Analyses of long-term fungal degradation of spruce bark reveals varying potential for catabolism of polysaccharides and extractive compounds.

The bark represents the outer protective layer of trees. It contains high concentrations of antimicrobial extractives, in addition to regular wood polymers. It represents a huge underutilized side stream in forestry, but biotechnological valorization is hampered by a lack of knowledge on microbial bark degradation. Many fungi are efficient lignocellulose degraders, and here, spruce bark degradation by five species, Dichomitus squalens, Rhodonia placenta, Penicillium crustosum, Trichoderma sp. B1, and Trichoderma reesei, was mapped, by continuously analyzing chemical changes in the bark over six months. The study reveals how fungi from different phyla degrade bark using diverse strategies, regarding both wood polymers and extractives, where toxic resin acids were degraded by Basidiomycetes but unmodified/tolerated by Ascomycetes. Proteome analyses of the white-rot D. squalens revealed several proteins, with both known and unknown functions, that were specifically upregulated during growth on bark. This knowledge can accelerate improved utilization of an abundant renewable resource.

Correction: Investigation of cellulose dissolution in morpholinium-based solvents: impact of solvent structural features on cellulose dissolution.

[This corrects the article DOI: 10.1039/D3RA03370H.].

Resin acids play key roles in shaping microbial communities during degradation of spruce bark.

The bark is the outermost defense of trees against microbial attack, largely thanks to toxicity and prevalence of extractive compounds. Nevertheless, bark decomposes in nature, though by which species and mechanisms remains unknown. Here, we have followed the development of microbial enrichments growing on spruce bark over six months, by monitoring both chemical changes in the material and performing community and metagenomic analyses. Carbohydrate metabolism was unexpectedly limited, and instead a key activity was metabolism of extractives. Resin acid degradation was principally linked to community diversification with specific bacteria revealed to dominate the process. Metagenome-guided isolation facilitated the recovery of the dominant enrichment strain in pure culture, which represents a new species (Pseudomonas abieticivorans sp. nov.), that can grow on resin acids as a sole carbon source. Our results illuminate key stages in degradation of an abundant renewable resource, and how defensive extractive compounds have major roles in shaping microbiomes.

Anion-Specific Adsorption of Carboxymethyl Cellulose on Cellulose.

Integration of fiber modification step with a modern pulp mill is a resource efficient way to produce functional fibers. Motivated by the need to integrate polymer adsorption with the current pulping system, anion-specific effects in carboxymethylcellulose (CMC) adsorption have been studied. The QCM-D adsorption experiments revealed that CMC adsorption to the cellulose model surface is prone to anion-specific effects. A correlation was observed between the adsorbed CMC and the degree of hydration of the co-ions present in the magnesium salts. The presence of a chaotropic co-ion such as nitrate increased the adsorption of CMC on cellulose compared to the presence of the kosmotropic sulfate co-ion. However, anion-specificity was not significant in the case of salts containing zinc cations. The hydration of anions determines the distribution of the ions at the interface. Chaotropic ions, such as nitrates, are likely to be distributed near the chaotropic cellulose surface, causing changes in the ordering of water molecules and resulting in greater entropy gain once released from the surface, thus increasing CMC adsorption.

Investigation of cellulose dissolution in morpholinium-based solvents: impact of solvent structural features on cellulose dissolution.

A series of N-methylmorpholinium salts with varying N-alkyl chains and Cl-, OAc- and OH- as counter ions have been synthesized and investigated for their ability to dissolve cellulose, aiming at elucidating solvent structural features affecting cellulose dissolution. Synthesis procedures have been developed to, to a high extent, rely on conversions in water and microwave-assisted reactions employing a reduced number of work-up steps and ion-exchange resins that can be regenerated. Water solutions of morpholinium hydroxides proved capable of dissolving cellulose, with those of them possessing alkyl chains longer than ethyl showing surprising dissolution ability at room-temperature. Morpholinium acetates behaved as ionic liquids, and were also capable of dissolving cellulose when combined with DMSO. The obtained cellulose solutions were characterized according to their chemical and colloidal stability using 13C NMR spectroscopy, size exclusion chromatography and flow sweep measurements, while the ethanol coagulates were investigated in terms of crystallinity using solid state NMR. In contrast, the morpholinium chlorides obtained were hygroscopic with high melting points and low solubility in common organic solvents e.g., acetone, DMSO and DMAc, thus lacking the ability to swell or dissolve cellulose.

Cellulose interactions with CO2 in NaOH(aq): The (un)expected coagulation creates potential in cellulose technology.

Upon the search for a suitable processing method for cellulose, the dissolution in NaOH(aq) presents a real green potential, including its ability to sorb environmental CO2(g) affecting dissolution. Here, CO2(g) was delivered in a controlled way to the cellulose/NaOH(aq) system during the in-situ analysis with FTIR, pH and temperature meters, resulting in efficient coagulation of cellulose. Surprisingly, the coagulation occurred with a minimal loss of alkalinity. This was considered an effect of a specific interaction between the dissolved polymer and gas, resulting in the introduction of carbonate species, highly influential in the coagulation process. The process repeated at 25 °C and 5 °C suggested a strong influence of temperature. The conversion routes of the CO2(g) coagulant were also related to the dissolved state of the polymer. The recovered cellulose appeared competitive with EtOH recovery in terms of structure. The presented finding put a perspective on the utilisation of both the coagulation process and final materials in cellulose technology.

Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum.

Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments. The enzymes were named CbTan1-3, and we show that each one exhibits a unique substrate preference on a range of galloyl ester model substrates; CbTan1 and 3 demonstrated preference toward galloyl esters linked to glucose, while CbTan2 was more promiscuous. All enzymes were also active on oak bark extractives. Furthermore, we solved the crystal structure of CbTan2 and produced homology models for CbTan1 and 3. In each structure, the catalytic triad and gallate-binding regions in the core domain were found in very similar positions in the active site compared with other bacterial tannases, suggesting a similar mechanism of action among these enzymes, though large inserts in each enzyme showcase overall structural diversity. In conclusion, the varied structural features and substrate specificities of the C. butyricum tannases indicate that they have different biological roles and could further be used in development of new valorization strategies for renewable plant biomass.

Calcium Ion-Induced Structural Changes in Carboxymethylcellulose Solutions and Their Effects on Adsorption on Cellulose Surfaces.

The adsorption of carboxymethylcellulose (CMC) on cellulose surfaces is one of the most studied examples of the adsorption of an anionic polyelectrolyte on a like-charged surface. It has been suggested that divalent ions can act as a bridge between CMC chains and the surface of cellulose and enhance the CMC adsorption: they can, however, also alter the structure of CMCs in the solution. In previous investigations, the influence of cations on solution properties has been largely overlooked. This study investigates the effect of Ca2+ ions on the properties of CMC solutions as well as the influence on cellulose nanofibers (CNFs), which was studied by dynamic light scattering and correlated with the adsorption of CMC on a cellulose surface probed using QCM-D. The presence of Ca2+ facilitated the multichain association of CMC chains and increased the hydrodynamic diameter. This suggests that the adsorption of CMCs at high concentrations of CaCl2 is governed mainly by changes in solution properties rather than by changes in the cellulose surface. Furthermore, an entropy-driven mechanism has been suggested for the adsorption of CMC on cellulose. By comparing the adsorption of CMC from H2O and D2O, it was found that the release of water from the cellulose surface is driving the adsorption of CMC.

Fundamental aspects of the non-covalent modification of cellulose via polymer adsorption.

The increasing need for new material applications based on cellulose demands increased functional diversity and thus new functionalisation/modification approaches. The non-covalent modification of cellulose fibres via the adsorption of functional polymers has emerged as a promising route for tailoring the properties of material. This review focuses on fundamental aspects of polymer adsorption on cellulose surfaces, where the adsorption of polyelectrolytes and non-polyelectrolytes are treated separately. Adsorption studies on model surfaces as well as cellulose macro-fibres are reviewed. A correlation of the adsorption findings with the Scheutjens-Fleer polymer adsorption theory is provided, allowing the fundamentals behind the polymer adsorption phenomenon and its context in utilization of cellulose fibres to be understood.

Scattering studies of the size and structure of cellulose dissolved in aqueous hydroxide base solvents.

Combining NaOH with other hydroxide bases with superior dissolution properties can be a means of improving dissolution of cellulose. However, this raises questions about how the size and structure of cellulose vary when dissolved in different hydroxide bases. Here, cellulose in aqueous solutions of NaOH, Tetramethylammonium hydroxide (TMAH), Benzyltrimethylammonium hydroxide (Triton B) and previously studied equimolar solutions of NaOH/TMAH and NaOH/Triton B were investigated using small angle X-ray scattering, static and dynamic light scattering. The results show that cellulose in NaOH(aq) is largely aggregated and that the more hydrophobic TMAH and Triton are capable of molecularly dissolving cellulose into worm-like conformations, stiffer than in NaOH. The dissolution properties of mixtures are highly dependent on the compatibility of the individual bases; in line with previous observations of the properties of the solutions which now could be correlated to the structure of the cellulose on a nano- and microscale.

On the interference of urea with CO2/CO32- chemistry of cellulose model solutions in NaOH(aq).

The CO2/CO32- chemistry of the cellulose/NaOH(aq) solutions has been recently reported to comprise a CO2 incorporation through formation of a transient cellulose carbonate intermediate along with cellulose - CO32- interactions. This work explores on molecular interactions arising when this chemistry is brought together with urea, the most common stabiliser of these solutions. 1H, 13C and steady-state heteronuclear Overhauser effect NMR studies on the cellulose analogues (methyl-ß-glucopyranoside (ß-MeO-Glcp) and microcrystalline cellulose), combined with pH and ATR-FTIR measurements, reveal concurrent interactions of urea with both CO2 and CO32-- leading to increased uptake of CO2 and a buffering effect. Yet, regardless of the presence of urea, the route of conversion from CO2 to CO32-, whether going through reaction with the carbohydrate alkoxides or OH-, is likely to determine the chemical environment of the formed CO32-. These findings shed a new light on rather overlooked, albeit prominent, interactions in these solutions with the readily absorbed air CO2, essential for further development and implementation, whether towards regenerated and modified cellulose or CO2-capturing concepts.

Probing Interactions in Combined Hydroxide Base Solvents for Improving Dissolution of Cellulose.

To further understand cellulose-solvent interactions in aqueous hydroxide solutions, cellulose behavior in aqueous solutions of NaOH combined with tetramethylammonium hydroxide (TMAH) or benzyltrimethylammonium hydroxide (Triton B), as well as urea, was investigated. The rheological properties of the solutions were assessed through flow sweeps at different temperatures, and the intermolecular interactions were probed using solvatochromic dyes. The results showed that NaOH combined with TMAH had synergistic effects on cellulose dissolution and was a better solvent for cellulose than the combination of NaOH with Triton B, in spite of the superior dissolution ability of Triton B alone. This somewhat unexpected finding shows that the base pair needs to be selected with care. Interestingly, addition of urea had no significant effect on the solvatochromic parameters or dissolution capacity of solutions of Triton B but rendered improved stability of solutions containing NaOH and/or TMAH. It seems that both urea and Triton B weaken the hydrophobic assembly effect of these solutions, but urea is excluded from interacting with cellulose in the presence of Triton B. This study provides further insight into dissolution of cellulose and the possibility of utilizing combinations of hydroxide bases to achieve improved solution properties.

Hydrothermal pretreatment of wood by mild steam explosion and hot water extraction.

The aim of this work was to compare the two most common hydrothermal pre-treatments for wood - mild steam explosion and hot water extraction - both with the prospect of enabling extraction of hemicelluloses and facilitating further processing. Although both involve autohydrolysis of the lignocellulosic tissue, they are performed under different conditions: the most prominent difference is the rapid, disintegrating, discharge employed in the steam explosion opening up the structure. In this comparative study, the emphasis was placed on local composition of the pre-treated wood chips (of industrially relevant size). The results show that short hot water extraction treatments lead to significant variations in the local composition within the wood chips, while steam explosion accomplishes a comparably more even removal of hemicelluloses due to the advective mass transport during the explosion step.

Nano-cellulosic materials: the impact of water on their dissolution in DMAc/LiCl.

The dissolution behaviour of disassociated cellulosic materials in N,N-dimethylacetamide/lithium chloride (DMAc/LiCl) was investigated. The parameters monitored were chromatographic elution profiles and recovered mass by means of gel permeation chromatography (GPC) with RI detection. In order to elucidate the impact of the disassembly on cellulosic fibres, comparative studies were performed with the non-disassociated cellulose counterparts. The importance of the presence of water was addressed by Karl Fischer titration and solvent exchange experiments. Morphological changes during the dissolution process were studied by scanning electron microscopy (SEM). Dissolution of fibrillated cellulosic materials is impeded compared to the non-fibrillated material. This is a consequence of the high-surface-area fibrils prone to retain high amounts of water. Dissolution behaviour of nano-crystalline cellulosic materials appeared to be source-dependent. Due to the absence of entangled networks, these materials retain only water bound at the surface of the nano-crystallites, indicative of both the exposed surface area and solubility. The small cellulose nano-particles extracted from dissolving pulp show lower solubility compared to the large NCC particles from cotton.

Electron Beam Irradiation of Cellulosic Materials-Opportunities and Limitations.

The irradiation of pulp is of interest from different perspectives. Mainly it is required when a modification of cellulose is needed. Irradiation could bring many advantages, such as chemical savings and, therefore, cost savings and a reduction in environmental pollutants. In this account, pulp and dissociated celluloses were analyzed before and after irradiation by electron beaming. The focus of the analysis was the oxidation of hydroxyl groups to carbonyl and carboxyl groups in pulp and the degradation of cellulose causing a decrease in molar mass. For that purpose, the samples were labeled with a selective fluorescence marker and analyzed by gel permeation chromatography (GPC) coupled with multi-angle laser light scattering (MALLS), refractive index (RI), and fluorescence detectors. Degradation of the analyzed substrates was the predominant result of the irradiation; however, in the microcrystalline samples, oxidized cellulose functionalities were introduced along the cellulose chain, making this substrate suitable for further chemical modification.

Synthesis, antitumor evaluation and DNA binding studies of novel amidino-benzimidazolyl substituted derivatives of furyl-phenyl- and thienyl-phenyl-acrylates, naphthofurans and naphthothiophenes.

A series of amidino-substituted benzimidazoles, related to furyl-phenyl- and thienyl-phenyl-acrylates, naphthofurans and naphthothiophenes were prepared, their antitumor evaluation and interactions with ct-DNA have been investigated. All tested compounds show differential and strong antitumor activity without apparent difference depending on their structures. Interestingly, the MCF-7 tumor cell line is highly sensitive to all compounds. Compounds 6-9 showed noticeable selectivity in regard to normal fibroblasts (WI 38). Compounds 4-9 interact with ct-DNA by more binding modes, whose mutual distribution is dependent on the compound/DNA ratio. The "acyclic"4-6 and "cyclic" compound 7 interact mostly within the minor groove of DNA, although partial intercalation of 6 and 7 cannot be excluded. The "cyclic" compounds 8 and 9 intercalate between DNA base pairs at high excess of DNA over compounds.