- Open Access
Effects of hot-washing process on structure and enzymatic hydrolysis of treated steam explosion corn stover
© The Author(s) 2016
Received: 24 April 2016
Accepted: 28 July 2016
Published: 18 August 2016
Cellulase adsorption of lignocellulosic materials is the key link during enzymatic hydrolysis. Hot-washing process (above lignin glass transition temperature) was used to change the physical structure of lignin, decrease covalent connection between cellulose and lignin, reduce the concentration of inhibitor, and explore the feasibility of enzymatic hydrolysis. The general objective of the paper was conducted to determine whether the hot-washing process has the potential to change the mechanism of lignin on enzyme hydrolysis.
Hot-washing was carried out at 151 °C for 20 min. The ratio of acid insoluble lignin to acid soluble lignin was increased, while the formation of spherical lignin droplets on the cell wall surface was decreased. Enzymatic digestibility of hot-washed filter cakes showed enhanced digestibility over the control samples. The concentration of fermentation inhibitor (acetic acid, formic acid, furfural and 5-hydroxymethylfurfural) obviously decreased after hot-washing process.
Hot-washing process significantly increased the adsorption ability of cellulase on the substrates and digestibility of biomass without removing much of the insoluble lignin content. Lignin distribution and/or physical property composition play a role.
Lignocellulosic biomass is recognized as a high-potential feedstock for bio-ethanol production. It contains polymers of cellulose, hemicellulose, and lignin which bound together in a complex structure. Ethanol can be made from cellulose via four main consecutive steps: pretreatment, enzymatic hydrolysis, fermentation, and separation. One of the major limitations in the cellulosic ethanol production is the enzymatic hydrolysis step (Sun and Cheng 2002), where fermentable sugars are released from biomass using enzymes. The success of enzymatic hydrolysis depends on the accessibility of the cellulose component to hydrolytic enzymes (Stauner et al. 2013). In steam explosion pretreatment, hemicellulose is solubilized, whereas lignin and cellulose remain mostly insoluble. Residual lignin consolidating the polysaccharide matrix interferes with the action of enzyme, leading to decreased hydrolysis yields and rates (Zhang et al. 2015). Enzyme adsorption onto lignin surface is considered as a major inhibitory mechanism in the enzymatic hydrolysis of cellulose (Sun et al. 2014), which is largely determined by the glassy state structure of lignin (Piccolo et al. 2010). Many pretreatment strategies, such as hot-water pretreatment, focus on removing a significant fraction of lignin from biomass to enable better hydrolysis. However, lignin is partially depolymerized and solubilized during hot-water pretreatment, but complete delignification is not possible, because of the recondensation of soluble components originating from lignin (Alvira et al. 2010).
The “hot-wash” concept developed at National Renewable Energy Laboratory (NREL) uses hot water or hot dilute acid at temperatures above the lignin liquid/glass condensation temperature (T g) to wash out the solubilized lignin. The previous works have characterized the glass transition behavior of lignin, where it transforms from a hard or glass-like state into a rubbery or viscous state upon heating (Ko et al. 2015). Hot-water pretreatments reaching temperatures above the range for lignin phase transition cause lignin to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls upon cooling (Donohoe et al. 2008). During cooling after pretreatment, coalesced lignin could harden and either became trapped within the cell wall layers or settle out of the bulk liquid phase, potentially depositing back onto the biomass surface. Typically, the solid fraction is separated by filtration. The solid fraction allows solubilized lignin to condense and precipitate out on the cellulosic residue interfering with the enzymatic hydrolysis of cellulose to glucose (Selig et al. 2007). Flow-through hot-water pretreatment, where the solids residence time is longer than that of the liquid, has been shown to effectively dissolve more lignin compared with pretreatments, in which liquid and solids have the same residence time (Mosier et al. 2005). This method also generates solids that are more reactive on enzymatic hydrolysis, because of removing the solubilized lignin. However, continuous flow-through operation is thought to use an excessive amount of water and energy (Liu and Wyman 2005).
In this study, a pressure filter reactor was designed for solid–liquid separation during hot-water treatment. The separation temperature was above T g. Experiments were conducted to determine if the observed droplet formations do emerge from the biomass into the bulk liquid phase during pretreatment and whether or not they deposit back onto the biomass surface after pressure filtration. To understand the mechanism of lignin on enzyme hydrolysis, it is crucial to investigate the adsorption of cellulase on pretreated substrates. In addition, experimental sets were run in an attempt to determine whether the hot-washing process has the potential to affect the enzymatic saccharification of cellulose in the pretreated biomass.
Steam explosion corn stover (ECS) was supplied by Henan Tianguan Group Co., Ltd. The composition of raw and pretreated solids was analyzed following the NREL standard procedures (Sluiter et al. 2008). All the experiments were performed in duplicate, with the average value reported. The pretreated solids were air-dried and milled to pass 40 mesh screen for compositional analysis.
Pretreatment of ECS
Enzymatic hydrolysis of cellulosic substrates
Cellic CTec 2 (the cellulase product for the production of cellulosic ethanol from Novozymes) was measured to have a cellulolytic activity of 117 FPU/mL as determined in our laboratory. Filter paper activity (FPA) measurement was carried out according to QB2583-2003.
Enzymatic hydrolysis was performed in 0.05 M sodium acetate buffer solution (pH 4.80) to control the same pH. Cellulosic substrates by hot-washing process were added to a 500 mL round-bottomed flask with a stir and diluted with acetate buffer solution, to get a final solid concentration of 250 g per liter. The enzyme loading of cellulose was 10 IU/g cellulose. The reaction mixtures were put in a water bath at 50 °C for 48 h.
Cellulase adsorption kinetics studies
In which, ACS is the adsorption capacity of substrate.
Cellulase adsorption isotherm studies
Sugar, furfural, 5-hydroxymethylfurfural (HMF), acetic acid, and formic acid were analyzed by high-performance liquid chromatography (HPLC) using an HPX-87H (300 × 7.8 mm) column operating at 60 °C. The eluent, 5 mmol/L H2SO4, was used at a flow rate of 0.5 mL/min using a refractive index detector. All samples were centrifuged for 5 min to remove the water insoluble substances and the supernatant filtered through a 0.22 mm filter before analysis.
The T g measurements was conducted using a simultaneous thermal analyzer (NETZSCH, STA409PC, USA). Scanning Electron Microscope (SEM) analysis was used to investigate physical changes in the native and pretreated materials. Fourier transform infrared spectroscopy (FTIR) was conducted using a Spectrum One FTIR system (Perkin Elemer) with a universal attenuated total reflection (ATR) accessory. Sample spectra were obtained using an average of 32 scans over the range between 400 and 4000/cm with a spectral resolution of 4/cm.
Results and discussion
Glass transition temperature (T g)
Chemical compositions of samples before and after hot-washing process (dry wt%)
The main drawback of steam explosion pretreatment is the partially hemicelluloses degradation and the generation of some toxic compounds that can affect the following hydrolysis and fermentation steps (Oliva et al. 2003). The possible toxic compounds in ECS were organic acids, such as acetic acid and formic acid, furan derivatives, such as furfural, and HMF (Table 1). Several detoxification methods have been studied to reduce the inhibitory effect caused by these compounds on enzymes and yeasts. For example, after hot-water pretreatment, the solid fraction was washed with warm deionized water (80 °C) to remove inhibitors of cellulase enzymes (Zeng et al. 2012). However, owing to the additional cost in the overall process, detoxification should be avoided if possible. Due to filtering step under the high temperature, these compounds were separated from solid into the liquid phase. The concentration of these obviously decreased in solid samples after hot-washing process. The yield of formic acid in residual substrates decreased by about 90 %, and the concentration of acetic acid dropped from 0.29 to 0.035 % in SECS. The content of furfural and HMF was well eliminated.
Morphology of the treated and untreated materials
Fourier transform infrared spectroscopy (FTIR) analysis
Adsorption kinetics of biomass substrates
The first step in enzymatic hydrolysis of substrates is the adsorption of cellulase on the substrate cellulose surface. Furthermore, lignin is also reported to adsorpt cellulase enzymes in many studies (Kumar and Wyman 2009). Investigation of adsorption kinetics on lignocelluloses contributes to the understanding the enzymatic hydrolysis of lignocelluloses.
Adsorption isotherm of biomass substrates
Adsorption isotherm parameters of cellulase fitted by Langmuir adsorption model on different substrates at 4°
E max (U/g)
K ad (mL/U)
Adj R 2
427.5 ± 19.53
0.013 ± 0.008
373.63 ± 12.92
0.027 ± 0.014
364.04 ± 16
0.028 ± 0.018
1168.88 ± 206.75
0.0064 ± 0.013
Hot-washing process had a significant effect on ESC’s physic-mechanical properties. It suggests that upon melting under T g, lignin in biomass becomes fluid, coalesce, and has potential to move throughout the cell wall matrix. Hot-washing process significantly increased the adsorption ability of enzymes onto the substrates and digestibility of biomass without removing much of the insoluble lignin content. The amount of lignin alone was not sufficient to explain the different enzymatic hydrolysis characteristics of the fractions. Lignin distribution and/or physical property composition play a role. The concentration of fermentation inhibitor (acetic acid, formic acid, furfural, and HMF) obviously decreased after hot-washing process. Future work is focused on the industrial equipment, which can be carried out to pressure filtration under high temperature.
XY wrote this manuscript. HLL, JYC, and SQF contributed general advice. XJM edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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