Optimization of FucoPol bioreactor production and exopolysaccharide applications

FucoPol is an exopolysaccharide (EPS) produced by the bacterium Enterobacter A47, composed of sugars (fucose, galactose, glucose and glucuronic acid) and acyl groups (succinyl, pyruvyl and acetyl), and with a high average molecular weight (>106 Da). The first main goal of this thesis was to investigate the effect of the phosphorus (P) concentration on FucoPol production. Production assays were performed under different concentrations of this nutrient: from 0.8 to 2.6 g.L-1(standard concentration). The conditions implemented did not affect Enterobacter A47 growth, but a reduction of the EPS synthesis was observed: 24% less polymer when 2 and 1.3 g.L-1 of P were used, and 44% for 0.8 g.L-1 of phosphorus. A slight reduction to 2.0 g.L-1 in the phosphorus concentration did not affect the polymer’s composition, neither the molecular weight. However, the EPS obtained from the assays with fewer P showed less fucose content and was richer in glucose. Secondly, this thesis aimed to assess the flocculation properties of FucoPol. The flocculation rate of FucoPol was determined using a kaolin clay suspension (5 g.L-1) in the presence of CaCl2. Flocculation rate values above 70% were achieved with a low bioflocculant dosage of 1 mg.L-1, for pH values in the range 3-5, and temperature within 15-20 ºC. The bioflocculant was also shown to be stable after freezing/thawing and heating up to 100 ºC, for 20 min. The work performed in this thesis also had as an objective to explore the metal binding efficiency of the polysaccharide. FucoPol revealed a good performance in the biosorption of cobalt, copper and zinc. The polysaccharide was an excellent biosorbent of lead so this metal was chosen for further studies, namely the impact of EPS dosage, Pb2+ initial concentration, pH and temperature. Optimal FucoPol concentration of 5 mg.L-1 was found to uptake 18645 mgmetal.g-1EPS from an 100 mg.L-1 Pb2+ solution, at pH 2.3. Moreover, FucoPol presented a great sorption performance in the range of temperatures between 5 and 45 oC

Biosorption of heavy metals by the bacterial exopolysaccharide fucopol

FCT/MCTES (UIDB/04378/2020). Patricia Concordio-Reis acknowledges FCT/MCTES for PhD grant SFRH/BD/131947/2017.Despite the efforts for minimizing the usage of heavy metals, anthropogenic activities still generate high amounts of wastewater containing these contaminants that cause significant health and environmental problems. Given the drawbacks of the conventional physical and chemical methods currently used, natural biosorbents (microbial cells or their products) arise as promising environmentally friendly alternatives. In this study, the binding efficiency of the polysaccharide secreted by Enterobacter A47, FucoPol, towards lead (Pb2+), cobalt (Co2+), copper (Cu2+) and zinc (Zn2+) cations was demonstrated. FucoPol revealed a higher performance for the biosorption of Pb2+, with a maximum overall metal removal of 93.9 ± 5.3% and a specific metal uptake of 41.1 ± 2.3 mg/gEPS, from a Pb2+ solution with an initial concentration of 10 mg/L, by a 5 g/L FucoPol solution. The overall metal removal decreased considerably (≤31.3 ± 1.6%) for higher Pb2+ concentrations (48 and 100 mg/L) probably due to the saturation of FucoPol's binding sites. Pb2+ removal was also less efficient (66.0 ± 8.2%) when a higher FucoPol concentration (10 g/L) was tested. Pb2+ removal efficiency of FucoPol was maximized at pH 4.3, however, it was affected by lower pH values (2.5-3.3). Moreover, the FucoPol's sorption performance was unaffected (overall metal removal: 91.6-93.9%) in the temperature range of 5-40 °C. These findings demonstrate FucoPol's great potential for utilization as a biodegradable and safe biosorbent for treating waters and wastewaters contaminated with Pb2+.publishersversionpublishe

Exopolysaccharide production by the marine bacterium Alteromonas macleodii Mo169 using fruit pulp waste as the sole carbon source

project LA/P/0140/202019 of the Associate Laboratory Institute for Health and Bioeconomy – i4HB. Publisher Copyright: © 2023 The Author(s)A sugar-rich apple pulp waste generated from fruit processing for juice production was used as the sole carbon source for the cultivation of Alteromonas macleodii Mo169, a marine bacterium known for its EPS-secreting ability. The strain efficiently utilized the glucose and fructose present in the apple pulp waste, reaching biomass and EPS production of 9.20 ± 0.61 and 3.51 ± 0.08 g L−1, respectively, in 24-hour bioreactor cultivation. Two high molecular weight (Mw) fractions (1.7 ± 0.0 and 0.74 ± 0.0 MDa) were detected in the sample recovered from the cell-free supernatant by dialysis. The compositional analysis revealed the presence of glucose (31.1 ± 0.2 mol%), arabinose (23.9 ± 0.1 mol%), mannose (17.3 ± 0.1 mol%), glucosamine (10.3 ± 0.5 mol%), galactose (8.7 ± 0.0 mol%) and galacturonic acid (8.7 ± 0.0 mol%), as well as a high content in sulphate (6.0 ± 0.5 wt%). Given the presence of a high Mw polysaccharide in the apple pulp waste, probably pectin, a fraction of the detected sugar monomers might be attributed to that polymer, which was recovered together with A. macleodii Mo169 EPS. Concomitant with EPS synthesis, there was a viscosity build-up in the cultivation broth, which developed a shear-thinning fluid behaviour not observed in the initial medium. Therefore, this study demonstrates that apple pulp waste can be efficiently converted into a novel polysaccharide by A. macleodii Mo169 in a sustainable bioprocess. Moreover, the EPS sugar and acyl composition, together with its good thickening capacity, render the biopolymer of interest for use in several applications.publishersversionpublishe

Deacetylation and Desuccinylation of the Fucose-Rich Polysaccharide Fucopol: Impact on Biopolymer Physical and Chemical Properties

FucoPol is an acylated polysaccharide with demonstrated valuable functional properties that include a shear thinning fluid behaviour, a film-forming capacity, and an emulsion forming and stabilizing capacity. In this study, the different conditions (concentration, temperature, and time) for alkaline treatment were investigated to deacylate FucoPol. Complete deacetylation and desuccinylation was achieved with 0.02 M NaOH, at 60 ºC for 15 min, with no significant impact on the biopolymer’s sugar composition, pyruvate content, and molecular mass distribution. FucoPol depyruvylation by acid hydrolysis was attempted, but it resulted in a very low polymer recovery. The effect of the ionic strength, pH, and temperature on the deacetylated/desuccinylated polysaccharide, d-FucoPol, was evaluated, as well as its emulsion and film-forming capacity. d-FucoPol aqueous solutions maintained the shear thinning behaviour characteristic of FucoPol, but the apparent viscosity decreased significantly. Moreover, contrary to FucoPol, whose solutions were not affected by the media’s ionic strength, the d-FucoPol solutions had a significantly higher apparent viscosity for a higher ionic strength. On the other hand, the d-FucoPol solutions were not affected by the pH in the range of 3.6–11.5, while FucoPol had a decreased viscosity for acidic pH values and for a pH above 10.5. Although d-FucoPol displayed an emulsification activity for olive oil similar to that of FucoPol (98 +- 0%) for an oil-to-water ratio of 2:3, the emulsions were less viscous. The d-FucoPol films were flexible, with a higher Young0s modulus (798 +- 152 MPa), a stress at the break (22.5 +- 2.5 MPa), and an elongation at the break (9.3 +- 0.7%) than FucoPol (458 +- 32 MPa, 15.5 +- 0.3 MPa and 8.1 +- 1.0%, respectively). Given these findings, d-FucoPol arises as a promising novel biopolymer, with distinctive properties that may render it useful for utilization as a suspending or emulsifier agent, and as a barrier in coatings and packaging filmsinfo:eu-repo/semantics/publishedVersio

Design, Characterization and Biological Properties

Funding Information: This work was financed by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of projects UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO, the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB, project UIDP/04129/2020 of LEAF—Linking Landscape, Environment, Agriculture and Food—Research Center, and projects LA/P/0037/2020, UIDP/50025/2020 and UIDB/50025/2020 of the Associate Laboratory Institute of Nanostructures, Nanomodelling and Nanofabrication—i3N. Diana Araújo and Catarina Roma-Rodrigues were funded by FCT/MCTES, with grant numbers SFRH/BD/140829/2018 and SFRH/BPD/124612/2016, respectively. Publisher Copyright: © 2023 by the authors.FucoPol, a fucose-rich polyanionic polysaccharide, was used for the first time for the preparation of hydrogel membranes (HMs) using Fe3+ as a crosslinking agent. This study evaluated the impact of Fe3+ and FucoPol concentrations on the HMs’ strength. The results show that, above 1.5 g/L, Fe3+ concentration had a limited influence on the HMs’ strength, and varying the FucoPol concentration had a more significant effect. Three different FucoPol concentrations (1.0, 1.75 and 2.5 wt.%) were combined with Fe3+ (1.5 g/L), resulting in HMs with a water content above 97 wt.% and an Fe3+ content up to 0.16 wt.%. HMs with lower FucoPol content exhibited a denser porous microstructure as the polymer concentration increased. Moreover, the low polymer content HM presented the highest swelling ratio (22.3 ± 1.8 g/g) and a lower hardness value (32.4 ± 5.8 kPa). However, improved mechanical properties (221.9 ± 10.2 kPa) along with a decrease in the swelling ratio (11.9 ± 1.6 g/g) were obtained for HMs with a higher polymer content. Furthermore, all HMs were non-cytotoxic and revealed anti-inflammatory activity. The incorporation of FucoPol as a structuring agent and bioactive ingredient in the development of HMs opens up new possibilities for its use in tissue engineering, drug delivery and wound care management.publishersversionpublishe

Characterisation of Films Based on Exopolysaccharides from Alteromonas Strains Isolated from French Polynesia Marine Environments

LA/P/0140/202019 UID/AGR/04129/2020 LA/P/0037/2020This work assessed the film-forming capacity of exopolysaccharides (EPS) produced by six Alteromonas strains recently isolated from different marine environments in French Polynesia atolls. The films were transparent and resulted in small colour alterations when applied over a coloured surface (ΔEab below 12.6 in the five different colours tested). Moreover, scanning electron microscopy showed that the EPS films were dense and compact, with a smooth surface. High water vapour permeabilities were observed (2.7–6.1 × 10−11 mol m−1 s−1 Pa−1), which are characteristic of hydrophilic polysaccharide films. The films were also characterised in terms of barrier properties to oxygen and carbon dioxide. Interestingly, different behaviours in terms of their mechanical properties under tensile tests were observed: three of the EPS films were ductile with high elongation at break (ε) (35.6–47.0%), low tensile strength at break (Ꞇ) (4.55–11.7 MPa) and low Young’s modulus (εm) (10–93 MPa), whereas the other three were stiffer and more resistant with a higher Ꞇ (16.6–23.6 MPa), lower ε (2.80–5.58%), and higher εm (597–1100 MPa). These properties demonstrate the potential of Alteromonas sp. EPS films to be applied in different areas such as biomedicine, pharmaceuticals, or food packaging.publishersversionpublishe

Characterization and Biotechnological Potential of Extracellular Polysaccharides Synthesized by Alteromonas Strains Isolated from French Polynesia Marine Environments

Marine environments comprise almost three quarters of Earth’s surface, representing the largest ecosystem of our planet. The vast ecological and metabolic diversity found in marine microorganisms suggest that these marine resources have a huge potential as sources of novel commercially appealing biomolecules, such as exopolysaccharides (EPS). Six Alteromonas strains from different marine environments in French Polynesia atolls were selected for EPS extraction. All the EPS were heteropolysaccharides composed of different monomers, including neutral monosaccharides (glucose, galactose, and mannose, rhamnose and fucose), and uronic acids (glucuronic acid and galacturonic acid), which accounted for up to 45.5 mol% of the EPS compositions. Non-carbohydrate substituents, such as acetyl (0.5–2.1 wt%), pyruvyl (0.2–4.9 wt%), succinyl (1–1.8 wt%), and sulfate (1.98–3.43 wt%); and few peptides (1.72–6.77 wt%) were also detected. Thermal analysis demonstrated that the EPS had a degradation temperature above 260 °C, and high char yields (32–53%). Studies on EPS functional properties revealed that they produce viscous aqueous solutions with a shear thinning behavior and could form strong gels in two distinct ways: by the addition of Fe2+, or in the presence of Mg2+, Cu2+, or Ca2+ under alkaline conditions. Thus, these EPS could be versatile materials for different applications

Development of new bioactive materials based on microbial exopolysaccharides of marine origin

The arising of novel bio-based products and processes is vital for the sustainable development of society. The exploitation of marine resources towards the development of new ecological products with commercial interest is a promising solution. In this thesis, marine biodiversity was explored for the development of new and improved biomaterials and/or bioactive compounds based on marine exopolysaccharides (EPS). Marine microorganisms, including both microalgae and bacteria, were prospected for their EPS production capacity. The results obtained in this study showed that marine microalgae have an enormous potential as producers of EPS with unique chemical compositions, including high contents in sulphate and unusual sugars. These characteristics are often associated with biological activities, which supports the potential of microalgal EPS to be employed as bioactive compounds. Indeed, in this work, the EPS produced by microalga Heterocapsa AC210 demonstrated antioxidant and anti-inflammatory properties. Additionally, the EPS produced by bacteria isolated from unusual marine environments showed interesting compositions, including high uronic acids contents, and relevant functional properties. These biopolymers presented thickening, gel and film forming capacities, suggesting that they might be successfully used as structuring biomaterials. This work showed that it was possible to use low-cost feedstocks for the cultivation of the bacterium Alteromonas macleodii Mo169, resulting in higher productivities and distinctive EPS compositions. The EPS produced by this strain also revealed potential in the nanotechnology field, as it might be used for the ecological synthesis of gold, silver, and selenium nanoparticles with wound healing and antioxidant properties. Overall, this work supports that natural EPS bioprospected from marine microorganisms can be used for the development of bio-based products with application in high-value markets, thus, contributing to a sustainable future powered by marine biotechnology.O aparecimento de novos produtos e processos de origem biológica é vital para o desenvolvimento sustentável da sociedade. Uma solução promissora é a exploração dos recursos marinhos para o desenvolvimento de novos produtos ecológicos com interesse comercial. Esta tese explora a biodiversidade marinha com o objetivo de desenvolver novos biomateriais e/ou compostos bioactivos a partir de exopolissacáridos (EPS) marinhos. A capacidade de produzir EPS foi avaliada para vários microrganismos marinhos, incluindo microalgas e bactérias. Os resultados obtidos neste trabalho demonstraram que as microalgas marinhas têm um enorme potencial na produção de EPS com composições químicas únicas, que incluem um elevado conteúdo em sulfato e monossacáridos invulgares. Estas características estão frequentemente associadas a atividades biológicas, reforçando a ideia de que os EPS das microalgas têm potencial para serem utilizados como compostos bioactivos. De facto, neste trabalho, o EPS produzido pela microalga Heterocapsa AC210 demonstrou ter propriedades antioxidantes e anti-inflamatórias. Além disso, os EPS produzidos por bactérias isoladas de ambientes marinhos incomuns apresentaram composições interessantes, incluindo altos teores de ácidos urónicos, e propriedades funcionais relevantes. Estes biopolímeros mostraram ter capacidade espessante, bem como a capacidade de formar géis e filmes, o que sugere que possam ser utilizados como biomateriais estruturais. Este trabalho demonstrou ser possível utilizar substratos de baixo custo no cultivo da bactéria Alteromonas macleodii Mo169, o que resultou em produtividades superiores e EPS com composições distintas. O EPS produzido por esta cultura também revelou ter potencial na área da nanotecnologia, dado que pode ser utilizado na síntese ecológica de nanopartículas de ouro, prata e selénio com propriedades regenerantes e antioxidantes. No geral, este trabalho apoia a convicção de que os EPS naturais obtidos de microrganismos marinhos podem ser utilizados para o desenvolvimento de bioprodutos com aplicações em mercados de elevado valor comercial, contribuindo assim para um futuro sustentável baseado na biotecnologia marinha

Characterization and Biotechnological Potential of Extracellular Polysaccharides Synthesized by <i>Alteromonas</i> Strains Isolated from French Polynesia Marine Environments

Marine environments comprise almost three quarters of Earth’s surface, representing the largest ecosystem of our planet. The vast ecological and metabolic diversity found in marine microorganisms suggest that these marine resources have a huge potential as sources of novel commercially appealing biomolecules, such as exopolysaccharides (EPS). Six Alteromonas strains from different marine environments in French Polynesia atolls were selected for EPS extraction. All the EPS were heteropolysaccharides composed of different monomers, including neutral monosaccharides (glucose, galactose, and mannose, rhamnose and fucose), and uronic acids (glucuronic acid and galacturonic acid), which accounted for up to 45.5 mol% of the EPS compositions. Non-carbohydrate substituents, such as acetyl (0.5–2.1 wt%), pyruvyl (0.2–4.9 wt%), succinyl (1–1.8 wt%), and sulfate (1.98–3.43 wt%); and few peptides (1.72–6.77 wt%) were also detected. Thermal analysis demonstrated that the EPS had a degradation temperature above 260 °C, and high char yields (32–53%). Studies on EPS functional properties revealed that they produce viscous aqueous solutions with a shear thinning behavior and could form strong gels in two distinct ways: by the addition of Fe2+, or in the presence of Mg2+, Cu2+, or Ca2+ under alkaline conditions. Thus, these EPS could be versatile materials for different applications

Sustainable use of agro-industrial wastes as potential feedstocks for exopolysaccharide production by selected Halomonas strains.

peer reviewedLarge quantities of waste biomass are generated annually worldwide by many industries and are vastly underutilized. However, these wastes contain sugars and other dissolved organic matter and therefore can be exploited to produce microbial biopolymers. In this study, four selected Halomonas strains, namely, Halomonas caseinilytica K1, Halomonas elongata K4, Halomonas smyrnensis S3, and Halomonas halophila S4, were investigated for the production of exopolysaccharides (EPS) using low-cost agro-industrial wastes as the sole carbon source: cheese whey, grape pomace, and glycerol. Interestingly, both yield and monosaccharide composition of EPS were affected by the carbon source. Glucose, mannose, galactose, and rhamnose were the predominant monomers, but their relative molar ratio was different. Similarly, the average molecular weight of the synthesized EPS was affected, ranging from 54.5 to 4480 kDa. The highest EPS concentration (446 mg/L) was obtained for H. caseinilytica K1 grown on cheese whey that produced an EPS composed mostly of galactose, rhamnose, glucose, and mannose, with lower contents of galacturonic acid, ribose, and arabinose and with a molecular weight of 54.5 kDa. Henceforth, the ability of Halomonas strains to use cost-effective substrates, especially cheese whey, is a promising approach for the production of EPS with distinct physicochemical properties suitable for various applications