Mark F. Davis

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 content in natural Populus variants affects sugar release

The primary obstacle to producing renewable fuels from lignocellulosic biomass is a plants recalcitrance to releasing sugars bound in the cell wall. From a sample set of wood cores representing 1,100 individual undomesticated Populus trichocarpa trees, 47 extreme phenotypes were selected across measured lignin content and ratio of syringyl and guaiacyl units (S/G ratio). This subset was tested for total sugar release through enzymatic hydrolysis alone as well as through combined hot-water pretreatment and enzymatic hydrolysis using a high-throughput screening method. The total amount of glucan and xylan released varied widely among samples, with total sugar yields of up to 92% of the theoretical maximum. A strong negative correlation between sugar release and lignin content was only found for pretreated samples with an S/G ratio < 2.0. For higher S/G ratios, sugar release was generally higher, and the negative influence of lignin was less pronounced. When examined separately, only glucose release was correlated with lignin content and S/G ratio in this manner, whereas xylose release depended on the S/G ratio alone. For enzymatic hydrolysis without pretreatment, sugar release increased significantly with decreasing lignin content below 20%, irrespective of the S/G ratio. Furthermore, certain samples featuring average lignin content and S/G ratios exhibited exceptional sugar release. These facts suggest that factors beyond lignin and S/G ratio influence recalcitrance to sugar release and point to a critical need for deeper understanding of cell-wall structure before plants can be rationally engineered for reduced recalcitrance and efficient biofuels production.

Efficient Degradation of Lignocellulosic Plant Biomass, without Pretreatment, by the Thermophilic Anaerobe “Anaerocellum thermophilum” DSM 6725

ABSTRACT Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that “Anaerocellum thermophilum” DSM 6725, an anaerobic bacterium that grows optimally at 75°C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75°C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70°C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.

Comparative genome analysis of lignin biosynthesis gene families across the plant kingdom.

BackgroundAs a major component of plant cell wall, lignin plays important roles in mechanical support, water transport, and stress responses. As the main cause for the recalcitrance of plant cell wall, lignin modification has been a major task for bioenergy feedstock improvement. The study of the evolution and function of lignin biosynthesis genes thus has two-fold implications. First, the lignin biosynthesis pathway provides an excellent model to study the coordinative evolution of a biochemical pathway in plants. Second, understanding the function and evolution of lignin biosynthesis genes will guide us to develop better strategies for bioenergy feedstock improvement.ResultsWe analyzed lignin biosynthesis genes from fourteen plant species and one symbiotic fungal species. Comprehensive comparative genome analysis was carried out to study the distribution, relatedness, and family expansion of the lignin biosynthesis genes across the plant kingdom. In addition, we also analyzed the comparative synteny map between rice and sorghum to study the evolution of lignin biosynthesis genes within the Poaceae family and the chromosome evolution between the two species. Comprehensive lignin biosynthesis gene expression analysis was performed in rice, poplar and Arabidopsis. The representative data from rice indicates that different fates of gene duplications exist for lignin biosynthesis genes. In addition, we also carried out the biomass composition analysis of nine Arabidopsis mutants with both MBMS analysis and traditional wet chemistry methods. The results were analyzed together with the genomics analysis.ConclusionThe research revealed that, among the species analyzed, the complete lignin biosynthesis pathway first appeared in moss; the pathway is absent in green algae. The expansion of lignin biosynthesis gene families correlates with substrate diversity. In addition, we found that the expansion of the gene families mostly occurred after the divergence of monocots and dicots, with the exception of the C4H gene family. Gene expression analysis revealed different fates of gene duplications, largely confirming plants are tolerant to gene dosage effects. The rapid expansion of lignin biosynthesis genes indicated that the translation of transgenic lignin modification strategies from model species to bioenergy feedstock might only be successful between the closely relevant species within the same family.

Sustained photosynthetic conversion of CO2 to ethylene in recombinant cyanobacterium Synechocystis 6803

Ethylene is the most widely produced petrochemical feedstock globally. It is currently produced exclusively from fossil fuels, and its production is the largest CO2-emitting process in the chemical industry. In this study, we report on a photobiological process for sustained production of ethylene from CO2. The efe gene encoding an ethylene-forming enzyme from Pseudomonas syringae pv. Phaseolicola was previously expressed in cyanobacterial strains, but was not stable. We modified the gene sequence to enhance its stability, and expressed it in Synechocystis sp. PCC 6803, leading to continuous ethylene production. The same ethylene production rate was sustained across four successive sub-cultures without apparent loss of ethylene-forming ability. Up to 5.5% of the fixed carbon was directed to ethylene synthesis, surpassing the published carbon-partition rate into the TCA cycle. Nitrogen- and phosphorus-enriched seawater can support both growth and ethylene production. Factors limiting ethylene production, including efe expression levels, light intensity and nutrient status, were identified and alleviated, resulting in a peak production rate of 5650 μL L−1 h−1 (7125 μg L−1 h−1, 252 μmol L−1 h−1, or 171 mg L−1 day−1), which is higher than that reported for other algae biofuels and chemicals. This study suggests that Synechocystis, expressing the modified efe gene, has potential to be an efficient biological catalyst for the uptake and conversion of CO2 to ethylene.

Identification of QTLs influencing wood property traits in loblolly pine (Pinus taeda L.). II. Chemical wood properties

Abstract Chemical wood property traits were analyzed for the presence of quantitative trait loci (QTLs) in a three-generation outbred pedigree of loblolly pine (Pinus taeda L.). These traits were assayed using pyrolysis molecular beam mass spectrometry and include mass spectrum peak intensities associated with carbohydrates, α-cellulose and hemicellulose sugars, and lignin. Models for projection to latent structures (PLS) were used to also estimate the chemical composition of cell walls (i.e., α-cellulose, galactan and lignin) from mass spectrum data using multivariate regression. Both earlywood and latewood fractions from the fifth annual ring were analyzed for each trait. An interval mapping approach designed for an outbred pedigree was used to estimate the number of QTLs, the magnitude of QTL effects, and their genomic position. Eight unique QTLs influencing cell wall chemistry were detected from multiple peak intensities and/or PLS estimates using the one- and two-QTL models. Significant differences in chemical contents were observed among the populations from North Carolina vs Oklahoma, and results from QTL×environment analyses suggest that QTLs interact with environmental location. QTLs should be verified in larger experiments and in different genetic and environmental backgrounds. QTL mapping will help towards eventually identifying genes having a major effect on chemical wood properties.

Variation of S/G ratio and lignin content in a Populus family influences the release of xylose by dilute acid hydrolysis.

Wood samples from second generation Populus cross were shown to have different lignin contents and S/G ratios (S: syringyl-like lignin structures; G: guaiacyl-like lignin structures). The lignin contents varied from 22.7% to 25.8% and the S/G ratio from 1.8 to 2.3. Selected samples spanning these ranges were hydrolyzed with dilute (1%) sulfuric acid to release fermentable sugars. The conditions were chosen for partial hydrolysis of the hemicellulosic fraction to maximize the expression of variation among samples. The results indicated that both lignin contents and S/G ratio significantly affected the yield of xylose. For example, the xylose yield of the 25.8% lignin and 2.3 S/G (hihg lignin, high S/G) sample produced 30% of the theoretical yield, whereas the xylose yield of the 22.7% lignin and 1.8 S/G (low lignin, low S/G) was 55% of the theoretical value. These results indicate that lignin content and composition among genetic variants within a single species can influence the hydrolyzability of the biomass.

Quantitative genetic analysis of biomass and wood chemistry of Populus under different nitrogen levels.

The genetic control of carbon allocation and partitioning in woody perennial plants is poorly understood despite its importance for carbon sequestration, biofuels and other wood-based industries. It is also unclear how environmental cues, such as nitrogen availability, impact the genes that regulate growth, biomass allocation and wood composition in trees. We phenotyped 396 clonally replicated genotypes of an interspecific pseudo-backcross pedigree of Populus for wood composition and biomass traits in above- and below-ground organs. The loci that regulate growth, carbon allocation and partitioning under two nitrogen conditions were identified, defining the contribution of environmental cues to their genetic control. Sixty-three quantitative trait loci were identified for the 20 traits analyzed. The majority of quantitative trait loci are specific to one of the two nitrogen treatments, demonstrating significant nitrogen-dependent genetic control. A highly significant genetic correlation was observed between plant growth and lignin/cellulose composition, and quantitative trait loci co-localization identified the genomic position of potential pleiotropic regulators. Pleiotropic loci linking higher growth rates to wood with less lignin are excellent targets to engineer tree germplasm improved for pulp, paper and cellulosic ethanol production. The causative genes are being identified with a genetical genomics approach.

Genetic Resources for Maize Cell Wall Biology

Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wall-related genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward- and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysis-molecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions.

Association genetics of traits controlling lignin and cellulose biosynthesis in black cottonwood (Populus trichocarpa, Salicaceae) secondary xylem

• An association genetics approach was used to examine individual genes and alleles at the loci responsible for complex traits controlling lignocellulosic biosynthesis in black cottonwood (Populus trichocarpa). Recent interest in poplars as a source of renewable energy, combined with the vast genomic resources available, has enabled further examination of their genetic diversity. • Forty candidate genes were resequenced in a panel of 15 unrelated individuals to identify single nucleotide polymorphisms (SNPs). Eight hundred and seventy-six SNPs were successfully genotyped in a clonally replicated population (448 clones). The association population (average of 2.4 ramets per clone) was phenotyped using pyrolysis molecular beam mass spectrometry. Both single-marker and haplotype-based association tests were implemented to identify associations for composite traits representing lignin content, syringyl : guaiacyl ratio and C6 sugars. • Twenty-seven highly significant, unique, single-marker associations (false discovery rate Q < 0.10) were identified across 40 candidate genes in three composite traits. Twenty-three significant haplotypes within 11 genes were discovered in two composite traits. • Given the rapid decay of within-gene linkage disequilibrium and the high coverage of amplicons across each gene, it is likely that the numerous polymorphisms identified are in close proximity to the causative SNPs and the haplotype associations reflect information present in the associations between markers.