Combined steam-explosion toward vacuum and dilute-acid spraying of wheat straw. Impact of severity factor on enzymatic hydrolysis

Renewable Energy 78 (2015) 516e526

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Combined steam-explosion toward vacuum and dilute-acid spraying of wheat straw. Impact of severity factor on enzymatic hydrolysis Maache-Rezzoug Zoulikha a, Maugard Thierry b, Zhao Jean-Michel Qiuyu b, Armelle Nouviaire a, Rezzoug Sid-Ahmed a, * Laboratoire des Sciences de l’Ing
enieur pour l’Environnement, LaSIE, UMR CNRS 7356, Universit
e de La Rochelle, Avenue Michel Cr
epeau, 17042 La Rochelle, France b Equipe Approches Mol
eculaires Environnement-Sant
e, UMR CNRS 7266, LIENSs, Universit
e de La Rochelle, France a

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 June 2014 Accepted 16 January 2015 Available online

This study deals with the development of an eco-friendly hydrothermal pretreatment of wheat straw. This pretreatment involves moderate temperatures without generation of liquid fractions allowing to reduce the energy input consumption and the environmental impact and enhance the enzymatic hydrolysis. For this purpose, the synergistic effect of impregnation of wheat straw by dilute sulfuric acid
tente instantanne
e Contro ^ le
e) pretreatment on enzymatic hydrolysis has been and DIC (in french: De investigated. DIC process involves subjecting the lignocellulosic biomass to saturated steam pressure (0.3 e0.7 MPa), followed by a sudden decompression toward vacuum (5 kPa). The optimization of processing conditions was carried out using a full-factorial design, in respect to processing temperature (133 e165
C), residence time (5e40 min) and sulfuric acid concentration (0.70e2.20%). These processing conditions were converted into a single combined severity factor (CS), relating pH, temperature and residence time of pretreatment, ranged from 2.22 to 1.25. The efficiency of proposed pretreatment was measured through the enzymatic digestibility of lignocellulose and the responses parameters of experimental design were the monomeric glucose and xylose. The most influential factor on biomass bioconversion was sulfuric acid concentration followed by temperature and processing time. The optimum values were 2.2%, 165
C and 40 min, for acid concentration, temperature and residence time, respectively. Strong correlations were observed between the enzymatic hydrolysis and conditions of pretreatment through the analysis of CS. The intense conditions have contributed to disrupt the biomass structure make it less dense leading to a higher specific surface area (ABET). Thus, increasing in accessibility of cellulose to enzymes has improved the rate and yields of bioconversion. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Wheat straw H2SO4 spraying DIC hydrothermal pretreatment Enzymatic hydrolysis Response surface methodology

1. Introduction The growing demand for energy and diminishing fossil fuel reserves have stimulated the interest in finding alternative renewable energy sources such as agricultural waste. For ethanol production, a special attention is currently given to the utilization of four major lignocellulosic biomass feedstocks: straw (wheat and barley), corn stover, hardwood and softwood. Their interest is that they are widespread, inexpensive and readily available to be used for conversion to fuel ethanol as an alternative to starch or sugarcontaining substrates.

* Corresponding author. E-mail address: [email protected] (R. Sid-Ahmed). http://dx.doi.org/10.1016/j.renene.2015.01.038 0960-1481/© 2015 Elsevier Ltd. All rights reserved.

Processing of lignocellulosic substrate to ethanol consists in different successive steps: pretreatments, hydrolysis, fermentation and product separation. In an enzymatic hydrolysis-based process, pretreatment play a key role in process efficiency since it disrupts cell wall physical barriers and renders cellulose more accessible to enzymes [1,2]. Several approaches have been used for developing this crucial step and published studies have included the mechanisms involved, advantages and drawbacks as well as economic assessments [3]. The pretreatments can be physical, physicochemical or chemical and can be combined between them, with the objective to obtain a high monosaccharide yield from enzymatic hydrolysis and generate small quantities of co-products and inhibitors for the subsequent fermentation [4]. The crystallinity of cellulose, its accessible surface area and protection by lignin and hemicellulose, degree of cellulose polymerization and degree of

M.-R. Zoulikha et al. / Renewable Energy 78 (2015) 516e526

acetylation of hemicelluloses are the main factors considered as affecting the rate of biological degradation of lignocelluloses by enzymes [5]. Steam explosion is a hydrothermal pretreatment and has been widely studied and cited in the literature [6,7]. This method has been recognized as one of the most effective pretreatment for breaking the crystalline structure of lignocellulose through chemical effects and mechanical shear induced by sudden explosive decompression [8]. Overall, the biomass is heated with saturated steam at high pressure (1e5 MPa, 180e264
C) for periods up to 90 min in absence (autohydrolysis) or presence of acid catalyst [9,10], followed by a sudden decompression to atmospheric pressure [11]. As a result of high pressure steaming, the partial autohydrolysis of hemicelluloses and the destructuring effect consisting in the defibration of cellulose caused by the sudden decompression, are largely dependent on treatment temperature [12]. Indeed, temperature is a key parameter which strongly impacts both the release of sugars and the formation of degradation compounds. High temperatures favor the release of C6 sugars from the highly crystalline cellulose but also lead to decrease in C5 sugars content in hydrolyzate due to its conversion to furfural and other degradation compounds [13]. In order to moderate the processing conditions without reducing pretreatment efficiency, modified explosion preatreatment have been developed as acidcatalyzed pretreatment [14,15] or ammonia fiber explosion (AFEX) [16]. Impregnation of biomass with acid catalyst such as dilute H2SO4 [17e20] or ammonium chloride [21] before steam explosion has been shown to decrease both temperature and time requirements while achieving optimal fractionation, sugar recovery, and enzymatic hydrolysis. The pretreatment used in this study (DIC process) and developed in our laboratory [22] is close to steam explosion but with lower processing conditions of tempertaure and processing pressures [20,23]. The other difference is that DIC treatment involves two additional steps; the first is the setting up of vacuum before injection of saturated steam, contributing to reduce air resistance and thus intensify the steam diffusion into the biomass. Consequently, the time required to reach steam equilibrium temperature is shortened [24]. The second step consists to an abrupt decompression which is carried out towards vacuum (5 kPa) instead of atmospheric pressure as for steam explosion. When the pressure drops suddenly, an autovaporization occurs, which is an adiabatic transition, water rashly escapes and disrupts the structure of biomass. This transition is accompanied by a rapid cooling to stop degradation reactions. The combined severity factor (CS) has been often adopted to compare the efficiency of various pretreatments on physicochemical changes in biomass [13,25,26]. This factor, initially used to control the pulping processes in the paper industry [27], was used by many authors for the comparison of steam explosion pretreatment severities [28,29] and their consequences on lignocellulosic biomass composition. The concept of CS can be applied for optimization of pretreatments by calculating an expression (CS ¼ LogðRO Þ  pH), that connects the effects of pH, temperature, and residence time. It was already used for the optimization of dilute acid pretreatment followed by enzymatic hydrolysis of corn stover [30,31], wheat straw [32], rape straw [33,34] and barley straw [35,36]. This study aims to investigate the efficiency of acid impregnation of wheat straw, used as a model substrate, and DIC hydrothermal pretreatment followed by enzymatic hydrolysis. Moderate temperatures (133e165
C), corresponding to pressures ranging from 0.3 to 0.7 MPa were used, contributing to reduce sugar loss, energy consumption and formation of inhibitors for both enzymes and fermenting microorganisms. The procedure adopted in this study is an impregnation by spraying. It differs from classic

517

immersion method, which generates a liquid fraction and high volumes of acid mobilized. Classical method require an additional steps of separation between liquid and solid phases as well as the recycling of acidic residues or their neutralization. According to Barakat et al. [37], the consumption of thermal energy in pretreatments is directly proportional to liquid/biomass ratio. Therefore, reducing the ratio allows improving energy efficiency and reducing waste production and water consumption. The optimization of processing conditions was carried out using a full-factorial design with the glucose and xylose concentration after enzymatic hydrolysis and the ratio xylose/glucose as responses parameters. Measurements of specific surface area of pretreated wheat straw at different processing conditions were also investigated using BET (Brenauer-Emmett-Teller) method [38] by krypton adsorption. The study also focused on the research of two groups of correlations: between CS factor and intial hydrolysis rate and reducing sugar for the first group and between specific surface area and the same responses for the second group. 2. Materials and methods 2.1. Chemicals and feedstock The chemicals were purchased from Sigma-Aldrich (France) and deionized water was obtained through a Milli-Q system (Millipore, France). All of the experiments were performed in triplicate. The wheat straw was sourced from a local farm (Poitou-Charentes region, France). In order to homogenize the material size, wheat straw at moisture content of 10.29% (g H2O/100 g dry wheat straw) was briefly crushed in a Gindomix (GM 200) Retsch crusher (7500 g) and calibrated in a sieve to obtain particle sizes between 1000 and 8000 mm. 2.2. Enzymes Celluclast 1.5 L, the enzyme concentrate used for cellulose hydrolysis, was commercial Trichoderma reesei cellulase preparation contains endo-glucanases, exo-glucanases, cellobiohydrolases and b-glucosidases. This preparation was a brownish liquid with density of approx. 1.20 g/mL and contained 191 mg protein/mL (calculated by Smith assay [39]). The cellulasic activity of concentrate was 96 FPU/mL. One unit of FPU is defined as the amount of enzyme which releases 1 mmol of glucose equivalents from Whatman n
1 filter paper in 1 min. Optimum conditions of activity were between 4.5 and 6 for pH and 50e60
C for temperature [40]. 2.3. Dilute-acid spraying method Aqueous solutions of dilute H2SO4 at concentration ranging from 0.7 to 2.2% (v/v) was sprayed at room temperature over the wheat straw through a nozzle, creating a mist above the solid. These levels were fixed basing on the literature according which the concentration of the solutions of impregnation typically varied between 1 and 3% [41,42]. The quantity of sprayed solution was adjusted to reach a final moisture content of 40% (w/w) (40 g H2O/ 100 g dry) with solutions corresponding to 0.7, 1.45 and 2.2% of sulfuric acid (v/v). The acid-sprayed straw was mixed manually in sealed bags to ensure a homogenous distribution and stored at 4
C for 24 h prior to DIC hydrothermal pretreatment (Fig. 1) to promote the impregnation process. 2.4. DIC hydrothermal pretreatment The detailed procedure and equipment of DIC hydrothermal pretreatment have been described previously [22,11]. Briefly, as

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Fig. 1. Flow chart of the dilute acid catalyst combined to steam explosion by DIC process.

described in Fig. 2, the experimental setup is composed from: a processing reactor where the sample was treated at high temperature/high steam pressure, a vacuum system which comprises mainly a stainless steel vacuum tank with a volume (1600 L), 133 fold greater than that of the reactor (12 L), a vacuum pump, and a

steam generator supplying steam into the reactor. As indicated in Fig. 1, the acid-sprayed straw (18 g) at moisture content of 40 g H2O/ 100 g dry wheat straw was introduced in the reactor at atmosphric pressure (Fig. 2a). A reduced pressure of 5 kPa was then created in the reactor (Fig. 2b), contributing in acceleration of the transfer

Fig. 2. Experimental set-up of DIC hydrothermal pretreatment and typical pressure-temperature-time profile for run 3 as example.

M.-R. Zoulikha et al. / Renewable Energy 78 (2015) 516e526

phenomenon, associated to the simultaneous heat and mass transfer in the material. The pretreatment was initiated and controlled by steam injection (Fig. 2c) which is maintained at fixed pressure and processing time (Fig. 2d), corresponding to the residence time at maximum temperature, according to the experimental design. The temperature rises rapidly from room temperature to steam equilibrium temperature (Fig. 2e). Main heating is the result of the transfer of latent heat of steam condensation during direct contact between biomass and saturated steam. After this period, there is no heat transfer and the exchange is dominated by the absorption of condensed steam [11]. The increase in moisture content depends on the amount of condensed water which is linked to pressure level and processing time [43,44]. The pressure was then released by an abrupt decompression towards a vacuum (5 kPa). A pneumatic valve separating the reactor from the vacuum tank opens in less than 1 s. After reducing pressure, the atmospheric air was injected to return to atmospheric pressure to retrieve the sample. Only the solid fraction of wheat straw was recovered at the output of reactor, with moisture around 400 g H2O/100 g dry wheat straw, due to water retention of steam condensation during hydrotreatment. Pretreated wheat straw samples were dried in oven at 40
C for 24 h and stored at room temperature for subsequent enzymatic hydrolysis to avoid any degradation. 2.5. Severity parameter calculation The effect of the acid-catalyzed hydrothermal severity on the fractionation process was determined using CS, the combined severity factor. CS is an index that integrates changes in temperature, time and acidity into a single value, which facilitates comparisons of different conditions [44e46]. It was defined as:

CS ¼ LogðRO Þ  pH

(1)


T  100 with RO ¼ t  EXP 14:75

(2)

where t (min), T (
C), 14.75 and 100 are respectively, the residence time, process temperature an empirical parameter related to activation energy value and the reference temperature. pH in Eq. (1) was defined as the acidity of pretreated solid fraction, measured in aqueous solution after decantation of solid material, according to the protocol defined in the Section 2.6.2. 2.6. Analysis The chemical composition of raw material was determined by acid hydrolysis and saponification as described previously [40]. The native straw contained the following composition (g/100 g dry wheat straw): 31% cellulose, 43% hemicellulose, 22% lignin and 4% ashes. This composition is consistent with a wide range of reported values in literature [6,15]. 2.6.1. Moisture content The moisture contents of straw were determined by drying samples in an oven at 105
C during 24 h, according to the A.F.N.OR standard method [47]. 2.6.2. pH measurements The pH of untreated and pretreated wheat straw was measured by a pH probe attached to a pH meter (InoLab® pH 720, WTW). Approximately 0.1 g of dry material was added to distilled water with a solideliquid ratio of 1:20. The suspension was stirred

519

mechanically for 5 min and pH measured in the liquid phase after decantation of the solid phase. 2.6.3. Specific surface area The measurements of specific surface area of wheat straw were determined by widely used method of adsorption of krypton using BET method. After have been oven-dried at 40
C, the samples (0.3 g) were degassed during 36 h at room temperature in order to minimize structural changes. The BET surface area was determined at a relative pressure of about 0.999 using a Micromeritics ASAP 2020 analyzer (Micromeritics Instrument, USA). 2.7. Cellulose hydrolysis Celluclast-1.5 L (480 FPU/L) was added to 50 mM citratephosphate buffer (pH ¼ 4.6) and then mixed to the substrate (10 g/L). The experiments were carried out in 100 ml Erlenmeyer flasks containing 10 mL total reaction volume (buffer enzyme mixture). The flasks were sealed and incubated in a rotary shaker at 600 rpm at 50
C during 20 h. To follow the hydrolysis, a flask was withdrawn at different times and the liquid phase (hydrolyzate) immediately heated for 5 min in a boiling water bath to precipitate the proteins and prevent further hydrolysis. The mixture was then centrifuged at 14000 rpm for 2 min to remove solids. The cellulose hydrolysis yield of samples was determined by the 3.5-dinitrosalicylic acid (DNS) method [40]. 2.8. GC/MS Carbohydrates monomers characterization After enzymatic hydrolysis of untreated and treated wheat straw, the concentrations of glucose and xylose were determined by GC/MS using a Varian CP-3800 GC/Varian Saturn 2000. 400 mL of pyridine and 400 mL of BSTFA: TMCS (99:1) was added to 2 mg of sample. The solution was mixed for 2 h at room temperature and then injected into a DB-1701 J&W Scientific column (30 m  0.32 mm  1 mm) at a flow of 1 mL/min. The helium pressure was 0.6 bar. The temperature of the injector was set at 250
C. The temperature rise in oven was programmed for a first step at 150
C, then an increment of 10
C/min up to 200
C with a final step at 200
C for 35 min. The ionization was performed by Electronic Impact (EI, 70 eV), the trap temperature was set at 150
C and the target ion was fixed at 40e650 m/z. The concentration of glucose and xylose were determined after enzymatic hydrolysis of wheat straw and all data on sugar concentrations provided below refer to hydrolyzates after 8 h of enzymatic hydrolysis. 2.9. Experimental design and surface methodology The relationships between responses and process variables have been estimated using a full-factorial design. The experimental design required eight (23) experiments corresponding to factorial points. Four central points were added to estimate the experimental error and to prove the suitability of models. The experiments were run in random order to minimize effects of unexpected variability due to extraneous factors. The three independent variables are coded according to Eq. (3):

xi ¼

Xi  Xi0 DXi

i¼13

(3)

where xi and Xi are respectively the dimensionless and the actual values of independent variable i, Xi0 the actual value of independent variable i at central point and DXi the step change of Xi corresponding to a unit variation of the dimensionless value. Acid concentration (x1), processing temperature (x2) and time (x3) are

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chosen as independent variables and varied between 0.7 and 2.2%, 133 and 165
C, 5 and 40 min, respectively (Table 1). The studied responses are related to the coded independent variables xi, xj according to the second order polynomial expressed in Eq. (4).

Y ¼ b0 þ

X

b i xi þ

X

bij xi xj

Y2 ¼ 4.353 þ 1.096 x1 þ 0.032 x2 þ 0.128 x3 e 0.003 x1x2 þ 0.004 x1x3 0.001 x2x3

(6)

Y3 ¼ 11.075 e 2.056 x1 e 0.062 x2 e 0.287 x3 þ 0.0141 x1x2 þ 0.005 x1x3 þ 0.002 x2x3

(7)

(4) where Y1, Y2 and Y3 represent the concentration of glucose (Eq. (5)) and xylose (Eq. (6)) released after 8 h of enzymatic hydrolysis from DIC pretreated residues and the ratio glucose/xylose (Eq. (7)). The quality of developed models was evaluated based on the coefficient of correlation (R2) and on the lack-of fit value. It can be seen that R2 were systematically close to 85% and p-value of lack-of-fit higher than 0.05 (not significant) suggesting that the predicted models reasonably represent the observed values and therefore the responses were sufficiently explained by the models.

With b0 the interception coefficient, bi linear terms and bij interaction terms. xi and xj are the coded values of the independent variables. The Fisher's test for variance analysis (ANOVA), effected on the experimental data make it possible to estimate the statistical significance of proposed models. Response surfaces as represented by Figs. 3 and 4 were drawn using the analysis design procedure of Statgraphics Plus for Windows software (Centurion version). 3. Results and discussion 3.1. Statistical analysis and models fitting

3.2. Effect of processing parameters on sugars released

The complete design matrix with experimental conditions values and analysis responses data are displayed respectively in Tables 1 and 2. The significance of linear and interaction terms are given in Table 3 through variance analysis (ANOVA). Based on pvalues, all linear terms have significant effects (p < 0.05) on glucose and xylose concentrations released after enzymatic hydrolysis of pretreated wheat straw. The concentration of H2SO4 (x1) had the most significant effect followed by processing temperture (x2) and residence time (x3) according to this order x1 > x2 > x3 and x1 > x3 > x2 for glucose and xylose concentrations, respectively. For these parameters, the interaction temperature-processing time was statistically significant indicating an antagonistic effect between these variables (x2x3) on responses parameters. Moreover, the interaction between sulfuric acid concentration and processing time was also significant with a positif effect on xylose concentration. The linear effect of acid concentration as well as the interactions time-temperature and acid concentration-temperature were statistically significant on glucose/xylose ratio. The regression analysis was carried out to fit experimental data by the mathematical models, aiming at an optimal region for two responses parameters. The predicted models were described by the following equations using actual values:

In general, yields or concentrations of xylose and glucose released following both the pretreatment and enzymatic hydrolysis processes are an indicator of pretreatment efficiency for ethanol bioconversion. Fig. 3 showed via three-dimensional response surface plot, the response models mapped against two experimental factors while the third factor was set constant at its central value, 1.45% and 22.5 min, respectively. Fig. 3A shows that the DIC treated wheat straw in presence of acid (1.45%) favors the glucose release from celulose by enzymatic hydrolysis, mainly when temperature was at its high level (165
C). Indeed, the interaction effect of processing time-temperature was significant (p-value ¼ 0.0258). At low residence time (5 min) and regardless the temperature applied, the concentration of glucose released was relatively moderate, about 2 g/L. While at its high level (40 min) a rising up to 3.5 g/L was observed with temperature, indicating that the heating of biomass during a sufficient processing time combined to acid catalyst plays a role in degradation of hemicelluloses and lignin by chemical effect while the rapid decompression process disrupts their structure. A moderate interaction (p-value ¼ 0.066) was observed between temperature and H2SO4 concentration (Fig. 3B). The glucose concentration increased from 0.98 to 2.1 g/L with increasing of acid concentration for lower temperature (133
C), while it increased from 1.1 to 3.8 g/L for the highest temperature (165
C). The xylan solubilization is more dependent on acid loading than on temperature (Table 3). The impregnation of wheat straw under weak acid

Y1 ¼ 3.811 e 1.624 x1 e 0.022 x2 e 0.159 x3 þ 0.017 x1x2 þ 0.001 x1x3 þ 0.0149 x2x3

(5)

Table 1 Full-factorial design with coded levels (x1: H2SO4 concentration (%), x2: Temperature (
C), x3: pretreatment time (min) for independent variables. pH values of wheat straw before DIC pretreatment were summarized below. Run

Coded factor

Processing conditions Pressure (MPa)

Temperature (
C)

Time (min)

3.99 3.99 3.99 3.99 2.25 2.25 2.25 2.25 2.67 2.67 2.67 2.67

0.3 0.3 0.7 0.7 0.3 0.3 0.7 0.7 0.5 0.5 0.5 0.5

133 133 165 165 133 133 165 165 152 152 152 152

5 40 5 40 5 40 5 40 22.5 22.5 22.5 22.5

6.54 ± 0.03

e

e

e

x1

x2

x3

H2SO4 (%)

a

1 2 3 4 5 6 7 8 9 10 11 12

1 1 1 1 þ1 þ1 þ1 þ1 0 0 0 0

1 1 þ1 þ1 1 1 þ1 þ1 0 0 0 0

1 þ1 1 þ1 1 þ1 1 þ1 0 0 0 0

0.70 0.70 0.70 0.70 2.20 2.20 2.20 2.20 1.45 1.45 1.45 1.45

Untreated

e

e

e

e

a

pH

pH, acidity of solid fraction measured in aqueous solution after decantation before DIC hydrothermal treatment. The average and standard deviation of pH were calculated by five replications.

M.-R. Zoulikha et al. / Renewable Energy 78 (2015) 516e526

521

Fig. 3. Simultaneous effects of processing temperature with sulfuric acid concentration of impegnated wheat straw (A) and with processing time (B) on the glucose concentration, at the central point, 22.5 min and 1.54%, respectively. For each variation of two parameters the third was set at its central value. Data with 95% confidence intervals.

condition before D.I.C treatment has maximized the xylose conversion. On the other hand, Table 3 indicates that the interaction time-processing temperature has a negative significant effect on xylose released (p-value ¼ 0.0005) and that pretreatment for longer times was detrimental to xylose recovery. Fig. 4A showed that the glucose/xylose ratio decreased from 2.6 to 1.3 with increasing of processing time when temperature was fixed to its low level (133
C). An opposite trend was observed for high temperature level (165
C) where the ratio rather increased from 0.9 to 2.5. This result indicates that a similar ratio can be obtained for high and low DIC hydrothermal pretreatment conditions. The more favorable situation is that corresponding to a low xylose and high glucose production, since the xylose degradation products can inhibit the further fermentation of glucose, but also the minimization of energy demand in the process. The temperature-acid concentration being a significant interaction. Fig. 4B showed that the variation of glucose/xylose ratio was greater when temperature was at its high level (165
C), the increasing H2SO4 concentration promoted the glucose/xylose ratio. The best glucose/xylose ratio of 3.3 was obtained for the highest pretreatment conditions for the three processing parameters. The ratio of glucose to xylose can be critical importance for optimization of lignocellulosic biomass pretreatment, depending on the type of biofuel (ethanol or hydrogen) that the substrate is intended to get and the sugar consumption patterns by used microorganisms during fermentation [5].

In general, increasing temperature and lowering pH are known to increase the pretreatment efficiency [48]. According to Ramos [14], the best pretreatment options are those that combine both physical and chemical methods. The acid impregnation before steam explosion has been pointed out as the key factor by many authors [26,29], in particular for the step of cellulose digestion. Zimbardi et al. [18] showed by investigating the synergistic effect of impregnation by sulfuric acid and steam explosion that the impregnation has greatly improved sugar solubility; 93.5% of cellulose digestibility was achieved at 190
C and 3 wt% of acid loading compared to 39% obtained at same conditions without acid. Furthermore, Emmel et al. [17] showed on preimpregnated chips of Eucalyptus grandis by sulfuric acid, with either 0.087 and 0.175% (w/ w) and 210
C steam treatment that shorter residence times (2 min) increased the xylose recovery while longer residence times (5 min) lowered it. 3.3. Influence of combined severity factor on pretreatment efficiency Hemicellulosic sugars recovery in the pretreated solids is interesting to obtain higher total fermentable sugar production. The purpose of pretreatment is not only to remove xylose from hemicellulose but also to increase the rate of enzymatic hydrolysis of cellulose to glucose. In order to estimate the efficiency of acid impregnation followed by DIC hydrothermal pretreatment on

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Fig. 4. Three-dimensional response surface for glucose/xylose ratio versus (A) temperature, acid concentration and (B) temperature, processing time, at the central point of 22.5 min and 1.54%, respectively. Data with 95% confidence intervals.

enzymatic hydrolysis, we used combined severity factor (CS) defined in Section 2.5 for which the values are presented in Table 2. As can be seen, CS values are low, ranging from 2.22 to 1.25, largely below those cited in literature on steam explosion of straw

impregnanted by dilute acid. According to Pedersen and Mayer [27], CS vary between 3.0 and 4.4 for acidic steam explosion of wheat straw in preparing the lignocellulosic biomass for enzymatic bioconversion. Overall CS values illustrate two trends around the

Table 2 Combined severity factor (CS), specific surface area (ABET), pH values and enzymatic hydrolysis parameters measured on DIC pretreated wheat straw. Run

a

pH

CS

ABET (ma/g)

Enzymatic hydrolysis conversion (g/L) 3.5-dinitrosalicylic acid analysis R2

R1 1 2 3 4 5 6 7 8 9 10 11 12 Untreated a

3.91 3.87 3.81 4.28 2.20 2.29 2.27 2.27 2.84 2.50 2.49 2.83

± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.03 0.13 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

2.22 1.28 1.20 0.76 0.51 0.30 0.35 1.25 0.05 0.38 0.39 0.05

0.77 0.55 0.49 0.92 0.87 1.06 1.07 1.68 1.22 1.30 1.15 1.20

0.07 0.03 0.04 0.59 0.44 1.66 1.75 1.97 1.17 1.30 1.40 1.21

± ± ± ± ± ± ± ± ± ± ± ±

e

0.76 ± 0.08

0.05 ± 0.01

0.01 0.00 0.01 0.00 0.06 0.09 0.24 0.05 0.07 0.03 0.04 0.18

1.11 1.48 1.68 2.62 3.12 4.93 4.69 6.07 4.47 4.90 4.35 4.51

GC/MS analysis R3

± ± ± ± ± ± ± ± ± ± ± ±

0.10 0.00 0.11 0.00 0.01 0.01 0.01 0.09 0.06 0.01 0.06 0.10

1.03 ± 0.02

R4

R5

R6

0.003 0.002 0.003 0.004 0.002 0.000 0.014 0.018 0.000 0.004 0.002 0.004

0.94 0.97 1.06 1.70 2.31 2.56 2.67 4.66 2.48 2.87 2.41 2.56

0.42 0.51 0.62 0.96 0.81 2.30 2.02 1.41 1.99 2.03 1.94 1.95

2.24 1.90 1.71 1.77 2.85 1.11 1.32 3.30 1.25 1.41 1.24 1.31

0.012 ± 0.003

0.35

e

e

0.014 0.029 0.028 0.030 0.041 0.046 0.043 0.049 0.051 0.048 0.034 0.049

± ± ± ± ± ± ± ± ± ± ± ±

pH, acidity measured in aqueous solution after decantation of DIC pretreated solid fraction. R1: Reducing sugars concentration (g/L) at 0 h of enzymatic hydrolysis. R2: Reducing sugars concentration (g/L). R3: Enzymatic hydrolysis rate (g/L.min). R4: Glucose concentration (g/L). R5: Xylose concentration (g/L). R6: Glucose/Xylose ratio. All sugar concentrations except R1 were analyzed after 8 h of enzymatic hydrolysis. The average and standard deviation values of pH, ABET and R1, R2, R3 were calculated by five and three replications, respectively.

M.-R. Zoulikha et al. / Renewable Energy 78 (2015) 516e526 Table 3 Analysis of variance showing the effect of the three independent variables as linear and cross products terms on three selected responses.

Glucose concentration (g/L)

Xylose concentration (g/L)

Glucose/Xylose ratio

Source

DF

Sum of squares

x1 x2 x3 x1x2 x1x3 x2x3 Lack-of-fit Pure error R2 x1 x2 x3 x1x2 x1x3 x2x3 Lack-of-fit Pure error R2 x1 x2 x3 x1x2 x1x3 x2x3 Lack-of-fit Pure error R2

1 1 1 1 1 1 2 3 0.92 1 1 1 1 1 1 2 3 0.82 1 1 1 1 1 1 2 3 0.86

7.08 1.36 1.05 0.32 0.69 0.31 0.76 0.12

175.04 33.82 26.14 8.00 17.05 7.61 9.36

0.0009** 0.0101* 0.0145* 0.0663 0.0715 0.0258* 0.0513

2.03 0.18 0.21 0.01 0.03 0.43 2.60 0.05

1200.07 69.52 126.81 8.05 14.96 252.89 7.68

0.0001** 0.0036** 0.0015** 0.0658 0.0306* 0.0005** 0.0661

1.67 0.00 0.00 3.14 0.46 30.24 1.22

0.0231* 0.9667 0.8354 0.0092** 0.0958 0.0003** 0.4093

0.117 0.000 0.000 0.221 0.033 2.132 0.095 0.210

F-ratio

p-value

**p < 0.01; *p < 0.05; DF: degree of freedom.

central point of factorial design, CS < 0 and CS > 0. Negative CS values were obtained for low pretreatment conditions with pH of pretreatment solid substrate about 4 (runs 1, 2, 3 and 4) excluding run 5 for which the low pH (2.3) was mainly due to the high level of acid catalyst concentration (þ1). CS close to 0 coincides mostly with the conditions chosen for the central points of factorial design (runs 9, 10, 11 and 12) and for runs 6 and 7 for which one of processing parameter was at level (1). The highest value of CS (1.25) was obtained for most intense pretreatment conditions (run 8.) The comparison of pH values measured on impregnated wheat straw by H2S04 dilute solution before (pH1) and after (pH2) DIC hydrothermal pretreament (Tables 1 and 2) showed that the pH of solid substrate has not changed significantly. In a previous study, Maache-rezzoug et al. [11] showed in absence of acid catalyst, that DIC hydrothermal pretreatment did not alter the pH substrate, confirming that our conditions remained moderate despite the presence of H2S04. In this study, addition of H2S04 in spraying solution was performed to compensate the low processing temperatures and consequently to avoid sugars degradation and formation of inhibitor components on ethanol fermentability. The combined severity factor facilitates the comparison of various conditions on the pretreatment effectiveness but its usage must be completed by integrating other parameters that influence on efficiency of enzymatic hydrolysis, as recommended by Pedersen and Meyer [27]. It is clear that the pH, temperature and processing time are the main factors affecting the effectiveness of pretreatment [29,49] but the mechanical effect induced by the sudden decompression toward vacuum can modify the exchange surface and therefore the accessibility of biomass to enzymatic attack. Indeed, direct physical contact between enzymes and cellulose is a primary requirement for enzymatic hydrolysis [5]. The increase in porosity, generally expressed by accessible surface area is among the main objectives of all pretreatment processes [50]. In Table 2, BET area values increased from 0.77 to 1.68 m2/g for untreated and pretreated straw in more severe conditions (CS ¼ 1.25), respectively. Maache-

523

Rezzoug et al. [11] showed by SEM, changes in morphological and structural properties of wheat straw cellulose fibers following DIC hydrothermal pretreatment. SEM images of untreated wheat straw showed a large part of compact areas, arranged tightly, with a mean circumference of ellipsoidal alveoles of about 58.2 mm. After pretreatment (0.7 MPa and 15 min), the structure has been expanded and the circumference of alveoles was about 92 mm. Some defibrillation and dispersion were also visible in the microstructure. Baharani et al. [24] have shown that changes in physical properties of biomass also depend on the mechanical effect induced by the abrupt decompression towards vacuum. The sudden transition from thermodynamic equilibrium reached during the pressurization phase toward an another equilibrium state generates vapor from biomass by flash vaporization. This phenomenon disrupts the biomass structure by individual defibrating of cellulose microfibrils and produced a microalveolation in matrix, promoting its specific surface area. 3.4. Correlations between the effectiveness parameters of pretreatment on hydrolysis Fig. 5A shows initial hydrolysis rate calculated up to 15 min of enzymatic hydrolysis as function of CS. The initial hydrolysys rate was positively correlated to CS with a significant increase for positif values of CS. In these conditions, enzymatic hydrolysis rate of approximately 0.046 g/L.min was multiplied by 4 compared to untreated sample. Therefore, reducing sugars released after 8 h were strongly correlated (R2 ¼ 0.96) with the pretreatment severity (Fig. 5B), since the reducung sugar concentration depend on the initial rate of hydrolysis. A same positive correlation was observed by Wiman et al. [51] between the rate of enzymatic hydrolysis of spruce chips steam-pretreated under various severity conditions, for CS ranged from 1.5 to 2.5. The maximum reducing sugar concentration (6.07 g/L) was observed for severes pretreatment conditions, corresponding to CS ¼ 1.25. For this CS value, the glucose released mainly from cellulosic fraction, quantified by GC/MS (Table 2), also reached its maximum (4.66 g/L). The low correlation observed between specific surface area (ABET) measured on pretreated wheat straw and the initial rate of enzymatic hydrolysis (Fig. 5C) seems mainly due to the significant disparity of ABET of the run 1, which is low compared to the others. The low severe pretreatment conditions applied during run 1 (CS ¼ - 2.2) appears to be not sufficient to have a positive impact on the biomass structure which remained identical to that of untreated straw. Wiman et al. [51] interpreted this low correlation by the inaccessibility of the smallest pores during BET measurement, supported by the fact that bound and even free water, which occupies the fine pores, is hard to remove. By contrast, a clear correlation was found between reducing sugar concentration released after 8 h of enzymatic hydrolysis and specific surface area (Fig. 5D). The higher the ABET values the higher the reducing sugar released. Kumar and Wyman [52] showed that the hydrolysis rate is controlled by enzymes adsorption onto cellulose, which is conditioned by enzyme accessibility to cellulose active sites and thus by the available biomass surface area. Pierre et al. [40] compared the adsorption isotherms of cellulases of native and DIC pretreated wheat straw (700 kPa and 15 min). Adjusted data using Langmuir adsorption model showed that at low enzyme concentrations ([EF]  20 g/L), the pretreatment increased the proteins adsorption. The adsorption coefficient (Kad) was decreased by six-fold compared to untreated wheat straw, confirming the fact that the treatment produced a more accessible substrate to cellulases. The results of hemicellulose and cellulose conversion after enzymatic hydrolysis are shown as functions of CS in Fig. 6. Significant amounts of xylose were released from hemicelluloses for

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0.06 C

Initial rate (g/L.min)

A 0.05

0.04 0.03 0.02 y = 0.01x + 0.04 R² = 0.82

Run 1

0.01

Run 1

y = 0.03x + 0.01 R² = 0.54

Reducing sugars concentration (g/L)

0.00 7 B

D

6 5

4 3 2

Run 1

y = 4.43x - 0.88 R² = 0.83

y = 1.63x + 4.05 R² = 0.96

1

Run 1

0 -3

-2

-1 CS

0

1

2 0.0

0.5

1.0 ABET (m2/g)

1.5

2.0

Fig. 5. Effect of combined severity factor (CS) and BET specific surface area (ABET) on initial rate of enzymatic hydrolysis of pretreated wheat straw (A and B, respectively) and on reducing sugar concentration liberated after 8 h of enzymatic hydrolysis (C and D, respectively). The line represents linear regression of data with 95% confidence intervals and untreated wheat straw.

CS higher than 0.76, and the conversion was enhanced at intermediate pretreatment conditions, corresponding to central point of factorial design. A maximum concentration of 2.30 g/L was reached for CS ¼ 0.31 (acid catalyst concentration at level þ1, temperature 1 and processing time þ1), beyond which the xylose concentration decreased, reflecting a probable sugar degradation. Apart from the mechanical effect induced by the abrupt decompression towards a vacuum on biomass destructuring during DIC hydrothermal pretreatment, the abrupt transition produces an intense cooling whose advantage is to stop thermal reactions, contributing to avoid degradation of xylose that is less resistant than glucose. In this study, it should be noted that only a residual solid fraction was recovered after DIC hydrothermal pretreatment

which undergoes enzymatic hydrolysis and therefore there is no loss of xylose by solubilization in a liquid phase. Linde et al. [49] observed on steam pretreated wheat straw impregnated by acid aqueous solution (0.2% H2SO4) that the total xylose recovery after pretreatment was more sensitive to time and temperature than glucose. Increasing of these parameters decreased the recovery of xylose due to degradation to furfural. According to Lee et al. [34], the decline in hemicelluloses hydrolysis yield was attributed to the partial conversion of XMG (Xylan, Manan, and Galatan) yield in degraded products primarily furfural and HMF, and their yields are an indication of the severity of pretreatment. Panagiotopoulos et al. [36] argued that the concentration of HMF released in the hydrolyzate of barley straw was kept low (<0.1 g/L) at a CS smaller than 0.0 and gradually increased to a maximum of 0.58 g/L, when CS increased from 0.0 to 1.1. 4. Conclusion

Fig. 6. Glucose and xylose concentrations of DIC treated wheat straw as a function of combined severity factor (CS). The line represents linear regression of data with 95% confidence intervals and the glucose concentration of untreated wheat straw.

The mechnaical effect induced by the abrupt falls in pressure during DIC pretratment and the subsequent cooling effect showed the potential of combined dilute acid and DIC pretreatment on enhancing enzymatic hydrolysis of wheat straw. Spraying H2SO4 aqueous solution on the substrate has contributed to make more efficient the DIC hydrothermal pretreatment despite moderate temperature conditions, thus enhancing the sugars recovery by enzymatic hydrolysis. The concentration of H2SO4 (x1), processing temperature (x2) and time (x3) exerted a significant effect according to this order x1 > x2 > x3 and x1 > x3 > x2 respectively on glucose and xylose recovery. This study also showed that the proposed pretreatment had an impact on the accessibility to cellulose active sites of wheat straw. For high pretreatment condition (CS ¼ 1.25), ABET value increased from 0.76 (native wheat straw) to 1.68 m2/g, promoting the release of reducing sugars after enzymatic hydrolysis.

M.-R. Zoulikha et al. / Renewable Energy 78 (2015) 516e526

The maximum reducing sugar concentration (6.07 g/L) and glucose released (4.66 g/L) mainly from cellulosic fraction was maximized with the same CS value. Acknowledgments The authors acknowledge the financial support of University of
de
ration de recherche en environnement La Rochelle (FREDD: fe pour le developpement durable) through FREDD/PRES 2013 project. Acronyms CS DIC ABET R1 R2 R3 R4 R5 R6 x1 x2 x3

combined severity factor
tente hydrothermal treatment termed in french as “de
e contro ^ le
e” instantanne specific surface area (m2/g) reducing sugars concentration (g/L) at 0 h of enzymatic hydrolysis reducing sugars concentration (g/L) enzymatic hydrolysis rate (g/L.min) glucose concentration (g/L) xylose concentration (g/L) glucose/xylose ratio H2SO4 concentration processing temperature processing time

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