How Resilient is Wood Xylan to Enzymatic Degradation in a Matrix with Kraft Lignin?

Despite the potential of lignocellulose in manufacturing value-added chemicals and biofuels, its efficient biotechnological conversion by enzymatic hydrolysis still poses major challenges. The complex interplay between xylan, cellulose, and lignin in fibrous materials makes it difficult to assess underlying physico- and biochemical mechanisms. Here, we reduce the complexity of the system by creating matrices of cellulose, xylan, and lignin, which consists of a cellulose base layer and xylan/lignin domains. We follow enzymatic degradation using an endoxylanase by high-speed atomic force microscopy and surface plasmon resonance spectroscopy to obtain morphological and kinetic data. Fastest reaction kinetics were observed at low lignin contents, which were related to the different swelling capacities of xylan. We demonstrate that the complex processes taking place at the interfaces of lignin and xylan in the presence of enzymes can be monitored in real time, providing a future platform for observing phenomena relevant to fiber-based systems.

Engineering Saccharomyces cerevisiae for targeted hydrolysis and fermentation of glucuronoxylan through CRISPR/Cas9 genome editing.

BACKGROUND: The abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers. RESULTS: The aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-ß-xylanase and ß-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA ß-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations. CONCLUSION: This work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Physicochemical Properties of 20 Ionic Liquids Prepared by the Carbonate-Based IL (CBILS) Process.

Ionic liquids (ILs) are an emerging materials' class with applications in areas such as energy storage, catalysis, and biomass dissolution and processing. Their physicochemical properties including surface tension, viscosity, density and their interplay between cation and anion chemistry are decisive in these applications. For many commercially available ILs, a full set of physicochemical data is not available. Here, we extend the knowledge base by providing physicochemical properties such as density (20 and 25 °C), refractive index (20 and 25 °C), surface tension (23 °C, including polar and dispersive components), and shear viscosity (ambient atmosphere, shear rate 1-200 s-1), for 20 commercial ILs. A correlation between the crystal volume, dispersive surface tension, and shear viscosity is introduced as a predictive tool, allowing for viscosity estimation. Systematic exploration of cation/anion alkyl side chain lengths reveals the impact on the IL's physicochemical attributes. Increasing the anion's headgroup decreases surface tension up to 35.7% and consequently shear viscosity. We further demonstrate that the dispersive part of the surface tension linearly correlates with the refractive index of the ionic liquid. While we provide additional physicochemical data, the screening and modeling efforts will contribute to better structure property predictions enabling faster progress in design and applications of ILs.

Humidity Response of Cellulose Thin Films.

Cellulose-water interactions are crucial to understand biological processes as well as to develop tailor made cellulose-based products. However, the main challenge to study these interactions is the diversity of natural cellulose fibers and alterations in their supramolecular structure. Here, we study the humidity response of different, well-defined, ultrathin cellulose films as a function of industrially relevant treatments using different techniques. As treatments, drying at elevated temperature, swelling, and swelling followed by drying at elevated temperatures were chosen. The cellulose films were prepared by spin coating a soluble cellulose derivative, trimethylsilyl cellulose, onto solid substrates followed by conversion to cellulose by HCl vapor. For the highest investigated humidity levels (97%), the layer thickness increased by ca. 40% corresponding to the incorporation of 3.6 molecules of water per anhydroglucose unit (AGU), independent of the cellulose source used. The aforementioned treatments affected this ratio significantly with drying being the most notable procedure (2.0 and 2.6 molecules per AGU). The alterations were investigated in real time with X-ray reflectivity and quartz crystal microbalance with dissipation, equipped with a humidity module to obtain information about changes in the thickness, roughness, and electron density of the films and qualitatively confirmed using grazing incidence small angle X-ray scattering measurements using synchrotron irradiation.

Visualizing Degradation of Cellulose Nanofibers by Acid Hydrolysis.

Cellulose hydrolysis is an extensively studied process due to its relevance in the fields of biofuels, chemicals production, and renewable nanomaterials. However, the direct visualization of the process accompanied with detailed scaling has not been reported because of the vast morphological alterations occurring in cellulosic fibers in typical heterogeneous (solid/liquid) hydrolytic systems. Here, we overcome this distraction by exposing hardwood cellulose nanofibers (CNFs) deposited on silica substrates to pressurized HCl gas in a solid/gas system and examine the changes in individual CNFs by atomic force microscopy (AFM). The results revealed that hydrolysis proceeds via an intermediate semi-fibrous stage before objects reminiscent of cellulose nanocrystals were formed. The length of the nanocrystal-like objects correlated well with molar mass, as analyzed by gel permeation chromatography, performed on CNF aerogels hydrolyzed under identical conditions. Meanwhile, X-ray diffraction showed a slight increase in crystallinity index as the hydrolysis proceeded. The results provide a modern visual complement to >100 years of research in cellulose degradation.

Highly Norbornylated Cellulose and Its "Click" Modification by an Inverse-Electron Demand Diels-Alder (iEDDA) Reaction.

A facile, catalyst-free synthesis of a norbornylated cellulosic material (NC) with a high degree of substitution (2.9) is presented by direct reaction of trimethylsilyl cellulose with norbornene acid chloride. The resulting NC is highly soluble in organic solvents and its reactive double bonds were exploited for the copper-free inverse-electron demand Diels-Alder (iEDDA) "click" reaction with 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine. Reaction kinetics are comparable to the well-known Huisgen type 1,3-dipolar cycloaddition of azide with alkynes, while avoiding toxic catalysts.

Chemical Engineering Laboratory Projects in Student Teams in Real Life and Transformed Online: Viscose Fiber Spinning and Characterization.

Chemical engineering education comprises a complexity of technical skills that include learning processes that are currently relevant in industry. Despite being a rather old industrial process, the manufacturing of viscose fibers still accounts for the major fraction of all human-made cellulosic fibers worldwide. Here we describe a laboratory setup to introduce chemistry and engineering students into the principles of cellulose fiber spinning according to the viscose process. The setup for fiber spinning is kept simplistic and allows the experiments to be performed without professional spinning equipment. However, all of the steps are performed analogously to the industrial process. The professional setting in process and chemical engineering involves work on projects and in teams. Hence, we have incorporated the fiber spinning laboratory experiment in the context of working in teams on projects. We will also present our experience on transferring a real-life laboratory experiment online, as this is required at times that online education is preferred over real-life teaching.

Structural Order in Cellulose Thin Films Prepared from a Trimethylsilyl Precursor.

Biopolymer cellulose is investigated in terms of the crystallographic order within thin films. The films were prepared by spin-coating of a trimethylsilyl cellulose precursor followed by an exposure to HCl vapors; two different source materials were used. Careful precharacterization of the films was performed by infrared spectroscopy and atomic force microscopy. Subsequently, the films were investigated by grazing incidence X-ray diffraction using synchrotron radiation. The results showed broad diffraction peaks, indicating a rather short correlation length of the molecular packing in the range of a few nanometers. The analysis of the diffraction patterns was based on the known structures of crystalline cellulose, as the observed peak pattern was comparable to cellulose phase II and phase III. The dominant fraction of the film is formed by two different types of layers, which are oriented parallel to the substrate surface. The stacking of the layers results in a one-dimensional crystallographic order with a defined interlayer distance of either 7.3 or 4.2 Å. As a consequence, two different preferred orientations of the polymer chains are observed. In both cases, polymer chain axes are aligned parallel to the substrate surface, and the orientation of the cellulose molecules are concluded to be either edge-on or flat-on. A minor fraction of the cellulose molecules form nanocrystals that are randomly distributed within the films. In this case, the molecular packing density was found to be smaller in comparison to the known crystalline phases of cellulose.

2-Methoxyhydroquinone from Vanillin for Aqueous Redox-Flow Batteries.

We show the synthesis of a redox-active quinone, 2-methoxy-1,4-hydroquinone (MHQ), from a bio-based feedstock and its suitability as electrolyte in aqueous redox flow batteries. We identified semiquinone intermediates at insufficiently low pH and quinoid radicals as responsible for decomposition of MHQ under electrochemical conditions. Both can be avoided and/or stabilized, respectively, using H3 PO4 electrolyte, allowing for reversible cycling in a redox flow battery for hundreds of cycles.

How Bound and Free Fatty Acids in Cellulose Films Impact Nonspecific Protein Adsorption.

The effect of fatty acids and fatty acid esters to impair nonspecific protein adsorption on cellulose thin films is investigated. Thin films are prepared by blending trimethylsilyl cellulose solutions with either cellulose stearoyl ester or stearic acid at various ratios. After film formation by spin coating, the trimethylsilyl cellulose fraction of the films is converted to cellulose by exposure to HCl vapors. The morphologies and surface roughness of the blends were examined by atomic force microscopy revealing different feature shapes and sizes depending on the blend ratios. Nonspecific protein adsorption at the example of bovine serum albumin toward the blend thin films was tested by means of surface plasmon resonance spectroscopy in real-time. Incorporation of stearic acid into the cellulose leads to highly protein repellent surfaces regardless of the amount added. The stearic acid acts as a sacrificial compound that builds a complex with bovine serum albumin thereby inhibiting protein adsorption. For the blends where stearoyl ester is added to the cellulose films, the cellulose:cellulose stearoyl ester ratios of 3:1 and 1:1 lead to much lower nonspecific protein adsorption compared to pure cellulose, whereas for the other ratios, adsorption increases. Supplementary results were obtained from atomic force microscopy experiments performed in liquid during exposure to protein solution and surface free energy determinations.

Interaction of Tissue Engineering Substrates with Serum Proteins and Its Influence on Human Primary Endothelial Cells.

Polymer-based biomaterials particularly polycaprolactone (PCL) are one of the most promising substrates for tissue engineering. The surface chemistry of these materials plays a major role since it governs protein adsorption, cell adhesion, viability, degradation, and biocompatibility in the first place. This study correlates the interaction of the most abundant serum proteins (albumin, immunoglobulins, fibrinogen) with the surface properties of PCL and its influence on the morphology and metabolic activity of primary human arterial endothelial cells that are seeded on the materials. Prior to that, thin films of PCL are manufactured by spin-coating and characterized in detail. A quartz crystal microbalance with dissipation (QCM-D), a multiparameter surface plasmon resonance spectroscopy instrument (MP-SPR), wettability data, and atomic force microscopy are combined to elucidate the pH-dependent protein adsorption on the PCL substrates. Primary endothelial cells are cultured on the protein modified polymer, and conclusions are drawn on the significant impact of type and form of proteins coatings on cell morphology and metabolic activity.

Adsorption Studies of Organophosphonic Acids on Differently Activated Gold Surfaces.

In this study, the formation of self-assembled monolayers consisting of three organophosphonic acids (vinyl-, octyl-, and tetradecylphosphonic acid) from isopropanol solutions onto differently activated gold surfaces is studied in situ and in real time using multiparameter surface plasmon resonance (MP-SPR). Data retrieved from MP-SPR measurements revealed similar adsorption kinetics for all investigated organophosphonic acids (PA). The layer thickness of the immobilized PA is in the range of 0.6-1.8 nm corresponding to monolayer-like coverage and correlates with the length of the hydrocarbon chain of the PA molecules. After sintering the surfaces, the PA are irreversibly attached onto the surfaces as proven by X-ray photoelectron spectroscopy and attenuated total reflection infrared and grazing incidence infrared spectroscopy. Potential adsorption modes and interaction mechanisms are proposed.

Enzymes as Biodevelopers for Nano- And Micropatterned Bicomponent Biopolymer Thin Films.

The creation of nano- and micropatterned polymer films is a crucial step for innumerous applications in science and technology. However, there are several problems associated with environmental aspects concerning the polymer synthesis itself, cross-linkers to induce the patterns as well as toxic solvents used for the preparation and even more important development of the films (e.g., chlorobenzene). In this paper, we present a facile method to produce micro- and nanopatterned biopolymer thin films using enzymes as so-called biodevelopers. Instead of synthetic polymers, naturally derived ones are employed, namely, poly-3-hydroxybutyrate and a cellulose derivative, which are dissolved in a common solvent in different ratios and subjected to spin coating. Consequently, the two biopolymers undergo microphase separation and different domain sizes are formed depending on the ratio of the biopolymers. The development step proceeds via addition of the appropriate enzyme (either PHB-depolymerase or cellulase), whereas one of the two biopolymers is selectively degraded, while the other one remains on the surface. In order to highlight the enzymatic development of the films, video AFM studies have been performed in real time to image the development process in situ as well as surface plasmon resonance spectroscopy to determine the kinetics. These studies may pave the way for the use of enzymes in patterning processes, particularly for materials intended to be used in a physiological environment.

Exploring Nonspecific Protein Adsorption on Lignocellulosic Amphiphilic Bicomponent Films.

In this contribution, we explore the interaction of lignocellulosics and proteins aiming at a better understanding of their synergistic role in natural systems. In particular, the manufacturing and characterization of amphiphilic bicomponent thin films composed of hydrophilic cellulose and a hydrophobic lignin ester in different ratios is presented which may act as a very simplified model for real systems. Besides detailed characterizations of the films and mechanisms to explain their formation, nonspecific protein adsorption using bovine serum albumin (BSA) onto the films was studied using a quartz crystal microbalance with dissipation (QCM-D). As it turns out, the rather low nonspecific protein adsorption of BSA on cellulose is further reduced when these hydrophobic lignins are incorporated into the films. The lignin ester acts in these blend films as sacrificial component, probably via an emulsification mechanism. Additionally, the amphiphilicity of the films may prevent the adsorption of BSA as well. Although there are some indications, it remains unclear whether any kind of protein interactions in such systems are of specific nature.

Designing Hydrophobically Modified Polysaccharide Derivatives for Highly Efficient Enzyme Immobilization.

In this contribution, a hydrophobically modified polysaccharide derivative is synthesized in an eco-friendly solvent water by conjugation of benzylamine with the backbone of the biopolymer. Owing to the presence of aromatic moieties, the resulting water-soluble polysaccharide derivative self-assembles spontaneously and selectively from solution on the surface of nanometric thin films and sheets of polystyrene (PS). The synthetic polymer modified in this way bears a biocompatible nanolayer suitable for the immobilization of horseradish peroxidase (HRP), a heme-containing metalloenzyme often employed in biocatalysis and biosensors. Besides the detailed characterization of the polysaccharide derivative, a quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) are used to investigate the binding efficiency and interaction of HRP with the tailored polysaccharide interfaces. Subsequent enzyme activity tests reveal details of the interaction of HRP with the solid support. The novel polysaccharide derivative and its use as a material for the selective modification of PS lead to a beneficial, hydrophilic environment for HRP, resulting in high enzymatic activities and a stable immobilization of the enzyme for biocatalytic and analytic purposes.

Photolithographic patterning of cellulose: a versatile dual-tone photoresist for advanced applications.

In many areas of science and technology, patterned films and surfaces play a key role in engineering and development of advanced materials. Here, we present a versatile toolbox that provides an easy patterning method for cellulose thin films by means of photolithography and enzymatic digestion. A patterned UV-illumination of trimethylsilyl cellulose thin films containing small amounts of a photo acid generator leads to a desilylation reaction and thus to the formation of cellulose in the irradiated areas. Depending on the conditions of development, either negative and positive type cellulose structures can be obtained, offering lateral resolutions down to the single-digit micro meter range by means of contact photolithography. In order to highlight the potential of this material for advanced patterning techniques, cellulose structures with sub-µm resolution are fabricated by means of two-photon absorption lithography. Moreover, these photochemically structured cellulose thin films are successfully implemented as dielectric layers in prototype organic thin film transistors. Such photopatternable dielectric layers are crucial for the realization of electrical interconnects for demanding organic device architectures.

Silanol-based surfactants: synthetic access and properties of an innovative class of environmentally benign detergents.

Herein, environmentally friendly surfactants based on new silanols as substitutes for the isoelectronic phosphonates were explored. Surface tensions of aqueous solutions are significantly reduced, particularly with those silanols that feature a high ratio of organic moiety to silanol. Besides their use as surfactants, their potential as coating agents for hydrophilic oxide surfaces was investigated for the example of glass substrates. In the solid-state sheet structures with silanol, double layers are present, in which the sheet spacing varies with the alkyl-chain length.

Triggering protein adsorption on tailored cationic cellulose surfaces.

The equipment of cellulose ultrathin films with BSA (bovine serum albumin) via cationization of the surface by tailor-made cationic celluloses is described. In this way, matrices for controlled protein deposition are created, whereas the extent of protein affinity to these surfaces is controlled by the charge density and solubility of the tailored cationic cellulose derivative. In order to understand the impact of the cationic cellulose derivatives on the protein affinity, their interaction capacity with fluorescently labeled BSA is investigated at different concentrations and pH values. The amount of deposited material is quantified using QCM-D (quartz crystal microbalance with dissipation monitoring, wet mass) and MP-SPR (multi-parameter surface plasmon resonance, dry mass), and the mass of coupled water is evaluated by combination of QCM-D and SPR data. It turns out that adsorption can be tuned over a wide range (0.6-3.9 mg dry mass m(-2)) depending on the used conditions for adsorption and the type of employed cationic cellulose. After evaluation of protein adsorption, patterned cellulose thin films have been prepared and the cationic celluloses were adsorbed in a similar fashion as in the QCM-D and SPR experiments. Onto these cationic surfaces, fluorescently labeled BSA in different concentrations is deposited by an automatized spotting apparatus and a correlation between the amount of the deposited protein and the fluorescence intensity is established.

Photoregeneration of trimethylsilyl cellulose as a tool for microstructuring ultrathin cellulose supports.

Microstructured thin films based on cellulose, the most abundant biopolymer on Earth, have been obtained by UV-irradiation of acid-labile trimethylsilyl cellulose thin films in the presence of N-hydroxynaphtalimide triflate as photoacid generator. We demonstrate that this photoregeneration process can be exploited for the manufacture of cellulose patterns having feature sizes down to 1 µm, with potential applications in life sciences.

Fusion of cellulose microspheres with pulp fibers: Creating an unconventional type of paper.

Cellulose microspheres (CMS) are a type of spherical regenerated cellulose particles with versatile properties which have been used as carrier materials in medical and technical applications. The integration of CMS into paper products opens up novel application scenarios for paper products in a wide range of fields. However, the incorporation of CMS carriers into paper products is challenging and hitherto no reports do exist in literature. Here, we present a feasibility study to incorporate up to 50 w.% CMS in paper hand sheets using retention aids. Our primary observations highlight the successful formation of uniform paper hand sheets retaining its tensile strengths at elevated CMS concentrations. Sheets with high CMS contents exhibit an increase in density and display enhanced surface smoothness - an outcome of a CMS layer forming atop the fiber base - which effectively bridges voids and rectifies surface irregularities as supported by Gurley testing, infinite focus microscopy and scanning electron microscopy. While our primary objective centered on the general feasibility to manufacture CMS-containing papers, the resulting composite scaffold carries significant potential as a platform for innovative, functional paper-based materials.