Accepted Manuscript Alkaline and alkaline peroxide pretreatments at mild temperature to enhance enzymatic hydrolysis of rice hulls and straw Emir Cabrera, María J. Muñoz, Ricardo Martín, Ildefonso Caro, Caridad Curbelo, Ana B. Díaz PII: DOI: Reference:

S0960-8524(14)00821-9 BITE 13511

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

25 March 2014 25 May 2014 27 May 2014

Please cite this article as: Cabrera, E., Muñoz, M.J., Martín, R., Caro, I., Curbelo, C., Díaz, A.B., Alkaline and alkaline peroxide pretreatments at mild temperature to enhance enzymatic hydrolysis of rice hulls and straw, Bioresource Technology (2014), doi:

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Alkaline and alkaline peroxide pretreatments at mild temperature to enhance enzymatic hydrolysis of rice hulls and straw Emir Cabreraa,, María J. Muñozb, Ricardo Martínb, Ildefonso Caro b, Caridad Curbeloa, Ana B. Díazb


Departamento de Ingeniería Química, Instituto Superior Politécnico José Antonio

Echeverría, Cujae, Ave. 114 No. 11901, Marianao 19390, Cuba. b

Departamento de Ingeniería Química y Tecnología de Alimentos, Universidad de

Cádiz, Campus Río San Pedro, s/n, Puerto Real, 11510 - Cádiz, Spain.

ABSTRACT The current study explores alkaline and alkaline peroxide pretreatments in order to achieve a method to improve saccharification of agricultural residues for ethanol production. The effects of reagent concentration and pretreatment time at 30ºC and atmospheric pressure on biomass dissolution after the pretreatment and enzymatic hydrolysis of the pretreated biomass were investigated. In fact, although all pretreatments tested improved enzymatic hydrolysis of native residues, the best results were not achieved for the highest biomass loss. The maximum conversions to reducing sugars in the hydrolysis stage of 77.5% and 92.6% were obtained for rice hulls and straw pretreated by alkaline peroxide (4%, 24 h) and alkaline (1%, 48 h) methods, respectively. For both pretreated residues, the reduction to more than half the recommended enzyme loading allowed obtaining more than 94% the reducing sugars attained with the recommended dose.


Keywords: lignocellulosic residue, sodium hydroxide pretreatment, alkaline peroxide pretreatment, enzymatic hydrolysis, bioethanol.

1. Introduction Bioethanol is one of the renewable energy sources in use today. The availability of large volumes of industrial and agricultural residues, together with the need to prevent environmental pollution raises the potential to use them as feedstock for ethanol production. These residues have been considered for ethanol production since they are inexpensive, abundant and have high cellulose and hemicellulose content (Sarkar et al., 2012; Suhardi et al., 2013). A key issue for utilization of lignocellulosic biomass is the disruption of the complex polymer matrix to liberate the monosaccharides (Mosier et al., 2005). However, several investigations have shown high resistance to polymeric degradation (Hsieh et al., 2009; Alvira et al., 2010). Thus, the development of pretreatment methods that increase the material digestibility for the subsequent enzymatic hydrolysis becomes a focus in this field of research. Pretreatment is one of the most expensive and least technologically mature steps in the process of converting biomass to fermentable sugars. Although many different types of pretreatments were tested in different conditions over the years (Gould, 1984; Chiaramonti et al., 2012), advances are still needed for efficiency improvement, cost reduction and the satisfaction of environmental requirements (Viikari et al., 2012). Therefore, technology of bioethanol production has a great potential to become competitive through the research of pretreatments. Each pretreatment has a specific effect on cellulose, hemicellulose and lignin fractions. Thus, a method and its conditions should be chosen according to the process


configuration selected for the subsequent hydrolysis and fermentation steps (Mosier et al., 2005; Alvira et al., 2010). Moreover, particular advantage has the use of reagents with low environmental impact and avoidance of special reaction chambers. Among chemical pretreatments, alkaline and alkaline peroxide methods in mild conditions have shown high effectiveness on several agricultural residues (Kumar and Wyman, 2009; Alvira et al., 2010). Basically, the main advantage of these methods is the use of readily available and environmentally benign chemicals at low concentrations. Furthermore, they make possible to operate at low temperature and atmospheric pressure, not being necessary expensive specialized reactors. Apart from that, they have lower operation cost than other highly effective pretreatments such as ionic liquids and organic solvents. On the other hand, they show some disadvantages as the cost of reagents and the relatively long pretreatment time required at ambient conditions (Menon and Rao, 2012). The mechanism by which these pretreatments enhance enzymatic saccharification appears to involve a release or redistribution of lignin and hemicellulose from the lignocellulosic matrix, a decrease in cellulose crystallinity and a dramatic increase in the degree of cellulose hydration (Hendriks and Zeeman, 2009; Shen et al., 2011). Nevertheless, possible loss of fermentable sugars and production of inhibitory compounds must be taken into consideration in both methods to optimize the pretreatment conditions (Alvira et al., 2010; Banerjee et al., 2011). Moreover, the addition of an oxidant agent like hydrogen peroxide to alkaline pretreatment can improve the performance by favouring lignin removal (Alvira et al., 2010; Alvarez-Vasco and Zhang, 2013). Hydrogen peroxide is commonly used in papermaking for the oxidative cellulose bleaching. As a result of its decomposition, the


highly reactive oxygen species superoxide and hydroxyl radical are produced as previously described Gould et al. (1985).

The oxidative action of the H2O2-derived radicals is thought to contribute to the depolymerization of lignin by attacking lignin side chains and fragmenting the lignin macrostructure into a number of low-molecular-weight compounds (Gould, 1985; Lachenal et al., 1988). However, in this process the pH is one of the most important parameters for efficient application of peroxide (Mancera et al., 2010; Maziero et al., 2012). In fact, depending on the pH adopted during lignin oxidation, no significant changes in chemical structure might be observed, since the oxidizing agent acts only in the aliphatic part of the macromolecule (Sun et al., 2000). Given that there is not a universal and economically viable pretreatment, the aim of the present work is to achieve a versatile method to pretreat diverse agricultural residues. For this purpose, the efficiency of alkaline and alkaline peroxide methods under similar conditions was evaluated on two complex lignocellulosic substrates, rice hulls and rice straw. Indeed, not only do these residues differ in their composition but they have also shown different pretreatment performances in previous works. In this investigation, the pretreatments efficacy was evaluated through the conversion to reducing sugars achieved after the enzymatic hydrolysis of the pretreated solids.


2. Materials and Methods Rice hulls and straw were provided by the Spanish company Herba Ricemills. The residues were first milled (Wonder Max blender) and then sieved to collect the fraction of 0.2 - 1 mm size, which was the one used in all experiments. Next, they were homogenized to avoid compositional differences and stored in plastic bags until use. Native materials showed moisture contents of 7.1% and 7.9% for rice hulls and straw, respectively.

2.1. Pretreatment Alkaline and alkaline peroxide pretreatments were carried out by mixing 2.5 g of residue with dissolutions of sodium hydroxide or alkaline hydrogen peroxide adjusted to pH 11.5 (± 0.2) with 5 M NaOH to reach a solid/liquid ratio of 1:20 (w/v). Sodium hydroxide (99% w/w) and hydrogen peroxide (30% w/v) were purchased from Panreac (Barcelona, Spain). Suspensions were prepared in 250 mL erlenmeyer flasks, which were incubated in a convection oven (Binder FD-53, Germany) at 30ºC without mechanical motion and pH readjustment during the pretreatment. Pretreatments were performed at several sodium hydroxide (0.5, 1, 3% w/w) and hydrogen peroxide (0.5, 1, 1.5, 2, 3, 4% w/v) concentrations for different pretreatment times (12, 24, 48 h) in order to study the effects of these factors on the subsequent enzymatic hydrolysis. Not only were low temperature and reagent concentration considered to maintain low costs but also to reduce environmental pollution. After each pretreatment, the slurry was filtered through a Whatman No. 1 filter paper (Sigma-Aldrich, Madrid, Spain) to separate solid


and liquid fractions. The liquid was collected to determine the total carbohydrates released, and the residual solid was washed with distilled water (100 – 150 mL) to remove undesired chemicals up to reach pH 7. Subsequently, the washed solid was dried at 40ºC for 24 h in a convention oven (Binder FD-53, Germany) in order to calculate the weight loss after the pretreatment. Finally, the dried solid was subjected to enzymatic hydrolysis to evaluate the efficacy of pretreatment methods proposed.

2.2. Enzymatic hydrolysis Enzymatic saccharification of pretreated materials was evaluated by conversion of cellulose and hemicellulose to monomeric sugars. For the hydrolysis, it was added to each 100 mL Erlenmeyer flask the pretreated dried solid, and 25 mL of a liquid fraction constituted by 0.05 mol/L sodium citrate buffer at pH 5 (Panreac, Barcelona, Spain) and a cocktail of enzymes kindly supplied by Novozymes (Novozymes Cellulosic Ethanol Enzyme Kit, Novozymes, Denmark). The cocktail was a mixture of cellulase, xylanase and β-glucosidase with recommended loadings of 17 FPU, 20 IU and 14 IU per gram of dry biomass, respectively (Novozymes A/S, 2010). The buffer dissolution was supplemented with 0.25 mL of 0.2 mg/mL sodium azide (Sigma-Aldrich, Madrid, Spain) to prevent microbial contamination. The hydrolysis mixture was incubated at 50ºC in an orbital shaker (New Brunswick Scientific, Excella E-24R) at 150 rpm for 72 h. After incubation, samples were collected and centrifuged for reducing sugar analysis. Rice hulls and straw without any pretreatment were also subjected to enzymatic hydrolysis as a control.


2.3. Analytical methods The chemical compositions of untreated residues were analyzed according to NREL methods (Sluiter et al., 2008). Sugars were quantified using an HPLC (Alliance 2695, Waters, Milford, MA, USA) equipped with a refractive index detector (Waters 2414, Milford, MA, USA) and an ion-exchange column (Aminex HPX-87H, Bio-Rad, Hercules, CA, USA). H2SO4 (0.005 mol/L) was used as the mobile phase at 0.036 L/h at a column temperature of 60°C. Composition values were calculated on dry-weight basis. Total reducing sugars generated from enzymatic hydrolysis were determined by using the modified dinitrosalicylic acid method in microtiter plates (Gonçalves et al., 2010). The conversion to reducing sugars (CRS%) in the enzymatic hydrolysis was calculated based on total cellulose and hemicellulose content of untreated residue as follows (Yoo et al., 2011):

where YRS = reducing sugars yield (g/g of untreated residue), C = amount of cellulose (g/g of untreated residue), H = amount of hemicellulose (g/g of untreated residue), 1.1 = conversion factor for polymer to monomer sugars. The total amount of carbohydrates released in pretreatments were determined by the phenol-sulfuric acid method (Nielsen, 2010) and its percentage was calculated based on total cellulose and hemicellulose content of untreated residues. The simple effect of each studied factor (reagent concentration and pretreatment time) on enzymatic saccharification was examined by looking at significant (p

Alkaline and alkaline peroxide pretreatments at mild temperature to enhance enzymatic hydrolysis of rice hulls and straw.

The current study explores alkaline and alkaline peroxide pretreatments in order to achieve a method to improve saccharification of agricultural resid...
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