This study introduces a decellularization-inspired strategy to isolate the tissue scaffold from the green macro-algae Ulva fenestrata as a platform for bio-based film production. A top-down approach was developed to remove cytosolic components while preserving the native hierarchical architecture. By combining chemical and mechanical treatments, it was shown that the addition of surfactant and mechanical treatment improved decellularization efficiency and scaffold integrity. The surfactant Cocamidopropyl betaine (CAPB) increased pigment extraction threefold during solvent treatment. Combined with ultrasonication, a synergistic effect enabled high extraction efficiency at solvent concentrations as low as 5-10 % and accelerated kinetics to equilibrium within 180 min. Auger-based mechanical pretreatment further enhanced extraction by promoting pigment removal before solvent-surfactant treatment. Biopolymer anatomy mapping by optotracing with fluorescent reporter molecules showed tissue-dependent recovery: In blade tissue, thinner-walled regions were more affected, whereas thicker-walled tissues retained integrity through an intermediate lamella. In rhizoidal tissue, fibrils from the median layer were additionally isolated. In both tissues, impairment of the outermost layer enhanced decellularization efficiency. Carbotrace 680 stained blade cell walls, while rhizoidal cell walls required Carbotrace 630, highlighting compositional differences. Fully algae-derived, self-standing films from decellularized Ulva were produced, reaching tensile strengths up to 39.7 MPa. Blade-derived films showed the highest performance, while rhizoidal films were more heterogeneous due to fibril inclusion. This demonstrates decellularization as a sustainable, low-input route to utilize macroalgal architectures for bio-based material development.

Austenitic stainless steel (AISI 316L) is the dominant material in food processing equipment due to high corrosion resistance and mechanical durability. The shift from animal-to plant-based food processing introduces new challenges for material performance, as plant-derived biomolecules may interact differently with food-contact surfaces than animal proteins. These interactions can modify interfacial properties, with consequences for fouling, corrosion, and metal migration. Despite its importance, such effects remain scarcely studied, with only a few reports on e.g. whey and casein proteins. Knowledge on rice-derived biomolecules is particularly limited, even though rice proteins and starches are increasingly relevant in gluten-free and plant-based systems. This study examines the adsorption kinetics and interfacial properties of rice protein concentrates (RPC) and rice starch (RS) dissolved in artificial tap water (ATW) onto 316L stainless steel. In situ quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to quantify adsorption dynamics, complemented by atomic force microscopy (AFM) and carbohydrate-specific opto-tracing (Carbotrace 680) to detect, and visualize adsorption patterns. Atomic absorption spectroscopy (AAS) was used to determine metal migration and evaluated with respect to European Union specific release limits (SRLs) for food-contact materials. Electrochemical measurements including open circuit potential (OCP), potentiodynamic polarization (PDP), and cyclic potentiodynamic polarization (CPDP) were employed to assess the effects of adsorption on the corrosion behavior. By demonstrating how rice-derived biomolecules interact with stainless steel and influence corrosion and metal migration, this study addresses a critical knowledge gap in the literature. The insights advance fundamental understanding of food biomolecule–metal interactions and support the design of more durable, compliant, and safe food-contact materials.

Staphylococcus aureus infections represent a clinical challenge due to their propensity to form biofilms and the increasing prevalence of antibiotic resistance. The ability of S. aureus to form biofilm affects clinical outcome, but techniques to study extracellular matrix (ECM) in S. aureus biofilms are lacking. Here, we present an agar-based method in which the optotracer EbbaBiolight 680 (Ebba680) is used to visualize ECM formation alongside evaluation of colony growth dynamics in agar colonies. As models for colony biofilms, we use drop inoculation for macrocolony formation or spread-plating for single-cell derived colonies. Kinetic fluorescence spectroscopy combined with time-lapse microscopy showed bright fluorescence signals, revealing different spatial-temporal appearance of ECM in macrocolonies versus single-cell derived colonies. In contrast, the microstructure was conserved between the two types of colonies. Detailed characterization of the biofilm microstructures by confocal microscopy revealed Ebba680 binding targets interspersed between cells as well as in a cap-like structure formed on the outer surface of the biofilm. Accessory gene regulator (agr) controlled expression of Ebba680 binding target(s) and the binding of Ebba680 to synthetic fibrillated phenol soluble modulins (fPSMs) suggests these functional amyloids act as targets for Ebba680 in the biofilm ECM. By upgrading ColTapp, an application developed for colony radius quantification, to also analyze fluorescence images, concurrent analysis of Ebba680-stained ECM and colony growth was achieved. This provided a new dimension to the assessment of colony biofilms. Detailed phenotypic characterization of clinical isolates is critical for treatment decision making, and enhanced screening which includes ECM as presented here has potential to facilitate treatment decisions in problematic staphylococcal infections.

Abstract [en]The macroalga Ulva fenestrata plays a key role in marine ecosystems and has increasing potential in aquaculture. However, its three-dimensional tissue architecture remains underexplored. This study applies multimodal fluorescence microscopy combined with optotracing to spatially map biopolymers and structural features in native Ulva tissue. Using Carbotrace 680, cellulose was localized in situ within the cell walls, while oligo/polyaromatic compounds were visualized across multiple scaffold layers via autofluorescence. Lambda scanning validated the fluorescence detection settings for cellulose (Exitation wavelength (Ex.) 561 nm, Emission wavelength (Em.) 570–631 nm), oligo/polyaromatics (Ex. 405 nm, Em. 408–505 nm), and chlorophyll (Ex. 639 nm, Em. 649–693 nm). Spatially resolved biopolymer anatomy maps were generated for blade and rhizoidal tissues, and 3D tissue models were constructed. The outermost blade layer exhibited a sandwich-like architecture, and a previously undescribed median layer was identified separating the two cell layers. This layer was >11 times thicker in rhizoidal tissue than in blade tissue, comprising 56 % and 7 % of the total thickness, respectively. Spectral differences in rhizoidal cells indicated cellular heterogeneity. Collectively, the observed biopolymer and architectural differences may reflect tissue-specific functional specialization of the macroalga. This imaging-based approach provides new perspectives on algal biology and supports the multisectoral valorization of Ulva.

The strive toward sustainability increases the demand for bio-based material production, forcing expansion of the biorefinery feedstock supply from forest wood to non-woody materials such as agricultural residues. As a model organism for legume crops, the aptness of agricultural lupins as a lignocellulose feedstock is investigated. Principle chemical analysis combined with optotracing, in which the fluorescent tracer molecule Carbotrace 680 generates a visual map of the native tissues’ lignocellulose anatomy at sub-cellular resolution, enables informed design of a mild recovery process. A streamlined conversion approach is then designed, yielding lignin-containing microfibrillated cellulose. By monitoring defibrillation and delignification throughout the extraction process, the use of optotracing for non-destructive fiber analytics at unprecedented details across all hierarchical structures of lignocellulosic materials is demonstrated. This crop valorization is a prime illustration of a holistic use of lupin biomass, with seeds serving as plant-based food sources, and other parts as sources for lignocellulose-based materials, thereby expanding both the biorefinery concept and feedstock supply.

Biofilms, comprised of cells embedded in extracellular matrix (ECM), enable bacterial surface colonization and contribute to pathogenesis and biofouling. Yet, antibacterial surfaces are mainly evaluated for their effect on bacterial cells rather than the ECM. Here, a method is presented to separately quantify amounts and distribution of cells and ECM in Salmonella biofilms grown on electroactive poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS). Within a custom-designed biofilm reactor, biofilm forms on PEDOT:PSS surfaces electrically addressed with a bias potential and simultaneous recording of the resulting current. The amount and distribution of cells and ECM in biofilms are analyzed using a fluorescence-based spectroscopic mapping technique and fluorescence confocal microscopy combined with advanced image processing. The study shows that surface charge leads to upregulated ECM production, leaving the cell counts largely unaffected. An altered texture is also observed, with biofilms forming small foci or more continuous structures. Supported by mutants lacking ECM production, ECM is identified as an important target when developing antibacterial strategies. Also, a central role for biofilm distribution is highlighted that likely influences antimicrobial susceptibility in biofilms. This work provides yet a link between conductive polymer materials and bacterial metabolism and reveals for the first time a specific effect of electrochemical addressing on bacterial ECM formation.

Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) are a significant global health challenge. The UPEC biofilm lifestyle is believed to play an important role in infection recurrency and treatment resistance, but our understanding of how the extracellular matrix (ECM) components curli and cellulose contribute to biofilm formation and pathogenicity is limited. Here, we study the spatial and temporal development of native UPEC biofilm using agar-based detection methods where the non-toxic, optically active fluorescent tracer EbbaBiolight 680 reports the expression and structural location of curli in real-time. An in vitro screen of the biofilm capacity of common UPEC strains reveals significant strain variability and identifies UPEC No. 12 (UPEC12) as a strong biofilm former at 28 °C and 37 °C. Non-interventional microscopy, including time-lapse and 2-photon, reveal significant horizontal and vertical heterogeneity in the UPEC12 biofilm structure. We identify region-specific expression of curli, with a shift in localization from the bottom of the flat central regions of the biofilm to the upper surface in the topographically dramatic intermediate region. When investigating if the rdar morphotype affects wettability of the biofilm surface, we found that the nano-architecture of curli guided by cellulose, rather than the rdar macrostructures, leads to increased hydrophobicity of the biofilm. By providing new insights at exceptional temporal and spatial resolution, we demonstrate how non-interventional analysis of native biofilms will facilitate the next generation of understanding into the roles of ECM components during growth of UPEC biofilms and their contribution to the pathogenesis of UTI.

The urinary tract is a hydrodynamically challenging microenvironment and uropathogenic Escherichia coli (UPEC) must overcome several physiological challenges in order to adhere and establish a urinary tract infection. Our previous work in vivo revealed a synergy between different UPEC adhesion organelles, which facilitated effective colonization of the renal proximal tubule. To allow highresolution real-time analysis of this colonization behavior, we established a biomimetic proximal-tubule-on-chip (PToC). The PToC allowed for single-cell resolution analysis of the first stages of bacterial interaction with host epithelial cells, under physiological flow. Time-lapse microscopy and single-cell trajectory analysis in the PToC revealed that while the majority of UPEC moved directly through the system, a minority population initiated heterogeneous adhesion, identified as either rolling or bound. Adhesion was predominantly transient andmediated by P pili at the earliest time-points. These bound bacteria initiated a founder populationwhich rapidly divided, leading to 3D microcolonies. Within the first hours, the microcolonies did not express extracellular curli matrix, but rather were dependent on Type 1 fimbriae as the key element in the microcolony structure. Collectively, our results show the application of Organ-on-chip technology to address bacterial adhesion behaviors, demonstrating a well-orchestrated interplay and redundancy between adhesion organelles that enables UPEC to form microcolonies and persist under physiological shear stress.

Effective treatment of bacterial infections requires methods that accurately and quickly identify which antibiotic should be prescribed. This review describes recent research on the development of optotracing methodologies for bacterial and biofilm detection and diagnostics. Optotracers are small, chemically well-defined, anionic fluorescent tracer molecules that detect peptide- and carbohydrate-based biopolymers. This class of organic molecules (luminescent conjugated oligothiophenes) show unique electronic, electrochemical and optical properties originating from the conjugated structure of the compounds. The photophysical properties are further improved as donor-acceptor-donor (D-A-D)-type motifs are incorporated in the conjugated backbone. Optotracers bind their biopolymeric target molecules via electrostatic interactions. Binding alters the optical properties of these tracer molecules, shown as altered absorption and emission spectra, as well as ON-like switch of fluorescence. As the optotracer provides a defined spectral signature for each binding partner, a fingerprint is generated that can be used for identification of the target biopolymer. Alongside their use for in situ experimentation, optotracers have demonstrated excellent use in studies of a number of clinically relevant microbial pathogens. These methods will find widespread use across a variety of communities engaged in reducing the effect of antibiotic resistance. This includes basic researchers studying molecular resistance mechanisms, academia and pharma developing new antimicrobials targeting biofilm infections and tests to diagnose biofilm infections, as well as those developing antibiotic susceptibility tests for biofilm infections (biofilm-AST). By iterating between the microbial world and that of plants, development of the optotracing technology has become a prime example of successful cross-feeding across the boundaries of disciplines. As optotracers offers a capacity to redefine the way we work with polysaccharides in the microbial world as well as with plant biomass, the technology is providing novel outputs desperately needed for global impact of the threat of antimicrobial resistance as well as our strive for a circular bioeconomy.

Antimicrobial resistance is a medical threat of global dimensions. Proper antimicrobial susceptibility testing (AST) for drug development, patient diagnosis and treatment is crucial to counteract ineffective drug use and resistance development. Despite the important role of bacterial biofilms in chronic and device-associated in-fections, the efficacy of antibiotics is determined using planktonic cultures. To address the need for antibiotics targeting bacteria in the biofilm lifestyle, we here present an optotracing-based biofilm-AST using Salmonella as model. Our non-disruptive method enables real-time recording of the extracellular matrix (ECM) components, providing specific detection of the biofilm lifestyle. Biofilm formation prior to antibiotic challenge can thus be confirmed and pre-treatment data collected. By introducing Kirby-Bauer discs, we performed a broad screen of the effects of antibiotics representing multiple classes, and identified compounds with ECM inhibitory as well as promoting effects. These compounds were further tested in agar-based dose-response biofilm-AST assays. By quantifying the ECM based on the amount of curli, and by visualizing the biofilm size and morphology, we achieved new information directly reflecting the treated biofilm. This verified the efficacy of several antibiotics that were effective in eradicating pre-formed biofilms, and it uncovered intriguing possible resistance mecha-nisms initiated in response to treatments. By providing deeper insights into the resistances and susceptibilities of microbes, expanded use of the biofilm-AST will contribute to more effective treatments of infections and reduced resistance development.