Coproduction of Oligosaccharides and Glucose from Corncobs by

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Coproduction of Oligosaccharides and Glucose from Corncobs by Hydrothermal Processing and Enzymatic Hydrolysis Gil Garrote,* Remedios Ya´ n˜ ez, Jose´ Luis Alonso, and Juan Carlos Parajo´ Department of Chemical Engineering, Faculty of Science, UniVersity of Vigo (Campus Ourense), As Lagoas, 32004 Ourense, Spain

Corncobs were subjected to nonisothermal reaction in aqueous conditions to reach temperatures in the range 202-216 °C. The liquors were assayed for composition to assess the conversion of xylan into oligosaccharides and reaction byproducts, whereas the spent solids from treatments were subjected to enzymatic hydrolysis. Enzymatic assays were carried out according to an incomplete, factorial, centered design of experiments, in which the following independent variables were considered: liquor to solid ratio (in the range 6-16 g/g), enzyme to substrate ratio (in the range 6-28 filter paper units/g), and β-glucosidase activity/cellulase activity ratio (in the range 1-9 IU/FPU). The concentration/time data were fitted to an equation selected from the literature, and the model parameters were expressed as a function of the operational variables considered in the experimental design. Under selected conditions, high oligosaccharide yields and near quantitative cellulose conversion into glucose were obtained. 1. Introduction Lignocellulosic materials (LCM) are a largely available, cheap, and renewable resource potentially suitable for the sustainable production of a variety of chemicals. LCM are made up of structural components (cellulose, hemicelluloses, and lignin) and nonstructural components (including extractives, inorganic components, and proteins). The benefit of LCM can be achieved through the “biomass refinery” or “biorefinery” concept,1,2 consisting of the sequential “fractionation” of the considered raw material by chemical treatments suitable for causing the solubilization of at least one structural component. In this context, processing of LCM in aqueous media (autohydrolysis or hydrothermal reaction) allows the selective depolymerization and solubilization of the hemicellulosic fraction. During hydrothermal treatments, hydronium ions (from water autoionization and from in situ generated acids) catalyze the breakdown of hemicellulose chains into oligosaccharides, leading to the formation of soluble oligosaccharides,3,4 whereas cellulose and acid-insoluble lignin remain in solid phase. When xylan-containing LCM are employed as feedstocks for autohydrolysis, xylooligosaccharides (XO) are the main reaction products. XO find a variety of applications in the pharmaceutical, food, and feed industries. As ingredients of functional foods, XO show prebiotic activity, mainly based on the modulation of gut microflora.5-9 A review on the biological properties of XO has been reported recently.10 Owing to the selective hemicellulose solubilization achieved in autohydrolysis treatments, the spent solids from the reaction media show increased contents of cellulose and lignin and can be subjected to further processing for either lignin depolymerization11-14 or cellulose conversion into glucose by enzymatic hydrolysis.15-19 This latter approach is gaining interest because the enzymatic hydrolysis media are easily fermentable (for example, into biofuels), and the cost of enzymes (which were reported to account for more than 50% of the total costs20) has been reduced drastically in the past few years.21 * To whom correspondence should be addressed. Tel.: +34988387075. Fax: +34988387001. E-mail: [email protected].

This work deals with the two-step processing of corncobs (autohydrolysis followed by enzymatic hydrolysis). In autohydrolysis experiments carried out at various severities (measured by the final temperature of nonisothermal experiments), the conversion of hemicellulose into XO was assessed, and the corresponding spent solids were subjected to enzymatic hydrolysis. Generalized models describing the effect of the selected operational variables on the enzymatic hydrolysis are developed. 2. Materials and Methods 2.1. Raw Material. Corncob samples collected from local plantations were milled to pass through a 8 mm screen, since in preliminary studies no diffusional limitations were observed for this particle size. Samples were air-dried, homogenized in a single lot to avoid differences in composition among aliquots, and stored. 2.2. Analysis of Raw Material. Aliquots of raw material were ground to particle sizes 99

0.979 37.6 >99

0.992 105.2 >99

0.988 68.0 >99

coefficient

a Coefficients significant at the 99% confidence level based on the Student’s t test. b Coefficients significant at the 95% confidence level based on the Student’s t test. c Coefficients significant at the 90% confidence level based on the Student’s t test.

The results determined for the dependent variables GMAX and t1/2G and the values of the R2 parameter are listed in Table 3, whereas Table 4 shows the values of the regression coefficients, their statistical significance (based in the Student’s t test), and the parameters measuring the correlation (R2) and statistical significance of models (Ficher’s F parameter). The Holtzapple model provided a satisfactory interpretation of individual enzymatic hydrolysis experiments, as can be seen from the R2 values listed in Table 3. On the basis of the values of the t parameter, it can be inferred that the most influential variable on GMAX was LSR, whereas ESR caused the major effects on t1/2G. GMAX achieved its maximum value (GPOT) for almost all the experiments carried out at LSR ) 11 and 17 g/g and varied in the range 57.1% -96.6% of GPOT in experiments performed at LSR ) 6 g/g (experiments 5 and 6 of Table 3, respectively). Figure 3 shows the calculated dependence of GMAX and t1/2G on LSR and ESR for CCR ) 9 IU/FPU. GMAX decreased when LSR increased, whereas ESR showed little influence on this variable. Oppositely, t1/2G was not significantly affected by LSR but decreased strongly with ESR to achieve minimum values in the range 6.1-8.6 h for ESR about 22 UPF/g. Lower values of CCR led to increased values of both GMAX and t1/2G.

3.2.2. Enzymatic Hydrolysis of Spent Solids Obtained from Hydrothermal Treatments at TMAX ) 205, 208, 212, and 216 °C. On the basis of the above findings, the enzymatic hydrolysis of the rest of the spent autohydrolysis solids (obtained in experiments carried out to achieve maximal temperatures of 205, 208, 212, or 216 °C) was assessed by means of simpler factorial designs, involving just the two most influential operational variables (LSR and ESR) and keeping constant the least influential variable (CCR) at 9 IU/UPF. The mathematical interrelationship between the dependent variables (GMAX and t1/2G) and the independent variables (LSR and ESR, or x1 and x2) is given again by eq 2, but the sums are now extended just for i or k from 1 to 2 (k g i), since x3 has been fixed (CCR ) 9 IU/UPF corresponds to x3 ) 1). Table 5 shows the operational conditions assayed in this set of experiments, expressed in terms of the dimensional and dimensionless operational variables, as well as the results determined for the dependent variables and for the statistical coefficient R2. The values of this latter coefficient confirmed the ability of the Holtzapple model for reproducing the experimental data. Table 6 lists the results calculated for the regression parameters, their statistical significance, and the parameters F and

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Figure 4. Calculated dependence of the kinetic parameter t1/2G on LSR and ESR for enzymatic hydrolyses carried out at CCR ) 9 IU/FPU using the spent solids from hydrothermal treatments performed at TMAX of 205, 208, 212, and 216 °C.

R2, whereas Figures 4 and 5 show the calculated dependence of GMAX and t1/2G on LSR and ESR. The values of the regression parameters confirmed that LSR caused the major effects on GMAX and that t1/2G was mainly affected by ESR. The values of t1/2G decreased sharply when ESR increased, with little dependence on LSR. On the other hand, t1/2G decreased for substrates treated at higher severities, particularly in samples obtained at temperatures in the range 208-212 °C. GMAX achieved about 70% of GPOT for ESR g 17 FPU/g and LSR ) 6 g/g in experiments made with the spent solids obtained in treatments at 205 and 216 °C. Under similar conditions, the spent autohydrolysis solids treated at intermediate severities (maximal temperatures of 208 and 212 °C) led to GMAX near GPOT. Near stoichiometric glucose yields were also predicted for spent solids obtained in treatments at 205, 208, and 212 °C operating at LSR ) 11 g/g and ESR g 17 FPU/g, whereas the solid treated at 216 °C presented a comparatively low susceptibity (GMAX in the range 63-69% of GPOT). Operating at the highest LSR (16 g/g), the models predicted

GMAX in the range 94-100% of GPOT, except when the substrate treated at 205 °C was reacted at an enzyme charge of 6 FPU/g. In a related study dealing with corn stover treated with hot water under isothermal conditions at controlled pH for 5-20 min, increasing the treatment temperature from 190 up to 200 °C did not result in improved hydrolysis.32 In this work, the maximum cellulose conversion into glucose (measured by the ratio GMAX/GPOT) led to values near 1 for substrates treated at TMAX in the range 208-212 °C, enabling the production of media with glucose concentrations in the range 80-100 g/L. Comparatively, corn stover subjected to AFEX pretreatment led to cellulose-to-glucose conversions up to 85% after 48 h,33 whereas substrates subjected to acid prehydrolysis (in media containing 1% weight:volume catalyst) allowed almost stoichiometric yields after 96 h of enzymatic hydrolysis.34 3.2.3. Xylose and Acetic Acid Generation during Enzymatic Hydrolysis. Xylose and acetic acid were generated as reaction byproducts during the enzymatic hydrolyses. Xylose was the only byproduct in the experiments made with the spent

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Figure 5. Calculated dependence of GMAX on LSR (liquor to solid ratio) and ESR (enzyme to substrate ratio) for enzymatic hydrolyses carried out at a enzyme activity ratio CCR ) 9 IU/FPU using the spent solids from hydrothermal treatments performed at TMAX of 205, 208, 212, and 216 °C.

autohydrolysis solid treated at 216 °C, revealing total cleavage of acetyl groups during aqueous treatments. 3.2.4. Validation of the Model. The suitability of the generalized calculation procedure developed in this work was checked by plotting the experimental glucose concentrations versus the calculated ones (see Figure 6). About 80% of data were predicted with deviations e10%, and the average of the absolute values of deviations was 0.64 g/L. Figure 6 confirms that the predictions are particularly accurate for glucose concentrations g50 g/L, which correspond to the most interesting conditions for practical applications. 3.3. Comparison of Operational Conditions. The autohydrolysis conditions leading to a maximum oligosaccharide concentration corresponded to the treatment carried out at TMAX ) 202 °C, which led to autohydrolysis liquors containing 23.2 g of oligosaccharides/100 g of oven-dried raw material (see section 3.1). Oppositely, the maximum glucose concentration in the enzymatic hydrolysis step corresponded to the solid treated at TMAX ) 212 °C, operating at LSR ) 6 g/g, ESR ) 28 FPU/

g, and CCR ) 9 IU/FPU (experiment 3 of Table 5), in which 97.2 g of glucose/L, 8.1 g of xylose/L, 11.0 g of arabinose/mL, and 0.8 g of acetic acid/L were obtained. Excluding the contribution of the enzymatic concentrates to the sugar concentrations, the corresponding data are 93.5 g of glucose/L, 5.2 g of xylose/L, and 0.34 g of acetic acid/L. This finding is in agreement with the results reported for the acid prehydrolysis/ enzymatic hydrolysis of corn stover, for which different optima in each step of the process have been reported.35 Hydrothermal treatments at intermediate severity (for example, at TMAX ) 208 °C) could be considered as a suitable compromise for obtaining high amounts of both oligosaccharides and glucose, as harsher treatments (for example, TMAX ) 212 °C) would result in significantly lower oligosaccharide concentrations. Under the conditions of experiment 3 of Table 5 (LSR ) 6 g/g, ESR ) 28 FPU/g, and CCR ) 9 IU/FPU), a solution containing 87 g of glucose/L can be obtained from spent solids obtained in aqueous treatments at TMAX ) 208 °C after 48 h of hydrolysis, with 82% cellulose conversion into glucose. Under

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ethanol by environmentally friendly technologies”, reference PGIDIT06REM38301PR. G.G. thanks the Spanish “Ministerio de Educacio´n y Ciencia” for his “Ramo´n y Cajal” contract. Nomenclature

Figure 6. Comparison of experimental and calculated values of the glucose concentration.

the considered autohydrolysis conditions, 19.5 g of oligosaccharides/100 g of oven-dried raw material (corresponding to 62.7% of xylan conversion) were obtained. 4. Conclusions Hydrothermal processing of corncobs is suitable for obtaning both oligosaccharide-containing liquors and spent solids susceptible to enzymatic hydrolysis. Under selected conditions, up to 23.2 g of oligosaccharides/100 g of oven-dried raw material was obtained in autohydrolysis treatments. When spent solids from autohydrolysis carried out at different severities were employed as substrates for enzymatic hydrolysis, near theoretical cellulose conversion was achieved under a variety of experimental conditions, leading to media containing up to 97.2 g of glucose/L. The glucose concentration/time series of data were fitted to the Holtzapple model, and the kinetic parameters (GMAX and t1/2G) were assumed to be a function of the independent variables considered in the enzymatic hydrolysis step (solid to liquid ratio, enzyme to substrate ratio, and cellobiose activity to cellulase activity ratio). Empirical models were developed to allow a generalized interpretation of experimental data. The susceptibility of spent solids from autohydrolysis increased with the severity of treatments for TMAX in the range 202-212 °C, but harsher treatments resulted in less reactive substrates. The optimal autohydrolysis conditions for producing oligosaccharides (TMAX ) 202 °C) and for obtaining maximum glucose concentration upon enzymatic hydrolysis of the spent solids (TMAX ) 212 °C) were different, but intermediate conditions provide a reasonable compromise: for example, when performing the autohydrolysis step up to a TMAX ) 208 °C, oligosaccharide and glucose concentrations accounting for 62.7% and 82% conversion of xylan and cellulose, respectively, can be obtained. Acknowledgment Authors are grateful to Novozymes for the enzyme supply and to “Xunta de Galicia” for the financial support of this work, in the framework of the Research Project “Production of

AcH ) acetic acid concentration in enzymatic hydrolysis assays (g/L) AcHMAX ) maximum acetic acid concentration; kinetic parameter (g/L) AcHPOT ) potential acetic acid concentration in enzymatic hydrolysis (g/L) Ar ) arabinose concentration in enzymatic hydrolysis assays (g/L) ArMAX ) maximum arabinose concentration; kinetic parameter (g/L) ArPOT ) potential arabinose concentration in enzymatic hydrolysis (g/L) b0j, bij, bikj ) regression coefficients CCR ) β-glucosidase activity/cellulase activity ratio (IU/FPU) ESR ) enzyme to sustrate ratio (FPU/oven-dried g of spent solids) FPU ) filter paper units G ) glucose concentration in enzymatic hydrolysis assays (g/ L) GMAX ) maximum glucose concentration; kinetic parameter (g/ L) GPOT ) potential glucose concentration in enzymatic hydrolysis assays (g/L) IU ) international units of β-glucosidase activity LCM ) lignocellulosic material LSR ) liquid to solid ratio (g of liquid/g of oven-dried solid) NVC ) nonvolatile compounds (g of nonvolatile compounds/ 100 g of dry raw material) SY ) solid yield (g of solid recovered after treatment/100 g of dry raw material) t ) duration of enzymatic hydrolysis (h) t1/2G ) parameter of the Holtzapple model for enzymatic hydrolysis (h) TMAX ) maximum temperature achieved in hydrothermal treatment (°C) VC ) volatile compounds (g of volatile compounds/100 g of dry raw material) X ) xylose concentration in enzymatic hydrolysis (g/L) x1 ) dimensionless liquor to solid ratio x2 ) dimensionless enzyme to substrate ratio x3 ) dimensionless β-glucosidase activity/cellulase activity ratio XMAX ) maximum xylose concentration; kinetic parameter (g/ L) XPOT ) potential xylose concentration in enzymatic hydrolysis (g/L) y1 ) generalized nomenclature for GMAX y2 ) generalized nomenclature for t1/2G Literature Cited (1) Myerly, R. C.; Nicholson, M. D.; Katzen, R.; Taylor, J. M. The forestry refinery. Chemtech 1981, 11, 186. (2) Arato, C.; Pye, E. K.; Gjennestad, G. The lignol approach to biorefining of woody biomass to produce ethanol and chemicals. Appl. Biochem. Biotechnol. 2005, 121-124, 871. (3) Garrote, G.; Domı´nguez, H.; Parajo´, J. C. Interpretation of deacetylation and hemicellulose hydrolysis during hydrothermal treatments on the basis of severity factor. Process Biochem. 2002, 37, 1067. (4) Garrote, G.; Domı´nguez, H.; Parajo´, J. C. Production of substituted oligosaccharides by hydrolytic processing of barley husks. Ind. Eng. Chem. Res. 2004, 43, 1608.

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ReceiVed for reView September 5, 2007 ReVised manuscript receiVed October 12, 2007 Accepted October 19, 2007 IE071201F