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La Société Canadienne de Génie Agroalimentaire et de Bioingénierie La société canadienne de génie agroalimentaire, de la bioingénierie et de l’environnement

Paper No. CSBE13-068

NMMO treatment: An effective pretreatment for improvement of lignocelluloses by increasing enzyme

accessibility

Amir Goshadrou Department of Bioresource Engineering, McGill University, Montreal, QC H9X 3V9, Canada

Keikhosro Karimi Department of Chemical Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran

Mark Lefsrud Department of Bioresource Engineering, McGill University, Montreal, QC H9X 3V9, Canada

Written for presentation at the CSBE/SCGAB 2013 Annual Conference

University of Saskatchewan, Saskatoon, Saskatchewan 7-10 July 2013

ABSTRACT Recalcitrant structure of lignocellulosic materials is a major obstacle for their efficient conversion to fermentable sugars. Several pretreatments have been investigated to enhance digestibility of lignocelluloses by improving substrate accessibility to the hydrolytic enzymes. Pretreatment with cellulose solvents such as N-Methylmorpholine-N-oxide (NMMO) can be applied in relatively milder conditions compared to common thermal pretreatments. Besides, NMMO is a non-toxic and fully biodegradable chemical with possibility of nearly complete recycling. The current study aimed to improve enzymatic hydrolysis yield of aspen wood, a widely available type of hardwood, by NMMO pretreatment. Native aspen wood was pretreated at 120 ºC for 1, 3, and 5 h and subsequently subjected to enzymatic hydrolysis at pH 4.8 using 15 FPU/g DM cellulase (Celluclast 1.5L, Novozyme, Denmark) and 30 IU/g DM β-glucosidase (Novozyme 188, Novozyme, Denmark). The substrate accessibility to cellulase was measured by evaluating the protein adsorption capacity using the Langmuir equilibrium isotherm. The results showed that the NMMO pretreatment could significantly increase the substrate accessibility to the cellulase (1.86-fold), and enhance the hydrolysis yield of aspen wood to 90%.

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Keywords: Lignocelluloses, NMMO Pretreatment, Digestibility, Enzyme accessibility.

INTRODUCTION Lignocelluloses are the most abundant polymeric carbohydrate in the world that could be used for sustainable biofuel production (Nigam & Singh, 2011). Owing to the recalcitrant structure of lignocellulosic materials, the pretreatment is known as a key step to remove lignin barrier and decrease the cellulose crystallinity, so that it becomes more accessible to the enzymatic complex (Yang & Wyman, 2008). There are several pretreatment methods, which use energy such as heat or radiation or chemicals such as acids, bases or solvents or their combinations (Agbor et al., 2011). Pretreatment with cellulose dissolution reagents can be generally applied in milder conditions than the thermal pretreatments. Pretreatment using some cellulosic solvents (e.g., phosphoric acid and NaOH/urea) have been shown to be effective prior to enzymatic hydrolysis process. However, chemical neutralization after the pretreatment is a major drawback of such acidic or basic solvents (Goshadrou et al., 2013b; Shafiei et al., 2010; Zhao et al., 2007).

N-Methylmorpholine-N-oxide (NMMO) is among the direct solvents for cellulose and dissolves cellulose by breaking intermolecular interactions without chemical derivatization. It is non-toxic and fully biodegradable and used in the Lyocell process which is one of the modern and environmentally friendly industrial fiber-making technologies. After dissolution in NMMO, cellulose can be regenerated by rapid precipitation with an anti-solvent such as water. Furthermore, the process works under moderate conditions and over 98% of the solvent can be recycled (Liebert, 2008; Zhao et al., 2007).

Since the enzymatic hydrolysis is inherently a heterogeneous catalytic process, the intimate contact between the cellulose and cellulase is the prerequisite step for hydrolysis to take place and the cellulose accessibility to cellulase is critical (Rollin et al., 2011; Wang et al., 2012).

The present study is aimed to evaluate the effect of NMMO pretreatment on improvement of the substrate accessibility to cellulase and sugar yield in enzymatic hydrolysis of aspen wood wastes.

MATERIALS AND METHODS

Raw material Aspen wood (Populus tremula) wastes were collected in summer 2011 from a local forest (Mazandaran, Iran). The wood was dried, milled, and screened to achieve a particle size in the range of 20-80 mesh.

NMMO pretreatment A commercial-grade NMMO solution in water (BASF, Ludwigshafen, Germany) was heated and concentrated to 85% NMMO by vacuum evaporation. An amount of 15 g wood was added to 185 g NMMO solvent and mixed every 15 min at 120 °C for 1, 3, and 5 h. The pretreated substrate was then regenerated by adding 150 mL boiling distilled water, followed by vacuum filtration and washing until a clear filtrate appeared. All the treated and untreated materials were oven dried at 40 °C for 48 h, and stored at 4 °C until use (Goshadrou et al., 2013b).

Composition analysis Polymeric carbohydrates and lignin contents of the untreated and pretreated woody biomass were determined using the method presented by the National Renewable Energy Laboratory. The method was based on two-stage acid hydrolysis of the wood and quantification of monomeric sugars in the hydrolysate using HPLC. The acid-soluble and acid-insoluble fractions of lignin were determined by UV/Vis spectroscopy and gravimetric analysis, respectively (Sluiter et al., 2008).

Morphological analysis All the untreated and pretreated samples were coated with a thin layer of gold (BAL-TEC SCD 005) and then the SEM images were captured using a Philips XL30 scanning electron microscope at an acceleration voltage of 15 kV.

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Enzyme adsorption The substrate accessibility to cellulase (SAC) was conducted by measuring the maximum adsorbed cellulase on the untreated and pretreated aspen wood. The substrate was mixed with 5 ml sodium citrate buffer solution (50 mM, pH 4.8) contained different amounts of cellulase to make a suspension with a final dry solid content of 2% (w/v). The mixture was continuously stirred at 0 ºC and 150 rpm for 90 min. The liquid samples were centrifuged at 5000×g for 15 min and subjected to protein analysis by Bio-Rad protein assay (Richmond, CA, USA). The maximum enzyme adsorption capacity was estimated by applying the Langmuir equilibrium isotherm over the experimental data (Goshadrou et al., 2013a; Kumar & Wyman, 2008).

fd

fdmaxa EK1

EKEE+

= (1)

In which Ea is concentration of adsorbed protein (mg g-1 substrate), Emax is maximum protein adsorption by the substrate (mg g-1 substrate), Ef is free protein concentration in liquid phase (mg ml-1), and Kd is the affinity parameter (ml mg-1).

Enzymatic hydrolysis Two commercial enzymes, cellulase from Trichoderma reesei (Celluclast 1.5 L, 90 FPU/ml, Sigma) and cellobiase from Aspergillus niger (Novozyme 188, 744 CBU/ml, Sigma) were used for enzymatic hydrolysis woody biomass. The hydrolysis experiments were conducted at 50 °C for 72 h in 50 mL sodium citrate buffer media (pH 4.8) using 20 FPU cellulase and 50 IU β-glucosidase per grams of dry substrates. The medium was supplemented with sodium azide (0.5 g/l) to prevent microbial contamination and samples were periodically taken for sugar analysis. The substrate enzymatic digestibility (SED) was calculated using the following equation:

( ) ( )F1.111ionconcentrat Substrate

100Lg glucose Produced % SED××

×= (2)

where F in denominator is the biomass glucan fraction from composition analysis.

RESULTS AND DISCUTION

Effects of NMMO pretreatment on wood composition The results of composition analysis for untreated and different NMMO pretreated materials are presented in Table 1. The untreated wood mainly contained glucan (49.0%) and xylan (14.9%). The glucan fraction in the pretreated materials was increased while the pretreatment partially removed xylan and other polysaccharides (data not shown). Xylan removal during NMMO pretreatment was more pronounced for longer pretreatment times, which is most likely due to the high sensitivity of hemicelluloses to severe conditions. Furthermore, the acid insoluble part of lignin was reduced from 24.6% to 18.5-19.9% after NMMO pretreatment and increasing the pretreatment time slightly assisted lignin removal yield. However, the acid soluble lignin content of aspen wood was almost unchanged after pretreatment.

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Table 1. Chemical composition (%) of untreated and NMMO pretreated aspen wood.

Substrate Glucan Xylan Acid Soluble Lignin Acid Insoluble Lignin

Untreated 49.0 14.9 1.0 24.6 NMMO pretreated (1 h) 51.3 13.2 0.8 19.9 NMMO pretreated (3 h) 52.2 12.7 0.9 19.4 NMMO pretreated (5 h) 53.0 11.9 0.8 18.5

Effects of NMMO pretreatment on wood morphological structure The SEM images of untreated and NMMO pretreated wood are demonstrated in Figure 1. The morphological structure of the substrate was significantly improved after pretreatment and NMMO could extensively disintegrate the fibrillar pattern of aspen wood. Furthermore, the NMMO pretreated sample exhibited a highly disordered and rough surface, and sponge-like structure which provide more surfaces area for enzymatic attack.

Figure 1. SEM images of untreated and NMMO pretreated aspen wood.

Effects of NMMO pretreatment on SAC Langmuir adsorption isotherm was applied to describe cellulase adsorption on the untreated and pretreated substrates, and the maximum extent of adsorbed protein was considered proportional to the surface area available for the cellulase enzyme. The most important results including are shown in Figure 2. The determination coefficient (R2) was in the range of 0.958-0.969.

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Figure 2. Protein adsorption capacity of untreated and different NMMO pretreated aspen wood.

Among the samples, the untreated aspen wood showed the minimum SAC value of 101.6 mg g-1, thus minimum accessibility to enzyme. Pretreatment with NMMO for 1 and 3 h significantly increased the SAC by about 1.4- and 1.6-fold, respectively, whereas extending the pretreatment time up to 5 h enhanced the SAC by maximum 1.9-fold. Therefore, NMMO pretreatment could substantially produce pores which were accessible to cellulase (larger than 51 A°).

Effect of NMMO pretreatment on SED All the treated and untreated materials were subjected to 72 h enzymatic hydrolysis and the most important results are summarized in Table 2. As shown, digestibility of the untreated wood was only 5.3% of theoretical glucose yield.

Table 2. SED (%) of the untreated and different NMMO pretreated materials.

Substrate SED (%)

Untreated 5.3 NMMO pretreated (1 h) 80.4 NMMO pretreated (3 h) 88.6 NMMO pretreated (5 h) 92.9

Comparatively, the NMMO pretreated materials exhibited to be highly digestible. The final hydrolysis yield significantly increased after 1 h pretreatment with NMMO and more than 80% of the biomass cellulose content was converted to glucose. Moreover, increasing the NMMO pretreatment time significantly enhanced substrate enzymatic digestibility and the maximum SED value (about 93%) was obtained after 5 h pretreatment of aspen wood with NMMO.

CONCLUSION The present study was undertaken to examine the effects of NMMO pretreatment on enzymatic hydrolysis of the hardwood Aspen. Pretreatment with NMMO could significantly increase the substrate accessibility to cellulase and improve its morphological structure. The aforementioned modifications led to over 17-fold enhanced enzymatic digestibility of the aspen wood.

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