In this study, novel bio-inks formulated with horseradish peroxidase, HRP, and some additives were successfully developed for piezoelectric inkjet application. The optimized bio-ink formulation had a reliable jetting performance and maintained the biofunctionality before and after printing. The bio-ink also demonstrated a good storage life for up to 40 days at 4 oC with a negligible loss of biofunctionality. However, it was observed that some additives used in the bio-ink for obtaining necessary operational characteristics had detrimental effects on the enzyme activity. Especially, it was found that various viscosity modifiers typically used in commercial inkjet inks significantly impaired HRP activity prior to printing. Sodium Carboxymethyl Cellulose was shown to be an effective viscosity modifier that had no adverse effect on the biological activity of the HRP enzyme. Using a confocal scanning fluorescent microscope, a method for characterizing the spatial distribution of the active enzyme within the cellulosic paper substrates after inkjet printing was developed. Interestingly, it was found that the active printed HRP enzyme was mostly located in the cell walls of the cellulosic fibers instead of near the pigments or fillers. In an effort to better understand the fundamental interactions between the enzyme and the immobilization substrates, HRP adsorption isotherms on various substrate surfaces were obtained using the depletion method. The substrates included not only pulp fibers with varying degree of hydrophobicity and pigment and latex (the key materials used in papermaking), but also other types of cellulosic fibers of different morphologies, crystallinities, porosities, or surface charge densities. The influence on enzyme adsorption and inactivation behaviour of these substrates was compared with that of polystyrene beads (dialysed), which has been well studied in the literature. Results from this thesis indicated that hydrophobic interactions between the enzyme and the substrate surfaces had a major impact on the HRP adsorption behavior, while electrostatic interactions played a minor role. However, strong hydrophobic interactions could lead to enzyme inactivation. Research findings from this study suggested that cellulosic pulp fibers could be tailor-made into excellent enzyme immobilization supports by using existing fiber surface modification techniques.

In this study, novel bio-inks formulated with horseradish peroxidase, HRP, and some additives were successfully developed for piezoelectric inkjet application. The optimized bio-ink formulation had a reliable jetting performance and maintained the biofunctionality before and after printing. The bio-ink also demonstrated a good storage life for up to 40 days at 4 oC with a negligible loss of biofunctionality. However, it was observed that some additives used in the bio-ink for obtaining necessary operational characteristics had detrimental effects on the enzyme activity. Especially, it was found that various viscosity modifiers typically used in commercial inkjet inks significantly impaired HRP activity prior to printing. Sodium Carboxymethyl Cellulose was shown to be an effective viscosity modifier that had no adverse effect on the biological activity of the HRP enzyme. Using a confocal scanning fluorescent microscope, a method for characterizing the spatial distribution of the active enzyme within the cellulosic paper substrates after inkjet printing was developed. Interestingly, it was found that the active printed HRP enzyme was mostly located in the cell walls of the cellulosic fibers instead of near the pigments or fillers. In an effort to better understand the fundamental interactions between the enzyme and the immobilization substrates, HRP adsorption isotherms on various substrate surfaces were obtained using the depletion method. The substrates included not only pulp fibers with varying degree of hydrophobicity and pigment and latex (the key materials used in papermaking), but also other types of cellulosic fibers of different morphologies, crystallinities, porosities, or surface charge densities. The influence on enzyme adsorption and inactivation behaviour of these substrates was compared with that of polystyrene beads (dialysed), which has been well studied in the literature. Results from this thesis indicated that hydrophobic interactions between the enzyme and the substrate surfaces had a major impact on the HRP adsorption behavior, while electrostatic interactions played a minor role. However, strong hydrophobic interactions could lead to enzyme inactivation. Research findings from this study suggested that cellulosic pulp fibers could be tailor-made into excellent enzyme immobilization supports by using existing fiber surface modification techniques.

Motivation - Neue Anwendungen für cellulosische Fasern z.B. in 3D-Verpackungslösungen, im Leichtbau oder in Konstruktionswerkstoffen - Technologisch effiziente Herstellbarkeit wird gefordert (z.B. integriertes Umformen und Fügen) - Verfügbarkeit geeigneter Simulationswerkzeuge unabdingbar (z.B. mittels FE-Analyse)

Dans un contexte de développement durable, des matériaux thermoplastiques multiphasés biosourcés issus de dimères d’acides gras ont été élaborés pour développer une « eco-membrane » durable pour le bâtiment. Différentes formulations intégrant des polymères thermoplastiques biosourcés, polyuréthane thermoplastique (TPU) et polyamide (DAPA), des micro-charges minérales et des renforts cellulosiques ont été élaborées et analysées. Les relations « structures-propriétés » de ces systèmes multiphasés ont été étudiées. Les architectures macromoléculaires proches ont permis d’obtenir un certain degré de compatibilité entre les polymères. Les viscosités à l’état fondu du TPU et DAPA sont comparables, ce qui permet une bonne dispersion des phases du mélange lors de la mise en œuvre. Les propriétés mécaniques et la stabilité dimensionnelle des mélanges sont améliorées par ajout de micro-charges minérales. L’impact du facteur de forme des charges sur le module d’Young a été analysé et modélisé avec un modèle micro-mécanique. Des essais du type charge-décharge ont également été menés afin d’étudier le comportement à la fatigue des biocomposites. Enfin, les interactions et les affinités entre les renforts et polymères ont été approchées. Des modifications chimiques ont été développées à la surface des fibres afin de diminuer leurs caractères hydrophiles et d’améliorer la compatibilisation avec la matrice. L’impact de ces modifications sur l’interface fibres-matrice a ensuite été analysé. Les différentes études réalisées ont permis de sélectionner pas à pas les différents constituants de l’éco-membrane pour réaliser in fine un prototype prometteur. / In the context of sustainable development, renewable multiphase systems from thermoplastics based on dimers of fatty acids were prepared to develop a renewable waterproofing material for building applications. Formulations based on thermoplastics, i.e. thermoplastic polyurethane (TPU) and polyamide (DAPA), mineral micro-fillers and cellulosic fibers were prepared with a special focus on the morphology-property relationships of the multiphase systems obtained thereof. The close macromolecular architectures promote a certain degree of compatibility between the polymers. Comparable viscosities in the melt state ensure a good dispersion of the matrices within each other during processing. The mechanical properties and dimensional stability were improved with micro-fillers. The effect of the aspect ratio of the fillers on the elastic properties was investigated and micro-mechanical modelling of the Young’s Modulus was performed. The fatigue behavior of the biocomposites was also examined through loading and unloading tests. Finally, interactions and affinities between the fibers and polymers were characterized. Chemical modifications were carried out on the surface of the fibers to decrease their hydrophilic nature and improve the fiber-matrix adhesion. The effect of the chemical modification was then investigated. Step-by-step, the studies carried out ensured the selection of the optimal components for a renewable waterproofing material enabling the production of a promising prototype.

Motivation - Neue Anwendungen für cellulosische Fasern z.B. in 3D-Verpackungslösungen, im Leichtbau oder in Konstruktionswerkstoffen - Technologisch effiziente Herstellbarkeit wird gefordert (z.B. integriertes Umformen und Fügen) - Verfügbarkeit geeigneter Simulationswerkzeuge unabdingbar (z.B. mittels FE-Analyse)

Les papiers récupérés sont de plus en plus utilisés pour fabriquer du papier recyclé de haute blancheur. La ligne de recyclage inclut entre autres les opérations de désencrage et de blanchiment, le peroxyde d’hydrogène (H2O2) étant le réactif blanchissant le plus utilisé en milieu alcalin (stade P). Son efficacité est toutefois limitée car il est peu ou moyennement réactif sur les fonctions azoïques des colorants papetiers. L’objectif de cette étude était donc d’améliorer l’élimination des colorants azoïques lors d’un stade P.L'amélioration du stade P utilisé en délignification des pâtes chimiques a fait l’objet de nombreuses études. En particulier, l’activation ou la catalyse de H2O2 par des complexes cuivre(II)-phénanthroline (Cu-Phen) présentant des résultats très intéressants, des essais de blanchiment de pâtes désencrées et colorées ont été entrepris. Le système H2O2/Cu-Phen s’est également révélé efficace en décoloration, mais le mécanisme n’avait alors pas été étudié.L’objectif de ce travail était donc de déterminer dans quel cas et pourquoi le complexe cuivre(II)-phénanthroline était capable d’améliorer la décoloration de fibres cellulosiques colorées. Trois questions intermédiaires se sont posées : (1) quel effet Cu-Phen a-t-il sur le colorant isolé? (2) la décoloration d’une pâte de fibres colorées par H2O2 est-elle améliorée par Cu-Phen? (3) par quel mécanisme la décoloration est-elle rendue plus efficace?Le travail a donc été organisé en trois études : (1) caractérisation des colorants sélectionnés et du complexe en l’absence d’oxydant et examen des interactions entre les deux, (2) étude et optimisation paramétrique de H2O2/Cu-Phen pour la décoloration de pâtes colorées, (3) étude du mécanisme d’oxydation en milieu aqueux, en l’absence et en présence de cellulose.Ce travail s’est appuyé sur de nombreuses techniques analytiques (spectroscopies RMN, FTIR, UV-vis et RPE ; ESI-MS) et des calculs de spéciation. Il a prouvé que le complexe Cu-Phen en présence de H2O2 améliorait la décoloration de colorants azoïques, avec ou sans fibres cellulosiques. Il a été mis en évidence que la phénanthroline agissait comme un stabilisant permettant d’ajuster la solubilité, la stabilité et le potentiel d’oxydo-réduction du cuivre(II), mais qu’elle n’était pas indispensable. De plus, le pH du milieu est également un paramètre clé, jouant à la fois sur l’activité du complexe et sur ses interactions avec le substrat. Le substrat (colorant mais aussi cellulose) s’est trouvé fortement dégradé par ce système H2O2/Cu-Phen. Les résultats corroborent l’hypothèse de l’oxydation du substrat par les radicaux hydroxyles issus de la décomposition de H2O2, plutôt que par H2O2 lui-même, ce mécanisme étant l’une des étapes d’un cycle catalytique.La recherche menée et les résultats obtenus sont applicables non seulement à l’amélioration du blanchiment des pâtes désencrées, mais aussi au traitement d’effluents colorés dans les industries du papier et du textile. / Today, recovered papers are reused for the manufacture of bright paper after deinking and fiber bleaching, which generally starts with an alkaline hydrogen peroxide stage (H2O2). However, the efficiency of H2O2 is often limited due to its low reactivity on the azo groups of paper dyes contained in recovered papers. The goal of this study was to improve the removal of these azo dyes by H2O2.The improvement of H2O2 bleaching has been studied thoroughly in the context of chemical pulp delignification. In particular, the activation or catalysis of H2O2 by copper(II)-phenanthroline complexes (Cu-Phen) was found to be very effective. This inspired a preliminary bleaching study on deinked pulp and dyed pulp, and resulted in significant improvement of dye removal, which gave birth to our project.The purpose of this work was to determine to what extent copper(II)-phenanthroline could improve the hydrogen peroxide color-stripping of dyed cellulosic fibers, and how. To answer this question, three intermediate issues were addressed: (1) does Cu-Phen alone have an effect on the dye? (2) does Cu-Phen improve the color-stripping of a dyed pulp by H2O2? (3) how does the H2O2/Cu-Phen system enhance the dye-color-stripping efficiency?This work was thus divided into three studies: (1) the selected dyes and the complex were characterized in the absence of any oxidant and the interactions between the two were examined, (2) the H2O2/Cu-Phen system was applied on two dyed pulps to assess their color-stripping potential and to attempt to optimize it, and (3) the oxidation mechanism was investigated via trials in aqueous solution, with and without cellulose.Using several analytical techniques (NMR, FTIR, UV-vis and EPR spectroscopy; ESI-MS) and speciation calculations, this work proved that the Cu-Phen complex enhanced H2O2 for the color-stripping of azo dyes, with and without fibers. It also provided evidence that phenanthroline acted as a stabilizer to adjust the solubility, stability and redox potential of copper(II), but may not be indispensable. The substrate (dyes but also cellulose) was strongly degraded by the H2O2/Cu-Phen system. The results of the mechanistic study supported the hypothesis of substrate oxidation by radicals produced via decomposition of H2O2 rather than by hydrogen peroxide itself. This mechanism, strongly dependent on the pH, is probably part of a catalytic cycle.Finally, along with further research proposed based on our conclusions, this thesis should contribute to the improvement of deinked pulp bleaching as well as wastewater treatment in the pulp and textile industries.

Tesis por compendio / [ES] En esta tesis se obtuvieron fracciones celulósicas y extractos activos de paja de arroz mediante diferentes técnicas de extracción y purificación, utilizando agua como disolvente. Estas fracciones se incorporaron en películas de almidón y PLA, y en bicapas de almidón-PLA, para obtener materiales de envasado activos para extender la vida útil de diferentes alimentos. Los extractos obtenidos por combinación de ultrasonidos y calentamiento a reflujo (USHT) y con agua subcrítica (SWE) (a 160 °C (SWE-160) y 180 °C (SWE-180)) presentaron un alto contenido fenólico (37, 51 y 83 mg GAE.g-1 de extracto seco, respectivamente) y actividad antioxidante (6,3, 2,0 y 1,2 mg de extracto seco.mg-1 DPPH, respectivamente). Los extractos SWE fueron activos contra L. innocua y E. coli. Se aislaron fibras de celulosa (FC) del residuo insoluble de las diferentes extracciones. A pesar de las ligeras diferencias en el grado de purificación, los nuevos métodos dieron lugar a FCs con rendimientos más elevados que el método alcalino (35-39 % frente al 29 %), con cristalinidad (60-69 %), comportamiento térmico y relación de aspecto (20-60) similares. Se incorporaron fibras USHT al 1, 3 y 5 % (p/p) en películas termoprocesadas de almidón de maíz, y de almidón de maíz modificado térmicamente. La incorporación al 3 % dio lugar a películas con mejores propiedades funcionales. Los extractos activos (al 4, 6 y 8 % en peso) en la matriz de almidón, con o sin CF (3 %), dieron lugar a películas más extensibles y menos resistentes, con mayor capacidad de barrera al oxígeno. Las bolsas monodosis de aceite de girasol con estas películas activas y reforzadas redujeron eficazmente la oxidación. Las películas activas de PLA termoprocesado, con diferentes proporciones (2, 4 y 6 % p/p) del extracto USHT, presentaron color y propiedades mecánicas ligeramente peores, pero con mayor barrera al oxígeno. La cinética de liberación de los antioxidantes incorporados en simulantes alimentarios de diferente polaridad (A: productos acuosos; y D1: sistemas de aceite en agua), mostró que las películas con un 6% de extracto, tuvieron una capacidad antioxidante similar en simulantes con distinta polaridad. Se obtuvieron bicapas biodegradables laminando PLA, con y sin extracto USHT (6 %), y almidón termoplástico, con o sin FC USHT (3 %). Las bicapas mostraron mejor capacidad de barrera global que las monocapas. Sin embargo, la migración de compuestos entre capas dio lugar a películas menos rígidas y resistentes de lo esperado de la contribución del film de PLA. Las bicapas activas y reforzadas alargaron la vida útil de carne de cerdo refrigerada. Las películas de PLA con un 6 % (p/p) de los diferentes extractos (USHT, SWE-160 y SWE-180) presentaron menor resistencia mecánica y capacidad de barrera al vapor de agua, pero una mayor capacidad de barrera al oxígeno y a la luz UV. Además, estas películas, especialmente con el extracto SWE-180, alargaron la vida útil de la carne de cerdo refrigerada. Las FCs obtenidas con los diferentes métodos de purificación también se utilizaron para producir aerogeles de celulosa. Las diferencias composicionales afectaron a la microestructura de los aerogeles. La capacidad de absorción y de retención de agua de los aerogeles estuvo dentro de los rangos descritos para este tipo de materiales. Por tanto, fue posible obtener fracciones valorizadas de la paja de arroz, extractos activos y fibras celulósicas, útiles en el desarrollo de materiales biodegradables activos a base de almidón y PLA. Son necesarios más estudios para validar la seguridad alimentaria de los materiales, así como para el desarrollo de otras aplicaciones en el campo de la industria alimentaria o farmacéutica. / [CA] En aquesta tesi doctoral es van obtenir fraccions cel·lulòsiques i extractes actius de palla d' arròs mitjançant l'aplicació de diferents tècniques d'extracció i purificació, utilitzant aigua com a dissolvent. Aquestes fraccions es van incorporar a pel·lícules de midó i PLA, i bicapes de midó-PLA, per obtenir materials d'envasament actius útils per allargar la vida útil de diferents aliments. Els extractes combinant ultrasons i escalfament a reflux (USHT) i amb aigua subcrítica (SWE) (a 160 °C (SWE-160) i 180 °C (SWE-180)) van mostrar un alt contingut fenòlic (37, 51 i 83 mg GAE.g-1 d'extracte sec, respectivament) i activitat antioxidant (6,3, 2,0 i 1,2 mg d'extracte sec.mg-1 DPPH, respectivament). Els extractes SWE van mostrar activitat antibacteriana contra L. innocua i E. coli. Es van aïllar fibres de cel·lulosa (FC) de les fraccions insolubles de l'extracció. Malgrat les lleugeres diferències en el grau de purificació, els nous mètodes van donar lloc a FCs amb rendiments més elevats que el mètode alcalí (35-39 % enfront del 29 %), amb cristal·linitat (60-69 %), comportament tèrmic i relació d'aspecte (20-60) similars. Es van incorporar fibres USHT a l'1, 3 i 5 % (p/p) en pel·lícules de midó de dacsa, i de midó de dacsa modificat per tractament tèrmic. La incorporació al 3 % va donar lloc a pel·lícules amb les millors propietats funcionals. Els extractes actius (al 4, 6 i 8 % en pes) a la matriu de midó, amb o sense CF (3 %), proporcionaren pel·lícules més extensibles i menys resistents, però amb més capacitat de barrera a l'oxigen. Les bosses monodosi de l'oli de gira-sol amb aquestes pel·lícules actives i reforçades van reduir eficaçment l'oxidació. Les pel·lícules actives de PLA amb diferents proporcions (2, 4 i 6 % p/p) de l'extracte USHT, presentaren color i propietats mecániques lleugerament pitjors, però amb major capacitat de barrera a l'oxigen. La cinètica d'alliberament dels antioxidants incorporats, en simulants alimentaris de diferent polaritat (A: productes aquosos; i D1: sistemes d'oli en aigua), va mostrar que les pel·lícules amb un 6% d'extracte, tenien una capacitat antioxidant similar en simulants amb diferent polaritat. Es van obtenir bicapes biodegradables laminant PLA, amb i sense extracte USHT (6 %), i midó termoplàstic, amb o sense FC USHT (3 %). Les bicapes van mostrar millor capacitat de barrera global que les monocapes. No obstant això, la migració de compostos entre capes va donar lloc a pel·lícules menys rígides i resistents respecte a l'esperat de la contribució de la pel·lícula de PLA. Les bicapes actives i reforçades van allargar la vida útil de carn de porc refrigerada. Les pel·lícules de PLA amb un 6 % (p/p) dels diferents extractes (USHT, SWE-160 i SWE-180) van presentar menor resistència mecànica i capacitat de barrera al vapor d'aigua, però una major capacitat de barrera a l'oxigen i a la llum UV. A més, aquestes pel·lícules, especialment amb l'extracte SWE-180, van allargar la vida útil de la carn de porc refrigerada. Les FCs obtingudes amb els diferents mètodes de purificació també es van utilitzar per produir aerogels de cel·lulosa. Les diferències composicionals van afectar la microestructura dels aerogels. La capacitat d' absorció i de retenció d'aigua dels aerogels va estar dins dels rangs descrits per a aquest tipus de materials. Per tant, va ser possible obtenir fraccions valoritzades de la palla d' arròs, extractes actius i fibres cel·lulòsiques, útils en el desenvolupament de materials biodegradables actius a base de midó i PLA. Són necessaris més estudis per validar la seguretat alimentària dels materials, així com per al desenvolupament d' altres aplicacions en el camp de la indústria alimentària o farmacèutica. / [EN] This Doctoral thesis focused on obtaining cellulosic fractions and bioactive extracts from rice straw (RS) by applying different extraction and purification techniques, using water as a green solvent. These fractions were incorporated into starch and poly (lactic acid) (PLA)-based films, as well as into starch-PLA bilayers, to obtain active packaging materials useful for extending the shelf life of different food matrices. The extracts obtained by applying a combined ultrasound-reflux heating method (USHT) and subcritical water extraction (SWE) (at 160 °C (SWE-160) and 180 °C (SWE-180)) exhibited high phenolic content (37, 51, 83 mg GAE.g-1 dry extract, respectively) and antioxidant activity (6.3, 2.0, and 1.2 mg dry extract.mg-1 DPPH, respectively). The SWE extracts showed antibacterial activity against L. innocua and E. coli. Cellulose fibres (CF) were isolated from the extraction insoluble fractions and compared with those obtained from the traditional alkaline method. Despite slight differences in the degree of purification, the new methods gave rise to CFs with higher yields than the alkaline method (35-39% vs. 29%), with similar crystallinity (60-69%), thermal behaviour, and aspect ratios. USHT fibres were incorporated into corn starch (1, 3, and 5% wt.), and corn starch-modified by heat treatment, obtained by thermoprocessing. The incorporation of CF at 3% gave rise to films with the best functional properties. When active extracts were incorporated at different ratios (4, 6, and 8% wt.) into the starch matrix, with or without CF (3%), the films were more stretchable and less resistant, but with higher oxygen barrier capacity. Likewise, mono-dose bags of these active and reinforced films effectively reduced the oxidation of packed sunflower oil. Active, plasticised PLA films were produced, incorporating USHT extract (2, 4, and 6% wt.) by thermoprocessing, which exhibited colour and slightly worsened tensile behaviour, but with higher oxygen barrier capacity. The release kinetics of the incorporated antioxidant compounds in food simulants of different polarity (A: aqueous products; and D1: oil-in-water systems) showed that films containing 6% of extract delivered similar antioxidant capacity regardless of the food simulant polarity. Biodegradable bilayers were obtained by laminating plasticised PLA, with and without USHT extract (6%), and thermoplastic starch, with or without USHT CFs (3%). The bilayers exhibited improved overall barrier capacity with respect to the monolayers. However, the interlayer compound migration led to less stiff and resistant films with respect to that expected from the PLA film contribution. The active and reinforced bilayers were able to extend the shelf life of the packed pork meat during cold storage. PLA films with 6% wt. of the different extracts (USHT, SWE-160, and SWE-180) were produced, which exhibited lower mechanical resistance and water vapour barrier capacity, but improved oxygen barrier capacity and intense UV light-blocking effect. Furthermore, the active PLA films, especially with SWE-180 extract, extended the shelf life of cold storage pork meat. The CFs obtained with the different purification methods were also used to produce cellulose aerogels. The differences in the chemical composition of the CFs affected the aerogels' microstructure. The water absorption and retention capacity of the aerogels were within the previously reported ranges for this type of material. Thus, it was possible to obtain valorised fractions of rice straw, active extracts and cellulosic fibres, useful in developing active biodegradable materials based on starch and PLA. Further studies are needed to validate the food safety of the materials, as well as for developing other applications in the food or pharmaceutical industry. / The authors thank the Agencia Estatal de Investigación (Spain) for the financial support through projects PID2019-105207RB-I00/AEI/10.13039/501100011033 and Generalitat Valenciana [grant number GrisoliaP/2019/115]. / Vieira De Freitas, PA. (2022). Valorisation of rice straw by obtaining active compounds and cellulosic materials for the development of biodegradable food packaging systems [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/191380 / Compendio