John N. Saddler

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.

An overview of second generation biofuel technologies.

The recently identified limitations of 1st-generation biofuels produced from food crops (with perhaps the exception of sugarcane ethanol) have caused greater emphasis to be placed on 2nd-generation biofuels produced from ligno-cellulosic feedstocks. Although significant progress continues to be made to overcome the technical and economic challenges, 2nd-generation biofuels production will continue to face major constraints to full commercial deployment. The logistics of providing a competitive, all-year-round, supply of biomass feedstock to a commercial-scale plant is challenging, as is improving the performance of the conversion process to reduce costs. The biochemical route, being less mature, probably has a greater cost reduction potential than the thermo-chemical route, but here a wider range of synthetic fuels can be produced to better suit heavy truck, aviation and marine applications. Continued investment in research and demonstration by both public and private sectors, coupled with appropriate policy support mechanisms, are essential if full commercialisation is to be achieved within the next decade. After that, the biofuel industry will grow only at a steady rate and encompass both 1st- and 2nd-generation technologies that meet agreed environmental, sustainability and economic policy goals.

Substrate Pretreatment: The Key to Effective Enzymatic Hydrolysis of Lignocellulosics?

Although the structure and function of cellulase systems continue to be the subject of intense research, it is widely acknowledged that the rate and extent of the cellulolytic hydrolysis of lignocellulosic substrates is influenced not only by the effectiveness of the enzymes but also by the chemical, physical and morphological characteristics of the heterogeneous lignocellulosic substrates. Although strategies such as site-directed mutagenesis or directed evolution have been successfully employed to improve cellulase properties such as binding affinity, catalytic activity and thermostability, complementary goals that we and other groups have studied have been the determination of which substrate characteristics are responsible for limiting hydrolysis and the development of pretreatment methods that maximize substrate accessibility to the cellulase complex. Over the last few years we have looked at the various lignocellulosic substrate characteristics at the fiber, fibril and microfibril level that have been modified during pretreatment and subsequent hydrolysis. The initial characteristics of the woody biomass and the effect of subsequent pretreatment play a significant role on the development of substrate properties, which in turn govern the efficacy of enzymatic hydrolysis. Focusing particularly on steam pretreatment, this review examines the influence that pretreatment conditions have on substrate characteristics such as lignin and hemicellulose content, crystallinity, degree of polymerization and specific surface, and the resulting implications for effective hydrolysis by cellulases.

Substrate and Enzyme Characteristics that Limit Cellulose Hydrolysis

The ability and, consequently, the limitations of various microbial enzyme systems to completely hydrolyze the structural polysaccharides of plant cell walls has been the focus of an enormous amount of research over the years. As more and more of these extracellular enzymatic systems are being identified and characterized, clear similarities and differences are being elucidated. Although much has been learned concerning the structures, kinetics, catalytic action, and interactions of enzymes and their substrates, no single mechanism of total lignocellulosic saccharification has been established. The heterogeneous nature of the supramolecular structures of naturally occurring lignocellulosic matrices make it difficult to fully understand the interactions that occur between enzyme complexes and these substrates. However, it is apparent that the efficacy of enzymatic complexes to hydrolyze these substrates is inextricably linked to the innate structural characteristics of the substrate and/or the modifications that occur as saccharification proceeds. This present review is not intended to conclusively answer what factors control polysaccharide biodegradation, but to serve as an overview illustrating some of the potential enzymatic and structural limitations that invariably influence the complete hydrolysis of lignocellulosic polysaccharides.

The effect of initial pore volume and lignin content on the enzymatic hydrolysis of softwoods

Four Douglas-fir pulps, a refiner mechanical pulp (RMP), sulphonated RMP, delignified RMP and a kraft pulp were used to determine whether the lignin content and initial pore volume affected cellulase adsorption and substrate hydrolysis. When compared on the basis of lignin content, it was apparent from the cellulase treatment of the sulphonated RMP that the proportion of lignin did not affect enzyme adsorption when the fibres were sufficiently swollen. However, it was observed that the initial adsorption of cellulase does not always translate to fast and complete hydrolysis. Pore volume data revealed that although modification of lignin resulted in a dramatically increased fibre saturation point the median pore width was not increased accordingly. In contrast, the delignified RMP had a higher median pore width and was hydrolyzed more completely, suggesting that steric hindrance from the residual lignin may be the rate limiting characteristic in this situation. Hydrolysis of the kraft pulp indicated that the larger average particle size of this substrate might have been an inhibiting factor, since it was hydrolyzed more slowly than the delignified RMP despite having a higher median pore width and lower lignin content.

Effects of Sugar Inhibition on Cellulases and β-Glucosidase During Enzymatic Hydrolysis of Softwood Substrates

A quantitative approach was taken to determine the inhibition effects of glucose and other sugar monomers during cellulase and beta-Glucosidase hydrolysis of two types of cellulosic material: Avicel and acetic acid-pretreated softwood. The increased glucose content in the hydrolysate resulted in a dramatic increase in the degrees of inhibition on both beta-Glucosidase and cellulase activities. Supplementation of mannose, xylose, and galactose during cellobiose hydrolysis did not show any inhibitory effects on beta-Glucosidase activity. However, these sugars were shown to have significant inhibitory effects on cellulase activity during cellulose hydrolysis. Our study suggests that high-substrate consistency hydrolysis with supplementation of hemicellulose is likely to be a practical solution to minimizing end-product inhibition effects while producing hydrolysate with high glucose concentration.

Comparative Sugar Recovery and Fermentation Data Following Pretreatment of Poplar Wood by Leading Technologies

Through a Biomass Refining Consortium for Applied Fundamentals and Innovation among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, leading pretreatment technologies based on ammonia fiber expansion, aqueous ammonia recycle, dilute sulfuric acid, lime, neutral pH, and sulfur dioxide were applied to a single source of poplar wood, and the remaining solids from each technology were hydrolyzed to sugars using the same enzymes. Identical analytical methods and a consistent material balance methodology were employed to develop comparative performance data for each combination of pretreatment and enzymes. Overall, compared to data with corn stover employed previously, the results showed that poplar was more recalcitrant to conversion to sugars and that sugar yields from the combined operations of pretreatment and enzymatic hydrolysis varied more among pretreatments. However, application of more severe pretreatment conditions gave good yields from sulfur dioxide and lime, and a recombinant yeast strain fermented the mixed stream of glucose and xylose sugars released by enzymatic hydrolysis of water washed solids from all pretreatments to ethanol with similarly high yields. An Agricultural and Industrial Advisory Board followed progress and helped steer the research to meet scientific and commercial needs.

The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility.

The influence of cellulose accessibility and protein loading on the efficiency of enzymatic hydrolysis of steam pretreated Douglas-fir was assessed. It was apparent that the lignin component significantly influences the swelling/accessibility of cellulose as at low protein loadings (5FPU/g cellulose), only 16% of the cellulose present in the steam pretreated softwood was hydrolyzed while almost complete hydrolysis was achieved with the delignified substrate. When lignin (isolated from steam pretreated Douglas-fir) was added back in the same proportions it was originally found to the highly accessible and swollen, delignified steam pretreated softwood and to a cellulose control such as Avicel, the hydrolysis yields decreased by 9 and 46%, respectively. However, when higher enzyme loadings were employed, the greater availability of the enzyme could overcome the limitations imposed by both the lignins restrictions on cellulose accessibility and direct binding of the enzymes, resulting in a near complete hydrolysis of the cellulose.

Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process.

Past technoeconomic modeling work has identified the relatively large contribution that enzymatic hydrolysis adds to the total cost of producing ethanol from lignocellulosic substrates. This cost was primarily due to the high concentration of enzyme and long incubation time that was required to obtain complete hydrolysis. Although enzyme and substrate concentration and end‐product inhibition influenced the rate of hydrolysis, the effect was less pronounced during the initial stages of hydrolysis. During this time most of the cellulases were adsorbed onto the unhydrolyzed residue. By recycling the cellulases adsorbed to the residual substrate remaining after an initial 24 h, a high rate of hydrolysis, with low overall residence time and minimal cellulase input, could be achieved for several rounds of enzyme recycle. A comparison of the front end (pretreatment, fractionation, and hydrolysis) of a softwood/hardwood to ethanol process indicated that the lignin associated with the softwood‐derived cellulose stream limited the number of times the cellulose containing residue could be recycled.

Weak lignin-binding enzymes: a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics.

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.