Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.

Additive manufacturing of three-dimensional objects on the nanoscale is a very relevant topic but still a highly challenging task. Among the pool of nanofabrication techniques, focused electron beam induced deposition (FEBID) has recently developed from a trial-and-error laboratory method to a predictable 3D nanoprinting technology with unique advantages. This perspective article first introduces the basic principles of 3D-FEBID, followed by an overview of historical developments with a particular emphasis on the last three years. Here, we examine different aspects of 3D nanoprinting such as the instrumental setup, fundamental growth mechanisms, simulations, computer aided design software solutions, material properties, and application studies. For each aspect, the individual challenges and limitations are discussed. In addition, we share our outlook about possible solutions and studies currently under investigation. As a perspective, we also address the most urgent milestones of the future and speculate on applications ranging from optics to mechanics, magnetics, and electronics, all of them benefiting from the recently improved 3D FEBID synthesis technique.

LPMO (lytic polysaccharide monooxygenase) represents a unique paradigm of cellulosic biomass degradation by an oxidative mechanism. Understanding the role of LPMO in deconstructing crystalline cellulose is fundamental to the enzyme’s biological function and will help to specify the use of LPMO in biorefinery applications. Here we show with real-time atomic force microscopy that C1 and C4 oxidizing types of LPMO from Neurospora crassa (NcLPMO9F, NcLPMO9C) bind to nanocrystalline cellulose with high preference for the very same substrate surfaces that are also used by a processive cellulase (Trichoderma reesei CBH I) to move along during hydrolytic cellulose degradation. The bound LPMOs, however, are immobile during their adsorbed residence time ( ~ 1.0 min for NcLPMO9F) on cellulose. Treatment with LPMO resulted in fibrillation of crystalline cellulose and strongly ( ≥ 2-fold) enhanced the cellulase adsorption. It also increased enzyme turnover on the cellulose surface, thus boosting the hydrolytic conversion.

Scanning probe microscopy (SPM) has become an essential surface characterization technique in research and development. By concept, SPM performance crucially depends on the quality of the nano-probe element, in particular, the apex radius. Now, with the development of advanced SPM modes beyond morphology mapping, new challenges have emerged regarding the design, morphology, function, and reliability of nano-probes. To tackle these challenges, versatile fabrication methods for precise nano-fabrication are needed. Aside from well-established technologies for SPM nano-probe fabrication, focused electron beam-induced deposition (FEBID) has become increasingly relevant in recent years, with the demonstration of controlled 3D nanoscale deposition and tailored deposit chemistry. Moreover, FEBID is compatible with practically any given surface morphology. In this review article, we introduce the technology, with a focus on the most relevant demands (shapes, feature size, materials and functionalities, substrate demands, and scalability), discuss the opportunities and challenges, and rationalize how those can be useful for advanced SPM applications. As will be shown, FEBID is an ideal tool for fabrication/modification and rapid prototyping of SPM-tipswith the potential to scale up industrially relevant manufacturing.