Bioenerg. Res. DOI 10.1007/s12155-017-9833-8
Study of the Ultrastructure of Eucalyptus globulus Wood Substrates Subjected to Auto-Hydrolysis and Diluted Acid Hydrolysis Pre-treatments and Its Influence on Enzymatic Hydrolysis Cristian Arévalo 1,2 & Juanita Freer 2,3 & Pamela A. Naulin 4 & Nelson P. Barrera 4 & Eduardo Troncoso 1 & Juan Araya 1,2 & Carlos Peña-Farfal 5 & Rosario del P. Castillo 1
# Springer Science+Business Media New York 2017
Abstract Dilute acid hydrolysis (DAH) and auto-hydrolysis (AU) have demonstrated to be optimal pre-treatments for the generation of biofuels from wood. Recent studies have highlighted the importance of ensuring the accessibility of cellulose enzymes during the enzymatic hydrolysis (EH) of pre-treated materials. In this work, the microscopic and nanoscopic structures of Eucalyptus globulus samples pretreated by AU and DAH were evaluated by different techniques to understand the effect of the ultrastructure of samples on the enzymatic conversion and cellulose accessibility for bioethanol production. Microscopic techniques revealed changes in the physical characteristics of pre-treated fibers, coalescence at microscopic level, and differences in the chemical distribution of lignocellulosic components depending on the severity and type of pre-treatment. The atomic force microscopy-based nanoscopic study of samples showed Electronic supplementary material The online version of this article (doi:10.1007/s12155-017-9833-8) contains supplementary material, which is available to authorized users. * Rosario del P. Castillo
[email protected] 1
Laboratory of Infrared Spectroscopy and Chemometrics, Department of Instrumental Analysis, Faculty of Pharmacy, University of Concepcion, Concepción, Chile
2
Biotechnology Center, University of Concepcion, Concepción, Chile
3
Faculty of Chemical Sciences, University of Concepcion, Concepción, Chile
4
Laboratory of Structural Biology and Nanophysiology, Department of Physiology, Pontificia Universidad Católica de Chile, Santiago de Chile, Chile
5
LABEL, Faculty of Pharmacy, University of Concepcion, Concepción, Chile
differences in the effect of the pre-treatments on the ultrastructure of samples, with DAH pre-treatment producing major changes in the secondary cell wall with respect to AU samples at comparable severities, and a positive effect of the DAH ultrastructure changes to increase the EH yield. Keywords FT-IR microimaging . Atomic force microscopy . Eucalyptus globulus . Wood ultrastructure . Auto-hydrolysis . Diluted acid hydrolysis
Introduction The use of non-agricultural lignocellulosic materials for the production of second-generation biofuels, such as bioethanol, is a sustainable alternative to reduce our dependence on fossil fuels and decrease the greenhouse effect produced by petroleum derivatives [1]. Among these materials, lignocellulosic biomass, such as agricultural residues (e.g., corn stover and wheat straw), forestry wastes, wastepaper, yard waste, and wood, may be used to produce bioethanol. Lignocellulosic biomass is a highly heterogeneous material. The structure of woody feedstock is complex; the cell walls are composed of cellulose and hemicellulose held together by lignin, which change depending on the type, age, and part of the wood source. Wood can be converted into ethanol by applying a pre-treatment to disrupt its complex structure, followed by the enzymatic hydrolysis (EH) of cellulose to obtain glucose. In this process, the interactions among the three components (i.e., cellulose, hemicellulose, and lignin) are the limiting factors for obtaining fermentable sugar [2]. Pre-treatments are applied to wood to decrease its recalcitrance to EH. The pre-treatments may alter the content, location, and molecular weight of lignin; hydrolyze
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hemicelluloses; decrease the crystallinity of cellulose; or increase the pore size or surface area [3, 4]. Because of the natural recalcitrance of wood, the major focus of industrial engineering approaches is on developing low-cost, lowenvironmental repercussion strategies for cellulose depolymerization and effective saccharification [4–6]. Regarding the factors that affect EH, minimal efforts were exerted to evaluate the physical characteristics of cellulosic materials and elucidate the enzymatic mechanism of hydrolysis some decades ago [5]. Studies examining the potential correlation between the recalcitrance of biomass and various physical or chemical properties were conducted. For example, the effect of the removal and re-localization of lignin on the enzyme’s accessibility to the fibers’ surfaces and the effect of the presence of hemicelluloses on the EH yield (EHY) have been investigated [7, 8]. Other factors, such as the porosity of the substrate relative to the size of the enzyme, may also be limiting factors of EH [1, 5, 9, 10]. Geleynse et al. [11] developed a method to determine biomass recalcitrance in Douglas fir at three levels: chemical composition, pre-treatment yield, and sugar release from the enzymatic hydrolysis; however, they show that trees did not follow any major trend between any particular factor and the overall degree of recalcitrance. Thus, the multi-level data generated from this type of screening can be used to identify specific specimens which exhibit particularly unusual traits in regards to recalcitrance, but more in-depth studies of the physical and chemical differences of trees are necessary. Evidence suggests that increasing the cellulose accessibility to cellulase (CAC) is particularly critical for improving EH [12–14]. CAC is considered a key factor for the efficient bioconversion of lignocellulosic to fermentable sugars. CAC has been evaluated by several methods that measure porosity (i.e., the physically accessible volume) using probing molecules, such as water (i.e., the water retention value [WRV] method), or by differential scanning calorimetry and nuclear magnetic resonance (NMR) porosimetry [14–16]. Other methods are based on measuring the adsorption of molecules on a lignocellulosic substrate. The molecules that have been used in these methods include nitrogen (the Brunauer-Emmett-Teller [BET] method) [17], dyes (Simon’s staining method) [5, 18], and protein or cellulose, which are used to determine the amount of enzymes adsorbed by the lignocellulosic substrate [19]. These methods are indirect measurements of CAC and appear to be good approaches on the surface. However, despite their application, the chemical characteristics of the substrates remain uncertain. Moreover, the distribution of lignocellulosic components on the surfaces of the substrates after pre-treatment is unknown. Thus, although the potential of cellulose as biomass is acknowledged, a better understanding of the biomass morphology, structure, and chemical composition on the microscale and nanoscale is necessary to optimize the production processes [20]. The ultrastructure of lignocellulosic compounds can be evaluated using diverse methods [21]. Currently, scanning
electron microscopy (SEM) and transmission electron microscopy (TEM) are used for the nanoscale characterization of lignocellulosic materials [22–24]. The images acquired using these techniques can reveal microfibrils and successive layers within the cell walls. Additionally, atomic force microscopy (AFM) has been widely used in studies of lignocellulosic materials due to the versatility of this technique [20, 23, 25–27]. AFM can work on soft and hard surfaces, from micrometric to nanometric scale, in air or liquid environment, and data collection is tridimensional (x-, y-, and z-axes) (for more details and application of AFM, see review [28]). Furthermore, AFM can be used in topographic studies, such as height and roughness measurements, to determine changes on surface morphology after application of different pre-treatments [20] and spectroscopic studies to evaluate mechanical and chemical characteristics through quantifying the adhesion force between the tip (naked or chemically functionalized) and the surface [29–32]. Compared to the other techniques, adhesion force measurements via AFM are largely unaffected by macroscopic chemical heterogeneity and roughness, and are surface specific [33]. It has been shown that AFM is capable of identifying microstructural variability in the polarity of the wood surface [29–31]. On the other hand, Kristensen et al. [27] used AFM and infrared spectroscopy to evaluate steam exploited samples of wheat straw. It was found that the vast majority of all fiber surfaces (more than 90%) were covered with a layer of globular deposits. These deposits were established to be re-localized lignin. Upon delignification of pre-treated fibers, the cellulose fibrillar structure of the cell walls was found to be intact. The skeletal structure conservation of the cell wall through pretreatment is not in accordance with the general perception that pre-treatments must disrupt the cell wall in order to increase its accessibility to enzymes. Therefore, in the present study, we investigated the microscopic and nanoscopic structures of lignocellulosic substrates subjected to dilute acid hydrolysis (DAH) [34] and auto-hydrolysis (AU) pre-treatments to characterize the ultrastructure of these substrates and their influence on the EH process. The topochemical characteristics of the pretreated wood samples were evaluated by diverse analytical and microscopic techniques, including SEM, confocal laser scanning microscopy (CLSM), Fourier transform IR (FT-IR) microspectroscopy with multivariate techniques (FT-IR imaging), and nanoscopic analysis by AFM. Results can be useful to understand the CAC of substrates for bioethanol production.
Experimental Samples and Pre-treatments Wood chips of Eucalyptus globulus (approximately 2 cm × 2 cm × 0.2 cm) were used as the feedstock for the pre-treatments (i.e., DAH and AU). These pre-treatments were
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conducted under different experimental conditions obtained from experimental designs based on the conditions reported by Marzialetti et al. [35] in the case of auto-hydrolysis and Amenaghawon et al. [36] in the case of diluted acid hydrolysis (DAH). In both cases, the ranges of applied conditions were oriented to get suitable pre-treated materials destined to biofuel production and to get some similarities in the chemical composition of some samples from the two pre-treatments, especially regarding the cellulose availability in order to compare differences in the ultrastructure. The severity factor (R0) was calculated according to Eq. (1) [37] as a global value including heating, net reaction, and cooling stages for each reaction, where T is the temperature (°C) for each time t, Tref = 100 °C, ω = 14.75 °C, and tf is the pre-treatment total time in minutes. Owing to the wide variation range of R0, the severity was expressed in terms of S0 as is defined in Eq. (2). R0 ¼ ∫0 expð tf
T−T ref ω
Þ dt
S 0 ¼ logðR0 Þ
ð1Þ ð2Þ
Table 1 shows the conditions used for each pre-treatment with the respective value of S0 and the percentage of the integrated area of Eq. (1) corresponding to the net reaction (without cooling and heating steps). In the case of AU, severities lower than that reported in this table showed wood practically intact while severities higher than that reported decrease the solid recovery from the samples.
Wood Characterization Pre-treated wood was characterized by conventional methods, with samples subjected to two hydrolysis steps—4% H2SO4 at room temperature followed by 72% H2SO4 at 121 °C—as described by Castillo et al. [3]. The concentrations of carbohydrates (glucose, cellobiose, and hemicelluloses) as well as acetic acid in the soluble fraction were determined by highperformance liquid chromatography (HPLC) in a Merck Hitachi instrument with an Aminex HPX-87H column at 45 °C and eluted at 0.6 mL min−1 with 5 mM H2SO4, using a refractive index detector. Glucose, cellobiose, acetic acid, and xylose were used as external calibration standards. The factors used to convert sugar monomers to anhydromonomers were 0.90 for glucose to glucan and 0.95 for cellobiose to glucan. The sum of the anhydromonomers from glucose and cellobiose gave the amount of total glucan in wood. The acetyl concentration was calculated by multiplying acetic acid content by a factor of 0.72. The hemicelluloses were measured from one peak (which includes xylose, mannose, and galactose) and converted to monomeric units using a factor of 0.88. The insoluble lignin content was determined by gravimetric method by recovering the solid residue on the Gosh filter. The soluble lignin content
was determined by an absorciometric method measuring the absorbance of the liquid fraction at 205 nm and considering the extinction coefficient of lignin which is 110 g L−1 cm−1. Insoluble and soluble lignin was added to obtain the total lignin content. Lignin, glucans, hemicelluloses, and acetyl content were expressed as weight percentages with respect to the dry weight of the pre-treated material (% w/w). Enzymatic Hydrolysis Portions of the wet pre-treated samples equivalent to 3 g (with precision of 0.0001) of the dry weight were weighed in 50-mL flasks. EH was performed with a commercial preparation of the cellulase enzyme complex (NS-22128 CCN03128; 71 FPU mL−1, Novozymes A/S, Bagsværd, Denmark), supplemented with β-glucosidase (NS-22128 DCN00216; 265 CBU mL−1, Novozymes A/S, Bagsværd, Denmark). The amounts of enzyme were 20 FPU of cellulase and 40 CBU of β-glucosidase per gram of pre-treated material. EH was performed in an air shaker at 60 °C with constant shaking. Samples were collected at 24, 36, 48, and 72 h to estimate the maximal experimental yield. Seventy-two hours was chosen to report the EHY determined in terms of the glucose content measured by high-performance liquid chromatography (HPLC) using an Aminex HPX-87H column at 45 °C and 5 mmol L−1 H2SO4 as the mobile phase with a flow rate of 0.6 mL min−1. The EHY was reported as the percentage of glucose in the sample collected at 72 h relative to the glucose content in the pre-treated material (characterized as described in the BWood Characterization^ section). Experiments were conducted in triplicate for each sample, and the average of the triplicate measurements is reported. FT-IR Microimaging: Image Acquisition and Chemometric Analysis The acquired microscopic IR images were analyzed according to the methodology described by Castillo et al. [38]. First, fibers of dried pre-treated materials were analyzed using an FT-IR Spectrum Frontier/Spotlight 400 Microscopy System (PerkinElmer, Inc.) with a linear array of mercury cadmium telluride (MCT) detectors. Areas between 50 μm × 50 μm and 200 μm × 200 μm were scanned in attenuated total reflectance (ATR) mode, with germanium crystal as the highreflective material. The pixel size of the images was 1.56 μm × 1.56 μm. The spectral range was 4000–748 cm−1, and the spectral resolution was 8 cm−1. Sixteen scans were acquired per spectrum. The spectra extracted from the images were submitted to the removal of atmospheric noise and baseline correction and were transformed to absorbance units before chemometric analysis. The images were subjected to multivariate analysis via principal component analysis (PCA) to detect the chemical components on the surfaces of the pre-
Bioenerg. Res. Table 1 Set of experiments of DAH and AU pre-treatments on Eucalyptus globulus chips
Pre-treatment DAH
AU
a
Experiment code
Conditions
%S.R.a
S0
Area of net reaction (%)b
DAH-1
120 °C/0.10%/1:4
97.9
3.22
67.0
DAH-2 DAH-3
170 °C/0.10%/1:4 120 °C/0.75%/1:4
83.3 97.6
4.72 3.26
71.1 68.9
DAH-4 DAH-5
170 °C/0.75%/1:4 120 °C/0.10%/1:8
65.9 97.4
4.74 3.33
72.9 77.4
DAH-6
170 °C/0.10%/1:8
82.6
4.71
72.8
DAH-7
120 °C/0.75%/1:8
94.9
3.24
71.45
DAH-8 DAH-9
170 °C/0.75%/1:8 145 °C/0.43%/1:6
73.7 94.1
4.83 4.02
65.7 67.3
DAH-10 DAH-11
113 °C/0.43%/1:6 177 °C/0.43%/1:6
96.8 71.0
2.76 5.03
75.9 70.9
AU-1
190 °C/70 min
66.1
5.89
89.4
AU-2 AU-3
175 °C/81 min 160 °C/70 min
64.1 81.0
5.46 4.93
92.8 90.9
AU-4 AU-5 AU-6
190 °C/15 min 160 °C/15 min 175 °C/4 min
70.9 94.1 86.4
5.33 4.40 4.39
78.4 76.0 63.2
Solid recovery percentage
2
Calculated as percentage of area of net reaction at maximum temperature with respect to the total integrated area of Eq. (1)
treated materials. PCA was applied using Spectrum Image (PerkinElmer Inc.) software, and the singular value decomposition (SVD) algorithm was performed in MATLAB (MathWorks, Inc.) after unfolding a hypercube with two spatial coordinates and one spectral coordinate in a matrix and the mean centering of the data for pre-processing. Confocal Laser Scanning Microscopy Microscopic images were obtained via CLSM following a previously described method in [38] using a confocal spectral Zeiss LSM 780 microscope with an Ar 488-nm laser and a Plan-Neofluar 20×/0.50 objective. Emission signals were collected in the range of 490–530 nm. Fibers were suspended in nanopure water and shaken before analysis to disaggregate the pulps into individual fibers. Drops of this suspension were placed on glass slides for analysis. Cross cryostat sections of fibers were analyzed under the same laser and emission conditions but using a 40×/0.50 objective.
Resin 828, and images were acquired in tapping mode; scan areas were within a range of 1 or 5 μm2 with a resolution of 512 × 512 pixels (pixel size is variable depending on scan area size, 1.9 and 9.7 nm2, respectively) with drive frequency ∼318 kHz, target amplitude 0.5 V, drive amplitude ∼100 mV, scan rate 0.5 Hz, gain 5, and set point ∼350 mV to obtain four different channel information: height, Zsensor, amplitude, and phase. Subsequently, atomic force spectroscopy was performed to topographic images of 5 μm2 to obtain adhesion force maps. Five maps were obtained from five different areas per sample. Scan areas had a resolution of 100 × 100 pixels (pixel size was 50 nm2). The parameters of force-distance curves were as follows: force distance 0.5 μm, scan rate 0.5 Hz, and trigger point 0.5 V. Force map analyses were focused on structures containing particular distributions of lignin or cellulose, cell wall, lamella media, and coalescent materials. AFM analyses were applied also on native wood in order to see the main changes in chemical component distribution after pre-treatments.
Atomic Force Microscopy
Results and Discussion AFM images were acquired in an AFM microscope (Asylum Research MFP-3D-SA) using Asylum silicon tips AC160TS with resonance frequency of 300 kHz, elasticity constant (k) of 42.0 N/m, and radius of 9 ± 2 nm. Ultrathin (
