution and Pulp Properties for the Different Feedstocks

original (% (bof)) MSEW liquor (% (bof)) fraction dissolved pulp intrinsic viscosity (mL/g) pulp cellulose DP

spruce chips

SW biomass

“cellulose” hemicellulose

“cellulose” hemicellulose

39.6 1.5

22.6 19.4

30.9 5.4

19.7 17.0

0.04 573

0.86

0.18 629

0.86

2100

lignin (% (bof)) sulfur (% (bof)) sulfur (S/C9)

28.9 0.004 −

lignin (% (bof)) sulfur (% (bof)) sulfur (S/C9)

29.8 0.024 0.005

pulp

MSEW liquor

Spruce 5.0 17.8 0.084 0.76 0.10 0.25 Softwood Biomass 9.7 16.7 0.13 0.75 0.083 0.27

LCC

EVAP liquor

12.7 0.062 0.029

4.1 0.36 0.52

11.0 0.097 0.049

4.2 0.36 0.51

been identified as the primary factor influencing the degree of lignin sulfonation,3 it can be concluded that hydrosulfite anions are not present in a significant amount during the fractionation of softwood biomass, despite its relatively high ash content. The very low amount of formed aldonic acids is a further confirmation of this. An ion balance provides more insight as to why very little SO2 is converted into hydrosulfite. The total concentration of sulfonic acid groups (sum in liquor and residual solids) in the fractionation systems for both spruce and softwood biomass is close to 0.12 mol L−1, while the charge of all metal cations (originating from ash; see Table 1) in the case of spruce is about 0.027 mol L−1 and in the case of softwood biomass is about 0.14 mol L−1. Therefore, it is evident that all/ most of the cations will be associated with the strong lignosulfonic acid groups, leaving SO2/sulfurous acid mostly in undissociated form, i.e., confirming the near absence of hydrosulfite anions. It should be noted, however, that a further increase in the ash content of the present softwood biomass would lead to significant concentrations of unwanted hydrosulfite anions which are known to oxidize sugars to hard-torecover aldonic acids. However, even the present amount of ash in softwood biomass results in a notable decrease in acidity and associated slower fractionation, since all of the formed lignosulfonic acids appear to be neutralized.

3260

cellulose sugars from both spruce chips and softwood biomass are dissolved in the MSEW liquor while cellulose is mostly preserved in the pulp. This is in agreement with previous research.2 Some apparent dissolution of cellulose in the case of softwood biomass can result from a wrong assumption of the mannose-to-glucose ratio for biomass GGM or from the presence of noncellulosic glucans (starch, callose, laricinan, etc.21). The intrinsic viscosity of spruce chips and softwood biomass pulps is 573 and 629 mL g−1, respectively, which corresponds to viscosity-average degree of polymerization (DP) values of 2100 and 3260.23 The low intrinsic viscosity and DP values suggest extensive cellulose hydrolysis at conditions of 12% SO2, L:W = 3:1, 150 °C, and 30 min (DPw of native cellulose in wood is about 10 00024). The DP value obtained at the present fractionation conditions (L:W = 3:1) for softwood biomass is in agreement with that obtained earlier at L:W = 6:1 (32406). The higher DP values for softwood biomass pulp are 4354



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Table 4. Mass Balance for Original Spruce Chips, Pulp, and Conditioning Liquorsa solid (fiber) phase composition (% bof)) solid yield carbohydrates cellulose glucan in hemicelluloses arabinan xylan mannan galactan extractives lignin acid insoluble acid soluble ash acetyl groups uronic acids proteins total in solid (fiber) phase liquor composition (% (bof)) dry solids contentb carbohydrates arabinose xylose mannose galactose glucose lignin acid insoluble acid soluble LCC Ca lignosulfonate precip furfural HMF ash acetic acid formic acid xylonic acid mannonic acid glucuronic acid galacturonic acid 4-O-Me-glucuronic acid total in liquorc total in all fractionation streams (% (bof))

spruce chips

pulp

100 62.2 39.6 2.6 1.1 5.6 10.8 2.5 1.5 28.9 28.3 0.6 0.3 1.1 3.0 0.3 97.3

48.7 41.3 38.1 0.4 0.0 1.2 1.6 0.0 1.3 5.0 4.7 0.3 0.0 0.0 0.2 n.m. 47.8

SEW

MSEW

EVAP

STR

LIME

CATOX

29.4 9.9 0.5 2.2 4.7 1.0 1.5 11.6 10.1 1.5

43.2 17.1 0.8 3.7 8.2 1.7 2.7 17.8 15.8 2.0

22.9 16.0 (−6%) 0.8 3.6 7.6 1.5 2.5 4.1 (−77%) 1.3 2.8 12.7

21.2 15.8 (−8%) 0.8 3.5 7.5 1.5 2.5 3.7 (−79%) 0.9 2.8 12.7

0.1 0.0 0.2 0.5 0.0 n.d 0.0 0.3 0.4 0.1 23.1 70.9

0.2 0.1 0.5 1.0 0.0 n.d 0.0 0.3 0.6 0.2 37.8 85.6

0.0 (−99%) 0.1 (−) 0.4 0.4 0.0 (−) n.d 0.0 0.9 1.9 0.6 37.1 84.9

n.d. (>99%) 0.1 (−) 0.4 0.0 0.0 (−) n.d 0.0 0.8 1.5 0.5 35.5 83.3

25.0 15.4 (−10%) 0.8 3.4 7.3 1.5 2.4 3.7 (−79%) 0.9 2.9 12.7 1.8 n.d. (>99%) 0.1 (−) 3.4 0.0 0.0 (−) n.d 0.0 0.7 1.3 0.4 39.6 87.4

23.5 15.4 (−10%) 0.8 3.4 7.3 1.5 2.4 3.0 (−83%) 0.3 2.7 12.7 1.8 n.d. (>99%) 0.1 (−) 2.4 0.0 0.0 (−) n.d 0.0 0.7 1.3 0.4 37.8 85.6

a

n.d., not detected; n.m., not measured. bDry solids content refers to those of liquors and excludes the precipitated materials (LCC and Ca lignosulfonate). cTotal in liquor includes the precipitated materials; values in parentheses show carbohydrate and ABE fermentation inhibitors (lignin, furfural, HMF, formic acid) percent losses/reduction after each conditioning step relative to MSEW liquor composition.

Rational for the Conditioning Scheme. The MSEW liquor resulting from the fractionation is rich in dissolved and hydrolyzed hemicellulose sugars; however, it cannot be directly subjected to fermentation due to the following: 1. high content of SO2 (estimated at about 36 g L−1) which needs to be recovered 2. high content of ethanol (estimated at about 220 g L−1) which needs to be recovered 3. high content of oligomers (35−80% of total carbohydrates) 4. high acidity (pH 1.1/1.6) In order to remove and recover SO2 and ethanol, MSEW liquors were subjected to vacuum evaporation (see Figure 2). During this step 99.9% of ethanol and around 95% of SO2 are

removed, which leads to a 4.4-fold/3.3-fold volume reduction. Some water is also removed. However, after this step about 5 g L−1 SO2 (Table S3 in the Supporting Information) and 1 g L−1 ethanol still remained in the liquor. Therefore, an additional stepsteam strippingwas applied which further reduces the concentrations of SO2 to about 0.1 g L−1 and ethanol to 0.3 g L−1. A small volume reduction (by a factor of about 1.1) is observed during this step. These two consecutive high temperature treatments of the acidic liquorevaporation and steam strippingalso decrease the oligomeric sugar content to about 30%. Further, lime treatment was applied to bring the pH to neutral and a 1.1-fold volume increase took place after addition of washings of the formed precipitate with alkaline water. Finally, the catalytic oxidation step (3.3/5.1 mg of 4355



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Table 5. Mass Balance for Softwood Biomass, Pulp, and Conditioning Liquorsa solid (fiber) phase composition (% (bof)) solid yield carbohydrates cellulose glucan in hemicelluloses arabinan xylan mannan galactan extractives lignin acid insoluble acid soluble ash acetyl groups uronic acids proteins total in solid (fiber) phase liquor composition (% (bof)) dry solids contentb carbohydrates arabinose xylose mannose galactose glucose lignin acid insoluble acid soluble LCC Ca lignosulfonate precip furfural HMF ash acetic acid formic acid xylonic acid gluconic acid glucuronic acid galacturonic acid 4-O-Me-glucuronic acid total in liquorc total in all fractionation streams (% bof)

softwood biomass

pulp

100 51.6 31.2 1.8 4.7 6.0 7.3 3.1 4.8 29.8 28.7 1.1 2.6 0.7 3.0 2.6 95.1

55.2 28.2 25.5 0.3 0.0 1.2 1.1 0.1 1.0 9.7 9.5 0.3 0.6 0.4 0.7 n.m. 40.6

SEW

MSEW

EVAP

STR

LIME

CATOX

21.8 9.8 1.1 2.7 2.9 1.8 1.4 9.5 7.9 1.7

38.8 18.9 1.9 5.3 5.6 3.4 2.7 16.7 14.0 2.7

23.8 14.0 (−26%) 1.5 4.0 4.1 2.4 2.0 4.2 (−75%) 1.9 2.3 11.0

22.1 13.9 (−27%) 1.5 4.0 4.1 2.4 1.9 4.0 (−76%) 1.9 2.1 11.0

0.0 0.0 0.4 0.1 0.0 n.d. 0.1 0.0 1.3 0.5 21.7 62.3

0.1 0.0 1.2 0.3 0.0 n.d. 0.2 0.0 2.9 1.1 41.4 82.0

n.d. (>99%) 0.0 (−) 0.6 0.6 0.0 (−) n.d. 0.2 0.0 2.2 0.9 33.7 74.3

n.d. (>99%) 0.0 (−) 0.6 0.0 0.1 (n.a.) n.d. 0.2 0.0 2.2 0.9 32.9 73.5

22.4 13.1 (−31%) 1.4 3.8 3.8 2.2 1.8 2.6 (−84%) 0.9 1.8 11.0 3.2 n.d. (>99%) 0.0 (−) 2.2 0.0 0.1 (n.a.) n.d. 0.2 0.0 2.0 0.9 35.3 75.9

22.3 13.0 (−31%) 1.4 3.8 3.8 2.2 1.8 2.5 (−85%) 1.1 1.4 11.0 3.2 n.d. (>99%) 0.0 (−) 2.2 0.0 0.1 (n.a.) n.d. 0.2 0.0 2.0 0.9 35.1 75.7

a

n.d., not detected; n.m., not measured; n.a., not applicable. bDry solids content refers to those of liquors and excludes the precipitated materials (LCC and Ca lignosulfonate). cTotal in liquor includes the precipitated materials; values in parentheses show carbohydrate and ABE fermentation inhibitors (lignin, furfural, HMF, formic acid) percent losses/reduction after each conditioning step relative to MSEW liquor composition.

L−1, and the corresponding total amount of lignin is 4.1/4.2% (bof). This major reduction in the total amount of lignin is associated with the LCC precipitate removed after vacuum evaporation. The amount of precipitate formed is equal to 12.7/11.0% (bof), whereas the difference in total lignin before and after evaporation is equal to 13.7/12.5% (bof). According to the sulfur analysis results the LCCs contain 0.49/0.83% sulfur. This content is lower than/comparable to the sulfur content of the char (0.8−1.0% sulfur) obtained during vacuum evaporation of SEW spent liquor produced from spruce fractionated at conditions of L:W = 6:1, 3% SO2, 150 °C, and 120 min.13 The degree of sulfonation of the LCCs is 0.029/ 0.049 S/C9 (Table 3). Lignin remaining in the solution is characterized by a substantially higher degree of sulfonation of

FeSO4·7H2O added to 155/255 mL of LIME liquor) was needed to decrease the SO2 concentration to 10 ppm or below required for fermentation. A 1.1-fold volume reduction was observed during this final conditioning step. Lignin Behavior during Conditioning. Lignin concentrations in each process liquor originating from spruce chips/ softwood biomass are presented in Tables S1 and S2, respectively (Supporting Information). The lignin concentration in the SEW spent liquor from spruce chips/softwood biomass is 59.8/60.6 g L−1, and the corresponding total amount of lignin is 9.9/9.8% (bof). Addition of the pulp washings produces MSEW liquor having a lignin concentration of 21.6/ 20.7 g L−1 and a corresponding amount of lignin of 17.8/16.7% (bof). The lignin concentration in EVAP liquor is 22.0/17.2 g 4356



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products to sugar losses is very small. Concentrations (Tables S1 and S2 in the Supporting Information) and amounts for the latter are presented in Tables 4 and 5 on a feedstock basis. On the basis of the data in Tables 4 and 5, it can be concluded that no significant sugar losses occur during the STR, LIME, and CATOX stages. Therefore, the largest total sugar mass balance loss occurs during vacuum evaporation and all the other liquor conditioning steps have a minor effect on sugar losses. Monomeric and Oligomeric Sugars. The concentrations of monomeric and oligomeric sugars in each liquor produced during the conditioning process are presented in Tables S1 and S2 (Supporting Information). On average, 37.0/79.4% oligomers were found in the SEW liquor. Addition of the pulp washings increases the oligomer content to 49.0% in spruce chips MSEW liquor (in agreement with refs 2 and 6). Addition of the pulp washings does not change the oligomer/ monomer ratio in softwood biomass MSEW liquor. Vacuum evaporation reduces the oligomers to 37.1/57.6% and 31.3/ 34.9% in the EVAP and STR liquor, respectively. Liming by Ca(OH)2 further reduced the oligomers to 20.3/29.1% possibly by entrapment of some of the oligomers in the precipitate, while in CATOX liquor essentially the same percentage (19.6/ 28.6%) of oligomers was present. For all conditioning stages it is seen that the content of monomeric sugars in liquors derived from softwood biomass (compared to spruce) is lower than that derived from spruce wood chips. This can be explained by the lower acidity during SEW fractionation, vacuum evaporation, and steam stripping as evidenced by the pH values in Tables S1 and S2 (Supporting Information). The lower acidity of the softwood biomass liquors is due to the relatively high ash (inorganics) content of this feedstock (as discussed earlier). The oligomer analysis of the spruce chips/softwood biomass CATOX liquor shows that monomers consist mainly of arabinose, xylose, and galactose, while glucomannan is most resistant to hydrolysis containing 27.6/39.5% mannose and 33.5/44.6% glucose in the oligomeric form. This is in agreement with the lower acid hydrolysis rate of methyl mannopyranosides and glucopyranosides compared to methyl xylopyranosides and galactopyranosides.26 Overall, it is demonstrated that in the course of the conditioning the monomeric sugar concentration is approximately doubled, i.e., from 35.3/22.6 g L−1 in SEW spent liquor to 72.6/50.8 g L−1 in the final CATOX liquor. This increase is beneficial for the fermentation and economy of the biofuel production process. Furanic Compounds. The furfural and hydroxymethylfurfural (HMF) analysis results are listed in Tables S1 and S2 (Supporting Information) and Tables 4 and 5. The concentrations of these compounds in SEW liquor are 0.5/ 0.2 and 0.2/0.1 g L−1, respectively, or 0.1/0.0 and 0.0/0.0% (bof). Accordingly, pentose and hexose sugar dehydration during SEW fractionation is minimal. After addition of the pulp washings furfural and HMF concentrations decrease due to dilution, i.e., at 0.2/0.1 and 0.1/0.0 g L−1, respectively, in MSEW liquor. After vacuum evaporation, the furfural concentration in the EVAP liquor is at zero/nondetect levelsdespite the associated volume reduction by a factor of 4.4/3.3because of evaporation of furfural as an azeotrope (35% furfural in water at 97.85 °C27). The HMF concentration, on the other, hand increases to 0.5/0.2 g L−1 in EVAP liquor because HMF is not volatile by steam.28 Also, more HMF may be formed during vacuum evaporation from the C6 sugars. Hence a part of the earlier identified sugar losses occurring

0.52/0.51 S/C9 (Table 3), corresponding to the common values for acid sulfite dissolved lignin (0.5−0.725). This indicates that lignin is sulfonated nonuniformly, and the less sulfonated portion precipitates when ethanol is removed. The formation of LCC precipitates instead of precipitated condensed lignin (char) is probably due to addition of the pulp washings to SEW spent liquor. It seems that the lower acidity of the MSEW liquor compared to SEW liquor prevents lignin condensation during vacuum evaporation. The lignin concentration in STR, LIME, and CATOX liquors is progressively reduced with the lignin amount on feedstock of 3.7/4.0, 3.7/2.6, and 3.0/2.5% (bof) respectively. About 90% of the lignin present in CATOX liquors remains in soluble form after double stage H2SO4 acid hydrolysis. Lignin concentrations in the CATOX liquors are 16.5 and 10.6 g L−1, respectively (Tables S1 and S2 in the Supporting Information). Total Sugars. Total sugar concentrations, i.e., the sums of the monomers and oligomers (but calculated as monomers) for each of the five hemicellulose derived neutral sugars are also presented in Tables S1 and S2 (Supporting Information). Accordingly, the total sugar concentrations in SEW, MSEW, EVAP, STR, LIME, and CATOX liquors are 56.8/69.4, 23.2/ 26.2, 95.9/63.8, 95.1/69.2, 88.2/58.8, and 94.8/61.6 g L−1, respectively. The differences in sugar concentrations between various conditioning steps are mostly governed by the corresponding volume changes. However, some sugar losses occur during evaporation and liming (see the section Total Sugar Mass Balance (as Anhydro Sugars)). Total Sugar Mass Balance (as Anhydro Sugars). The anhydro sugar mass balance for each stage of the conditioning process is presented in Tables 4 and 5. The total amount of anhydro sugars based on the original amount in wood/ softwood biomass in the SEW spent liquor is 9.9/9.8%. Anhydro sugars in MSEW liquor are present at a level of 17.1/ 18.9% (bof). Their amount is nearly double the amount of anhydro sugars found in SEW spent liquor, indicating that a substantial amount of dissolved sugars remains in the pulp after drainage when using a L/W ratio of 3 L/kg (bof). It also indicates that the majority of these sugars are efficiently removed by washing. The numerical difference between the carbohydrate content in the original feedstock and pulp is 20.9/ 23.4% (bof) (Tables 4 and 5), and this amount is fairly close to the anhydro sugar content of the MSEW spent liquor (17.1/ 18.9% (bof)). The difference observed can be mostly explained by the residual organics still remaining in the pulp after washing. Previously it has been shown that no significant loss in sugars occurs for SEW cooking of spruce chips and softwood biomass.2,5,8 The amount of anhydro sugars in the EVAP liquor is reduced to 16.0/14.0% (bof). For spruce chips EVAP liquor the sugar losses amount to only 6% relative to MSEW liquor; however, for softwood biomass EVAP liquor the sugar losses are much higher, i.e., at 26% relative to MSEW liquor. These sugar losses are presumably due to coprecipitation of sugars with lignin (as LCC precipitates) in both cases. It seems, though, that the lower acidity of the softwood biomass EVAP liquor (pH 1.6 vs pH 0.8 for spruce EVAP liquor) results in suppressed hydrolysis of the LCC precipitate. Thus less sugars are released from the LCC precipitate during this conditioning step with a profound effect on sugar losses. The major sugar degradation products are furfural (pentose dehydration) and hydroxymethylfurfural (HMF; hexose dehydration). Other degradation products include formic and aldonic acids. However, the overall contribution of sugar degradation 4357



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CATOX liquor. The sulfate anion concentration in the MSEW liquor of 0.9/0.8 g L−1 is due to oxidation of SO2 in the SEW stage. During evaporation, steam stripping, and liming the amount of sulfate (bof) remains approximately constant. However, after the final catalytic oxidation step the sulfate anion concentration increases from 3.1/2.7 to 3.8/3.4 g L−1 in the CATOX liquor due to oxidation of SO2 by oxygen in the presence of the iron catalyst (FeSO4·7H2O) leading to elimination of SO2. The SO2 mass balance calculations based on Table S3 (Supporting Information) show that it is possible using the present procedure to reduce the SO2 content of the liquor by 4 orders of magnitude from 12% to 10 ppm which includes the final 10-fold reduction of 126/109 ppm SO2 to 10 ppm/ nondetect levels by the liming and catalytic oxidation. ABE Fermentation. The conditioned liquors were treated with ASS 01G Cl− FINEX anionic resins at a resin-to-liquor ratio of 1:1.5 prior to fermentation trials to further remove lignin. ABE fermentation tests using a patent pending fermentation column that uses wood pulp as cell immobilization material31 show that the conditioned liquors are fermentable by Clostridia bacteria upon 4-fold dilution and supplementation of glucose to a level of 35 g L−1 glucose. This supplementation takes into account the glucose feed stream, obtained by enzymatic hydrolysis of the pulp fibers, which will be mixed with the conditioned SEW spent liquor. Acetone, butanol, and ethanol are produced at a ratio of 3:6:1 and a maximum total concentration of approximately 13 g L−1. A solvent total yield of around 0.25 g/g sugars is obtained (maximum theoretical yield is around 0.4 g/g sugars). Ongoing work with metabolically engineered Clostridia bacteria demonstrates that it is possible to produce 2-propanol instead of acetone in the mixture of solvents. The former would be more beneficial for the present process because acetone cannot be used as a biofuel.

during vacuum evaporation may be due to formation and evaporation of furfural and formation of HMF. Evidence of HMF degradation by the formation of formic acid is observed during vacuum evaporation of spruce chips EVAP liquor, as discussed under Other Compounds Present in Conditioned Liquors. Furfural was not detectable in the STR, LIME, and CATOX liquors for spruce chips/softwood biomass, confirming that furfural is rapidly removed during steam stripping. The HMF concentration in STR, LIME, and CATOX liquors is gradually reduced to 0.3/0.2 g L−1 in CATOX liquor, well below the harmful levels for Clostridia bacteria (1.5 g L−1 29). Other Compounds Present in Conditioned Liquors. The acetic acid concentration is 2.4/0.7 g L−1 or 0.5/0.1% (bof), and the formic acid concentration is 0.2/0.0 g L−1 in SEW liquor (Tables S1 and S2 in the Supporting Information, Tables 4 and 5). After addition of the pulp washings acetic and formic acid concentrations are at 1.3/1.8 and 0.1/0.0 g L−1, respectively, in MSEW liquor. On the original feedstock basis the values are 1.0/0.3 and 0.0/0.0%, respectively. After vacuum evaporation, the acetic acid concentration in the EVAP liquor is increased to 2.1/3.5 g L−1 due to the associated volume reduction. Based on original feedstock, this is only 0.4/0.6%. The formic acid concentration in spruce EVAP liquor increases to 0.2 g L−1 due to volume reduction. Minimal HMF degradation to formic acid occurs during vacuum evaporation of softwood biomass EVAP liquor because of its lower acidity. Some additionalstill insignificantHMF degradation is observed during steam stripping of softwood biomass EVAP liquor. Concentrations of formic and acetic acids are at negligible levels in the subsequent process liquors for spruce chips/softwood biomass. Total Mass Balance. The total mass balance based on comparison of the identified components in the original spruce chips/softwood biomass, pulp, and conditioning liquors is presented in Tables 4 and 5. From these tables it is seen that the dry solids content of all liquors corresponds reasonably well to the total identified components. However, there are some discrepancies when comparing total mass balances after each conditioning step. For example, the sum of the identified components in the pulp and MSEW spent liquor is 85.6/82.0%, which is less than the original 97.3/95.1% total for the original spruce chips and softwood biomass. The sum of the mass of the components in the EVAP liquor, pulp, and LCC precipitate decreases to 84.9/74.3% mainly due to the earlier discussed sugar losses. At the end of the conditioning process, the total solids identified account for 85.6/75.7% of the original spruce chips/softwood biomass weight. The amount of Ca lignosulfonate precipitate formed after liming is also included in the mass balance calculations as it is mostly lignosulfonate. Inorganic Sulfur Species. The quantification of sulfite and sulfate anions is presented in Table S3 (Supporting Information). It is shown that the SO2 concentration in the fresh cooking liquor is approximately 120 g kg−1, representing a charge of 33.3% (feedstock). This value is based on the originally added weight of SO2 when taking into account the density of the fresh cooking liquor of 0.957 kg L−1. The measured concentration of SO2 in the SEW and MSEW liquors is low because significant SO2 losses to the atmosphere occur during the handling of the liquors and the solids. It was shown earlier that all free SO2 (i.e., SO2 not bound to lignin) can be recovered by distillation.2,30 The SO2 concentration is 101/87 ppm in the STR liquor, decreases to 20/25 ppm in the LIME liquor, and finally decreases to 10 ppm/nondetect levels in the

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CONCLUSIONS Our study shows that the proposed industry optimized scheme for SEW fractionation of spruce chips/softwood biomass and spent liquor conditioning for ABE fermentation can be successfully applied to produce conditioned liquors rich in hemicellulose monosugars. At the same time the conditioning process allows removal of major inhibitors for ABE fermentation, including recovery of the fractionation chemicals, i.e., ethanol and SO2. Fermentation tests confirm that the conditioned liquors can be successfully fermented to produce ABE solvents at a high yield. It is, however, evident that the conditioning scheme can be further optimized in the following areas: 1. One area for optimization is more efficient fractionation of heterogeneous feedstocks such as softwood biomass. The present study demonstrates that the higher ash (inorganics) content of biomass compared to stem wood can have a significant effect on the hydrolysis of sugars to monomers and other aspects of the conditioning process. 2. More complete lignin removal during conditioning so that the conditioned liquor can be utilized without dilution for ABE fermentation is a second area for optimization. However, it should be noted that if ethanol is produced instead of ABE solvents, the lignin inhibition would not be an obstacle, as fermentation by yeast is routinely applied in sulfite mills in the presence of dissolved lignosulfonates. 4358



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Proceedings), Hamburg, Germany, Aug 16−19, 2010; DeGruyter: Berlin, 2011; pp 205−208. (12) Wang, L.; Chen, H. Increased fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production by removal of fermentation inhibitors. Process Biochem. (Oxford, U. K.) 2011, 46 (2), 604−607. (13) Sklavounos, E.; Iakovlev, M.; Yamamoto, M.; Teräsvuori, L.; Jurgens, G.; Granström, T.; van Heiningen, A. Conditioning of SO2ethanol-water spent liquor from spruce for the production of chemicals by ABE fermentation. Holzforschung 2011, 65 (4), 551−558. (14) Iakovlev, M.; Sixta, H.; van Heiningen, A. SO2-Ethanol-Water (SEW) pulping: II. Kinetics for spruce, beech, and wheat straw. J. Wood Chem. Technol. 2011, 31 (3), 250−266. (15) Iakovlev, M.; van Heiningen, A. SO2 ethanol-water (SEW) pulping: I. Lignin determination in pulps and liquors. J. Wood Chem. Technol. 2011, 31 (3), 233−249. (16) Janson, J. Analytik der Polysaccharide in Holz und Zellstoff. Faserforsch. Textiltech. 1974, 25, 375−382. (17) Sundberg, A.; Sundberg, K.; Lillandt, C.; Holmbom, B. Determination of hemicelluloses and pectins in wood and pulp fibers by acid methanolysis and gas chromatography. Nord. Pulp Pap. Res. J. 1996, 11 (4), 216−219. (18) Mariotti, F.; Tomé, D.; Mirand, P. P. Converting Nitrogen into proteinBeyond 6.25 and Jones’ Factors. Food Sci. Nutr. 2008, 48, 177−184. (19) Rakkolainen, M.; Iakovlev, M.; Teräsvuori, A.-L.; Sklavounos, E.; Jurgens, G.; Granström, T. B.; van Heiningen, A. SO2-ethanol-water fractionation of forest biomass and implications for biofuel production by ABE fermentation. Cellul. Chem. Technol. 2010, 44 (4−6), 139− 145. (20) Werkelin, J.; Lindberg, D.; Bostöm, D.; Skrifvars, B. J.; Hupa, M. Ash-forming elements in four Scandinavian wood species part 3: Combustion of five spruce samples. Biomass Bioenergy 2011, 35, 725− 733. (21) Fengel, D.; Wegener, G. WoodChemistry, Ultrastructure, Reactions; Kessel Verlag: Remagen, Germany, 2003. (22) Iakovlev, M.; Päak̈ könen, T.; van Heiningen, A. Kinetics of SO2ethanol-water pulping of spruce. Holzforschung 2009, 63 (6), 779− 784. (23) Da Silva Perez, D.; van Heiningen, A. R. P. Seventh European Workshop on Lignocellulosics and Pulp (EWLP, Proceedings); Åbo Akademi University: Turku, Finland, 2002; pp 393−396. (24) Rydholm, S. A. Pulping Processes; John Wiley and Sons, Inc.: New York, 1965; p 108. (25) Rydholm, S. A. Pulping Processes; John Wiley and Sons, Inc.: New York, 1965; p 467. (26) Feather, M. S.; Harris, J. F. The acid-catalyzed hydrolysis of glycopyranosides. J. Org. Chem. 1965, 30 (1), 153−157. (27) Zeitsch, K. J. The Chemistry and Technology of Furfural and Its Many By-products; Elsevier: Amsterdam, 2000; p 75. (28) Sjöström, E. Wood Chemistry: Fundamentals and Applications; Academic Press: New York, 1993, p231. (29) Teräsvuori, L. Unpublished results, 2010. (30) Iakovlev, M.; You, X.; Sklavounos, E.; van Heiningen, A.; Sixta, H. Unpublished results, 2012. (31) Survase, S.; Sklavounos, E.; Jurgens, G.; van Heiningen, A.; Granström, T. Continuous acetone−butanol−ethanol fermentation using SO2 ethanol−water spent liquor from spruce. Bioresour. Technol. 2011, 102 (23), 10996−11002.

The suggested conditioning scheme also allows producing two lignin streams: high molecular weight/low sulfonated lignin obtained after ethanol evaporation and low molecular weight/ highly sulfonated lignin remaining in soluble form. Possible applications of these fractions are also the subject of future research.

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ASSOCIATED CONTENT

S Supporting Information *

Tables listing compositions of the liquors derived from spruce chips and softwood biomass and IC analysis results for spruce/ softwood biomass liquors. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +358 50 512 4224. E-mail:evangelos.sklavounos@aalto.fi. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Myrtel Kåll, Heikki Tulokas, and Xiang You for their assistance with the IC, HPAEC, and viscosity/kappa number analyses. Financial support by TEKES (Finnish Funding Agency for Technology and Innovation), ABB, American Process Inc., Kemira, Neste Oil, Stora Enso, and UPM through the SEWIBE project is greatly appreciated. We thank Prof. Raimo Alén for providing Schö n iger combustion apparatus. We also thank Dr. Tom Granström, Dr. German Jurgens, and Dr. Shrikant Survase for performing the ABE fermentation tests.

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REFERENCES

(1) Schorning, P. Studies of sulphite pulping of wood without base. Faserforsch. Textiltech. 1957, 8, 487−494. (2) Iakovlev, M.; van Heiningen, A. Efficient fractionation of spruce by SO2-Ethanol-Water treatment: closed mass balances for carbohydrates and sulfur. ChemSusChem 2012, 5, 1625−1637. (3) Iakovlev, M.; van Heiningen, A. Kinetics of fractionation by SO2Ethanol-Water (SEW) treatment: understanding the deconstruction of spruce wood chips. RSC Adv. 2012, 2, 3057−3068. (4) Retsina, T.; Pylkkänen, V. Back to the biorefinery: a novel approach to boost pulp mill profits. Paper360° 2007, No. Feb, 18−19. (5) Yamamoto, M.; Sklavounos, E.; Survase, S.; Jurgens, G.; Granström, T.; van Heiningen, A. Biobutanol from forest residues a process utilizing SO2 -ethanol-water fractionation and ABE fermentation. In The 4th Nordic Wood Biorefinery Conference (NWBC Proceedings), Helsinki, Finland, Oct 23−25, 2012; VTT: Espoo, Finland, 2012; pp 205−210. (6) Yamamoto, M.; Iakovlev, M.; van Heiningen, A. Unpublished results, 2012. (7) Iakovlev M. SO2-Ethanol-Water Fractionation of Lignocellulosics. Doctoral Thesis, Aalto University, Finland, 2011. http://lib.tkk.fi/ Diss/2011/isbn9789526043142/isbn9789526043142.pdf. (8) Yamamoto, M.; Iakovlev, M.; van Heiningen, A. Total mass balances of SO2−ethanol−water (SEW) fractionation of forest biomass. Holzforschung 2011, 65 (4), 559−565. (9) Jurgens, G.; Survase, S.; Berezina, O.; Sklavounos, E.; Linnekoski, J.; Kurkijärvi, A.; Väkevä, M.; van Heiningen, A.; Granström, T. Butanol production from lignocellulosics. Biotechnol. Lett. 2012, 1−20. (10) Dürre, P. Fermentative butanol production: bulk chemical and biofuel. Ann. N. Y. Acad. Sci. 2008, 1125, 353−362. (11) Sklavounos, E., van Heiningen, A. SO2-ethanol-water fractionation of spruce and spent liquor conditioning for ABE fermentation. In The 11th European Workshop on Lignocellulosics and Pulp (EWLP, 4359


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