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- Acid-Based Deep Eutectic Solvents for Structural Modification of Sulphite Pulp Cellulose: A Potential Route Toward Advanced MaterialsPublication . Moran, Guadalupe; Costa Trigo, Iván; Domínguez, José Manuel; Vilaseca, FabiolaABSTRACT: The transition toward renewable and environmentally responsible materials has intensified interest in cellulose-based systems for use in sustainable packaging applications. Although cellulose offers biocompatibility, structural versatility, and tuneable physicochemical properties, conventional modification routes rely on harsh chemicals and generate environmentally burdensome effluents. In this study, an efficient and a potentially green strategy for cellulose modification was developed using acid-based deep eutectic solvents (DES) composed of choline chloride and lactic, acetic, or citric acid at different molar ratios. Under mild conditions (110 degrees C, 4 h), DES pretreatment reduced glucan content in sulphite pulp from 99% to 79-93%, depending on the hydrogen bond donor (HBD), while suggesting an apparent increase in relative crystallinity, from approximately 82% to 90%, as estimated by the Segal method. FTIR, XRD, and morphological analyses revealed the disruption of the hydrogen bonding network, enhanced fibrillation, and residual DES-derived functional groups detectable by FTIR. Although DES pretreatment increased structural order, it also reduced enzymatic digestibility due to the higher proportion of crystalline domains. Overall, the results demonstrate that acidic DES constitutes a sustainable and recyclable medium capable of modulating cellulose structure and generating materials with enhanced physicochemical properties. These findings suggest that DES-modified cellulose could serve as a potential reinforcement platform for future biodegradable packaging and bioplastic formulations, enabling the development of high-performance, renewable, and environmentally compliant packaging materials.
- A two-stage cultivation strategy for the industrial production of the novel microalga Chlorococcum amblystomatisPublication . Correia, Nádia; Trovão, Mafalda; Santo, Gonçalo E.; Guerra, Inês; Cunha, P.; Fonseca, Joana; Pereira, Hugo; Costa, Monya M.; Ferreira, Sara; Santos, Tamára; Barros, Ana; Cardoso, Helena; Silva, Joana; Gouveia, Luisa; Varela, JoãoABSTRACT: This study presented the industrial-scale validation of a two-stage growth strategy for a poorly explored yet industrially relevant chlorophyte microalga, Chlorococcum amblystomatis, addressing the gap between laboratory research and practical application. By using cultures grown heterotrophically as inoculum for industrial-scale photoautotrophic reactors, this approach significantly accelerated scale-up. The proposed strategy reduced the overall scale-up time by approximately 5.5-fold compared to the conventional pipeline, without compromising productivity or biochemical quality. A maximum biomass concentration of 70.96 +/- 0.35 g L-1 was achieved in 7-L benchtop fermenters under heterotrophic conditions, representing the highest value reported to date for this microalga. The biomass obtained presented a protein content of 32.42 +/- 2.30% of dry weight, which increased to 54.55 +/- 2.04% upon cultivation under photoautotrophic conditions, as well as polyunsaturated fatty acids, which increased from 50.12 +/- 3.27 to 67.74 +/- 0.35% of total fatty acids, with alpha-linolenic acid as the predominant fatty acid. Conversely, the saturated fatty acids content (38.26 +/- 1.63% of TFA) decreased to 22.37 +/- 0.02% of TFA in the phototrophic stage. Pigment contents also increased significantly during the phototrophic stage, with chlorophylls rising from 10.04 +/- 1.68 to 44.91 +/- 5.11 mg g(-)1 and carotenoids reaching notable concentrations, including lutein (5.34 +/- 0.10 mg g-1), beta-carotene (5.92 +/- 0.39 mg g-1), and neoxanthin (3.53 +/- 0.75 mg g-1). Among the industrial photoautotrophic systems tested, the closed tubular photobioreactors were the most suitable configuration for C. amblystomatis cultivation, achieving higher biomass productivity, enhanced protein content and pigment accumulation compared to open raceway ponds. Overall, this work demonstrates a scalable, efficient, and time-saving cultivation strategy for C. amblystomatis, supporting its potential for industrial biotechnological applications.
- A Strategy to Improve Porous MOF Structural Stability for the Effective Removal of S/N Pollutants from (Bio)fuelsPublication . Silva, Dinis; Mirante, Fátima; Santos-Vieira, Isabel; Granadeiro, Carlos; Silva, Luis Cunha; Balula, Salete S.ABSTRACT: The need to mitigate environmental pollution drives the continuous quest for advanced desulfurization and denitrogenation methods in refineries. The presence of sulfur and nitrogen compounds in fossil fuels and biofuels contributes largely to atmospheric polllution. Oxidative catalysis presents a promising way for addressing these challenges, offering efficient and environmental friendly pathways. This work investigates the heterogeneous catalytic performance of the polyoxometalates, plenary Keggin PW12 structure and the derivative lacunary PW11 structure, supported on the porous MIL-100(Fe) metal-organic framework, for simultaneous oxidative desulfurization and denitrogenation. Both PW11@MIL-100(Fe) and PW12@MIL-100(Fe) catalysts demonstrated rapid and complete desulfurization within 60 min of reaction using a sustainable H2O2 oxidant. Remarkably, PW11@MIL-100(Fe) exhibited higher catalytic efficiency and demonstrated high recycle capacity. In fact, notable results were attained to remove the most noxious pollutants in (bio)fuels, offering insights for the development of effective catalytic materials with high viability for the energy industry.
- Microalgae-Based Treatment of Winery WastewaterPublication . Lopes da Silva, Teresa; Lopes, Tiago; Reis, AlbertoABSTRACT: Winery wastewater (WWW) is a high-strength effluent characterized by high organic load, nutrient content, and seasonal variability, posing significant environmental challenges. This review critically evaluates microalgae-based systems for WWW treatment, addressing their performance, scalability, and role within circular bioeconomy frameworks. Analysis of recent studies shows that microalgae-based systems can achieve high pollutant removal efficiencies, including up to ~ 90–92% chemical oxygen demand removal, high removal of total organic carbon and nitrogen (typically above 80%), and up to 91–95% ammonium removal. These systems also enable the production of biomass rich in proteins (up to ~ 58.8% dry weight) and valuable compounds such as pigments, supporting multiple valorization pathways. A key finding is that, despite strong technical performance, large-scale implementation remains limited. The main constraints are associated with wastewater variability, presence of inhibitory compounds, and operational challenges in biomass recovery, particularly energy-intensive harvesting and downstream processing. Techno-economic and life cycle analyses indicate that standalone systems are rarely economically viable under current conditions, and that feasibility depends on integration into multiproduct biorefineries and existing infrastructures. Regulatory uncertainty and the need for stakeholder acceptance further constrain deployment. Overall, microalgae-based WWW treatment represents a promising but system-dependent solution, requiring integrated technological, economic, and policy frameworks to achieve scalable implementation.
- From waste to circularity: the potential of different treatments of poultry manure and forestry residues in a hot-spot production region in PortugalPublication . d'Espiney, Ana; Verworner, Bengt; Pinheiro, Helena; Isabel, Marques; Oehmichen, Katja; Engler, Nils; Majer, Stefan; Stinner, Walter; Thrän, DanielaABSTRACT: Purpose The management of biogenic residues in a way that extends the lifetime of carbon within the systems is a focus of the present work. Different waste management systems are assessed in terms of GHG emissions and removals, aiming to identify that with the lowest impact on climate change. Methods A life cycle comparative analysis is performed, assessing the mitigation of the impact on climate change of the current waste management system (A) of the top two residues of a Portuguese region – composting of poultry manure and open air burning of forestry residues – considering two bioenergy solutions: biochemical conversion of poultry manure and thermochemical conversion of wood residues (B); biochemical conversion for both residues together (C), implying fungal pre-treatment of the wood residues, which adds a food product to the supply chain. Data were retrieved from literature and from authors’ experimental work. Assumptions underlying the methodological framework (e.g., cut offs inherent to comparative assertions) are discussed. A sensitivity analysis covers uncertainties underpinning bioenergy systems assessments (e.g., unperfect substitution and rebound effect phenomena). Results and discussion The highest impact on climate change is observed with system A, greenhouse gas (GHG) emissions 2 to 3 times higher than those of the other two systems. System A also performs poorly in conserving the biomass, e.g., forestry residues burning results in carbon being directly released to the atmosphere with no valuable intermediate use. In the base scenario, system B results in the lowest GHG emissions of the three systems, producing biochar, which contributes to carbon sequestering, and biodiesel. Nevertheless, system C can result in the lowest GHG emissions, in a scenario considering unperfect substitution with renewable sources being selected to match the biodiesel supply of system B. Conclusions System A is least preferable among the three options. Between systems B and C, ranking depends on whether unperfect substitution is considered. System B performs better in all scenarios, except with unperfect substitution, where system C outranks B. Moreover, system C achieves a higher yield of digestate, a product providing other ecosystem services. Thus, in future research, the inclusion of other impact categories in LCA can reveal a better overall environmental performance for system C.
- Nanobubble-enhanced oxygen transfer in bacterial nanocellulose production: Comparative evaluation with static and airlift systemsPublication . Rodrigues, Ana Cristina; Martins, Daniela; Carvalho, Ricardo; Marques, Susana; Belo, Isabel; Espina, Begona; Dourado, Fernando; Gama, MiguelABSTRACT: Despite the unique properties of bacterial nanocellulose (BNC), oxygen limitation during large-scale production impairs microbial metabolism and cellulose synthesis, leading to high production costs and limited commercial success. Static fermentation can achieve high titers, but industrially it is operationally challenging. Agitated systems like airlift (AL) bioreactors, allow faster production but typically yield lower titers. This study pioneered the use of an agitated bioreactor equipped with a nanobubble (NB) generator, and its performance was compared with that of static and AL systems, employing a newly isolated Komagataeibacter sp. strain from kombucha cultivated in Eucalyptus bark hydrolysate and corn steep liquor. Key monitored parameters included dissolved oxygen, cell density, pH, sugar and lactic acid contents, and BNC production. The obtained BNC was characterized for its crystallinity, thermal stability, degree of polymerization, morphology and fiber size. The AL-and NB-derived BNC exhibited a denser network structure, lower crystallinity index, and lower polymerization degrees than that from static culture. NB technology generated stable nanobubbles (size: 95.8 f 12.9 nm; zeta potential:-14.2 f 8.6 mV). At 1 L. min-1 airflow, compared to AL, the NB bioreactor achieved a 6-fold higher volumetric mass transfer coefficient (kLa 35.9 f 1.2 h-1) and oxygen transfer rate (OTR: 309.7 f 10.2 mg.L-1.h-1). It supported greater cell density but maintained a similar BNC volumetric productivity to that of the AL (0.023 g.L-1.h-1), and moderately higher (near 280%) than that of static culture (0.0082 g.L-1.h-1). Thus, the improved oxygen levels provided by the NB system favored biomass growth rather than BNC production, suggesting that further optimization is needed to redirect carbon flux toward BNC production.
- Sustainable Production of Poly(3-hydroxybutyrate) Using Eucalyptus Bark: Integration with Green Downstream ProcessingPublication . Matias, João; Rodrigues, Thomas; Torres, Cristiana A. V.; Marques, Susana; Ribeiro, Belina; Gírio, Francisco; Reis, Maria A.; Freitas, FilomenaABSTRACT: This study integrates the valorization of a lignocellulose material into poly(3-hydroxybutyrate), P(3HB), with biopolymer extraction from bacterial cells with the enzyme alcalase. The work focused on Burkholderia thailandensis DSM 13276 as the P(3HB) producer and on eucalyptus bark, a byproduct from the pulp industry, as the sole feedstock for bacterial cultivation. The eucalyptus bark was hydrolyzed by a cellulolytic enzymatic cocktail following steam explosion and further subjected to ultrafiltration for enzyme recovery. The resulting hydrolysate supported good cell growth, achieving a cell dry weight of 7.67 +/- 0.16 g/L within 72 h of cultivation, and high P(3HB) content (60.0 +/- 2.19 wt %) in the bacterial cells, clearly favoring biopolymer synthesis over cell growth, as demonstrated by the polymer and growth yields (0.190 gP(3HB)/gsugar and 0.026 gX/gsugar, respectively). High extraction efficiency (96%) and biopolymer purity (100 +/- 3.38%) were reached by enzymatic treatment, resulting in a sample with properties aligned with those of commercial P(3HB) in terms of molecular mass distribution, crystallinity, and thermal properties. These findings demonstrate the successful use of a sustainable feedstock together with the application of environmentally friendly technologies based on the use of enzymes for both lignocellulosic saccharification and biopolymer recovery to develop high-quality bioplastics, advancing the goals of a circular bioeconomy.
- Applicability Assessment of a Microbial Proteolytic Fermentation Broth to Leather Processing and Protein Stain RemovalPublication . Lageiro, Maria Manuela; Moura, Maria João; Simões, Fernanda; Alvarenga, Nuno; Reis, AlbertoABSTRACT: Microbial proteases are fundamental towards the eco-sustainability of proteolysis at the industrial scale. A proteolytic broth was obtained from a bioreactor fermentation of a proteolytic Bacillus strain isolated from an industrial alkaline bath. Broth proteolytic activity was applied to leather tanning and to the removal of protein stains. The hide tanned with the microbial proteolytic fermentation broth showed better physical properties than the one tanned with commercial pancreatic proteases of the same activity (780 LVU). Proteinaceous stains on cotton fabric were removed more efficiently using the Bacillus proteolytic broth than water or a commercial detergent. Blood and egg yolk disappeared in less than 30 min. The removal of soya and English sauce stains was even faster. Broth proteolytic activity was characterised by caseinolytic (5200 LVU), collagenolytic (10.0 U mg-1), elastolytic (3.7 U mg-1), and keratinolytic (0.7 U mg-1) activities, which were compared with those of a commonly used commercial protease. Alkaline protease activity in the broth was demonstrated by a 20% increase in caseinolytic activity from pH 5 to 8. Besides the demonstrated applications in the leather and detergent industries, the produced alkaline microbial proteases can also be used in the treatment of proteinaceous wastes and effluents, offering potential environmental benefits reinforcing and impacting the bioeconomy.
- Enhancing microalgal biohydrogen production: Unlocking higher yields with hydrothermal pretreatment with niobium phosphatePublication . Silva, Thiago; Jesus Junior, Maurino Magno; Neves de Araujo, Matheus; Castro, Laressa Santos; Fuess, Lucas Tadeu; Rodrigues, Fábio de Ávila; Zaiat, Marcelo; Reis, Alberto; Calijuri, Maria LuciaABSTRACT: Microalgae cultivated in wastewater hold promise as a substrate for biohydrogen (bioH2) production. However, their rigid cell walls pose a challenge to fermentability. In this context, this study evaluated hydrothermal pretreatment with niobium phosphate (NbP) at 100-180 degrees C for 0-70 min, using up to 75 % NbP (relative to the dry weight of microalgal biomass). The hydrothermal pretreatment at 180 degrees C for 10 min with 75 % NbP released 7431 mg total carbohydrates (CHt) L-1, increasing the availability of fermentable substrates in subsequent dark fermentation (DF). When this pretreated biomass was subsequently fermented at pH 5.0 (sample PB5), bioH2 production reached 1.03 mmol H2 mol-1 CHt, with a maximum cumulative output of 0.17 mmol H2 and a CHt conversion efficiency of 83.6 %. In contrast, pH 5.5 and 6.0 reduced bioH2 yields and promoted methanogenic activity, while no pH control resulted in negligible bioH2 evolution. In conclusion, hydrothermal pretreatment with niobium phosphate and pH improvement synergize to enhance hydrogenogenesis, integrating wastewater treatment and renewable biohydrogen production.
- Enhancing the Biorefinery of Chestnut Burrs, Part II: Influence of Pretreatment with Choline Chloride–Urea-Diluted Deep Eutectic Solvent on Enzymatic HydrolysisPublication . Costa Trigo, Iván; Moran, Guadalupe; Pérez Guerra, Nelson; Oliveira, Ricardo; Domínguez, José ManuelABSTRACT: Agro-industrial chestnut waste derived from chestnut processing is usually discharged without further use. However, these residues are attractive due to their high-value composition, rich in sugars and lignin. Among these residues, chestnut burrs (CB) represent a promising feedstock for biorefinery applications aimed at maximizing the valorization of their main constituents. In this study, we propose an environmentally friendly approach based on deep eutectic solvents (DES) formed by choline chloride and urea (ChCl/U) (1:2, mol/mol) for the selective deconstruction of lignocellulosic architecture, followed by enzymatic hydrolysis to release second-generation (2G) fermentable sugars. Pretreatments were applied to raw CB, washed CB (W-CB), and the obtained solid fraction after prehydrolysis (PreH). Structural and morphological modifications, as well as crystallinity induced by DES pretreatment, were characterized using attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD). Remarkable results in terms of effectiveness and environmental friendliness on saccharification yields were achieved for PreH subjected to DES treatment for 8 h, reaching approximately 60% glucan and 74% xylan conversion under the lower enzyme loading (23 FPU/g) and liquid-to-solid ratio (LSR) of 20:1 studied. This performance significantly reduces DES pretreatment time from 16 h to 8 h at mild conditions (100 degrees C), lowers the LSR for enzymatic hydrolysis from 30:1 to 20:1, and decreases enzyme loading from 63.5 FPU/g to 23 FPU/g, therefore improving process efficiency and sustainability.