water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides

Accepted Manuscript Acetone/water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides Constantinos Katsimpouras, Grigorios Dedes, Perrakis Bistis, Dimitrios Kekos, Konstantinos G. Kalogiannis, Evangelos Topakas PII: DOI: Reference:

S0960-8524(18)31263-X https://doi.org/10.1016/j.biortech.2018.09.018 BITE 20434

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

27 July 2018 1 September 2018 3 September 2018

Please cite this article as: Katsimpouras, C., Dedes, G., Bistis, P., Kekos, D., Kalogiannis, K.G., Topakas, E., Acetone/water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.09.018

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Acetone/water oxidation of corn stover for the production of bioethanol and prebiotic oligosaccharides Constantinos Katsimpourasa, Grigorios Dedesa, Perrakis Bistisa, Dimitrios Kekosa, Konstantinos G. Kalogiannisc, Evangelos Topakasa,b*

a

Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of

Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece. b

Biochemical and Chemical Process Engineering, Division of Sustainable Process

Engineering, Department of Civil, Environmental and Natural Resources Engineering, Luleå University of Technology, SE-97187 Luleå, Sweden. c

Chemical Process and Energy Resources Institute (CPERI), CERTH, 6th km Harilaou-

Thermi road, 57001, Thermi, Thessaloniki, Greece.

Abstract Ethanol production at high-gravity promise to achieve concentrations over the threshold for an economical distillation process and concurrently reduce water consumption. However, a persisting limitation is the poor mass transfer conditions resulting in low ethanol yields and concentrations. Hereby, the combination of an acetone/water oxidation pretreatment process (AWO) with a liquefaction/saccharification step, using a

*

Corresponding author at: Industrial Biotechnology & Biocatalysis Group, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou Str., Zografou Campus, Athens 15780, Greece. E-mail address: [email protected] (E. Topakas). 1

free-fall mixer, before simultaneous saccharification and fermentation (SSF) can realize ethanol concentrations of up to ca. 74 g/L at a solids content of 20 wt.%. The free-fall mixer achieved a biomass slurry’s viscosity reduction by 87 % after only 2 h of enzymatic saccharification, indicating the efficiency of the mixing system. Furthermore, the direct enzymatic treatment of AWO pretreated corn stover (CS) by a GH11 recombinant xylanase, led to the production of xylooligosaccharides (XOS) with prebiotic potential and the removal of insoluble fibers of hemicellulose improved the glucose release of AWOCS by 22 %.

Keywords: Bioethanol; Biorefinery; High gravity; Organosolv oxidation pretreatment; prebiotics

1. Introduction The production of renewable fuels from agricultural waste residues, such as corn stover (CS), has become imperative in order to face growing global energy demands and diminishing fossil fuels reserves. Lignocellulosic biomass could serve as an attractive source of fermentable carbohydrates, which in turn may be used as feedstock for the production of liquid fuels for the transport sector or other value-added chemicals (Jørgensen et al., 2007a). The proper conversion of each fraction of lignocellulosic biomass, namely cellulose, hemicellulose and lignin, into a range of products would realize the biorefinery concept. The sustainability aspect of biorefineries is the main motive of the concept, and every biorefinery should be evaluated from an economic, environmental and social sustainability point of view. The economic feasibility of 2nd generation biofuels production is quite challenging; therefore, the coproduction of value-added products could create the required revenue (de Jong and Jungmeier, 2015).

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A wide spectrum of products with high market value could be produced such as succinic, itaconic, fumaric and lactic acid, prebiotic oligosaccharides, and aromatic compounds from lignin such as xylene, benzene and toluene among others (De Bhowmick et al., 2018). More than 7.3 × 1015 kg d−1 of lignocellulosic biomass residues are estimated of being produced worldwide (Tuck et al., 2012). CS is one of the major representative agricultural residues globally, treated improperly, typically combusted directly, thereby not only wasting energy but causing serious environmental problems. Appropriate utilization of such agricultural residues, handling them as valuable resources of carbon, hydrogen, oxygen and inorganics rather than as low grade fuels is becoming more and more important (Qiu et al., 2010). However, the enzymatically produced fermentable sugars from lignocellulosic biomass is not a trivial task, mainly due to its recalcitrant nature (Himmel et al., 2007). Lignin is considered to be a crucial factor limiting the enzymatic hydrolysis of biomass by acting as a physical barrier between cellulases and cellulose (Zhao et al., 2012). In addition to that, irreversible adsorption of cellulases onto the lignin matrix has been reported to have a negative impact on enzymatic hydrolysis of biomass (Lu et al., 2016). Hence, delignification pretreatment seems to be a necessary process step in order to achieve high conversions and efficiency of the lignocellulosic carbohydrates. Pretreatment is a critical step in order to alter the structure of lignocellulosic biomass (remove lignin, hemicellulose, and disrupt crystalline structure of cellulose among others), which is intended to be utilized in enzymatic deconstruction processes. It is also the most challenging and costly process step of the emerging biorefinery technology. Developing a feedstock agnostic pretreatment process, that can handle different types of biomass of increased variability, complicates the task even more (Goh et al., 2011).

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Hence, several different pretreatment methods have been developed as a means of overcoming the challenges of biomass fractionation. Recently, the acetone/water oxidation (AWO), is developed as a new pretreatment method that exhibits the benefits of wet oxidation, such as relatively low temperatures and low yield of degradation products, while achieving higher lignin removal. Compared to other biomass pretreatment methods such as kraft, the AWO pretreatment uses less toxic chemicals which can be easily recycled, requires lower water usage, does not employ sulfur containing compounds, which can form toxic chemicals that are released in the environment, and has an improved lignin removal efficiency. Compared to other novel pretreatment methods such as ionic liquids, it has lower energy demands concerning liquids separations and recycle, and is dependent on the use of cheap and common bulk chemicals such as acetone as opposed to expensive ones as in the case of ionic liquids (Klein-Marcuschamer et al., 2011). Moreover, the lignin retrieved from AWO pretreatment is sulfur free and has low carbohydrates content making it a suitable candidate for phenols, thermoplastics and carbon fibers production (Lora and Glasser, 2002). Oxygen delignification using a mixture of acetone/water achieved high delignification degree of up to 95.0 % and 96.4 % in the case of hot water extracted sugar maple and eucalyptus, respectively (Gong et al., 2012). Delignification degrees of up to 93.6 % were also obtained in the case where Paulownia species underwent AWO pretreatment, while a promising lignin stream was obtained (Gong and Bujanovic, 2014). Moreover, the optimization of AWO of beechwood resulted in 95.8 % lignin removal, while the pretreated material was successfully utilized to produce high cellulosic ethanol concentrations of up to ca. 76 g/L at high initial solids-content of 20 wt.% (Katsimpouras et al., 2017a). A key advantage of this technology is the use of medium pressure O 2 rather than inorganic

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acids that are corrosive and are difficult to handle downstream (Ruiz Cuilty et al., 2018). Operating at high solids loadings of pretreated material is considered to be essential in order to reduce energy demands for product recovery processes, such as distillation for ethanol recovery and minimize waste water. High concentrations of glucose could be realized by performing enzymatic hydrolysis of pretreated biomass under high gravity conditions and consequently produce highly concentrated ethanol (Szijártó et al., 2011). However, cellulose conversion decreases when solids-loading increases following a linear correlation (Katsimpouras et al., 2017b) . One of the main factors impeding efficient cellulose conversion at high solids-loadings is mass transfer limitations due to improper mixing, hindering the ability of the enzymes to reach the substrate (Du et al., 2017). The application of a liquefaction/saccharification step before fermentation with the use of an alternative mixing system have been reported to adequately deal with issues related to poor mass transfer conditions (Katsimpouras et al., 2017a, 2017b; Matsakas and Christakopoulos, 2013; Paschos et al., 2015). Besides slurry rheology, increased end-product inhibition has been also accused for causing suboptimal enzyme function (Koppram et al., 2014). Relative to increased sugars concentration that inhibits enzymatic activity, simultaneous saccharification and fermentation (SSF) has been shown as the most proper fermentation mode for ethanol production at high gravity conditions (Koppram et al., 2014). Besides ethanol, lignocellulosic materials could be also used for the production of xylooligosaccharides (XOS) either by chemical methods or enzymatic hydrolysis of a vulnerable substrate, such in case of hemicellulose. XOS have gained a lot of interest mainly due to their potential to function as prebiotics and preserve a balanced intestinal microflora, which is associated with improved health and well-being (Lin et al., 2016).

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The biological properties of XOS that have been reported are manifold, from immunomodulatory and anti-microbial activity to growth regulatory activity in aquaculture and poultry (Aachary and Prapulla, 2011). Enzymatic production of XOS often includes the alkali extraction of xylan and subsequently its enzymatic hydrolysis by an endoxylanase. Moreover, autohydrolysis treatments in non-isothermal regime have been reported to produce XOS by solubilizing hemicellulose in the pretreatment liquor (Aguilar-Reynosa et al., 2017). In this manuscript, we described the ethanol production process from AWO pretreated CS at high solids loading of 20 wt.% using a free-fall mixer in order to perform the liquefaction/saccharification step before the SSF. To do so, the effect of pretreatment pressure on the pretreated samples’ composition was examined. Subsequently, the generated samples were evaluated in terms of enzymatic digestibility and fermentability. In addition, enzymatic saccharification parameters, such as the enzyme loading and duration of the liquefaction/saccharification step were investigated. We also highlighted the importance of the mixing system in order to improve cellulose conversion at high gravity. The combined enhancement of all these actions resulted in the production of highly concentrated ethanol surpassing the threshold for low-cost distillation process significantly. Finally, the production of XOS from the pretreated CS by direct enzymatic treatment, using a recombinant G11 family endoxylanase, was investigated in an attempt to utilize the remaining hemicellulolytic fraction under the biorefinery approach.

2. Materials and methods 2.1. Materials

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CS (94 % dry matter content) was the same that was used in previous study (Katsimpouras et al., 2017b). The non-xylose fermenting S. cerevisiae strain Ethanol Red® was kindly provided by Fermentis (Marcq-en-Barœl, France). The enzyme cocktail Cellic® CTec2 was a generous offer from Novozymes A/S (Bagsværd, Denmark).

2.2. Analytical procedures Composition of CS, total reducing sugars (TRS), glucose concentration and samples generated during the SSF were analyzed as described previously (Katsimpouras et al., 2017a). Filter paper activity and protein content of Cellic® CTec2 were found to be 84 FPU/mL and 90 mg/mL, respectively, following the assays that were described by Ghose (Ghose, 1987) and Bradford (Bradford, 1976). Apparent viscosities of biomass slurries were determined employing an Anton Paar Physica MCR rheometer (Anton Paar Gmbh, Styria, Austria), as described previously (Katsimpouras et al., 2016a). Hydrolysis products of AWOCS under the action of endoxylanase were analyzed by high-performance anion exchange chromatography (HPAEC) using a CarboPac PA-1 (4×250 mm) column equipped with an ED40 electrochemical detector (Dionex, Sunnyvale, CA, USA). The mobile phase was 100 mM NaOH and 500 mM NaOAc in 100 mM NaOH at a flow rate of 1 mL/min.

2.3. Corn stover pretreatment AWO of CS was performed as described previously (Katsimpouras et al., 2017a). The solid feedstock was fed into the reactor and the acetone/water mixture were then added at a liquid to solid ratio (LSR) of 25 on a weight basis. In our previous work

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(Katsimpouras et al., 2017a), an LSR of 10 was employed for treating Beechwood sawdust. CS was much less dense, its higher volume requiring a higher LSR in order to achieve proper mass transfer coefficients and efficiently remove its lignin. It was found that acetone to water ratio 1:1 (w/w), temperature and duration of 175 °C and 2 h, respectively, gave the best results in terms of degree of delignification and cellulose recovery, yielding a pulp that was well suited for high gravity fermentation. Heat up and cool down times were approximately 15 min for each stage. In addition, only around 8 min were needed for the system to go from 100 °C to 175 °C and from 175 °C to 100 °C, minimizing the time the biomass spent under these dynamic conditions. After the solid fraction was separated from the liquid one through vacuum filtration, it underwent washing with acetone and drying overnight. The parameter studied was the initial pressure. Specifically, the effect of three different pressures on CS composition and digestibility was investigated. The pressure was regulated with a N2/O2 mixture containing 40 vol% O2 at 8.5 atm, 20 atm, and 40 atm at 20 °C. Final pressure at the reaction temperature of 175 °C was 24, 42 and 74, respectively. All runs were carried out twice and the mean values are reported.

2.4. Screening of pretreated samples Enzymatic hydrolysis of AWOCS in order to assess different pretreatment conditions and the effect of solids-content on cellulose hydrolysis was performed in Erlenmeyer flasks (small scale). Solids content was 2-16 wt.% in 100 mM citrate-phosphate buffer pH 5.0 and enzyme loads were from 3 to 18 mg/g dry matter (DM). Enzymatic saccharification was carried out for 72 h at 50 °C and 200 rpm. Given the enzymatic digestibility of the AWOCS samples, a secondary screen was designed to investigate the

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fermentability of the acquired enzymatic hydrolysates. Conversion of AWOCS samples to ethanol was tested at the conditions described for batch SSF in paragraph 2.5.

2.5. Simultaneous saccharification and fermentation In the first approach of batch SSF, sample prehydrolysis was carried out in Erlenmeyer flasks (small scale) at 50 °C for 12 h before the SSF process. Solids-content was from 10 wt.% to 20 wt.% using an enzyme loading of 9 mg/g DM in 100 mM citrate-phosphate buffer pH 5.0. After the prehydrolysis step, the temperature was adjusted at 35 °C and Ethanol Red ®, corresponding to 15 mg/g DM, was added in order to start the fermentation process. Samples were collected at 0, 8, 24, 48, 72, 96, and 120 h, considering yeast addition as starting point, and were analyzed for ethanol. Each experiment was carried out in duplicates. Fed-batch was conducted at three different final solids-contents of 16 wt.%, 18 wt.% and 20 wt.%. The feeding of pretreated AWOCS was performed in batches at prehydrolysis times of 2, 4, and 6 h into the 100-mL Erlenmeyer flasks, while the total prehydrolysis step duration was 12 h. In the case where final solids-content was 16 wt.%, the initial solids-content was 8.7 wt.%, increased to 14.3 wt.% after 2 h and finally reached 16 wt.% after 4 h of prehydrolysis. From initial 8.9 wt.%, the solidscontent increased to 12.8 wt.% after 2 h, to 16.4 wt.% after 4 h, and to 18 wt.% after 6 h of prehydrolysis. Finally, from initial 9.1 wt.%, the solids-content increased to 14.9 wt.% after 2 h, to 18.4 wt.% after 4 h, and to 20 wt.% after 6 h of prehydrolysis. It should be noted that the addition of cellulolytic enzymes (9 mg/g DM) occurred at the start of the prehydrolysis step. The fermentation conditions for fed-batch SSF were the same as batch SSF.

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The third approach was batch SSF where the prehydrolysis step was conducted using an alternative mixing system. Liquefaction/saccharification of selected AWOCS sample at initial solids content of 20 wt.% employing the custom-made free fall mixer (large scale) was performed, as described previously (Katsimpouras et al., 2017a; Paschos et al., 2015). The duration of liquefaction/ saccharification step was either 6 or 12 h with an enzyme loading of 9 mg/g DM of Cellic® CTec2 at 100 mM citrate-phosphate buffer pH 5.0. The fermentation experiments were carried out in 100-mL Erlenmeyer flasks with 25 g of liquefacted CS slurry at pH 5.0 and incubation temperature of 35 °C under stirring at 80 rpm (each trial was carried out in duplicates). Ethanol Red®, corresponding to 15 mg/g DM, was used for the fermentation. The equation of Zhang and Bao (2012) for the calculation of ethanol yield at high gravity conditions was employed.

2.6. Production of xylooligosaccharides The enzymatic hydrolysis of selected AWOCS for the production of XOS was performed by using a GH family 11 endo-1,4-β-xylanase (FoXyn11a), which had been previously cloned from Fusarium oxysporum and expressed in Pichia pastoris (Moukouli et al., 2011). The recombinant P. pastoris harboring foxg-09638 gene was grown in agitating Erlenmeyer flasks (180 rpm) in BMGY medium at 30 °C for 18 h and then inoculated into the production BMMY medium to an O.D. 600 = 1 with the addition of methanol required every 24 h to maintain induction, as previously described (Katsimpouras et al., 2016b). After 5 d, the culture broth was centrifuged and the cell-free supernatant was concentrated using an Amicon ultrafiltration apparatus (30 kDa exclusion size, Diaflo PM-30 membrane, Amicon chamber 8400), (Merck Millipore, Darmstadt, Germany).

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The endoxylanase activity was assayed against 0.5 % (w/v) beechwood xylan, for 10 min in 0.1 M citrate-phosphate buffer pH 5.0. Reducing sugars that were released by beechwood xylan hydrolysis, were determined by DNS using xylose for constructing the calibration curve. One unit (U) of xylanase activity was defined as the amount of enzyme that released 1 μmol of reducing sugars per minute under standard assay conditions. Hydrolysis of AWOCS at solids-content of 5 wt.% was carried out in 100-mL Erlenmeyer flasks using 15 mg/g DM of FoXyn11a for 24 h at 50 °C with constant stirring. Afterwards, the supernatant was separated from the remaining solids by vacuum filtration and the hydrolysis products were determined by HPAEC-PAD. The remaining solid material was utilized as feedstock for ethanol production.

3. Results and discussion 3.1. Composition of solid pulp from AWO pretreatment The composition of untreated CS was determined according to the National Renewable Energy Laboratory (NREL) procedure and it was found to be 40.7±0.5 wt.% cellulose, 23.4±0.2 wt.% hemicellulose, 23.7±0.7 wt.% lignin, and 6.2 wt.% ash. CS underwent a pretreatment step, namely AWO, to generate a glucan rich material that could serve as a potential feedstock for ethanol production. A pretreated material with high glucan and low lignin content is of great importance in order to perform high gravity processes and subsequently obtain high ethanol concentrations. In this work, AWO pretreatment was conducted at 175 °C for 2 h with an acetone solution of 50 vol.% and at an LSR of 10. The autoclave was pressurized at 8.5 atm, 20 atm, and 40 atm at 20 °C with a N2/O2 mixture. The composition of the solid pulps (AWOCS-8.5, AWOCS-20, and AWOCS-40) from AWO pretreatment at different pretreatment

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pressures are presented in Fig. 1a. The use of a mixture of water and acetone had a significant effect on pretreated CS composition, especially on hemicellulose and lignin content. The mechanisms involved in lignin depolymerization and removal have been discussed previously (Katsimpouras et al., 2017a). In short, a synergistic effect between hemicellulose hydrolysis and lignin depolymerization is observed. The water hydrolyzed hemicellulose, disrupting its linkages with lignin which is subsequently released, while the acetone solubilized the released, partly depolymerized, lignin and removed it from the solid biomass that in turn facilitated the further disruption of ligninhemicellulose bonds. Moreover, the use of oxygen resulted in the cleaving of the carbon-carbon and ether linkages of the building blocks of lignin, which have been found to be very reactive under wet oxidation conditions (Gong et al., 2012). This led to further depolymerization of lignin which was easily dissolved and removed by the organic solvent acetone. In addition, the pressure increase led to a further decrease of hemicellulose and lignin content. Fig. 1b shows that high cellulose recovery of about 83 % was achieved in every case, while lignin removal of up to 86 % was noted in the case of AWOCS-40. Lignin removal is an important step towards a material rich in glucan and susceptible to cellulolytic enzymes. Moreover, the action of enzymes which are responsible for the release of fermentable sugars from biomass polysaccharides is hindered by lignin, mostly due to their non-productive binding to it (Rahikainen et al., 2013). The resulting pulp at pressure of 40 atm at 20 °C had a cellulose content of up to 79.8 wt.%, rendering it a promising feedstock for downstream processes, such as enzymatic hydrolysis and fermentation.

3.2. AWOCS samples evaluation and solids-content effect on cellulose conversion

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Initially, the impact of enzyme loading on enzymatic hydrolysis was investigated aiming at achieving high glucose release while minimizing the enzyme addition. Low enzyme loadings are of great importance in order to design a cost effective cellulosic ethanol process (Klein-Marcuschamer et al., 2012). Furthermore, high concentrations of commercial enzymatic cocktails often come with relatively high amounts of preservatives such as glycerol and sorbitol, which have been reported to have a negative effect on enzymatic hydrolysis (Selig et al., 2012). As expected, there was a correlation between glucose concentration and enzyme loading. However, after enzyme dosage of 9 mg/g DM, further enzyme addition led to no significant increase in neither glucose concentrations nor hydrolysis yields that were achieved following enzymatic hydrolysis of AWOCS sample. Hence, the enzyme dosage of 9 mg/g DM (8.4 FPU/g DM) was selected for the rest of the processes. Enzymatic hydrolysis of AWOCS-8.5, AWOCS-20, and AWOCS-40 was carried out at solids-content of 10 wt.% in order to test their digestibility in terms of glucose release. Among the pretreated samples, AWOCS-40, exhibited the highest glucose concentration of 84 g/L after 72 h of enzymatic hydrolysis (Fig. 2a). The maximum glucose concentration that was obtained from AWOCS-40 was 33 % and 11 % higher, compared to that of AWOCS-8.5 and AWOCS-20, respectively. The effectiveness of AWO pretreatment was underlined, since a maximum of a 10-fold increase in glucose release was observed compared to that achieved by untreated CS. Based on the final glucose concentrations, the lignin removal had a positive impact on enzymatic hydrolysis. The next step was to test the fermentability of the enzymatic hydrolysates and compare the final ethanol concentrations. All three pretreated samples were fermented at solids-content of 10 wt.% employing a batch-SSF process with a prehydrolysis step

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of 12 h. From the tested samples, AWOCS-40 exhibited the highest ethanol concentration and yield of ca. 29 g/L and 64 %, respectively (Fig. 2b). Screening experiments revealed that the composition in lignin of pretreated biomass played a significant role to produce enhanced glucose or ethanol concentrations. Therefore, taking into consideration both the enzymatic saccharification and fermentation screening, it was determined that the pretreatment at initial pressure of 40 atm at 20 °C led to a highly digestible material that could be utilized for bioethanol production. Hence, AWOCS-40 was selected for SSF experiments at high gravity conditions either at small scale or at large scale using the custom made free-fall mixer. The performance of the selected AWO pretreated sample at different solids-contents was also investigated. The effect of increasing initial solids-content (from 2 wt.% to 20 wt.%) on glucose release (g/L) and cellulose conversion (%) both at small scale (Erlenmeyer flasks) and large scale (free-fall mixer) is presented in Fig. 3. Enzymatic hydrolysis exhibited a negative linear correlation with increasing solids-content especially after 4 wt.%, while before that the cellulose conversion seemed to be stable. This negative effect on yields has been also observed in other studies and has been attributed to mass and heat transfer limitations, and high inhibitors concentrations in the slurry due to inadequate mixing (Katsimpouras et al., 2017a). However, when the enzymatic saccharification was performed using the free-fall mixer, a significant increase in cellulose conversion was noted reaching 64 % after 12 h. In that case, the cellulose conversion was fairly lower than that achieved with Erlenmeyer flasks when the solids content was 6 wt.% (68 %). These results exhibited the effectiveness of this alternative high gravity mixing system, which in combination with the AWOCS seems to be a promising technology for an ethanol production process.

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3.3. SSF of AWOCS Pretreated CS (AWOCS-40) was fermented at different solids contents of 10 wt.%, 12 wt.%, and 14 wt.% following a batch SSF process where the amount of biomass was fed all at once at the start of a 12-h prehydrolysis step. As expected, the ethanol concentration was improved with increasing solids content (Fig. 4). Maximum ethanol concentration of ca. 40 g/L was obtained at solids content of 14 wt.% after 96 h of SSF exhibiting a productivity of 0.42 g/L·h, while at solids content of 10 wt.% and 12 wt.% ethanol concentration was found to be ca. 29 g/L and 33 g/L with productivities of 0.30 g/L·h and 0.34 g/L·h, respectively. It is noteworthy that the ethanol concentration of ca. 40 g/L was realized at relatively low solids loading (14 wt.%) using Erlenmeyer flasks with known problems coming from functioning at high solids content. In order to examine higher solids loadings, the process was changed from batch SSF to fed-batch SSF. Several approaches of fed-batch enzymatic saccharification have been reported in literature to serve a variety of purposes such as enzyme recycle, to decrease the effect of inhibitors in the hydrolysate during SSF and relieve mixing issues at high-solids content. A feeding strategy of AWOCS-40 during the prehydrolysis step before the SSF was employed in order to increase solids content during saccharification (up to 20 wt.%) and thus improve ethanol production by overcoming mass transfer limitations due to improper mixing. The fed-batch SSF results are presented in Fig. 4 in comparison with batch SSF results at similar solids content. Maximum ethanol concentration, following fed-batch approach, was obtained at 20 wt.% and was found to be ca. 58 g/L while also high ethanol concentrations were achieved in lower solids loadings of 16 wt.% and 18 wt.% (ca. 41 g/L and 52 g/L, with productivities of 0.57 g/L·h and 0.54 g/L·h, respectively).

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The feeding strategy that was followed in the current study led to ethanol productivities of over 2 g/L·h in the first 12 h of SSF, which were about 2-fold higher than those achieved during batch SSF. The ethanol yields were determined to be up to 56 %. For comparison reasons batch SSF was also performed at the same solids contents of 16, 18, and 20 wt.% where lower ethanol concentrations (28 g/L, 33 g/L, and 39 g/L, respectively) and consequently productivities (1.17, 1.38, 1.6 g/L·h, respectively) were observed after the first 24 h in contrast with fed-batch SSF. The results revealed that fed-batch enzymatic saccharification approach seemed to handle high initial solids content more effectively than feeding the material all at once by achieving higher ethanol concentrations at shorter time. Comparable results were obtained by Li et al. (2014) where fed-batch SSF for ethanol production from liquid hot water pretreated CS resulted in higher ethanol concentrations and yields compared to batch SSF. However, even with the employment of a different feeding system in order to minimize the negative effect of high gravity enzymatic hydrolysis, the ethanol yields were not significantly improved when using fermentation flasks. In this context, an alternative mixing system should be sought that can lead to both high ethanol concentrations and yields when operating at high-solids content.

3.4. SSF of AWOCS using a free-fall mixer In an attempt to obtain higher ethanol concentrations and yields, a free-fall mixer was employed for the liquefaction and saccharification of AWOCS-40 at high gravity conditions (20 wt.%). The concentrations of glucose and TRS were 93.7 and 107.5 g/L, respectively, after 6 h of liquefaction/saccharification (Fig. 5a). As expected, higher enzymatic saccharification was achieved after 12 h resulting in 131.1 and 151.8 g/L of glucose and TRS, respectively. Conversion of cellulose (%) was 46.5 % after 6 h and

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65.1 % after 12 h of enzymatic treatment. The calculation was conducted based on glucose release. According to these results, a liquefaction/saccharification step of 6 h seemed sufficient for the following fermentation, since glucose release (g/L) was high enough to possibly obtain a concentrated ethanol solution, suitable for an economical distillation process (Jørgensen et al., 2007b). The initial apparent viscosity was measured during liquefaction and was found to be 1356 Pa·s. It quickly decreased to 176 Pa·s after 2 h of enzymatic liquefaction. The viscosity decreased about 87 % rendering the initial viscous material into a slurry that was easy to handle and suitable for submerged fermentation. When the duration of liquefaction/saccharification step was 6 h or 12 h, maximum ethanol concentrations of 70.6 g/L (after 72 h of SSF) and 73.8 g/L (after 96 h of SSF) were realized, respectively (Fig. 5b). Ethanol concentration over 40 g/L (55.2 g/L) was obtained after 30 h (24 h of SSF with 6 h of prehydrolysis), exhibiting a high ethanol productivity of 2.3 g/L/h. However, ethanol concentration was found to be 39.2 g/L (24 h of SSF) after 12 h of prehydrolysis despite the fact that initial glucose concentration was higher, exhibiting an ethanol productivity of 1.6 g/L/h. Under high gravity conditions, increased levels of initial glucose are considered to impede fermentation (Koppram et al., 2014). High productivities were exhibited by both the 6 and 12-h liquefacted/saccharified AWOCS (0.98 and 0.77 g/L/h, respectively) at maximum ethanol concentrations. Therefore, high final cellulosic ethanol concentrations were achieved, indicating that AWO seems to be an efficient strategy to convert CS into a highly digestible feedstock for the production of bioethanol. The 6-h liquefied AWOBW seemed to be appropriate for a large-scale ethanol fermentation process owing to the higher ethanol productivity, while the prolongation of the prehydrolysis step by 6 h led to a maximum ethanol production enhancement by only 3.2 g/L.

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The combined use of AWO pretreatment and a free-fall mixer generated an ethanol production process that handled issues relevant to high initial solids content and consequently resulted in high ethanol concentrations. In literature, there are several reports where high bioethanol concentrations were realized under high gravity conditions and to assess the results obtained from this study, a comparison appears to be essential. During this study, only the solid fraction of pretreatment was used for fermentation. Thus, comparison to works where solely the solid residue was employed for enzymatic hydrolysis and bioethanol production was conducted. In the case where CS was utilized for bioethanol production, in our previous work, ethanol concentrations of up to 75.5 g/L were achieved using steam exploded CS employing longer prehydrolysis duration (24 h) and higher enzyme loading (12 mg/g DM or 11.2 FPU/g DM), and 62.3 g/L from organosolv pretreated CS using 8.4 FPU/g DM (prehydrolysis duration was 24 h) (Katsimpouras et al., 2017b). In every case, solids content was 24 wt.%. Liu et al. (2014) reported ethanol concentration of 59.8 g/L from steam exploded CS (20 wt.% solids-loading) employing 36 h of prehydrolysis and an enzyme loading of 17.7 FPU/g DM. Nguyen et al. (2016) obtained 58.8 g/L ethanol by SSF at 15.5 wt.% solids with an enzyme loading of ~9 FPU/g glucan applied a novel pretreatment process called co-solvent enhanced lignocellulosic fractionation on CS, while after optimizing the SSF process, 79.2 g/L of ethanol was reported at 20 wt.% solids-content (Nguyen et al., 2017). Aguilar-Reynosa et al. (2017) reported 38.1 g/L ethanol concentration in SSF from microwave pretreated corn residues at 12.5 % solids loading and 9.4 FPU/g DM. Furthermore, under high gravity conditions, ethanol concentrations from 47.9 g/L to 80.0 g/L have been exhibited using different agricultural residues, such as rice and wheat straw (Paschos et al., 2015; Sun and Tao, 2013), forest residues such as beechwood, redcedar and birch (Katsimpouras et al., 2017a; Matsakas et al., 2018;

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Ramachandriya et al., 2013), and grasses such as sweet sorghum bagasse and Miscanthus (Kang et al., 2015; Matsakas and Christakopoulos, 2013). Nutrient addition and/or medium sterilization was incorporated in the majority of the reports conferred, together with enzyme loadings of about 15 FPU/g DM and long prehydrolysis steps, contributing to high final bioethanol titers but at the same time increasing process’ overall cost. Ethanol concentrations up to 73.8 g/L (corresponding to ethanol yields up to 71.0 %) were realized by spending a quite low enzyme load of 8.4 FPU/g DM without the supplement of nutrients during SSF. It is notable that the process realized already 70.9 g/L after only 2.5 days (including prehydrolysis duration). Hence, AWO could be applied effectively on CS in order to produce a solid feedstock, rich in cellulose, while the lignin from the pretreatment’s liquor could possibly be upgraded towards fine chemicals.

3.5. Hemicellulosic fraction utilization towards the production of XOS Aiming in the utilization of the hemicellulosic fraction that would also have a beneficial result in the cellulose enzymatic hydrolysis, xylanase treatment of AWOCS40 was employed. For this purpose, a fungal GH11 endoxylanase from F. oxysporum was used to produce a mixture of XOS, which are known to stimulate growth or activity of bifidobacteria in the colon demonstrating prebiotic effects (Vardakou et al., 2008b). Specifically, XOS with low degree of polymerization (DP), such 2-3 have been found to be more beneficial to lactobacilli and bifidobacteria in the human gastrointestinal tract (Mathew et al., 2017). Xylanases that belong in GH11 family are considered as “true xylanases”, since all members are exclusively active against xylan backbone (see Topakas et al., 2013 for a review). GH11 are known for their preference in long-chain XOS, preferentially cleaving unsubstituted regions of arabinoxylan in contrast to GH10

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xylanases, however, highly complex decorations at the +2 subside of arabinoxylan backbone are being exploited as specificity determinants producing decorated XOS (Vardakou et al., 2008a). Linear XOS up to DP 6 were detected, while the presence of cellobiose (C2) was indicated exhibiting a retention time (Rt) of 16 min. The enzymatic release of XOS prior to AWOCS utilization for the production of bioethanol offers a dual advantage; a direct production of prebiotics in the supernatant that could easily be separated by filtration or low speed centrifugation and a more digestible feedstock, since the insoluble fibers of hemicellulose is being removed, making cellulose more vulnerable to enzymatic attack. This enhancement was demonstrated by the elevated glucose release after enzymatic hydrolysis of AWOCS-40 sample that underwent treatment with the GH11 xylanase FoXyn11a. The enzymatic hydrolysis was performed at solids loading of 16 wt.% using 9 mg/g DM as described in paragraph 2.4. After 12 h of enzymatic hydrolysis, the glucose concentration of 84.2 g/L was achieved by the xylanase treated sample, while in the case of AWOCS-40, glucose concentration was lower and was found to be 68.9 g/L. The increase of 22 % in glucose release is evidence of the significant role that insoluble and closely associated with cellulose, hemicellulose fibers play in regard to lignocellulosic biomass recalcitrance. The presence of C2 in the mixture of XOS was not expected, however, its existence promotes the prebiotic activity of the low DP XOS, since it has been shown to increase the lactic acid bacteria and bifidobacteria populations (van Zanten et al., 2012).

4. Conclusions In this study, the described process led to ethanol concentrations of up to ca. 74 g/L at high-gravity. Thus, the application of a liquefaction/ saccharification step prior to

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SSF, the use of an alternative mixing system and the AWO pretreatment process deliver a promising bioethanol production process by realizing almost two-fold higher ethanol concentrations than the limit for the economical ethanol recovery. Moreover, a mixture of XOS with prebiotic potential was generated from the treatment of AWOCS with a recombinant GH11 xylanase. The xylanase treated material exhibited improved glucose release by 22 % after enzymatic hydrolysis with Cellic® CTec2, indicating the significance of insoluble hemicellulosic fibers in plant cell wall recalcitrance.

E-supplementary data for this work can be found in the e-version of this paper online.

Acknowledgments The authors are grateful to Novozymes A/S and to Lesaffre for the generous gifts of Cellic® CTec2 and Ethanol Red®, respectively.

Funding: This work was supported by the Greek State Scholarships Foundation (Research Projects for Excellence IKY/Siemens).

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Figure captions Fig. 1. Effect of pretreatment’s pressure at 20 °C on pulp composition and constituents cellulose, hemicellulose and lignin recoveries. (a) Pulp composition vs pressure at 20 °C (atm); untreated CS (UCS) is included for comparison, and (b) cellulose, hemicellulose and lignin recoveries in solid pulp vs pressure at 20 °C. All pulp composition determination experiments were carried out in triplicates.

Fig. 2. Screening of AWOCS samples, generated at three different pressures (8.5, 20 and 40 atm at 20 °C) of the AWO process, with (a) glucose release (g/L) and (b) ethanol production (g/L) as response factors. Solids-content was 10 wt.% and enzyme loading 9 mg/g DM.

Fig. 3. Effect of solids content on glucose concentration (g/L) and cellulose conversion (%) of AWOCS after 12 h of enzymatic hydrolysis in Erlenmeyer flasks (solids-content from 2-16 wt.%) and free-fall mixer (solids-content 20 wt.%) using an enzyme loading of 9 mg/g DM.

Fig. 4. Time course of ethanol production (g/L) during batch SSF (B-SSF) at solidscontent from 10 wt.% to 14 wt.%, and fed-batch SSF (FB-SSF) at solids-content from 16 wt.% to 20 wt.%.

Fig. 5. (a) Time course of glucose and TRS concentration (g/L) during the liquefaction/ saccharification step of AWOCS and (b) ethanol production (g/L) and glucose consumption (g/L) from the 6-h and 12-h liquefacted/saccharified AWOCS at 20 wt.% solids-content employing the free-fall mixer.

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Table 1. Pulp composition of untreated and after AWO pretreatment CS. Constituents

Untreated* (wt.%) Acetone/water oxidation (wt.%) 8.5 atm 20 atm 40 atm Cellulose 40.7±0.5 66.8±0.2 71.4±1.2 79.8±0.9 Hemicellulose 23.4±0.2 17.0±0.3 14.7±0.3 9.7±0.1 Lignin 23.7±0.7 12.1±0.9 8.6±0.6 7.7±0.7 Klason lignin 20.2±0.7 10.6±0.9 7.3±0.6 6.8±0.7 Acid soluble lignin 3.5±0.0 1.5±0.0 1.3±0.0 0.9±0.0 Total 87.8±0.3 95.8±0.8 94.7±0.9 97.2±0.2 *Ash content in untreated corn stover: 6.2 wt.%

Highlights 

Acetone/water oxidation pretreatment was applied on corn stover



87 % biomass’ viscosity reduction after enzymatic hydrolysis by free-fall mixer



Maximum ethanol concentration and yield of up to ca. 74 g/L and 71 %, respectively



Production of xylooligosaccharides using a recombinant GH11 xylanase



Improved glucose release by 22 % from the xylanase treated material

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(a)

(b)