Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones naringenin and dihydrotricin, and the flavone tricin, incorporated into the lignin polymer of papyrus (Cyperus papyrus L.) rind. These flavonoids were released from the rind lignin by Derivatization Followed by Reductive Cleavage (DFRC), a chemical degradative method that cleaves the β-ether linkages, indicating that at least a fraction of each was integrated into the lignin as β-ether-linked structures. Due to the particular structure of tricin and dihydrotricin, whose C-3ʹ and C-5ʹ positions at their B-rings are occupied by methoxy groups, these compounds can only be incorporated into the lignin through 4ʹ–O–β bonds. However, naringenin chalcone and naringenin have no substituents at these positions and can therefore form additional carbon–carbon linkages, including 3ʹ– or 5ʹ–β linkages that form phenylcoumaran structures not susceptible to cleavage by DFRC. Furthermore, Nuclear Magnetic Resonance analysis indicated that naringenin chalcone can also form additional linkages through its conjugated double bond. The discovery expands the range of flavonoids incorporated into natural lignins, further broadens the traditional definition of lignin, and enhances the premise that any phenolic compound present at the cell wall during lignification could be oxidized and potentially integrated into the lignin structure, depending only on its chemical compatibility. This study indicates that papyrus lignin has a unique structure, as it is the only lignin known to date that integrates such a diversity of phenolic compounds from different classes of flavonoids. This discovery will open up new ways to engineer and design lignins with specific properties and for enhanced value.

Million tons of cellulosic paper have been used for insulating coils in oil-filled electrical power transformers, thereby assuring the electricity supply for our societies. The high working temperatures in transformers constantly degrade paper insulators throughout their service life of up to 40 years. We approached the structural changes in oil-immersed cellulosic paper samples upon thermal stress in a study that compared unbleached softwood Kraft paper used as insulator paper with pure cotton cellulose paper. The model experiments used a thermal treatment in transformer oil at 170 °C for up to 14 days. The samples were characterized by means of¹³C CP/MAS NMR spectroscopy, mainly based on deconvolution of the C4 resonance. An automated, fast, and reproducible C4 resonance deconvolution employing the “Peak Analyzer” tool of OriginPro 2020 (OriginLab Corporation, USA) was developed and used to exploit¹³C CP/MAS NMR spectroscopy for the characterization of thermally stressed paper samples. Our results show that thermally induced structural changes depend heavily on the composition of paper, that hornification and coalescence of fibrils take place, and that the allomorph composition of cellulose crystallites is altered under the given conditions.Graphical abstract

Endoglucanases are increasingly being touted as the ultimate solution for reducing energy consumption during the refining process in the pulp and paper industry. However, due to the high variety of endoglucanases in different enzyme formulations, these perform heterogeneously when applied to different pulps. In this study, the effect of four endoglucanases on softwood and hardwood pulp was studied using confocal laser scanning microscopy (CLSM) after addition of fluorescently labelled carbohydrate binding modules (CBMs). Nuclear magnetic resonance (NMR) analysis and high-performance liquid chromatography quantification of released oligo- and monosaccharides was performed for in-depth mechanistical investigation. Changes in the crystallinity levels caused by enzymatic degradation of amorphous regions were monitored by incubation with two different CBMs from Caldicellulosiruptor bescii and from Thermobifida fusca with high preference to either amorphous or crystalline regions of cellulose, respectively. When dosed at identical activity on the endoglucanase specific CellG5 substrate, CLSM analysis indicated the highest decrease of amorphous regions for those endoglucanases which were also most active in laboratory refining trials and which released highest amounts of cellooligomers from pulp. Using ¹³C-NMR analysis, an increase in para-crystalline cellulose caused by enzyme application was observed. Release of reducing sugars was determined at identical CellG5 dosage, indicating a high variance between the enzymes, especially when complex enzyme formulations were used. Scanning electron microscopy images were obtained for visualization of the endoglucanase activity. The results of mechanistical studies indicate that reduction of amorphous moieties of pulp by endoglucanases is especially beneficial for the refining process.

Papyri belong to the oldest writing grounds in history. Their conservation is of the highest importance in preserving our cultural heritage, which is best achieved based on an extensive knowledge of the materials’ constituents to choose a tailored conservation approach. Thermogravimetric Analysis (TGA) has been widely employed to quantify cellulose and lignin in papyrus sheets, yielding reported lignin contents of 25% to 40%. In this work, the TGA method conventionally used for papyrus samples was repeated and compared to other lignin determination approaches (Klason-lignin and acetyl bromide-soluble lignin). TGA can lead to a large overestimation of the lignin content of commercial papyrus sheets (~27%) compared to the other methods (~5%). A similar overestimation of the lignin content was found for the pith and rind of the native papyrus plant. We concluded that the TGA method should, therefore, not be used for lignin quantification.

Oil-immersed paper insulation and paper pressboards for structural support are widely used in electrical power transformers. Cellulose thus fulfills an essential task for the smooth power supply of our societies. However, the prevailing temperatures in such equipment, combined with a targeted service life of several decades, pose a serious challenge to the long-term integrity of cellulosic paper insulation. Therefore, numerous studies have been conducted to obtain kinetic data on the degradation processes that contribute to the thermally induced decomposition of cellulose. These studies usually rely on the assessment of the average degree of polymerization by viscosity measurements. In this work, we applied and optimized more advanced methods for the characterization of cellulosic materials based on gel permeation chromatography for the special case of thermally stressed unbleached Kraft paper samples. This allowed studying the molar mass distributions of paper polymers upon exposure to heat, as well as the investigation of changes in their conformation in solution and the observation of thermally induced cross-linking. In combination with group-selective fluorescence labeling, it was possible to track over time the changes in molar mass-dependent profiles of carbonyl and carboxyl groups of authentic Kraft insulator paper samples under thermal stress. In addition, changes of the hemicellulose composition were quantified. We hope that this analytical approach to the in-depth characterization of thermally stressed insulator paper will prove useful for future studies of this important cellulose product, and that our findings will contribute to a better understanding of the thermal decomposition of paper in general. Graphical abstract