Effects of Dilute Acid Pretreatment Parameters on Sugar Production

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Effects of Dilute Acid Pretreatment Parameters on Sugar Production during Biochemical Conversion of Switchgrass Using a Full Factorial Design Angele Djioleu, and Danielle Julie Carrier ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00441 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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ACS Sustainable Chemistry & Engineering

Effects of Dilute Acid Pretreatment Parameters on Sugar Production during Biochemical Conversion of Switchgrass Using a Full Factorial Design Angele Djioleu and Danielle Julie Carrier* Department of Biological and Agricultural Engineering, 203 White Engineering Hall, University of Arkansas, Fayetteville AR 72701 USA * Corresponding Email: [email protected], Phone: +1 (865) 974-7266

ACS Paragon Plus Environment

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ABSTRACT Pretreatment is an important unit operation in the production of cellulosic fuels because it governs the efficiency of the overall conversion process. The effect of dilute acid pretreatment parameters on prehydrolyzate xylose and glucose concentrations, as well as on glucan content and digestibility of pretreated switchgrass, was determined using a full factorial design. Temperature varied from 140°C to 180°C; time ranged from 10 to 40 min; and sulfuric acid concentrations changed from 0.5% or 1% (v/v). Results showed that pretreatment temperature was a significant factor on all response variables. Moreover, the prehydrolyzate xylose levels that reached the maximum of 21.71 g/L, were also significantly impacted by pretreatment time, while remaining insensitive to changes in acid concentration. A three-way interaction between all the pretreatment factors significantly affected prehydrolyzate glucose concentrations, which ranged from 1.52 g/L to 11.34 g/L. The glucan content of pretreated switchgrass decreased from 64.3% to almost zero as pretreatment conditions increased in severity, while switchgrass digestibility rose up to 95.8%. Temperature-acid concentration interaction significantly impacted glucan content, whereas digestibility was affected by temperature-time interaction. Overall, this study provided useful insights to select dilute acid pretreatment conditions to convert switchgrass into fermentable sugars. Keywords: Switchgrass, Dilute acid pretreatment, statistical analysis, Xylose, Glucose


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INTRODUCTION For the past few decades, industrialized societies have appreciated the potential that lignocellulosic materials can offer for crafting solutions to mitigate their energy, environmental, and sustainability needs.1-3 Through biochemical processes, the carbohydrate polymers of lignocellulosic biomass can be hydrolyzed into monomeric sugars, which can be further converted to fuels and chemicals via fermentation processes.1-3 However, due to its natural recalcitrance, lignocellulosic biomass first needs to be pretreated in order to facilitate the hydrolysis of its carbohydrate polymers into their monomeric moieties.1-3 Among the leading pretreatment techniques, dilute acid pretreatment (DAP) has gained popularity because of its resulting high monomeric sugar yields on a wide range of lignocellulosic materials.2,4 Unfortunately, the use of acid at elevated temperatures during DAP also promotes undesirable degradation of sugars, hereby reducing sugar yields.5,6 Moreover, generated degradation compounds produced during DAP have been reported to inhibit cellulolytic enzymes.7,8 In order to remove these degradation products, pretreated biomass is usually washed prior to saccharification with large quantities of water that could reach 30 times the amount of biomass to be converted.9 It is understood by the biofuels community that a washing step using colossal volumes of water would be a difficult practice to implement at deployment scale.9 Switchgrass (Panicum virgatum, L.) has been recognized by the US Department of Energy as an important herbaceous energy crop that could be used for fuels and chemical production. Its potential is mainly due to its high productivity across a wide geographic range, low resource requirements, an ability to grow on marginal land, and its resistance to severe weather.10-12 There is a large body of work that highlights the impact of individual DAP


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parameters on sugar yields from bioconversion of switchgrass;13-17 however, there is a gap in knowledge with respect to the interaction between DAP parameters on ensuing sugar yields. Additionally, comparatively little attention has been given to the impact of pretreatment conditions on glucose formation during DAP of switchgrass. It is important to acknowledge that some cellulose hydrolysis does occur during DAP, especially at conditions necessary to produce amenable biomass to enzyme saccharification and high combined xylose and glucose yields.14,17,18 Therefore, in order to prevent loss of sugar during DAP and ensure effective saccharification of pretreated biomass, this study examined the impact of individual DAP parameters, as well as their interaction, on resulting concentrations of xylose and glucose in pretreatment hydrolyzates, and the digestibility of the ensuing pretreated switchgrass using a full factorial design experiment. MATERIALS AND METHODS Biomass Switchgrass (Panicum virgatum, L.), specifically the Alamo cultivar, was harvested on July 4, 2009, one year after being planted at the University of Arkansas Agricultural Research and Extension Center in Fayetteville, AR (36.0625° N, 94.1572° W). The collected biomass was air-dried and ground to pass through a 20-mesh screen with a Thomas Willey® mini mill (Swedesboro, NJ). Ground biomass was stored at 4°C in sealed containers until used for compositional analysis and dilute acid pretreatment (DAP). Compositional analysis The mass of extractives, structural carbohydrates, and acid insoluble lignin (AIL) contained in raw biomass was established by protocols from the National Renewable and Energy


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Laboratory (NREL).19,20 All determinations were done in duplicate to yield average results and standard deviations presented in Table 1. Glucan content in dilute acid pretreated switchgrass was determined using the protocol outlined by Sluiter et al.20 Dilute acid pretreatment (DAP) Switchgrass was pretreated with sulfuric acid (H2SO4) (EMD Chemicals, Gibbstown, NJ) in a 1-L Parr 4525 reactor (Moline, IL) according to processing conditions depicted in Figures 1 and 2. The biomass (25 g) and the acid were loaded in the reactor at a 1:10 mass ratio and the agitation was set at 144 RPM. The biomass-acid mixture was heated to the reaction temperature for 10-15 min. Pretreatment time was started when the experimental temperature was reached. The reaction was stopped by circulation of cold water through a cooling coil until the reactor reached a safe handling temperature. The ensuing slurry was then separated by vacuum filtration into a solid and liquid portion through a perforated Büchner funnel lined with a Whatman No 1 filter paper from Sigma-Aldrich (St Louis, MO). The liquid hydrolyzate (prehydrolyzate) was stored at -20°C until used for analysis. The solid hydrolyzate, consisting of pretreated switchgrass, was washed at room temperature with water prepared with a Direct-Q filtering system (Millipore, Billerica, MA). Washed pretreated switchgrass was then separated by vacuum filtration through a perforated Büchner funnel lined with a Whatman No 1 filter paper and stored at -20°C until needed for compositional analysis and enzyme saccharification. All pretreatment experiments were performed in duplicate. Enzyme saccharification of pretreated switchgrass Glucan contained in pretreated washed switchgrass was hydrolyzed with Accelerase 1500®, an industrial enzyme cocktail generously donated by DuPont Industrial Biosciences (Cedar Rapids, IA). Total cellulase activity of the enzyme cocktail was determined to be 25


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FPU/mL based on the IUPAC protocol.21 Enzyme saccharification assay was performed in 50mL brown amber bottles, in which aliquots of pretreated switchgrass containing an equivalent of 0.1 g of glucan (dry basis) were mixed with enzyme at a loading of 15 FPU/ g of glucan, 5 mL of 50 mM sodium citrate buffer (pH = 4.8), and water to make a total solution volume of 10 mL. Amber bottles containing assay mixture were placed for 48 h in an agitated water bath (Thermo Scientific, Nashville, TN) set at 50°C and 100 RPM. The resulting slurry was centrifuged at 1286 g for 2 min (IEC Spinette centrifuge, Needham, MA); the liquid fraction (enzymatic hydrolyzate) was collected and stored at -20°C until further analysis. Assays were performed in duplicate. Characterization of liquid hydrolyzate Monosaccharides, furfural and hydroxymethylfurfural (HMF) of liquid hydrolyzates from compositional analysis and DAP were analyzed by high performance liquid chromatography (HPLC) as described in Djioleu et al.22 Glucose in enzymatic hydrolyzates was detected with a glucose analyzer, YSI 2900 (YSI Inc., Yellow spring, OH), as described by Mohanram et al.23 Digestibility Calculation The digestibility of the washed pretreated switchgrass samples was calculated based on equation 1: % =

!

× 0.9 × 100 1

Experimental design JMP Pro 11 from SAS Institute Inc. (Cary, NC) was used to develop a 3x4x2 full factorial design, resulting in 24 distinct experimental conditions listed in Table S1, in supporting information. The factors of interest and their levels were: pretreatment temperature, X1 = 140 °C, 160 °C, 180 °C; time, X2 = 10, 20, 30, 40 min; and H2SO4 concentration, X3 =0.5% and 1%. The


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levels for each factor were selected based on values commonly used in literature for DAP of switchgrass.13-17 The response variables investigated were the concentration of xylose and glucose in the prehydrolyzate, glucan content and digestibility of the pretreated switchgrass. Experimentally obtained values for the response variables were subjected to a linear regression analysis to determine main and interaction effects of factors using the Least Square method. The general form of the linear center polynomial obtained from the regression analysis is shown in Equation 1. The center polynomial was used to prevent interaction effects in overwhelming the main effects by ensuring that the test of main effects was independent of the test of interaction effects (JMP, Statistical Discovery from SAS). Models and regression coefficients were validated with an analysis of variance (ANOVA). Significance for any statistical results was established for P-value < 0.05. = '( + '* +* + ', +, + '- . ': +, − 25 .

/0 1(.23

/0 1(.23 (.,3

(.,3

4 + '5 +* − 160 +, − 25 + '3 +* − 160 .

4 + '2 +* − 160 +, − 25 .

/0 1(.23 (.,3

4

/0 1(.23 (.,3

4+

(2)

In equation (2) y = predicted response variable β0-7 = regression coefficient X1 = pretreatment temperature X2 = pretreatment time X3 = H2SO4 concentration used for pretreatment RESULTS AND DISCUSSION Effects of dilute acid pretreatment conditions on chemical composition of prehydrolyzate The analysis of dilute acid prehydrolyzates, in terms of monosaccharides, furfural and HMF, were similar to those reported previously.14-17 As expected, xylose and glucose were the


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main fermentable sugars present in the prehydrolyzate. Furfural and HMF, which are products of xylose and glucose degradation5,6, respectively, were also detected. The later two compounds were monitored in this study in an attempt to examine the variation in xylose and glucose levels in prehydrolyzates. The concentrations of the compounds in the prehydrolyzate were dependent on DAP conditions. Figure 1 shows how the concentrations of xylose, glucose, furfural and HMF varied with pretreatment temperature and time with the acid concentration maintained at 0.5% (v/v). With 0.5% (v/v) acid, the prehydrolyzate xylose concentrations declined as the pretreatment temperature was increased; the maximum xylose concentration recorded was 21.7 ± 2.8 g/L and was obtained when the switchgrass was pretreated at 140°C for 20 min. When using a 0.5% (v/v) acid concentration, a pretreatment time of 40 min yielded the lowest xylose concentrations for all examined pretreatment temperatures; the xylose concentrations dropped from 13.9 ± 0.8 g/L to nearly zero when the pretreatment temperature was increased from 140°C to 180°C. The decrease of xylose in the prehydrolyzate was attributed to its degradation to furfural. In this study, furfural concentrations were observed to increase when pretreatment temperatures were raised. This observation mirrored findings reported in other investigations of DAP of sunflower hulls,24 distiller grains,25 and corn fiber biomass.25 The presence of glucose in the prehydrolyzate indicated that DAP conditions promoted the release of glucose during pretreatment. Hydrolysis of non structural carbohydrates and amorphous portions of structural cellulose have been reported to occur under acidic conditions, especially when severe temperatures are used.1,26 This could possibly explain the source of glucose in the prehydrolyzate. Overall, the accumulation of glucose in the prehydrolyzate increased as temperature was raised, regardless of pretreatment length. It was important to note


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that increasing reaction temperature had varying effects on glucose concentrations. For example, at a pretreatment time of 40 min, an increase in temperature from 140°C to 160°C resulted in an increase in glucose concentration by 39.6%, while an increase in temperature from 160°C to 180°C increased glucose prehydrolyzate concentrations by 260.5%. The presence of HMF in the prehydrolyzate indicated that glucose was degraded as it was released into the prehydrolyzate, confirming the importance of preventing premature cellulose hydrolysis during DAP. The lowest glucose concentration of 1.5 ± 0.3 g/L was obtained at 180°C and 10 min, while the highest concentration of 11.2 ± 1.29 g/L occurred at 180°C and 30 min. The composition of switchgrass prehydrolyzates, with respect to pretreatment temperature and time at 1% (v/v) acid concentration, is presented in Figure 2. The effect of pretreatment temperature and time on the production of fermentable sugars at a 1% (v/v) acid was different than that observed with 0.5% (v/v) acid for glucose. Glucose concentrations increased with temperature when the biomass was pretreated for less than 30 min. For pretreatments lasting longer than 30 min, the glucose concentrations rose with temperature up to 160°C, but declined with further increases in temperature. The results presented in this work demonstrated that increasing acid concentrations intensified glucose disappearance. However, glucose disappearance could not solely be attributed to its degradation to HMF, as HMF prehydrolyzate concentrations obtained with a 1% (v/v) acid concentration were similar to those produced in 0.5% (v/v) acid. Simultaneous degradation of HMF to compounds, such as formic acid, levulinic acid, or humin compounds,5,6,27 could possibly clarify the observed discrepancy. Glucose decreases in prehydrolyzates could also be due to its condensation into humin compounds6,27 or its recombination with acid soluble lignin, a reaction observed by Xiang et al.28 in dilute acid prehydrolyzate of biomass.


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The amount of glucan that remained in each sample of pretreated switchgrass was determined as a percentage of the weight of the pretreated biomass on a dry basis. The 24 pretreated switchgrass samples were hydrolyzed using cellulolytic enzymes. The glucose recovered from biomass saccharification was used to calculate the digestibility of pretreated samples as shown in Equation 1. Variations in glucan content and digestibility of pretreated switchgrass with respect to pretreatment temperature, time, and acid concentration are presented in Figures 3 and 4, respectively. Results in Figure 3 show that for acid concentrations 0.5% (v/v) and 1% (v/v) represented in Figure 3A and Figure 3B, respectively, the glucan content of pretreated biomass declined as a function of increases in pretreatment temperatures. When pretreatment was performed with 1% (v/v) acid, the pretreatment time did not affect the resulting glucan content. Cellulose degradation of biomass during DAP was more prominent at higher acid concentrations, especially at temperatures above 160°C. This could possibly explain why the glucan content of the pretreatment biomass decreased from 55% to near zero when the temperature was increased from 160°C to 180°C in 1% (v/v) acid. Results in Figure 4 illustrate how digestibility of pretreated biomass was affected by DAP conditions. The samples pretreated at 180°C with 1% (v/v) acid were not submitted for enzymatic hydrolysis as they were severely damaged and had very low glucan content (< 13%). In general, switchgrass susceptibility to enzyme saccharification increased with pretreatment temperature, regardless of the acid concentration used (0.5% (v/v) in Figure 4A or 1% (v/v) in Figure 4B). However, when pretreating in 1% (v/v) acid for at least 40 min, biomass digestibility decreased by 6.1% points, when temperatures increased from 140°C to 160°C. These results indicate that prolonged pretreatment of switchgrass at high temperature and high acid concentration could impede biomass digestibility. Such a decline in digestibility could possibly


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be attributed to lignin re-deposition on the biomass, which has been reported to occur in acidic media at elevated temperature and prevent adsorption of cellulolytic hydrolysis enzymes.26,29 Such an occurrence would corroborate the observed decline in glucose concentrations reported above, particularly if lignin droplets coalesced with glucose, resulting in glucose and lignin combinations. It is also important to appreciate that, although using harsher pretreatment temperatures could improve biomass digestibility, this unfortunately would reduce the amount of glucan available for saccharification. Consequently, higher digestibility may not necessarily correlate with higher glucose yields. Statistical impact of DAP conditions on xylose, glucose, glucan content and digestibility The results presented in Figures 1-4 show that the composition of switchgrass DAP prehydrolyzates, as well as glucan contents and digestibility, varied as a function of DAP conditions. However, the significance of the effects of each individual DAP parameter or their interactions on the response variables were not unambiguously established. Such analysis could supplement current literature on switchgrass conversion to biofuels and biobased chemicals. A full factorial model, an experimental strategy where all factors of the experiments are simultaneously varied with all possible combinations of factors levels tested, can allow for the determination of individual and interaction effects of factors on a response variable.30 Such a model was employed to estimate the influence of pretreatment temperature, time and acid concentration on xylose and glucose concentration in the prehydrolyzate, glucan content and digestibility of the pretreated biomass. Using experimentally obtained data, a linear regression model was developed for each response variable. This regression analysis for digestibility did not include pretreatment temperature of 180°C because digestibility data on biomass pretreated at this temperature with 1% (v/v) acid were not available. An analysis of variance demonstrated


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that the developed linear models for xylose, glucose, glucan content and digestibility were significant, as their P-values were below 0.05. R-squared values for the developed models were 94%, 73%, 86% and 86% for xylose, glucose, glucan content and digestibility, respectively. The main and interaction effects of DAP parameters on response variables were estimated as the regression coefficients for the models developed for each response variable. Regression coefficients were also validated with statistical analysis and are listed in Table 2. The results in Table 2 indicate that pretreatment temperature had the highest significant impact on all the response variables investigated. Pretreatment time was another significant factor determining xylose concentration in the prehydrolyzate; however, no significant interaction effect between pretreatment temperature and time was detected for xylose. Surprisingly, acid concentration did not significantly affect (P > 0.05) xylose level in the 13

prehydrolyzate. Similar findings were also observed by Esteghlalian et al.

reporting that

increasing acid concentration resulted in decreased xylose concentrations in switchgrass dilute acid prehydrolyzates only at pretreatment temperatures greater than 180°C. Moreover, Lau et al.31 reported that the acid concentration exponents in the modified Arrhenius equations used to model the effects of temperature and acid concentration on the hydrolysis rate of switchgrass hemicellulose oligomers and xylose were significantly low (P > 0.05), to the point that the hydrolysis rates of these compounds were not sensitive to changes in acid concentration. Morinelly et al.15 made a similar observation when switchgrass was pretreated with acid concentrations ranging from 0.25-0.75 % at temperatures of 150-175°C, attributing this lack of acid effect on xylose concentration to the neutralizing capacity of switchgrass generated by its ash and mineral content. It is important to realize that the lack of significant impact that acid had on xylose concentration ensuing from biomass acid pretreatment cannot be generalized. Other


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investigators have reported the inverse trend when determining the effect of DAP conditions on xylose concentration from sunflower hulls,24 rapeseed straw,32 and corncobs.33 On the other hand, the results in Table 2 also show that acid concentration significantly affected the glucan content in pretreated switchgrass individually and through a two-way interaction with pretreatment temperature. Additionally, a three-way interaction between all the studied DAP factors was observed to affect glucose concentration in prehydrolyzates. Unlike glucan content, digestibility was affected by a two-way interaction between pretreatment temperature and time. The fact that glucan content was simultaneously affected by pretreatment temperature and acid concentration on one hand, and digestibility was affected by pretreatment temperature and time on the other hand, could cause the overall glucose yield from DAP and enzymatic saccharification to be significantly affected by all three studied factors. Thus, other elements, such as equipment capability, would also be important in selecting pretreatment conditions, as all three pretreatment factors were found to be critical to the production of glucose from switchgrass conversion. The effect of two-way interactions between experiment factors on a response variable can be displayed with a surface plot. Figure 5 presents the surface plots for the predicted xylose and glucose concentrations of switchgrass dilute acid prehydrolyzates, glucan content and digestibility of pretreated biomass, obtained from their respective model. Varying the two factors with significant effect on the response variable and keeping the third factor constant obtained surface plot for each response variable. For example, the surface plot for xylose was obtained by varying pretreatment temperature and time at acid concentration of 0.75% (v/v). From the surface plots, it was determined that finding DAP conditions that simultaneously achieve high production of xylose and glucose from switchgrass conversion


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would be challenging. This is mainly due to the fact that pretreatment temperatures yielding high prehydrolyzate xylose concentration (Figure 5A) resulted in biomass samples with low susceptibility to enzymatic saccharification (Figure 5D), regardless of their high glucan content (Figure 5C). Additionally, while prolonged pretreatment time at low temperatures would preserve glucan content in pretreated biomass samples and increase their amenability to enzyme saccharification (Figure 5D), especially at low acid concentration, the risk of xylose degradation under these pretreatment conditions would also increase. Although simultaneously maximizing xylose and glucose release could prove challenging, biomass conversion processes could be designed to have trade-offs for the release of both sugars.14,18 The conversion of lignocellulosic biomass to fermentable sugars is laborious and costly. Biomass pretreatment is an important unit operation in this process, since it will determine the overall conversion efficiency. Therefore, it is very important to choose productive pretreatment techniques and conditions. Results in this work illustrated how DAP parameters and their interactions impacted the conversion of switchgrass into fermentable sugars. Such an analysis could provide useful insights to make technoeconomic decisions when designing a conversion process for a biochemical refinery platform using switchgrass as the feedstock. In addition to the conversion of biomass to fermentable sugars, the results presented in this work could prove useful to the nanocellulose community; the studied processing parameters could be manipulated to yield cellulose and lignin with targeted physical and chemical properties. Acknowledgments The authors thank National Science Foundation award number 0822275, United States Department of Energy award number GO88036, and the Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville for their gracious financial


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support. The authors also acknowledge the Plant Powered Production (P3) Center. P3 is funded through the RII: Arkansas ASSET Initiatives (AR EPSCoR) I (EPS-0701890) 323 and II (EPS1003970) by the National Science Foundation and the Arkansas Science and Technology Center. Description of supporting information Table S1 in supporting information lists experimental conditions for the 24 experiments of dilute acid pretreatment; the resulting data was used to develop regression models.


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References 1) Kumar, P.; Barrett, D. M.; Delwiche, M. J.; Stroeve, P. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 2009, 48 (8), 3713-29. 2) Mosier, N.; Wyman, C.; Dale, B.; Elander, R.; Lee, Y. Y.; Holtzapple, M.; Ladisch, M. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 2005, 96 (6), 673-86. 3) Wyman, C. E. Biomass ethanol: technical progress, opportunities, and commercial challenges. Annu. Rev. Energy Env. 1999, 24 (1), 189-226. 4) Agbor, V. B.; Cicek, N.; Sparling, R.; Berlin, A.; Levin, D. B. Biomass pretreatment: fundamentals toward application. Biotechnol. Adv. 2011, 29 (6), 675-85. 5) Palmqvist, E.; Hahn-Hägerdal, B. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 2000, 74 (1), 25-33. 6) Rasmussen, H.; Sørensen, H. R; Meyer, A. S. Formation of degradation compounds from lignocellulosic biomass in the biorefinery: sugar reaction mechanisms. Carbohydr. Res. 2014, 385, 45-57. 7) Kothari, U. D.; Lee, Y. Y. Inhibition effects of dilute-acid prehydrolysate of corn stover on enzymatic hydrolysis of solka floc. Appl. Biochem. Biotechnol. 2011, 165(5-6), 1391404. 8) Jönsson, L. J.; Alriksoson, B.; Nilvebrant, N. Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnol. Biofuels 2013, 2, 1-10. 9) Frederick, N.; Zhang, N.; Ge, X., Xu, J.; Pelkki, M.; Martin, E.; Carrier, D. J. Poplar (Populus deltoides L.): The effect of washing pretreated biomass on enzymatic hydrolysis and fermentation to ethanol. ACS Sustainable chem. Eng. 2014, 1835-42. 10) McLaughlin, S. B.; Kszos, L. A. Development of switchgrass (Panicum virgatum) as a bioenergy feedstock in the United States. Biomass Bioenergy 2005, 28 (6), 515-35. 11) Parrish, D. J.; Fike, J. H. The biology and agronomy of switchgrass for biofuels. Cri. Rev. Plant Sci. 2005, 24 (5-6), 423-59.


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12) Sanderson, M. A.; Reed, R. L.; McLaughlin, S. B.; Wullschleger, S. D.; Conger, B. V.; Parrish, D. J.; Wolf, D. D.; Taliaferro, C.; Hopkins, A. A.; Ocumpaugh, W. R.; Hussey, M. A.; Read, J. C.; Tischler, C. R. Switchgrass as a sustainable bioenergy crop. Bioresour. Technol. 1996, 56 (1), 83-93. 13) Esteghlalian, A.; Hashimoto, A. G.; Fenske, J. J.; Penner, M. H. Modeling and optimization of the dilute-sulfuric-acid pretreatment of corn stover, poplar and switchgrass. Bioresour. Technol. 1997, 59 (2), 129-36. 14) Jensen, J. R.; Morinelly, J. E.; Gossen, K. R.; Brodeur-Campbell, M. J.; Shonnard, D. R. Effects of dilute acid pretreatment conditions on enzymatic hydrolysis monomer and oligomer sugar yields for aspen, balsam, and switchgrass. Bioresour. Technol. 2010, 101 (7), 2317-25. 15) Morinelly, J. E.; Jensen, J. R.; Browne, M.; Co, T. B.; Shonnard, D. R. Kinetic characterization of xylose monomer and oligomer concentrations during dilute acid pretreatment of lignocellulosic biomass from forests and switchgrass. Ind. Eng. Chem. Res. 2009, 48 (22), 9877-84. 16) Yat, S. C.; Berger, A.; Shonnard, D. R. Kinetic characterization for dilute sulfuric acid hydrolysis of timber varieties and switchgrass. Bioresour. Technol. 2008, 99 (9), 385563. 17) Shi, J.; Ebrik, M. A.; Wyman, C. E. Sugar yields from dilute sulfuric acid and sulfur dioxide pretreatments and subsequent enzymatic hydrolysis of switchgrass. Bioresour. Technol. 2011, 102 (19), 8930-8. 18) Lloyd, T. A.; Wyman, C. E. Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids. Bioresour. Technol. 2005, 96 (18), 1967-77. 19) Sluiter, A.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D. Determination of extractives in biomass. NREL/TP-510-42619; National Renewable Energy Laboratory: Golden, CO, 2005; http://www.nrel.gov/docs/gen/fy08/42619.pdf (accessed June 3, 2013). 20) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. Determination of structural carbohydrates and lignin in biomass. NREL/TP-510-42618; National Renewable Energy Laboratory: Golden, CO, 2008; http://www.nrel.gov/biomass/pdfs/42618.pdf (accessed June 3, 2013). 21) Ghose, T. K. Measurement of cellulase activities. Pure Appl. Chem. 1987, 59 (2), 257-68. 22) Djioleu, A. C.; Martin, E. M.; Pelkki, M. H.; Carrier, D. J. Sugar yields from dilute acid pretreatment and enzymatic hydrolysis of sweetgum (Liquidambar styraciflua L.). Agric. Food Anal. Bacteriol. 2012, 2, 175-86.


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23) Mohanram, S.; Rajan, K.; Carrier, D. J.; Nain, L.; Arora, A. Insights into biological delignification of rice straw by Trametes hirsuta and Myrothecium roridum and comparison of saccharification yields with dilute acid pretreatment. Biomass Bioenergy 2015, 76, 54-60. 24) Kamireddy, S. R.; Kozliak, E. I.; Tucker, M.; Ji, Y. Determining the kinetics of sunflower hulls using dilute acid pretreatment in the production of xylose and furfural. Green Process. Synth. 2014, 3 (1), 69-75. 25) Noureddini, H.; Byun, J. Dilute-acid pretreatment of distillers’ grains and corn fiber. Bioresour. Technol. 2010, 101 (3), 1060-7. 26) Mansfield, S. D.; Mooney, C.; Saddler, J. N. Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnol. Progr. 1999, 15 (5), 804-16. 27) van Zandvoort, I.; Wang, Y.; Rasrendra, C. B.; van Eck, E. R.; Bruijnincx, P. C.; Heeres, H. J.; Weckhuysen, B. M. Formation, molecular structure, and morphology of humins in biomass conversion: influence of feedstock and processing conditions. ChemSusChem, 2013, 6(9), 1745-58. 28) Xiang, Q.; Lee, Y. Y.; Torget, R. W. Kinetics of glucose decomposition during diluteacid hydrolysis of lignocellulosic biomass. Appl. Biochem. Biotechnol. 2004, 113-116, 1127-39. 29) Selig, M. J.; Viamajala, S.; Decker, S. R.; Tucker, M. P.; Himmel, M. E.; Vinzant, T. B. Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose. Biotechnol. Progr. 2007, 23 (6), 1333-9. 30) Montgomery, D. C. Design and analysis of experiments; John Wiley & Sons: New York, 1984. 31) Lau, C. S.; Bunnell, K.; Carrier, D. J. Kinetic modeling of switchgrass-derived xylose oligomers degradation during pretreatment in dilute acid or in water. ACS Sustainable Chem. Eng. 2015, 3 (9), 2030-5. 32) Castro, E.; Díaz, M. J.; Cara, C.; Ruiz, E.; Romero, I.; Moya, M. Dilute acid pretreatment of rapeseed straw for fermentable sugar generation. Bioresour. Technol. 2011, 102 (2), 1270-6. 33) Cai, B. Y.; Ge, J. P.; Ling, H. Z.; Cheng, K. K.; Ping, W. X. Statistical optimization of dilute sulfuric acid pretreatment of corncob for xylose recovery and ethanol production. Biomass Bioenergy 2012, 36, 250-7.


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Tables Table 1: Composition of raw switchgrass (% dry weight)a Total extractives 4.79 ± 0.08 Glucan 35.69 ± 1.21 Xylan 24.17 ± 1.64 Arabinan 6.42 ± 0.69 b AIL 21.52 ± 2.01 a data are average and standard deviation of two replications b Acid insoluble lignin


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Table 2: Regression coefficient for prediction of xylose, glucose concentration in dilute acid prehydrolyzate of switchgrass, glucan content and digestibility of pretreated switchgrass Glucan content Term Xylose (g/L) Glucose (g/L) Digestibility (%) (%) Constant

84.83***

-14.58***

Temp1

-0.45***

0.12***

Time

-0.12* -0.59

Temp x Time

-0.00

Temp x Acid Time x Acid

Acid

2

202.52**

-118.19**

-0.96***

1.21***

0.02

-0.14

0.10

-0.35

-5.00*

0.72

0.00

-0.01

-0.04*

-0.06

-0.05

-0.36**

-0.34

0.06

-0.07

0.06

-0.17

0.00 Temp x Time x Acid -0.00 -0.01** *** ** * significant at p < 0.001; significant at p < 0.01; significant at p < 0.05 1 = temperature; 2 = concentration of sulfuric acid


0.00

20

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Figure captions Figure 1

Figure 2

Figure 3 Figure 4 Figure 5

Effect of dilute acid pretreatment conditions on compounds concentration in switchgrass prehydrolyzate with 0.5% (v/v) H2SO4. HMF = hydroxymethylfurfural Effect of dilute acid pretreatment conditions on compounds concentration in switchgrass prehydrolyzate with 1% (v/v) H2SO4. HMF = hydroxymethylfurfural Effect of dilute acid pretreatment conditions on glucan content in pretreated switchgrass. (A) = 0.5% H2SO4, (B) = 1% H2SO4 Effect of dilute acid pretreatment conditions on digestibility of pretreated switchgrass. (A) = 0.5% H2SO4, (B) = 1% H2SO4 Surface plot of predicted: (A) = xylose and (B) = glucose concentration in dilute acid prehydrolyzate of switchgrass, (C) = glucan content and (D) = digestibility of pretreated switchgrass. Acid concentration for xylose and digestibility was 0.75% (v/v); time for glucose and glucan content was 25 min


21


Figure 1 30

0.5%

10 min

Xylose (g/L)

25

20 min 30 min

20

40 min

15 10 5 0 14

Glucose (g/L)

12 10 8 6 4 2 0 9 8 Furfural (g/L)

7 6 5 4 3 2 1 0 1.6 130

140

150

160

170

180

190

180

190

1.4 1.2 HMF (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

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1 0.8 0.6 0.4 0.2 0 130

140

150 160 170 Pretreatment Temperature (C)


22

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Figure 2

Xylose (g/L)

30

1%

25

10 min

20

20 min 30 min

15

40 min

10

Glucose (g/L)

5 0 9 8 7 6 5 4 3 2 1 0 6

Furfural (g/L)

5 4 3 2 1 0 1.2 1 HMF (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


0.8 0.6 0.4 0.2 0 130

140

150 160 170 Pretreatment Temperature (C)

180


190

23


Figure 3 70

A

60

Glucan (%)

50 10 min

40

20 min

30

30 min 20

40 min

10 0

B

70 60 50 Glucan (%)

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

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40 30 20 10 0 130

140

150

160

170

180

190

Pretreatment Temperature (C)


24

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Figure 4 100

A

Diges bility (%)

80

60 10 min 40

20 min 30 min

20

40 min

0 130

140

150

160

170

180

190

100

B 80 Diges bility (%)

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


60 40 20 0 130

140

150

160

170

180

190

Pretreatment Temperature (C)


25


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Figure 5 (A)

(B)

(C)

(D)


26

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TOC (A)

(B)

(C)

(D)

SWITCHGRASS

Dilute Acid Pretreatment

(A) = Prehydrolyzate xylose concentration, (B) = Prehydrolyzate glucose concentration, (C) = Pretreated biomass glucan content, (D) = Pretreated biomass digestibility


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Table of content (TOC) graphic (A)

(B)

(C)

(D)

SWITCHGRASS

Dilute Acid Pretreatment

(A) = Prehydrolyzate xylose concentration, (B) = Prehydrolyzate glucose concentration, (C) = Pretreated biomass glucan content, (D) = Pretreated biomass digestibility

Manuscript Title: Effects of Dilute Acid Pretreatment Parameters on Sugar Production During Biochemical Conversion of Switchgrass Using a Full Factorial Design Authors: Djioleu, Angele; Carrier, Danielle Julie Description: The graphic highlights the effects of pretreatment parameters on the conversion of switchgrass into fermentable sugars via dilute acid pretreatment and enzyme saccharification


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Effects of Dilute Acid Pretreatment Parameters on Sugar Production

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