Biomass
Autohydrolysis pretreatment was performed at Brazilian Bioethanol Science and Technology Laboratory (CTBE) in a 350-L stainless steel batch reactor using 15 kg of raw bagasse at solid:liquid ratio of 1:10. The autohydrolysis pretreatment was performed at 190 °C during 10 min. After pretreatment, the liquid fraction was separated and the remaining solid fraction (pretreated bagasse) was washed with sufficient tap water to eliminate any soluble fractions until neutral pH was achieved. The washed solid in a wet state was stored in a cold room. Pretreated bagasse was characterized using a Laboratory Analytical Procedure published by National Renewable Energy Laboratory (NREL) [28] presenting the following composition (dry basis): glucan = 56.5 ± 0.3; xylan = 7.4 ± 0.2; total lignin = 28.1 ± 0.4; ash = 4.1 ± 0.2.
Mechanical refining (PFI refining and disc refining)
Mechanical refining was applied on the pretreated bagasse using both a PFI and disc refiner at North Carolina State University (NCSU). The PFI refiner is a batch processing piece of equipment where biomass is beaten between a roll with bars and a smooth-walled housing, both rotating in the same direction but at different speeds. The refining action is achieved through the differential rotational action and the application of loading between the roll and housing for a specified number of revolutions [29]. Each batch of refining was performed with 30 g (dry basis) of pretreated biomass at 10% insoluble solids content. Four refining intensities (2000, 4000, 6000 and 8000 revolutions) were evaluated.
The pretreated biomass was also refined using a 12-in. continuous disc refiner. The disc refiner is composed of two vertical disks with serrated and contoured surfaces. One disk rotates, while the other remains stationary. The pretreated biomass at 20% insoluble solids content was fed between the disks where a centrifugal force pushes the fibers out toward the perimeter of the disks. The abrasion experienced by fibers cuts, softens, rubs, and disperses them. The space between the disks can be widened or shortened to modify the refining intensity. In this study, two refining intensities were evaluated (disc gap = 0.002 and 0.005 in.).
The chemical composition of refined samples was assumed to be the same as the pretreated biomass presented in section “Biomass”. The mechanical refiners used in this study are closed-type equipment, where all the biomass processed is collected at the end of refining. In other words, there is no mass loss during the refining process, and therefore, no difference is expected in the composition between the pretreated biomass and refined samples.
Enzymatic hydrolysis
Enzymatic hydrolysis was performed at NCSU for all unrefined and refined samples in 50-mL tubes using 5 FPU/g (dry basis) of pretreated and washed substrate, 10% insoluble solids content, pH 4.8–5.0 and 50 °C. Novozymes Cellic CTec 2 supplemented with 1/9 Cellic HTec 2 was used as the enzyme cocktail. Sodium acetate buffer was used for pH control. The incubator (Fine PCR COMBI-D24) was maintained at 50 °C and 15 rpm. During enzymatic hydrolysis, samples were taken at 24, 48, 72 and 96 h. Each sample was centrifuged for 10 min at 4400 rpm using an Eppendorf Centrifuge 5702. The supernatant was used for sugar determination (glucose and xylose) and for calculation of carbohydrate conversion during enzymatic hydrolysis [30].
Macroscopic morphology (fiber quality analyzer and light scattering)
The HiRes fiber quality analyzer (FQA) from OpTest Equipment Inc. uses circular polarized light to measure the length and width of particles. During the measurements, a very dilute fiber suspension is pumped to a flow cell, where an infrared light source is located. The polarized light passes through the flow cell. If the polarized light strikes a fiber, a phase shift will occur, which will allow the light to pass through a second polarizer and reach the camera located on the opposite side of the flow cell. Only highly organized structure, such as cellulosic fibers, is able to cause a phase shift in the polarized light. Therefore, FQA will not detect air bubbles, ink or scale. Samples were diluted (~ 1 mg/L) and dispersed before each analysis using a British disintegrator for 15,000 revolutions. Particles with sizes ranging from 0.03 mm to 10.0 mm were measured and 10,000 particles were analyzed for each FQA run. Fiber width was measured for particles with size bigger than 0.2 mm. Particle length was measured as the true contoured length and reported as the length-weighted length (Lw).
$$\left( {L_{\text{w}} = \frac{{\mathop \sum \nolimits n_{i} L_{i}^{2} }}{{\mathop \sum \nolimits n_{i} L_{i} }}} \right),$$
where n is the number of fibers and L is the contour length.
Particle size distribution was evaluated by light scattering using a Beckman Coulter LS13320 instrument with Universal Liquid Module at CTBE. Particles with sizes ranging from 0.38 to 2000 µm were measured. Dilute aqueous suspensions of the refined particulates were injected into the instrument, passing through a window illuminated by 780 nm laser light. The scattered light is detected by 126 photodetectors distributed in scattering angle. The Fraunhofer optical model (which assumes spherical particles) encoded in the instrument software was employed to convert detected light intensities into particle size distributions. Unrefined pretreated bagasse could not be characterized by light scattering because particle lengths exceeded the instrument limit (2 mm).
Pore structure (water retention value and calorimetric thermoporometry)
Water retention value (WRV) was performed at NCSU to estimate the swelling capacity of fibers by measuring the amount of the water retained in a wet and swollen sample after centrifugation. With the centrifugation and consequent elimination of excess water located in fiber lumens and in spaces between adjacent fibers, the remaining water will be mostly located on the outer surfaces of fibers and within the cell wall. WRV measurements were executed according to the TAPPI standard procedure [31]. A pulp suspension was placed into a filtering glass tube of medium porosity (22 mm diameter) to yield 1400 g/m2 (approximately 0.5233 OD g). The filtering glass tube was centrifuged for 30 min at 0.9 relative centrifugal force to gravity. After centrifugation, samples were oven dried at 105 °C. The weights of the wet centrifuged sample (mwc) and the oven-dried sample (mod) were measured to calculate WRV = (mwc − mod)/mod.
Calorimetric thermoporometry using differential scanning calorimetry (DSC) was performed at CTBE to assess the pore area and porosity profile as a function of pore size. DSC experiments are able to differentiate three categories of water absorbed in biomass: non-freezing bound water (NFBW), freezing bound water (FBW), free water (FW) [31]. FW is bulk water and it is measured with ice melting at 0 °C. FBW is confined inside the biomass capillaries and it is measured with ice melting below 0 °C [32]. Water present in the capillaries has a depressed melting temperature due to the curved interfaces in cavities. This temperature has a relationship with the pore diameter, which allows the evaluation of pore size. NFBW is located at the first layers of water adjacent to the biomass surface. As the water movement is restricted by its association with the surface, NFBW does not freeze [32]. The main output from the technique is the FBW profile, given as cumulative distribution as a function of pore diameter in the range of 1–200 nm. Thermoporometry analysis was executed using a DSC TA Q200 with an auto sampler and RCS90 cooling unit, according to the procedure described previously [33] and recently updated [19].
Crystalline structure (X-ray diffraction (XRD), solid-state nuclear magnetic resonance (NMR), sum frequency generation (SFG) spectroscopy)
X-ray diffraction was used at NCSU to estimate the crystallinity index (CI) of the samples. The wide angle diffraction data were acquired using a Rigaku SmartLab X-ray diffractometer (CuKα radiation). The diffraction angle of 2θ was measured from 5° to 41° with a step size of 0.05° and 5 s of exposure at each step. Crystallinity index was calculated, based on the peak height method [20, 34] as the ratio between the estimated intensity of the crystalline peak (I200 − Iam) and the total intensity (I200). I200 is the maximum intensity of (200) lattice diffraction (2θ around 22.5°) and Iam is the intensity at the valley between (200) and (110)/(1–10) peaks (2θ around 18.4°), where intensity from amorphous components may have a notable contribution (in addition to the contribution from the tails of the adjacent peaks).
13C solid-state NMR measurements were carried out to evaluate crystalline structure of the samples using a Bruker Avance II 500 MHz with 4 mm MAS probe [35]. The instrument was operated at frequency of 125.76 MHz and spinning speed of 5 kHz. Signals were scanned 3000 times with pulse delay of 3 s and contact time of 2 ms.
The SFG experiment was performed at Pennsylvania State University using the broadband SFG spectroscopic system [36, 37]. The synchronized 800 nm and tunable infrared (2.5–10 µm) are needed to make the SFG process possible. The SFG intensity was normalized with the IR profile and each SFG spectrum was averaged from 4 different locations on each sample. The SFG spectra were collected from 2750 to 3650 cm−1.
Microscopic morphology (stereomicroscopy, confocal scanning laser microscopy (CSLM), and transmission electron microscopy (TEM)
The microscope work was performed at NREL. For stereo microscopic analysis, whole pieces of refined and unrefined samples were examined without further processing. Images were captured on a Nikon SMZ1500 stereomicroscope and captured with a Nikon DS-Fi1 CCD camera operated by a Nikon Digital Sight system (Nikon Instruments, Melville, NY).
For CSLM and TEM analysis, samples were preserved for structural characterization using microwave processing as described previously [38]. Briefly, samples were fixed in 2.5% gluteraldehyde buffered in 0.1 M sodium cacodylate buffer (EMS, Hatfield, PS) under vacuum. The samples were dehydrated with ethanol and acetone. After dehydration, the samples were infiltrated with LR White resin (EMS, Hatfield, PA) at room temperature for several hours to overnight. The samples were transferred to flat-bottomed capsules and the resin polymerized by heating to 60 °C for 48 h. LR White embedded samples were sectioned to ~ 300 nm for CSLM or ~ 60 nm for TEM with a Diatome diamond knife on a Leica EM UTC ultramicrotome (Leica, Wetzlar, Germany).
For CSLM imaging, the semi-thin-sectioned samples were positioned on glass microscope slides and stained with 0.1% acriflavine. Images were captured using a 60 × 1.4 NA Plan Apo lenses on a Nikon C1 Plus microscope (Nikon, Tokyo, Japan), equipped with the Nikon C1 confocal system, excited using 488 nm from a tunable Argon laser operated via Nikon’s EZ-C1 software.
For TEM imaging, the ultra-thin sections were positioned on 0.5% Formvar-coated copper slot grids (SPI Supplies, West Chester, PA). Grids were post-stained for 3 min with 2% aqueous uranyl acetate and 3 min with 1% KMnO4 to selectively stain for lignins. Images were taken with a 4 mega-pixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hilsboro, OR).