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The bacterial BcsA-B cellulose synthase synthesizes cellulose II when immobilized on nickel surfaces. To explore how lignin interacts with cellulose, lignin was polymerized either during or after cellulose formation from surface immobilized BcsA-B, leading to significant alterations in cellulose and lignin morphology. To facilitate lignin detection, polymerized coniferyl alcohol monolignols were detected by lignin autofluorescence. This compound was previously demonstrated as a suitable lignin monolignol exhibiting autofluorescence that enables lignin detection even at low concentrations. Cellulose and lignin coated nickel surfaces were studied under confocal microscopy, as well as by atomic force microscopy and X-ray diffractometry (XRD). As a consequence of simultaneous lignin and cellulose deposition on the surface, cellulose microfibril morphology was altered. XRD data indicated that cellulose crystal size was significantly decreased by polymerized lignin incorporation, suggesting that simultaneous lignin polymerization prevents the proper assembly of cellulose microfibrils. In contrast, polymerizing lignin after cellulose synthesis yielded a cellulose that was very similar in character to control (lignin-free) samples in terms of crystal morphology.

The manuscript initially failed to cite the work of Dr. Cong et al., which was the basis for this effort. We apologize for this error.

The polyaromatic structure of lignin has long been recognized as a key contributor to the rigidity of plant vascular tissues. Although lignin structure was once conceptualized as a highly networked, heterogeneous, high molecular weight polymer, recent studies have suggested a very different configuration may exist in planta. These findings, coupled with the increasing attention and interest in efficiently utilizing lignocellulosic materials for green materials and energy applications, have renewed interest in lignin chemistry. Here we focus on quinone methides (QMs)—key intermediates in lignin polymerization—that are quenched via reaction with cell-wall-available nucleophiles. Reactions with alcohol and uronic acid groups of hemicelluloses, for example, can lead to lignin-carbohydrate crosslinks. Our work is a first step toward exploring potential QM reactions with nucleophilic groups in cell wall proteins. We conducted a model compound study wherein the lignin model compound guaiacylglycerol-β-guaiacyl ether 1, was converted to its QM 2, then reacted with amino acids bearing nucleophilic side-groups. Yields for the QM-amino acid adducts ranged from quantitative in the case of QM-lysine 3, to zero (no reaction) in the cases of QM-threonine (Thr) 10 and QM-hydroxyproline (Hyp) 11. The structures of the QM-amino acid adducts were confirmed via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and density functional theory (DFT) calculations, thereby extending the lignin NMR database to include amino acid crosslinks. Some of the QM-amino acid adducts formed both syn- and anti-isomers, whereas others favored only one isomer. Because the QM-Thr 10 and QM-Hyp 11 compounds could not be experimentally prepared under conditions described here but could potentially form in vivo, we used DFT to calculate their NMR shifts. Characterization of these model adducts extends the lignin NMR database to aid in the identification of lignin-protein linkages in more complex in vitro and in vivo systems, and may allow for the identification of such linkages in planta.

The present study characterized the products formed from the reaction of amino acids and in turn, proteins, with lignin resulting in cross-coupling. When added to reaction mixtures containing coniferyl alcohol, horseradish peroxidase and H2O2, three amino acids (Cys, Tyr, and Thr) are able to form adducts. The low molecular weight products were analyzed by HPLC and from each reaction mixture, one product was isolated and analyzed by LC/MS. LC/MS results are consistent with bond formation between the polar side-chain of these amino acids with Cα. These results are consistent with the cross-coupling of Cys, Tyr and Thr through a quinone methide intermediate. In addition to the free amino acids, it was found that the cross-coupling of proteins with protolignin through Cys or Tyr residues. The findings provide a mechanism by which proteins and lignin can cross-couple in the plant cell wall.

The current study uses nuclear magnetic resonance, Fourier-transform infrared spectroscopy and Raman spectroscopy to investigate the evolution of refined softwood and hardwood lignins under various pyrolytic exposures. Little chemical change occurred at pyrolysis temperatures of 250 and 300 °C, whereas significant mass loss and chemical change was observed at 400 and 500 °C. These losses were mainly attributed to evolution of methoxyl, hydroxyl, and propyl groups. Mass loss plateaued following pyrolysis at 500 °C, but rearrangements continued to occur at higher temperatures, resulting in char that became increasingly polyaromatic in nature. Following brief pyrolytic exposures at 500 and 600 °C, the refined hardwood and softwood lignins yielded coal-like products. Lignin pyrolyzed at higher temperatures yielded chars with greater order, similar in composition to coke. These coal and coke-like products are called “lignin-based carbon” (LBC). The polyaromatic nature of the LBC after high temperature pyrolysis was perceived as the result of radical formation and recombination, leading to fused aromatic structures, which occurs more readily at higher temperatures.

Lignin that had been extracted from Kraft black liquor was investigated as a fusing binder in briquetted anthracite fines for a foundry coke substitute. Cupola “heat zone” pyrolytic temperatures of 300–1550 °C were appraised, with the focus on 900 °C. Briquettes with favorable strength were made with 86–92% anthracite fines, 2.3–8.6% lignin, 4.5% silicon metal powder, and 0.9% hydrolyzed collagen (denatured collagen) by mass. Briquettes were pyrolyzed under a nitrogen atmosphere or a starved air condition to simulate a cupola pyrolytic heat zone, and then crushed after this pyrolysis so as to discern their unconfined compressive (UC) strength. These tests mimicked key features of the crushing load that coke endures in a cupola. After 30 min of 900 °C pyrolysis, UC strength reached 2200–3000 kPa (320–440 psi), when these briquettes contained 4.5% softwood lignin or 2.3% hardwood lignin. With ⩾6.5% hardwood lignin, the UC strength after 900 °C pyrolysis reached 6000–6500 kPa. When no lignin was incorporated into the briquette, the UC strength after 900 °C pyrolysis was a mere 200 kPa. Denatured collagen quantity affected lignin heat zone strength, despite by itself losing strength around 300 °C: with 4.5% lignin present, 1.8% denatured collagen doubled the strength of 0.45% denatured collagen briquettes. Adding tannic acid to the briquettes greatly increased the UC strength as well. Lignin provided strength up to 1400 °C. Moreover above 1100 °C, silicon carbide nanowires greatly enhanced UC strength relative to lignin alone. Briquettes with lignin gained UC strength very quickly when flash pyrolyzed to 900 °C, which is important in the cupola. The results herein showed that the bindered briquettes burned at an equal rate as did coke when these were burned at 1100 °C in air. The briquettes also contained an energy density that was 38% higher by volume than that of coke. Harnessing these high temperature pyrolytic lignin fusing reactions creates a valuable foundry coke substitute for the future, as well as large scale applications for otherwise underutilized industrial streams of lignin.

Lignin degradation is of critical importance in biomass utilization of woody plants, because the presence of lignin limits hydrolytic enzyme access to cellulose and hemicellulose. To date, strategies for lignin removal have ranged from treatment with lignin-degrading fungi to genetic manipulations that reduce lignin content or modify monolignol composition. However, reducing lignin content without compromising tree fitness can be difficult. An alternate approach to modify lignin composition by introducing in situ peptide cross-links is reported here. The hypothesis was that introducing in situ peptide cross-links would not change overall lignin content or distribution in the cell wall, yet should render the lignin more susceptible to protease digestion. To test this, a transgene encoding a high tyrosine-content peptide was designed to express in lignifying tissues of hybrid poplar. Relative to wildtypes, the transgenics had no change in total lignin content or overall plant morphology, although the wood in a number of lines had reduced storage modulus. As predicted, a number of transgenic lines were more susceptible to protease digestion than wildtypes, resulting in a higher polysaccharide release from the lignocellulose complexes. Although preliminary, these results suggest that this may be a viable means to facilitate lignin removal and potentially advance the utilization of woody biomass as a biofuel feedstock.

The work presented here investigates the cross-linking of various nucleophilic amino acids with lignin under aqueous conditions, thus providing insight as to which amino acids might cross-link with lignin in planta. Lignin dehydrogenation polymer (DHP) was prepared in aqueous solutions that contained tripeptides with the general structure XGG, where X represents an amino acid with a nucleophilic side chain. Fourier-transform infrared spectroscopy and energy dispersive X-ray spectroscopy showed that peptides containing cysteine and tyrosine were incorporated into the DHP to form DHP-CGG and DHP-YGG adducts, whereas peptides containing other nucleophilic amino acids were not incorporated. Scanning electron microscopy showed that the physical morphology of DHP was altered by the presence of peptides in the aqueous solution, regardless of peptide incorporation into the DHP. Nuclear magnetic resonance (NMR) spectroscopy showed that cysteine-containing peptide cross-linked with lignin at the lignin α-position, whereas in the case of the lignin-tyrosine adduct the exact cross-linking pathway could not be determined. This is the first study to use NMR to confirm cross-linking between lignin and peptides under biomimetic conditions. The results of this study may indicate the potential for lignin-protein linkage formation in planta, particularly between lignin and cysteine- and/or tyrosine-rich proteins.

Our study has identified pathways and gene candidates that may be associated with the greater flexibility and digestibility of the poplar cell walls. With the goal of facilitating lignin removal during the utilization of woody biomass as a biofuel feedstock, we previously transformed a hybrid poplar clone with a partial cDNA sequence encoding a tyrosine- and hydroxyproline-rich glycoprotein from parsley. A number of the transgenic lines released more polysaccharides following protease digestion and were more flexible than wild-type plants, but otherwise normal in phenotype. Here, we report that overexpression of the tyrosine-rich peptide encoding sequence in these transgenic poplar plants did not significantly alter total lignin quantity or quality (S/G lignin ratio), five- and six-carbon sugar contents, growth rate, or susceptibility to a major poplar fungal pathogen, Septoria musiva. Whole-genome microarray analysis revealed a total of 411 differentially expressed transcripts in transgenic lines, all with decreased transcript abundance relative to wild-type plants. Their corresponding genes were overrepresented in functional categories such as secondary metabolism, amino acid metabolism, and energy metabolism. Transcript abundance was decreased primarily for five types of genes encoding proteins involved in cell-wall organization and in lignin biosynthesis. The expression of a subset of 19 of the differentially regulated genes by qRT-PCR validated the microarray results. Our study has identified pathways and gene candidates that may be the underlying cause for the enhanced flexibility and digestibility of the stems of poplar plants expressing the TYR transgene.