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.

Today’s sustainability challenges demand more than new materials - they require new ways of thinking about the resources we already have to support zero waste strategies. This thesis explores the valorization of underutilized biomasses - specifically the terrestrial crop Lupinus angustifolius (Lupin) and the marine macroalga Ulva fenestrata (Ulva) - as alternative feedstocks for bio-based materials. These two biomasses were selected for their dual functionality: both are already cultivated for food applications, yet their residual non-edible fractions remain largely unexplored. By combining structural biology, bioprocess engineering, materials science, and bioimaging, the thesis establishes a comprehensive, interdisciplinary framework for biomass characterization and conversion. Biopolymer mapping using multimodal fluorescence imaging and optotracing revealed the tissue architecture and native biopolymer distribution in Lupin residues and Ulva thalli. From Lupin, lignocellulose was extracted through mild alkaline pretreatment and defibrillated into lignin-containing microfibrillated cellulose (L-MFC). In Ulva, complex structural features, including oligo-/polyaromatic-rich layers and rhizoidal fibrillar structures, were discovered, prompting a redefinition of its tissue terminology. A decellularization-inspired approach was then developed to recover tissue scaffolds from Ulva, leveraging its naturally thin, two-cell-layered structure to remove cellular content while preserving scaffold integrity. Finally, two material design strategies were employed: a bottom-up approach for Lupin-derived L-MFC films, exploiting their nanoscale fibrillar network for structural organization, and a top-down approach for Ulva-based films, preserving the intrinsic tissue scaffold architecture. The resulting materials demonstrated structural integrity while preserving key biopolymer networks. Across the entire biomass-to-material workflow, multimodal fluorescence imaging combined with optotracing was integrated and adapted as a novel analytical tool, providing non-destructive, real-time and high-resolution information.

Dagens hållbarhetsutmaningar kräver mer än bara nya material – de kräver ett nytt sätt att tänka kring resurser vi redan har. Denna avhandling utforskar möjligheten att använda underutnyttjade biomassor – specifikt jordbruksgrödan Lupinus angustifolius (lupin) och den marina makroalgen Ulva fenestrata (Ulva) – som alternativa råvaror för biobaserade material. Dessa två biomassor valdes utifrån sin dubbla funktionalitet: båda odlas redan idag för livsmedelsändamål, medan resterande oätliga delar förblir till stor del outforskade.Genom att kombinera strukturbiologi, bioprocessteknik, materialvetenskap och avbildningsteknik etableras ett interdisciplinärt ramverk för karakterisering och omvandling av biomassa. Kartläggning och avbildning av biopolymerer med multimodal fluorescensmikroskopi och optotracing avslöjade vävnadsarkitekturen och den naturliga fördelningen av biopolymerer i olika växtdelar hos lupin och Ulva. Från lupin extraherades lignocellulosa via mild alkalisk förbehandling som sedan defibrillerades till lignininnehållande mikrofibrillerad cellulosa (L-MFC). I Ulva upptäcktes komplexa strukturer, inklusive oligo-/polyaromatiska lager och fibrillära strukturer i rhizoidzonen, vilket föranledde en omdefinition av dess vävnadsterminologi. En metod inspirerad av vävnadsdecellularisering utvecklades därefter för att isolera en cellväggsstruktur från Ulva. Cellinnehållet avlägsnades under milda betingelser för att bevara dess naturligt endast två cellager tunna struktur och integritet. Slutligen applicerades två olika strategier för materialdesign: en bottom-up-metod för att skapa filmer av lupin-baserad L-MFC, där dess fibrillära nätverk nyttjades för strukturell organisering, och en top-down-metod för Ulva-baserade filmer där den ursprungliga vävnadsarkitekturen bevarades. De resulterande materialen uppvisade god strukturell integritet samtidigt som viktiga biopolymernätverk bevarades. Multimodal fluorescensavbildning och optotracing integrerades och anpassades som ett nytt analytiskt verktyg, vilket möjliggjorde icke-förstörande, realtids- och högupplöst analys genom hela processen från biomassa till material.

The production of lignocellulosic-based materials requires leveraging alternative biomasses such as agricultural residues. This study investigates Lupinus angustifolius as a renewable feedstock for lignin-containing microfibrillated cellulose (L-MFC) and explores the role of lignin and hemicellulose in producing lupin-derived L-MFC films. By utilizing mild alkaline pre-treatments (0.5 or 1.5 M NaOH at 90 or 140 °C) and avoiding extensive delignification, four lignocellulose systems with varying lignin and hemicellulose content are generated. To produce L-MFC, these systems were further subjected to a defibrillation process, including TEMPO oxidation and mechanical homogenization. TEMPO oxidation partly removed the lignin while preserving hemicellulose, leading to altered biopolymer ratios. Confocal fluorescence microscopy combined with optotracing enabled real-time visualization of the defibrillation process and spatial biopolymer mapping. Complementary excitation-emission matrix (EEM) spectroscopy provided fluorescence fingerprints for tracking molecular-level changes during processing. The resulting lupin-derived L-MFCs were processed into self-standing films using Rapid Köthen drying. Films with higher residual lignin displayed increased hydrophobicity and mechanical strength with a tensile index of up to 110 Nm/g and a Young's modulus of up to 9700 MPa. This study demonstrates that partial preservation of native lignocellulose architecture offers a viable pathway for sustainable lupin-derived L-MFC film production.

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.

The seaweed Saccharina latissima is often blanched to lower iodine levels, however, it is not known how blanching affects protein extraction. We assessed the effect of blanching or soaking (80/45/12 degrees C, 2 min) on protein yield and protein extract characteristics after pH-shift processing of S. latissima. Average protein yields and extract amino acid levels ranked treatments as follows: blanching-45 degrees C -control > soaking -blanching -80 degrees C. Although blanching-45 degrees C decreased protein solubilization yield at pH 12, it increased isoelectric protein precipitation yield at pH 2 (p < 0.05). The former could be explained by a higher ratio of large peptides/proteins in the blanched biomass as shown by HP -SEC, whereas the latter by blanching-induced lowering of ionic strength, as verified by a dialysis model. Moreover, blanching-45 degrees C yielded a protein extract with 49 % less iodine compared with the control extract. We recommend blanching-45 degrees C since it is effective at removing iodine and does not compromise total protein extraction yield.

Seaweed biomass is a renewable resource with multiple applications. Sea-based cultivation of seaweeds can provide high biomass yields, low construction, operation, and maintenance costs and could offer an environmentally and economically sustainable alternative to land-based cultivations. The biochemical profile of sea-grown biomass depends on seasonal variation in environmental factors, and the optimization of harvest time is important for the quality of the produced biomass. To identify optimal harvest times of Swedish sea-based cultivated sea lettuce (Ulva fenestrata), this study monitored biomass yield, morphology, chemical composition, fertility, and biofouling at five different harvesting times in April - June 2020. The highest biomass yields (approximately 1.2 kg fw [m rope](-1)) were observed in late spring (May). The number and size of holes in the thalli and the amount of fertile and fouled tissue increased with prolonged growth season, which together led to a significant decline in both biomass yield and quality during summer (June). Early spring (April) conditions were optimal for obtaining high fatty acid, protein, biochar, phenolic, and pigment contents in the biomass, whereas carbohydrate and ash content, as well as essential and non-essential elements, increased later in the growth season. Our study results show that the optimal harvest time of sea-based cultivated U. fenestrata depends on the downstream application of the biomass and must be carefully selected to balance yield, quality, and desired biochemical contents to maximize the output of future sea-based algal cultivations in the European Northern Hemisphere.

The production of lignocellulosic-based materials necessitates leveraging alternative biomasses such as agricultural residues. This study investigates Lupinus angustifolius as a renewable feedstock for lignin-containing microfibrillated cellulose (L-MFC) and explores the role of lignin and hemicellulose in producing lupin-derived L-MFC films. By utilizing mild alkaline pre-treatments (0.5 or 1.5 M NaOH at 90 or 140 °C) and avoiding extensive delignification, four lignocellulose systems with varying lignin and hemicellulose content are generated. To produce L-MFC, these systems were further subjected to a defibrillation process, including TEMPO oxidation and mechanical homogenization. TEMPO oxidation removed partly lignin while preserving hemicellulose, leading to altered biopolymer ratios. Confocal fluorescence microscopy combined with optotracing enabled real-time visualization of the defibrillation process and spatial biopolymer mapping. Complementary excitation-emission matrix (EEM) spectroscopy provided fluorescence fingerprints for tracking molecular-level changes during processing. The resulting lupin-derived L-MFCs were successfully processed into self-standing films using Rapid Köthen drying. Films with higher residual lignin displayed increased hydrophobicity and mechanical strength, especially the R0.5-140-RK system, which achieved a tensile index of ~110 Nm/g and Young’s modulus of ~9700 MPa. This study demonstrates that partial preservation of native lignocellulose architecture offers a viable pathway for sustainable lupin-derived L-MFC film production.

This study presents a decellularization-inspired strategy for isolating the native cell wall scaffold of the tissue of the green macroalgae Ulva fenestrata as platform for bio-based film material. Advancing from conventional bottom-up biopolymer extraction, this top-down approach aims to preserve the inherent hierarchical architecture of the cell wall while reducing chemical use and processing steps. Several extraction protocols were evaluated, revealing that surfactant addition and mechanical pretreatment significantly enhanced decellularization efficiency and scaffold integrity. A high-throughput fluorescence spectroscopy-based method was established for real-time monitoring of pigment extraction kinetics. Cytosolic components were removed, evidenced by the presence of free-floating particles and sheet-like structures, suggesting outer layer detachment. Particle size distribution revealed three distinct fractions, with the smallest likely representing cytosolic remnants.  Biopolymer anatomy mapping revealed that tissue recovery varied with biomass architecture: In the blade tissue, thinner-walled tissues were more susceptible to treatment, whereas thicker-walled tissues retained integrity through an intermediate lamella. Additionally, in both blade and rhizoidal tissues, impairment of the outermost layer significantly enhanced decellularization efficiency. While Carbotrace 680 effectively stained Ulva cell walls in blade tissue, it showed no affinity for the rhizoidal cell walls, which instead were stained by Carbotrace 630, highlighting differences in tissue architecture. Recovered material was fractionated by size for film fabrication into fine, coarse, and mixed films. Tensile testing demonstrated distinct mechanical profiles among the film categories. This sustainable, biomimetic method offers a promising route for developing bio-based films directly from Ulva tissue, preserving native structure while enabling material tunability through process parameters and particle size selection.

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.