Bioethanol production from autohydrolyzed Eucalyptus globulus by Simultaneous Saccharification and Fermentation operating at high solids loading

Fuel 94 (2012) 305–312

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Bioethanol production from autohydrolyzed Eucalyptus globulus by Simultaneous Saccharification and Fermentation operating at high solids loading Aloia Romaní, Gil Garrote ⇑, Juan Carlos Parajó Department of Chemical Engineering, Faculty of Science, University of Vigo, Campus Ourense, As Lagoas, 32004 Ourense, Spain Centro de Investigación, Transferencia e Innovación (CITI), University of Vigo, Tecnopole, San Cibrao das Viñas, Ourense, Spain

a r t i c l e

i n f o

Article history: Received 13 April 2011 Received in revised form 2 December 2011 Accepted 6 December 2011 Available online 21 December 2011 Keywords: Autohydrolysis Bioethanol Eucalyptus wood Pretreatment Simultaneous Saccharification and Fermentation

a b s t r a c t Eucalyptus globulus wood samples were subjected to non-isothermal autohydrolysis in order to solubilize hemicelluloses, leading to treated solids of increased cellulose content and enzyme digestibility. Autohydrolyzed solids obtained under a variety of operational conditions were assayed as substrates for bioethanol production by Simultaneous Saccharification and Fermentation (SSF). SSF was optimized using the Response Surface Methodology. The experimental plan included as independent variables the autohydrolysis severity (So defined as the logarithm of the severity factor), the liquor to solid ratio and the enzyme to substrate ratio. The dependent variables considered were the maximum ethanol concentration achieved in individual experiments, the volumetric productivity, the maximum ethanol conversion, the yield of solids after SSF and the cellulose recovery in solids coming from SSF. Operating at high solids loading (So = 4.67, LSR = 4 g/g, ESR = 16 FPU/g), media containing up to 67.4 g ethanol/L and corresponding to 91% of the stoichiometric amount calculated from the cellulose content of wood were obtained. Wood processing resulted in the generation of soluble products from hemicelluloses, in the generation of up to 291 L ethanol/1000 kg oven-dry wood from cellulose, and in a solid material mainly made up of lignin. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction The increasing world population and the growing per capita energy demand boost the interest in producing energy from renewable feedstocks, alternative to fossil resources [1]. Lignocellulosic materials (LCMs) are clean and cheap raw materials for obtaining energy and chemicals. Other advantages of LCM as potential energy sources include their large availability, independence of geographic location, improvement of local economy derived from cultivation, neutral carbon balance and renewable character [2]. Biomass-derived biofuels are considered as the only suitable alternative to oil-derived fuels [3]. Second generation bioethanol, produced from LCM, could replace up to 30% of gasoline [4,5]. The heterogeneous and complex structure of LCM make their fractionation and further benefit difficult. LCM contain nonstructural components (extractives, moisture, etc.) and structural components (cellulose, a polysaccharide made up of glucose units, with high crystallinity and polymerization degree; hemicelluloses, made up of various sugar units, which can be substituted; and lignin, a polymer of phenolic nature). ⇑ Corresponding author at: Department of Chemical Engineering, Faculty of Science, University of Vigo, Campus Ourense, As Lagoas, 32004 Ourense, Spain. Tel.: +34 988387075; fax: +34 988387001. E-mail address: [email protected] (G. Garrote). 0016-2361/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fuel.2011.12.013

LCM utilization may be based on the biorefinery concept: the feedstocks can be fractionated (by reactions involving the depolymerization of at least one of the structural components), and the resulting fractions can be employed for specific applications, including bioethanol production from polysaccharidederived sugars. The manufacture of second generation bioethanol from LCM can be carried out by processes involving three major steps [6]: (a) pretreating the raw material (for example, by a fractionation treatment) to increase its susceptibility to further processing (and, in some cases, to obtain valuable byproducts), (b) enzymatic hydrolysis of cellulose to sugars, and (c) biological conversion of sugars to ethanol. Pretreatment is one of the most influential stages on the production of second-generation bioethanol, owing to its high incidence in the operational costs [5,7]. An optimal pretreatment should fulfill was many as possible of the following requirements [7–13]: (a) simple and economic operation; (b) particle size reduction, if necessary, should be achieved at low cost; (c) reduced consumption of energy, water and chemicals;

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(d) (e) (f) (g) (h)

limited corrosion effects; ability for reaching favorable effects on the LCM structure; reduced polysaccharide losses; recovery of valuable products from hemicelluloses; limited generation of unwanted products from polysaccharides (for example, dehydration products such as furfural or 5-hydroxymethylfurfural) or lignin; (i) production of pretreated solids with enhanced cellulose contents and high susceptibility toward enzymatic hydrolysis; (j) recovery of high-quality lignin or lignin-derived compounds; and (k) limited waste generation.

There is not a general agreement on which pretreatment can be considered as the best one [14]. Pretreatments intending hemicellulose solubilization have been considered, either operating with water [15–17] or with acidic solutions [18–20]. Autohydrolysis (also called hot water processing or hydrothermal pretreatment) is one of the approaches that meet several of the above conditions. This method is carried out by heating an aqueous suspension of the LCM, and results in the partial hydrolysis of hemicellulose into soluble fragments (by means of the catalytic action of hydronium ions from water ionization and from in situ generated acids). Operating under suitable conditions, hemicelluloses can be extensively converted into soluble saccharides [21], whereas the treated solids (with increased proportions of cellulose and lignin) show an increased susceptibility to cellulolytic enzymes [6,22–24], enabling their utilization as substrates for bioethanol production. The fermentative production of ethanol from pretreated LCM can be carried out operating either in two sequential steps of hydrolysis and fermentation (Separate Hydrolysis and Fermentation, SHF) or in a single stage (Simultaneous Saccharification and Fermentation, SSF). SSF presents advantages derived from the decreased product inhibition, limited operational costs and decreased contamination risks [25]. SSF also compare favorable with SHF in terms of experimental results [26]. From a general perspective, the loadings of solids and enzymes are major variables affecting the economic features of bioethanol manufacture. The importance of the type of pretreatment and its repercussion on the overall process has been emphasized in literature [27], as well as the fact that a correct choice of pretreatment conditions increases the bioconversion of Eucalyptus globulus, from about 40% up to near 100% [12]. High solids loading (HSL) is a key operational strategy for both economic and technical reasons [28,29]. Increased concentrations of solids enable higher potential bioethanol concentrations, reducing the size equipment, the consumption of energy in heating and distillation, and the downstream processing duties. According to literature, the threshold for economic profitability corresponds to bioethanol concentrations in the fermentation broth in the range 4–5 volume percent [28]. Achieving this threshold entails the utilization of media containing 15–20% solids (on dry basis). However, high solid loadings may result in limited cellulose conversions in enzymatic hydrolysis [29] or in SSF stages, owing to mass transfer limitation. In the manufacture of bioethanol by technologies involving enzymatic hydrolysis, the cost of enzymes has been identified as the second contributor to the operational costs, just after the raw material [30], and can account for 20% of total costs [31]. Because of this, the optimization of enzyme spending has been one of the topics considered in literature [12,25,28,32,33]. However, it can be noted that enzyme loadings below a given threshold may result in increased SSF duration [33] and limited cellulose conversion. Eucalyptus is a fast growing species with favorable features as a raw material for bioethanol production. E. globulus wood has been

considered by Romaní et al. [12] as a substrate for bioethanol production by SSF by a method based on hydrothermal pretreatment and bioconversion at low-intermediate solids loading, reaching a maximum ethanol concentration of 27 g/L. Similar concentrations (up to 29 g/L) have been achieved using acid-catalyzed pretreatments [34], whereas higher concentrations (up to 35 g/L) were achieved with organosolv-processed substrates [35]. To our knowledge, no information has been reported dealing with the simultaneous assessment of variables measuring the pretreatment conditions and two variables (loadings of solids and enzymes) of major economic importance. The experimental plan developed in this work provides relevant information for preliminary technical and economic analysis. This work deals with the optimization of bioethanol production from autohydrolyzed E. globulus wood, operating at high substrate loadings in SSF mode. The effects of substrate pretreatment conditions, liquor to solid ratio employed in SSF, and enzyme loading on selected operational variables (ethanol concentration, ethanol yield and exhausted solid composition) were assessed in selected experiments. 2. Materials and methods 2.1. Raw materials E. globulus wood samples (kindly provided from ENCE, Pontevedra, Spain) were milled to pass a 8 mm screen, air-dried and stored in a dark and dry place until use. 2.2. Analysis of the raw material For analytical purposes, raw material was milled to a particle size less than 0.5 mm and analyzed by the following standard methods: extractives (TAPPI T-264-om-88m), moisture (TAPPI T264-om-88m), ashes (T-244-om-93) and quantitative acid hydrolysis (QAH) of extractive-free wood with 72% w/w sulphuric acid (T-249-em-85). The liquid phases from QAH were assayed for glucose, xylose, arabinose and acetic acid by HPLC using a Refractive Index detector and a BioRad Aminex HPX-87H column, eluted with 0.01 M H2SO4 at a flow rate of 0.6 mL min1. The results measured the wood contents of cellulose, xylan, arabinan and acetyl groups, respectively. The solid residue from QAH was considered as Klason lignin. The experimental results are shown in Table 1. 2.3. Autohydrolysis of E. globulus wood Raw material and water were mixed in a 3.75 L stainless steel reactor (Parr Instruments Company, Moline, Illinois, USA) at a proportion of 8 g of water/g of oven-dry wood. Wood moisture was considered as water for media formulation. The reactor was fitted with four-blade turbine impellers, heated by an external fabric mantle, and cooled by tap water circulating through an internal loop. The suspension was heated following the standard temperature profile

Table 1 Eucalyptus globulus wood composition (g/100 g wood in oven-dry basis ± standard deviation). Content Cellulose Xylan Arabinan Acetyl groups Klason lignin Extractives Ash

44.7 ± 0.4 16.0 ± 0.2 1.09 ± 0.06 2.96 ± 0.05 24.7 ± 0.01 2.96 ± 0.15 0.23 ± 0.03

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[32] until reaching temperatures in the range 210–230 °C. In order to quantify the effects of the heating profile on reaction, the processing conditions are also reported in terms of severities. Severity (denoted So) was calculated as the logarithm of the severity factor R0 (measured in min), which has been proposed to assess the joint effects of temperature and time in the hydrothermal processing of LCM (see [32] for further details). The experimental conditions employed in this work corresponded to severities within the range 4.08–4.67. When the desired temperature was achieved, the medium was cooled, and the liquor was recovered by filtration.

(120 rpm) at 35 °C for 96 h (pH = 5). SSF media were prepared at the desired solid and enzyme loadings, by mixing the necessary amounts of substrate and commercial enzyme concentrates with 10 mL of nutrient solution (containing 5 g peptone/L, 3 g malt extract/L and 3 g yeast extract/L) and 10 mL of inoculum (cell concentration in the resulting media, 1.85 g/L). Samples were withdrawn from SSF media at preset times (0, 3, 6, 11, 24, 31, 48, 72 or 96 h), centrifuged (6000 rpm) and analyzed by HPLC for ethanol and sugars. The ethanol conversion at time t (ECt, g ethanol/100 g potential ethanol) was calculated as:

2.4. Analysis of liquors and solids from autohydrolysis

ECt ¼ 100 

Aliquots of autohydrolysis liquors were filtered through membranes of 0.45 lm pore size and used for direct HPLC determination of glucose, xylose, arabinose, acetic acid, hydroxymethylfurfural and furfural. A second sample of liquors was subjected to quantitative posthydrolysis (by triplicate, with 4% H2SO4 at 121 °C for 30 min), filtered though 0.45 lm membranes and analyzed by HPLC. The increase in the concentration of monosaccharides and acetic acid caused by posthydrolysis measured the oligomer concentration and their degree of substitution by acetyl groups [21]. Autohydrolyzed solids were washed with distilled water, and used for compositional analysis and for measuring the autohydrolysis solid yield (denoted YA, and defined as g of autohydrolyzed solids/100 g oven-dry wood). Samples of autohydrolyzed solids were milled to particle a size less than 0.5 mm and assayed for composition using the same methods cited above for raw material. The corresponding data are shown in Table 2.

where Et is the ethanol concentration (g/L) at time t, 92/180 is the stoichiometric factor for glucose conversion into ethanol and GPOT is the potential glucose, calculated as:

2.5. Microorganism, medium and yeast cultivation The strain used in the experimental design was Saccharomyces cerevisiae CECT-1170, obtained from the Spanish Collection of Type Cultures (Valencia, Spain). Cells were grown at 32 °C for 24 h in a medium containing 10 g glucose/L, 5 g peptone/L, 3 g malt extract/L and 3 g yeast extract/L. Biomass concentrations in the media were measured by dry cell weight. 2.6. Simultaneous Saccharification and Fermentation (SSF) of autohydrolyzed solids For optimization purposes, SSF assays (using autohydrolyzed solids as substrates) were carried out according to a factorial design of experiments (conditions listed in Table 3). The enzymes used in this study were ‘‘Celluclast 1.5L’’ cellulases (from Trichoderma reesei) and NS50010 b-glucosidase (from Aspergillus niger), both kindly supplied by Novozymes (Madrid, Spain). Cellulase activity was determined using the Filter Paper assay [36], whereas b-glucosidase activity was determined using p-nitrophenyl-b-D-glucoside as a substrate [37], and expressed in International Units (IU). Assays were carried out in 250 mL Erlenmeyer flasks (100 mL) placed in an orbital shaker Table 2 Composition of solis from the autohydrolysis stage. TMAX (°C) So (dimensionless)

210 4.08

220 4.38

Total recovery of solids (g/100 g raw material, oven dry basis) 71.8 69.1 Solid yield (YA)

230 4.67 70.7

Composition of autohydrolyzed solids (g/100 g autohydrolyzed solids, oven dry basis) Cellulose Xylan Arabinan Acetyl groups Klason lignin

61.5 ± 0.2 1.76 ± 0.01 0.00 0.31 ± 0.05 31.3 ± 0.2

64.1 ± 0.6 1.15 ± 0.04 0.00 0.15 ± 0.04 31.3 ± 0.3

60.1 ± 0.3 0.62 ± 0.04 0.00 0.05 ± 0.01 36.5 ± 0.3

GPOT ¼

Et 92 GPOT  180

Gn 180 q   KL 100 162 LSR þ 1  100

ð1Þ

ð2Þ

where Gn is the cellulose content of spent solid (g cellulose/100 g oven-dry autohydrolyzed solids basis), 180/162 is the stoichiometric factor for cellulose hydration upon hydrolysis, q is the density of the reaction medium (average value, 1005 g/L), LSR is the liquid solid ratio in the considered experiment, and KL is the Klason lignin content of the substrate (g Klason lignin/100 g oven-dry autohydrolyzed solid). 2.7. Analysis of spent solids from Simultaneous Saccharification and Fermentation (SSF) At the end of SSF experiments, the spent solids were centrifuged, washed with water, filtered and used for yield determination (YRS, g of residual solids from SSF/100 g oven-dry autohydrolyzed solids). Samples of spent solids from SSF were milled to a particle size less than 0.5 mm and assayed for composition using the same methods cited above for the raw material. 2.8. Fitting of data and modelling The experimental data were fitted to the proposed models using commercial software (Microsoft Excel, Redmon, Washington, USA). Response Surface Methodology (RSM) was used for optimization purposes. 3. Results and discussion 3.1. Experimental design and selection of operational variables and variation ranges The suitability of autohydrolysis processing for obtaining suitable substrates for enzymatic hydrolysis was confirmed in a previous work [12]. In this article, preliminary experiments allowing the production of second-generation bioethanol from autohydrolyzed E. globulus wood were described: SSF performed with substrates autohydrolyzed at So = 4.67 led to a good ethanol conversion (up to 86.4%), but at a limited concentration (26.7 g ethanol/L), conditioned by the values of the major operational variables (liquor to solid ratio and enzyme loading). On the other hand, the range of autohydrolysis conditions leading simultaneously to good enzymatic susceptibility and limited incidence of sugar-decomposition reaction corresponded to So in the range 4.0–4.7: So < 4.0 resulted in poor cellulase digestibility, whereas So > 4.7 led to increased cellulose losses (and so, in decreased ethanol yield based on wood weight). Samples treated at So = 4.08 led to the maximal recovery of hemicellulose-derived saccharides (including oligomers

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Table 3 Operational conditions assayed (expressed as dimensional and dimensionless independent variables) and results (EMAX, maximum ethanol concentration, g/L; QPMAX volumetric productivity at t of EMAX, g/(L h); ECMAX, maximum ethanol conversion, g ethanol/100 g potential ethanol; YRS, residual solid yield, g residual solid after SSF/100 g autohydrolyzed material, on dry basis; RecCel, cellulose recovery, g cellulose in residual solid after SSF/100 g cellulose in autohydrolyzed material, on dry basis) in experimental design carried out to optimize the SSF of autohydrolyzed Eucalyptus globulus wood. Exp. no

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Dimensional, independent variables

Dimensionless, normalized, independent variables

Dimensional dependent variables

LSR (g/g)

ESR (UPF/g)

So ()

x1

x2

x3

EMAX (g/L)

QPMAX (g/L/h)

ECMAX (g/100 g)

YRS (g/100 g)

RecCel (g/100 g)

16 16 4 4 16 4 4 16 10 10 16 4 10 10 10 10 10

16 4 16 4 16 16 4 4 10 10 10 10 16 4 10 10 10

4.67 4.67 4.67 4.67 4.08 4.08 4.08 4.08 4.67 4.08 4.37 4.37 4.37 4.37 4.37 4.37 4.37

1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0

1 1 1 1 1 1 1 1 0 0 1 1 0 0 0 0 0

1 1 1 1 1 1 1 1 0 0 0 0 1 1 0 0 0

18.7 7.0 67.4 33.8 16.8 64.4 33.5 6.3 25.8 23.6 16.2 62.3 31.6 13.9 27.0 27.5 28.4

0.39 0.10 0.94 0.35 0.23 0.67 0.35 0.13 0.45 0.25 0.22 0.87 0.44 0.15 0.38 0.47 0.49

90.6 33.8 91.1 45.6 79.8 86.0 44.7 30.2 80.0 71.8 73.7 79.8 92.2 40.7 78.9 80.4 82.7

43.8 61.0 34.3 53.5 41.9 40.4 58.0 62.9 37.5 41.7 40.0 35.2 31.9 50.9 37.6 37.6 35.4

2.5 28.5 2.1 29.2 5.2 8.3 31.3 32.1 1.3 6.2 2.2 3.1 1.1 22.9 1.7 1.6 1.4

and sugars) in authohydrolysis liquors: 19.8 g of hemicellulosic saccharides (measured as sugars) per 100 g of oven-dry wood were recovered, most of them in oligomeric form (xylooligosaccharides). This kind of compounds can be either utilized directly [38], or subjected to a posthydrolysis stage (catalyzed by acids o enzymes) to yield xylose solutions (which could be employed, for example, as fermentation substrates to obtain bioethanol, acetone–butanol or xylitol). On the basis of the above ideas, the So range selected in this work was 4.08–4.67. Table 2 lists the values considered for So, as well as the experimental results determined for solid yield (YA) and composition of autohydrolyzed solids, which enable the formulation of material balances to the pretreatment stage. YA varied in the range 69.1–71.8 g/100 g oven-dry wood, reaching values close to the joint contribution of cellulose and lignin in wood (69.5 g/100 g). In oven-dry basis, autohydrolyzed solids contained 60.1–64.1 cellulose/100 g, 31.3–36.5 g lignin/100 g, and 0.7–2.0 g hemicelluloses/100 g (calculated as the joint contributions of xylan, arabinan and acetyl groups). The data showed that 98–94% of cellulose and Klason lignin in wood were recovered in autohydrolyzed solids, in comparison with less than 5% of hemicellulose recovery. This finding confirmed the ability of autohydrolysis treatments to achieve extensive hemicellulose solubilization with limited losses of lignin and cellulose. Table 3 shows the structure of the experimental design employed in this work. Severity (So or x1), liquor to solid ratio (LSR or x2) and enzyme to substrate ratio (ESR or x3) were considered as operational variables; whereas the dependent variables were EMAX (maximum ethanol concentration, g/L), QPMAX (volumetric productivity measured at time leading to the maximum ethanol concentration, g/[L h]), ECMAX (maximum ethanol conversion, g ethanol/100 g potential ethanol), YRS (residual solid yield, g of residual solid from SSF/100 g oven-dry autohydrolyzed solids) and RecCel (cellulose recovery, g cellulose in solids from SSF/ 100 g cellulose in oven-dry autohydrolyzed solids). For calculation purposes, the independent variables were converted into dimensionless, normalized ones (with variation range from 1 up to 1) using the following equation:

xi ¼ 2 

X ij  X ime X imax  X imin

ð3Þ

where x stands for the dimensionless dependent variable, X denotates the corresponding dependent variable (So, LSR and ESR corresponding to X1, X2 and X3, respectively), the subscripts i, j, me, min and max correspond to the independent variable considered, the value that the variable takes in the experiment considered, and to the mean, minimum and maximum values of their variation ranges, respectively. The interrelationship between dependent and independent variables was established by empirical models following the generalized expression:

yj ¼ b0j þ

X i

bij xj þ

XX i

bikj xj xk

ð4Þ

k

where yj is the dependent variable considered (j: 1–5), xi or xk (i or k: 1–3, k P i) are the dimensionless, independent variables defined by Eq. (3), and b0j . . . bikj are the regression coefficients, calculated from the experimental data by multiple regression using the least-squares method. Table 3 shows the experimental values achieved for the experimental variables, whereas Table 4 lists the sets of regression coefficients b0j . . . bikj and their significance (based on the Student’s t-test), as well as the statistical parameters measuring the correlation (R2) and significance of models (based on the Fisher’s F-test). 3.2. Time course of Simultaneous Saccharification and Fermentation experiments Fig. 1 shows the time course of representative SSF experiments. As a general trend, the ethanol concentrations increased sharply along the first fermentation stages, leading to ethanol conversions after 24 h (EC24) from 26% (in experiment 7, carried out under conditions defined by So = 4.08, LSR = 4 g/g and ESR = 4 FPU/g) up to 83% (in experiment 13, carried out under conditions defined by So = 4.37, LSR = 10 g/g and ESR = 16 FPU/g). After 48 h, the ethanol conversion (EC48) varied from 30% (in experiment 8, carried out under conditions defined by So = 4.08, LSR = 16 g/g and ESR = 4 FPU/g) up to 90% (in experiment 1, carried out under conditions defined by So = 4.67, LSR = 16 g/g and ESR = 16 FPU/g). Longer fermentation times resulted in progressively lower increases in ethanol production, up to achieve conversions in the range 90–92% in several experiments. The highest ethanol concentrations were reached in experiments 3, 12 and 6, carried out at the lowest LSR (4 g/g). The

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** ***

QPMAX (g/L/h)

ECMAX (g/100 g)

YRS (g/100 g)

RecCel (g/100 g)

b0 b1 b2 b3 b11 b22 b33 b12 b13 b23 R2 F Significance level

27.75*** 19.64*** 10.44*** 0.81 11.43*** 5.06*** 3.12* 5.30** 0.11 0.51 0.993 115 >99%

0.423** 0.210*** 0.159*** 0.059* 0.140** 0.113* 0.060 0.064* 0.019 0.057* 0.950 14.2 >99%

0.796*** 0.039*** 0.245*** 0.028*** 0.020 0.123*** 0.028* 0.025*** 0.010 0.014* 0.996 210 >99%

0.358*** 0.028*** 0.094*** 0.015** 0.025** 0.064*** 0.046*** 0.002 0.013** 0.003 0.990 76.9 >99%

0.0110* 0.0035 0.1248*** 0.0196*** 0.0194** 0.1128*** 0.0298*** 0.0034 0.0024 0.0041 0.995 167 >99%

Coefficients significant at the 90% confidence level. Coefficients significant at the 95% confidence level; Coefficients significant at the 99% confidence level;

maximum concentration (67.4 g ethanol/L) was obtained in experiment 3, which was performed using a substrate pretreated at So = 4.67, under SSF conditions defined by: duration = 72 h, LSR = 4 g/g, and ESR = 16 FPU/g. In most experiments, the highest ethanol concentrations were obtained after 72 h. As an average, the ethanol conversions achieved after 24 and 48 h were 71% and 93%, respectively. Byproducts such as xylose, glycerol and acetic acid were found at little concentrations: for example, the media contained just 0–2 g xylose/L, owing to the limited substrate content of residual xylan. For comparison, Table 5 lists experimental results obtained in this work and data reported in related studies. In an earlier work dealing with hydrothermally processed E. globulus wood [12], concentrations to 27 g ethanol/L and cellulose conversions up to 86% were achieved under conditions defined by LSR = 10–20 g/g and ESR = 6.2–10.3 FPU/g. Using Eucalyptus grandis pretreated with acids, Silva et al. [34] reached media containing 28.7 g ethanol/L operating under HSL conditions (LSR = 5 g/g), but high enzyme loadings (ESR = 30 FPU/g) were necessary. Muñoz et al. [35] employed organosolv-delignified Eucalyptus wood to obtain substrates containing 84% cellulose, which was processed by SSF (under conditions defined by LSR = 10 g/g and ESR = 20 FPU/g) to obtain media containing up to 35 g ethanol/L, but some cellulose was lost in the pretreatment step. In general, operating at solid loadings above 10–20 weight percent may result in decreased conversions [39,40]. Manzanares et al. [28] using hardwood pretreated with liquid hot water or by acid prehydrolysis reported significant decreased in ethanol yields when the solid loading increased from 9% up to 23%. A similar behavior has been reported for corncobs pretreated first with acidic solutions and then under alkaline conditions: increasing the solids loading from 7.5% up to 19% decreased the ethanol yield from 90% to 77% [41]. In experiments with steam exploded corn stover, increasing the solid loading in SSF from 15% up to 30% decreased the ethanol conversion from 76.5% to 52.1% [42]. The highest ethanol concentrations have been reported for susceptible substrates of agricultural origin, such as steam exploded corn stover. Using this feedstock, ethanol concentrations up to 64.6 g/L were reached operating at a high solids loading (LSR = 3.33 g/g) in a reactor fitted with a modified helical impeller [42]. Corn cobs subjected to consecutive pretreatments (prehydrolysis with sulphuric acid and further treatment with NaOH) resulted in a substrate with high cellulose content (91.1%), which was employed as a substrate for SSF (operating at LSR = 4 g/g in fed-batch

Ethanol concentration (g/L)

EMAX (g/L)

60 Exp 1

50

Exp 2

40

Exp 3

30

Exp 4

20

Exp 5

10 0 70

Ethanol concentration (g/L)

*

Parameter

70

60 Exp 6

50

Exp 7

40

Exp 8

30

Exp 9

20

Exp 10

10 0 70

Ethanol concentration (g/L)

Table 4 Regression coefficients and statistical parameters measuring the correlation and significance of the models.

60 Exp 11

50

Exp 12

40

Exp 13

30

Exp 14

20

Exp 15 to 17

10 0 0

20

40

60

80

100

t (h) Fig. 1. Time course of ethanol concentration in SSF experiments (see Table 3 for experimental conditions of individual experiments). Experiments 15–17 were carried out under the same experimental conditions, and the average values are shown.

mode for 96 h, with ESR = 23 FPU/g). The resulting media contained up to 84.7 g ethanol/L, with ethanol conversions below 70% based on treated solids, as no data were provided for assessing the ethanol conversion based on raw material [41]. Comparatively, the approach assessed in this work presents advantages derived from no utilization of chemicals different from water and wood, lower operational costs derived from the single pretreatment stage and the possibility of obtaining simultaneously high ethanol concentrations and high ethanol conversions: for example, experiment 3 (carried out at LSR = 4 g/g) resulted in EMAX = 67.4 g ethanol/L and ECMAX = 91.1%). It can be noted that the pretreatment considered in this work does not result in significant cellulose losses and result in substrates highly susceptible to enzymatic hydrolysis. Both aspects improve the amount of ethanol that can be produced per kg of raw material. Comparatively, other reported pretreatments are less selective and result in partial cellulose solubilization [35]. 3.3. Response Surface Methodology assessment Response Surface Methodology is a valuable tool for optimization. The dependence of the maximum ethanol concentration

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Table 5 Experimental data obtained in this work and results reported in related studies. Raw material

Eucalyptus globulus Eucalyptus grandis Eucalyptus globulus Olive tree pruning Olive tree pruning Corn cobs

Pretreatment

Autohydrolysis at So = 4.67 Acid hydrolysis (1.2% H2SO4, 121 °C, 45 min) Delignification (60% ethanol) at H = 125001 Autohydrolysis (210 °C, 10 min)

Acid hydrolysis (1% H2SO4, 180 °C, 10 min) Acid hydrolysis (conc. formic acid, 60 °C, 6 h) + delignification (15% aq. ammonia, 60 °C, 12 h)

Corn stover

Steam explosion (200 °C, 4 min)

Barley straw

Steam explosion (210 °C, 5 min)

Corn stover

Ethanol production

Steam explosion (205 °C, 6 min)

Eucalyptus Autohydrolysis at So = 4.67 globulus Rt 1 H ¼ 0 expð43:2  16115=TÞdt; T: temperature (K), t: time (min). 2 ESR in FPU/g glucan.

(EMAX, g ethanol/L) on the operational variables was well interpreted by the empirical model, as it can be confirmed from the results of the fitting parameters included in Table 4. The average deviation between experimental and calculated values was 2.6%. On the basis of the results determined for the model coefficients (see Table 4), it can be concluded that LSR and ESR were the most influential variables on EMAX, and that little effects were caused by So. Fig. 2 shows the calculated dependence of EMAX on LSR and ESR for samples pretreated at So = 4.43 (the most favorable autohydrolysis conditions, according to the model predictions). ESR was more influential on EMAX when LSR was near its lowest value. This finding can be justified on the basis of mass transfer principles, as low LSR (i.e., when operating at a high solid loading) the heterogeneous enzymatic reaction can be limited by hydrodynamic factors. The highest EMAX (69.7 g ethanol/L) was predicted for conditions defined by LSR = 4 g/g, ESR = 16 FPU/g and So = 4.43. Under the conditions of assay 3 (which led to the maximum experimental concentration), the model predicted EMAX = 67.8 g/L, close to the experimental value (67.4 g ethanol/L). This confirmed the ability of the model for reproducing the experimental results on a quantitative basis, particularly within the operational range leading to high ethanol concentration, which concentrates the conditions of practical interest. Interestingly, the model confirmed the possibility of obtaining high EMAX operating at limited enzyme loadings: for example, using substrates pretreated at So = 4.43 in SSF performed at LSR = 4 g/g, enzyme to substrate ratios as low as 4.5 FPU/g (near the minimum value employed in this work) allowed EMAX > 40 g/L, a value that has been considered as the threshold for economical feasibility [43]. The volumetric productivities in SSF experiments were higher at the beginning of experiments (with typical values in the range 0.68–2.32 g/(L h) after 4 h), owing to the favorable kinetics of cellulose hydrolysis in the early reaction stages. However, these results lack practical interest, and the variable employed in

Reference

LSR (g/ g)

ESR (FPU/ g)

EMAX (g/ L)

ECMAX (g/ 100 g)

20 10 5

10.3 6.2 30

15.1 26.7 28.7

86.4 77.7

10 11 5.9 4.4 11 5.9 4.4 13.3 5.3 6.7 5 4 3.3 20 10 6.7 10 6.7 5 4 3.3 4

20 15

35 15.7 19.7 7.1 12.7 21.9 24.9 29.4 62.7 24.7 31.0 39.3 40.6 9.6 22.0 29.4 16 25 33 41 49 67.4

0-10

15

22.82 7

13.6

20

16

10-20

20-30

[12] [34] [35]

76 46 11 57 49 38 89.7 77.3 76.5 68.0 64.8 52.1

[28]

[28] [41]

[42]

[44]

[45]

91.1

30-40

40-50

This work

50-60

60-70

70 60 50 40

E MAX (g/L)

30 20 10

16 13,6 11,2 8,68 6,28 ESR (FPU/g)

0 4

6,4

8,8

LSR (g/g)

11,2

13,6

4 16

Fig. 2. Calculated dependence of EMAX (maximum ethanol concentration, g ethanol/ L) on LSR (liquor to solid ratio, g liquor/g oven-dry substrate) and ESR (enzyme to substrate ratio, FPU/g oven-dry substrate). Data corresponding to substrates pretreated at So = 4.43.

modelling (QPMAX) corresponded to the volumetric productivity reached under conditions leading to the highest ethanol concentration (EMAX). The variation range determined for QPMAX was 0.10–0.94 g/(L h), whereas the values calculated for the model coefficients (see Table 4) indicated that QPMAX was principally affected by LSR and ESR, with a variation pattern similar to the one previously described for EMAX (even if a stronger influence of ESR can be observed in this case). The value predicted for QPMAX under

311

A. Romaní et al. / Fuel 94 (2012) 305–312 78-80

80-82

82-84

84-86

86-88

88-90

90-92

92-94

94

0-1

1-2

2-3

3-4

4-5

5-6

6-7

7-8

8-9

9

92

8

90 88

EC MAX (% ) 86 84 82 80 4 6.4

4.08 4.20 4

8.8

8.8

the conditions of experiment 3, 0.96 g/(L h), was again close to the experimental result, 0.94 g/(L h), confirming the ability of the model for providing a quantitative assessment of this variable. The dependence of the maximum ethanol conversion (variable ECMAX, defined as g ethanol/100 g potential ethanol) on So and LSR was interpreted by the empirical model through the response surface shown in Fig. 3 (drawn for ESR = 16 FPU/g). Considering the coefficients in Table 4, in can be seen that ESR caused the major effects on ECMAX, with a limited influence of So. Comparatively, the effects caused by LSR were of minor importance. The highest value predicted for ECMAX (93.4%) corresponded to operational conditions defined by LSR = 8.8 g/g, ESR = 16 FPU/g and So = 4.53. This model prediction was in the range of the highest experimental ECMAX value (92.2%), which was reached under conditions defined by LSR = 10 g/g, ESR = 16 FPU/g and So = 4.37. The empirical model showed a remarkable ability for reproducing the experimental results, with just 0.8% average deviation between experimental and calculated data. According to the model predictions, no matter of the LSR considered (within the considered range), ECMAX above 80% could be achieved operating at ESR = 9 FPU/g, whereas ECMAX > 90% would require ESR = 13 FPU/g. In terms of ethanol yield (measured as the kg ethanol produced per 100 kg oven-dry wood), the experimental values varied in the range 7.6–23.3 kg/100 kg. These results corresponded to experiments 8 (carried out at LSR = 16 g/g, ESR = 4 FPU/g and So = 4.08) and 13 (carried out at LSR = 10 g/g, ESR = 16 FPU/g and So = 4.37), respectively. Considering that the stoichiometric amount of ethanol achievable from Eucalyptus wood cellulose is 25.4 kg/100 kg wood, the best result reported in this work corresponds to an overall efficiency of the pretreatment–saccharification–fermentation process (measured respect to the theoretical ethanol yield) of 91%. The spent solids from SSF were characterized to measure its potential for further applications. Lignin was their major component (50.4–88.3 g lignin/100 g oven-dry spent solids, with an average value of 71.7 g/100 g), whereas cellulose accounted for 2.1–33.2 g cellulose/100 g oven-dry spent solids (average value, 12.5 g/100 g). Other components of spent solids (residual hemicelluloses, yeast biomass and enzymes) were of minor importance for the purposes of this study.

4.55 13.6

16

Fig. 3. Calculated dependence of ECMAX (maximum ethanol conversion, g ethanol/ 100 g potential ethanol) on LSR (liquor to solid ratio, g liquor/g oven-dry substrate) and So. Data calculated for ESR = 16 FPU/g.

4.43

LSR (g/g) 11.2

LSR (g/g)

4.08

4.20

So (-)

13.6

4.32 6.4

11.2 4.32

4.43

4.55

4.67

78

7 6 5 RecCel (%) 4 3 2 1 0

S o (-)

4.67 16

Fig. 4. Calculated dependence of RecCel (cellulose recovery, g cellulose recovered in solid residue after SSF/100 g cellulose in raw material) on LSR (liquor to solid ratio, g liquor/g oven-dry substrate) and So. Data calculated for ESR = 16 FPU/g.

The spent solid yield (YRS, g of residual solid after SSF/100 g ovendry raw material) varied in the range 32–63 g/100 g, and decreased markedly with increasing ESR. Comparatively, YRS was scarcely affected by LSR and So. The cellulose recovered in spent solids, measured by variable RecCel (defined as g cellulose in spent solids/100 g cellulose in oven-dry wood) varied in the range 1.1–32.1 g/100 g, most results being in the range 1.1–3.1 g/100 g. The limited values determined RecCel were indicative of extensive cellulose hydrolysis. The empirical modelling of experimental data led to the set of coefficients included in Table 4, from which it can be observed that the major variations of RecCel are associated to ESR. Fig. 4 shows the dependence of de RecCel on So and LSR, calculated for ESR = 10 FPU/g. Values of RecCel near zero were predicted for a range of operational conditions, whereas results below 10 g/100 g were achieved under a variety of operational conditions.

4. Conclusions The experimental data confirm that autohydrolysis processing of E. globulus wood followed by SSF processing of the resulting solid phase is a suitable framework for the production of second generation bioethanol. Operating under suitable conditions, autohydrolyzed solids highly susceptible to cellulases can be obtained together with hemicellulose-derived products. Optimization of the whole process (including pretreatment conditions and SFF bioconversion) was assessed by the Response Surface Methodology, resulting in the identification of operational conditions (corresponding to high solid loadings) enabling simultaneously high ethanol conversion (up to 91%) and high volumetric concentration (67.4 g ethanol/L). Under selected conditions, the cellulosic fraction of wood was converted into bioethanol to yield 291 L bioethanol/metric ton of oven-dry wood. Acknowledgment Authors are grateful to ‘‘Xunta de Galicia’’ for the financial support of this work, in the framework of the Research Project with reference ‘‘Use of forest residues for biofuels production’’ (reference 08REM002383PR).

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