http://hdl.handle.net/10498/6779 2026-11-04T19:16:39Z http://hdl.handle.net/10498/39647 Aprovechamiento de subproductos agroalimentarios con propiedades farmacoactivas para la impregnación supercrítica de polímeros biomédicos Verano Naranjo, Lidia En España se implantan cada año miles de dispositivos médicos, incluidos stents, prótesis articulares, tornillos de fijación ósea, andamios para ingeniería de tejidos, etc. Aunque la tecnología ha avanzado, aún existen riesgos de rechazo y complicaciones relacionados con la integración de estos materiales en el organismo. Para minimizar estos problemas, la investigación biomédica busca desarrollar materiales biocompatibles que no solo sean tolerados, sino que favorezcan la regeneración tisular. Entre las alternativas más prometedoras destacan los polímeros bioabsorbibles, capaces de degradarse de forma controlada tras cumplir su función terapéutica, evitando la necesidad de una segunda intervención para su retirada. Dentro de esta tipología de materiales, los llamados de “tercera generación” están diseñados para liberar fármacos de manera progresiva en el tejido diana y estimular la endotelización, ya que contienen principios activos incluidos en el polímero durante su manufactura. Aunque tradicionalmente se han empleado compuestos sintéticos para este fin, en los últimos años ha crecido el interés por sustancias bioactivas de origen natural (compuestos farmacoactivos procedentes de plantas, bacterias, hongos, algas o invertebrados marinos) con propiedades terapéuticas diversas. La incorporación de estas sustancias en matrices poliméricas suele realizarse mediante métodos convencionales que implican el uso de disolventes orgánicos a menudo resultan en una baja eficiencia. Como alternativa, la técnica de impregnación con CO₂ supercrítico facilita la incorporación homogénea de los compuestos bioactivos en la matriz polimérica, prescindiendo del empleo de disolventes orgánicos tóxicos. Esto resulta en una optimización de la eficiencia de carga y, crucialmente, minimiza el riesgo de residuos solventes en el producto final, lo que incrementa su biocompatibilidad y mitiga el impacto ambiental asociado a los procesos convencionales. Esta tesis doctoral propone investigar la impregnación supercrítica de sistemas poliméricos biocompatibles con extractos naturales, obtenidos de subproductos agroalimentarios. El empleo de estas fuentes secundarias fomenta la reincorporación de estos materiales en la cadena de valor promoviendo los principios de la economía circular. Se trata de un enfoque de carácter fundamental que busca optimizar los parámetros del proceso y evaluar su potencial para el desarrollo futuro de sistemas poliméricos funcionalizados en aplicaciones biomédicas. El estudio tiene tres pilares fundamentales: la evaluación de los extractos, la selección de polímeros, y la impregnación supercrítica de los mismos para aplicación biomédica. Para esta investigación se seleccionaron tres extractos naturales: obtenidos a partir de hojas de mango (Mango Leaf Extract - MLE), hojas de olivo (Olive Leaf Extract - OLE) y hollejo de uva tinta (Grape Pomace Extract - GPE). Estos compuestos se obtuvieron mediante extracción a alta presión mejorada por disolventes (Enhanced Solvent Extraction - ESE), utilizando las condiciones optimizadas en estudios previos. Se caracterizó el perfil fenólico de los extractos, así como su actividad antioxidante, antiinflamatoria y antimicrobiana, confirmando su potencial como agentes bioactivos para aplicaciones biomédicas. De forma paralela, se realizó un screening de distintos polímeros de uso biomédico y se evaluaron los efectos del contacto con CO₂ supercrítico sobre la integridad y el hinchamiento permanente de poliuretano termoplástico (TPU), polidioxanona (PDO), policarbonato (PC), ácido poliláctico (PLA), acrilonitrilo butadieno estireno (ABS), policaprolactona (PCL) y polietileno tereftalato modificado con glicol (PETG). A partir de estos ensayos se seleccionaron PLA y PDO por su biodegradabilidad y su idoneidad para dispositivos de baja porosidad, como los stents. Respecto a los ensayos de impregnación, inicialmente se utilizó ketoprofeno como molécula modelo para establecer las condiciones operacionales del proceso de impregnación y definir las bases de liberación posterior del fármaco. Se estudió los efectos de la presión (100–400 bar) y la temperatura (35–55 °C) como variables principales del proceso, encontrando que un aumento de ambas variables promovía el hinchamiento del polímero y el rendimiento de la impregnación, favoreciendo una impregnación más profunda del soluto. Las condiciones óptimas para alcanzar la máxima tasa de impregnación con un hinchamiento polimérico controlado se establecieron en 200 bar y 75 °C, el rendimiento de impregnación alcanzó un valor del 8,5 ± 0.4%. Finalmente, se detectó una cinética de liberación multifásica, cuya velocidad y duración de cada fase estaban determinadas intrínsecamente por las condiciones de impregnación. Posteriormente, se estudió la impregnación de extractos naturales. En este trabajo se presentan los resultados experimentales del estudio de dos sistemas: PLA impregnado con GPE y PDO impregnado con OLE. En cuanto a la impregnación de PLA con GPE, se emplearon dos extractos: E-GPE, obtenido con etanol como cosolvente, y HE-GPE, obtenido con una mezcla hidroalcohólica. Además, se compararon dos técnicas de impregnación: la impregnación supercrítica convencional (Supercritical Solvent Impregnation - SSI) y una nueva metodología propuesta, denominada impregnación por inmersión presurizada (Pressurized Soaking Impregnation - PSI), que constituye una opción intermedia entre la SSI y la inmersión tradicional (soaking). El análisis de los resultados demostró que el E-GPE superó al HE-GPE, evidenciando un mayor rendimiento de impregnación y una superior bioactividad en los filamentos resultantes. La técnica PSI indujo una impregnación predominantemente superficial en la matriz polimérica y confirió una aparente mayor capacidad antioxidante inmediata, lo que se atribuye a una cinética de liberación más rápida de los compuestos impregnados. Por el contrario, las tasas de impregnación más elevadas se obtuvieron con la técnica SSI, siendo el máximo rendimiento alcanzado específicamente con E-GPE bajo 100 bar y 55 °C (7,7 ± 1,5 %). El siguiente sistema que se evaluó fue la impregnación de PDO con OLE mediante la metodología SSI. A pesar de registrar tasas de impregnación reducidas, la mayor proporción en el extracto de su polifenol principal, la oleuropeína, facilitó la detección y cuantificación mediante cromatografía líquida, permitiendo así una evaluación más precisa de la impregnación y la liberación del compuesto. En este caso, se obtuvieron condiciones óptimas de impregnación en los valores máximos estudiados: 400 bar y 75 °C (OLE: 1,5 ± 0,3%; oleuropeína: 706,4 ± 37,2 μg g-1 de PDO; hidroxitirosol: 16,9 ± 0,5 μg g-1 de PDO). Se estudiaron los perfiles de liberación de oleuropeína e hidroxitirosol, observando que este último puede generarse in situ como producto de degradación de otros compuestos, como la oleuropeína. La modelización cinética de la liberación de la oleuropeína confirmó un proceso dominante por difusión. Durante el ensayo de liberación in vitro se mantuvo la capacidad antioxidante y antiinflamatoria proporcionadas por los compuestos fenólicos del extracto. Respecto a los ensayos celulares, se realizaron estudios de adhesión y crecimiento celular sobre dispositivos de PLA impregnados con GPE, aunque los resultados no fueron concluyentes. Si bien se confirmó la no toxicidad del sistema y se observó un cambio morfológico hacia células más alargadas, indicando un microambiente ligeramente más favorable, no se detectaron beneficios cuantificables en términos de proliferación, apoptosis o capacidad funcional de las células. Por tanto, la conclusión principal obtenida con el alcance de esta tesis es que es posible funcionalizar polímeros biomédicos con extractos naturales procedentes de subproductos agroalimentarios. Se ha demostrado que los polímeros adquieren propiedades antioxidantes y antiinflamatorias de los extractos; sin embargo, sería necesario estudiar con mayor detalle el efecto terapéutico en condiciones fisiológicas y en modelos in vivo con distintos sistemas polímero-extracto.; Each year, thousands of medical devices (e.g. stents, joint prostheses, bone fixation screws and tissue engineering scaffolds) are implanted in Spain. Although technology has advanced, risks of rejection and complications related to the integration of these materials into the organism still persist. To minimize these issues, biomedical research seeks to develop biocompatible materials that are not only tolerated but also capable of promoting tissue regeneration. Among the most promising alternatives are bioabsorbable polymers, which can degrade in a controlled manner after fulfilling their therapeutic function, thus avoiding the need for a second intervention for their removal. Within this type of material, the so-called "third-generation" devices are designed to progressively release drugs into the target tissue and stimulate reendothelialization, as they contain active principles incorporated into the polymer during manufacturing. Although synthetic compounds have traditionally been used for this purpose, interest in naturally-derived bioactive substances of natural origin (pharmacoactive compounds derived from plants, bacteria, fungi, algae, or marine invertebrates) with diverse therapeutic properties has grown in recent years. The incorporation of these substances into polymeric matrices is usually carried out using conventional methods that involve the use of organic solvents, often resulting in low incorporation efficiency. As an alternative, the supercritical CO2-assisted impregnation technique facilitates the homogeneous incorporation of the bioactive compounds into the polymeric matrix, dispensing with the use of toxic organic solvents. This technique significantly enhances the loading efficiency and, crucially, minimizes the levels of residual organic solvents in the final product. These improvements boosts its overall biocompatibility and reduces the environmental burden associated with conventional processing methods. This doctoral thesis proposes the functionalization of biocompatible polymeric systems via supercritical fluid impregnation utilizing natural extracts sourced from agri-food by-products. The use of these secondary sources promotes the reincorporation of these materials into the value chain, fostering the principles of the circular economy. This is a fundamental research approach that seeks to optimize process parameters and evaluate the potential for the future development of functionalized polymeric systems in biomedical applications. The study is based on three fundamental pillars: the evaluation of the extracts, the selection of polymers, and their supercritical impregnation for biomedical applications. For this research, three natural extracts were selected: obtained from Mango Leaf Extract (MLE), Olive Leaf Extract (OLE), and Grape Pomace Extract (GPE). These compounds were obtained via Enhanced Solvent Extraction (ESE), using conditions optimized in previous studies. The phenolic profile of the extracts, as well as their antioxidant, anti-inflammatory, and antimicrobial activities, were characterized, confirming their potential as bioactive agents for biomedical applications. In parallel, a screening of various polymers for biomedical use was performed, and the effects of contact with supercritical CO2 on the integrity and permanent swelling of Thermoplastic Polyurethane (TPU), Polydioxanone (PDO), Polycarbonate (PC), Polylactic Acid (PLA), Acrylonitrile Butadiene Styrene (ABS), Polycaprolactone (PCL), and Polyethylene Terephthalate Glycol-modified (PETG) were evaluated. Based on these tests, PLA and PDO were selected due to their biodegradability and suitability for low-porosity devices, such as stents. Regarding the impregnation assays, ketoprofen was initially used as a model molecule to establish the operational conditions of the impregnation process and to define the bases for subsequent drug release. The effects of pressure (100–400 bar) and temperature (35–55 °C) were studied as main process variables. The results indicated that an increase in both variables promoted polymer swelling and the impregnation yield, favoring a deeper penetration of the solute. The optimal conditions to achieve the highest impregnation rate with a controlled polymeric swelling were established at 200 bar and 75 °C, where the impregnation yield reached a value of 8.5 ± 0.4 %. Finally, a multiphase release kinetic was detected, whose velocity and duration of each phase were intrinsically determined by the impregnation conditions. Subsequently, the impregnation of natural extract impregnation was evaluated, focusing on the results of two systems: PLA impregnated with GPE and PDO impregnated with OLE. Regarding the impregnation of PLA with GPE, two extracts were employed: E-GPE, obtained with ethanol as a cosolvent, and HE-GPE, obtained with a hydroalcoholic mixture. Furthermore, two impregnation techniques were compared: conventional Supercritical Solvent Impregnation (SSI) and a newly proposed methodology called Pressurized Soaking Impregnation (PSI), which constitutes an intermediate option between SSI and traditional soaking. The analysis of the results demonstrated that E-GPE outperformed HE-GPE, evidencing a higher impregnation yield and superior bioactivity in the resulting filaments. The PSI technique induced a predominantly superficial impregnation in the polymeric matrix and conferred an apparently greater immediate antioxidant capacity, which is attributed to a faster release kinetic of the impregnated compounds. Conversely, the highest impregnation rates were obtained with the SSI technique, with the maximum yield specifically achieved with E-GPE under 100 bar and 55 °C (7.7 ± 1.5 %). The next system evaluated was the impregnation of PDO with OLE using the SSI methodology. Despite registering reduced impregnation rates, the greater proportion of its main polyphenol, oleuropein, in the extract facilitated its detection and quantification via liquid chromatography, thus allowing a more precise evaluation of the impregnation and compound release. In this case, optimal impregnation conditions were obtained at the maximum values studied: 400 bar and 75 °C (OLE: 1.5 ± 0.3 %; oleuropein: 706.4 ± 37.2 μg mg-1 of PDO; hydroxytyrosol: 16.9 ± 0.5 μg g-1 of PDO). The release profiles of oleuropein and hydroxytyrosol were studied, observing that the latter can be generated in situ as a degradation product of other compounds, such as oleuropein. The kinetic modeling of oleuropein release confirmed a diffusion-dominated process. During the in vitro release assay, the antioxidant and anti-inflammatory capacities provided by the phenolic compounds of the extract were maintained. Regarding the cellular assays, adhesion and cell growth studies were performed on PLA devices impregnated with GPE, although the results were not conclusive. While the non-toxicity of the system was confirmed and a morphological change towards more elongated cells was observed, indicating a slightly more favorable microenvironment, no quantifiable benefits were detected in terms of proliferation, apoptosis, or functional capacity of the cells. Therefore, the main conclusion obtained within the scope of this thesis is that it is feasible to functionalize biomedical polymers with natural extracts derived from agri-food by-products. It has been demonstrated that the polymers acquire antioxidant and anti-inflammatory properties from the extracts; however, it would be necessary to study the therapeutic effect in greater detail under physiological conditions and in in vivo models with different polymer-extract systems. 2026-01-01T00:00:00Z http://hdl.handle.net/10498/39361 Application of physicochemical and biological pretreatments to enhance volatile fatty acids production via Dark Fermentation from agri-food waste ROUABHIA, AMER In the field known as the Green Economy, one of the most important pillars for developing a future, more sustainable production model is the incorporation of sustainable production processes as substitutes for conventional ones. In this regard, Volatile Fatty Acids (VFAs) constitute the so-called carboxylate platform, which plays a central role in biorefinery processes. The benchmark bioprocess for VFA production is Dark Fermentation (DF), a mode of anaerobic digestion in which the methanogenic phase is inhibited. This approach is framed within the biorefinery concept, where residual biomass is processed comprehensively and sustainably to obtain multiple value-added products, thereby promoting a circular economy model and reducing the environmental impact of agro-industrial waste management. The use of residual biomass as raw material contributes to lowering the production costs of these processes. However, it necessitates the application of costly pretreatments to ensure adequate solubilization of the organic matter present in the biomass. Residual biomass is a cheap carbon source with high volume availability and decentralized geographical distribution, but its use requires the optimization of applicable pretreatments. Waste and byproducts from the agro-food sector -which is immensely important both nationally and in the autonomous community of Andalusia- already represent one of the main sources of renewable residual biomass. They can be valorized through bioprocesses to obtain high value-added compounds. This Doctoral Thesis aimed to collect a significant sample of the principal types of agro-food biomass generated in the Andalusian autonomous community, also considering their diverse physicochemical characteristics. The following substrates were selected for this study: - Rice Husks (RH) - Sugar Beet Pulp (SBP) - Brewers' Spent Grain (BSG) - Orange Peel (OP) The goal is to optimize the valorization of agro-food substrates by applying different pretreatment strategies, including physical (ultrasonic sonication and microwave), biological, and chemical (hydrothermal) pretreatments, using only water as a solvent. These pretreatments will allow for improved solubilization of organic compounds in the biomass and the extraction of bioproducts of interest, such as reducing sugars, proteins, and polyphenols, which have high potential for application in the biotechnology industry. Following pretreatment, the substrates will be subjected to Dark Fermentation, an acidogenic anaerobic digestion process aimed at the production of Volatile Fatty Acids (VFAs) and a biogas with a high hydrogen content. Additionally, although not originally planned in the Doctoral Thesis project, the application of Enzyme Hydrolysis (EH) was also tested to enhance the valorization strategy. So, the general objective can be expressed as: "To develop and optimize physicochemical and biological pretreatments applied to agro-food residual biomass to favor the solubilization of organic matter with the purpose of maximizing the production of volatile fatty acids through a dark fermentation process and to evaluate the different alternatives." And the Specific objectives were: 1. Evaluate and determine the most suitable pretreatment (hydrothermal, microwave, ultrasonic sonication, and biological) for solubilizing and hydrolyzing the organic matter present in four agro-food sector substrates (rice husks, sugar beet pulp, brewers' spent grain, and orange peel) in terms of organic matter solubilization. 2. Evaluate the Volatile Fatty Acid production capacity of a dark fermentation process using the pretreated substrates. 3. Select the most favorable overall alternative for the generation of volatile fatty acids. The development of these partial objectives was carried out sequentially and coordinated with the experimental stages of the Doctoral Thesis work plan. As a result of the work conducted, four scientific articles corresponding to the contents of the Doctoral Thesis have been published in JCR® indexed journals. The DOIs for these publications are listed below: - Article 1: https://doi.org/10.1016/j.jenvman.2023.118332 - Article 2: https://doi.org/10.1021/acs.energyfuels.5c03953 - Article 3: https://doi.org/10.3390/app15158736 - Article 4: https://doi.org/10.1016/j.biombioe.2025.108634 The investigation began with the (Article 1) of a passively aerated biological pretreatment applied to all four residues to evaluate its capability for initial organic matter solubilization. This low-energy approach proved highly effective for biomasses with specific fiber profiles, with OP achieving the highest solubilization yield (sCOD 58.6%) under optimized conditions. These findings confirmed the feasibility of a biological pre-hydrolysis stage to enrich substrate quality and reduce the severity of subsequent processes. The second research axis involved a direct comparison between Microwave-assisted (MW) and Hydrothermal Reactor (HTR) pretreatments (Article 2) to quantify the recovery of Total Reducing Sugars (TRS), proteins (PR), and VFAs. MW pretreatment emerged as the most energy-efficient technique for rapidly solubilizing accessible biomasses such as OP and SBP. Conversely, HTR generated significantly higher extraction yields of TRS and VFAs for more complex substrates (RH, BSG), underscoring that pretreatment effectiveness depends not only on energy input but also on target compounds and biomass structural characteristics. The final phase investigated the integration of Ultrasound (US) and Hydrothermal Pretreatment (HTP) to optimize the sequential production of biohydrogen (H2) and (VFA) within a biorefinery framework, as detailed in Article 3 and Article 4. pretreatment effectively enhanced the solubilization of organic matter, serving as a crucial first-stage intensification method prior to (DF). Specifically, the integrated strategy combining (US) pretreatment, dark fermentation, and enzyme hydrolysis was developed to maximize the output of (H2), (VFA), and Total Reducing Sugars (TRS) from the four feedstocks. Optimized HTP conditions were critically assessed to determine the optimal reaction time necessary to maximize (H2)production from the lignocellulosic feedstocks. This research conclusively demonstrated that combining these tailored physicochemical strategies maximizes the overall biohydrogen and VFA yields, underscoring the necessity of multi-step pretreatment designs for achieving high-efficiency bioconversion. The main conclusions of the work have been: *Effective pretreatment strategies for the valorization of agro-industrial biomasses depend on a careful balance between structural disruption of the biomass, precursor release, and process energy cost. *Pretreatment optimization based on substrate: Method selection must be based on the intrinsic complexity of the biomass. Microwave is preferable for simple substrates (OP, SBP) due to its speed and lower energy consumption (40.1 kJ/g). Hydrothermal pretreatment is necessary to maximize the solubilization of recalcitrant biomasses (BSG, RH) and achieve high VFA yields, although it requires higher energy (70.85 kJ/g). *Decoupling organic matter solubilization and biohydrogen production pretreatment must avoid excessive release of proteins, as this diverts dark fermentation metabolism and limits H2 production. Moderately pretreated SBP (HTP for 20 min) is superior to OP for biohydrogen production. *US pretreatment as the best option for sequential processes: Integration of ultrasound with enzyme hydrolysis of the solid phase of the pretreated effluent is an effective biorefinery strategy. Ultrasound pretreatment reduces recalcitrance (although it uses green solvents with less delignification capacity), allowing subsequent enzyme hydrolysis to significantly increase TRS yield, especially for SBP (4.5-fold increase). *Lignin as the main problem: RH was shown to be consistently resistant to all pretreatments, underlining that its high lignin content (14.0%) and low enzymatic accessibility limit its use as an efficient substrate for dark fermentation without more severe chemical treatments. *The integration of pretreatment, dark fermentation, and enzyme hydrolysis is demonstrated to be an effective strategy for the sustainable conversion of agro-industrial residues into biofuels and value-added biochemicals. 2026-01-29T00:00:00Z http://hdl.handle.net/10498/38195 Estudio a escala piloto de la desulfuración anóxica de biogás empleando un efluente nitrificado a partir de lixiviados de vertedero Torres Herrera, Sandra Biogas generated in landfills is a valuable source of renewable energy, the use of which is hindered by the presence of compounds that affect its quality, primarily hydrogen sulfide (H2S). This compound is corrosive and toxic, and its combustion generates sulfur oxides, necessitating its reduction or removal for most applications. Biological technologies offer a sustainable alternative to physicochemical methods, among which is anoxic desulfurization. However, this technology relies on electron acceptors such as nitrate/nitrite, which, if sourced from chemical reagents, increase process costs. Therefore, this Doctoral Thesis addresses this challenge from a circular economy perspective by proposing and validating a pilot-scale integrated system that couples two waste treatment processes: biogas purification and landfill leachate treatment. The study proposes using the nitrified effluent from ammonium-rich leachate as a low-cost source of nitrate/nitrite for anoxic biogas desulfurization, thereby reducing operational costs and facilitating the management of this complex waste stream. A key byproduct of the anoxic desulfurization process is elemental sulfur. This sulfur can be recovered from suspended-growth bioreactors for use as a fertilizer or as a raw material in the chemical industry. To optimize both the bioprocess and byproduct recovery, an analytical method was first developed and validated for the quantification of biogenic sulfur (S0) using gas chromatography with a pulsed flame photometric detector (GC-PFPD), combined with a simple sample preparation based on filtration and toluene extraction. This method exhibited an error of less than 5% when applied to samples from a laboratory-scale stirred-tank anoxic desulfurization bioreactor. Additionally, a recovery method for biogenic sulfur was optimized via coagulation-flocculation, achieving removal efficiencies exceeding 97% with a cationic flocculant at a dosage of 0.82 mg/L (0.5 mg flocculant per g S0) at a stirring speed of 30 rpm and a pH of 8.0. In subsequent tests with biogenic sulfur from biological samples with high solids and organic matter content, the required flocculant dosage increased 40 times (to 20 mg flocculant per g S0). To generate the low-cost electron acceptor, the nitrification of real landfill leachate was studied at a pilot scale in a 1 m3 sequencing batch reactor (SBR). Over the 61-week operational period, a total volume of 8,950 L of landfill leachate was treated, with an average ammonium concentration of 1,410±362 mg N-NH4+/L. The system demonstrated high robustness and resilience, with the reactor stably generating nitrate and removing over 83.9% of the ammonium under controlled pH (7.4±7.7) and temperature (25±27°C) conditions. A maximum ammonium elimination capacity o161 g N-NH4+/(m3·d) (at 99.9% efficiency) and a nitrate production rate of up to 95.4 g N-NO3-/(m3·d) were achieved. High leachate salinity was identified as a critical factor requiring control via dilution to prevent process inhibition. To assess the biomass's sensitivity to increasing leachate concentrations, its activity was evaluated through respirometric tests, and the data were fitted to a Haldane-type inhibition model. The maximum substrate oxidation rates obtained in these laboratory-scale tests were 8.89 mg N/(gVSS·h) for ammonium and 5.91 mg N/(gVSS·h) for nitrite. Finally, the integration of both processes was evaluated through the long-term operation of a pilot-scale (1 m3) anoxic bioscrubber system fed with real landfill biogas (total volume: 264,812 m3) and the nitrified effluent (total volume: 2.4 m3). The system demonstrated high stability, achieving H2S removal efficiencies exceeding 85% (with a maximum of 97%) under optimal conditions (pH 7.5±8.0 and T > 22°C). The system's operational limits were determined, reaching a maximum elimination capacity of 50.8 g S-H2S/(m3·h) with an elemental sulfur yield of 60%. Microbial community analysis revealed a predominance of the genus Thioalkalispira-Sulfurivermis as the primary driver of desulfurization in the bioreactor. The system proved resilient to operational disturbances, such as recirculation pump failures and fluctuations in the biogas flow rate. This study provides valuable insights into the practical application of anoxic bioscrubbers for landfill biogas biodesulfurization, highlighting key control factors and offering a foundation for enhancing the efficiency and sustainability of biogas purification technologies under real-world constraints. 2025-11-11T00:00:00Z http://hdl.handle.net/10498/36722 Production of bioplastic precursors (polyhydroxyalkanoates) from macroalgae Romero Vargas, Agustín In recent years, there has been increasing interest in the use of macroalgae to obtain high value-added products through biotechnological processes. This natural biomass has many advantages, such as the fact that they do not need land or fresh water to be cultivated. Moreover, they are not energy crops, so they do not compete directly with those that are a food resource, such as maize or wheat. Another aspect that makes them interesting is that around half of their dry weight are carbohydrates, such as cellulose or laminarin in the case of brown algae, in addition to other glucose polysaccharides that can be found depending on the species. These unconventional carbon sources can be used as substrates in fermentation processes to produce compounds of industrial interest, such as polyhydroxyalkanoates (PHAs), precursors of bioplastics, and thus contribute to reducing the impact of conventional plastics on the environment. However, due to the integral nature of the macroalgae structure, the accessibility of microorganisms to organic matter is restricted, so it is necessary to study the most appropriate way to solubilise it. Studies on the solubilisation of organic matter in macroalgae are scarce and very recent, so methodologies described for lignocellulosic matter, which have been widely studied, are usually applied. Among the techniques used, the ones that offer the best yields include a chemical pretreatment stage and an enzymatic hydrolysis or saccharification stage. The former is used to weaken the structure of the biomass and facilitate accessibility to the carbohydrates of which it is composed. In the second, the bioconversion of polysaccharides to fermentable monosaccharides takes place. In this Doctoral Thesis, the invasive species Rugulopteryx okamurae was used as algal biomass, a brown alga whose origin is in the coasts of East Asia, but which has reached the Mediterranean coast and is causing serious environmental problems, affecting native species, and having great repercussions on the local economy. To minimise its impact, it was evaluated the use of this macroalga as a raw material to produce PHAs, by fermentation with a pure culture of Cupriavidus necator, using as substrate sugar hydrolysates obtained from the alga without pretreatment, and previously pretreated with acid dilutions and heat input. Firstly (article 1), the dietary fibre content of the biomass without pretreatment was analysed and compared with that of 10 other species. It was found that R. okamurae contained the richest protein fraction among the species compared, with 49.05 % (w/w), and a hydrolysable polysaccharide content of 27.29 % (w/w), a high value compared to the algae compared. Thus, the enzymatic hydrolysis stage was studied in order to obtain the highest amount of fermentable sugars. For this purpose, the main variables in the saccharification process were evaluated, in batch mode, and using algae without pretreatment. Different ratios of biomass loading, 6, 8 and 10 % (w/v), enzyme doses, 18, 30 and 50 FPU/g biomass, and stirring rates, 150 and 250 rpm, were tested. Furthermore, the condition at which the highest concentration of fermentable sugars was achieved was tested in Fed-Batch mode. For a better comparison of the results, a new kinetic model was developed based on the conventional model, which considers first order kinetics for the enzymatic reaction. A new term was added to the proposed model to account for the effect of enzyme diffusion through the inert solid biomass. The proposed model showed a better fit to the experimental data, compared to the conventional model, especially for prolonged hydrolysis times, obtaining the highest sugar concentration (13.7 g/L) after 165 hours of hydrolysis, in the condition: 10 % (w/v) biomass loading, an enzyme dose of 50 FPU/g dry biomass, and a stirring rate of 250 rpm, in Batch mode. Secondly (article 2), a study of the effect of hydrothermal acid pretreatment on the enzymatic saccharification process was carried out. The acid (HCl) concentrations studied were 0.05 N, 0.1 N and 0.2 N, for reaction times of 15, 30 and 60 minutes. In order to determine the most suitable pretreatment conditions among those analysed, the amount of reducing sugars (RS) generated after the saccharification process was evaluated, taking into account the severity of the pretreatment. Thus, the optimum pretreatment conditions were found when 0.2 N HCl was used for 15 minutes, achieving 20.7 g/L RS, of which 92.6 % was glucose. In addition, the severity of pretreatment on dietary fibre composition was analysed, and it was observed that increasing the severity resulted in an impoverishment of the protein fraction and an enrichment of the hydrolysable polysaccharide fraction. The latter is positive from the point of view of its application in biorefinery, as it favours the fractionation of the biomass and the macroalgae can therefore be better utilised. Finally (article 3), fermentations were carried out to evaluate the capacity of the R. okamurae algal biomass to be used as feedstock for the production of PHAs. The hydrolysates obtained from the non-pretreated algae, and pretreated under optimal conditions, were used as culture medium for fermentation to PHAs with C. necator, and the profiles of substrate consumption, biomass generation and biopolymer production were compared with those obtained with fermentations carried out in synthetic medium. Thus, those fermentations that were carried out with hydrolysate of pretreated algae showed the best values of yields (0.774 g PHB/g RS), and percentage of PHB accumulation (89.8 % CDW), reaching values comparable to the highest values reported in the scientific literature so far. From the results obtained in this Doctoral Thesis it was possible to estimate the amount of PHB that could be produced using macroalgae as feedstock. The estimation was based on a real case of R. okamurae disposal, specifically on the coast of Ceuta (Spain), where, in 2016, more than 5,000 tonnes of algal waste deposited along 15 km of coastline had to be removed. It was calculated, that up to 9.3 tons of PHB could be produced using such amount of residual biomass and applying the pretreatment, saccharification and fermentation conditions proposed in this Doctoral Thesis. Thus, based on the results obtained in this Doctoral Thesis, it would be interesting to carry out a technical-economic analysis for the production of PHB from R. okamurae, since the production costs depend to a large extent on the raw material, and for this case, the process could be cost-effective. 2024-10-01T00:00:00Z