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- 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.
- Advancing Sustainable Production of High-Performance Cellulose PulpsPublication . Moran, Guadalupe; Costa Trigo, Iván; Bastida, Gabriela Adriana; Mazega, André; Duran, Josep; Domínguez, José Manuel; Vilaseca, FabiolaABSTRACT: Highlights What are the main findings? Enzymatic hydrolysis pretreatment of industrial pulps Pulp composition influencing the enzymatic performance Enhanced conditions for high-performance cellulose pulps What is the implication of the main finding? Sustainable methodology to produce cellulose pulps Lower environmental impact and alignment with circular economic principles Improvements in tensile strength, air permeability, hydrophobicity, and internal bondingHighlights What are the main findings? Enzymatic hydrolysis pretreatment of industrial pulps Pulp composition influencing the enzymatic performance Enhanced conditions for high-performance cellulose pulps What is the implication of the main finding? Sustainable methodology to produce cellulose pulps Lower environmental impact and alignment with circular economic principles Improvements in tensile strength, air permeability, hydrophobicity, and internal bondingAbstract With a growing demand for renewable resources in high-performance materials, sustainable methods are preferred for their lower environmental impact and alignment with circular economy principles. Among these, enzymatic hydrolysis remains relatively underexplored yet shows strong potential for cellulose fibrillation, offering a promising route that may lower energy requirements by minimizing the need for extensive refining compared to conventional mechanical or chemical approaches. In this study, enzyme cocktails rich in cellulase and xylanase were applied to three industrial pulps, sulphite, bleached Kraft eucalyptus and thermomechanical pine, to produce high-performance cellulose pulps. Treatments were carried out using varying enzyme loads (5-40 filter paper units per gram of dry pulp, FPU/gdp) and reaction times (1-16 h). The resulting chemical composition, structural morphology, and physical-mechanical properties were systematically evaluated. The findings revealed that pulp composition strongly influenced enzymatic treatment, affecting surface fibrillation, fibre aggregation, swelling, and fibre shortening. Under optimized conditions, enzymatic pretreatment significantly enhanced paper performance, with improvements in tensile strength, air permeability, hydrophobicity, and internal bonding. Overall, enzymatic hydrolysis represents a sustainable solution and a strategy which could reduce energy expenditures to high-performance cellulose pulps, suitable as reinforcing fibres in packaging applications.
- Improving Carbon Fixation and Acetate Production from Syngas Fermentation: On-Demand Versus Continuous FeedingPublication . Pacheco, Marta; Silva, Tiago; silva, carla; Moura, PatríciaABSTRACT: Syngas fermentation is a promising carbon capture and utilization (CCU) technology for producing carboxylic acids while transforming low-cost waste gas into high-value products. This study evaluates the two bioreactor feeding strategies for synthesis gas (syngas) fermentation by Eubacterium callanderi (formerly Butyribacterium methylotrophicum) strain Marburg-on-demand feeding (ODF) and continuous feeding (CF)-with a synthetic syngas mixture of 23 vol% CO2, 29 vol% CO, 32 vol% H2, and 16 vol% CH4, mimicking the syngas from lignocellulosic gasification. The ODF assay achieved a maximum syngas consumption rate of 112 mL/h, yielding 24.1 g/L acids, namely 22.9 g/L acetate and 1.3 g/L butyrate. CF of syngas at 223 mL/h required more gas (62.9 L) to produce 22.7 g/L total acids, from which 19.0 g/L acetate and 3.7 g/L butyrate were achieved. The CF-specific production rate (gproduct/gdry_cell_weight/hour) reached 0.5 g/gDCW/h (acetate) and 0.17 g/gDCW/h (butyrate), outperforming ODF with 0.3 and 0.02 g/gDCW/h, respectively. ODF minimized gas wastage and enabled CH4 accumulation inside the bioreactor up to approximately 78 vol%, while CF led to CO2 accumulation, indicating a need for more efficient CO2 utilization strategies, such as sequential fermentations. This work highlights the critical impact of the two feeding options studied with regard to scaling up the carbon-efficient production of carboxylic acids, and indicates that both strategies can have potential applications. ODF is ideal for increasing carbon fixation and achieving, simultaneously, gas cleaning, while CF fermentations are better suited to maximizing the acid production rate.
- Assessment of piggery wastewater treatment in vertical flow constructed wetlands: role of plants and aerationPublication . de Oliveira Corrêa, Diego; Ferreira, Alice; Ribeiro, Belina; Gogoi, Jayanta; Karan, N.; Nalwad, A.; Ganguly, A.; Mutnuri, S.; Gouveia, LuisaABSTRACT: Piggery wastewater (PWW), rich in pollutants, poses significant environmental risks if not properly treated. Natural treatment processes, such as constructed wetlands (combined action of plants, substrates, and microbes) and microalgae cultivation, offer sustainable and low-cost alternatives for managing these effluents while enabling resource recovery. This study represents an initial step toward optimizing key operational conditions, such as aeration (passive and active), vegetation presence, and the use of single or sequential Vertical Flow Constructed Wetlands (VFCWs), for the treatment of piggery wastewater, using different experimental setups, at a laboratory scale. Indoor experiments were conducted over an 8-week period to optimize operational conditions for the treatment of PWW. The VFCWs, arranged in two stages and operated in series through gravity flow, were fed daily and monitored weekly. The best removal rates of total Kjeldahl nitrogen (42.9 %), ammoniacal nitrogen (50.3 %), and chemical oxygen demand (20.5 %), were observed in the second stage of VFCW without aeration and with plant. Nitrate and phosphorus levels increased during the experiment, likely due to microbial activity within the substrate and plant root zones. The final goal is to treat the piggery wastewater from a rural farm in India, produce electricity (by a Microbial Fuel Cell), to generate an effluent suitable for microalgae cultivation, with the produced biomass intended for use either as a biostimulant to enhance cereal crops included in pig diets or as a direct nutritional supplement in pig feed.
- Extraction and assessment of the colouring capacity of Arthrospira platensis-derived pigmentsPublication . Villaro-Cos, Silvia; Gouveia, Luisa; Vladic, Jelena; Sanchez-Zurano, Ana; Martínez-García, Irene; Lafarga, TomásABSTRACT: This study presents a zero-waste biorefinery approach for the sequential extraction of phycocyanin and chlorophyll from Arthrospira platensis, followed by the valorisation of the remaining biomass as a plant biostimulant. Natural deep eutectic solvents were screened for phycocyanin recovery, with the mixture proline:glycerol:sorbitol:water (1:1:1:13 molar ratio) showing the highest potential (1.15 g⋅100 g-1; p < 0.05). An initial ultrasound-assisted cell wall disruption step significantly enhanced phycocyanin yield by 400–450 % relative to the untreated control (p < 0.05). A response surface methodology optimised extraction achieved a recovery yield of 8.26 g⋅100 g-1 at 39.7 °C and 127.9 min. The phycocyanin-rich extract was used to mimic the blue colour of commercial blue gin, with a minimal colour difference (ΔE) of 4.53. Subsequent chlorophyll extraction from the phycocyanin leftovers yielded an extract that successfully coloured a commercial green alcohol-free apple liquor (ΔE = 3.93) and green gin (ΔE = 1.65). Finally, the residual biomass demonstrated a significant biostimulant capacity, increasing the germination index of various seeds by 80–150 % compared to water (p < 0.05). This work highlights the potential of A. platensis as a sustainable source for natural colourants and agricultural inputs.
- Pretreatment Tunes scCO2 Extract Composition and Bioactivity in Three Microalgae: Chemometric and Molecular Docking InsightsPublication . Vladic, Jelena; Radman, Sanja; Besu, Irina; Stanojkovic, Tatjana; Zloh, Mire; Jerkovic, Igor; Karadžić Banjac, Milica; Ivkovic, Milena; Pereira, Hugo; Gouveia, LuisaABSTRACT: This study explores the impact of enzymatic (ENZ), microwave (MW), and ultrasound (US) pretreatments on supercritical CO2 (scCO2) extraction efficiency, chemical composition, and cytotoxic activity of Tetraselmis sp., Tetradesmus obliquus, and Chlorococcum sp. Pretreatments significantly enhanced extraction yields, with ENZ being most effective for Tetraselmis and Chlorococcum, and MW for T. obliquus. UPLC-HRMS profiling revealed species- and pretreatment-specific shifts: ENZ and US improved pigment recovery in Tetraselmis, while MW enriched carotenoids and chlorophyll derivatives. In Chlorococcum, MW boosted pigment diversity, whereas ENZ and US favored fatty acid derivatives in the extracts. Multivariate analysis confirmed significant compositional changes, particularly after ENZ and MW pretreatments. Tetraselmis extracts, especially those pretreated with MW, exhibited the strongest cytotoxic activity and highest selectivity indices against HeLa and MDA-MB-453 cancer cell lines. Correlation analysis identified compounds such as 2,3-dihydroxypropyl stearate, fucoxanthin, and (3 beta)-3-hydroxystigmast-5-en-7-one as strongly linked to cytotoxicity. Molecular docking further showed that abundant compounds in Tetraselmis extracts have high predicted affinities for cancer-related targets (e.g., BCL2, EGFR, PDK1). The results suggest that cytotoxic effects arise from both specific bioactive compounds and their synergistic interactions. These findings show that pretreatments can purposefully tune scCO2 extracts and provide a data-driven basis for designing more sustainable microalgal extraction workflows.