Arthur J. Ragauskas

Lignin Valorization: Improving Lignin Processing in the Biorefinery

Background Lignin, nature’s dominant aromatic polymer, is found in most terrestrial plants in the approximate range of 15 to 40% dry weight and provides structural integrity. Traditionally, most large-scale industrial processes that use plant polysaccharides have burned lignin to generate the power needed to productively transform biomass. The advent of biorefineries that convert cellulosic biomass into liquid transportation fuels will generate substantially more lignin than necessary to power the operation, and therefore efforts are underway to transform it to value-added products. Production of biofuels from cellulosic biomass requires separation of large quantities of the aromatic polymer lignin. In planta genetic engineering, enhanced extraction methods, and a deeper understanding of the structure of lignin are yielding promising opportunities for efficient conversion of this renewable resource to carbon fibers, polymers, commodity chemicals, and fuels. [Credit: Oak Ridge National Laboratory, U.S. Department of Energy] Advances Bioengineering to modify lignin structure and/or incorporate atypical components has shown promise toward facilitating recovery and chemical transformation of lignin under biorefinery conditions. The flexibility in lignin monomer composition has proven useful for enhancing extraction efficiency. Both the mining of genetic variants in native populations of bioenergy crops and direct genetic manipulation of biosynthesis pathways have produced lignin feedstocks with unique properties for coproduct development. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery and enables catalytic modifications for desired chemical and physical properties. Outlook Potential high-value products from isolated lignin include low-cost carbon fiber, engineering plastics and thermoplastic elastomers, polymeric foams and membranes, and a variety of fuels and chemicals all currently sourced from petroleum. These lignin coproducts must be low cost and perform as well as petroleum-derived counterparts. Each product stream has its own distinct challenges. Development of renewable lignin-based polymers requires improved processing technologies coupled to tailored bioenergy crops incorporating lignin with the desired chemical and physical properties. For fuels and chemicals, multiple strategies have emerged for lignin depolymerization and upgrading, including thermochemical treatments and homogeneous and heterogeneous catalysis. The multifunctional nature of lignin has historically yielded multiple product streams, which require extensive separation and purification procedures, but engineering plant feedstocks for greater structural homogeneity and tailored functionality reduces this challenge. The Lignin Landscape Lignin is a chemically complex polymer that lends woody plants and trees their rigidity. Humans have traditionally either left it intact to lend rigidity to their own wooden constructs, or burned it to generate heat and sometimes power. With the advent of major biorefining operations to convert cellulosic biomass into ethanol and other liquid fuels, researchers are now exploring how to transform the associated leftover lignin into more diverse and valuable products. Ragauskas et al. (10.1126/science.1246843) review recent developments in this area, ranging from genetic engineering approaches that tune lignin properties at the source, to chemical processing techniques directed toward extracting lignin in the biorefinery and transforming it into high-performance plastics and a variety of bulk and fine chemicals. Research and development activities directed toward commercial production of cellulosic ethanol have created the opportunity to dramatically increase the transformation of lignin to value-added products. Here, we highlight recent advances in this lignin valorization effort. Discovery of genetic variants in native populations of bioenergy crops and direct manipulation of biosynthesis pathways have produced lignin feedstocks with favorable properties for recovery and downstream conversion. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for future targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery, and this coupled with genetic engineering will enable new uses for this biopolymer, including low-cost carbon fibers, engineered plastics and thermoplastic elastomers, polymeric foams, fungible fuels, and commodity chemicals.

Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300–400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

Ionic Liquid as a Green Solvent for Lignin

Abstract This study examined the application of select ionic liquids (ILs) as aprotic green solvents for lignin. Dissolution experiments were carried out employing lignin isolated from pine kraft pulp. Up to 20 wt% lignin could be dissolved in [hmim][CF3SO3], [mmim][MeSO4] and [bmim][MeSO4]. For the [bmim]+‐containing ionic liquids, the order of lignin solubility for varying anions was: [MeSO4]−>Cl−∼Br−⋙[PF6]−, indicating that the solubility of lignin was principally influenced by the nature of the anions. Ionic liquids containing large, non‐coordinating anions [PF4]− and [PF6]− were unsuitable as a solvent for lignin. 13C nuclear magnetic resonance (NMR) analyses of lignin and model compounds showed that 13C signals using ionic liquid as a solvent were shifted up‐field by δ 0.1 to 1.9 ppm in comparison to 13C NMR data acquired using dimethyl sulfoxide (DMSO) as the solvent.

Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments

The production of cellulosic ethanol from biomass is considered a promising alternative to reliance on diminishing supplies of fossil fuels, providing a sustainable option for fuels production in an environmentally compatible manner. The conversion of lignocellulosic biomass to biofuels through a biological route usually suffers from the intrinsic recalcitrance of biomass owing to the complicated structure of plant cell walls. Currently, a pretreatment step that can effectively reduce biomass recalcitrance is generally required to make the polysaccharide fractions locked in the intricacy of plant cell walls to become more accessible and amenable to enzymatic hydrolysis. Dilute acid and hydrothermal pretreatments are attractive and among the most promising pretreatment technologies that enhance sugar release performance. This review highlights our recent understanding on molecular structure basis for recalcitrance, with emphasis on structural transformation of major biomass biopolymers (i.e., cellulose, hemicellulose, and lignin) related to the reduction of recalcitrance during dilute acid and hydrothermal pretreatments. The effects of these two pretreatments on biomass porosity as well as its contribution on reduced recalcitrance are also discussed.

Pseudo-lignin and pretreatment chemistry

The formation of pseudo-lignin by the combination of carbohydrate and lignin degradation products has been proposed to be responsible for the increased Klason lignin content in biomass pretreated under acidic conditions. Direct evidence for the presence of pseudo-lignin has never been presented. The formation of additional lignin-like material may be detrimental to enzymatic hydrolysis due to the non-productive binding of enzymes with lignin. To investigate the chemistry of pseudo-lignin formation, dilute acid pretreatments were performed on delignified hybrid poplar biomass under conditions of varying severity. The results show a progressive increase in the Klason lignin content of the acid pretreated material with increasing pretreatment severity. NMR and FT-IR spectroscopic characterization shows the development of aliphatic, unsaturated and carbonyl carbon functionalities in the samples pretreated at higher severities. Given the very low Klason lignin content of the starting material, acid catalyzed dehydration of carbohydrates is responsible for the formation of pseudo-lignin.

Pretreatment and Lignocellulosic Chemistry

Lignocellulosic materials such as wood, grass, and agricultural and forest residues are promising alternative energy resources that can be utilized to produce ethanol. The yield of ethanol production from native lignocellulosic material is relatively low due to its native recalcitrance, which is attributed to, in part, lignin content/structure, hemicelluloses, cellulose crystallinity, and other factors. Pretreatment of lignocellulosic materials is required to overcome this recalcitrance. The goal of pretreatment is to alter the physical features and chemical composition/structure of lignocellulosic materials, thus making cellulose more accessible to enzymatic hydrolysis for sugar conversion. Various pretreatment technologies to reduce recalcitrance and to increase sugar yield have been developed during the past two decades. This review examines the changes in lignocellulosic structure primarily in cellulose and hemicellulose during the most commonly applied pretreatment technologies including dilute acid pretreatment, hydrothermal pretreatment, and alkaline pretreatment.

Structural Characterization and Comparison of Switchgrass Ball-milled Lignin Before and After Dilute Acid Pretreatment

To reduce the recalcitrance and enhance enzymatic activity, dilute H2SO4 pretreatment was carried out on Alamo switchgrass (Panicum virgatum). Ball-milled lignin was isolated from switchgrass before and after pretreatment. Its structure was characterized by 13C, HSQC, and 31P NMR spectroscopy. It was confirmed that ball-milled switchgrass lignin is of HGS type with a considerable amount of p-coumarate and felurate esters of lignin. The major ball-milled lignin interunit was the β-O-4 linkage, and a minor amount of phenylcoumarin, resinol, and spirodienone units were also present. As a result of the acid pretreatment, there was 36% decrease of β-O-4 linkage observed. In addition to these changes, the S/G ratio decreases from 0.80 to 0.53.

Recent advances in understanding the role of cellulose accessibility in enzymatic hydrolysis of lignocellulosic substrates.

Cellulose accessibility has been proposed as a key factor in the efficient bio-conversion of lignocellulosic biomass to fermentable sugars. Factors affecting cellulose accessibility can be divided into direct factors that refer to accessible surface area of cellulose, and indirect factors referring to chemical composition such as lignin/hemicellulose content, and biomass structure-relevant factors (i.e. particle size, porosity). An overview of the current pretreatment technologies special focus on the major mode of action to increase cellulose accessibility as well as multiple techniques that could be used to assess the cellulose accessibility are presented in this review. The appropriate determination of cellulose accessibility before and after pretreatment can assist to understand the effectiveness of a particular pretreatment in overcoming lignocellulosic recalcitrance to improve substrate enzymatic digestibility.

Pseudo-lignin formation and its impact on enzymatic hydrolysis

Pseudo-lignin, which can be broadly defined as aromatic material that yields a positive Klason lignin value and is not derived from native lignin, has been recently reported to form during the dilute acid pretreatment of poplar holocellulose. To investigate the chemistry of pseudo-lignin formation, GPC, FT-IR and 13C NMR were utilized to characterize pseudo-lignin extracted from dilute-acid pretreated α-cellulose and holocellulose. The results showed that pseudo-lignin consisting of carbonyl, carboxylic, aromatic and aliphatic structures was produced from dilute acid pretreated cellulose and hemicellulose. Pseudo-lignin extracted from holocellulose pretreated at different conditions had similar molecular weights (Mn∼1000 g/mol; Mw∼5000 g/mol) and structural features (carbonyl, carboxylic, aromatic and methoxy structures). These characterizations have provided the pseudo-lignin formation mechanisms during pretreatment. The presence and structure of pseudo-lignin is important since pseudo-lignin decreases the enzymatic conversion.

Lignin Pyrolysis Components and Upgrading—Technology Review

Biomass pyrolysis oil has been reported as a potential renewable biofuel precursor. Although several review articles focusing on lignocellulose pyrolysis can be found, the one that particularly focus on lignin pyrolysis is still not available in literature. Lignin is the second most abundant biomass component and the primary renewable aromatic resource in nature. The pyrolysis chemistry and mechanism of lignin are significantly different from pyrolysis of cellulose or entire biomass. Therefore, different from other review articles in the field, this review particularly focuses on the recent developments in lignin pyrolysis chemistry, mechanism, catalysts, and the upgrading of the bio-oil from lignin pyrolysis. Although bio-oil production from pyrolysis of biomass has been proven on commercial scale and is a very promising option for production of renewable chemicals and fuels, there are still several drawbacks that have not been solved. The components of biomass pyrolysis oils are very complicated and related to the properties of bio-oil. In this review article, the details about pyrolysis oil components particularly those from lignin pyrolysis processes will be discussed first. Due to the poor physical and chemical property, the lignin pyrolysis oil has to be upgraded before usage. The most common method of upgrading bio-oil is hydrotreating. Catalysts have been widely used in petroleum industry for pyrolysis bio-oil upgrading. In this review paper, the mechanism of the hydrodeoxygenation reaction between the model compounds and catalysts will be discussed and the effects of the reaction condition will be summarized.