Strategies for Fermentable Sugar Production by Using Pressurized Acid Hydrolysis for Rice Husks

Giovanni B. Pedroso, Michael R. Philippsen, Loisleini F. Saldanha, Raiara B. Araujo, Ayrton F. Martins

Strategies for Fermentable Sugar Production by Using Pressurized Acid Hydrolysis for Rice Husks

Giovanni B. Pedroso, Michael R. Philippsen, Loisleini F. Saldanha, Raiara B. Araujo, Ayrton F. Martins

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This study investigated the use of leftover biomass (rice husks) as the raw material for the biotechnological production of platform chemicals and biopolymers. Following the biorefinery concept, different acid hydrolysates were studied and resulted into a wide range of treatment strategies. Chemometrics were applied throughout the procedures in multivariate experimental conditions. By using the best hydrolytic conditions of 6.0% H3PO4, 135 oC (45 MPa) and reaction time of 62 min, 21.0 g/L sugar hydrolysates were produced; by using the best hydrolytic condition of 4.5% HNO3, 135 oC/35 min, 16.1 g/L sugar hydrolysates were produced; and with the hydrolysates use of 1.5% H2SO4and 1.5% HCl,135 oC/62 min, 18.2 and 17.8 g/L sugar hydrolysates were produced, respectively. The highest productivity, in terms of fermentable sugars, reached 68% of integral cellulose/hemicellulose fraction and surpassed those found in the literature, with regard to the processing of rice husks, by considering just one step process. Sulfuric hydrolysate, detoxified with active carbon, was used to prove this proposal viability, resulting in a fermentation substrate for.(ATCC10020) and.(LMG196) strains (natural producers of bioproducts), which certified the feasibility of the proposal. The production of fermentable sugars from leftover biomass should encourage a search for new bioconversion routes, which can result in economic and environmental benefits and a spread of knowledge.

biorefinery; multivariate design; pressurized acid hydrolysis; rice husk; sugar

The employment of renewable lignocellulosic materialsas feedstock for biotechnological conversion is integrated with the biorefinery concept. Currently, this is a fundamental issue, which has attracted a great deal of attention worldwide and required considerable resources. Each renewable process, which involves generated chemical inputs, excluding petrochemicals, has been strongly supported owing to its energy savings, as well as its environmental and economic benefits (Balagurumurthy et al, 2014; Choi et al, 2015).

Nowadays, new production channels are looking for affordable bio-feedstock and a favorable investment/ productivity ratio (Cheali et al, 2015; Arefieva et al, 2017). Provided it does not compete with food production, the lignocellulosic biomass has a great potential value, since it undergoes a cracking in the refractory matrix, which allows its components to be extracted (Menon and Rao, 2012; Kumari and Asthir, 2016; Rabemanolontsoa and Saka, 2016).

The promising leftover biomass rice husks (RHs) are the subproduct of rice processing, which is a basic foodstuff for half of the world’s population. RH possesses a significant economic value since it makes up 20% of the weight of the unrefined grain and still does not have any other purpose. Brazil, which is a rice-exporting country, produced 1.163 × 107t rice grain in 2017 and 2018, and 70.9% of this production was grown in the State of Rio Grande do Sul (CONAB, 2018). In the harvest season alone, more than 1.65 × 106t RHs are disposable, and it is an economic priority to find a use for this abundant lignocellulosic biomass, as a means of meeting environmental challenges and creating added value (Bevilaqua et al, 2013, 2015; Pedroso et al, 2017).

In general, RH is an unwanted by-product, apart from its appreciable content of carbohydrates (37.1% cellulose, 19.5% hemicellulose and 17.6% lignin), which can promise economic returns (Ng et al, 2015). However, before it can serve as a source of fermentable sugars, physicochemical, enzymatic and/or chemical action must be taken, in particular, the employment of acid hydrolysis (Dagnino et al, 2013), which continues to yield the best results (de Vasconcelos et al, 2013). Even though hydrolysis is not a new technique, new strategies are continually arising, owing to demands for a more efficient conversion of hydrolysate carbohydrates (Timung et al, 2015; Demirel et al, 2018).

The most widely used acid hydrolysis processes make use of either diluted sulfuric or hydrochloric acids (1%–10%), since they can effectively convert lignocellulosic materials into several monosaccharides (e.g. glucose, xylose and arabinose) under conditions of relatively low severity (Ang et al, 2013; Chen et al, 2018; Demirel et al, 2018). The use of HNO3and H3PO4in RH hydrolytical processes has not been sufficiently exploited, as their residuals can provide benefits during the fermentation and downstream stages by acting as important nutrients, and, in the case of phosphoric acid, generating fewer inhibitors (de Vasconcelos et al, 2013). The detoxification procedure, which follows the hydrolysis, is carried out more easily since there is a lower concentration of interferers, like furfural, or acetic and formic acids (Lenihan et al, 2010). The use of microwaves (MW) can improve the hydrolytical reactions and raise the yield of fermentable sugars without influencing the formation of interferers (Germec et al, 2017; Chen et al, 2018).

In view of this, the use of RH as a sustainable source of cellulose and hemicellulose can be beneficial,since it represents both a means of alleviating problems caused by the illicit release in the environment, as well as a useful alternative to the production of high-value platform chemicals, mostly through fermentation processes (Lenihan et al, 2010; Ang et al, 2013). Thus, the strategy here was to exploit the best chemical conversion of the leftover RH in hydrolysates with highly fermentable sugar concentration. The strict employment of chemometrics was vital to define the experimental conditions required for the best techno-economic result, for the bioconversion of RH into added value products, while also avoiding the use of nutritional supplements.

MATERIALs AND METHODS

Reagents

All the reagents used were of analytical or superior grade, and included the following: hydrochloric, formic, nitric, sulfuric and phosphoric acids (Merck, Darmstadt, Germany), glacial acetic acid (J. T. Baker, Xalostoc, Mexico), yeast extract and nutrient broth (Acumedia, Lansing, USA), activated carbon (≥ 99%, Exodo, Hortolandia, SP, Brazil), anhydrous D-glucose (96%), arabinose (≥ 98%), xylose (≥ 99%), mannose (≥ 98%), fructose (≥ 98%), sodium hydroxide and deuterium oxide from Sygma-Aldrich (St. Louis, MO, USA). The solutions injected in the chromatographic system were prepared with ultrapure water (MerckMillipore, Billerica, MA, USA), and acetonitrile was high performance liquid chromatography (HPLC) grade (St. Louis, MO, USA).

Experimental design and data analysis

The multivariate technique used for the hydrolytical stage comprised a Central Composite Rotational Design (CCRD) with three factors (concentration, time and temperature), and involved 17 experiments (Supplemental Table 1), 3 of which were at the central point (designed as 15, 16 and 17). The levels of each factor (-α, -1, 0, 1, α) are listed in Table 1 for each stage of the procedure.

The multivariate approach ensures confidence in the data generated, since it entails evaluating the largest sampling space possible, and reducing reagent usage and time wasted. The values found for the response to each factor were analyzed according to the least- squares regression method before employing.

Table 1. Central composite rotational design values for yield determination of glucose,xylose and arabinose through acid hydrolysis of rice husks.

whereis the predicted response of the model;1,2, ..., xare the codified values of each experimental value;0is the intercept;1, ...,βare the linear factors;11,22, ...,βare the quadratic factors;12,23, ...,(n-1)nare the interactions between the coefficients;is the random error with mean zero and2. The error limits were defined as half of the range of the confidence interval at 95%.

Statistical analysis included analysis of variance, normality test, outlier detection, model adjustment by observed versus predicted analysis, and linear and quadratic regression models with response surface methods.

Pretreatment of rice husks

The RH samples were ground in a laboratorial knife mill (MA1680, Marconi, S?o Paulo, Brazil) in order to increase the surface area and reduce the crystallinity of the lignocellulose fraction. Following this, the biomassparticles were classified, and the particles, with a diameter within the range of 288–312 μm, were separated, rinsed with distilled water and dried at 60 oC for 24 h.

The composition of the RH was determined before comminution and after the optimized hydrolysis process. The RH was characterized in accordance with the reports (Sluiter et al, 2005a, b, 2008).

Optimization of acid hydrolysis

The pretreated biomass was submitted to a hydrolytic process in a pressurized reactor (Berghof, Eningen, Germany), provided with a stainless steel mortar (DAB-3), PTFE reaction vessel (Teflon?TFM, 250 mL) and heating block DAH902. The procedure included the addition of 1.0 g pretreated RH and 10 mL diluted acid solution (with separate concentrations of H2SO4, HCl, HNO3and H3PO4) to the reaction vessel, which was subsequently closed, and submitted to the heating stage (calculated pressure varied from 4.3 to 5.4 MPa).

Optionally, the hydrolytical process was conducted in a lab stove, as well as in a MW oven (Anton Paar, Graz, Austria), using the same proportion of RH/acidic solution. When the MW oven was used, hermetically-sealed quartz vessels (80 mL) enclosed in polyether ether ketone flasks were used.

The MW heating operated at 2.45 GHz to a limit of 1 400 W, using a ramp of 5 min and infrared digital temperature control (± 1 oC). The pressure was also digitally monitored in 1.2–2.7 MPa.

The variables monitored were acid concentration (0.6%–6.0%), reaction time (28–62 min) and temperature (128 oC–162 oC). The results of the experimental design were analyzed with the aid of Statistica 8.0 software (StatSoft, Tulsa, OK, USA).

Optimization of hydrolysate detoxification

The subproducts of hydrolysis, which act as inhibitors in the fermentation stage, must be blocked/removed in the detoxification stage. To achieve this, the multivariate approach entails the one block factorial design (Table 2), including concentration of the detoxification agent (1.0%, 25% and 4.0%), which was previously optimized according to Rambo et al (2013) and Pedroso et al (2017), and temperature (40 oC, 60 oC and 80 oC). Active carbon, calcium oxide (CaO) and calcium hydroxide [Ca(OH)2] were evaluated as detoxifiers.

The addition of CaO and [Ca(OH)2] leads to the formation of insoluble derivatives of the inhibitors (formic and acetic acids), which are precipitate and separated (Heredia-Olea et al, 2012). Following this, all the solutions were passed through a qualitative cellulose filter (0.45 μm) and then vacuum filtered through a cellulose nitrate membrane (47 mm × 0.45 μm). With regard to the detoxification with activated carbon, the pH of the hydrolysate must be adjusted. All the samples were kept at 4 oC–8 oC until autoclaving (15 min, 120 oC) (Rambo et al, 2013; Bevilaqua et al, 2017).

Preparation of micro-organism and inoculum

The filamentous fungus (yeast)(ATCC10020), which is a well-known organic acid generator, was obtained from the National Institute for Quality Assurance in Health, Brazil. The lyophilized culture was reactivated in accordance with established method (INCQS, 2015) and, afterwards, kept (and repiqued) in potato agar dextrose, and then incubated at 30 oC for 5 d. The inoculum volume was defined as 10% of total volume, and the cellular concentration had an absorbance of 0.3 (620 nm), equivalent to 1 × 109CFU/mL.

Table 2. Factorial design for the best detoxification conditions of rice husk hydrolysates.

The strain of.(ATCC10020) was employed to evaluate the fermentability of the hydrolysates, following the guidelines of Pedroso et al (2017), and using sulfuric hydrolysate, at 30 oC, adding 10 g/L yeast extract (pH 6.0, adjusted by 6 mol/L NaOH) and 100 r/min orbital stirring, cultivated in 20 mL medium in 125 mL flasks.

The bacterial strain of(LMG196), which is recognized as biopolymers producer, was obtained from tropical culture collection Andre Tosello, being kept in nutrient broth after reactivation as established method (INCQS, 2015). After activation and before being used, the bacterial culture was kept at 30 oC and repiqued in nutrient broth for 24 h. The inoculum volume was defined in the same way, as 10% of the total, and the cellular concentration had an absorbance of 0.6 (620 nm), equivalent to 1 × 109CFU/mL. Both microorganisms were kept isolated, free of cross contamination.

Analytical determination by HPLC-RID

The concentration of the reagents, subproducts and sugars, involved in the hydrolytic and fermentative processes, were determined by HPLC coupled to a refractive index detector (RID-10A) (Shimadzu, S?o Paulo, Brazil). The chromatograph was equipped with a LC-20AT pump, a DGU-20A5r degasser, an autosampler SIL-20A, and a communication module CBM-20A, and LC Solution Software. The injection volume was 20 μL. The samples were diluted in ultrapure water (1:5) and, afterwards, passed through syringe filters (PTFE or MCE 13 mm × 0.22 μm).

Validation of analytical method

The chromatographic analytical conditions were validated to establish the linearity range and the coefficient of determination, as well as the quantification limit (LOQ). The signal/noise ratio (S/N) was set at > 10 for LOQ (INMETRO, 2017).

Scanning electron microscopy (SEM) analysis

SEM images were used to evaluate the disaggregation of the lignocellulose caused by the pressurized acid hydrolysis, which was carried out with the aid of a Sigma 300 VP microscope (Carl Zeiss, England), fitted with a field emission gun, made of tungsten filament coated with zircon oxide, in a Gemini column. The images were obtained with a variable pressure secondary electron detector with different resolutions.

RESULTS

Analytical method

The figure-of-merit of the chromatographic method is shown in Table 3. The best separation was obtained with a chromatographic Aminex HPX-87H column (300.0 mm × 7.8 mm), using mobile phase of 0.005 mol/L H2SO4, isocratic mode, at 0.5 mL/min flow at 50 oC.

The occasional occurrence of matrix effects was tested, showing no statistical difference through-test. The linearity of the calibration curve was verified by linear regression at seven concentration levels in triplicate by evaluating the residuals and ensuring if the estimate was random and unbiased.

The stability of the samples was assessed in different conditions. The shelf time was determined for the analytical solutions and hydrolysates, over a period of a month. The decay of the analytes was < 5% in the first two weeks for both the analytical solutions and hydrolysates.

Characterization of rice husks

Natural and residual RHs were used to measure the percentiles (fractions), as well as the highest carbohydrate content of the cellulose and hemicellulose fractions (Table 4).

Table 3. Analytical figure-of-merit for the high performance liquid chromatography coupled to a refractive index detector (HPLC-RID) determinations.

Chromatographic run time corresponding to the analyte signal.

LOD, Limit of detection; LOQ, Limit of quantification.

Table 4. Characterization of rice husk (RH) and resulting hydrolysis products and sub-products.

Having knowledge of the chemical composition of the RH is of the utmost importance to quantify the yield and the efficiency of the treatments. The results showed that the sugar content (54.1% of the RH) was released into the hydrolysate by means of the pressurized acid hydrolysis strategy (around 68.2% of all the fermentable monosaccharides). RH have a high content of ash (> 15%), which makes hydrolysis difficult.

Optimization of hydrolytical process

By using a pressurized system, the phosphoric hydrolysates can reach concentrations of 0.1–1.5 g/L for glucose, 3.1–18.3 g/L for xylose, and 0.8–1.4 g/L for arabinose, with a total yield of 21.0 g/L (210 mg/g RH sugars); with HNO3, the hydrolysate concentrations ranging from 1.0–2.1 g/L for glucose, 6.1–13.2 g/L for xylose, 0.5–1.9 g/L for arabinose, resulting in 16.1 g/L sugars (161 mg/g). In contrast, when HCl was used, the hydrolysate showed 1.5–12.0 g/L for glucose, 0–14.0 g/L for xylose, 0.3–2.5 g/L for arabinose, making a total of 17.1 g/L (171 mg/g RH sugars). Finally, when H2SO4was used, the hydrolysate showed 1.2–11.9 g/L glucose, 4.0–13.6 g/L xylose, and 0.4–2.0 g/L arabinose, with a total yield of 17.0 g/L (168 mg/g RH sugars) (Fig. 1).

By compiling the ANOVA for each hydrolysis reaction, it is possible to evaluate the best procedure, while acquiring a better understanding of the significant factors (Table 5).

The use of MW and HCl for hydrolysis results in glucose concentrations ranging of 0.1–8.4 g/L, xylose of 0–11.9 g/L, arabinose of 0.2–1.8 g/L, with total yield of 15.2 g/L (152 mg/g in RH). In the case of H2SO4, the concentrations ranged from 1.0 to 9.2 g/L for glucose, 0 to 11.9 g/L for xylose, and 0.2 to 1.3 g/L for arabinose, resulting in 14.3 g/L sugars (143 mg/g in RH) (Fig. 2).

Fig. 1. Experimental design for pressurized hydrolysis strategies.

Table 5. Polynomial equations and model adjustment values for the pressurized acid hydrolysis of rice husks by ANOVA analysis.

SE, Squareerror.

1,2and3represent acid concentration, time and temperature, respectively.

The hydrolysates obtained using the lab stove and HCl, showed results ranging from 0 to 2.6 g/L for glucose, 0 to 12.1 g/L for xylose, and 0.2 to 1.9 g/L for arabinose, with a total yield of 15.5 g/L (155 mg/g in RH). When H2SO4was used, the concentrations ranged from 0 to 5.5 g/L for glucose, 0 to 13.2 g/L for xylose, and 0 to 2.2 g/L for arabinose, resulting in 16.9 g/L sugars (169 mg/g in RH).

By compiling the ANOVA, a better evaluation of the MW and the lab stove procedures can be made, as well as of the significant factors (Table 6).

With the exception of the following: (a) release of arabinose by phosphoric hydrolysis (= 0.445); (b) MW xylose release (< 0.600); and (c) arabinose release by sulfuric hydrolysis (= 0.438). All the-values indicate that the multivariate design factors are significant, which means that the applied models can be classified as suitable (Supplemental Figs. 1, 2 and 3), allowing the use of contour surfaces as shown in Fig. 3.

Fig. 2. Total 17 experimental designs for the hydrolysis strategies using microwave oven and the laboratory (lab) stove.

Table 6. Polynomial equations and model adjustment values for the acid hydrolysis of rice husks by ANOVA analysis in the microwave (MW) oven and laboratory (lab) stove.

AcidSugarP-valueR2Adjusted R2Mean SEP-valuePolynomial equationa Hydrochloric (Lab stove)Glucose< 0.0050.6200.5040.0220.069y = 1.092 + 0.221x1 – 0.247x12 + 0.427x2 – 0.391x32 Xylose< 0.0050.8300.6980.1230.013y = 11.516 + 1.705x1– 2.093x12 + 2.914x2– 2.512x22 + 1.086x3– 2.940x32– 1.517x2x3 Arabinose< 0.0050.7810.6500.0250.202y = 1.637 + 0.173x1– 0.159x12 + 0.342x– 0.171x22– 0.293x32– 0.120x1x3 Sulfuric (Lab stove)Glucose< 0.0050.5910.4050.0440.032y = 1.905 + 0.172x1– 0.691x12 + 0.279x2– 0.611x22 + 0.761x3 Xylose< 0.0050.9190.8570.1290.026y = 12.86 + 1.484x1– 2.962x12 + 2.459x2– 3.086x22 + 1.348x3– 3.886x32 + 1.687x2x3 Arabinose< 0.0050.9510.9220.0650.864y = 1.913 + 0.158x1– 0.289x12 + 0.325x2– 0.386x22 + 0.265x3 + 0.518x32 Hydrochloric (MW oven)Glucose< 0.0050.7390.6210.5690.185y = 3.562 + 0.636x2 + 0.895x22– 0.642x3– 0.856x32– 2.149x1x3 Xylose> 0.0050.5640.3033.7300.004y = 7.835 – 1.662x1– 1.178x22– 1.795x3 – 1.101x32 – 1.546x1x2 – 1.408x1x3 Arabinose< 0.0050.6340.4680.1360.686y = 1.127 – 0.218x1– 0.222x3– 0.137x32 + 0.235x1x2 + 0.089x1x3 Sulfuric(MW oven)Glucose< 0.0050.8290.7521.0910.349y = 6.971 + 1.002x1– 1.720x12 + 0.790x22 + 2.003x32 + 0.941x1x3 Xylose> 0.0050.5040.2062.0700.002y = 4.461 – 1.377x1– 1.749x12– 0.992x2– 1.347x3– 1.184x1x2– 1.035x2x3 Arabinose> 0.0050.4380.1810.1700.001y = 0.786 – 0.205x1 + 0.137x12– 0.111x3 + 0.146x1x + 0.181x2x3

SE, Squareerror.

1,2and3represent acid concentration, time and temperature, respectively.

The SEM images of the RH samples can help to achieve higher sugar yields as a result of different strategies chosen afterwards. The finding about the reduction of crystallinity of the RH matrix, which assists the complete release of the sugars that is desired, was also monitored by HPLC-RID, which pointed out the best hydrolysis conditions (Fig. 4).

Fig. 3. Example of contour surfaces showing the optimal region of glucose production.

A, Hydrolysis at a higher temperature and HCl concentration. B, Hydrolysis aiming for xylose production in a less severe optimal region.

Fig. 4. Scanning electron microscopy images for rice husks.

A and B arenatural and residual rice husks treated by pressurized acid hydrolysis with 1.5% H2SO4(65 min, 135 oC), respectively.

Table 7. Characterization of hydrolysates submitted to different detoxification processes.

Values are Mean ± SE (= 3).

Assessment of hydrolysate fermentability

The optimized hydrolysate was then analyzed in order to determine its composition, comparing values with the detoxified hydrolysate (Table 7). These results represent a comparison between the detoxification strategies with active carbon, CaO and Ca(OH)2.

The bacterial strain.(LMG196) and the yeast strain.(ATCC10020) were chosen to verify the detoxification procedure (for 60 min under stirring), as recognized producers of bioproducts. The response factor selected was sugar consumption (monitored by HPLC-RID) (Supplemental Fig. 4).

The experiments using 4% active carbon (7.0 effect, level +1) provided the best results (96% consumption of total sugars) (Table 8).

Both the inoculated strains (.ATCC10020 and.LMG196) in the detoxified hydrolysate showed a total consumption of sugars in 72 h (Fig. 5). This experiment confirms that the monosaccharides are highly fermentable and that the detoxification was effective enough.

DISCUSSION

The employed analytical method provided qualified information and can be considered to be fit-for- purpose. The coefficients for determining the calibrationcurve of the optimized method (Table 3) were satisfactory, which means there was a suitable quantification of all the products and subproducts, without any overlapping chromatographic peaks.

Fig. 5. Kinetics of sugar consumption (g/L) duringandfermentation of the rice husk hydrolysates.

The RH characterization (Table 4) showed similar results as Dagnino et al (2013) and Pedroso et al (2017). In addition, owing to the complex matrix of lignocellulosic feedstock (Karimi and Taherzadeh, 2016), identical biomasses can generate different values, when different analytical methods are employed.

By the resulting yields from the hydrolysis optimization (68.2% of all the fermentable monosaccharides were liberated at one step can be concluded that the hydrolysate may be the sole source of carbon for fermentation (even RH being highly recalcitrant), and the exclusion of the supplements reduces costs because synthetic media are expensive (Kuenz et al, 2012). Thus, on the basis of the monosaccharide concentrations produced under optimized conditions, it can also be established that the RH hydrolysate might be produced by any studied acid. Boonterm et al (2016) have adopted this approach and employed SEM images for a better examination of the effects of hydrolytic treatment on lignocellulosic biomasses.

Phosphoric hydrolysis

The Central Composite Rotational Design regarding the three strategies chosen was used to study the response pattern and to determine the optimum combination of variables. The statistical treatment of the variables, along with the response values, expressed as yield of sugar (glucose, xylose and arabinose), were analyzed for each quantifiable monosaccharide (Supplemental Fig. 1). Similarly, the same approach was adopted for RH pressurized hydrolysis with nitric and phosphoric acid, although neither of the hydrolysates proved to be fermentable enough.

Table 8. Polynomial equations and model adjustment values for the rice husk hydrolysate detoxification by ANOVA analysis.

1and2represent acid concentration and time, respectively.

Glucose production was statistically affected (< 0.05), especially by the phosphoric acid concentration (1, 0.419). In the case of xylose, a higher production was possible by raising the acid concentration linearly (1, 2.502), as well as quadratically (12, 1.314), and by including the interaction of acid concentration with the temperature (13, -2.901) (Supplemental Fig. 1).

Nitric hydrolyses

Both nitric acid concentration and temperature were significant quadratically significant (12, 0.191;32, 0.152), and the temperature linearly (3, 0.127), which suggested that a higher severity level is preferable in terms of glucose productivity (Supplemental Fig. 1). However, in the case of xylose, lower temperatures were preferable (3, -1.894), as well as the combination of temperature with acid concentration (13, -1.159). Hence, it can be concluded that the optimization range should be chosen with regard to only one monosaccharide, because there is no simultaneous best condition for glucose and xylose. The production of the least important arabinose is supported linearly by the reduction of all the significant factors (1, 0.114;2, 0.113;3, 0.304), which corroborates the optimized conditions required for the xylose production.

Hydrochloric hydrolysis

In the pressurized HCl hydrolysis, the highest temperatures (3, 2.395) and acid concentration (1, 1.628) were required for the highest rates of glucose production; the same can be said for the interaction between temperature and reaction time (23, 1.520), which means, more severe conditions. In the case of xylose productivity, again lower temperatures were preferable (3, -3.205), as well as a lower acid concentration (1, -3.117); which thus means that it is not feasible to ensure optimized xylose and glucose production at the same time (Fig. 3). The arabinose production is similar to that of xylose, because the temperature (3, -0.420) and the acid concentration (1, -0.330) are linearly negative (Supplemental Fig. 1).

When in the lab stove, the HCl hydrolysis glucose production was affected by the reaction time (2, 0.427) and temperature (12, 0.391). When MW was used, the interaction between acid concentration and temperature was the predominant factor (13, -2.149).

With regard to glucose production, hydrolysis by electric-resistive heating in a pressurized system were found to be superior to MW heating, which provides evidence that pressure is of paramount importance, despite the better energy transfer of MW.

In the case of the xylose production, there were no significant factors with regard to MW heating, which means a situation of too high severity, and that an in-depth investigation is needed. In the case of lab stove hydrolysis, the quadratic pattern was significant for all the factors, which suggests that the central points (i.e. moderate severity) lie in the optimal region (Supplemental Fig. 2).

Sulfuric hydrolysis

The sulfuric hydrolysis resulted in complex models with several significant interactions between the factors affecting productivity, in particular, the acid concentration (1, 1.176) and temperature (3, 3.075). In contrast, xylose had a higher rate of productivity at a lower temperature (3, -3.523) and acid concentration (1, -1.811). In the case of arabinose, it was mainly the temperature (3, -0.384) that was significant.

When the lab stove was used, a higher temperature (3, 0.761) and moderate acid concentration (12, 0.691) were required for glucose production. The same applies to MW hydrolysis and, thus, a slightly higher temperature (3, 1.002) increases productivity, the central point lies in the optimal region with moderate temperature (32, -1.720) and acid concentration (12, 2.003). The same was observed when lab stove heating was used, but in this case, it affected the production of xylose and arabinose.

In all the three hydrolytic strategies, the production of xylose slightly exceeded that of glucose, and the lab stove hydrolysis was the less severe option, producing xylose rather than glucose, which may be advantageous, sometimes.

Efficiency of hydrolytical strategies

The severity of hydrolysis is of crucial importance for the production of monosaccharides, which, in general, increases to a maximum threshold and then rapidly declines. The usual lack of multivariate strategies in studies, then, decreases both the yield and the proposal merit. Reports dealing with lignocellulosic biomasses, generally, reach high yields (about 70%) by using both acid and enzymatic hydrolysis (or, yet, with additional treatments), without any measure to avoid toxic compounds that inhibits fermentation steps. Farther, recent studies dealing with RH but without the hydrolysate fermentability reported yields of 15 g/L, reached 21.0 g/L fermentable sugar production and also presented a feasible bioconversion route (Loow et al, 2016; Temiz and Akpinar, 2017).

The H3PO4and HNO3catalysts were only examined in pressurized procedures, and there was a low total sugar yield because of the negative interaction between the variables. It was clear that both catalysts were more efficient with xylose, while HCl and H2SO4were better with glucose production.

Sulfuric hydrolysate was chosen for the evaluation of the detoxification because: 1) it had the best preliminary fermentation performance; 2) the higher toxicity of nitric hydrolysate; 3) the equivalent performance of sulfuric and phosphoric hydrolysates; and 4) sulfuric hydrolysate had a better performance than hydrochloric hydrolysate (inhibition by the chlorides).

Effect of detoxification conditions

Several secondary reactions can occur during the RH acid hydrolysis, and result in subproducts and interferers, many of which can act adversely during the bioconversion stage (Kundu et al, 2015). In fact, the toxicity caused by inhibitors can mean it not feasible to make use of the hydrolysate as a fermentation substrate (Pedroso et al, 2017).

Table 7 shows that detoxification with CaO and Ca(OH)2is efficient enough to eliminate acetic and formic acids by forming insoluble salts (de Vasconcelos et al, 2013). It is likely that the employment of the RH phosphoric hydrolysate may even lead to the formation of nutrients (phosphates) as reported by Pedroso et al (2017).

Although much is known about the active carbon adsorption capacity, the detoxification procedure of RH hydrolysates had until then not been found in the literature (Rambo et al, 2013). Although the temperature was not significant (> 0.05), when 80 oC was used for the detoxification, there were benefits to the bacterial metabolism, which indicates an increase in efficiency (Table 8).

Although success has been achieved through the addition of calcium compounds (82% of total sugar consumption), the performance was only affected by the time [2, 13.25 for CaO;2, -10.75 for Ca(OH)2)], and the rate of efficiency does not depend on the concentration. The general result was inferior to that using active carbon, probably because remaining traces of insoluble salts. Table 7 illustrates the minimal loss of sugars during the detoxification, e.g. when activated carbon is used, significant for the choice of the best strategy.

The detoxification stage is mandatory to the bacterial fermentation, which must be cost-effective and simple, without any loss of efficiency and sugars. The use of active carbon was definitely a key strategy in making the fermentation of RH hydrolysates feasible.

There were some metabolic differences between the microorganisms (Fig. 5). While bacteria continued to consume sugars in the first 12 h, yeasts had a longer lag phase during which there was a slow depletion of the carbon source. Both bacteria and yeasts preferred to consume the glucose of the hydrolysates. Other experiments in our lab seek to exploit the use of RH hydrolysates for bioconversion in other value-added products.

Additionally, the present chemometric treatment of the data resulted in the high fermentability of the RH hydrolyzate, consumption of 96% sugars, without supplementation of the culture medium, indicating that the present proposal obtained a fermentable medium that is less toxic and has less cost than those reported in other studies, with a high feasible yield of bioconversion.

CONCLUSIONS

In this study, three hydrolysis strategies, followed by three different detoxification procedures, were compared to determine the best approach to the bioconversion of RH into fermentable monosaccharides, which are conceivably the precursors of bioproducts. Owing to the multivariate designs and chemometric data treatment, it was possible to compare the RH hydrolysis by using four different inorganic acids. A leftover biomass was used successfully as fungi (.) and bacteria (.) fermentable substrates. The analytical methodology allowed accurate quantification, providing qualified information (figure-of-merit and a fit-for-purpose method). The findings are significant in terms of best sugar yields, greatly reduced lignocellulosic crystallinity and degree of fermentability, while being exempt from any nutritional supplements. These strategy proposals can transform a leftover biomass in a feedstock for biorefineries, combining cleaner production with green engineering.

ACKNOWLEDGEMENTS

The authors would like to express their gratitude to the scholarship from the Brazilian National Council of Technological and Scientific Development and for the financial support from the Research Support Foundation of the State of Rio Grande do Sul (Grant No. 001897- 25.51/13S-I4).

SUPPLEMENTAL DATA

The following materials are available in the online version of this article at http://www.sciencedirect.com/science/ journal/16726308; http://www.ricescience.org.

Supplemental Table 1. Central composite rotational design of acid hydrolysis for rice husks under different strategies.

Supplemental Fig. 1. Evaluation of significant model effects (hydrolysis in heat block) after its adjustment by elimination of non-significant effects.

Supplemental Fig. 2. Evaluation of significant model effects (hydrolysis in lab stove) after its adjustment by elimination of non-significant effects.

Supplemental Fig. 3. Evaluation of significant model effects (hydrolysis in microwave oven) after its adjustment by elimination of non-significant effects.

Supplemental Fig. 4. Evaluation of significant model effects (hdrolysate detoxification) after its adjustment by elimination of non-significant effects.

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15 August 2018;

29 October 2018

Ayrton F. Martins(martins@quimica.ufsm.br; ayrton@pq.cnpq.br)

Copyright ? 2019, China National Rice Research Institute. Hosting by Elsevier B V

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Peer review under responsibility of China National Rice Research Institute

http://dx.doi.org/10.1016/j.rsci.2019.08.006

(Managing Editor: Wang Caihong)