Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production
Introduction
Second-generation bioethanol, produced from lignocellulosic materials, is a promising alternative to fossil fuel for vehicular transportation (Ragauskas et al., 2006, Carroll and Somerville, 2009). The benefits of cellulosic ethanol include, but are not limited to, reduced greenhouse gases emission, value-added utilization of agricultural and forest residues, enhancement of the rural economy, and improved national energy independence and security (Farrell et al., 2006, Scharlemann and Laurance, 2008). A typical process for cellulosic ethanol production consists of three steps: feedstock pretreatment to enhance cellulases accessibility to cellulose, enzymatic saccharification of the cellulose-to-glucose, followed by fermentation of the glucose to ethanol. One of the barriers to the commercialization of cellulosic ethanol is the lack of economical and effective technologies for the feedstock pretreatment. Pretreatment is a necessary operation required to achieve optimal bioconversion for all forms and types of lignocellulosic feedstocks to ethanol, but is particularly important for the more recalcitrant softwoods. The significance and importance of the pretreatment step cannot be overemphasized, as the effectiveness of the pretreatment affects the upstream selection of biomass, the yield of fermentable sugars, and the chemical and morphological characteristics of the pretreated substrate which in turn govern downstream hydrolysis/saccharification (Wyman et al., 2005).
An effective pretreatment method should be economical (in terms of both capital and operating costs) and effective for a variety of lignocellulosic biomass. Specifically, it should require minimal feedstock preparation and preprocessing prior to pretreatment, maximally recover all lignocellulosic components in usable forms with minimal formation of fermentation inhibitors, and produce a readily digestible cellulosic substrate that can be easily hydrolyzed with a low loading of enzymes. In the last several decades, research and development efforts have made significant progress in pretreatment technologies for lignocellulosic feedstocks (Lynd et al., 2002, Mosier et al., 2005, Wyman et al., 2005, Chandra et al., 2007, Hendriks and Zeeman, 2009). Many pretreatment technologies, such as lime, dilute acid, hot water, ammonia, steam explosion, and organosolv pretreatments (Nguyen et al., 2000, Pan et al., 2005a, Chandra et al., 2007, Sendich et al., 2008, Wingren et al., 2008, Gupta and Lee, 2009, Kim et al., 2009, Monavari et al., 2009, Sierra et al., 2009), have achieved varying levels of success.
Dilute-acid pretreatment (DA), one of the most investigated pretreatment methods, is conducted typically using sulfuric acid at high temperature (160–200 °C) (Schell et al., 2003, Lloyd and Wyman, 2005, Wyman et al., 2005). DA enhances the digestibility of lignocellulose mainly by dissolving hemicellulose and partially pre-hydrolyzing cellulose. DA can achieve satisfactory levels of cellulose saccharification for agricultural residues and some hardwood species, but is not effective for softwoods. The low pH value of the dilute-acid process causes serious equipment corrosion problems. In addition, high temperature and low pH lead to formation of significant amounts of fermentation inhibitors, notably furfurals from hemicellulosic sugars. Furthermore, dilute-acid pretreatment causes extensive condensation of lignin, which diminishes the commercial value of lignin for co-products development (Shevchenko et al., 1999, Nguyen et al., 2000).
Sulfur dioxide (SO2)-catalyzed steam explosion is another acidic pretreatment at milder conditions (weak acid and short reaction time), which reduces equipment corrosion and minimizes the generation of fermentation inhibitors. The process has been extensively studied by Zacchi’s and Saddler’s groups (Galbe and Zacchi, 2002, Chandra et al., 2007) and is one of the most investigated pretreatments for woody biomass, including softwoods. Feedstock is first treated using gaseous SO2 and then subjected to steam explosion. The process works well with agricultural residues and some hardwoods, but performs less satisfactorily with softwoods. Two-stage steam explosion can improve the enzymatic digestibility and overall sugar recovery from softwoods, but the advantages are partially outweighed by increased energy consumption and operation cost (Galbe and Zacchi, 2002). The toxicity of gaseous SO2 is another concern in application. Furthermore, the mild SO2 treatment is unable to sulfonate or dissolve lignin significantly. The lignin is extensively condensed during the subsequent steam explosion (Shevchenko et al., 1999), a key factor imparting recalcitrance of steam exploded softwood substrate (Pan et al., 2005b).
Recently, we developed and reported a novel pretreatment method, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), for robust conversion of woody biomass including softwood to sugars (Wang et al., 2009, Zhu et al., 2009a). The pretreatment consists of a short chemical treatment of feedstock followed by mechanical size reduction (fiberization). Wood chips or other feedstocks first react with a solution of a sulfite salt (e.g., Na, Mg, or Ca) at 160–180 °C and pH 2–4 for about 30 min, and are then fiberized (size-reduced) using a disk mill to generate fibrous substrate for subsequent saccharification and fermentation. SPORL produced readily digestible substrates and excellent recovery of fermentable hemicellulose sugars with low amount of fermentation inhibitors. Energy consumption for post-SPORL size reduction of wood chips was about 30 Wh/kg, equivalent to those consumed for size reduction of agricultural biomass. In addition, direct pretreatment of commercial wood chips afforded a low liquid-to-wood ratio (<3) (Zhu et al., 2009), which can lead to considerable thermal energy savings and produce a concentrated hemicellulose sugar stream. Because the lignin dissolved in SPORL pretreatment hydrolysate is sulfonated (lignosulfonate), it has a variety of commercial applications within the established market. It should be pointed out that SPORL pretreatment is compatible with SO2-catalyzed steam explosion. One can conduct sulfite-catalyzed steam explosion by using sulfite in addition to SO2 as catalysts to significantly improve cellulose enzymatic saccharification, especially for softwoods.
To further understand the fundamentals of the SPORL pretreatment, in this study, we systematically compared SPORL with the DA for the pretreatment of spruce, a softwood that is notoriously more recalcitrant than other types of biomass (hardwood and herbage) to bioconversion processes. The experimental flowchart is summarized in Fig. 1. The chemical changes of cell-wall components were investigated before and after the SPORL and DA pretreatments. The enzymatic digestibility of the pretreated substrates was evaluated. Mass balances of carbohydrates in the two pretreatments were established. The structural changes of lignin and carbohydrate during the pretreatments were examined using NMR spectrometry. Additionally, the formation of fermentation inhibitors during the pretreatments was compared. The fermentability of the spent pretreatment liquors of the SPORL and DA was evaluated using an in vitro ruminal fermentation assay.
Section snippets
Materials
Fresh spruce chips were generously provided by the Wisconsin Rapids Mill of Stora Enso North America (now New Page Corporation, Miamisburg, OH). After being air-dried, the chips were ground to pass a 40-mesh (0.42 mm opening) screen using a Wiley mill. Chemical composition of the spruce wood is presented in Table 1. Commercial enzymes, Celluclast and β-glucosidase produced by Novozymes, were purchased from Sigma–Aldrich (St. Louis, MO) and used as received. All the chemical reagents used in this
Changes in cell-wall components during SPORL and DA pretreatments
As described in Section 2, ground spruce wood (−40-mesh) rather than wood chips was used as the feedstock, and mechanical size reduction was not included in either SPORL or DA pretreatment. The consideration of doing so is that a refining step adds difficulties to mass balance evaluations. In addition, only physical size changes and no (significant) chemical reactions are expected during the mechanical size reduction. Both SPORL and DA chemical pretreatments were carried out at the same
Conclusion
SPORL pretreatment significantly removed the recalcitrance of spruce wood and allowed nearly complete enzymatic hydrolysis (>90%) within 24 h with a cellulase loading of 15 FPU/g cellulose. SPORL removed the recalcitrance not only by dissolving hemicellulose and depolymerizing cellulose, but also by partially (32%) dissolving lignin and sulfonating the residual lignin in substrate, which presumably reduced the hydrophobic interactions between lignin and the enzymes. SPORL achieved a significantly
Acknowledgements
The support of this work is provided by USDA Forest Service’s biomass program (2008) to J.Y. Zhu and X.J. Pan and partially by Graduate School of the University of Wisconsin-Madison to X.J. Pan. The authors thank R. Gleisner and D. Mann for their assistance in operating digester for the pretreatments. We thank the US Dairy Forage Research Center for the NMR access.
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