Effect of mechanical disruption on the effectiveness of three reactors used for dilute acid pretreatment of corn stover Part 2: morphological and structural substrate analysis
© Ciesielski et al.; licensee BioMed Central Ltd. 2014
Received: 27 November 2013
Accepted: 17 March 2014
Published: 1 April 2014
Lignocellulosic biomass is a renewable, naturally mass-produced form of stored solar energy. Thermochemical pretreatment processes have been developed to address the challenge of biomass recalcitrance, however the optimization, cost reduction, and scalability of these processes remain as obstacles to the adoption of biofuel production processes at the industrial scale. In this study, we demonstrate that the type of reactor in which pretreatment is carried out can profoundly alter the micro- and nanostructure of the pretreated materials and dramatically affect the subsequent efficiency, and thus cost, of enzymatic conversion of cellulose.
Multi-scale microscopy and quantitative image analysis was used to investigate the impact of different biomass pretreatment reactor configurations on plant cell wall structure. We identify correlations between enzymatic digestibility and geometric descriptors derived from the image data. Corn stover feedstock was pretreated under the same nominal conditions for dilute acid pretreatment (2.0 wt% H2SO4, 160°C, 5 min) using three representative types of reactors: ZipperClave® (ZC), steam gun (SG), and horizontal screw (HS) reactors. After 96 h of enzymatic digestion, biomass treated in the SG and HS reactors achieved much higher cellulose conversions, 88% and 95%, respectively, compared to the conversion obtained using the ZC reactor (68%). Imaging at the micro- and nanoscales revealed that the superior performance of the SG and HS reactors could be explained by reduced particle size, cellular dislocation, increased surface roughness, delamination, and nanofibrillation generated within the biomass particles during pretreatment.
Increased cellular dislocation, surface roughness, delamination, and nanofibrillation revealed by direct observation of the micro- and nanoscale change in accessibility explains the superior performance of reactors that augment pretreatment with physical energy.
KeywordsBiomass conversion Dilute acid pretreatment Severity factor Quantitative image analysis Delamination Nanofibrillation
Lignocellulosic biomass is a renewable, naturally mass-produced form of stored solar energy, and interest in its cost-effective conversion to a liquid fossil fuel alternative has increased steadily over the last decade . Challenges to widespread industrialized biomass conversion processes are presented by the structure and composition of the plant cell wall: this complex, composite material comprises multiple biopolymers that impart structural integrity as well as chemical and biological resistance. This inherent recalcitrance greatly impedes enzymatic digestion of the cellulose in biomass and increases conversion costs . Several different thermochemical pretreatment processes have been developed to address this challenge, however the optimization, cost reduction, and scalability of these processes remain as obstacles to the adoption of biofuel production processes at the industrial scale . The process variables used in current pretreatment technologies include concentration of chemical additives, pH, temperature, and reaction time. The performance and costs of pretreatment are directly related to the choice of pretreatment technology and process conditions as well as the maximum expected sugar yield . Additionally, as we demonstrate in this study, the type of reactor in which pretreatment is carried out can also profoundly alter the micro- and nanostructure of the pretreated materials and dramatically affect the subsequent efficiency, and thus cost, of enzymatic conversion of cellulose.
Here we investigate three reactor configurations that are commonly used for pretreatment of biomass at the National Renewable Energy Laboratory (NREL): the ZipperClave® (ZC), steam gun (SG), and horizontal screw (HS) reactors. A more detailed comparison of these reactors is presented by Wang and colleagues in Part 1 of this study. In brief, all three reactors facilitate high temperature, pressurized, dilute acid catalyzed processes, but employ distinct operational modes: the ZC and SG are batch-type reactors wherein the biomass is pretreated and discharged at the end of pretreatment; the HS reactor is a flow-through process in which acid-impregnated biomass is continuously fed into the reactor. With both the SG and HS reactors, the pressurized biomass is explosively decompressed to ambient conditions at the end of pretreatment, while the ZC releases steam pressure into a heat exchanger gradually through a globe valve.
Composition of pretreated samples and percentage of glucan released
Composition of pretreated (2% H2SO4, 160°C, 5 min) samples
Glucan released (%)
Results and discussion
Corn stover biomass pretreats differently under the same nominal conditions (2 wt% H2SO4, 160°C, 5 min) in differently designed pretreatment reactors
The motivation for the rest of the analysis presented here is based on the large range in digestibility of the pretreated corn stover samples from different pretreatment reactors. After 96 h of enzymatic digestion, biomass treated in the SG reactor and HS reactors achieved much higher cellulose conversions (88% and 95%, respectively), compared to 69% for the ZC pretreated material. All of the pretreatments did a reasonable job of depolymerizing and removing xylan and none of the minor differences in the composition of the pretreated samples is enough to explain the difference in digestibility (Table 1).
At the macro-scale, pretreated biomass particles exhibit color changes, clumping, and particle size variability
These data confirm that pretreatment in all three reactors has reduced the overall particle size distribution measured as the area of a two-dimensional projection of the biomass particles and eliminated the bimodal particle size distribution found for the control material (Figure 1a”). Treatment in the SG and HS reactors in particular have shifted the distribution of biomass particles to <1 mm2. The observation that the ZC reactor leaves long fibers intact is also confirmed by the particle aspect ratio analysis (Figure 1b”), where more particles with an aspect ratio greater than 10 were found.
By this analysis, the HS reactor succeeded in producing the smallest and most consistently size-reduced particles. Smaller particles, by definition, will have an increased surface area that should promote digestibility. Also, smaller particles with low aspect ratios should have improved mixing behavior. Particles with higher aspect ratios, such as those treated in the ZC reactor, have been shown to impede mixing and reduce the consistency of pretreatment and saccharification within larger batches and at higher solids loadings [7, 8].
Micro-scale evaluation reveals changes in cell wall thickness, cell-cell dislocations, and cell wall fragmentation
Surface analysis reveals changes in cell wall roughness due to pretreatment
A surface plot visualizing the surface roughness and a chart displaying the averages of the eighteen analyzed regions is shown in Figure 4. The changes in surface roughness measured by this technique trend strongly with the changes in digestibility among the reactor samples and may contribute an important characteristic of effective pretreatment.
Extensive delamination and increasing intra-cell wall void space is evident at the nanoscale
Correlations between structural properties and digestibility
Glucan release (%)
Xylan content (%)
Lignin content (%)
Degree of polymerization
Cell wall thickness
Correlation coefficient (R)
Correlation coefficient squared (R2)
Description of the biomass feedstock handling, dilute acid pretreatment in each of the different reactors, compositional analysis, and enzymatic digestibility are described in detail in the companion study, ‘Effect of mechanical disruption on the effectiveness of three reactors used for dilute acid pretreatment of corn stover Part 1: chemical and physical substrate analysis’ in this issue.
Approximately 100 mg of dried biomass sample was placed inside a plastic envelope and spread to minimize particle overlap or contact. The particles were scanned using an Epson Stylus Photo RX500 (Seiko Epson, Nagano, Japan) flatbed scanner set at 2,400 dpi and captured as TIFF files.
Control and pretreated corn stover samples from the three reactors (ZC, SG, and HS) were examined wet and without further processing by stereomicroscopy. A subset of each sample was washed and dried by freeze-drying and imaged dry to reanalyze clumping. 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, USA).
Scanning electron microscopy (SEM)
Imaging by SEM was performed using a FEI Quanta 400 FEG instrument (FEI, Hillsboro, OR, USA) under low vacuum (0.40 to 0.65 Torr) operating with the gaseous solid-state detector (GAD) collecting secondary electrons. Samples were washed and prepared for imaging by freeze-drying. Dry samples were mounted on aluminum stubs using carbon tape and sputter coated with 6 nm of gold. Imaging was performed at beam accelerating voltages from 12.5 to 25 keV.
Sample preparation for microtomy
Pretreated corn stover samples were prepared using microwave processing for electron microscopy as described previously . Briefly, samples were fixed 2 × 6 min (with intermittent power) in 2.5% glutaraldehyde buffered in 1X PBS buffer (EMS, Hatfield, PA, USA) under vacuum. The samples were dehydrated with increasing concentrations of acetone and infiltrated with Eponate 812 resin (EMS) by incubating at room temperature for several hours to overnight in increasing concentrations of resin. The samples were transferred to flat-bottomed capsules and the resin polymerized at 60°C overnight. Embedded samples were sectioned to 300 nm for light microscopy and to approximately 60 nm for TEM with a Diatome diamond knife (Diatome, Hatfield, PA, USA) on a Leica EM UTC ultramicrotome (Leica, Wetzlar, Germany).
Confocal scanning laser microscopy (CSLM)
Semi-thin (300 nm) sectioned samples were positioned on glass microscope slides and stained with 0.1% acriflavine for confocal scanning laser microscopy of cell walls. Images were captured using a Nikon C1 Plus microscope (Nikon, Tokyo, Japan), equipped with the Nikon C1 confocal system with four lasers (403 nm, 561 nm, 643 nm, and Argon tunable 458/477/488/515 nm), and operated via Nikon’s EZ-C1 software.
Transmission electron microscopy (TEM)
Ultra-thin sections were positioned on 0.5% formvar coated copper slot grids (SPI Supplies, West Chester, PA, USA). Grids were post-stained for 1 min with 1% aqueous KMnO4. Images were captured with a 4 megapixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA, USA) on a FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI).
Macro-scale particle geometry descriptors
Particle size was characterized by image processing to calculate size and shape descriptors including area, perimeter, aspect ratio, and roundness. Similar methods for quantifying the size and shape distribution of biomass particles using image analysis have been reported previously . In this study, images were thresholded to segregate biomass particles from background and the resulting binary file was processed by watershed analysis to disconnect remaining overlapping particles. Then, the Analyze Particles tool in ImageJ was employed to calculate and output the size and shape descriptors of the biomass particles . A custom MATLAB (MathWorks, Natick, MA, USA) script was used to generate the histograms and tables presented in Figure 1 from the text output from ImageJ particle analysis. The numbers of biomass particles analyzed were 3,899 (control), 8,530 (ZC), 9,333 (SG), and 6,769 (HS).
Cell wall thickness
yields a measurement of cell wall thickness (denoted as CWT) at each pixel in MAT(x,y). By this method, thousands of cell wall thickness measurements are obtained from each image. A graphical display of the key image processing steps used to obtain these measurements is presented in Additional file 1: Figure S1. In the case of disjoined cells, like those shown in Figure 3c, the measurement represents one half the actual cell wall thickness. For adjoined cells, the distance map value is the cell wall thickness for each individual cell. These image operations were performed with ImageJ, and the numbers of cells and individual measurements of their walls were 13,183 measurements of 54 cells (control), 15,959 measurements of 50 cells (ZC), 16,259 measurements of 51 cells (SG), and 20,179 measurements of 52 cells (HS), respectively.
SEM roughness factor
A quantitative estimate of variations in surface roughness may be simply calculated as the standard deviation in pixel intensity from selected and normalized regions of interest from SEM micrographs as demonstrated previously by the analysis of surface roughness of paper fibers . While this is not as direct a measurement of surface roughness as that provided by scanning probe techniques, the contrast generated in an SEM micrograph is directly related to changes in slope of the particle surface , and thus provides a relative measure of exposed surface area within a sample set. Six 0.5 μm square regions of interest were analyzed from each of three separate SEM micrographs from each sample condition. Pixel values within selected ROIs were re-scaled to maximize the dynamic range of grayscale values from 0 to 255 to normalize ROIs for quantitative comparison.
Intra-cell wall void space and nanofibrillation
where T is the threshold value, and and σ v are the mean and standard deviation, respectively, of the pixel values from a known void region. Graphical examples showing example ROIs of intra-wall regions, as well as the binary images determined using this thresholding method, are shown in Figure 6b.
Increased dislocation, surface roughness, delamination, and nanofibrillation revealed by direct observation of the micro- and nanoscale change in accessibility explains the superior performance of the SG and HS reactors. Reactor designs that augment pretreatment with physical disruption better overcome biomass recalcitrance. We propose that the enhanced disintegration seen in biomass from the SG and HS reactors is primarily due to mechanical work done on the biomass by rapidly expanding fluid. Both the SG and HS reactors employ an explosive discharge of the pretreated solids. In addition, the HS reactor has a continuous screw feed that imparts mixing and mechanical sheer. This mechanophysical energy disrupts cross-linked matrices and, in addition to acid hydrolysis and temperature, results is an unprecedented degree of cell wall nanofibrillation and explains the increased xylan removal.
Confocal scanning laser microscopy
National Renewable Energy Laboratory
Region of interest
Scanning electron microscopy
Transmission electron microscopy
The pretreatment, digestibility, and surface characterization work was supported by the US Department of Energy Bioenergy Technologies Office (BETO). The quantitative image analysis development was supported as part of the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, Award Number DE-SC0000997. NREL is a national laboratory of the US Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.
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