Gregg T. Beckham

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.

Lignin valorization through integrated biological funneling and chemical catalysis.

Significance For nearly a century, processes have been used to convert biomass-derived carbohydrates, such as glucose, into fuels and chemicals. However, plant cell walls also contain an aromatic polymer, lignin, that has not been cost-effectively converted into fuels or commodity chemicals. With the intensive development of lignocellulosic biorefineries around the world to produce fuels and chemicals from biomass-derived carbohydrates, the amount of waste lignin will dramatically increase, warranting new lignin upgrading strategies. In nature, some microorganisms have evolved pathways to catabolize lignin-derived aromatics. Our work demonstrates that the utilization of these natural aromatic catabolic pathways may enable new routes to overcome the lignin utilization barrier that, in turn, may enable a broader slate of molecules derived from lignocellulosic biomass. Lignin is an energy-dense, heterogeneous polymer comprised of phenylpropanoid monomers used by plants for structure, water transport, and defense, and it is the second most abundant biopolymer on Earth after cellulose. In production of fuels and chemicals from biomass, lignin is typically underused as a feedstock and burned for process heat because its inherent heterogeneity and recalcitrance make it difficult to selectively valorize. In nature, however, some organisms have evolved metabolic pathways that enable the utilization of lignin-derived aromatic molecules as carbon sources. Aromatic catabolism typically occurs via upper pathways that act as a “biological funnel” to convert heterogeneous substrates to central intermediates, such as protocatechuate or catechol. These intermediates undergo ring cleavage and are further converted via the β-ketoadipate pathway to central carbon metabolism. Here, we use a natural aromatic-catabolizing organism, Pseudomonas putida KT2440, to demonstrate that these aromatic metabolic pathways can be used to convert both aromatic model compounds and heterogeneous, lignin-enriched streams derived from pilot-scale biomass pretreatment into medium chain-length polyhydroxyalkanoates (mcl-PHAs). mcl-PHAs were then isolated from the cells and demonstrated to be similar in physicochemical properties to conventional carbohydrate-derived mcl-PHAs, which have applications as bioplastics. In a further demonstration of their utility, mcl-PHAs were catalytically converted to both chemical precursors and fuel-range hydrocarbons. Overall, this work demonstrates that the use of aromatic catabolic pathways enables an approach to valorize lignin by overcoming its inherent heterogeneity to produce fuels, chemicals, and materials.

Adipic acid production from lignin.

Lignin is an alkyl-aromatic polymer present in plant cell walls for defense, structure, and water transport. Despite exhibiting a high-energy content, lignin is typically slated for combustion in modern biorefineries due to its inherent heterogeneity and recalcitrance, whereas cellulose and hemicellulose are converted to renewable fuels and chemicals. However, it is critical for the viability of third-generation biorefineries to valorize lignin alongside polysaccharides. To that end, we employ metabolic engineering, separations, and catalysis to convert lignin-derived species into cis,cis-muconic acid, for subsequent hydrogenation to adipic acid, the latter being the most widely produced dicarboxylic acid. First, Pseudomonas putida KT2440 was metabolically engineered to funnel lignin-derived aromatics to cis,cis-muconate, which is an atom-efficient biochemical transformation. This engineered strain was employed in fed-batch biological cultivation to demonstrate a cis,cis-muconate titer of 13.5 g L−1 in 78.5 h from a model lignin-derived compound. cis,cis-Muconic acid was recovered in high purity (>97%) and yield (74%) by activated carbon treatment and crystallization (5 °C, pH 2). Pd/C was identified as a highly active catalyst for cis,cis-muconic acid hydrogenation to adipic acid with high conversion (>97%) and selectivity (>97%). Under surface reaction controlling conditions (24 °C, 24 bar, ethanol solvent), purified cis,cis-muconic acid exhibits a turnover frequency of 23–30 s−1 over Pd/C, with an apparent activation energy of 70 kJ mol−1. Lastly, cis,cis-muconate was produced with engineered P. putida grown on a biomass-derived, lignin-enriched stream, demonstrating an integrated strategy towards lignin valorization to an important commodity chemical.

Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.

Cellulose is a crystalline polymer of β1,4-D-glucose that is difficult to deconstruct to sugars by enzymes. The recalcitrance of cellulose microfibrils is a function of both the shape of cellulose microfibrils and the intrinsic work required to decrystallize individual chains, the latter of which is calculated here from the surfaces of four crystalline cellulose polymorphs: cellulose Iβ, cellulose Iα, cellulose II, and cellulose III(I). For edge chains, the order of decrystallization work is as follows (from highest to lowest): Iβ, Iα, ΙΙΙ(Ι), and II. For cellulose Iβ, we compare chains from three different locations on the surface and find that an increasing number of intralayer hydrogen bonds (from 0 to 2) increases the intrinsic decrystallization work. From these results, we propose a microkinetic model for the deconstruction of cellulose (and chitin) by processive enzymes, which when taken with a previous study [Horn et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18089] identifies the thermodynamic and kinetic attributes of enzyme and substrate engineering for enhanced cellulose (or chitin) conversion. Overall, this study provides new insights into the molecular interactions that form the structural basis of cellulose, which is the primary building block of plant cell walls, and highlights the need for experimentally determining microfibril shape at the nanometer length scale when comparing conversion rates of cellulose polymorphs by enzymes.

A perspective on oxygenated species in the refinery integration of pyrolysis oil

Pyrolysis offers a rapid and efficient means to depolymerize lignocellulosic biomass, resulting in gas, liquid, and solid products with varying yields and compositions depending on the process conditions. With respect to manufacture of “drop-in” liquid transportation fuels from biomass, a potential benefit from pyrolysis arises from the production of a liquid or vapor that could possibly be integrated into existing refinery infrastructure, thus offsetting the capital-intensive investment needed for a smaller scale, standalone biofuels production facility. However, pyrolysis typically yields a significant amount of reactive, oxygenated species including organic acids, aldehydes, ketones, and oxygenated aromatics. These oxygenated species present significant challenges that will undoubtedly require pre-processing of a pyrolysis-derived stream before the pyrolysis oil can be integrated into the existing refinery infrastructure. Here we present a perspective of how the overall chemistry of pyrolysis products must be modified to ensure optimal integration in standard petroleum refineries, and we explore the various points of integration in the refinery infrastructure. In addition, we identify several research and development needs that will answer critical questions regarding the technical and economic feasibility of refinery integration of pyrolysis-derived products.

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism

Significance Plant cell walls contain significant amounts of the polysaccharides cellulose and hemicellulose, which can be depolymerized by enzymes to sugars and upgraded to renewable fuels and chemicals. Traditionally, enzymes for biomass depolymerization were based on naturally occurring hydrolytic enzymes, until the recent discovery of another natural enzymatic paradigm for carbohydrate deconstruction. Namely, lytic polysaccharide monooxygenases (LPMOs), long thought to be hydrolases or carbohydrate-binding modules, were revealed to be oxidative, copper-containing enzymes. These enzymes are receiving significant attention as they could revolutionize biomass deconstruction to upgradeable intermediates for renewable energy applications. Here, we apply quantum mechanical calculations to elucidate the oxidative reaction mechanism to offer predictions into how LPMOs function. Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η1-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η1) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.

Applications of computational science for understanding enzymatic deconstruction of cellulose

Understanding the molecular-level mechanisms that enzymes employ to deconstruct plant cell walls is a fundamental scientific challenge with significant ramifications for renewable fuel production from biomass. In nature, bacteria and fungi use enzyme cocktails that include processive and non-processive cellulases and hemicellulases to convert cellulose and hemicellulose to soluble sugars. Catalyzed by an accelerated biofuels R&D portfolio, there is now a wealth of new structural and experimental insights related to cellulases and the structure of plant cell walls. From this background, computational approaches commonly used in other fields are now poised to offer insights complementary to experiments designed to probe mechanisms of plant cell wall deconstruction. Here we outline the current status of computational approaches for a collection of critical problems in cellulose deconstruction. We discuss path sampling methods to measure rates of elementary steps of enzyme action, coarse-grained modeling for understanding macromolecular, cellulosomal complexes, methods to screen for enzyme improvements, and studies of cellulose at the molecular level. Overall, simulation is a complementary tool to understand carbohydrate-active enzymes and plant cell walls, which will enable industrial processes for the production of advanced, renewable fuels.

Lignocellulose degradation mechanisms across the Tree of Life

Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.

Towards lignin consolidated bioprocessing: simultaneous lignin depolymerization and product generation by bacteria

Lignin represents an untapped resource in lignocellulosic biomass, primarily due to its recalcitrance to depolymerization and its intrinsic heterogeneity. In Nature, microorganisms have evolved mechanisms to both depolymerize lignin using extracellular oxidative enzymes and to uptake the aromatic species generated during depolymerization for carbon and energy sources. The ability of microbes to conduct both of these processes simultaneously could enable a Consolidated Bioprocessing concept to be applied to lignin, similar to what is done today with polysaccharide conversion to ethanol via ethanologenic, cellulolytic microbes. To that end, here we examine the ability of 14 bacteria to secrete ligninolytic enzymes, depolymerize lignin, uptake aromatic and other compounds present in a biomass-derived, lignin-enriched stream, and, under nitrogen-limiting conditions, accumulate intracellular carbon storage compounds that can be used as fuel, chemical, or material precursors. In shake flask conditions using a substrate produced during alkaline pretreatment, we demonstrate that up to nearly 30% of the initial lignin can be depolymerized and catabolized by a subset of bacteria. In particular, Amycolatopsis sp., two Pseudomonas putida strains, Acinetobacter ADP1, and Rhodococcus jostii are able to depolymerize high molecular weight lignin species and catabolize a significant portion of the low molecular weight aromatics, thus representing good starting hosts for metabolic engineering. This study also provides a comprehensive set of experimental tools to simultaneously study lignin depolymerization and aromatic catabolism in bacteria, and provides a foundation towards the concept of Lignin Consolidated Bioprocessing, which may eventually be an important route for biological lignin valorization.

Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.