Ind. Eng. Chem. Res. 2007, 46, 7711-7720
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SO2-Catalyzed Steam Fractionation of Aspen Chips for Bioethanol Production: Optimization of the Catalyst Impregnation Isabella De Bari,* Francesco Nanna, and Giacobbe Braccio ENEA-National Agency for New Technology, Energy, and EnVironment, Policoro (MT) 75025, Italy
The pretreatment step has a key role in the enzymatic conversion of lignocellulosic biomass to bioethanol. Steam pulping of biomass has long been recognized as being effective in producing high biomass fractionation. In this step, the preliminary impregnation of biomass with acid catalysts, including SO2, has been shown to further improve the hydrolytic effect and increase the digestibility of the fibers by enzymes. This work focused on developing an experimental setup for the SO2 impregnation, enabling accurate control of the process variables. The final purpose was the assessment of a method to minimize the quantity of SO2 used, and that could be applicable to continuous operations. Aspen chips were impregnated with the acid catalyst at room temperature in a stainless steel batch reactor, and the influence of the biomass humidity and contact time on SO2 uptake was explored. After the catalyst adsorption, biomass was fed into the steam explosion batch reactor and steamed at 205 °C for 3 and 10 min to obtain slurries. The addition of catalyst (∼0.9% w/w raw material dry matter) reduced the degree of polymerization of the cellulose by 50%, on average. The highest yield of xylose (10.3 g/100 dry chips) was obtained via water extraction following a steam pretreatment of 3 min. The amount of monomer xylose was 80% of the total extracted dry matter. Simultaneous saccharification and fermentation (SSF) of the washed fibers yielded 37 g of glucose/100 g dry chips, 96% of which fermented to ethanol. Introduction Recently, escalating international tensions have resulted in a progressive increase in oil prices, thus renewing interest in the production and use of biofuels (bioethanol, biodiesel). The major benefit of replacing fossil fuels with these new-generation fuels produced from biomass is that they can be obtained from locally available bioresources, such as dedicated crops and/or lignocellulosic residues. In southern Europe, hardwoods are a valuable bioresource for ethanol production. Our previous work on ethanol production through the enzymatic conversion of steam-pretreated aspen was focused mainly on the cellulose conversion. At the same time, it anticipated the importance of optimizing also the recovery of hemicellulose to make the overall process more convenient.1 Since the 1990s, ENEA has investigated the steam explosion (SE) treatment, using the continuous pilot digester patented by StakeTech (Canada). The efficiency of this technology is due to the combination of the hydrothermal and mechanical effects that cause autohydrolysis reactions in which part of hemicellulose and lignin are converted to soluble oligomers. However, some lignocellulosic biomass, especially that with high lignin content, are often recalcitrant to SE pretreatment, thus requiring severe process conditions (high steaming temperature and/or prolonged treatment time). These may help to overcome this drawback but, at the same time, reduce the sugars recovery, mainly at the expense of the thermolabile carbohydrates of hemicellulose. Thus, recovery and utilization of the water-soluble hemicellulose component has been thoroughly investigated recently. Some publications of the mid-1980s already proved that biomass impregnation with sulfuric acid, combined with mild SE pretreatments, improves the enzymatic hydrolizability of water-insoluble fibers and yields higher sugars recovery from water-soluble hemicellulose.2,3 To date, pretreatment with H2SO4 impregnation is among the * To whom correspondence should be addressed. Tel.: +39 835974313. Fax: +39 835974210. E-mail address: isabella.debari@ trisaia.enea.it.
processes that have been scaled up.4 However, especially in case of hardwood varieties, impregnation with gaseous SO2 proved more effective, compared to H2SO4, because it ensures higher recovery of the most abundant component (glucose).5 Furthermore, it is also less corrosive than acid solutions. Remarkable advantages in using SO2 impregnation have also been claimed recently for several varieties of softwoods,6-8 corn fiber,9 and sugar cane bagasse.10 Overall, depending on the composition of the raw material, several concentrations of catalyst have been explored, in combination with a wide range of pretreatment conditions. The major part of the methods for the catalyst impregnation described in the literature consists of bench-scale procedures. Generally, they utilize plastic bags filled with SO2, where biomass remains in contact with the catalyst from a few dozen minutes to overnight. However, if the impregnation is done outside the steam reactor, the actual level of SO2 retained in the biomass is expected to be lower than projected, because of its loss during the transfer into the digester. Alternatively, the addition of SO2 directly into the steam reactor was considered unsuitable, because of the high temperature of the reactor.12 Thus, as consequence of the aforementioned remarks, different experimental setups could not make even nominally equivalent SO2-catalyzed pretreatments comparable. Additional topics to be addressed include the following: Does the biomass moisture have a role in the SO2 uptake? What is the minimum gas exposition time to achieve the highest uptake? What is the saturation threshold of the chips? These variables may be relevant for scaling the process up to larger scale. To the best of our knowledge, only one recent publication explicitly made the distinction between SO2 loaded and SO2 adsorbed.12 However, no detailed descriptions were given about the impregnation conditions. The objective of this work is the development of an experimental setup for the SO2 impregnation that could be also considered for continuous operations. This was made using a pressurizable reactor that was built for the occasion which
10.1021/ie0701120 CCC: $37.00 © 2007 American Chemical Society Published on Web 10/06/2007
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Figure 2. Experimental apparatus for SO2 impregnation.
Figure 1. Flow sheet of SO2-catalyzed steam pretreatment of aspen chips.
enables an accurate control of the process operations. Although the system operates in a batch manner that is not interfaceable with the technology of the continuous SE digester, it represents a first attempt of scaling up the corresponding bench-scale operation. The effect of the minimum concentration of the catalyst in the pores of the chips during the steam pretreatment was then assessed based on the cellulose enzymatic digestibility and the recovery of highly hydrolyzed hemicellulose. This research was started in mid-2002 as completion of the previous investigation on steam treatment of aspen chips for ethanol production.1 Further research on different biomass feedstocks is ongoing. Experimental Section The Process Flow-Sheet. The bioconversion of aspen chips into ethanol has been accomplished according to the process flow sheet outlined in Figure 1. Chips were first humidified and then impregnated with SO2, using a batch reactor. The catalyst excess was desorbed and the SO2-saturated chips were exploded in a 10-L steam reactor at 205 °C at two treatment times: 3 and 10 min. Steam pretreatments without any catalyst were performed for comparison. The steam-exploded slurries were washed with warm water and the fibers were converted to bioethanol via simultaneous saccharification and fermentation (SSF). The water-extracted oligomers were subjected to a further hydrolysis (hereinafter referred to as post-hydrolysis) with different amounts of sulfuric acid. The concentrations of monosaccharides were determined before and after the posthydrolysis. The effect of the catalyst impregnation on the substrate was discussed in terms of both the cellulose degree of polymerization (DP) and the ratio of monomer-to-total sugars in the water extracts. In addition to the sugar compositions, the concentration of the main degradation byproducts in the different conditions was also investigated. Finally, the fate of the inorganic catalyst after SE was determined by analyzing the concentration of sulfites and sulfates generated by the pretreatment. Feedstock. Aspen chips were retrieved from local sawmills and did not contain bark. At the moment of their use, the average moisture was 15%.
Acid Impregnation Setup. The impregnation with SO2 was performed in a 30-L reactor appositely built (Figure 2). It consists of a stainless steel vessel, hermetically sealed and equipped with a manometer and an inner perforated basket to facilitate the biomass loading and unloading. Taking into account the volume of the basket and the total volume occupied by the chips (the bulk density is roughly 600 kg/m3), the maximum volume for the gas expansion was around 24-25 L. The entire apparatus was located on a high resolution industrial weighing platform provided by Sartorius Corporation (ISI 10). The device has a maximum weight capacity of 150 Kg and was calibrated in the interval 0-40 kg by the following accurate checking procedures: UNI CEI EN 45501. The expanded uncertainty taken at a confidence level of 95% was 1.5 g. Before starting the tests, gas leaks were checked. Almost 2 kg of biomass dry matter (DM) was used for each impregnation cycle. After loading, the vessel was sealed and the gas was injected until a weight increase of ∼4%-5% of the biomass DM was displayed. Presteaming and prehumidification of the raw material were both considered as possible options aimed at improving the SO2 uptake. Presteaming was performed in a vertical laboratory autoclave at 120 °C (1 bar) for 5-10 min. Biomass Moistening. The moisture content in biomass is one of the most important variables that can affect the SE pretreatment efficiency. The optimal content for woody feedstock usually is in the range of 35%-40%. In particular, if the SE process is conducted in continuous mode, this humidity range ensures the formation of a “biomass cap” of such a consistence that it can seal the steam gun. In addition to assisting the hydrothermal effect, water in biomass could have a further role when acid catalysts are used. In fact, these species are highly water-soluble and higher levels of the gaseous catalyst could, in principle, be adsorbed at higher moisture contents. Furthermore, water could better drain the acid within the fibers thus ensuring a more homogeneous action which is determining especially for short steaming times. For this reason, the effect of biomass water content in the range of 30%-50% on the SO2 uptake has been investigated. In the continuous pretreatment pilot station at ENEA, biomass can be automatically weighed and humidified up to a set value, by means of several water jets through the biomass conveyor screw. To reproduce this continuous operation on the batch digester used for the present investigation, humidification was done by spraying known amounts of water on the chips spread out on a plastic film. The chips then were repeatedly mixed to ensure a homogeneous water distribution. The substrate imbued with water was stored for 1 h in polyethylene bags, to allow the water to penetrate the chips. After this procedure, the DM percentage was uniform
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across the feedstock, with an acceptable error of 1%-2%. The maximum amount of water adsorbed using this procedure was 38%. Water saturation of wood was achieved by soaking the wood in water for 10 min. The resultant moisture was 51%. Overall, the effect of the biomass DM on the SO2 uptake was investigated over three moisture contents: 29%, 38%, and 51%. More than water, the use of steam to increase the biomass humidity could contribute to clear the chips pores, making the biomass-gas interaction more effective. Tests were made to homogeneously inject steam through the biomass bed without exploding it. Despite several attempts, the experimentation failed, because the autoclave configuration used generated a moisture gradient through the biomass bed height. (The data are available in the Supporting Information.) The chips that were closer to the steam source at the bottom of the autoclave acted as an adsorbent and prevented the upper biomass layers from being humidified. Therefore, only humidification with water was considered. The humidity value corresponding to each setup was calculated as mean of four tests. The variations between the samples and within the samples were of the same order of magnitude (1%). After the pressure drop due to the SO2 adsorption in the chamber had reached a constant value, the vessel was opened and the chips were removed at regular intervals of time and monitored for their weight loss until steady state was achieved. Besides the weight measurements, also the pressure diminution were used to estimate the adsorbed gas moles through the compressibility factor equation of state (1):
P×V)z×n×R×T
(1)
where P is the pressure (in atmospheres), V the system volume (in liters), z, the compressibility factor for SO2 (0.269), R the gas constant (0.08207 liters atm mol-1 K-1), and T the temperature (in Kelvin). In some experiments, the formation of water condensation at the bottom of the vessel was observed. Usually, they corresponded to 1%-1.7% of the overall biomass moisture and, therefore, were neglected. The actual SO2 uptake was estimated based on the final constant weight. It was referred to as the equilibrium concentration of SO2 within the wood pores. The weight variations following the catalyst adsorption were between 15 and 130 higher than expanded uncertainty of the balance. Thus, the conclusions derived from them can be considered reliable. Pretreatment by Steam Explosion. Aspen chips were steamexploded within a 10-L batch reactor (StakeTech, Ltd., Canada). The treatment severity was quantified by a semiempirical parameter called the severity parameter (R0), which combined the treatment time and the temperature according to eq 2:13,14
R0 ) t exp
- 100 (T14.75 )
(2)
where t is the time (in minutes) and T the temperature (in degrees Celsius). The reactor can process 1 kg of biomass per cycle, and the volatiles released during the decompression phase were recovered by means of a condensing coil. The conditions that were used and the corresponding values of the R0 logarithm are summarized in Table 1. In the previous investigation on poplar, the highest cellulose fragmentation (lowest DP) was achieved in the range of severity parameters of log R0 ) 3.60-4.10.1 Therefore, the temperature and the two steaming times were chosen so that the corresponding severity parameters were
Table 1. Experimental Conditions (Steam Temperature and Residence Time) during the Steam Explosion of Aspen Chips with and without SO2 Impregnation
description of products
steam temp, T (°C)
residence time, t (min)
log R0
substrate ID
aspen chips + SO2 aspen chips + SO2 aspen chips without SO2 aspen chips without SO2
205 205 205 205
10 3 10 3
4.09 3.57 4.09 3.57
A B C D
consistent with the lower and upper extremes of the previous investigation. Each explosion test was repeated three times. The slurries collected from each run were thoroughly homogenized and stored at -18 °C until use (some photos of the biomass after the steam explosion pretratment are viewable in the Supporting Information). Chemical Analysis of the Process Streams. The biomass was analyzed before and after each pretreatment using the following standard procedures: modified ASTM D-1348-94 Method B for moisture content; ASTM D-1102-84 for ash content; modified CPPA G.13 and modified ASTM D-110784 for ethanol/toluene extractives; and modified TAPPI T 13 m-54 and ASTM D-1106-84 for Klason lignin in the extractives-free samples. The procedures details have already been described elsewhere.15,16 The carbohydrates were analyzed by NaOH gradient elution (2-200 mM) on a Dionex anion-exchange column (carbopac PA1, kept at 28 °C), followed by electrochemical detection on a ED40 system. The percentage compositions reported in the present paper are the means of six values (two replicates for three samples of each substrate). The water extracts were characterized for their content in soluble carbohydrates, main degradation byproducts, and sulfites and sulfates. The concentrations of 5-HMF (hydroxymethylfurfural), furfural, and acetic acid were determined by HPLCDAD analysis (HP 1100 series) equipped with a RP-C18 Phenomenex SYNERGI column (4µ Hydro RP 80 A). The quantification was conducted at two different wavelengths, 205 and 280 nm for acids and aldehydes, respectively. Sulfites and sulfates were quantified using an ionic chromatograph that was equipped with an AS 12 Ionpac column and a conductibility detector. The DP was calculated through viscosity measurements (ISO5351/1-1981standard method). The relationship between DP and intrinsic viscosity was expressed according to eq 3:17
DP ) 190[η]
(3)
Assessment of the Hemicelluloses Hydrolysis Extent. To quantify the monomer-to-total sugars ratio in the hemicelluloserich water streams, post-hydrolysis was performed by autoclaving those streams at 120 °C and 1 bar for 45 min. Increasing concentrations of H2SO4 (0-3% w/w) were used to ascertain the lowest amount of acid yielding the maximum sugars recovery. Enzymatic Digestibility of the Steam-Exploded Products. The enzymatic digestibility of the steam-exploded products was investigated by means of SSF tests, using 250-mL Erlenmeyer flasks that were kept at 35 °C in an orbital shaken incubator (180 rpm). Concentrated biomass suspensions (solid-to-liquid ratio of 0.13 g/g) were inoculated with 3 g/L Saccharomyces cereVisiae (Sigma, type I) and supplemented with yeast extract (2.5 g/L), (NH4)2HPO4 (0.25 g/L), and MgSO4‚7H2O (0.025 g/L). The tests were conducted in media buffered with 50 mM
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Figure 3. SO2 pressure decay inside the impregnation chamber for biomass batches with different levels of humidity.
acetic acid/sodium acetate solution. Two cellulolytic mixtures (Celluclast 1.5L and Novozym 188 (Novo Nordisk)) were used to hydrolyze the cellulose in the washed biomass. The enzyme preparations were added to each flask to obtain 12.6 FPU/g of substrate and 64.6 IU/g. The enzymes dosage for this biomass has been previously investigated and optimized.1 Samples for the analysis were withdrawn from the flasks and centrifuged at 3000 rpm for 5 min, and the surnatant was deproteinized with HClO4. The quantification of glucose and ethanol in the fermentation broths was performed soon afterward with the ionic chromatograph (DIONEX DX300) using a Nucleogel 300 OA column and an electrochemical detector (PED). The system was equipped with a fully automated autosampler that was provided with the Peltier cooling (10 °C), thus slowing down any possible further hydrolysis and fermentation. The fermentation yields were computed taking into account that the theoretical ethanol yield from glucose is considered to be 0.51 (g ethanol/g of glucose). Mannose was neglected because only traces remained in the steam-pretreated products after washing. The overall hydrolysis yields during the SSF tests were estimated by summing the residual glucose at the end of the process and the glucose that had been converted to ethanol (i.e., [glucose] ) [ethanol]/0.51). Results and Discussions Optimization of the Acid-Catalyzed Steam Pretreatment. The objective of the impregnation setup proposed in this paper was to improve the SO2 penetration within the fibers and to use the minimum possible level of catalyst. This is indented as the amount of catalyst that was effectively retained inside the porous lignocellulosic structure and, therefore, was available to catalyze the hydrolytic reactions. To the best of our knowledge, this topic has not been examined in detail in previous works; however, it is important for reducing catalyst consumption and cost. Figure 3 shows the SO2 pressure decay inside the adsorption chamber for substrates with different moistures. The initial pressure inside the chamber was 500 mbar. The final pressure after 24 h for all the conditions explored was 250 mbar. Only slight differences could be appreciated in the experimental data, mainly because of the statistical reproducibility. To render the graph legible, only one set of error bars was displayed. The uncertainties on the pressure measurements for the biomass moistures of 38% and 51% were of the same order of magnitude, namely, 4%-8%. In all the cases, the majority of the pressure decrease occurred in the first 15 min and it reached a constant value within 2 h. Figure 4 shows the percentage of SO2 desorbed from substrates after the vessel opening, and Figure 5 summarizes the amounts of SO2 in the substrates during the three impregnation stages (initial loading, adsorbed amount, deeply retained amount). The same experiments were performed using
Figure 4. Kinetics of SO2 desorption from chips with different levels of humidity.
Figure 5. Percentage of SO2 in biomass during the impregnation stages as a function of chips humidity.
acid-free moist chips to ascertain the extent of moisture loss and its influence on the calculation of the SO2 that is lost. The results show that the weight diminutions only due to the moisture loss were at least 12-fold lower than those due to both moisture and SO2 loss. (The data are available in the Supporting Information.) Also, the pressure diminutions could be used to calculate approximately the adsorbed gas moles through the compressibility factor equation of state accounting for the real gas behavior. The amounts estimated in this way were then compared with those corresponding to the weight measurements. The two approaches yielded discrepant results with the estimations by pressure being 0.4%-0.7% higher than those estimated by weight. Nevertheless, the general trend of adsorbed SO2 versus biomass humidity was the same as it is represented by the white bars in Figure 5. The discrepancy in the absolute values can be due to the approximations adopted in the calculation through the compressibility factor equation and/or to some instantaneous desorption occurred when the gas pressure was released. In any case, it is worth noting that both the weight and the pressure measurements indicated that only a part of the gas injected was actually adsorbed (at most, 60%) even after a prolonged gas-to-biomass contact time. In the following analysis, only the percentages relevant to the weight measurements were considered, because they were derived from direct measurements without any intermediate estimation. Because the data may have been affected by uncontrollable changes in the air temperature (25-28 °C) during the measurement, they are merely intended to capture a possible trend. The gas release kinetics shown in Figure 4 seems to be independent of the biomass moisture, suggesting, possibly, that the extent of the gas penetration was almost the same in the three cases. The trend in Figure 5 indicates that a higher moisture content results in greater adsorption of SO2 from the initial loading (white bars). The same trend, although less marked, also appears in the amounts of SO2 finally retained in the biomass matrix (dark gray in Figure 5). Overall, the largest amount of catalyst retained is 30% of the adsorbed amount. Therefore, we could infer that putting the biomass in contact with a certain level of gas dosed on the mass basis does not ensure that it will be integrally adsorbed, because it is also dependent on, among other things, the system pressure and the biomass humidity. This conclusion
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Figure 6. Possible modification to the scheme of the present ENEA’s pilot station for the acid impregnation. Scheme legend: 1, biomass feeder; 2, canopy valve (able to intercept dust and granules and ensuring an hermetic seal at high pressure); 3, impregnation chamber equipped with screw feeder; 4, ejector ; 5, gas-solid settler; and 6, steam-explosion gun.
could be relevant when the effect of increasing levels of catalyst is being investigated. In the present investigation, the biomass steam-treatment tests were performed using the equilibrium level of the catalyst, because it is the portion that has interacted more deeply with the chips and it most likely assists the biomass defiberization. This theory could partially explain the fact that some authors found similar hydrolysis yields when, under the same conditions of temperature and treatment time, the level of SO2 loaded was even doubled, from 3% to 6%.9 Furthermore, considering the rapid desorption of the catalyst when pressure is released, the use of the equilibrium amount could ensure a better reproducibility in the process conditions. The slurries collected at the outlet of the steam reactor were washed with warm water and the amounts of extracted SO32and SO42- species were determined. The chromatographic analysis indicated that the pretreated product from substrate A contained ∼1.4% (w/w) sulfates and that from substrate B contained ∼1% (w/w) sulfates. These amounts correspond to 0.9% and 0.7% SO2 equiv, respectively, which are congruent with the estimations of the SO2 retained in biomass done by the weight measurements. There were only traces of SO32species in the water extracts. An almost-complete in situ oxidation of sulfites to sulfates had likely occurred under the steam reactor conditions, and the major part of the sulfuric acid produced was not expelled among the volatiles and was likely retained in the form of salts. Also, this result indicates that the addition of SO2 did not generate sulfites that are potential fermentation inhibitors. On the other hand, it implies that sulfuric acid, which could be corrosive, has been produced during the process and confirms the necessity of using as little catalyst as possible. When applied to a continuous process, a screw feeder reactor could be used for the SO2 adsorption (Figure 6). The outlet of the impregnation chamber could be faced with a gassolid settler unit through which biomass is continuously conveyed to the steam reactor. The gas is introduced at this point and may flow countercurrent, with respect to the biomass, maintaining a continuous and more effective contact with the solid particles. The recovery and recycle of excess SO2 could be accomplished through an ejector device. In this way, a pore
Figure 7. Substrate degree of polymerization (DP), as a function of the steam-explosion severity parameters and the acid catalyst addition. Table 2. Dry Matter (DM) Percentages of Steam-Treated Chips and Water-Insoluble Fractions (WIFs) substrate
steam-treated product, DM (%)
WIF,a DM (%)
A B C D
23.87 ( 0.06 29.30 ( 0.06 31 ( 2 31.5 ( 0.3
75.96 ( 0.11 73.10 ( 0.09 85.34 ( 0.48 90 ( 1.7
a
Steam-pretreated substrates were washed at a solid-to-liquid ratio of
5%.
saturation condition would be ensured and the exceeding SO2 could be recycled. This configuration could be particularly versatile toward disparate types of feedings, considering that different feedstocks may require different saturation conditions. Table 2 summarizes the DM percentages that are relevant to the steam-pretreated substrates and lists the percentages of DM separated as a water-insoluble fraction (WIF). The trend of the WIFs indicates that the higher the severity, the lesser the WIF percentage. In Figure 7, the DP of the substrates treated with SO2 and those of the non-impregnated substrates are compared. Data are reported as a function of the severities explored. As expected, increasing the severity parameter leads to a deeper defiberization of the substrate. It is worth noting that the use of SO2 not only caused a 50% decrease of the DPs, compared to those without catalyst, but produced an overall defiberization that seemed less affected by the treatment time (DPB-DPA< DPD-DPC). This result underlines a synergistic effect between SO2 and the SE process.
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Table 3. Chemical Compositions of the Raw Material and WIFs after Steam Treatment and Water Washing Amount (%)a
a
component
raw material
substrate A
substrate B
substrate C
substrate D
glucan xylan arabinan galattan mannan lignin ash
47.7 ( 1.5 15.8 ( 0.3 0.530 ( 0.011 0.60 ( 0.05 1.7 ( 0.2 27.0 ( 0.7 1.21 ( 0.06
57.42 ( 1.0 0.79 ( 0.04 0.019 ( 0.008 0.070 ( 0.010 0.14 ( 0.02 39.1 ( 0.4 0.54 ( 0.02
64.4 ( 0.3 1.91 ( 0.11 0.037 ( 0.011 0.080 ( 0.011 0.25 ( 0.03 30.7 ( 0.4 0.30 ( 0.03
59.9 ( 1.1 2.7 ( 0.4 0.10 ( 0.02 0.21 ( 0.03 0.19 ( 0.02 32.8 ( 0.7 0.29 ( 0.04
52.9 ( 0.4 11.8 ( 0.3 0.211 ( 0.011 0.46 ( 0.06 1.40 ( 0.17 29.8 ( 0.2 0.49 ( 0.002
Data are reported as a percentage of the washed substrates DM.
Table 4. Composition of the Water-Soluble Fractions (WSFs), in Terms of the Total Amount of Extracted Matter, the Total Acidity Levels, the Main Degradation Byproductsa substrate
water extract
total acidityb
acetic acid
5-HMF
furfural
A B C D
24.04 ( 0.04 26.90 ( 0.07 14.66 ( 0.01 9.588 ( 0.008
6.95 ( 0.02 4.71 ( 0.01 3.94 ( 0.03 0.742 ( 0.003
4.33 3.48 3.65 0.74
0.2 0.09 0.06 0.009
2.01 1.38 1.03 0.29
a Data are expressed as a percentage of the steam-treated products DM. Measured by titration of water-extract acids and expressed as the equivalent weight of CH3CO2H/DM of steam exploded substrate × 100.
b
Chemical Composition of the Process Streams after Water Extraction. Table 3 summarizes the chemical compositions of the WIF after steam pretreatment and water washing. Under acidic conditions (A, B), the WIF contained lower amounts of xylan. A similar trend could be observed for the other hemicellulose-derived anhydrous sugars (namely, arabinan, galattan, and mannan, although higher experimental errors affected the determination of these minor compounds). This is partially due to the higher degradation of the hemicelluloses into secondary products and partially to the larger extent of hydrolysis to small water-soluble oligomers. When the amount of lignin in the WIF samples is expressed on the basis of 100 g of steam-exploded products DM, the values are 29.5, 22.4, 28.0, and 26.9 for samples A, B, C, and D, respectively. This means that the uncatalyzed treatment (C, D) did not solubilize lignin at low severity, and that the amount of lignin increased slightly at high severity, probably due to the formation of adducts with furfural, HMF, and carbohydrate degradation products (lignin-like). The results for the catalyzed treatments suggest that the acidic media could depolymerize and solubilize lignin at low severity, but at high severity, the formation of lignin-like byproducts increased the amount of lignin again. Table 4 lists the amounts of the water-soluble fractions (WSF) as a percentage of the SE slurries DM. Table 4 also reports the titrated acidity of the SE substrates, the percentage of acetic acid, and the composition of the furanic byproducts. The overall inhibitors concentration increased as the pretreatment conditions became more severe (as the sum of the temperature and acid effects), thus reflecting increasing substrate degradation. The small difference in the inhibitor concentrations of substrates B and C indicates that prolonged steaming without catalyst (substrate C) generates a concentration of the measured byproducts that is similar to that of the milder treatment with the addition of SO2 (substrate B). On the other side, the ratio of the inhibitors to the monomer sugars in substrate B was 1.7fold lower than that in substrate C, and this implies that the former pretreatment conditions yielded major benefits overall. The degradation of substrate D seemed much less pronounced, probably because of a poor structural defiberization already evident in the substrate appearance after the steam treatment.
Figure 8 displays the effect of increasing the concentration of H2SO4 on the recovery of the monosaccharides by the acidic post-hydrolysis of the WSFs. The data are reported as the concentration of each monosaccharide after the post-hydrolysis divided by the maximum concentration obtained. Besides the effect of H2SO4 concentration in the range of 0-3 wt %, the influence of the post-hydrolysis temperature is also shown. Specifically, the first bar reported for each carbohydrate represents the recovery of that specific sugar before applying either the acid or the autoclave heating. In the streams from the substrates exploded without SO2 (substrates C and D), the concentrations of the soluble monosaccharides significantly increased as the H2SO4 concentration increased. In fact, the maximum hydrolizability for arabinan, xylan, and galactan fell in the range of 0.5%-1% w/w H2SO4. In contrast, the streams from the substrates previously impregnated with SO2 (substrates A and B) already had high percentages of monomer sugars and a complete hydrolysis was achieved at 0.5% w/w H2SO4 (at the most). In these substrates, arabinan has already been released rapidly during the pretreatment and the post-hydrolysis conditions degraded it by more than 20%. More than the other carbohydrates, mannose in the water extracts from acidcatalyzed pretreatment (substrates A and B) showed a peakshape trend as a consequence of degradation side reactions. Comparing these results with the analogous investigation by Shevchenko et al.7 on the post-hydrolysis of the WSFs from softwood chips, some common features can be observed: (i) arabinan was already depolymerized during the steam explosion, and (ii) the ratio of oligomers to monomers was higher for mannan and glucan. On the other side, some differences can also be observed: in most cases, the solely autoclave heating during the post-hydrolysis did not meaningfully increase the monomer concentrations in the water extracts; arabinose degradation in the present investigation was more accentuated than that observed by the authors (0%-3%). In contrast, xylose degradation with 3% H2SO4 reported by them is >30%. These diversities in the sugars profiles during the post-hydrolysis could be due to the different hemicellulose compositions of the feedstocks used. More in detail, Douglas fir contains much more mannan (typically 13%) than aspen chips (1.7%), which, in contrast, are richer in xylan. This could have caused different pathways of the hemicellulose depolymerization and subsequent monomers degradation. Overall, in substrates A, B, and C, the amount of sugars was in the range of 60%-67% of the WSF, whereas in substrate D, it represented only 40% of the total. For each sugar, the maximum concentrations of monomers are summarized in Table 5. The highest recovery of all the hemicellulose-derived sugars (arabinose, xylose, and mannose) was achieved under the conditions of substrate B. It is worth noting that the difference in terms of the total amount of recovered monosaccharides was more between substrates C and D, compared to that between substrates A and B. This result
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Figure 8. Percentages of monosaccharides recovered after post-hydrolysis with increasing concentration of H2SO4 (0.5%, 1%, 1.5%, and 3% (w/w)). Error bars were calculated by applying the theory of errors propagation to the calculated ratios. Table 5. Composition of Monosaccharides Recovered through Post-Hydrolysis of the Water-Extracted Oligomersa Composition (%) substrate
arabinan
xylan
galattan
glucan
mannan
A B C D
0.25 0.35 0.09 0.13
7.25 10.52 6.79 3.50
0.50 0.47 0.32 0.12
7.76 5.07 2.73 0.23
0.21 0.67 0.26 0.16
a For each sugar, data reported in the table represent the maximum amount obtained (data expressed as a percentage of the steam-treated products DM.
Figure 9. Percent theoretical yields of glucose and ethanol produced during simultaneous saccharification and fermentation (SSF) of the washed fibers (data referred to the composition of the steam-exploded substrates).
still buttresses the fact that the impregnation with SO2 greatly amplified the effect of the thermal treatment of the SE condition only. SSF Tests. The use of the SSF for the substrate fermentation was widely documented, because the presence of yeast together with the enzymes reduces the accumulation of sugars and increases the process yield. Figure 9 displays the process yields obtained during the SSF of the four batches. Beside the specific trends for glucose and ethanol production, the white bars in the graphs show the overall hydrolysis yields (calculated as the sum of the free glucose and that converted to bioethanol). Because, during fermentation, the yeast biomass growth is negligible, it can be assumed that glucose consumption was mostly due to ethanol production. Furthermore, the chromatograms of the fermentation broths did not contain appreciable amounts of
coproducts (i.e., glycerol, lactic acid). The hydrolysis yield improved by increasing the severity factor and by adding SO2. The maximum hydrolysis yield was 84% of the theoretical glucose in the WIF from substrate A. The trend closely resembles that of DP, indicating that the use of catalyst enhanced the enzymatic accessibility of the fibers. However, with different kinetics, the hydrolysis yields of substrate B had a tendency to become closer to substrate A in the long run. This result still indicates that the effect of SO2 was somehow predominant over the hydrothermal effect of the SE process. Despite the hydrolysis trend, substrates A and B had different fermentability. The better working substrate was substrate B, with a fermentation yield of 75% of the theoretical value. Taking into account that both substrates A and B had been washed before SSF, one explanation for the higher ethanol yield of substrate B could lie in the lower lignin content (see Table 3). Also, we cannot exclude the influence of monomers derived from the lignin fragmentation (e.g., syringaldehyde), as already described in a previous publication.1 The SSF yields in the literature often are derived from various combinations of specific experimental conditions (type of biomass, pretreatment, enzyme dosages, microorganism type and loading) and specific bioconversion strategies (SSF instead of SHF). Thus, the final bioethanol yield may not be ascribed to either the efficiency of the pretreatment or the use of SSF or SHF, batch or fed batch process. Instead, the final yields are the result of the entire process flowsheet, and the comparison with different flowsheets could make the discussion of other findings in the literature difficult and, to a certain measure, unreliable. Despite the widespread literature on bioethanol production through acid-catalyzed steam pretreatment, only a few publications can be cited for a direct comparison with the results obtained through the present investigation.18,19 The bioethanol yield of the SSF experiments performed by Ballesteros et al.18 under laboratory conditions at 10% (w/v) poplar concentration and 15 FPU/g substrate of commercial enzyme was 71.2% of the theoretical yields (1.9% w/v) (that is to say, less than that obtained in the present investigation). Their SSF conditions seem comparable to those described here, although the pretreated product was bioconverted to ethanol without any intermediate washing step and SSF was assayed using KluyVeromyces marxianus CECT 10875 at 42 °C. Generally, it is reasonable that a potential reduction of the substrate fermentability, which was expected for the absence of the water-washing step, was counterbalanced by the higher enzymes performance at 42 °C. Cantarella et al.19 investigated the bioethanol produc-
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Table 6. Overall Mass Balance and Recovery Yields for Hexoses and Pentoses Sugars Composition (%) substrate
recovery yield after SE (% of original DM)
glucan
xylan
arabinan
galattan
mannan
A B C D
91 ( 6 91 ( 6 89 ( 6 96 ( 7
46.5 47.3 47.9 46.1
7.6 11.7 8.5 13.5
0.24 0.34 0.17 0.29
0.53 0.53 0.48 0.51
0.31 0.90 0.40 1.36
Enzymatic Hydrolysis Yield
Process Yield of Original Sugar Composition (%)
substrate
plateau hydrolysis yield (% of WIF)
glucose recovery (g/100 raw material)
overall hexan recovery
A B C D
84 75 57 15
37 36 29 8
95 97 98 96
tion from steam-pretreated and washed poplar at 10% (w/v) substrate concentration and a total enzymatic proteins-to-biomass ratio of 0.06 g/gDM. Saccharomyces cereVisiae (or Baker’s yeast) was used in the SSF tests. Under optimized conditions, the ethanol yield was 82% of the theoretical value, corresponding to 2.4% w/v. This result is higher than that reported in the present paper but it is comparable with that reported in a previous publication on bioethanol production from aspen chips treated in the continuous pilot digester (85%).1 As better discussed later, the difference could be due to the use of different plant scales and technologies (batch and continuous). Some other pretreatments, different from SE, were also described in the literature for the production of bioethanol from poplar. Meuniemer-Goddik et al.20 investigated the effect of dilute acid pretreatment (0.6% H2SO4, 10 min, and either 170 or 180 °C) on the SSF yields. Low biomass suspensions (5% w/v) were tested. The achieved bioethanol yields can be estimated from the reported data and are in the range of 41%-76% (0.6%1.3% w/v), depending on the treatment temperature. Chang et al.21 performed the oxidative lime pretreatment (lime + oxygen) of poplar and obtained 73% of the theoretical yield from the washed biomass (1.3% w/v). Overall, it is worth noting that, apart from the process yields, the flowsheets cited for the comparison with the present results yielded lower bioethanol concentrations in the final broth. Some further improvements could come from minimizing the effect of the inhibitors (for instance, through fed-batch fermentation). Mass Balance and Overall Carbohydrates Recovery. The overall compositions of the steam exploded slurries (both soluble and insoluble fractions) obtained from 100 g of raw material are listed in the upper part of Table 6. Interestingly, the overall compositions of the substrates A and C, and substrates B and D, did not exhibit significant differences. This result further confirms that the degradation of the substrates produced by SO2, at least at the concentrations used in the present paper, is less important than that caused by the treatment temperature. Considering that the products, A and C on one hand, and B and D on the other hand, showed different compositions of the WSF and WIF, it could be argued that the catalyst affected the extent of the carbohydrates fragmentation and the subsequent distribution of the oligomers between the insoluble fibers and the hemicellulose liquid stream. The overall recoveries of glucose and xylose, as obtained after the enzymatic hydrolysis of the WIFs and the acid posthydrolysis of the WSFs, are listed in the lower part of Table 6. For glucose, acid-catalyzed steam pretreatment at higher severity resulted in the highest yield (70% of the original). However, the highest yield of pentosan (65% of the original) was obtained from B.
glucose recovered through enzymatic hydrolysis
overall pentosan recovery
xylose recovered in the water extract
70 67 54 14
43 65 49 84
40 57 36 20
The use of acids, with the intention of producing high poplar defiberization at mild SE pretreatments, has been widely demonstrated. Despite the literature available, the comparison of the obtained results is not always immediate, because of the diversity of the protocols used (i.e., type of acid, impregnation conditions) or the lack of an overall mass balance. In one of the earliest publications on this subject, Brownell et al.3 showed that an impregnation with 0.2% (w/w) H2SO4 solution, followed by a steam treatment at 220 °C for 40 s (log R0 ) 3.67) yielded up to 68% pentosan and 93% hexosan. The overall procedure was performed in two steps and lasted for more than one week. The authors conducted an extensive investigation of the effect of the biomass moisture content on the optimum steam treatment time and, therefore, optimum pretreatment conditions. No details on the acid-to-biomass ratio or the actual catalyst uptake were given. In the present paper, the effect of the biomass moisture content was exclusively explored, in view of its possible influence on the catalyst uptake. All the feedstocks then were impregnated under the same moisture conditions, prior to the steam explosion. High recovery of pentosan (70%) has been reported also by Excoffier et al.,22 who worked with a batch plant at 217 °C for 120 s and used 0.4% (w/w) H2SO4 solution during an overnight impregnation. Under these conditions, rather low hydrolysis yields were obtained after 1 day of concentrated slurries hydrolysis (53% of the theoretical value). Ballerini et al.4 obtained recoveries of 89.5% hexosan and 60% pentosan during test runs in the continuous steam cracker (biomass flow rate up to 4 t/h). The chips were impregnated with a 3.85 ratio of H2SO4 to dry matter and treated for 150 s with a saturated steam at 16.2 bar. Severe conditions were used during this impregnation, which was performed by presoaking the crude feedstock for 8 h at 60 °C. The authors disclosed only the information about the yields reached in the single steps without giving any overall mass balance. Thus, the percentage of the initial glucose recovered through the enzymatic hydrolysis of poplar cannot be estimated. In the present paper, the maximum sugars recovery yields were 97% hexan and 65% pentosan. Based on the initial xylan content of the raw material, the maximum yield of xylose monomer, 57% of the theoretical, was obtained for the pretreatment B. Overall, the obtained results are comparable with those reported in the literature, despite the milder conditions (both thermal and acid) adopted in the present work. We previously explored the production of bioethanol by steam-treated aspen chips, using the steam explosion pilot digester run in the continuous modality without acid impregnation.1 Under the best conditions, namely, log R0 ) 4.13 (214 °C and 6 min), the steam-exploded product contained 52.2% glucan and 6.7% xylan (63% of which was water-soluble). The
Ind. Eng. Chem. Res., Vol. 46, No. 23, 2007 7719
hydrolysis yield of the WIF was 90%. In comparison to these results, the use of SO2 yielded almost the same glucan content in the steam-exploded pulp A, despite the thermal pretreatment conditions being milder than that previously settled. Conversely, the use of SO2 at the conditions of B almost doubled the xylan content in the steam-exploded product. However, although the glucan percentages in the pretreated products were comparable, the hydrolysis yields, obtained as a result of the SSF process, were slightly different. This finding could be explained taking into account that data were obtained from two different plant scales and process modalities. In fact, it was determined that the severity of the two plants are correlated through eq 4:16
log R0batch ) 1.5 × (log R0continuous - 1)
(4)
By applying this correlation, the severity parameters log R0batch ) 3.57 and log R0batch ) 4.09 used in the present work virtually correspond to the log R0continuous values from the literature (3.38 and 3.73, respectively). By comparing the hydrolysis yields referred to the same process scale, it can be verified that the yields linearly increase with the severity (log R0continuous) either when SO2 was added or when it was not. However, the use of the acid catalyst reduces the sensibility of the process yields to the log R0continuous by one-fifth. In fact, the cellulose hydrolysis yields obtained with the combination of log R0batch ) 4.09 (corresponding to log R0continuous )3.73) and ∼0.9% SO2, and log R0continuous ) 4.13 without SO2 are almost comparable (that is, 84% in the former and 90% in the latter). Conclusions Sequential SO2-catalyzed steam pretreatment and enzymatic hydrolysis of aspen chips was investigated for the purpose of using the minimum amount of SO2 that could be steadily retained by biomass. Under a gas pressure of 500 mbar, only 50% of the gas injected was actually adsorbed until the impregnation reactor was pressurized. Approximately 70%80% of this amount was adsorbed within the first 15 min, whereas the pressure drop did not attain a constant value before 120 min from the beginning of the injection. The impregnation curve of the substrate with the lowest moisture content (29%) reached the plateau after a longer time. Only 45%-60% of the adsorbed catalyst was retained soon after the reactor opening (that is to say, after releasing the exceeding gas). Allowing desorption, the system ultimately reached an equilibrium state in which the level of the catalyst embedded was 0.6-0.9% w/w raw material DM. This amount was slightly dependent on the biomass moisture in the range of 29%-51%. These evaluations were cross-checked by weight and pressure measurements, as well as by the analysis of the sulfates/sulfites in the steamexploded products. The findings could provide a starting point for developing larger-scale impregnation procedures and reactors. Generally, our results demonstrate that low concentrations of SO2 already have significant effects on improving the overall sugars recovery without increasing the amount of degradation byproducts significantly. At this low concentration, the use of the acid catalyst did not produce meaningful carbohydrates degradation, with respect to the steam treatment alone, whereas, on the other hand, its use almost halved the cellulose degree of polymerization (DP). The highest yield of xylose (65% of the original, more than 80% of which already identified as monomer), obtained by water extraction, corresponded to the mild pretreatment conditions (3 min). Simultaneous saccharification and fermentation (SSF) of the corresponding washed
fibers yielded 67% glucose of the raw material (75% of the water-insoluble fraction (WIF)), 96% of which fermented to ethanol. Increasing the steaming time to 10 min improved the glucose recovery by enzymatic hydrolysis up to 70% (84% of WIF) but reduced that of xylose and made the substrate less fermentable. The best ethanol yield achieved at substrate concentrations of 13% (solid-to-liquid ratio) was 75% of the WIF theoretical yield (corresponding to an ethanol level in the broth of 3.6% (w/v)). In comparison to our previous findings on the bioconversion of aspen chips, the use of SO2 under conditions that were milder than that formerly presented, yielded similar glucan content in the WIF, whereas it doubled the xylan level. Higher process yields could be expected by the “fine-tuning” of the process conditions and of the bioconversion strategies (fed batch). Acknowledgment The work was started within the European Union, Framework Program V (Project NNE5-1999-00272, “Production of Clean Hydrogen for Fuel Cells by Reformation of Bioethanol”, under Contract No. ERK6-CT-1999-0012) and, afterward, was cofinanced by MIUR (Italian Ministry of University and Scientific Research). The authors gratefully acknowledge Mr. G. Cardinale for the preparation of steam-exploded aspen, and Dr. E. Viola and F. Zimbardi for helpful discussions. Supporting Information Available: Photos of the biomass slurries, data concerning the biomass humidity gradient produced by the autoclave steaming, and data concerning the effect of moisture loss on the calculation of the SO2 adsorption. (PDF) This material is available free of charge via the Internet at http:// pubs.acs.org. Literature Cited (1) De Bari, I.; Viola, E.; Barisano, D.; Cardinale, M.; Nanna, F.; Zimbardi, F.; Cardinale, G.; Braccio, G. Ethanol Production at Flask And Pilot Scale from Concentrated Slurries of Steam-Exploded Aspen. Ind. Eng. Chem. Res. 2002, 41, 1745. (2) Schultz, T. P.; Templeton, M. C.; Biermann, C. J.; McGinnis, G. D. Steam Explosion of Mixed Hardwood Chips, Rice Hulls, Corn Stalks, and Sugar Cane Bagasse. J. Agric. Food Chem. 1984, 32, 1166. (3) Brownell, H. H.; Yu, E. K. C.; Saddler, J. N. Steam-Explosion PreTreatment of Wood: Effect of Chip Size, Acid, Moisture Content and Pressure Drop. Biotechnol. Bioeng. 1986, 28, 792. (4) Ballerini, D.; Desmarquest, J. P.; Porquie´, J. Ethanol Production from Lignocellulosics: Large Scale Experimentation and Economics. Bioresour. Technol. 1994, 50, 17. (5) Eklund, R.; Galbe, M.; Zacchi, G. The Influence of SO2 and H2SO4 Impregnation of Willow Prior to Steam Pretreatment. Bioresour. Eng. 1995, 52, 225. (6) Wu, M. M.; Chang, K.; Gregg, D.; Boussaid, A.; Beatson, R. P.; Saddler, J. N. Optimization of Steam Explosion to Enhance Hemicellulose Recovery and Enzymatic Hydrolysis of Cellulose in Softwoods. Appl. Biochem. Biotechnol. 1999, 47, 77-99. (7) Shevchenko, S. M.; Chang, K.; Robinson, J.; Saddler, J. N. Optimization of Monosaccharide Recovery by Post-Hydrolysis of the WaterSoluble Hemicellulose Component after Steam Explosion of Softwood Chips. Bioresour. Technol. 2000, 72, 207. (8) So¨derstro¨m, J.; Pilcher, L.; Galbe, M.; Zacchi, G. Two-Step Steam Pretreatment of Softwood with SO2 Impregnation for Ethanol Production. Appl. Biochem. Biotechnol. 2002, 5, 98-100. (9) Bura, R.; Mansfield, S. D.; Saddler, J. N.; Bothast, R. J. SO2Catalyzed Steam Explosion of Corn Fiber for Ethanol Production. Appl. Biochem. Biotechnol. 2002, 59, 98-100. (10) Martin, C.; Galbe, M.; Nilvebrant, N. O.; Jo¨nson, L. J. Comparison of the Fermentability of Enzymatic Hydrolyzates of Sugarcane Bagasse Pretreated by Steam Explosion Using Different Impregnation Agents. Appl. Biochem. Biotechnol. 2002, 699, 98-100.
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(11) Stenberg, K.; Tengborg, C.; Galbe, M.; Zacchi, G. Optimization of Steam Pretreatment of SO2- Impregnated Mixed Softwood for Ethanol Production. J. Chem. Technol. Biotechnol. 1998, 71, 299-308. (12) O ¨ hgren, K.; Galbe, M.; Zacchi, G. Optimization of Steam PreTreatment of SO2-Impregnated Corn Stover for Fuel Ethanol Production. Appl. Biochem. Biotechnol. 2005, 121, 1055. (13) Overend, R. P.; and Chornet, E. Fractionation of Lignocellulosics by Steam-Aqueous Pretreatments. Philos. Trans. R. Soc. A 1987, 321, 523. (14) Abatzoglou, N.; Chornet, E.; Belkacemi, K. Phenomenological Kinetics of Complex Systems: the Development of a Generalized Severity Parameter and its Application to Lignocellulosics Fractionation. Chem. Emg. Sci. 1992, 47, 1109. (15) De Bari, I.; Barisano, D.; Cardinale, M.; Nanna, F.; Viggiano, D. Air Gasification of Biomass in a Downdraft Fixed Bed: A Comparative Study of the Organic and Inorganic Product Distribution. Energy Fuels 2000, 14, 889. (16) Zimbardi, F.; Viggiano, D.; Nanna, F.; Demichele, M.; Cuna, D.; Cardinale, G. Steam Explosion of Straw in Batch and Continuous Systems. Appl. Biochem. Biotechnol. 1999, 117, 77-79. (17) Browing, B. Viscosity and Molecular Weight Method. Wood Chemistry; Wiley: New York, 1976. (18) 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 Biochem. 2004, 39, 1843. (19) Cantarella, M.; Cantarella, L.; Gallifuoco, A.; Spera, A.; Alfani, F. Comparison of Different Detoxification Methods for Steam-Exploded Poplar Wood as a Substrate for the Bioproduction of Ethanol in SHF and SSF. Process Biochem. 2004, 39, 1533. (20) Meunier-Goddik, L.; Bothwell, M.; Sangseethong, K.; Piyachomkwan, K.; Chung, Y.-C.; Thammasouk, K.; Tanjo, D.; Penner, M. H. Physicochemical Properties of Pretreated Poplar Feedstocks During Simultaneous Saccharification and Fermentation. Enzyme Microb. Technol. 1999, 24, 667. (21) Chang, V. S.; Holtzapple, W. E.; Holtzapple, B.; Holtzapple, M. T. Simultaneous Saccharification and Fermentation of Lime-Treated Biomass. Biotechnol. Lett. 2001, 23, 1327. (22) Excoffier, G.; Toussaint, B.; Vignon, M. R. Saccharification of Steam-Exploded Poplar Wood. Biotechnol. Bioeng. 1991, 38, 1308.
ReceiVed for reView January 17, 2007 ReVised manuscript receiVed June 18, 2007 Accepted August 1, 2007 IE0701120
