Steam Pretreatment of Rice Hulls to Release Fermentable Saccharides: An Approach to Improve Recovery of (Hemi)Cellulosic Sugars Through Multivariate Design

Sheila Montipó, Christian Roslander, Marli Camassola, Mats Galbe, Ola Wallberg

Research Paper

Steam Pretreatment of Rice Hulls to Release Fermentable Saccharides: An Approach to Improve Recovery of (Hemi)Cellulosic Sugars Through Multivariate Design

Sheila Montipó1, 2, Christian Roslander1, Marli Camassola2, Mats Galbe1, Ola Wallberg1

(Department of Chemical Engineering, Lund University, P. O. Box 124, Lund SE-221 00, Sweden; Biotechnology Institute, University of Caxias do Sul, Caxias do Sul 95070-560, Brazil)

The conversion of rice hulls into fermentable saccharides was explored through steam pretreatment employing 2.5% SO2. The interaction between temperature and time was assessed by means of the response surface method to achieve optimum contents of C6-sugars in water-insoluble solids (WIS) and C5-sugars in the liquor. Pretreatment carried out at 218 oC for 2.3 min released liquor containing 55.4 g/L of sugars (29.1 g/L of xylose). In parallel, the WIS was subjected to enzymatic saccharification using different solid and enzyme loads via an experimental design: assays using 22.0% WIS and 20.0 filter paper units (FPU)/g led to 90.6 g/L of glucose, corresponding to a yield of 86.4% and an overall yield of 72.4%. The data reported are the highest ever found for such raw material, making it attractive to compete with conventional lignocellulosic biomass.

lignocellulosic feedstock; sulfur dioxide; high solid loading; hydrolysis; central composite rotatable design

Current energy consumption is essentially dependent on nonrenewable petroleum-based fuels (Gupta et al, 2019). The energy captured in annually produced biomass by terrestrial plants is 3?4 times greater than the global energy demand, which was 583.9 EJ in 2019 and was predicted to increase to 860.0 EJ in 2040 (Guo et al, 2015; BP, 2020). Lately, much interest has been directed towards biofuels and particularly bioethanol from biomass, which offers several benefits including the mitigation of gaseous emissions besides the diversification of fuel supply, avoiding nonrenewable resources depletion (Abbas and Ansumali, 2010; Quispe et al, 2017).

The biorefinery platform approach aims to optimize the use of renewable lignocellulosic biomass to produce bioenergy, biofuels and bio-derived chemicals, being able to connect different processes and contributing to the short- and long-term sustainability (Kohli et al, 2019; Rosales-Calderon and Arantes, 2019). Many agricultural processing residues have low or no commercial value, but still represent a considerable amount of raw materials that are generally available for conversion without too complicated process designs (Galbe and Wallberg, 2019).

According to the Food and Agriculture Organization of the United Nations (FAO), the latest forecast for global rice production (milled equivalent) in 2019/ 2020 is subject to reaching a record of 5.19 × 108t, up 1.3% year on year and resulting in a 0.5 kg annual expansion in the per capita intake (FAO, 2019). During production of milled rice, large quantities of rice hulls (RH) are produced as by-product (nearly 20% of dry weight), indicating that this agro-industrial waste is definitely an abundant resource for the production of adsorbents, biofuels and value-added products such as chemicals, enzymes, food, textiles, bioplastics and pharmaceuticals, although most RH is underutilized or left unused (Huang and Lo, 2019; Zou and Yang, 2019). Furthermore, pyrolysis has been proposed as a process of utility of this by-product, resulting in ashes abundant(Soltani et al, 2015).

Attributing to its recalcitrant nature, direct conversion of untreated RH usually results in low production yield (Ang et al, 2013). Numerous natural factors are considered to contribute to the recalcitrance of the lignocellulosic feedstocks to chemicals or enzymes, including the epidermal tissue of the plant body, the arrangement and density of the vascular bundles, the relative amount of the sclerenchymatous tissue, the degree of lignification, the structural heterogeneity andcomplexity of the cell-wall constituents, the challenges for enzymes acting on an insoluble substrate and the inhibitors for further fermentation (Himmel et al, 2007). Consequently, several methods have been employed for pretreating RH. Among them, physico- chemical pretreatment carried out by impregnating the RH with alkalis or acids (e.g., sulphuric, hydrochloric, phosphoric, acetic and nitric acids, and sodium and calcium hydroxides) in a pressurized reactor stands out due to the cost-benefit (Ang et al, 2013; Montipó et al, 2016; Temiz and Akpinar, 2017; Pedroso et al, 2019). Very few studies have used one of the most promising technologies, steam pretreatment, for RH (Pi?eros-Castro et al, 2011; Wood et al, 2016). Typically, the existing studies are mainly concentrated on auto- hydrolysis without addition of chemical catalysts (Schultz et al, 1984; Wood et al, 2016; Tabata et al, 2017; Montipó et al, 2019, 2020).

Nonetheless, it is observed that the results available in the literature from the pretreatment step for RH are still very incipient, even with the use of catalysts, since the released sugars are present in a very low amount, or are partially or totally degraded due to high applied temperatures and/or high concentrations of chemicals, affecting the subsequent enzymatic hydrolysis (EH) and fermentation. Thereby, it is not possible to compete with traditional lignocellulosic biomass to produce biofuels and other bioproducts. Additionally, most research focuses only on the recovery of glucans in the solid fraction, not involving a balance study including xylans in the liquid fraction, thus enabling multiple subsequent applications of the raw materials, important for the construction of chemical building blocks.

In view of the RH recalcitrance and buffering capacity (Montipó et al, 2020), the main concern of this study was to make RH a more attractive biomass with application in biorefineries. The use of SO2- catalyzed steam pretreatment was investigated to obtain improved yield of fermentable saccharides since this technique has shown to be promising for distinct lignocellulosic feedstocks (Carrasco et al, 2010). To the best of our knowledge, there is no previous study involving the impregnation of RH with sulfur dioxide and subsequent use of steam pretreatment. Therefore, an experimental design was established aiming for a high recovery of hemicellulosic and cellulosic sugars. Solid and liquid fractions were separated and characterized, and the first one was also used for the study of EH using high solid content.

RESULTS

Macromolecular compositions of untreated RH, as percentage of dry weight, were found to be: extractives(5.6% ±0.5%), cellulose (31.5% ±0.3%), hemicellulose (20.6% ±0.1%), total lignins (23.2%±0.4%), ashes (16.0%±0.1%) and acetyl groups (1.8%±0.0%).

Steam pretreatment: Chemical composition conversion analysis

Water-insoluble solids (WIS)

The compositions of the pretreated RH, with coded and real values, are detailed in Table 1, as a percentage of dry weight. Generally, it was evidenced how the SO2- steam pretreatment favoured the increase of cellulose content, ranging from 39.2% to 45.1%, while contributing to the solubilisation of hemicellulose-derived sugars, since such fraction has been reduced to 2.6%.

As the sugars contained in the WIS were present as oligosaccharides, an EH was performed using the following conditions: 5% solids loading and an enzyme dosage of 15.0filter paper units(FPU)/g substrate (Table 1). Effective EH of pretreated RH was influenced by the operating conditions of the steam pretreatment, particularly temperatures above 218 oC (Pretreatments 2, 4 and 6), reaching yields in the order of 90.3%.

Liquor

Sugars formed during steam pretreatment and released in the filtrate are listed in Table 1. Xylose was in greater amounts in this fraction [Pretreatments 1?3, 5 and the central point (Pretreatments 9?11)], and experiments applying mild conditions were responsible for up to 30.4 g/L. At higher temperatures, maximum glucose levels were in the range of 18.6?19.5 g/L (Pretreatments 2, 4 and 6). Pretreatments 2, 3 and the central point provided high concentrations of xylose and glucose, reaching up to 48.5 g/L.

Table 1. Rice hull compositions obtained after SO2-catalyzed steam pretreatments.

The numbers in the brackets correspond to the values used in the experimental design (Central Composite Rotational Design), which are always fixed when there are two independent variables. In the present study, a planning 2=2was developed: 2 levels and 2 independent variables, with 4 combinations, 4 axial points (-1.41, +1.41) and 3 central points (0), totaling 11 pretreatments. Axial points were calculated as follows: α = ± 2/4, α = ± 1.41.Acid-insoluble lignin.cDetermined at 5% water-insoluble solids with 15.0 filter paper units (FPU)/gsubstrate.Expressed as percentage of the potential glucose in the pretreated raw material. 5-HMF, 5-hydroxymethylfurfural.

These data are mean values of two or three assessments.

The major degradation end-products found in the liquor are also reported in Table 1, and their presence was dependent on the applied variables: temperature and time. Pretreatments 4 and 6 contained the highest percentages of interfering compounds, reaching around5.0 g/L of acetic acid, 2.0 g/L of 5-hydroxymethylfurfural(5-HMF) and 4.0 g/L of furfural, these being related to more severe conditions. The other pretreatments produced moderate or lower amounts of by-products.

Total sugar recovery

Recovery of glucan and xylan is illustrated in Fig. 1, represented as percentage of the initial components in the RH. Generally, it was observed that the glucan loss rates were lower than those for xylan. Both glucan recovery from the solid fraction (GR-WIS) and xylan recovery from the liquid fraction (XR-LIQ), the main focuses of interest were evidenced. Most of the glucan was recovered in the WIS, with the recovery rates varying from 62.5% to 85.2%, and temperatures of 205 oC or lower favoured its recovery. The recovery rates of XR-LIQ ranged from 23.7% to 54.2% and the pretreatments at 205 oC for 5.0 min (central point), or 8.9 min (Pretreatment 8), as also Pretreatments 2 and 3, increased the recovery.

Pretreatment 8 was responsible for the total recovery of glucan in solid and liquid fractions, and Pretreatment 3 for 63.0% of xylan recovery. When the temperature was at the positive axial point, maximum losses were found: around 19.0% for glucan and 73.2% for xylan.

Analysis of overall sugar yields through multivariate design

For estimating the efficiency of the whole process, the overall yields of glucose and xylose, including the soluble sugars obtained after EH (5.0% of WIS and 15.0 FPU/g substrate of cellulase), are revealed inFig. 2. The released sugars in each fraction were presented as a percentage of the sugar values in the RH. Overall glucose yields reached 86.3%, and higher temperatures of 218 oC and 227 oC resulted in higher values. Overall xylose yields achieved 65.9% at 192 oC for 7.8 min (Pretreatment 3), and Pretreatments 2, 8 and the central points were also suitable.

Fig. 1. Sugar recovery yields obtained using different catalyzed steam explosion conditions.

GR-LIQ, Glucan recovery in the liquor; GR-WIS, Glucan recovery in the solid fraction; XR-LIQ, Xylan recovery in the liquor; XR-WIS, Xylan recovery in the solid fraction. 1?11 represent the pretreatment assays at 192 oC for 2.3 min, 218 oC for 2.3 min, 192 oC for 7.8 min, 218 oC for 7.8 min, 183 oC for 5.0 min, 227 oC for 5.0 min, 205 oC for 1.1 min, 205 oC for 8.9 min, 205 oC for 5.0 min, 205 oC for 5.0 min, 205 oC for 5.0 min, respectively.

In the case of glucose in the solid fraction (G-WIS), the maximum yields were around 68.2% using 218 oC for 2.3 min (Pretreatment 2). As for xylose in the liquor (X-LIQ), the maximum yield was 61.2% (central point), and Pretreatments 2 and 3 also reached similar values. Pretreatment 2, which used a higher temperature for a short time, as well as pretreatments at the central point, contributed to the effectiveness of the overall process, reaching up to 74.8 g/L of saccharides based on G-WIS and all of the sugars released in the liquor (Table 1). When RH was subjected toPretreatment 2, for example, 330.0 g sugars per kg RH were released (236.0 g G-WISand 94.0 g X-LIQ, equivalent to 682.0 g glucose per kg glucose present in the untreated RH and 551.0 g xylose per kg xylose in the untreated RH, respectively).

The models in equations (Overall yield(G-WIS)= 60.9 +8.5 ×; Overall yield(X-LIQ)= 57.2 – 6.9 ×? 6.8 ×2)were proposed to represent the optimal settings for overall sugar yields, G-WIS and X-LIQ. Only significant coefficients (<0.05) were included, depending on temperature () and time ().

Pareto diagrams in Fig. 3 supported the above equations and reported the results of the-test for each variable. The values of the variables that maximize the total sugar yields of G-WIS and X-LIQ, concomitantly, were done through the desirability method (Fig. 4-A). In accordance with analysis of variance (ANOVA), results were properly explained by the models once the calculatedvalues were higher than the referencevalues: for G-WIS,= 62.8 (1, 9; 0.05= 5.12;2= 87.5%), while for X-LIQ,= 25.6 (2, 8; 0.05= 4.46;2= 86.5%) (Table S1). The contour curves shown in Fig. 4-B and -C exemplified such behaviours as the percentage of each sugar liberated according to the original sugar content in the feedstock.

Fig. 2. Overall sugar yields from catalyzed steam pretreatment followed by enzymatic hydrolysis of solid fraction.

G-WIS, Glucose in the solid fraction; X-LIQ, Xylose in the liquor; G-LIQ, Glucose in the liquor; X-WIS, Xylose in the solid fraction. 1?11 represent the pretreatment assays at 192 oC for 2.3 min, 218 oC for 2.3 min, 192 oC for 7.8 min, 218 oC for 7.8 min, 183 oC for 5.0 min, 227 oC for 5.0 min, 205 oC for 1.1 min, 205 oC for 8.9 min, 205 oC for 5.0 min, 205 oC for 5.0 min, 205 oC for 5.0 min, respectively.

Fig. 3. Pareto chart of standardized effects for rice hulls in terms of temperature (T) and time (t) for overall yield responses of glucose released in solid fraction (A) and xylose released in liquor (B).

L, Linear variable; Q, Quadratic variable.

Fig. 4.Response surface method and contour curves.

A, Response surface method using the desirability function to maximize yields in a single-step.

B and C, Contour curves comprising the interactions between temperature and time for overall yields of glucose released in the solid fraction (B) and xylose released in the liquor after hydrolysis (C).

Experimental design of enzymatic saccharification

Based on the data above, WIS resulting from Pretreatment 2 (218 oC for 2.3 min) was used for the study of EH considering high load of solids (12.0% to 22.0%) combined with different cellulase doses (13.0 to 27.0 FPU/g), promoting the release of G-WIS. The average results of these assays, in terms of concentrations and yields, are given in Fig. 5.

For all equations (Glucose concentration (g/L) = 70.0 + 13.3 ×+ 4.4 ×;Glucose yield (%) = 86.4 + 5.2 ×; Overall glucose yield (%) = 72.4 + 4.4 ×), only significant coefficients (<0.05) were included, depending on solid loads () and enzyme loads (). Thereby, with a load of 22.0% WIS, it was possible to attain up to 90.6 g/L glucose (Fig. 5-A) employing 20.0 FPU/g pretreated RH within 48 h, corresponding to a yield in the order of 86.4% (Fig. 5-B) and an overall yield of 72.4% (Fig. 5-C), both being satisfactory and making RH attractive to compete with conventional lignocellulosic biomass.

It is noticeable that, by rising the percentage of solids, there was a decrease in yields, and this characteristic was remarkable mainly in the first 24 h (Fig. 5-B and -C). However, this variable was not significant for estimating the effects, and the yields were dependent only on the enzyme load. In both models, thevalues were around seven times superior to the referencevalue (1, 10; 0.05= 4.96). In the case of glucose yield, there was an increase of approximately 10.5% more when the enzyme load was changed from 13.0 to 27.0 FPU/g WIS. As for the global glucose yield, this increase represented 8.8% more.

Fig. 5. Time course of glucose concentration (A) and yields (B and C) obtained after 96 h of enzymatic hydrolysis at different solid loadings and enzyme dosages.

1, 13.5% water-insoluble solids (WIS) and 15.0 filter paper units (FPU)/g; 2, 20.5% WIS and 15.0 FPU/g; 3, 13.5% WIS and 25.0 FPU/g; 4, 20.5% WIS and 25.0 FPU/g; 5, 12.0% WIS and 20.0 FPU/g; 6, 22.0% WIS and 20.0 FPU/g; 7, 17.0% WIS and 13.0 FPU/g; 8, 17.0% WIS and 27.0 FPU/g; 9, 17.0% WIS and 20.0 FPU/g; 10, 17.0% WIS and 20.0 FPU/g; 11, 17.0% WIS and 20.0 FPU/g; 12, 17.0% WIS and 20.0 FPU/g.

DISCUSSION

In addition to the recognized recalcitrance of RH due to the high percentages of lignin and ash, the elevated contents of the ash and extractives contribute significantly to the pH-buffering capacity of this raw material, which will directly affect the subsequent pretreatment efficacy (Montipó et al, 2020). In view of the intrinsic characteristics of this feedstock, combined with the lack of effectiveness of the pretreatments, a new approach was necessary to increase the accessibility to hemicellulose.

Steam pretreatment catalyzed by sulfur dioxide for rice hulls

In an attempt to work around this challenge and make RH with more promising applications, steam pretreatment catalyzed with SO2is an appropriate strategy. This technique has been shown to be effective for the pretreatment of agricultural and woody biomasses, although it is recognized that different assay conditions are needed to treat each type feedstock (Bura et al, 2009).

It was decided to alter the moisture content by pre-soaking the material in warm water and, subsequently, impregnates it with sulfur dioxide. It is known that SO2uptake and effectiveness can be improved by saturating biomass void volumes with water (Ewanick and Bura, 2011).Additionally, it was evidenced that up to 3.0% SO2, there is a very pronounced effect of gas impregnation when temperature and time are constant, achieving high yields of glucose and xylose if combined with EH (Clark et al, 1989; Tao et al, 2011). Based on this, the SO2concentration was fixed to carry out an experimental planning exploring different temperature and time conditions in order to increase yields for both C6-sugars in the solid fraction and C5-sugars in the liquid fraction through steam pretreatment.

The current results (Table 1) were more promising than those obtained through non-catalyzed steam pretreatment concerning RH (Montipó et al, 2020). The SO2pretreatment process converted most of the hemicellulose carbohydrates in the feedstock to monosaccharides and oligosaccharides by hydrolysis reactions, which can be further hydrolyzed or directly fermented.In addition, the removal of hemicellulose from microfibrils exposed the crystalline cellulose core. Glucan in the hemicellulose, as well as a small fraction of the cellulose, are converted to glucose, but most glucan remains in an insoluble form that requires posterior EH (Himmel et al, 2007; Tao et al, 2011). As a result, losses of solid fraction were associated with changes in the lignocellulosic structure (Table 1).

An EH was performed using 5.0% solid loading and an enzyme dosage of 15.0 FPU/g, and this strategy was for assessing the pretreatment method. In reality, much higher WIS is necessary to reach high enough sugar concentrations. The maximum amount of glucose released (Table 1) appeared to be lower when compared with traditional lignocellulosic biomasses, such as sugarcane bagasse, corn stover and wheat straw. However, it should be noted that RH is a much more complex material than those mentioned, and the yields obtained for some of the pretreatments were very prosperous when compared with the data contained in the literature for the same feedstock. Tabata et al (2017) pretreated 200 g of RH in a 2-L steam apparatus using different conditions. WIS was enzymatically hydrolyzed by ONOZUKA R-10 containing hemicellulasesin a shaker at 42 oC. A total of 0.2 g of steam-pretreated RH was soaked in 5 mL of 50 mmol/L sodium acetate buffer and autoclaved; after which, 5 mL of enzyme (5 g/L of 50 mmol/L sodium acetate buffer) was added and incubated. The pretreatment at 236 oC for 5 min, and subsequent enzymatic saccharification, provided 6.5 g/L of glucose and 7.9 g/L of reducing sugars (Tabata et al, 2017).RH was also steam-pretreated by Wood et al (2016) using a 35-L reactor charged with 500 g feedstock. The pretreated biomass (220 oC, 10 min) was employed for the enzyme loading study using 5% of WIS digested with Cellic?CTec2, at 50 oC for 96 h in an orbital shaker. The pretreated material required a higher loading in excess of 10 FPU/g dry matter, reaching a glucose yield of around 60% (maximum theoretical) (Wood et al, 2016).

The concentration of 55.4 g/L (Pretreatment 2) of total fermentable saccharides (glucose, xylose, galactose, arabinose and mannose) released directly into the liquor after SO2-steam pretreatment was approximately five times higher than that obtained by Montipó et al (2019), in an autohydrolysis process of RH (205 oC, 11.5 min) using a steam reactor without considering the post-hydrolysis step. Pedroso et al (2019) carried out a series of pretreatments with diluted acids (sulfuric, hydrochloric, nitric and phosphoric) for RH, including pressurized reactor, laboratory oven, microwave, varying concentrations of acids, temperature and time. The most promising result occurred at the condition of 6.0% H3PO4, 135 oC and 62 min, utilizing a Berghof reactor, where 21.0 g/L of sugars (glucose, xylose and arabinose) were produced. In parallel, Ewanick and Bura (2011) pointed out that soluble glucose in the liquor was increased by 56%?170% in samples processed with SO2following soaking, compared to dry lignocellulosic biomasses.

Overall sugar yields

The determination based on the potential sugars of each pretreated fraction resulted in a glucose yield of 81.4% and a xylose yield of 92.9%. In a previous study carried out with corn stover, a commonly used biomass for the production of second generation ethanol due to its simple conversion of sugars during the pretreatment step, when compared with RH, for example,it was concluded that in the steam pretreatment (190 oC, 5 min) using SO2as a catalyst, a high overall yield of both glucose (87%) and xylose (78%), after EH, can be achieved. It was found that high temperature and short time in the pretreatment step favor a high glucose yield; in contrast, low temperature and long time favor a high xylose yield (?hgren et al, 2005).

To obtain a single pretreatment step for RH to combine satisfactory values of G-WIS and X-LIQ, after the hydrolysis action, an experimental design was developed to verify the interaction between the independent variables during the steam pretreatment catalyzed with SO2. For G-WIS, there was an increment in the overall yield when the temperature, the unique significant variable, migrated from -1.41 to 1.41. For the case of X-LIQ, the consecutive increase in temperature, linear and quadratic, had a negative effect on its global yield.

The desirability function involves transformation of each estimated response variable, to a desirability valued, where 0 ≤d≤ 1. The value ofdraises as the desirability of the corresponding response increases. The individual desirabilities are then combined using a geometric mean, wherevalue gives the overall assessment of the desirability of the combined response levels (Derringer and Suich, 1980). The profiles for predicted values (data not shown) and desirability (Fig. 4-A) revealed that the optimized pretreatment would be precisely for the central point (205 oC, 5 min), indicating that the maximum yields of 68.2% and 61.2% would be found in G-WIS (Pretreatment 2) and X-LIQ (Pretreatment 3), respectively. Pretreatment 2 and central points revealed a similar susceptibility to the recovery of cellulose contained in WIS, as well as hemicellulose reduction. However, the EH performed initially with WIS was facilitated in comparison to the pretreatments of the central point, resulting in higher yields. Concomitantly, Pretreatment 2 exhibited a greater tendency to recover sugars in the liquor.

Enzymatic hydrolysis with high solid load

The gradual rise in solid concentration is directly proportional to glucose production, much more than the increase related to enzyme dosages (Fig. 5-A). The augmentation in monomeric glucose concentrations based on the WIS contents was meaningful for all enzyme loads. The Central Composite Rotational Design(CCRD) employed aided to understand the results at 96 h, where the estimated effects for WIS and enzyme loads were significant (linear) in the study,however, the influence of the solids was responsible for boosting glucose concentration. In line with ANOVA,value was higher than the referencevalue, and2= 97.2%, again proving the fit of the model on the parameters (Table S1). The results of the effects indicated that an increase from -1.41 to 1.41 in the solid and enzyme loads caused an augment of at least 26.6 g/L and 8.7 g/L, respectively, in the glucose concentration.

The glucose obtained after EH carried out with a high load of WIS became more concentrated, a fact that must be decidedly considered for a subsequent biotechnological conversion process. Both monomeric glucose concentrations and yields achieved in the present study were higher than that involving a steam pretreatment without catalyst (20% solid loading and an enzyme dose of 20.0 FPU/g pretreated RH), representing an increase of until 253% and 220%, respectively (Montipó et al, 2019). Ewanick and Bura (2011) also evidenced that both sugarcane bagasse and switchgrass require SO2for an effective steam pretreatment. Six pretreatment processes (ammonia fiber expansion, dilute acid, lime, liquid hot water, soaking in aqueous ammonia and sulfur dioxide- impregnated steam) were compared by Tao et al (2011) to convert switchgrass into fermentable sugars and cellulosic ethanol, consistently, in a technoeconomic analysis. The SO2-steam pretreatment resulted in the highest overall monomer sugar yields and the highest ethanol yield per dry ton of raw material.

The soaking of RH for subsequent impregnation with SO2, followed by steam pretreatment, showed the functionality of the process. The multivariate design established the gradual increase in temperature as positively influencing the pretreatment. The recoveries of total carbohydrates in solid and liquid fractions were elevated, contributing to the overall yield of the process. The CCRD related to enzymatic saccharification also revealed the influence of the increase in the load of solids. Pretreatment carried out at 218 oC for 2.3 min, followed by enzymatic saccharification with 22% WIS was selected as the optimized condition of the process, resulting in 146.0 g/L of total sugars (solid and liquid fractions). Ultimately, it was possible to obtain high levels of G-WIS and X-LIQ from a one-step steam pretreatment, making the RH liable to conversion into further biotechnological processes. To the best of our knowledge, there are no reports in the literature of values of this order for such raw material. Even though the purchased cost of SO2is relatively high compared to other pretreatments, onsite SO2production is believed to be more economical in a large-scale cellulosic ethanol process and should be evaluated in future processes (Tao et al, 2011).

METHODS

Chemicals

The reagents and analytical standards used were: 5-HMF (99%), levulinic acid (98%), sodium hydroxide (50%), xylitol (100%) (Sigma Aldrich, Steinheim, Germany); arabinose (100%), ethanol (96%), furfural (> 99%), glacial acetic acid, glycerol (99.5%), lactic acid (90%) (VWR, Radnor, USA); calcium carbonate (100%), cellobiose (100%), citric acid monohydrate, formic acid (98%?100%), galactose (100%), glucose anhydrous (Merck, Darmstadt, Germany); ethanol (70%) (Solveco, Rosersberg, Sweden); mannose (99%) (Alfa Aesar, Heysham, the United Kingdom); sulfuric acid (72%), xylose (100%) (PanReac Applichem, Barcelona, Spain); and sulfur dioxide (100%) (Linde Gas, Solna, Sweden).

Steam pretreatment of rice hulls

RH was supplied by Volkmann Food, located in Sentinela do Sul/RS, Brazil. A total of 500.0 g dry matter (9.0 mm × 1.5 mm) was soaked with 10 L warm distilled water, and one day later it was filter-pressed using a Tinkturenpressen HP5M hydraulic press (Fischer Maschinenfabrik, Neuss, Germany). After assessing moisture content, the retained solid was transferred to a plastic bag and impregnated with 2.5% of SO2(amount based on the water content of wet RH) (Frankó et al, 2019) for about 30 min at room temperature (approximately 70% of the added SO2was absorbed). Pretreatments were performed in a steam pilot unit operated in batches, with a vessel volume of 10 L (Palmqvist et al, 1996). The impregnated RH was transferred to the reactor, which was directly heated to the desired temperature for a specific period with saturated steam. An experimental design was planned in order to attain the highest release of C6-sugars from WIS and C5-sugars from the liquor. Based on the previous experience with RH (Montipó et al, 2020), temperatures at 183 oC to 227 oC and holding time at 1.1 to 8.9 min were selected. A two levels and two independent variables (22)CCRD comprised these independent variables with three central point repetitions to inform about the behavior of the responses among the levels initially attributed to the factors and the quality of the process repeatability. The whole slurry was recovered in a collecting vessel, cooled and vacuum filtered to recover the solid and liquid fractions. The combined severity factor log(′0) based on temperature (, oC), reaction time (, min) and final pH was considered to analyze the severity of each experiment (Pedersen and Meyer, 2010):

Pretreated RH was washed with deionized water (1:3) at room temperature before compositional analysis and EH, whereas sugars and interfering compounds were quantified in the liquor. For this latter, a mild acid hydrolysis with 4% H2SO4at 121 oC for 60 min was carried out for the yield calculations (Sluiter et al, 2010). The overall yields of fermentable saccharides were selected as the dependent variables and the data were evaluated using Origin 8.1 (OriginLab, Northampton, Massachusetts, USA) and Statistica 8.0 (StatSoft, Inc., Tulsa, Oklahoma, USA) softwares.

Enzymatic hydrolysis

For all pretreatments, 5% RH-WIS was hydrolysed by Cellic?CTec2 (activity of 204.0 FPU/g) (Novozymes, Bagsv?rd, Denmark) using 15.0 FPU/g substrate in 20 mL suspension containing 50 mmol/L of sodium citrate buffer at pH 4.8. The flasks were incubated in a rotatory shaker (Lab-Therm/Lab-Shaker, Adolf Kühner AG, Birsfelden, Switzerland) at 50 oC and 150 r/min for 72 h. Hydrolysis was terminated by heating the hydrolysate in sealed tubes at 100 oC for 10 min. Based on the results from these initial experiments, the most suitable process was chosen to carry out a 22CCRD using different solid loadings (12% to 22%) and enzyme dosages (13 to 27 FPU/g) in citrate buffer to a total of 25 mL. The experiments were carried out in a rotary drum incubator (Hybridization Incubator combi-H12, Kreienbaum, Langenfeld, Germany) at 50 oC and samples were withdrawn at regular intervals for 96 h. The assessment of EH was based on glucose yield, which was related to grams of glucose released by EH per 100 g potential glucose present in the WIS. Overall glucose yield was related to grams of glucose released per 100 g glucose present in the RH. The data were evaluated using Origin 8.1 and Statistica 8.0 softwares.

Analytical methods

The chemical compositions ofand pretreated RH were determined according to the Laboratory Analytical Procedures techniques stated by the National Renewable Energy Laboratory (NREL, Golden, USA) for the standardisation of analytical methods for biomass (Sluiter et al, 2010).

Analytes were quantified via high performance liquid chromatography (HPLC) through a Shimadzu system (Shimadzu Corporation, Kyoto, Japan) coupled with a refractive index detector RID-10A, and equipped with LC-20AT pump, DGU- 20A3degasser, SIL-20AC auto sampler and CTO-20AC column oven. Carbohydrates were analyzed with a CarboSep CHO 782 column (Concise Separations, San Jose, CA, USA), at an oven temperature of 70 oC, and deionized water as mobile phase at 0.6 mL/min flow (Fig. S1). When necessary, samples were pH-adjusted to 5.0 with CaCO3. Organic acids and by-products concentrations were determined using an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, USA), at an oven temperature of 50 oC, and an eluent contained 5 mmol/L of H2SO4at a flow rate of 0.5 mL/min. Elution was in isocratic mode and the injection volume was 20 μL for all analysis.

ACKNOWLEDGEMENT

This study was supported by Coordination for the Improvement of Higher Education PersonnelFoundation, Ministry of Education in Brazil (Grant No. POS-DOC-88881.170076/2018-01).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1.Chromatograms obtained from different treatment samples.

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26 July 2020;

5 January 2021

Sheila Montipó (smontipo@ucs.br)

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http://dx.doi.org/10.1016/j.rsci.2021.07.010

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