Quantification of native lignin structural features with gel-phase 2D-HSQC0 reveals lignin structural changes during extraction.

Our ability to study and valorize the lignin fraction of biomass is hampered by the fundamental and still unmet challenge of precisely quantifying native lignin's structural features. Here, we developed a rapid elevated-temperature 1H-13C Heteronuclear Single-Quantum Coherence Zero (HSQC0) NMR method that enables this precise quantification of native lignin structural characteristics even with whole plant cell wall (WPCW) NMR spectroscopy, overcoming fast spin relaxation in the gel phase. We also formulated a Gaussian fitting algorithm to perform automatic and reliable spectral integration. By combining HSQC0 measurements with yield measurements following depolymerization, we can confirm the combinatorial nature of radical coupling reactions during biosynthesis leading to a random sequential organization of linkages within a largely linear lignin chain. Such analyses illustrate how this analytical method can greatly facilitate the study of native lignin structure, which can then be used for fundamental studies or to understand lignin depolymerization methods like reductive catalytic fractionation or aldehyde-assisted fractionation.

Production of Biomass-Derived p-Hydroxybenzamide: Synthesis of p-Aminophenol and Paracetamol.

As we work to transition the modern society that is based on non-renewable chemical feedstocks to a post-modern society built around renewable sources of energy, fuels, and chemicals, there is a need to identify the renewable resources and processes for converting them to platform chemicals. Herein, we explore a strategy for utilizing the p-hydroxybenzoate in biomass feedstocks (e. g., poplar and palm trees) and converting it into a portfolio of commodity chemicals. The targeted bio-derived product in the first processing stage is p-hydroxybenzamide produced from p-hydroxybenzoate esters found in the plant. In the second stage a continuous reaction process converts the p-hydroxybenzamide to p-aminophenol via the Hofmann rearrangement and recovers the unreacted p-hydroxybenzamide. In the third stage the p-aminophenol can be acetylated to form paracetamol, which is readily isolated by liquid/liquid extraction at >95 % purity and an overall p-hydroxybenzamide-to-paracetamol process yield of ~90 %. We explore how utilization of protecting groups alters the challenges in this process and expands the portfolio of possible products to include p-(methoxymethoxy)aniline and N-acetyl-p-(methoxymethoxy)aniline. These target compounds could become value-added renewably-sourced platform chemicals that could be used to produce biodegradable plastics, pigments, and pharmaceuticals.

Functional and Evolutionary Integration of a Fungal Gene With a Bacterial Operon.

Siderophores are crucial for iron-scavenging in microorganisms. While many yeasts can uptake siderophores produced by other organisms, they are typically unable to synthesize siderophores themselves. In contrast, Wickerhamiella/Starmerella (W/S) clade yeasts gained the capacity to make the siderophore enterobactin following the remarkable horizontal acquisition of a bacterial operon enabling enterobactin synthesis. Yet, how these yeasts absorb the iron bound by enterobactin remains unresolved. Here, we demonstrate that Enb1 is the key enterobactin importer in the W/S-clade species Starmerella bombicola. Through phylogenomic analyses, we show that ENB1 is present in all W/S clade yeast species that retained the enterobactin biosynthetic genes. Conversely, it is absent in species that lost the ent genes, except for Starmerella stellata, making this species the only cheater in the W/S clade that can utilize enterobactin without producing it. Through phylogenetic analyses, we infer that ENB1 is a fungal gene that likely existed in the W/S clade prior to the acquisition of the ent genes and subsequently experienced multiple gene losses and duplications. Through phylogenetic topology tests, we show that ENB1 likely underwent horizontal gene transfer from an ancient W/S clade yeast to the order Saccharomycetales, which includes the model yeast Saccharomyces cerevisiae, followed by extensive secondary losses. Taken together, these results suggest that the fungal ENB1 and bacterial ent genes were cooperatively integrated into a functional unit within the W/S clade that enabled adaptation to iron-limited environments. This integrated fungal-bacterial circuit and its dynamic evolution determine the extant distribution of yeast enterobactin producers and cheaters.

Functional and evolutionary integration of a fungal gene with a bacterial operon.

Siderophores are crucial for iron-scavenging in microorganisms. While many yeasts can uptake siderophores produced by other organisms, they are typically unable to synthesize siderophores themselves. In contrast, Wickerhamiella/Starmerella (W/S) clade yeasts gained the capacity to make the siderophore enterobactin following the remarkable horizontal acquisition of a bacterial operon enabling enterobactin synthesis. Yet, how these yeasts absorb the iron bound by enterobactin remains unresolved. Here, we demonstrate that Enb1 is the key enterobactin importer in the W/S-clade species Starmerella bombicola. Through phylogenomic analyses, we show that ENB1 is present in all W/S clade yeast species that retained the enterobactin biosynthetic genes. Conversely, it is absent in species that lost the ent genes, except for Starmerella stellata, making this species the only cheater in the W/S clade that can utilize enterobactin without producing it. Through phylogenetic analyses, we infer that ENB1 is a fungal gene that likely existed in the W/S clade prior to the acquisition of the ent genes and subsequently experienced multiple gene losses and duplications. Through phylogenetic topology tests, we show that ENB1 likely underwent horizontal gene transfer from an ancient W/S clade yeast to the order Saccharomycetales, which includes the model yeast Saccharomyces cerevisiae, followed by extensive secondary losses. Taken together, these results suggest that the fungal ENB1 and bacterial ent genes were cooperatively integrated into a functional unit within the W/S clade that enabled adaptation to iron-limited environments. This integrated fungal-bacterial circuit and its dynamic evolution determines the extant distribution of yeast enterobactin producers and cheaters.

Rapid Biocatalytic Synthesis of Aromatic Acid CoA Thioesters by Using Microbial Aromatic Acid CoA Ligases.

Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzyme A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady-state levels of aromatic acid thioester donors in plants. As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high-yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.

p-Coumaroylation of lignin occurs outside of commelinid monocots in the eudicot genus Morus (mulberry).

The presence of p-coumarate (pCA) in plant cell walls is generally considered to be a trait present only in commelinid monocots. Here, we show that this long-held overgeneralizing assumption is incorrect and that mulberry trees (Morus) are eudicot plants that have lignins derived in part from monolignol pCA esters. As in commelinid monocots, the lignin-bound pCA acylates the sidechain γ-hydroxyl of both coniferyl and syringyl units. This discovery expands mulberry's potential applications to include being a source of p-coumaric acid, a supplier of nutritious berries, a forage crop, a decorative plant, and the main food source for silkworms.

Metabolic engineering of p-hydroxybenzoate in poplar lignin.

Ester-linked p-hydroxybenzoate occurs naturally in poplar lignin as pendent groups that can be released by mild alkaline hydrolysis. These 'clip-off' phenolics can be separated from biomass and upgraded into diverse high-value bioproducts. We introduced a bacterial chorismate pyruvate lyase gene into transgenic poplar trees with the aim of producing more p-hydroxybenzoate from chorismate, itself a metabolic precursor to lignin. By driving heterologous expression specifically in the plastids of cells undergoing secondary wall formation, this strategy achieved a 50% increase in cell-wall-bound p-hydroxybenzoate in mature wood and nearly 10 times more in developing xylem relative to control trees. Comparable amounts also remained as soluble p-hydroxybenzoate-containing xylem metabolites, pointing to even greater engineering potential. Mass spectrometry imaging showed that the elevated p-hydroxybenzoylation was largely restricted to the cell walls of fibres. Finally, transgenic lines outperformed control trees in assays of saccharification potential. This study highlights the biotech potential of cell-wall-bound phenolate esters and demonstrates the importance of substrate supply in lignin engineering.

Evolution of p-coumaroylated lignin in eudicots provides new tools for cell wall engineering.

Ester-linked p-coumarate (pCA) is a hallmark feature of the secondary cell walls in commelinid monocot plants. It has been shown that pCA groups arise during lignin polymerisation from the participation of monolignol conjugates assembled by p-coumaroyl-CoA:monolignol transferase (PMT) enzymes, members of the BAHD superfamily of acyltransferases. Herein, we report that a eudicot species, kenaf (Hibiscus cannabinus), naturally contains p-coumaroylated lignin in the core tissues of the stems but not in the bast fibres. Moreover, we identified a novel acyltransferase, HcPMT, that shares <30% amino acid identity with known monocot PMT sequences. Recombinant HcPMT showed a preference in enzyme assays for p-coumaroyl-CoA and benzoyl-CoA as acyl donor substrates and sinapyl alcohol as an acyl acceptor. Heterologous expression of HcPMT in hybrid poplar trees led to the incorporation of pCA in lignin, but no improvement in the saccharification potential of the wood. This work illustrates the value in mining diverse plant taxa for new monolignol acyltransferases. Furthermore, the occurrence of pCA outside monocot lineages may represent another example of convergent evolution in lignin structure. This discovery expands textbook views on cell wall biochemistry and provides a new molecular tool for engineering the lignin of biomass feedstock plants.

A new approach to zip-lignin: 3,4-dihydroxybenzoate is compatible with lignification.

Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. This study shows, for the first time, that 3,4-dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants. We introduced a bacterial 3-dehydroshikimate dehydratase into hybrid poplar (Populus alba × grandidentata) to divert carbon flux away from the shikimate pathway, which lies upstream of lignin biosynthesis. Transgenic poplar wood had up to 33% less lignin with p-hydroxyphenyl units comprising as much as 10% of the lignin. Mild alkaline hydrolysis of transgenic wood released fewer ester-linked p-hydroxybenzoate groups than control trees, and revealed the novel incorporation of cell-wall-bound DHB, as well as glycosides of 3,4-dihydroxybenzoic acid (DHBA). Two-dimensional nuclear magnetic resonance (2D-NMR) analysis uncovered DHBA-derived benzodioxane structures suggesting that DHB moieties were integrated into the lignin polymer backbone. In addition, up to 40% more glucose was released from transgenic wood following ionic liquid pretreatment and enzymatic hydrolysis. This work highlights the potential of diverting carbon flux from the shikimate pathway for lignin engineering and describes a new type of 'zip-lignin' derived from the incorporation of DHB into poplar lignin.

Identification and characterization of a set of monocot BAHD monolignol transferases.

Plant BAHD acyltransferases perform a wide range of enzymatic tasks in primary and secondary metabolism. Acyl-CoA monolignol transferases, which couple a CoA substrate to a monolignol creating an ester linkage, represent a more recent class of such acyltransferases. The resulting conjugates may be used for plant defense but are also deployed as important "monomers" for lignification, in which they are incorporated into the growing lignin polymer chain. p-Coumaroyl-CoA monolignol transferases (PMTs) increase the production of monolignol p-coumarates, and feruloyl-CoA monolignol transferases (FMTs) catalyze the production of monolignol ferulate conjugates. We identified putative FMT and PMT enzymes in sorghum (Sorghum bicolor) and switchgrass (Panicum virgatum) and have compared their activities to those of known monolignol transferases. The putative FMT enzymes produced both monolignol ferulate and monolignol p-coumarate conjugates, whereas the putative PMT enzymes produced monolignol p-coumarate conjugates. Enzyme activity measurements revealed that the putative FMT enzymes are not as efficient as the rice (Oryza sativa) control OsFMT enzyme under the conditions tested, but the SbPMT enzyme is as active as the control OsPMT enzyme. These putative FMTs and PMTs were transformed into Arabidopsis (Arabidopsis thaliana) to test their activities and abilities to biosynthesize monolignol conjugates for lignification in planta. The presence of ferulates and p-coumarates on the lignin of these transformants indicated that the putative FMTs and PMTs act as functional feruloyl-CoA and p-coumaroyl-CoA monolignol transferases within plants.

pHBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar.

Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol-p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol-p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol-p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol-p-hydroxybenzoates and cell-wall-bound monolignol-p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

Oxidative Catalytic Fractionation of Lignocellulosic Biomass under Non-alkaline Conditions.

Biomass pretreatment methods are commonly used to isolate carbohydrates from biomass, but they often lead to modification, degradation, and/or low yields of lignin. Catalytic fractionation approaches provide a possible solution to these challenges by separating the polymeric sugar and lignin fractions in the presence of a catalyst that promotes cleavage of the lignin into aromatic monomers. Here, we demonstrate an oxidative fractionation method conducted in the presence of a heterogeneous non-precious-metal Co-N-C catalyst and O2 in acetone as the solvent. The process affords a 15 wt% yield of phenolic products bearing aldehydes (vanillin, syringaldehyde) and carboxylic acids (p-hydroxybenzoic acid, vanillic acid, syringic acid), complementing the alkylated phenols obtained from existing reductive catalytic fractionation methods. The oxygenated aromatics derived from this process have appealing features for use in polymer synthesis and/or biological funneling to value-added products, and the non-alkaline conditions associated with this process support preservation of the cellulose, which remains insoluble at reaction conditions and is recovered as a solid.

Overexpression of a Sugarcane BAHD Acyltransferase Alters Hydroxycinnamate Content in Maize Cell Wall.

The purification of hydroxycinnamic acids [p-coumaric acid (pCA) and ferulic acid (FA)] from grass cell walls requires high-cost processes. Feedstocks with increased levels of one hydroxycinnamate in preference to the other are therefore highly desirable. We identified and conducted expression analysis for nine BAHD acyltransferase ScAts genes from sugarcane. The high conservation of AT10 proteins, together with their similar gene expression patterns, supported a similar role in distinct grasses. Overexpression of ScAT10 in maize resulted in up to 75% increase in total pCA content. Mild hydrolysis and derivatization followed by reductive cleavage (DFRC) analysis showed that pCA increase was restricted to the hemicellulosic portion of the cell wall. Furthermore, total FA content was reduced up to 88%, resulting in a 10-fold increase in the pCA/FA ratio. Thus, we functionally characterized a sugarcane gene involved in pCA content on hemicelluloses and generated a C4 plant that is promising for valorizing pCA production in biorefineries.

Stacking AsFMT overexpression with BdPMT loss of function enhances monolignol ferulate production in Brachypodium distachyon.

To what degree can the lignin subunits in a monocot be derived from monolignol ferulate (ML-FA) conjugates? This simple question comes with a complex set of variables. Three potential requirements for optimizing ML-FA production are as follows: (1) The presence of an active FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme throughout monolignol production; (2) Suppression or elimination of enzymatic pathways competing for monolignols and intermediates during lignin biosynthesis; and (3) Exclusion of alternative phenolic compounds that participate in lignification. A 16-fold increase in lignin-bound ML-FA incorporation was observed by introducing an AsFMT gene into Brachypodium distachyon. On its own, knocking out the native p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (BdPMT) pathway that competes for monolignols and the p-coumaroyl-CoA intermediate did not change ML-FA incorporation, nor did partial loss of CINNAMOYL-CoA REDUCTASE1 (CCR1) function, which reduced metabolic flux to monolignols. However, stacking AsFMT into the Bdpmt-1 mutant resulted in a 32-fold increase in ML-FA incorporation into lignin over the wild-type level.

A bacterial biosynthetic pathway for methylated furan fatty acids.

Fatty acids play many important roles in cells and also in industrial processes. Furan fatty acids (FuFAs) are present in the lipids of some plant, fish, and microbial species and appear to function as second messengers in pathways that protect cells from membrane-damaging agents. We report here the results of chemical, genetic, and synthetic biology experiments to decipher the biosynthesis of the monomethylated FuFA, methyl 9-(3-methyl-5-pentylfuran-2-yl) nonanoate (9M5-FuFA), and its dimethyl counterpart, methyl 9-(3,4-dimethyl-5-pentylfuran-2-yl) nonanoate (9D5-FuFA), in two α-proteobacteria. Each of the steps in FuFA biosynthesis occurs on pre-existing phospholipid fatty acid chains, and we identified pathway intermediates and the gene products that catalyze 9M5-FuFA and 9D5-FuFA synthesis in Rhodobacter sphaeroides 2.4.1 and Rhodopseudomonas palustris CGA009. One previously unknown pathway intermediate was a methylated diunsaturated fatty acid, (10E,12E)-11-methyloctadeca-10,12-dienoic acid (11Me-10t,12t-18:2), produced from (11E)-methyloctadeca-11-enoic acid (11Me-12t-18:1) by a newly identified fatty acid desaturase, UfaD. We also show that molecular oxygen (O2) is the source of the oxygen atom in the furan ring of 9M5-FuFA, and our findings predict that an O2-derived oxygen atom is incorporated into 9M5-FuFA via a protein, UfaO, that uses the 11Me-10t,12t-18:2 fatty acid phospholipid chain as a substrate. We discovered that R. palustris also contains a SAM-dependent methylase, FufM, that produces 9D5-FuFA from 9M5-FuFA. These results uncover the biochemical sequence of intermediates in a bacterial pathway for 9M5-FuFA and 9D5-FuFA biosynthesis and suggest the existence of homologs of the enzymes identified here that could function in FuFA biosynthesis in other organisms.

Assessing the Viability of Recovery of Hydroxycinnamic Acids from Lignocellulosic Biorefinery Alkaline Pretreatment Waste Streams.

Invited for this month's cover is the research team from the D.O.E. Great Lake Bioenergy Research Center (GLBRC) at the University of Wisconsin-Madison. The cover image shows how a diverse team with expertise in many different fields works together in an integrated fashion to address complex problems. Only when the whole system, from field to the liquid fuels and co-products, is assessed, can we identify the key parameters needed to design an economically viable biorefinery-based economy. Cover art by Chelsea Mamott. The Full Paper itself is available at 10.1002/cssc.201903345.

Mechanistic Study of Diaryl Ether Bond Cleavage during Palladium-Catalyzed Lignin Hydrogenolysis.

Hydrogenolysis has emerged as one of the most effective means of converting polymeric lignin into monoaromatic fragments of value. Reported yields may be higher than for other methods and can exceed the theoretical yields estimated from measures of the content of lignin's most readily cleaved alkyl-aryl ether bonds in ß-ether units. The high yields suggest that other units in lignin are being cleaved. Diaryl ether units are important units in lignin, and their cleavage has been examined previously using simple model compounds, such as diphenyl ether. Herein, the hydrogenolysis of model compounds that closely resemble the native lignin 4-O-5 diaryl ether units was analyzed. The results provided unexpected insights into the reactivity and partial cleavage of these compounds. The models and lignin polymer produced not only monomers, but also unusual 1,3,5-meta-substituted aromatics that appear to be diagnostic for the presence and the cleavage of the 4-O-5 diaryl ether unit in lignin.

Assessing the Viability of Recovery of Hydroxycinnamic Acids from Lignocellulosic Biorefinery Alkaline Pretreatment Waste Streams.

The hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) add diversity to the portfolio of products produced by using grass-fed lignocellulosic biorefineries. The level of lignin-bound pCA in Zea mays was modified by the alteration of p-coumaroyl-CoA monolignol transferase expression. The biomass was processed in a lab-scale alkaline-pretreatment biorefinery process and the data were used for a baseline technoeconomic analysis to determine where to direct future research efforts to couple plant design to biomass utilization processes. It is concluded that future plant engineering efforts should focus on strategies that ramp up accumulation of one type of hydroxycinnamate (pCA or FA) predominantly and suppress that of the other. Technoeconomic analysis indicates that target extraction titers of one hydroxycinnamic acid need to be >50 g kg-1 biomass, at least five times higher than observed titers for the impure pCA/FA product mixture from wild-type maize. The technical challenge for process engineers is to develop a viable process that requires more than 80 % reduction of the isolation costs.

Anaerobic Degradation of Syringic Acid by an Adapted Strain of Rhodopseudomonas palustris.

While lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogeneous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a by-product of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions via the benzoyl coenzyme A (CoA) degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta-methoxylated phenolics, such as syringic acid. We isolated a strain of R. palustris (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the vanARB operon (rpa3619, rpa3620, rpa3621), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustrisIMPORTANCE Lignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for the production of aromatic-based biochemicals requires detailed knowledge of the metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid degradation reveal a previously unknown metabolic route for aromatic degradation in R. palustris This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire required to metabolize aromatic compounds from lignin and other renewable sources.

Electrochemical Aminoxyl-Mediated Oxidation of Primary Alcohols in Lignin to Carboxylic Acids: Polymer Modification and Depolymerization.

An electrochemical process has been developed for chemoselective oxidation of primary alcohols in lignin to the corresponding carboxylic acids. The electrochemical oxidation reactions proceed under mildly basic conditions and employ 2,2,6,6-tetramethyl-1-piperidine N-oxyl (TEMPO) and 4-acetamido-TEMPO (ACT) as catalytic mediators. Lignin model compounds and related alcohols are used to conduct structure-reactivity studies that provide insights into the origin of the reaction selectivity. The method is applied to the oxidation of lignin extracted from poplar wood chips via a mild acidolysis method, and the reaction affords a novel polyelectrolyte material. Gel permeation chromatography data for the oxidized lignin shows that this material has a molecular weight and molecular weight distribution very similar to that of the extracted lignin, but notable differences are also evident. Base titration reveals a significant increase in the acid content, and the oxidized lignin has much higher water solubility relative to the extracted lignin. Treatment of the oxidized lignin under acidic conditions results in depolymerization of the material into characterized aromatic monomers in nearly 30 wt% yield.