Experimental design and optimization

Central composite design (CCD) under Response Surface Methodology (RSM) was applied to optimise SE conditions for the two types of pretreatment. Temperature and residence time were selected as independent variables. The yields of arabinose, xylose and glucose in monomeric and oligomeric form released after pretreatment, and the same sugars in monomeric form after enzymatic hydrolysis, were combined in a single output and referred to as combined sugar yield (CSY). Oligomeric sugars were converted to equivalent monomeric sugars for totalling up CSY. CSY and monomeric glucose (as major sugar released) from EH were considered as the main response variables. The residual acetyl groups in the WIS, and the concentrations of hydroxymethyl furfural (HMF), furfural, acetic acid and formic acid in the pretreatment liquor, were set as constraints for the optimisation process and consequently targeted simultaneously for minimisation.

A two level, two factor full factorial design [12] with four axial points and three replicates at the centrepoint was applied for each of the two types of pretreatment of straw, as well as enzymatic

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hydrolysis of the pretreated material (WIS). The design led to a total number of eleven experiments in all cases. The matrixes of experimental design applied for water impregnated material were adapted to each of the feedstocks according to the results found in the preliminary studies performed with triticale straw and reported in Chapter 6 of this dissertation work. The range of conditions investigated for SO2-impregnation was selected based on studies on wheat straw [13] and corn stover [14]. Table 7-1 provides the experimental conditions, pretreatment severities and resulting pH values after pretreatment for each type of SE pretreatment and feedstock. The pretreatment experiments were performed in a random order. The coded values for the axial, factorial and centrepoints were -1.4142 (at the lowest point) and +1.4142 (at the highest point), -1 and +1, and 0 (zero), respectively as shown in Table 7.1. The uncoded values were calculated according to equation 7-1.

(7-1)

Where Xi is the uncoded value of the independent variable i, Xmin and Xmax are the uncoded minimum and maximum values (corresponding to -1 and +1 coded values), and xi is the code value to be translated. The second order polynomial model described by equation 7-2 was used for predicting the optimal pretreatment conditions.

(7-2)

Where Y is the estimated value of the response; n is the number of independent variables; β0 is an intercept, βi, βii and βij represent the regression coefficients for linear, quadratic and the interaction of two independent variables respectively; Xi, X²i and XiXj refers to linear, quadratic and two-way interaction effects, respectively.

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Table 7-1: Matrix of the experimental designs, and resulting pH values of the slurry and severities for uncatalyzed and SO2 impregnation pretreatments of the feedstocks.

(*) The effects of the operational variables temperature and residence time for water-impregnation and SO2-pretreatments were expressed in terms of the single parameter termed severity factor and combined severity factor, defined by the equations:

and , respectively.

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Predictive models were developed for CSY. Inhibitors formation and CSY were subjected to simultaneous optimisation using a desirability function, whereby the responses were assigned with values from 0 (less desired output) to 1 (more desired output), according to determined benchmarks (in range of the values obtained in the study, maximise, minimise or target a specific value) and the weight allocated to them (0-100%) [15]. The maximum weight (95%) for the optimisation was given to maximise CSY. In addition, the yields of HMF, furfural, formic and acetic acids were totalled and set as threshold value for minimisation, with a weighting of 20%. A residual acetyl group content in the WIS, yielding acetate concentrations below 5 g.L^-1 during a SSF at 20% of total solids, was also preferred. Additionally, the fitted models were statistically validated by conducting five pretreatment experiments at the predicted optimum conditions.

Statistical analysis was performed by Design Expert, version 8.0.2 (State Ease Inc., Minneapolis, United States). ANOVA analysis was also performed by Design Expert to determine the statistical significance of the independent variables on the process responses. Predictive polynomial equations were developed to describe the experimental yield of combined sugars for all of the feedstocks by type of pretreatment, using Matlab^ (Version R2013R). The equations for the CSYs were introduced as continuous functions to represent the contour plots described by the equations into the input range of the experimentation. An uncertainty of 5% (95% of statistical confidence) was set into the programming steps to reproduce graphical representations of at least 95% of the maximum value of the CSY response. Finally, pretreatment conditions in common for all three straw samples, incorporating conditions able to yield CSYs of at least 95% of the maxima observed for individual straw samples, were identified. These conditions were validated by running triplicate runs at severities in the overlapping pretreatment area, by using the M4 straw as control for validation.

7.2.5 Calculations

The effects of the operational variables temperature and residence time for water only pretreatment were expressed in terms of the single parameter termed severity factor [16], defined by the equation (7-3):

(7-3)

Where t is the residence time in min, and T is the pretreatment temperature in C.

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The effects of the temperature, time and acid concentration, in the case of SO2-impregnation pretreatment, were unified in the combined severity factor (CSF) expression [16] calculated by the following expression:

(7-4)

Where Log R’0 is defined by the equation (7.3) and pH corresponds to the pH of the environment in which the pretreatment takes place, often measured in the whole slurry resulting from pretreatment.

The yields of monomeric xylose, glucose and arabinose in pretreatment liquor, as well as in the wash fraction from both types of pretreatment, were calculated on the basis of the amount of material fed into the reactor for comparisons of outputs among pretreatment conditions for each straw. This was expressed as gram of sugar per 100 grams of dry raw material (DRM) by the equation 7-5. The sugar yields in liquor and wash were finally totalled and reported as sugar yields from pretreatment liquor.

(7-5)

Where sugar yield represents the yield of xylose, glucose or arabinose expressed in gram per 100 grams DRM. Sugar concentration is the concentration of the sugar from HPLC analysis (g.L^-1), volume of pretreatment liquor is the total volume obtained after removing the liquor from the whole slurry after pretreatment corrected with the residual MC left in the solids after liquid separation (L), and the denominator of the expression corresponds to the dry mass of the raw material fed into the steam gun unit.

Additionally, the yields were compared to the measured contents of each sugar in the raw material. This was expressed as percentage recovery of the maximum potential sugars present in the feedstock, using equation 7-6, to facilitate comparisons of results among types of pretreatments for each straw. All sugar-oligomer yields were converted to the respective equivalent monomeric sugar using conversion factors of 1.136 and 1.111 for pentoses and hexoses, respectively.

(7-6)