Biorefinery Scheme for Residual Biomass Using Autohydrolysis and

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A biorefinery scheme for residual biomass using autohydrolysis and organosolv stages for oligomers and bioethanol production Fatima Vargas, Elena Domínguez, Carlos Vila, Alejandro Rodríguez, and Gil Garrote Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b00277 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on September 5, 2016

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A biorefinery scheme for residual biomass using autohydrolysis and organosolv stages for oligomers and bioethanol production Fátima Vargas†, Elena Domínguez‡,§ , Carlos Vila‡,§, Alejandro Rodríguez†, Gil Garrote‡,§,*

†

Department of Chemical Engineering, Faculty of Science. University of Cordoba. Campus of Rabanales. Marie-Curie building (C-3). N-IV road, km. 396, 14071 Cordoba, Spain ‡

Department of Chemical Engineering. Faculty of Science, University of Vigo (Campus Ourense). As Lagoas. 32004 Ourense. Spain. §

CITI (Centro de Investigación, Transferencia e Innovación) – University of Vigo, Tecnopole, San Cibrao das Viñas, Ourense, Spain

KEYWORDS: autohydrolysis, barley straw, bioethanol, fed-batch SSF, high solids loading, organosolv.

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ABSTRACT: Straw is one of the main lignocellulosic waste produced during cereal crop cultivation. The abundance of barley straw makes it a good candidate for bioethanol production. This work deals with barley straw pretreatment by means of autohydrolysis in order to get xylooligosaccharides in the liquid phase, followed by an organosolv treatment using ethanol to increase the solid phase enzymatic susceptibility. Up to 17.4 g oligomers/L were obtained in the hydrothermal stage, in which practically all the cellulose and lignin remained in the solid phase. The solid phase from the hydrothermal-delignification was subjected to an experimental design in order to study the effect of pretreatment conditions in the bioethanol production, with values of solids concentrations in the range 7.7 to 20 weight % and values of enzyme loading in the range 14 FPU/g to 6 FPU/g. In the experiments carried out at a liquid to solid ratio = 4 g/g it is possible to obtain 31.6 g ethanol/L in just 9 h (corresponding to 100% ethanol conversion), with optimum results of 44.5 g ethanol/L in 46 h (90-93% glucose to ethanol conversion) and with a maximum concentration of 48.7 g ethanol/L in 89 h (79% conversion). The combination of a hydrothermal pretreatment (under conditions that lead to the recovery of high amounts of hemicellulosic by-products), followed by an organosolv treatment under mild conditions turns out to be suitable for second generation bioethanol production, applying a high solids loading, by means of fed-batch simultaneous saccharification and fermentation.

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INTRODUCTION Ethanol is nowadays the most widely used liquid biofuel alternative to fossil fuels.1 Biomass resources such as lignocellulosic materials could be used to provide a large-scale biomass to energy industry.2 Ethanol from lignocellulosic materials (LCM) is called second generation bioethanol.3 Second generation bioethanol would meant a net reduction of up to 85% of emissions of greenhouse gases.4 Straw is one of the main lignocellulosic wastes produced during cereal crop cultivation. The principal constituents of LCM are cellulose, hemicelluloses and lignin. Barley straw is an abundant by-product from barley production. The abundance of barley straw and its high carbohydrates content makes it a good candidate for the production of bioethanol.4 Barley straw is the second most abundant agricultural residue in Europe, after wheat straw.5 144 million metric tons of barley were produced in the world in 2013. Europe (59.7%), Asia (15.2%), and America (14.7%) are the main producing regions.6 The main byproduct of barley is straw, with a ratio of up to 530 kg straw/metric ton grain7 During the conversion of the LCM to bioethanol, the main task is the pretreatment step, which is necessary to break the tight lignocellulosic structure cellulose, hemicelluloses, and lignin, allowing amongst many other things the easier conversion into fermentable sugars.8 An ideal pretreatment should comply the following conditions: (1) simple and inexpensive operation, (2) reduce particle size easily, (3) low consumption of energy, water and chemicals, (4) little corrosion, (5) capable of altering the LCM structure, (6) with minimal polysaccharides losses, (7) allow producing large quantities of hemicelluloses that derive in high added value compounds, (8) generating little quantities 3


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of furans and phenolic acids (9) obtaining a solid fraction with high cellulose content and high enzyme susceptibility, (10) production of lignin and/or derivatives of high quality, and low waste (11). Taking this into account, autohydrolysis seems to be an ideal pretreatment. In the autohydrolysis or hydrothermal process (compressed hot water treatment), an aqueous suspension of LCM, without the addition of foreign chemicals, is heated causing hydrolytic degradation of hemicelluloses (through reactions catalyzed by hydronium ions, first generated from water autoionization and in subsequent reaction steps coming from organic acids generated in situ, as acetic acid). When autohydrolysis is conducted under suitable conditions, the obtained liquid phase is rich in hemicelluloses as xylooligomers, useful in several industries (food, pharmaceutical, etc.); while the solid phase (composed mainly of cellulose and lignin) is suitable for conversion to ethanol, and can be previously subjected to an organosolv delignification stage in order to increase its enzymatic susceptibility and removal of lignin.9 Organosolv treatments use organic solvents (with or without addition of mineral acid catalysts) to break bonds such as a-aryl ether and aryl glycerol-b-aryl ether in the lignin macromolecule, causing lignin alteration, including reduction in the average molecular mass.10 Alcohols, especially primary ones, such as methanol and ethanol, are the most commonly used solvents. Methanol and ethanol seem to be the most suitable alcohols for alcohol-based organosolv pretreatment due to its low cost and easy recovery. However ethanol pretreatment is safer than methanol pretreatment due to its lower toxicity.11 Simultaneous saccharification and fermentation (SSF) processes combine enzymatic hydrolysis of cellulose to monosaccharides with simultaneous fermentation of the obtained monosaccharides to ethanol.12 One of the main advantages of SSF process is that the final 4


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products of the enzymatic hydrolysis decrease as they are consumed by the microorganisms thus, they do not accumulate. This conversion diminishes the effect of any inhibition of final product in the enzymatic catalysis. SSF process can be achieved in one process step.13 The operation in fed-batch mode is a feasible method to maximize final ethanol concentration as well as to minimize the negative effects of inhibitors present in lignocellulosic hydrolysate 14. The fed-batch SSF process also offers several advantages including less water consumption and lower production cost through the reduced number and size of required equipment and utility 15, 16. This work aims to barley straw pretreatment using autohydrolysis in order to get xylooligosaccharides (XO) in the liquid phase, followed by an organosolv delignification (using ethanol) to increase the solid phase enzymatic susceptibility. Finally, the conversion into ethanol of the autohydrolysis solid phase followed by an organosolv pretreatment was compared through a Fed-Batch SSF process. EXPERIMENTAL Raw Material. Experiments were carried out using barley straw (from a local farm, Alcalá La Real, southern Spain). Barley straw was milled through a 2 mm screen, air dried, homogenized in a single lot and stored at room temperature in a dark and dry place until use. Chemical characterization of barley straw was carried out according to TAPPI standards (moisture by TAPPI 264 cm-07, ash by TAPPI T 211 om-12, extractives by TAPPI T 264 cm-07, composition by Quantitative Acid Hydrolysis (QAH) and Klason Lignin by TAPPI T 249 cm-09). Aliquots of liquid phase from QAH were filtered through 0.45µm membranes and analyzed by HPLC (Agilent Technologies 1100 Series HPLC with 5


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a Refractive Index Detector and a BioRad Aminex HPX-87H column, using 0.006 mol/L H2SO4 at 60 ºC as mobile phase) to determine the content of glucose, xylose, arabinose and acetic acid. The results allowed the determination of glucan, xylan, arabinan and acetyl groups of raw material, all the analyses were carried out by quadruplicate. The raw material composition expressed as g/100 g raw material on dry basis was (average values of quadruplicate ± standard deviation): glucan: 35.1 ± 0.1, xylan: 20.0 ± 0.1, arabinan: 4.61 ± 0.02, acetyl groups: 1.98 ± 0.03, Klason lignin: 19.8 ± 0.2, extractives: 6.8 ± 0.2, ash: 7.61 ± 0.02. These data show that hemicelluloses are composed by arabinoxylans. Autohydrolysis pretreatment. Non isothermal autohydrolysis was carried out in a stainless steel Parr reactor of 3.75 L (Parr Instruments Company, Moline, Illinois, USA) equipped with a four-blade rotor, heated by an external mantle and cooled by flowing water through an inner loop. Barley straw and water were mixed to achieve a Liquid to Solid Ratio (LSR) of 8 g liquid/g raw material odb (oven dry basis), corresponding to 11.1 wt. % of solids, and reacted at 150 rpm. The experiments were carried out in non isothermal mode, up to reach the maximum temperature (TMAX, ºC). The hardness of treatments can be measured as severity (So), the logarithm of the severity factor R0.17 So combined the effects of time and temperature during the entire hydrothermal processing. In a non isothermal process, taking into account the heating and cooling periods, So was calculated as:

(1)

6


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where: tMAX is the time (in min) needed to achieve maximum temperature (TMAX, °C); tF is the time (in min) required for the entire heating-cooling cycles; T(t) and T'(t) (°C) are the temperature profiles in heating and cooling, respectively, and ω and TREF are parameters whose values have been reported in literature 17 (ω = 14.75 °C; TREF = 100 ˚C). Three different severities were employed, with maximum temperatures of TMAX (195ºC, 205ºC and 215ºC), with values of So = 3.641, 3.936 and 4.230, respectively. These conditions were selected in order to obtain high amounts of xylooligosaccharides in liquid phase. Once TMAX was reached, the reactor was cooled, opened, and the solid and liquid phases were separated by solid-liquid filtration. The solid phase was washed with distilled water, filtered and centrifuged, its moisture content was quantified and assayed to determine the autohydrolysis yield (YA, autohydrolysis yield, g solid phase obtained after autohydrolysis/100 g raw material, on dry basis). An aliquot of autohydrolyzed barley straw was assayed for solid composition using the same methods as those applied for the raw material characterization. A first aliquot of liquid phase was analysed by HPLC to determine the concentration of glucose, xylose, arabinose, acetic acid, furfural and hydroxymethylfurfural. A second aliquot was subjected to quantitative posthydrolysis (4% w/w sulphuric acid, 121⁰C, 40 minutes) to convert oligomers into monomers and analyzed by HPLC. The increase in the concentrations of glucose, xylose, arabinose and acetic acid after posthydrolysis measures the concentration and composition of oligomers. A third aliquot was dried (at 105 ºC until constant mass) in order to quantify the non-volatile

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content (NVC, g non volatile compounds in liquid phase from autohydrolysis/100 g raw material, on dry basis). Organosolv pretreatment. Autohydrolyzed barley straw was subjected to organosolv process with ethanol/water mixtures under milder conditions. Experiments were carried out in a pressurized reactor (Parr Instruments Company, Moline, Illinois, USA) of 0.6 L equipped with a four- blade rotor, heated by an external mantle and cooled by flowing water through an inner loop. Experimental conditions were the following: Ethanol concentration was 40% (w/w), delignification temperature was 170 ºC, delignification time was 1 hour and liquid to solid ratio employed was 8 g liquid/g autohydrolyzed barley straw (oven dry basis). After carrying the delignification process out, the solid and liquid phases were separated by solid-liquid filtration and solid phase was first washed with an ethanol/water mixture, of the same concentration than the one used in the pretreament, and then with distilled water. Solid phases were assayed for organosolv yield, and aliquots were employed for chemical characterization (with the methods described in “raw material” section) and for Simultaneous Saccharification and Fermentation (SSF). Yeast cultivation and inoculum preparation. Saccharomyces cerevisiae CECT-1170 (Spanish Collection of Type Cultures, Valencia, Spain) was used in simultaneous saccharification and fermentation assays. Cells were grown at a set temperature of 32 ºC during 24h in 250 mL Erlenmeyer flasks containing 10 g glucose/L, 5 g peptone/L, 3 g malt extract/L and 3 g yeast extract/L. The concentration of biomass in the media was measured by dry cell mass. 8


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Simultaneous Saccharification and Fermentation in fed-batch mode (FB-SSF). FBSSF experiments were carried out in 250 mL Erlenmeyer flasks in orbital shakers (120 rpm and at 35ºC). The experiments started with the addition of 10 mL inoculum (leading to an initial yeast cell concentration of 0.83 ± 0.02 g/L), the desired amounts of enzymes (from 14 FPU/g material, odb to 6 FPU/g LCM, odb, depending on the experiment, supplemented with 5 IU/FPU, corresponding to 70 to 30 IU/g material, odb; respectively) and nutrients (5 g peptone/L, 3 g yeast extract/L and 3 g malt extract/L). Previous experiments were carried out in order to establish the best options for fed-batch SSF. These assays were performed with the substrate divided into 3 or 4 equal loadings, following two options: a) all the nutrients added at the beginning, b) the addition of nutrients were divided in 3 or 4 equal parts, in the same proportions used with the substrate. These previous experiments show that the best results were obtained working with the substrate and the nutrients fed in 4 additions. These conditions were settled as the most favourable conditions for FB -SSF. In each addition, the substrate and nutrients were fed always in equal amounts (33.3% or 25% of substrate and nutrients, for 3 or 4 additions, respectively). Solids and nutrients were added at the beginning of the fermentation (0 h), and after 34 h, 64 h and 90 hours. The 100% of enzymes were added at the beginning of the fermentation. Commercial enzymes “Celluclast 1.5 L” from Trichoderma reesei and “Novozym 188” β-glucosidase from Aspergillus niger were kindly provided by Novozymes. The enzymes activities of “Celluclast 1.5 L” and “Novozym 188” commercial concentrates were 70 FPU/mL and 630 UI/mL, respectively. Fed-batch SSF lasted up to 118 h. Samples were taken at desired times and analysed by HPLC. The maximum ethanol conversion after the “i” loading (ECi, g ethanol /100 g potential ethanol, with I = 1, 2, 3 or 4) was calculated as: 9


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(2) where Ei is the maximum ethanol concentration (g ethanol/L) after the“i” loading, and EPOT is the potential ethanol concentration (g ethanol/L) corresponding to the stoichiometric conversion of the glucan to ethanol, without degradation, calculated as: ீ௡

ଽଶ

‫ܧ‬௉ை் ൌ ଵ଴଴ ൉ ଵ଺ଶ ൉

ఘ ௅ௌோ೔ ା

ಸ೙ భబబ

(3)

where Gn is the glucan content of the solid subjected to FB-SSF (g glucan/100 g pretreated material, on dry basis), 92/162 is the stoichiometric factor for glucan to ethanol conversion, ρ is the density of the reaction medium (average value, 1005 g/L), and LSRi is the liquid to solid ratio in FB-SSF after the “i” loading. Experimental design Treated barley straw was subjected to simultaneous saccharification and fermentation. In order to get a deeper understanding of the process, an experimental design was performed. The independent variables were: Autohydrolysis severity: barley straw was hydrothermally pretreated at three different severities (So = 3.641, 3.936 and 4.230) and then subjected to an organosolv process under the same experimental conditions. Two variables related to saccharification and fermentation process have been studied: final values of liquid to solid ratio (LSR) and enzyme loading (EL). Values of liquid to solid ratio were selected to cover a range from medium to high solids loading (from 12 g/g to 4 g/g, corresponding to 7.7 to 20 wt. % of solids), and the values of enzyme loading correspond to medium to low enzyme dosage (from 14 FPU/g odb material to 6 10


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FPU/g odb material). Both ranges of variables, LSR and EL, are the most interesting from an economical and technical point of view to reach a compendium between residence time and the amount of ethanol produced, to make the process cost effective 18. Simultaneous saccharification and fermentation assays were carried out in fed batch mode (see previous section), which allowed an easy handling of high solids loading. Three parameters were selected in order to follow on the saccharification and fermentation process: 1) maximum ethanol concentration (denoted E, g ethanol/L), 2) maximum ethanol conversion (EC, g ethanol /100 g potential ethanol), and 3) volumetric productivity (denoted QP, measured at time leading to the maximum ethanol concentration, g/[L·h]). In this fed batch configuration process, these 3 parameters have different values in function of each solid loading (4 time ranges: 0 – 34 h, 34 – 64 h, 64 – 90 h, 90 h to end of essay). Based on this, the variables E, EC and QP for each of the 4 time ranges were considered as dependent variables. Table 1 shows the structure of this experimental design. For calculation purposes, the independent variables were expressed as dimensionless variables, with variation range from -1 to 1, using the following equation:

(4) where x is the dimensionless independent variable, X is the corresponding independent variable (So, LSR and EL corresponding to X1, X2 and X3, respectively), the subscript i corresponds to the independent variable considered (i = 1, 2 or 3), the subscript j corresponds to the value of independent variable in the experiment, and the subscripts me,

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min and max correspond 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:

y

j

= b0

j

+

∑ i

b ij x

j

+

∑ ∑ i

b ikj x j x k

(5)

k

where yj is the dependent variable considered (j: 1 to 12, see Table 1), xi or xk (i or k: 1 to 3, k ≥ i) are the dimensionless, independent variables defined by equation (4), and b0j....bikj are the regression coefficients, calculated from the experimental data by multiple regression using the least-squares method. Commercial software Microsoft Excel (Microsoft, USA) was used to treat the experimental data RESULTS AND DISCUSSION Pretreatment. Non isothermal autohydrolysis of eucalyptus carried out at log So = 3.6 produces the maximum amount of XO obtained in the liquid phase, but with a poor enzymatic susceptibility of the resulting solid phase (only 40% at 72 h); to get higher enzymatic susceptibilities, close to 100%, severities higher than log Ro= 4.7 are needed, and the amount of XO in the liquid phase is only the 7% of the maximum amount under these conditions.19 On the other hand, the use of organosolv stages with ethanol/water mixtures, easily implementable on a global bioethanol production process, can increase the enzymatic susceptibility.20 The coupling of a first autohydrolysis stage followed by an ethanol/water organosolv one can enable the simultaneous production of high amounts of XO in the autohydrolysis liquid phase and higher enzymatic susceptibilities of the solid phase from the organosolv stage. 12


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In this work, non isothermal autohydrolysis under conditions leading to the production of maximum amounts of XO in the liquid phase, followed by an organosolv ethanol/water treatment in mild conditions, were carried out. Table 3 presents the experimental conditions and the results determined for solid yields and composition of solid phase from both treatments and liquid phase from autohydrolysis. The solid yield (YA) varied between the range 64.9 – 58.2 g/100 g raw material odb, whereas the non volatile content in the liquid phase fluctuated between 26.0 – 29.9 g/100 g raw material odb. Regarding the composition of the solid phase, the glucan content varied from 48.1 to 56.4 g glucan/100 g solid odb, the xylan content between 12.4 and 3.6 g xylan/100 g solid odb, the arabinan content between 0.30 and 0.06 g arabinan/100 g solid odb and Klason lignin varied between 30.2 and 39.3 g lignin/100 g solid odb. Acetyl groups were not detected in the solid phases. In the liquid phase, oligomers were the most abundant compounds, with concentrations ranging 17.44 g/L to 9.46 g/L, composed essentially of xylose units (62.4-73.6 molar percent) with some arabinosyl moieties (4.1-8.3 molar percent), acetyl groups (7.5-13.0 molar percent) and glucose units (14.2-20.4 molar percent). Other compounds present in the liquid phase were some sugars such as arabinose (0.71 – 0.21 g/L), glucose (0.23 – 0.40 g/L) or xylose (1.04 – 2.66 g/L), acetic acid (1.88 – 3.05 g/L) and furans as furfural (0.29 – 1.83 g/L) or hydroxymethylfurfural (0.02 – 0.19 g/L). Regarding to material balances, about the 90% of the initial glucan remained in the solid phases (without a clear dependence on autohydrolysis severity) and the rest kept present predominantly in oligomeric form. This suggests that about the 90% of the glucan corresponds to cellulose (about 31.7 g/100 g raw material odb) and 3.4% to hemicellulosic 13


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polymers; other materials, such as olive wood, have been found to present comparable behaviour.21 Hemicelluloses content (measured as the addition of xylan, arabinan and acetyl groups) decreases with severity. At the lowest severity, the 69% of the hemicelluloses were solubilized, and at the highest severity the solubilisation increases up to 92%. On the other hand, lignin remains in the solid phases almost quantitatively. These facts show the selectivity of autohydrolysis processes in order to solubilize hemicelluloses. The hemicelluloses derived compounds, mainly xylooligomers and xylose, account up to the 60% of the initial xylan, similar to the maximum values achieved for other materials such as eucalyptus or gorse.21, 22 Table 3 shows the results related to the mild ethanol/water organosolv process that followed autohydrolysis. According to previous experiences,20 conditions for organosolv process were fixed in t = 1h, ethanol concentration = 40%, T = 170 ºC. According to the temperature profile of the reactor, an organosolv treatment carried out at 170 ºC is about 10 times lower in severity terms than one carried out at 200 ºC (a normal temperature for organosolv process of lignocellulosic materials). The aim of this stage was to improve the susceptibility of solids to enzymatic processing. Results showed that almost all cellulose is retained in the solid phase (more than 98%) and the content in hemicelluloses and lignin decreased. Time course of ethanol concentration and ethanol conversion in FB-SSF assays. Simultaneous saccharification and fermentation assays were carried out in fed-batch mode in order to handle high solids loading (up to LSR = 4 g/g corresponding to a consistency of 20% of solids), needed to obtain bioethanol concentrations higher than 30 – 40 g/L; concentrations considered as a minimum threshold for techno-economic viability. 23 14


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Figures 1 and 2 show the time-course of ethanol concentration of FB-SSF experiments. In relation to the process with different loadings, it can be observed that very high kinetic and conversions were achieved compared to bibliographic data 24, 25,. As can be seen the highest bioethanol concentrations were obtained after the third loading, and the fourth solid loading did not improve concentrations or conversions. At the beginning of the FB-SSF, the concentration raised very quickly, reaching bioethanol concentration values at 9 h between 6.1 g ethanol/L and 31.5 g ethanol/L that are in the threshold for techno-economic, proving the validity of the proposed process scheme (autohydrolysis-organosolv). The maximum ethanol concentration was achieved in experiment 11 (E=31.5 g ethanol/L, about 4.0% volume) with EC = 100% and Qp = 3.50 g ethanol/(L·h). As expected, ethanol concentration continued increasing at longer times but more slowly. After the second solids loading, the ethanol concentration reached its maximum value of E = 44.5 g ethanol/L in experiments 2 and 11, with EC of 93.3 and 89.7 g ethanol/100 g potential ethanol, respectively and Qp of 0.97 g ethanol/(L·h) in both experiments. Compared to bibliographic data (Table 6),better results were obtained in terms of concentration, conversion and volumetric productivity; results that can be technical and economically suitable based in the techno-economic studies of Wingren, et al. 14-

With the 3rd loading low increments of ethanol concentration were achieved but with the expected decreases in conversion and volumetric productivity. The 4th loading produced no improvement in the results.

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All the FB-SSF were carried out up to 118 h. Taking into account the whole time range, the higher ethanol concentration was achieved between 46 and 89 h in all the experiments, and the highest ethanol concentration was obtained in experiment 2 with E = 48.7 g ethanol/L. In all the experiments carried out at the highest solids loading (experiments 1, 2, 9 and 11), high ethanol concentrations were obtained with values between 34.8 (experiment 1 at 64 h, after the 2nd solids loading) and 48.7 g ethanol/L (experiment 2 at 89 h, after the 3rd loading). It is recommended the use high solids loadings (denomination that is often used when consistency is higher than 10%) in order to get a more efficient process from a techno-economic point of view. Operating under these conditions usually implies mixing and agitation problems which results in low conversions.26-28 The increment of the solids loading also tends to cause a drop in the conversion. Zhang et al.28 found a reduction from EC= 83% (operating at 7% consistency) down to EC = 63% (operating at 19% consistency). By carrying out hydrothermal pretreatments, it was also observed a decrease of the EC when increasing solids loading. With hardwood as olive tree pruning subjected to autohydrolysis conversion of EC=51.9% was obtained.29 Conversions about EC= 70% and values of Qp = 0.34 – 0.58 g ethanol/(L·h) were obtained in corn stover hydrothermal treatments, performing agricultural materials FB-SSF with high solids loadings.30, 31 In corn stover FB-SSF treatments subjected to SO2 catalyzed steam explosion at 10% consistency, obtained EC was 59% with values of Qp = 0.38 g ethanol/(L·h).32 Response surface methodology assessment. Response surface methodology (RSM) is a useful tool for a global study of all FB-SSF experiments and also for the optimization of the results. Selected response variables for their interest to be subjected to RSM were 16


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maximum ethanol concentration (E), maximum ethanol conversion (EC) and volumetric productivity under the conditions leading to the maximum ethanol concentration (Qp). In this type of assays, these three response variables can be obtained at different times. Qp was higher in the first stages of assays with maximum values of 1.79 and 4.78 g ethanol/(L·h) were obtained at 3 and 6 h in experiment 10 and 9 respectively). Although high Qp values are desirables, short times at which these values are obtained imply low values of concentrations and conversions, and therefore, a lack of practical interest. For these reasons, Qp was always referred to conditions leading to the maximum ethanol concentration. Since the SSF experiments were carried out in fed-batch mode, the application of RSM to set the three variables (E, EC and Qp) was done separately for each of the time periods after each of the solids loading and denoted consequently (see Table 1 for structure and nomenclature). As a general trend, E showed their maximum values after the 3rd loading (and after the 2nd loading in some experiments), with lower values after the 1st loading; and EC and Qp were higher after the 1st loading and decreased with increasing loadings. Table 2 shows the operational conditions of the experiments involved in the design (in terms of dimensional and dimensionless independent variables) and also the experimental values for dependents variables y1 to y12. Table 3 shows the values determined for the regression coefficients of the model for equation (5) and also the statistical parameters. The values of statistical parameters indicate the ability of the model for the reproduction of experimental data for all the considered dependent variables, and the average deviation between experimental and calculated values of experimental dependent variables which was only 3.1%, and especially under the high ethanol concentrations conditions, with more interest from a practical point of view. 17


Energy & Fuels

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According to the regression coefficients data shown in Table 3, the most influential independent variable on ethanol concentration (g ethanol/L) after each loading (denoted as E1, E2, E3 and E4) was LSR and to a lesser extent EL, with little influence of So. As supplementary material figures that show the dependence of calculated values with LSR and EL for the three values of S0 can be seen. The S0 and EL influence is higher at lower values of LSR, at which higher ethanol concentrations with high S0 and EL values are obtained. The maximum values of ethanol concentration predicted by the model are the following: E1 = 32.4 g ethanol/L, E2 = 47.1 g ethanol/L, E3 = 48.7 g ethanol/L and E4 = 43.7 g ethanol/L, (at So = 4.2, LSR = 4 g/g, corresponding to a 20 wt. % of solids, and EL = 14 FPU/g for all the cases). The ability of the model was confirmed, especially in conditions of high ethanol concentrations. Some of the most interesting conditions are those ones of experiments 2 and 11 after carrying out the 2nd loading, which produces high concentrations in relatively short times. In experiment 2 the experimental and calculated values were, respectively, 44.5 and 43.5 g ethanol/L and, in the experiment 11 were 44.5 and 45.2 g ethanol/L. It can be highlighted that it was possible to obtain concentrations higher than 40 g ethanol/L operating under a wide range of experimental conditions: at a consistency of 20 wt. % of solids and EL= 14 FPU/g, it was achieved for all the So values, while for EL values between 8-10 FPU/g, it was obtained just for So values≥ 3.8 – 4.1, compatible with the conditions that led to high XO and X amounts in the liquid phase in the autohydrolysis stage. Table 6 shows the comparison of the results obtained in this work with those ones obtained in similar works of bioethanol production from agricultural residues. Compared to the bibliographic data (Table 6), in this paper high concentrations 18


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Energy & Fuels

and conversions of ethanol are simultaneously obtained. It is particularly noteworthy that these results are obtained at very short times. In accordance with the coefficients in Table 2, the most influential variable in the ethanol conversion after the first loading (denoted as EC1) was EL, followed by LSR and So, getting values higher than the 90% for most of the experimental conditions. In EC2 and EC3 experimental variables, the three variables had a similar influence increasing their values when So and EL incremented and LSR diminished. This last fact is very favorable for the process because it indicates that the use of high solids loadings (desirable in order to obtain high ethanol concentrations) also leads to higher conversions. As indicated, standard deviations of ethanol concentration between experimental and calculated values are low (Table 3). Values of EC2 and EC3 higher than 80 or 90% can be achieved in a wide range of experimental conditions. Qp was calculated in the conditions leading to the highest ethanol concentration after each loading. Obviously, due to the calculation procedure of this variable the highest values are achieved at shorter times and as a general trend Qp1 > Qp2 > Qp3 > Qp4. According to the coefficient values in Table 2, the most influential variable for Qp was LSR (high values of LSR lead to low values of Qp), followed by EL (high values of EL lead to high values of Qp) and then by So (high values of So lead to high values of Qp). After the 2nd loading, the model predicts Qp 2 of 0.91 and 0.96 g ethanol/(L·h), in experiments 2 and 11, respectively. After the 3rd loading Qp 3 of 0.51 g ethanol/(L·h) was predicted by the model with values of E=48.9 g ethanol/Lf. These data confirm the ability of the model in the more practical interest conditions. CONCLUSIONS 19


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In this work, an abundant agricultural residue, barley straw, has been subjected to a biorefinery scheme in order to obtain second generation bioethanol and valuable hemicelluloses derived compounds. In the hydrothermal stage, up to 17.4 g oligomers/L can be obtained, remaining most of the cellulose and lignin in the solid phase. After carrying out a mild organosolv stage with ethanol/water, the solid phases were subjected to high solids loadings fed batch during the simultaneous saccharification and fermentation, in which glucan to ethanol conversions close to 100% and/or ethanol concentrations between 40 - 50 g ethanol/L were obtained. Under optimal conditions (So = 3.94, high solids loading with a liquid to solid ratio = 4 g/g, and 10 FPU/g), 48.7 g ethanol/L (with an ethanol conversion of 78.8%) was obtained after 90 h of fermentation. This hydrothermalorganosolv scheme leads to solids with high susceptibilities to the enzymatic hydrolysis. Under the same optimal conditions, 44.5 g ethanol/L (with an ethanol conversion of 93.3%) was obtained after 46 h of fermentation, implying a volumetric productivity value of 0.97 g ethanol/(L·h).

AUTHOR INFORMATION Corresponding Author * Telephone: +34988387075. Fax: +34988387001. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding sources 20


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Research Project ‘‘Advanced processes for second generation biofuels production’’ (reference EM2012/159) from Xunta de Galicia (Spain). ACKNOWLEDGEMENTS Authors are grateful to Xunta de Galicia (Spain) for the financial support of this work, in the framework of the Research Project ‘‘Advanced processes for second generation biofuels production’’ (reference EM2012/159).

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TABLES

Table 1. Structure of experimental design of this work (odb: on dry basis; FB-SSF: simultaneous saccharification and fermentation in fed batch mode) Nomenclature

Variable

Value or range

Units

Fixed variables LSRA

Liquid to solid ratio in autohydrolysis

8

g liquid/g raw material; odb

LSRO

Liquid to solid ratio in organosolv

8

g liquid/g autohydrolyzed material; odb

C

Ethanol concentration in pulping media

40

g ethanol/100 g ethanol + water

TD

Organosolv temperature

170

ºC

tD

Organosolv time (isothermal period)

1

h

Independent variables So

x1

Severity of autohydrolysis

3.64 – 3.94 – 4.23

Dimensionless (So = Log Ro; with Ro in min)

LSR

x2

Liquid to solid ratio in FB-SSF

4 – 8 – 12

g liquid/g material; odb

EL

x3

Enzyme loading in FB-SSF

6 – 10 – 14

Filter paper units/g material; odb

Dependent variables

30


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Energy & Fuels

E1

y1

Maximum ethanol concentration after the 1st loading

g ethanol/L

E2

y2

Maximum ethanol concentration after the 2nd loading

g ethanol/L

E3

y3

Maximum ethanol concentration after the 3rd loading

g ethanol/L

E4

y4

Maximum ethanol concentration after the 4th loading

g ethanol/L

EC1

y5

Maximum ethanol conversion after the 1st loading

g ethanol/100 g potential ethanol

EC2

y6

Maximum ethanol conversion after the 2nd loading


EC3

y7

Maximum ethanol conversion after the 3rd loading


EC4

y8

Maximum ethanol conversion after the 4th loading


QP 1

y9

Volumetric productivity at time of maximum E1

g ethanol/(L·h)

QP 2

y10


g ethanol/(L·h)

QP 3

y11


g ethanol/(L·h)

QP 4

y12


g ethanol/(L·h)

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Table 2. Operational conditions considered (expressed in terms of dimensional and dimensionless independent variables) and experimental results obtained for dependent variables y1 to y12 (see Table 3 for definitions and units) Independent variables Dependent variables

Dimensionless; normalized

Dimensional Exp

So (-)

LSR (g/g)

Consistency (wt. %)

EL (FPU/g)

x1

x2

x3

y1

y2

y3

y4

y5

y6

y7

y8

y9

y10

y11

y12

1

3.64

4

20

10

-1

-1

0

27.0

34.8

27.9

22.7

91.0

73.2

47.0

38.2

1.93

0.547

0.316

0.254

2

3.94

4

20

10

1

-1

0

30.2

44.5

48.7

42.6

100

93.3

78.8

58.5

2.16

0.968

0.549

0.361

3

3.64

12

7.7

10

-1

1

0

6.4

4.7

5.8

6.0

83.9

32.6

28.7

29.8

0.714

0.073

0.065

0.067

4

3.94

12

7.7

10

1

1

0

7.2

9.5

9.0

9.2

90.7

63.5

42.6

43.3

0.796

0.206

0.102

0.101

5

3.64

8

11.1

6

-1

0

-1

8.4

9.4

8.8

8.5

68.9

42.8

29.3

28.2

0.932

0.204

0.099

0.095

6

3.94

8

11.1

6

1

0

-1

10.1

13.2

13.4

13.0

81.5

58.0

42.6

41.2

1.13

0.286

0.151

0.144

7

3.64

8

11.1

14

-1

0

1

12.9

17.6

17.7

17.1

100

80.3

58.8

56.9

1.43

0.383

0.200

0.191

8

3.94

8

11.1

14

1

0

1

14.2

21.0

21.3

21.0

100

92.5

67.4

66.4

1.58

0.456

0.239

0.231

9

4.23

4

20

6

0

-1

-1

26.2

37.2

28.0

22.4

87.0

74.9

44.1

35.3

1.87

0.585

0.316

0.245

10

4.23

12

7.7

6

0

1

-1

6.1

6.4

5.6

6.1

74.7

41.8

25.8

28.2

0.674

0.138

0.063

0.067

11

4.23

4

20

14

0

-1

1

31.7

44.5

40.2

35.8

100

89.7

63.5

56.4

2.26

0.968

0.455

0.391

12

4.23

12

7.7

14

0

1

1

8.9

11.2

11.8

11.7

100

73.6

54.9

54.3

0.989

0.244

0.134

0.128

13

4.23

8

11.1

10

0

0

0

12.4

17.7

18.3

17.7

97.2

75.7

56.8

55.0

1.38

0.384

0.206

0.193

14

4.23

8

11.1

10

0

0

0

12.6

17.5

17.7

17.8

98.9

75.2

55.0

55.2

1.40

0.381

0.200

0.194

15

4.23

8

11.1

10

0

0

0

12.7

17.8

19.6

17.8

99.5

76.2

61.0

55.5

1.41

0.386

0.222

0.195

32


Page 33 of 39

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Energy & Fuels

Table 3. Regression coefficients and statistical parameters measuring the correlation and significance of the models

Parameter

y1

y2

y3

y4

y5

y6

y7

y8

y9

y10

y11

y12

b0

12.60***

17.67***

18.53***

17.76***

98.55***

75.73***

57.59***

55.20***

1.400***

0.3840***

0.2093***

0.1941***

b1

0.8676***

2.714***

4.037**

3.930**

3.549***

9.809***

8.430**

7.040***

0.08054***

0.08852**

0.04525**

0.02889***

b2

-10.82***

-16.16***

-14.09***

-11.31***

-3.592***

-14.95***

-10.18***

-4.111**

-0.6313***

-0.3009***

-0.1590***

-0.1109***

b3

2.128***

3.534***

4.400**

4.446***

10.98***

14.82***

12.84***

12.63***

0.2092***

0.1047***

0.04968**

0.04898***

b11

-0.8565***

-1.921***

-0.8882

-0.8841

-4.981***

-5.853

-2.944

-4.066*

-0.09182**

-0.0434

-0.01040

-0.02041***

b22

5.958***

7.605***

5.223**

3.226*

-2.144

-4.241

-5.393

-8.676***

0.09107**

0.1080***

0.05892**

0.02193***

b33

-0.3439**

-0.4440

-2.353

-2.001

-5.962***

-1.498

-5.127

-2.961

-0.04155

-0.007974

-0.02649

-0.008539

b12

-0.6153***

-1.236

-4.390**

-4.170**

-0.5269

2.714

-4.452

-1.711

-0.03664

-0.07197*

-0.04929**

-0.01816**

b13

-0.08373

-0.1015

-0.2554

-0.1643

-3.149**

-0.7539

-1.167

-0.8690

-0.009303

-0.002208

-0.002948

-0.002082

b23

-0.6638***

-0.6299

-1.499

-1.946

3.082**

4.242

2.428

1.223

-0.01930

-0.06937*

-0.01693

-0.02127***

R2

0.9998

0.996

0.977

0.974

0.985

0.957

0.941

0.978

0.997

0.977

0.977

0.997

F

2676

156.1

23.6

20.5

36.5

12.4

8.9

25.1

195.1

23.7

23.8

178.6

Significance level

>99%

>99%

>99%

>99%

>99%

>99%

>99%

>99%

>99%

>99%

>99%

> 99%

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

33


Energy & Fuels

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Page 34 of 39

Table 4. Barley straw non isothermal autohydrolysis: material balances and phases composition TMAX (maximum temperature; ºC)

195

205

215

So (Severity; dimensionless)

3.64

3.94

4.23

Material balance (g autohydrolyzed/100 g raw material; on dry basis) YA (Autohydrolysis yield)

64.9

59.7

58.2

NVC (Non-volatile content)

29.0

29.9

26.0

VC (volatile content)

6.10

10.4

15.8

Solid phase composition (g/100 g autohydrolyzed material; on dry basis) (In bracket g/100 g raw material) Glucan

48.1 (31.2)

52.2 (31.2)

56.4 (32.8)

Xylan

12.4 (8.10)

5.90 (3.50)

3.60 (2.10)

Arabinan

0.30 (0.20)

0.17 (0.10)

0.06 (0.00)

Acetyl groups

0.00 (0.00)

0.00 (0.00)

0.00 (0.00)

Klason lignin

30.2 (19.6)

34.3 (20.4)

39.3 (22.9)

Unknowns

9.00 (5.80)

7.50 (4.50)

0.60 (0.40)

Glucose

0.23

0.27

0.40

Xylose

1.04

1.93

2.66

Arabinose

0.71

0.46

0.21

Acetic Acid

1.88

2.44

3.05

HMF

0.02

0.05

0.19

Furfural

0.29

0.94

1.83

Oligomers

17.44

16.31

9.46

Liquid phase composition (g/L)

Oligomers composition (molar %) Glucose

14.2

14.8

20.4

Xylose

69.7

73.6

62.4

Arabinose

8.30

4.10

4.10

Acetyl groups

7.70

7.50

13.0

34 ACS Paragon Plus Environment

Page 35 of 39

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Energy & Fuels

Table 5. Organosolv treatment of autohydrolyzed barley straw: material balances and solid phases composition TMAX (maximum temperature; ºC)

195

205

215

So (Severity; dimensionless)

3.64

3.94

4.23

Material balance (g autohydrolyzed-delignified/100 g autohydrolyzed material; on dry basis) (In bracket g autohydrolyzed -delignified/100 g raw material) YO (Organosolv yield)

85.5 (55.5)

87.6 (52.3)

89.0 (51.8)

NVC (Non-volatile content)

11.5

8.90

10.2

VC (volatile content)

3.00

3.50

0.80

Solid phase composition (g/100 g autohydrolyzed-delignified material; on dry basis) (In bracket g/100 g raw material) Glucan

48.0 (31.1)

52.9 (31.6)

53.0 (30.8)

Xylan

8.60 (5.60)

4.40 (2.60)

3.00 (1.70)

Arabinan

0.10 (0.08)

0.10 (0.05)

0.10 (0.04)

Acetyl groups

0.00 (0.00)

0.00 (0.00)

0.00 (0.00)

Klason lignin

24.1 (15.6)

27.1 (16.2)

29.1 (16.9)

Unknowns

4.60 (3.00)

3.10 (1.80)

4.00 (2.30)

35 ACS Paragon Plus Environment

Energy & Fuels

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Page 36 of 39

Table 6. Data of bioethanol production from agricultural residues SSF conditions Raw material

Pretreatment

Operational conditions (enzyme loading // consistency // time)

Results E (g/L)

EC (%)

Ref

Barley straw

NaOH/water in twin-screw struder

8 PFU/g // 2.5% // 96 h 8 PFU/g // 20% // 96 h

5 28.7

73 53

33

Barley straw

NaOH/water in twin-screw struder

30 PFU/g glucan // 15% // 88 h 30 PFU/g glucan// 20% // 88 h

38.2 43.9

90 77

34

Barley straw

Prehydrolysis with H2SO4

15 PFU/g glucan // 6% // 72 h

18.5

88

35

Wheat straw


20 mg protein/g glucan // 6% // 72 h

21.6

54

36

Wheat straw

Autohydrolysis

30 FPU/g glucan // 3% // 96 h

14.9

82

37

Wheat straw

Aqueous ammonia

40 FPU/g glucan // 13% // 96 h

25.1

91

38

Corn stover


30 FPU/g glucan // 10% // 96 h

22.4

69

39

Corn stover

Autohydrolysis

30 FPU/g // 10% // 78 h

39.4

80

40

Barley straw

Autohydrolysis + etanol/water organosolv

10 FPU/g // 25% // 46 h 14 FPU/g // 25% // 46 h

44.5 44.5

93 90

This work

E: ethanol concentration (g ethanol/L); EC: ethanol conversion (g ethanol/100 g potential ethanol)

36


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Energy & Fuels

FIGURE LEGENDS

Figure 1. Time course of ethanol concentration in fed batch simultaneous saccharification and fermentation (experiments 1 to 7). Dotted lines correspond to the time in which loads are performed. Figure 2. Time course of ethanol concentration in fed batch simultaneous saccharification and fermentation (experiments 8 to 12 and average values of experiments 13 to 15; with average standard deviation of 2.6%). Dotted lines correspond to the time in which loads are performed.

1


Energy & Fuels

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Page 38 of 39

2


Page 39 of 39

Energy & Fuels

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3


Biorefinery Scheme for Residual Biomass Using Autohydrolysis and

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