Second generation bioethanol production combining simultaneous

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Second generation bioethanol production combining simultaneous fermentation and saccharification of ILs pretreated barley straw Marta Lara-Serrano, Felicia Saez Angulo, Maria Jose Negro, Silvia Morales-delaRosa, Jose M. Campos-Martin, and Jose Luis Garcia Fierro ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00953 • Publication Date (Web): 15 Apr 2018 Downloaded from http://pubs.acs.org on April 15, 2018

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ACS Sustainable Chemistry & Engineering

Second generation bioethanol production combining simultaneous fermentation and saccharification of ILs pretreated barley straw Marta Lara-Serrano[a], Felicia Sáez Angulo[b], María José Negro[b], Silvia Morales-delaRosa[a], Jose M. Campos-Martin*[a] and Jose L. G. Fierro[a] [a]

Sustainable Energy and Chemistry Group (EQS), Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie, 2, Cantoblanco, 28049 Madrid, Spain [b]

Department of Energy, Biofuels Unit, Centro de Investigaciones Energéticas,

Medioambientales y Tecnológicas, CIEMAT, Avda. Complutense 40, 28040 Madrid, Spain Corresponding Author: e-mail: [email protected]

ABSTRACT. Ionic liquids dissolution/precipitation pretreatment of barley straw was used to improve the yield to produce ethanol. The pretreatment with ionic liquid greatly improves the enzymatic hydrolysis compared to untreated samples, reducing the hydrolysis time to only 24 h, which is lower than untreated samples and samples treated with other methods. The treatment with 1-ethyl-3-methylimidazolium acetate produced a better hydrolysis yield with a glucose yield close to 100%. We produced ethanol with pretreated samples using simultaneous

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saccharification and fermentation (SSF) and pre-saccharification, saccharification and fermentation (PSSF). In both procedures we obtained very high and similar yields because the pretreatment enhances the enzymatic hydrolysis rate and no pre-saccharification is needed. We obtained 22.9 g ethanol/100 g pretreated barley straw, representing 97% of the potential ethanol from the cellulose, one of the highest yields reported in the literature.

KEYWORDS. ethanol • hydrolysis • saccharification • fermentation • Ionic Liquid

INTRODUCTION The transition from the present economy based on fossil fuels and products to a new economy based on the use of biomass is challenging. Biorefineries have an essential role to play in sustainable production of biofuels, bioproducts and biomaterials, as their primary purpose is to integrate industrial processing of biomass, thus making bioprocessing more efficient and competitive. The use of biorefineries would mitigate environmental issues and could become a solution to reducing national dependence on imported fossil fuels1. Lignocellulosic biomass has been proposed as a very promising feedstock to be used in biorefineries as a source of biofuels, chemicals, and other biomass-derived products with high added-value2. Furthermore, lignocellulosic materials obtained from energy crops, wood and agricultural residues represent the most abundant global source of renewable biomass 3. Among the lignocellulosic residues, barley wastes are the second largest biomass feedstock in Europe

4-5

. The projected

environmentally sustainable availability of cellulosic residue from barley crops for cellulosic biofuel production in the Europe Union has been estimated as 23 and 25 million tons in 2020 and


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2030, respectively 6. This estimate is based on the quantities of feedstock that can be harvested without undue adverse impacts on the environment or on existing uses that are treated as available for biofuel production. Due to the lignocellulosic nature of barley straw, pretreatment is a crucial stage in any lignocellulose-based biorefinery process, to break down fiber structure and make carbohydrates accessible to saccharification and fermentation processes7. Various biological, chemical, and physical pretreatment approaches have opened the recalcitrant structure of lignocellulosic biomass, increasing cellulose and hemicellulose to the hydrolytic enzymes 8. Particularly for barley straw, biomass dilute acid 9, extrusion

10

, un-catalyzed steam explosion

approaches of sequential stream and reactive extrusion explosion hydrothermal and organosolv pretreatments have been tested

13

12

11

, combined

and a combination of

. However, a pretreatment that

operates at moderate temperatures and atmospheric pressure is still needed. An interesting option is the use of ionic liquids (ILs), which is a fluid constituted exclusively by ions, considering as such the salts with a melting temperature below the boiling point of water (100 °C) and that are often hydrolytically stable. Some ILs can dissolve lignocellulosic materials and then precipitate with water14, and the recovered biomass is less structured and crystalline, which favors their conversion

15-16

. A disadvantage of using the IL is the high price, although the pretreatment of

dissolution/precipitation of the biomass samples offers the possibility of recycling them, partially overcoming this drawback. Previous results on ionic liquids (ILs) pretreatment have shown much promise in the use of 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]) for sugars obtained through enzymatic saccharification from barley straw17. However, literature on bioethanol production using ionic liquid pretreated with this substrate is scarce. To our best knowledge,


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there is no literature on bioethanol production combining bioethanol production by simultaneous fermentation and saccharification of barley straw using ILs pretreatment. In this work, the production of bioethanol fuel from ILs pretreated barley straw has been investigated. Bioethanol production of IL pretreated barley straw was performed by comparing two configuration strategies (Figure 1): Simultaneous saccharification and fermentation (SSF) and pre-saccharification, saccharification and fermentation (PSSF). We have selected two IL, 1ethyl-3-methyl imidazolium acetate ([Emim][Ac]) and 1-ethyl-3-methyl imidazolium chloride ([Emim][Cl]). [Emim][Ac] was selected as an IL for biomass pretreatment due to its low melting point, and it is liquid at room temperature. It has low viscosity and is easy to handle. The imidazolium group has relatively short alkyl chain substituents. According to the bibliography, the ILs with short-chain groups are less toxic than those with long-chain groups 18. On the other hand, the acetate ion is less corrosive than the ILs of halide anions, which have also been described as effective in cellulose dissolution. Pretreatment of cellulose with [Emim][Cl] improves the hydrolytic reactivity of the cellulose with mineral acids

16, 19

. Moreover, in the

present study, changes in chemical characteristics after pretreatment, enzymatic hydrolysis and bioethanol conversion were also investigated using FT-IR and scanning electron micrographs (SEMs).


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Figure 1. Scheme of the experimental procedure.

EXPERIMENTAL SECTION Raw Material Barley straw (10.9% moisture) was provided by CEDER (Soria, Spain). Biomass was coarsely crushed to approximately 1 mm particle size using a laboratory hammer mill, and stored until use. Barley straw pretreatment The treatment of lignocellulosic biomass involved the complete dissolution of barley straw (3 g) in two different ionic liquids (57 g) using a Mettler-Toledo EasyMax® 102 reactor equipped with mechanical stirring at 105 ºC until the complete dissolution around 7.5 h. The ionic liquids were 1-ethyl-3-methyl imidazolium acetate ([Emim][Ac]) and 1-ethyl-3-methyl imidazolium chloride ([Emim][Cl]). The temperature was optimized in previous works with cellulose 19. The study was based on the premise that the samples were dissolved when solids in suspension were not seen in the solution. Once samples were solubilized, the lignocellulosic component was precipitated by addition of deionized water (250 mL). The solid was separated by vacuum filtration with a nylon membrane (0.2 µm) and washed with deionized water to eliminate the ionic liquid from its fibers. X-ray Diffraction Analysis Barley straw samples, original and treated, were analyzed by X-ray diffraction (XRD). The profiles of samples were recorded using an X’Pert Pro PANalytical diffractometer equipped with a CuKα radiation source (λ=0.15418 nm) and an X’Celerator detector based on real time multiple strip (RTMS). The samples were ground and placed on a stainless-steel plate. The


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diffraction patterns were recorded in steps over a range of Bragg angles (2 θ) between 4° and 90° at a scanning rate of 0.02° per step and an accumulation time of 50 s. The diffractograms were analyzed with X’Pert HighScore Plus software.

SEM Analysis The scanning electron micrographs (SEMs) of raw barley straw and pretreated barley straw samples with IL were taken with a Hitachi S-3000 N instrument with a resolution of 3 nm to 25 kV and a voltage of 0.3 to 30 kV. The ESED detector is coupled to an X-ray dispersive energy analyzer from Oxford Instruments, model INCAx-sight, and the EDX analyzer is from Oxford Instruments, model INCAx-sight with a cathodoluminescence system CHROMA-CL2 from Gatan that allows an inclination of 90º in the samples. In addition, the samples were metallized with a thin layer of gold in a Quorum Q150T-S gold-sputter coater. Before the metallization the samples were treated with increasing concentrations of bioethanol to fix the structure and to dehydrate the samples (ethanol/water: 50%, 70%, 80%, 90% and finally 100% in ethanol). FTIR Analysis Raw material, [Emim][Ac] pretreated biomass, insoluble solid obtained after enzymatic hydrolysis and the PSSF and SSF processes were analyzed using FTIR to determine the chemical changes during pretreatment and biochemical processes. Dried biomass was analyzed in a JASCO FT-IR 6300 spectrophotometer equipped with a TGS detector. Absorbance spectra were recorded in the 4000-400 cm-1 spectral region at a resolution of 4 cm-1 and 180 scans. The samples were diluted in KBr 1% by weight. Enzymatic hydrolysis


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Solid fractions obtained after [Emim][Ac] and [Emim][Cl] pretreatment, were used as the substrate for enzymatic hydrolysis. Enzymatic hydrolysis tests were performed in 2 mL Eppendorf tubes at 50 ºC, pH 5 and 800 rpm in a microplate incubator (ThermoStar, 3 mm shaking amplitude). The saccharification process was performed using the commercial Cellic Ctec2 (Novozymes, Bagsvard, Demark). Cellic Ctec2 is a cellulase preparation that incorporates β-glucosidase activity and contains endoxylase activity. Pretreated barley straw was applied at 9% total solid (w/w) and an enzyme loading of 15 FPU/g glucan. Samples were taken after 24, 48, 72 and 96 h. The glucose and xylose concentration in the enzymatic hydrolysis media was measured using HPLC as described below in the analytical methods section. Additionally, blanks of the enzyme were analyzed using HPLC to subtract the sugar content present in the enzyme preparation. Enzymatic hydrolysis yields were calculated as the ratio of glucose and/or xylose divided by potential glucose and/or xylose content in [Emim][Ac] pretreated barley straw. Ethanol fermentation Fermentation of the solid residue obtained after [Emim][Ac] pretreatment was performed using two different configuration processes: either simultaneous saccharification and fermentation (SSF) or pre-saccharification, saccharification and fermentation (PSSF). Saccharomyces cerevisiae Ethanol Red (Fermentis, France) was used for fermentation in SSF and PSSF experiments. Active cultures for inoculation were prepared by growing the yeast on a rotary shaker at 150 rpm and 35 ºC for 16 h in a growth medium containing: yeast extract, 5 g/L; NH4Cl, 2 g/L; KH2PO4, 1 g/L; MgSO4·7H2O, 0.3 g/L; and glucose, 30 g/L. The preculture was centrifuged at 10,000 g for 5 min at 4 ºC, and the supernatant was discarded. The pellet was suspended in 0.05 M citrate buffer in a volume calculated to achieve an inoculum size of 1 g/L.


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The SSF experiments were performed in a 25 mL Erlenmeyer flask, each containing 10 mL of fermentation medium (growth medium described above), which were shaken at 150 rpm. During fermentation, flasks were capped by a rubber stopper punctured with a needle to allow CO2 outflow. Glucose was substituted by wet pretreated substrate. Enzymes were also added as in enzymatic hydrolysis tests. Experiments were conducted at 35 ºC, pH 5 for 72 h at a substrate concentration of 9% (w/w). Samples were withdrawn every 24 h and analyzed for ethanol and sugars. In PSSF experiments the pre-saccharification stage was run for 24 h at 50 ºC and then the temperature was reduced to 35 ºC and nutrients and yeast were added, which turned the process into an SSF. The experiments were run for another 48 h. Samples were withdrawn at 24 h during pre-saccharification and after 24 and 48 h of SSF and then analyzed for ethanol and sugars. The experiments were performed in duplicate and the average results were reported. Potential ethanol was determined as the maximum ethanol that could be produced from glucose in pretreated barley straw, assuming a 100% enzymatic hydrolysis yield and a theoretical conversion factor (0.51 g ethanol/g glucose). The solid was recovered by filtration after performing the various hydrolysis and fermentation sample procedures. The samples were then washed with distilled water and characterized by SEM analysis and FTIR. Analytical methods Raw material and solid pretreated barley straw were characterized using the National Renewable Energy Laboratory (NREL) standard methods for determining structural carbohydrates and lignin in biomass 20. Sugar concentrations were determined using high-performance liquid chromatography (HPLC) in a Waters 2695 liquid chromatograph with a refractive index detector. A CARBOSep CHO-


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682 LEAD column (Transgenomic, Omaha, NE) operating at 75 ºC with Milli-Q water (Millipore) as the mobile phase (0.5 mL/min) was used. Other structural carbohydrates content was calculated as the sum of xylan, arabinan, galactan and mannan content. Total lignin was calculated as the sum of acid soluble and insoluble lignin. All measurements were performed in triplicate and the results were presented as a percentage on an oven-dry weight basis. The composition of solids obtained after pretreatment were also determined according to NREL procedures, except for the extraction step. Sugar concentrations after enzymatic saccharification were measured using HPLC as described elsewhere

21

. The ethanol concentration was determined through gas chromatography using an

Agilent 7693B Series injector equipped with a flame ionization detector and a column Carbowax 20 M operating at 85 ºC as described earlier22.

RESULTS AND DISCUSSION Raw material and pretreated composition The compositions, determined in this work, of raw barley straw and solid materials obtained after pretreatment are depicted in Table 1. The results are given as a percentage of the total mass on a dry basis. Based on its total carbohydrate content of 60.1 wt. % (32.9 wt. % glucan and 27.2 wt. % hemicellulose), barley straw is considered a very promising substrate for sugar production. Barley straw composition determined in this work is similar to what has been reported previously 10, 23

. The lignin content of barley straw biomass (18.8 wt. %) is in the range of reported values

for other agricultural residues: corn stover24 or wheat straw21. Barley straw samples were dissolved in both ILs at 105 ºC and then recovered by adding water, which produced the precipitation of solid residues. These recovered solids were swollen


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compared to the untreated barley straw and had a dark brown appearance. The yield of regenerated biomass (solid obtained after solubilization and precipitation, divided by the original oven dried weight) account for 78.4% and 65.0% of [Emim][Ac] and [Emim][Cl] pretreatment, respectively. The value obtained with [Emim][Ac] pretreatment is in the range previously reported for barley straw and other herbaceous materials such as switchgrass, but lower than those obtained in poplar using [Emim][Ac] as the IL in the pretreatment step 25. The lower mass recovery with [Emim][Cl] is attributed to the acidic nature of chloride ILs, which can result in prehydrolysis of the cellulose and hemicellulose during the IL treatment 26-27. With regard to chemical characterization, pretreated solids showed similar compositions (Table 1). In both cases, there are solids somehow enriched in cellulose and acid-insoluble lignin in relation to untreated barley straw due to the solubilization of easily removable biomass components such as extractives, soluble ash and a fraction of hemicellulose. However, differences are observed in the composition of the solids recovered. For [Emim][Ac] pretreated material, almost 25% of the original hemicellulose was removed after pretreatment (Table 1). Reported losses due to solubilization of xylan were 15 and 32% at 120 ºC for poplar and switchgrass, respectively, which are similar to the results in a prior work in which the [Emim][Ac] was also used

25

. In contrast to hemicellulose, the glucan fraction in barley straw

was highly preserved during pretreatment (close to 100% recovery). The acid insoluble compounds (lignin) content increased significantly from 16.7% to 23.3%. In contrast, for the [Emim][Cl] pretreatment yield obtained after pretreatment, cellulose and xylan recoveries were lower than those found with the treatment with acetate IL (Table 2), primarily due to the low amount of regenerate biomass obtained. Solubilization of 18.7% of cellulose and 40.7% have


10

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been achieved after [Emim][Cl] pretreatment, probably due to the previously mentioned acidic properties of this IL 26


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Raw and pretreated barley straw composition. Mean value and standard deviation

Table 1

of three replications. Component wt. % ( dry basis)

[a]

Untreated barley straw

[Emim][Ac] pretreated barley straw

[Emim][Cl] pretreated barley straw

Extractives

13.4 ± 0.9

nd[a]

nd[a]

Glucan

32.91 ± 1.25

41.92 ± 0.41

41.17±0.28

Other structural carbohydrates

27.2

25.92

Xylan

22.06 ± 0.47

20.85 ± 0.17

20.12±0.11

Galactan

1.30 ± 0.01

1.28 ± 0.02

1.14±0.54

Arabinan+ mannan

3.87 ± 0.03

3.82 ± 0.01

2.49±0.08

Acid-insoluble lignin

16.67 ± 1.13

23.29 ± 0.2

22.27±0.11

Acid-soluble lignin

2.10 ± 0.03

2.25 ± 0.01

2.49±0.02

Acetyl-groups

0.02

nd[a]

nd[a]

Ash

3.89 ± 0.05

nd[a]

nd[a]

Glucan recovery (% referred to raw material)

---

~100

81.3

Xylan recovery (% referred to raw material)

---

74.0

59.3

23.48

nd: not determined

. X-ray Diffraction Analysis The X-ray diffraction patterns of the barley straw show important changes in the different treated samples (Figure 2). The diffractogram of untreated barley straw shows the typical peaks of the cellulose crystalline structure, only wider and less defined than those of pure cellulose, which implies a less ordered structure or a structure with smaller crystal sizes. Only a prominent peak at 23° corresponding to the reflection (200) and a very wide peak between 15-17° representing the combination of the two reflections corresponding to (110) and (11ത0) is shown. The diffraction peaks lose much intensity with the treatments of the sample. The loss of intensity is much greater when the barley straw is subjected to complete dissolution with [EMIM][Cl] and


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[EMIM][Ac]. In this sample, almost no diffraction peaks are observed, indicating a nearly total disappearance of its crystallinity. This study indicates that the original barley straw has a certain crystallinity that is lost after IL treatment. Other authors have also reported this behavior in different samples of lignocellulosic biomass after treating the samples with ILs19, 28.

SEM analysis of raw material and pretreated samples The SEM micrographs of the original and treated barley straw samples were completely different: the micrographs of the raw barley straw sample clearly showed the structure of vascular bundles (Figure 3), whereas the treated sample was completely amorphous (Figure 4 and Figure 5). In Figure 3, the raw barley straw shows the presence of its vascular bundles, forming hollows along the small pieces (0.250 µm). Ionic liquid pretreatment of the samples produces swelling of the barley straw vascular bundles compared to the raw material that remains intact. The surface of the resulting sample is porous, which is similar to the surface of cellulose samples in previous works19. This destruction of the structure of the biomass by dissolution with ILs has been observed by other authors such as Singh et al. in the pasture (Panicum virgatum) 29 and other treatments30-32. The destruction of the structure of the sample treated with [EMIM][Cl] is more evident than that observed for [EMIM][Ac], where a type of structure that remembers the original structure of the barley straw is observed. These results agree with the data obtained from X-ray diffraction. Both characterization data clearly indicate that the initial lignocellulosic structure of the barley straw is completely altered by treatment with IL after 7.5 h of dissolution and precipitation.


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Figure 2. XRD patterns of IL-treated and original barley straw samples.

Figure 3. SEM micrographs of the raw barley straw.


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Figure 4. SEM micrographs of barley straw pretreated with [EMIM][Ac] (at 105 ºC) and precipitated with deionized water.

Figure 5. SEM micrographs of barley straw pretreated with [EMIM][Cl] (at 105 ºC) and precipitated with deionized water.


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Enzymatic hydrolysis The main objective of pretreatment is to alter the structure of the fibers to increase the accessibility of enzymes to structural carbohydrates. To check the effect of IL dissolution/precipitation treatment, solids obtained after precipitation with antisolvent and washing were subjected to enzymatic saccharification for 96 h at 50 ºC. Enzymatic saccharification versus the time of pretreated barley straw is shown in Figure 6. Tests of untreated raw substrate (not shown) reached a maximum enzymatic hydrolysis yield of approximately 8% after 96 h of enzymatic hydrolysis. Experimental results using acetate and chloride IL base produced a significant improvement in the enzymatic accessibility when compared with the untreated barley straw (Figure 6). This observation is quite important because the pretreated materials are similar in composition (Table 1). These experiments show that enzymatic hydrolysis of treated samples with [Emim][Cl] or [Emim][Ac] results in a marked enhancement in polysaccharides hydrolysis by enzymes. The enzymatic hydrolysis yield depends on the IL. For the [Emim][Ac] pretreated sample the yield was nearly 100% and 71.6% for glucan and xylan, respectively (Table 2), whereas for the [Emim][Cl] pretreated sample the yield was clearly lower, 71.5% for glucan and 47.8% for xylan. The lower enzymatic hydrolysis yield of chloride IL was previously observed with barley straw 33, but remains unexplained. It may be related to the differ ent dissolution mechanisms of the ILs, but the presence of tiny quantities of acid sites in the solid after the precipitation is more plausible, because [Emim][Ac] IL has no acid impurities, in contrast to its [Emim][Cl] counterpart. The [Emim][Ac] pretreatment released 36.3 g glucose/100 g barley straw and 13.2 g xylose/100 g barley straw (Table 2), representing 81% of the major sugars present in barley straw biomass. Lower values were obtained with [Emim][Cl] pretreatment (13.2 g glucose/100 g


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barley straw and 7.07 g xylose/100 g barley straw), due to lower enzymatic and regenerated biomass yields.

Figure 6. Sugar production in enzymatic hydrolysis at 50 ºC and pH 5.


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Table 2.

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Main sugars yield after enzymatic hydrolysis. Parameter

[Emim][Ac]

[Emim][Cl]

EH glucose yield (%)

98.7

71.5

EH xylose yield (%)

71.6

47.8

Overall glucose yield (g glucose/100 g raw material)

36.3

21.5

Overall xylose yield (g xylose/100 g raw material)

13.2

7.4

The dissolution/precipitation treatment of barley straw with [Emim][Ac] clearly generates a material that can be hydrolyzed into monomeric sugars more easily compared to other pretreatment technologies, while rendering the enzymatic attack faster, as the initial hydrolysis rate is greatly increased. Approximately 93% of the total glucose and 97% of the total xylose produced by enzymatic hydrolysis were obtained in 24 h. This result was previously observed by Silva et al.34 in sugarcane bagasse pretreated with [Emim][Ac] (80% glucose yield within 6 h, achieving 90% within 24 h). The use of IL dissolution/precipitation can reach high cellulose conversion yields (>80%) in only 24 h, whereas other pretreatment technologies need a longer time (48-72 h). In addition, the cellulose digestibility obtained in this work was higher than that obtained by other authors. For instance, Mood et al.33, reported an enzymatic hydrolysis yield of 76% after 72 h saccharification using the same IL with barley straw. Duque et al.10 described an integrated alkaline-extrusion pretreatment of barley straw and obtained a glucan yield close to 90% after 72 h of hydrolysis, whereas xylan conversion was 71% of the theoretical value under optimum pretreatment conditions (6% NaOH/DM and 68 ºC). An enzymatic hydrolysis yield of 65% has been reported for dilute acid barley straw 9. [Emim][Ac] pretreatment of barley straw at 105 ºC for 7.5 h followed by enzymatic hydrolysis leads to a sugar yield of 51.8 g per 100 g raw material, which is readily available for conversion


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into bioethanol and equivalent to more than 77.5% from all potential sugars. These results support the great potential of [Emim][Ac] pretreatment for sugar production from barley straw. Comparable results were obtained by Saha and Cotta35 for barley straw after lime (0.1 g Ca(OH)2/g straw, 121 ºC, 1 h) and dilute acid (0.75% H2SO4, 121 ºC, 1 h) pretreatment. The yield in both pretreatments was 78% of the total sugar in terms of the percentage of the theoretical maximum total sugar yield. Due to the clearly better behavior of the pretreated biomass with [Emim][Ac] compared to the other IL, bioethanol production experiments have only been done with [Emim][Ac] pretreated samples.

Bioethanol Production Considering the good digestibility of [Emim][Ac] pretreated barley straw material, the solid was submitted to SSF and PSSF processes to evaluate the potential fermentation of this pretreated material. SSF process has been one of the most successful methods for lignocellulosic bioethanol production. During this process, the glucose released by the action of hydrolytic enzymes is converted directly to bioethanol by fermenting yeasts, minimizing the end-products inhibition of enzymes caused by cellobiose and glucose accumulation 22. The entire process was performed at 35 ºC and pH 5 for 72 h. In contrast, for the PSSF process, treated material was first incubated 24 h at 50 ºC with the cellulolytic enzymes to produce a relatively high concentration of glucose in dissolution, and then inoculated and incubated for an additional 48 h at 35 ºC (lower temperature) to use the high concentration of glucose in dissolution. Figure 7 illustrates the concentration profiles of glucose, xylose and bioethanol during the SSF and PSSF processes with [Emim][Ac] pretreated barley straw material. Due to the characteristics of both processes, the concentration profiles are different. The bioethanol concentration obtained


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in the SSF process (Figure 7 A) increases quickly, reaching the maximum with approximately 48 h remaining and decreasing slightly for longer times, whereas the glucose concentration was negligible for long reaction times because the glucose produced by the enzymatic hydrolysis is simultaneously consumed during ethanol formation process. However, in the PSSF process, the concentration profiles are slightly different (Figure 7 B). The glucose concentration increases in the first 24 h (when only enzyme is present in the medium). After the inoculation (24 h) the concentration of glucose decreases and the bioethanol concentration increases. A maximum bioethanol concentration of 18.5 g/L was obtained after 48 h of the SSF process, and a similar bioethanol concentration was achieved after 72 h of the PSSF process. In both processes, the glucose concentration was negligible at the end of the run. The consumption of xylose was very small because the S. cerevisiae Ethanol Red that was used is not efficient for C5 sugars processing to ethanol. The dissolution/precipitation treatment with [Emim][Ac] favors the formation of a material that easily hydrolyzes and the reaction conditions make presaccharification unnecessary because the glucose formed in SSF is sufficient to maintain the fermentation. This point implies that the use of SSF reduces 24 h from the production of bioethanol compared to PSSF. In a first approach, SSF is the most efficient procedure under the reaction conditions employed in this work, but in the case of higher concentration of solids PSSF will surpass the SSF., We are working in this point, but taking in account the high content of water in the precipitate, solid recovering after the IL treatment is a complicated issue.


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40

40

B

A 35

35

30

30

Concentration (g/L)

Concentration (g/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25 20 Ethanol

15 10

Glucose

Inoculation

25 20

Ethanol

15 10

Xylose

Xylose

5

5 Glucose

0 0

24

48

72

96

0 0

Time (h)

24

48

72

96

Time (h)

Figure 7. Time course of bioethanol production (g/L) and sugars consumption of [EMIM][Ac] pretreated barley straw submitted to A) SSF and B) PSSF at a 9%(w/w) solid loading mixture. The obtained bioethanol concentrations correspond to 17.9 g bioethanol/g barley straw (22.9 g bioethanol/100 g pretreated barley straw). Improvement of ethanol production for others lignocellulosic biomass, such as herbaceous residues or softwood, using the same ionic liquid has been reported. Results of barley straw pretreatment obtained in this work are comparable with results of rice straw using the same ionic liquid36: 19.2 g ethanol released after 48 h per 100 grams of initial straw has been for rice straw using [EMIN][Ac] at 120ºC for 5 h. This IL pretreatment ([Emim][Ac] at 120 ºC for 15 h) was also used with spruce wood, for which the total ethanol production was 17.1 and 19.6 g ethanol/100 g of initial wood for spruce chips and


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powder respectively37. However, the ethanol concentration obtained by these authors (5.6 and 6.8 g/L for chips and power respectively) represent utilization of 66.8% and 81.5% of the available glucose and mannose ofthe starting biomass. Therefore, ours results produced 3.3-2.7 times higher ethanol concentration (18.5 g/L) by using 1.8 times higher solid loading (9%, w/w) by means of SSF process. Compared to the reported values for barley straw with other pretreatment s, the overall bioethanol yield obtained in this work is excellent. For instance, Duque et al.38 reported an overall bioethanol yield 11 g/100 g barley straw when the biomass was pretreated by alkalineextrusion (6 g NaOH/ 100 g barley straw, 68 ºC, and 150 rpm) in a twin-screw extruder. GarcíaAparicio et al.11 reported a bioethanol yield after 72 h of 67.4%, which corresponds to a bioethanol concentration of 22 g/L and was achieved with SSF at 10% water insoluble solid obtained after steam explosion pretreatment of barley straw. More recently, García-Torreiro et al.39 reported 9.1 g/100 g barley straw after fungal pretreatment using white fungus Irpex lacteus and an SSF process at 10% (w/w) substrate loading. Higher bioethanol concentrations were obtained by Vargas et al13 using a hydrothermal pretreatment (autohydrolysis) followed by an organosolv pretreatment, applying a high solid loading, by means of fed-batch simultaneous saccharification and fermentation (44.5 g/L). Although a high bioethanol conversion was reached (90-93% glucose to bioethanol conversion), the overall bioethanol yield, calculated from data obtained by the authors, did not exceed 15 g bioethanol/100 g barley straw. This calculation considers solid recovery values in the pretreatment steps, so it provides more representative data of the overall process efficiency. Considering these data, the ethanol yield represents 97% of the potential bioethanol yield of the barley straw, one of the highest yields of those described in the literature. Despite the high yield of ethanol obtained from cellulose, some improvements can be


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expected. . On one hand, the use of different yeasts able to process C5 sugars can improve the ethanol production because hemicellulose represents about 45 % of the carbohydrates of the raw material. On the other hand, the increase in the ethanol concentration in the final fermentation medium, requires a deep study because the solid precipitate recovered after the IL treatment has a very high water content that makes difficult to reach high solids concentrations in the SSF or PSSF process.

SEM analysis of the final solid samples The solids obtained after hydrolysis or bioethanol production were analyzed using SEM. SEM micrographs (Figure 8, Figure 9 and Figure 10) clearly show that all structures of the original barley straw have disappeared. At higher magnification, all samples have a large porosity. For the sample after PSSF (Figure 10), aggregates of spherical moieties that correspond to residuals of inoculated yeast were observed between the porous voids.


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Figure 8. SEM micrographs of barley straw after enzymatic hydrolysis (EH).


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Figure 9. SEM micrographs of barley straw after the SSF process.

Figure 10.

SEM micrographs of barley straw after the PSSF process.


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FTIR Spectroscopy Changes in the composition of solids after pretreatment, hydrolysis and fermentation were monitored using infrared spectroscopy. Figure 11 depicts the IR spectra of barley straw and the solid collected after IL pretreatment, enzymatic hydrolysis, and the PSSF and SSF processes. The spectral region between 4000-2400 cm-1 shows a wide peak near 3380 cm-1 for all samples corresponding to the O-H stretching vibration. The intensity of this peak is higher in the starting sample and the IL treated samples because the presence of cellulose and hemicellulose results in a larger proportion of OH groups that decreases after the hydrolysis of fermentation. Peaks are detected between 3000 cm-1 and 2800 cm-1, which correspond to a C-H stretching vibration. The intensity of these peaks increases after the hydrolysis, indicating an enrichment of lignin, the component containing a greater proportion of C-H bonds with respect to O-H groups. The region between 1800 and 600 cm-1 is more complex with numerous peaks where several changes between components of the lignocellulosic biomass are observed 40-41. Although the spectra are quite complex, several signals attributed to the main components of the lignocellulosic biomass can be identified. The peaks near 1650 cm-1 and 1590 cm-1 can be reasonably assigned to absorbed O-H and conjugated C-O bonds, respectively. The weak peak at 1375 cm-1 is associated with C-H deformation in cellulose and hemicellulose structures. The peak at 1240 cm-1 corresponds to C-O stretching and could be due to the xylan or syringyl ring present in lignin. The peak at 1158 cm−1 can be attributed to a C–O–C vibration in cellulose and hemicellulose, the peak at 1125 cm−1 to an aromatic skeletal vibration or C–O stretching, the intense peak at 1048 cm−1 is due to the C–O stretching of cellulose and hemicellulose and the peak at 897 cm−1 is due to C–H def ormation of cellulose. The peak at 1508 cm-1 can be attributed to vibrations of the aromatic C-C skeletons of lignin, and the peaks at 1462 and 1425


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cm-1 to C-H asymmetric vibration and asymmetric deformation in lignin and carbohydrates,

2848

2900

3380

respectively, The peak at 1268 cm-1 is from the guaiacyl ring aromatic methoxyl groups40.

Barley Straw Untreated

Barley Straw Treated

PSSF SSF EH

4000

3800

3600

3400

3200

3000

2800

2600

1125

Barley Straw Untreated

897

1268 1240

1508 1462 1425 1375

1650 1590

1158

1048

Wavenumber (cm-1)

1730

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Barley Straw Treated

PSSF

SSF

EH

2000

1800

1600

1400

1200

1000

800

600

400

-1

Wavenumber (cm )

Figure 11. Infrared absorption spectra of untreated barley straw (black), solid fraction after pretreatment (blue), solid fraction collected after a) enzymatic hydrolysis EH (red), b) SSF (orange) and PSSF (green).


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Clear differences between the starting and IL pretreated substrates and sample recovered after hydrolysis and fermentation are observed. Often, the peak at 1505-1508 cm−1 is taken as a reference for lignin, whereas the 1730, 1158 or 897 cm−1 peaks become the polysaccharide reference. The spectra of barley straw and IL pretreated samples show a mixture of polysaccharide and lignin but clearly dominated by the former component. whereas the spectra of the solids collected after hydrolysis, PSSF and SSF were clearly dominated by the lignin signals. These observations clearly indicate that the solid residues obtained after hydrolysis/fermentation come basically from the lignin of the barley straw because the cellulose and hemicellulose have been transformed. Due to the chemical nature of residues collected after biochemical conversion (enriched-lignin residues), it would be interesting to investigate the convenience of these residues as precursors for lignin-based byproducts and biomaterials. CONCLUSIONS Dissolution/precipitation with IL pretreatment produces a tremendous change in the structure and crystallinity of barley straw (XRD and SEM). These structural changes

strongly enhance

the enzymatic hydrolysis with respect the raw substrate. The solid obtained with the pretreatment with [EMIM][Ac] can be hydrolyzed more efficiently than with its [EMIM][Cl] counterpart. High cellulose conversion yields (>80%) are reached in only 24 h in contrast to other pretreatment technologies that need more than 48-72 h. The pretreated samples can be used to produce bioethanol using SSF and PSSF in a very efficient way. The maximum bioethanol yield is similar for SSF and PSSF. The pretreatment with IL produces a more reactive substrate that can be hydrolyzed quickly, which makes it very appropriate for use in SSF, and it reaches the maximum concentration of bioethanol in only 48 h. The ethanol yield of 17.9 g bioethanol/100 g barley straw, representing 97% of the potential


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yield of bioethanol from cellulose present in the barley straw, is one of the highest yields reported in literature. Despite the high yield of ethanol obtained from cellulose, some improvements are expected and hence additional work is required. Studies such as recycling of IL; the valorization of C5 sugars fraction (for instance use of yeasts able to convert in ethanol); and use of the residual solids after enzymatic hydrolysis, SSF or PSSF which would be interesting precursors for lignin-based bio-products and biomaterials. ACKNOWLEDGEMENTS The authors acknowledge financial support from Comunidad de Madrid (Spain) (RESTOENE2-CM S2013/MAE-2882 project), CSIC (Spain) (201880E029 project) and MLS acknowledges the support of the European Social Fund and Comunidad de Madrid for her contract. REFERENCES 1. Cherubini, F., The biorefinery concept: Using biomass instead of oil for producing energy and chemicals. Energy Conversion and Management 2010, 51 (7), 1412-1421 DOI: 10.1016/j.enconman.2010.01.015 2. Uihlein, A.; Schebek, L., Environmental impacts of a lignocellulose feedstock biorefinery system: An assessment. Biomass and Bioenergy 2009, 33 (5), 793-802 DOI: 10.1016/j.biombioe.2008.12.001 3. Lin, Y.; Tanaka, S., Ethanol fermentation from biomass resources: Current state and prospects. Applied Microbiology and Biotechnology 2006, 69 (6), 627-642 DOI: 10.1007/s00253-005-0229-x 4. Kim, S.; Dale, B. E., Global potential bioethanol production from wasted crops and crop residues. Biomass and Bioenergy 2004, 26 (4), 361-375 DOI: 10.1016/j.biombioe.2003.08.002 5. Gupta, A.; Verma, J. P., Sustainable bio-ethanol production from agro-residues: A review. Renewable and Sustainable Energy Reviews 2015, 41, 550-567 DOI: 10.1016/j.rser.2014.08.032 6. Malins, S. S. a. C., Availability of cellulosic residues and wastes in the EU. The International Council on Clean Transportation. White paper. 2013, 7. Alvira, P.; Tomas-Pejo, E.; Ballesteros, M.; Negro, M. J., Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review. Bioresour Technol 2010, 101 (13), 4851-61 DOI: 10.1016/j.biortech.2009.11.093 8. Tomás-Pejó, E.; Alvira, P.; Ballesteros, M.; Negro, M. J., Pretreatment technologies for lignocellulose-to-bioethanol conversion. In Biofuels, Elsevier Inc.: 2011; pp 149-176. 9. Kim, S. B.; Lee, J. H.; Oh, K. K.; Lee, S. J.; Lee, J. Y.; Kim, J. S.; Kim, S. W., Dilute acid pretreatment of barley straw and its saccharification and fermentation. Biotechnology and Bioprocess Engineering 2011, 16 (4), 725-732 DOI: 10.1007/s12257-010-0305-7


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For Table of Contents Use Only SYNOPSIS Ionic liquid dissolution/precipitation pretreatment of barley straw yields the 97% of the potential bioethanol from the cellulose, one of the highest reported.


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Second generation bioethanol production combining simultaneous

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