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Hideno, A. (2018). "Thermal degradation behavior of ball-milled Miscanthus plants and its relationship to enzymatic hydrolysis," BioRes. 13(3), 6383-6395.

Abstract

Correlations were determined between the thermal degradation behaviors of ball-milled Miscanthus plants and their enzymatic digestibilities. Overall, thermal degradation temperatures of Miscanthus giganteus were higher than those of M. sinensis. The differential thermogravimetric (DTG) curve of M. giganteus had a characteristic shoulder peak near 292 °C as opposed to that of M. sinensis. The thermal degradation temperatures of both ball-milled samples decreased with increased ball-milling time, although the composition was not changed by ball milling. Remarkable changes in the DTG curves of M. sinensis and M. giganteus occurred with ball milling for more than 60 min and 120 min, respectively. These thermal degradation results were similar to the results for physicochemical pretreatments and enzymatic digestibilities. The thermal decomposition temperatures of both ball-milled samples at 20% weight loss were most negatively correlated with the enzymatic digestibilities with a value of approximately -1.0.


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Thermal Degradation Behavior of Ball-milled Miscanthus Plants and Its Relationship to Enzymatic Hydrolysis

Akihiro Hideno *

Correlations were determined between the thermal degradation behaviors of ball-milled Miscanthus plants and their enzymatic digestibilities. Overall, thermal degradation temperatures of Miscanthus giganteus were higher than those of M. sinensis. The differential thermogravimetric (DTG) curve of M. giganteus had a characteristic shoulder peak near 292 °C as opposed to that of M. sinensis. The thermal degradation temperatures of both ball-milled samples decreased with increased ball-milling time, although the composition was not changed by ball milling. Remarkable changes in the DTG curves of M. sinensis and M. giganteus occurred with ball milling for more than 60 min and 120 min, respectively. These thermal degradation results were similar to the results for physicochemical pretreatments and enzymatic digestibilities. The thermal decomposition temperatures of both ball-milled samples at 20% weight loss were most negatively correlated with the enzymatic digestibilities with a value of approximately -1.0.

Keywords: Thermal gravimetric analysis; Differential thermal gravimetric (DTG) curve; Miscanthus plants; Ball milling; Enzymatic hydrolysis

Contact information: Paper Industry Innovation Center, Ehime University, 127 Mendori-cho, Shikokuchuo, Ehime 799-0113, Japan; *Corresponding author: a-hideno@agr.ehime-u.ac.jp

INTRODUCTION

Lignocellulosic biomass consists of three main chemical components: cellulose, the most abundant organic material; lignin, the second-most abundant organic material; and hemicellulose, which mainly connects cellulose and lignin. Biorefinery processes that transform lignocellulosic biomass into useful fuels and chemicals are considered promising toward the establishment of a sustainable society and the reduction of carbon dioxide emissions. Recently, nanocellulose made from lignocellulose has attracted much attention as an advanced and environmentally friendly material (Yano 2010; Isogai et al. 2011). The isolation or separation of cellulose and other components is required to produce fuels (e.g., bioethanol), chemicals, and nanocellulose.

Pretreatment techniques have been widely investigated for the facile production of fermentable sugars and nanofibrillated cellulose (Mosier et al. 2005; Pääkko et al. 2007; Hendriks and Zeeman 2008; Lagerwall et al. 2014; Hideno 2016). These pretreatments include: physical treatments, such as ball milling and wet-disk milling; physicochemical treatments, such as hot compressed-water treatment and steam explosion; chemical treatments, such as organosolv or sulfuric acid treatments; and biological treatments, such as pulping by white-rot fungi.

Ball milling, the most basic and efficient technique, has been investigated by many researchers, although this process normally consumes considerable energy (Sun and Cheng 2002; Hideno et al. 2009; Avolio et al. 2012). The high enzymatic digestibility of ball-milled samples has been explained through their increased surface areas and decreased crystallinity (Sun and Chen 2002; Mosier et al.2005; Hendriks and Zeeman 2008).

Cellulases and hemicellulases are key enzymes in biorefinery processes such as fermentable sugars production (Himmel et al. 2007; Igarashi et al. 2011; Nonaka and Hideno 2014) and nanocellulose preparation (Pääkko et al. 2007) from pretreated biomass. Cellulases are enzymes that hydrolyze cellulose, and they generally consist of many enzyme molecules, including cellobiohydrolas I (CBH1), cellobiohydrolase II (CBHII), and endo--1,4 glucanase (EG).

Cellulase actions are strongly affected by the conditions and properties of the lignocellulosic biomass. Pretreatment processes such as ball milling and wet-disk milling are required to effectively hydrolyze lignocellulosic biomass by the cellulases (Himmel et al.2007; Hendriks and Zeeman 2008). The pretreatment probably affects not only the enzymatic digestibility but also the thermal-degradation behavior. However, the relationships between the enzymatic digestibilities and thermal-degradation behaviors of the pretreated lignocellulosic biomass are not well known.

Thermogravimetric analysis (TGA) has been widely used to characterize the thermal-decomposition behaviors of plant biomass (Antal, Jr. and Varhegyi 1995; Negro et al. 2003). In general, TGA is applied to obtain calorimetric results in the development of fuels such as wood pellets. To effectively use plant biomass, it is necessary to develop it not only as a fuel for direct combustion, but also to convert it into value-added chemicals through non-combustion technologies such as enzymatic hydrolysis.

In a previous report, it was suggested that the thermal degradation characteristics of ball-milled Japanese cypress may be related to its enzymatic digestibility, although further details were unavailable. It was also suggested that the differential thermogravimetric curves (DTG) of pretreated biomass are strongly affected by their composition and condition (Hideno 2016). Moreover, based on DTG data, it was indicated that the effects of ball milling on non-cellulosic substances, such as hemicellulose and lignin, were more significant than those on crystalline cellulose. We also previously described the enzymatic hydrolysis of two ball-milled Miscanthus species (M. giganteus and M. sinensis) (Hideno et al. 2013), which are promising bioenergy crops with high biomass production potential because they are C4 plants (Clifton-Brown et al. 2004; Anzoua et al. 2011).

It has been reported that the effects of ball milling on enzymatic digestibility differ for M. giganteus and M. sinensis. However, a detailed understanding of these differences was not developed. Although thermal analyses of Miscanthus plants have been published (Szabo et al. 1996; Elmay et al. 2015), the relationships between their thermal degradation behaviors and other degradation properties, such as enzymatic digestibility, have not been described in detail.

In this study, Miscanthus plants were thermogravimetrically characterized, and the relationships between the thermal degradation properties of the ball-milled grasses and their enzymatic digestibility were investigated in detail.

EXPERIMENTAL

Materials

M. sinensis and M. giganteus were used similarly in the author’s previous report (Hideno et al. 2013). Each sample was hand-cut to approximately 1 cm to 2 cm in length, and dry milled using a blender (ABSOLUTE3, Osaka Chemical Co., Ltd., Osaka, Japan). The dry-milled samples were electrically sieved (ANF-30; Nitto Kagaku Co., Ltd, Nagoya, Japan) for 30 min, and selected in size from 125 m to 500 m as the starting material.

Ball milling

Ball milling was performed using the same method described previously (Hideno et al. 2013). The sieved sample (approximately 3 g, 125 m to 500 m) and stainless balls (118 g) were placed in a stainless steel vessel, and ball milled at 400 rpm for 5 min to 240 min using a free-star ball-milling machine (Fritsch Japan Co., Yokohama, Japan), as shown in Fig. 1 (Hideno et al. 2013). These conditions were selected on the basis of our previous report.

Fig. 1. Schematic diagram of ball milling and representative ball-milled samples. The figure was modified from our previous report.

Methods

TGA

The TGA of the original and ball-milled Miscanthus samples was conducted based on the previous report (Hideno 2016). The sample (approximately 5 mg) was pressed and formed into a tablet (Ф 4.5 mm) by a hand-press machine. The TGA instrument (TG/DTA6200; Seiko Instrument Co., Chiba, Japan) was used under a nitrogen atmosphere at a flow rate of 100 mL min-1 based on a previous report (Uetani et al. 2014) as follows; room temperature (RT) to 110 °C (40 °C min-1), 110 °C for 10 min, 110 °C to 550 °C (10 °C min-1), 550 °C for 10 min. This range of temperatures is typically used for detecting the thermal degradation of cellulosic biomass. The thermogravimetric (TG) and DTG curves were plotted by calculations using Eqs. 1 and 2:

TG (%) = (thermal decomposition weight loss (g) / original weight (g)) × 100 (1)

DTG (% min-1) = TG (%) / time for increase in temperature (min) (2)

The weight of the sample at 120 °C was defined as 100% dry weight. Curve fitting for the peak separation in the DTG curves was accomplished using TA7000 software (Hitachi High-Tech Science Co., version 10.41, Tokyo, Japan) combined with Fityk (Fityk, version 0.9.4, Warsaw, Poland). The split Gaussian method and the Levenberg-Marquardt algorithm were used for the peak separation and fitting of the DTG curves, respectively.

Relationships between thermal degradation properties and enzymatic hydrolysis of ball-milled Miscanthus plants

Enzymatic hydrolytic data for the ball-milled Miscanthus plants were obtained in part from a previous report (Hideno et al. 2013) or by using the method from that report. These data were obtained by triplicate experiments. The AP-treated Miscanthus plants (final concentration 5% w/v) and the Accellerase® 1500 (10 mg-protein g-1; DuPont, Co., USA) were mixed well in the tube and they were incubated at approximately 50 °C for 72 h with agitation. The mixture was centrifuged at 20,000 g for 10 min. The supernatant was filtered out and subjected to high-pressure liquid chromatography equipped with an Aminex HPX-87P column and a Carbo-P micro-guard cartridge (BioRad, Hercules, CA, USA).

Correlation factors between the thermal degradation properties and enzymatic digestibility were calculated using Excel software (Microsoft Co., Excel 2013, Redmond, USA). Almost all of the ball-milled samples used in this study (M. giganteus: ball-milling times of 0, 30, 60, 120, and 180 min; M. sinensis: ball milled for 0, 30, and 60 min) were applied in the correlation factor calculations.

RESULTS AND DISCUSSION

Thermal Degradation Behavior of Raw and Ball-milled Miscanthus Plants

Judging from the TGA curves of the raw M. giganteus and M. sinensis (Fig. 2), the thermal decomposition of M. sinensis occurred at a lower temperature than that of M. giganteus. The thermal decomposition temperature of M. giganteus at 1% weight loss and the differential thermogravimetric peak were 225.5 °C and 341.5 °C, respectively, and were higher than those of M. sinensis at 209.6 °C and 327.2 °C, respectively. The DTG peaks at 341.5 °C and 327.2 °C were likely derived from the thermal degradation of cellulose. These differences in thermal degradation behavior between M. sinensis andM. giganteus are likely derived from their components. The cellulose, xylan, and lignin contents of M. giganteus are higher than those of M. sinensis (Hideno et al. 2013). Our results suggest that M. giganteushas more tolerance not only to physical and chemical pretreatment but also thermal degradation than M. sinensis.

The effects of ball milling on the DTG curves of the Miscanthusplants are shown in Fig. 3. Commonly observed features in the ball-milled samples were decreased peak heights and decreased thermal degradation temperatures, which shifted the DTG curves to the left.

Fig. 2. TG curves for M. giganteus and M. sinensis

Fig. 3. Changes in DTG curves for (a) M. giganteus and (b) M. sinensis during ball milling

In a previous report (Hideno et al. 2013), we showed that the enzymatic digestibilities of M. giganteus and M. sinensis were significantly increased by ball milling for more than 120 min and 60 min, respectively. In this study, the DTG curves of M. giganteus and M. sinensis are remarkably altered and shifted to lower temperatures after more than 120 and 60 min of ball milling, respectively. Thus, for both Miscanthus plants, the changes in their DTG curves with respect to ball-milling time can be correlated with the changes in their enzymatic digestibilities with respect to ball-milling time. Interestingly, the characteristic DTG shoulder peak of M. giganteusremained, and its area increased after ball milling, although the thermal-decomposition temperatures at the shoulder DTG peak’s top gradually decreased. For both samples, the DTG peak areas at lower temperatures (i.e., below 300 °C) gradually increased with increased ball-milling time. The shoulder peaks near 292 °C are probably derived from a hemicellulose, such as xylan, as previously suggested (Werner et al. 2014). In our previous report, the DTG peaks of Avicel (microcrystalline cellulose) were hardly changed by ball milling, although the crystallinity was lost after ball milling for more than 20 min (Hideno 2016). In contrast, the DTG peaks of both Miscanthusplants were remarkably affected by ball milling. This tendency of Miscanthus plants was the same as observed for ball-milled Japanese cypress (Hideno 2016). It is likely that not only crystalline cellulose but also portions of the hemicellulose and lignin components in the Miscanthus plants were broken and denatured by ball milling.

Separated and Curve-fitted DTG Curves of M. giganteus and M. sinensis

The DTG curves and curve-fitted DTG curves were compared for raw and ball-milled M. giganteus and M. sinensis (Fig. 4). These ball-milled samples were selected based on the results of the previous report (Hideno et al. 2013), which had shown remarkably improved enzymatic digestibility. Separating and curve-fitting the DTG peaks indicated that the DTG curves of the Miscanthus plants comprised of two main peaks with several trace peaks. As mentioned, M. giganteushad a characteristic DTG shoulder peak at approximately 292 °C while M. sinensis did not (Fig. 3). However, curve-fitting the DTG data for M. sinensis revealed a similar peak near 296 °C (Fig. 4 (b)), which was equivalent to the DTG shoulder peak of M. giganteus. In general, DTG curves derived from the thermal decomposition of xylan include two peaks: a small shoulder peak at approximately 230 °C, and a main peak at approximately 290 °C (Yi-Min et al. 2009; Collard and Blin 2014). The separated peaks at 292 °C (Fig. 4(a)) and 296 °C (Fig. 4(b)) were probably the main peak attributed to the thermal decomposition of xylan as speculated earlier. The author’s results indicated that the DTG peak derived from xylan in M. sinensiswas detected by separating and curve-fitting. Comparing the constituent sugars of both species, the glucose and xylose contents derived mainly from the cellulose and xylan in M. giganteus are higher than those in M. sinensis (Hideno et al. 2013). However, the ratio of xylan/cellulose in M. giganteus of approximately 0.45 was lower than that of M. sinensis (0.48). It may be possible to correlate not only the xylan content, but also the bonds between cellulose and xylan, with the DTG shoulder peak at approximately 292 °C to 296 °C. The area of each highest curve-fitted DTG peak could be correlated with the cellulose, similar to previous research results (Hideno 2016). As shown in Fig. 4, these main peaks gradually shifted to lower temperatures through ball milling, except for the separated peak at 301.7 °C in Fig. 4(c). The reason this peak shifted to higher temperature may have been due to the unexpected generation of chemical bonds between cellulose and hemicellulose by ball milling. The resistance towards the physicochemical treatment of M. giganteus was higher than that of M. sinensis judging from the ball-milling results (Hideno et al. 2013). Indeed, the thermal degradation temperature of M. giganteus was higher than that of M. sinensis. This may be related to the percentages of cellulose, xylan, and lignin in the grass because their contents in M. giganteus are greater than those in M. sinensis (Hideno et al. 2013). In particular, the lignin content is important for the thermal degradation of crystalline cellulose based on the previous report (Hilbers et al.2015). Alternatively, the amount of lignin and the bond strengths of the cellulose and hemicellulose may be discerned from the DTG curve. The author’s results indicate that the DTG curve and curve-fitted DTG curve can be applied as a markers to classify or select the grass species.