Semi-continuous cultivation of EPS-producing marine cyanobacteria: A green biotechnology to remove dissolved metals obtaining metal-organic materials.

Given the necessity for bioprocesses scaling-up, the present study aims to explore the potential of three marine cyanobacteria and a consortium, cultivated in semi-continuous mode, as a green approach for i) continuous exopolysaccharide-rich biomass production and ii) removal of positively charged metals (Cu, Ni, Zn) from mono and multi-metallic solutions. To ensure the effectiveness of both cellular and released exopolysaccharides, weekly harvested whole cultures were confined in dialysis tubings. The results revealed that all the tested cyanobacteria have a stronger affinity towards Cu in mono and three-metal systems. Despite the amount of metals removed per gram of biomass decreased with higher biosorbent dosage, the more soluble carbohydrates were produced, the greater was the metal uptake, underscoring the pivotal role of released exopolysaccharides in metal biosorption. According to this, Dactylococcopsis salina 16Som2 showed the highest carbohydrate productivity (142 mg L-1 d-1) and metal uptake (84 mg Cu g-1 biomass) representing a promising candidate for further studies. The semi-continuous cultivation of marine cyanobacteria here reported assures a schedulable production of exopolysaccharide-rich biosorbents with high metal removal and recovery potential, even from multi-metallic solutions, as a step forward in the industrial application of cyanobacteria.

Microbial Polysaccharides Extracted from Different Mature Muds of the Euganean Thermal District Show Similar Anti-Inflammatory Activity In Vivo.

The Euganean Thermal District, situated in North-East Italy, is one of Europe's largest and oldest thermal centres. The topical application of its therapeutic thermal muds is recognised by the Italian Health System as a beneficial treatment for patients suffering from arthro-rheumatic diseases. Polysaccharides produced by the mud microbiota have been recently identified as anti-inflammatory bioactive molecules. In this paper we analysed the efficacy of Microbial-Polysaccharides (M-PS) derived from mature muds obtained at different maturation temperatures, both within and outside the codified traditional mud maturation range. M-PSs were extracted from six mature muds produced by five spas of the Euganean Thermal District and investigated for their chemical properties, monosaccharide composition and in vivo anti-inflammatory potential, using the zebrafish model organism. Additionally, mature muds were characterized for their microbiota composition using Next-Generation Sequencing. The results showed that all M-PSs exhibit similar anti-inflammatory potential, referable to their comparable chemical composition. This consistency was observed despite changes in cyanobacteria populations, suggesting a possible role of the entire microbial community in shaping the properties of these biomolecules. These findings highlight the importance of scientific research in untangling the origins of the therapeutic efficacy of Euganean Thermal muds in the treatment of chronic inflammatory conditions.

A sulfated exopolysaccharide derived from Chlorella sp. exhibiting in vitro anti-α-D-Glucosidase activity.

There is a great scientific curiosity to discover all environments sheltering microalgae, especially those with exceptional characteristics from coldest to hottest ones, the purpose remains to explore the potential of the native microalgae flora and the research for new bioactive compounds. This study aimed to isolate a polysaccharide-producing microalga from an extreme ecosystem and to evaluate its capacity to inhibit the α-D-glucosidase enzyme. Chlorella strain is isolated from hypersaline Lake in the Algerian desert. The exopolysaccharide extraction was performed by the concentration of free-cell supernatant in a rotary evaporator. The infrared analysis showed a characteristic footprint of carbohydrates with particular functional groups, such as sulfate. Gas chromatography-mass spectrometry has revealed a hetero-exopolysaccharide composed of galactose 35.75%, glucose 21.13%, xylose 16.81%, fructose 6.96%, arabinose 5.10%, and glucuronic acid 2.68%. The evaluation of the anti-hyperglycemic activity demonstrated a significant α-D-glucosidase inhibition of 80.94 ± 0.01% at 10 mg mL-1 with IC50 equal to 4.31 ± 0.20 mg mL-1. This study opens a vast prospect to use exopolysaccharides as natural nutraceutical or food additive.

Shaping the phycosphere: Analysis of the EPS in diatom-bacterial co-cultures.

The phycosphere is a unique niche that fosters complex interactions between microalgae and associated bacteria. The formation of this extracellular environment, and the associated bacterial biodiversity, is heavily influenced by the secretion of extracellular polymers, primarily driven by phototrophic organisms. The exopolysaccharides (EPS) represent the largest fraction of the microalgae-derived exudates, which can be specifically used by heterotrophic bacteria as substrates for metabolic processes. Furthermore, it has been proposed that bacteria and their extracellular factors play a role in both the release and composition of the EPS. In this study, two model microorganisms, the diatom Phaeodactylum tricornutum CCAP 1055/15 and the bacterium Pseudoalteromonas haloplanktis TAC125, were co-cultured in a dual system to assess how their interactions modify the phycosphere chemical composition by analyzing the EPS monosaccharide profile released in the culture media by the two partners. We demonstrate that microalgal-bacterial interactions in this simplified model significantly influenced the architecture of their extracellular environment. We observed that the composition of the exo-environment, as described by the EPS monosaccharide profiles, varied under different culture conditions and times of incubation. This study reports an initial characterization of the molecular modifications occurring in the extracellular environment surrounding two relevant representatives of marine systems.

Cyanoremediation and phyconanotechnology: cyanobacteria for metal biosorption toward a circular economy.

Cyanobacteria are widespread phototrophic microorganisms that represent a promising biotechnological tool to satisfy current sustainability and circularity requirements. They are potential bio-factories of a wide range of compounds that can be exploited in several fields including bioremediation and nanotechnology sectors. This article aims to illustrate the most recent trends in the use of cyanobacteria for the bioremoval (i.e., cyanoremediation) of heavy metals and metal recovery and reuse. Heavy metal biosorption by cyanobacteria can be combined with the consecutive valorization of the obtained metal-organic materials to get added-value compounds, including metal nanoparticles, opening the field of phyconanotechnology. It is thus possible that the use of combined approaches could increase the environmental and economic feasibility of cyanobacteria-based processes, promoting the transition toward a circular economy.

Nanoformulations with exopolysaccharides from cyanobacteria: enhancing the efficacy of bioactive molecules in the Mediterranean fruit fly control.

The increasing demand for food has required intensive use of pesticides which are hazardous to the ecosystem. A valid alternative is represented by biopesticides; however, these molecules are often insoluble in water, and poorly bioavailable. Nanopesticides can be engineered to reach a selected target with controlled release of the active principle. In this work, capsaicin, an irritant alkaloid from hot chili peppers, and hydroxytyrosol, a phenolic compound obtained from extra-virgin olive oil by-products, were loaded into innovative nanocarriers. These were designed ad hoc combining exopolysaccharides from the cyanobacteria Neocyanospira capsulata, and a lipid component, i.e., egg phosphatidylcholine. The polysaccharide was chosen for chemical affinity with the chitin of insect exoskeleton, while the lipids were introduced to modulate the carrier rigidity. The newly formed nanosystems were characterized by physico-chemical techniques and tested for their possible use in pest control programs. The Mediterranean Fruit Fly Ceratitis capitata Wiedemann, 1824 (Diptera, Tephriditae), a pest of the Mediterranean Region causing high economic losses, was used as a model insect. We found that the nanoformulations nanocarriers prepared in this work, were able to increase the ovicidal effect of hydroxytyrosol. Moreover, the formulation encapsulating either hydroxytyrosol or capsaicin were able to reduce the number of females landing on treated apricots.

Phormidium ambiguum and Leptolyngbya ohadii Exopolysaccharides under Low Water Availability.

Cyanobacteria can cope with various environmental stressors, due to the excretion of exopolysaccharides (EPS). However, little is known about how the composition of these polymers may change according to water availability. This work aimed at characterizing the EPS of Phormidium ambiguum (Oscillatoriales; Oscillatoriaceae) and Leptolyngbya ohadii (Pseudanabaenales; Leptolyngbyaceae), when grown as biocrusts and biofilms, subject to water deprivation. The following EPS fractions were quantified and characterized: soluble (loosely bound, LB) and condensed (tightly bound, TB) for biocrusts, released (RPS), and sheathed in P. ambiguum and glycocalyx (G-EPS) in L. ohadii for biofilms. For both cyanobacteria upon water deprivation, glucose was the main monosaccharide present and the amount of TB-EPS resulted was significantly higher, confirming its importance in these soil-based formations. Different profiles of monosaccharides composing the EPSs were observed, as for example the higher concentration of deoxysugars observed in biocrusts compared to biofilms, demonstrating the plasticity of the cells to modify EPS composition as a response to different stresses. For both cyanobacteria, both in biofilms and biocrusts, water deprivation induced the production of simpler carbohydrates, with an increased dominance index of the composing monosaccharides. The results obtained are useful in understanding how these very relevant cyanobacterial species are sensitively modifying the EPS secreted when subject to water deprivation and could lead to consider them as suitable inoculants in degraded soils.

Assessing the Antitumor Potential of Variants of the Extracellular Carbohydrate Polymer from Synechocystis ΔsigF Mutant.

Cancer is a leading cause of death worldwide with a huge societal and economic impact. Clinically effective and less expensive anticancer agents derived from natural sources can help to overcome limitations and negative side effects of chemotherapy and radiotherapy. Previously, we showed that the extracellular carbohydrate polymer of a Synechocystis ΔsigF overproducing mutant displayed a strong antitumor activity towards several human tumor cell lines, by inducing high levels of apoptosis through p53 and caspase-3 activation. Here, the ΔsigF polymer was manipulated to obtain variants that were tested in a human melanoma (Mewo) cell line. Our results demonstrated that high molecular mass fractions were important for the polymer bioactivity, and that the reduction of the peptide content generated a variant with enhanced in vitro antitumor activity. This variant, and the original ΔsigF polymer, were further tested in vivo using the chick chorioallantoic membrane (CAM) assay. Both polymers significantly decreased xenografted CAM tumor growth and affected tumor morphology, by promoting less compact tumors, validating their antitumor potential in vivo. This work contributes with strategies for the design and testing tailored cyanobacterial extracellular polymers and further strengths the relevance of evaluating this type of polymers for biotechnological/biomedical applications.

Pomegranate peel as a promising source of pectic polysaccharides: A multi-methodological analytical investigation.

Polysaccharides from pomegranate peel (Wonderful and Purple Queen® varieties) were extracted by hot water and fractionated using ethanol. Three fractions (F1-F2-F3) were obtained for each sample. Polysaccharides' yield was higher for Purple Queen®: 13% dw. Polysaccharides of the three fractions were characterized by size exclusion chromatography (SEC), dynamic light scattering (DLS), 1H NMR, methylation, and acylation degree. Differently from SEC, DLS highlighted some differences between the polysaccharides's molecular sizes of the fractions. The highest methylation and acylation degree was observed for F3 of Purple Queen®: 74.0% and 18.6%, respectively. The percentage of galacturonic acid confirmed the presence of pectin in almost all the fractions recognized as homogalacturonan. Arabinan and arabinogalactan were also found in all the collected F3 samples, although in different proportions. The stepwise fractionation process followed by a multi-methodological analytical investigation was helpful to improve the knowledge of the pectic polysaccharides of pomegranate.

Microalgae-Based Biorefineries: Challenges and Future Trends to Produce Carbohydrate Enriched Biomass, High-Added Value Products and Bioactive Compounds.

Microalgae have demonstrated a large potential in biotechnology as a source of various macromolecules (proteins, carbohydrates, and lipids) and high-added value products (pigments, poly-unsaturated fatty acids, peptides, exo-polysaccharides, etc.). The production of biomass at a large scale becomes more economically feasible when it is part of a biorefinery designed within the circular economy concept. Thus, the aim of this critical review is to highlight and discuss challenges and future trends related to the multi-product microalgae-based biorefineries, including both phototrophic and mixotrophic cultures treating wastewater and the recovery of biomass as a source of valuable macromolecules and high-added and low-value products (biofertilizers and biostimulants). The therapeutic properties of some microalgae-bioactive compounds are also discussed. Novel trends such as the screening of species for antimicrobial compounds, the production of bioplastics using wastewater, the circular economy strategy, and the need for more Life Cycle Assessment studies (LCA) are suggested as some of the future research lines.

Phenolic compounds and polysaccharides in the date fruit (Phoenix dactylifera L.): Comparative study on five widely consumed Arabian varieties.

The study analysed polysaccharides and phenolic compounds in widely consumed but little studied date fruits varieties such as Sukkari, Ajwa, Segae, Barrny and Khalas harvested at Tamr stage. The total phenols were in similar amount in the five varieties and ranged from 20 to 50 mg/100 g DW. The decoction and successive centrifugation made it possible to collect two main polysaccharide fractions for all the selected fruits. For each variety the first fraction was more abundant, with a lower swelling capacity and a higher amount of galacturonic acid (28.3% to 40.1%). The second fraction was only soluble in alkaline solution, with an average galacturonic acid content of only 17%. The different structure of the two polysaccharide fractions was also confirmed by the composition in neutral sugars and the degrees of methylation and acetylation. The proposed extraction procedure could be applied for larger scale extraction of the date fruit polysaccharides.

Novel strategies towards efficient molecular biohydrogen production by dark fermentative mechanism: present progress and future perspective.

In the scenario of alarming increase in greenhouse and toxic gas emissions from the burning of conventional fuels, it is high time that the population drifts towards alternative fuel usage to obviate pollution. Hydrogen is an environment-friendly biofuel with high energy content. Several production methods exist to produce hydrogen, but the least energy intensive processes are the fermentative biohydrogen techniques. Dark fermentative biohydrogen production (DFBHP) is a value-added, less energy-consuming process to generate biohydrogen. In this process, biohydrogen can be produced from sugars as well as complex substrates that are generally considered as organic waste. Yet, the process is constrained by many factors such as low hydrogen yield, incomplete conversion of substrates, accumulation of volatile fatty acids which lead to the drop of the system pH resulting in hindered growth and hydrogen production by the bacteria. To circumvent these drawbacks, researchers have come up with several strategies that improve the yield of DFBHP process. These strategies can be classified as preliminary methodologies concerned with the process optimization and the latter that deals with pretreatment of substrate and seed sludge, bioaugmentation, co-culture of bacteria, supplementation of additives, bioreactor design considerations, metabolic engineering, nanotechnology, immobilization of bacteria, etc. This review sums up some of the improvement techniques that profoundly enhance the biohydrogen productivity in a DFBHP process.

Towards a mechanistic understanding of microalgae-bacteria interactions: integration of metabolomic analysis and computational models.

Interactions amongst marine microalgae and heterotrophic bacteria drive processes underlying major biogeochemical cycles and are important for many artificial systems. These dynamic and complex interactions span the range from cooperative to competitive, and it is the diverse and intricate networks of metabolites and chemical mediators that are predicted to principally dictate the nature of the relationship at any point in time. Recent advances in technologies to identify, analyze, and quantify metabolites have allowed for a comprehensive view of the molecules available for exchange and/or reflective of organismal interactions, setting the stage for development of mechanistic understanding of these systems. Here, we (i) review the current knowledge landscape of microalgal-bacterial interactions by focusing on metabolomic studies of selected, simplified model systems; (ii) describe the state of the field of metabolomics, with specific focus on techniques and approaches developed for microalga-bacterial interaction studies; and (iii) outline the main approaches for development of mathematical models of these interacting systems, which collectively have the power to enhance interpretation of experimental data and generate novel testable hypotheses. We share the viewpoint that a comprehensive and integrated series of -omics approaches that include theoretical formulations are necessary to develop predictive and mechanistic understanding of these biological entities.

In vivo anti-inflammatory and antioxidant effects of microbial polysaccharides extracted from Euganean therapeutic muds.

Therapeutic thermal mud produced by spas of the Euganean Thermal District (Italy) is used as a treatment for arthro-rheumatic diseases. Its production involves the growth of a specific microbiota embedded in a polysaccharidic matrix. Polysaccharides (Microbial-PolySaccharides, M-PS) released in the mud by the resident microorganisms were extracted and analyzed. The monosaccharidic composition analysis showed the presence of galacturonic acid, mannose, xylose, ribose and glucose and a high percentage of sulfated groups in the polymers. To assess their involvement in the therapeutic efficacy of the mud, the M-PS were tested using the model organism zebrafish (Danio rerio). The anti-inflammatory and antioxidant activities were evaluated after confirming the lack of toxic effects during development. Inflammatory state was induced chemically with copper sulfate, or through tail fin amputation procedure and UVB exposure. Recovery from inflammatory condition after exposure to M-PS was always observed with specific morphometric analyses, and further supported by qPCR. Genes linked with the inflammatory and oxidative stress response were investigated confirming the M-PS treatment's efficacy.

Cyanobacterial persistence and influence on microbial community dynamics over 15 years in induced biocrusts.

Biocrusts provide numerous ecological functions in drylands. Recovering biocrusts via cyanobacterial inoculation recently gathered interest for ecological restoration, yet it still lacks long-term experiments to unravel biocrust community dynamics. To examine how cyanobacterial inoculants influenced local microbial community and biocrust development, we observed a 2 km2 (Qubqi Desert, China) inoculation experiment after 10 and 15 years, following biocrust formation. Our results revealed that biocrust development was in line with ecological regime shift, providing evidence for biocrust community succession, from cyanobacteria- to moss-dominated types. Associated with biocrust development, microbial communities differed significantly with less specialists compared to shifting sands. Cyanobacterial community analysis showed that Microcoleus vaginatus and Scytonema javanicum are an ideal inoculating model, as they were still dominating the community after 15 years since inoculation, while other nitrogen-fixing cyanobacteria occurred profusely with biocrust development. Biocrust community composition combined with thickness, Chl-a and exopolysaccharide measurements revealed the large variation of cyanobacterial ecological functions along biocrust development, suggesting a main function shift: from carbon fixation associated with exopolysaccharide secretion in bare sandy soils to nitrogen fixation in developed biocrusts. This large-scale field study verifies that cyanobacterial inoculation accelerates biocrust development and forwards succession, shaping the biocrust community composition over a long time.

Scaling down the microbial loop: data-driven modelling of growth interactions in a diatom-bacterium co-culture.

An intricate set of interactions characterizes marine ecosystems. One of the most important is represented by the microbial loop, which includes the exchange of dissolved organic matter (DOM) from phototrophic organisms to heterotrophic bacteria. Here, it can be used as the major carbon and energy source. This interaction is one of the foundations of the entire ocean food-web. The carbon fixed by phytoplankton can be redirected to bacteria in two main ways; either (i) bacteria feed on dead phytoplankton cells or (ii) DOM is actively released by phytoplankton (a process resulting in up to 50% of the fixed carbon leaving the cell). Here, we have set up a co-culture of the diatom Phaeodactylum tricornutum and the chemoheterotrophic bacterium Pseudoalteromonas haloplanktis TAC125 and used this system to study the interactions between these two representatives of the microbial loop. We show that the bacterium can thrive on diatom-derived carbon and that this growth can be sustained by both diatom dead cells and diatom-released compounds. These observations were formalized in a network of putative interactions between P. tricornutum and P. haloplanktis and implemented in a model that reproduces the observed co-culture dynamics, revealing an overall accuracy of our hypotheses in explaining the experimental data.

Exopolysaccharide Features Influence Growth Success in Biocrust-forming Cyanobacteria, Moving From Liquid Culture to Sand Microcosms.

Land degradation in drylands is a drawback of the combined action of climate change and human activities. New techniques have been developed to induce artificial biocrusts formation as a tool for restoration of degraded drylands, and among them soils inoculation with cyanobacteria adapted to environmental stress. Improvement of soil properties by cyanobacteria inoculation is largely related to their ability to synthesize exopolysaccharides (EPS). However, cyanobacterial EPS features [amount, molecular weight (MW), composition] can change from one species to another or when grown in different conditions. We investigated the differences in growth and polysaccharidic matrix features among three common biocrust-forming cyanobacteria (Nostoc commune, Scytonema javanicum, and Phormidium ambiguum), when grown in liquid media and on sandy soil microcosms under optimal nutrient and water, in controlled laboratory conditions. We extracted and analyzed the released EPS (RPS) and sheath for the liquid cultures, and the more soluble or loosely-bound (LB) and the more condensed or tightly-bound (TB) soil EPS fractions for the sandy soil microcosms. In liquid culture, P. ambiguum showed the greatest growth and EPS release. In contrast, on the sandy soil, S. javanicum showed the highest growth and highest LB-EPS content. N. commune showed no relevant growth after its inoculation of the sandy soil. A difference was observed in terms of MW distribution, showing that the higher MW of the polymers produced by P. ambiguum and S. javanicum compared to the polymers produced by N. commune, could have had a positive effect on growth for the first two organisms when inoculated on the sandy soil. We also observed how both RPS and sheath fractions reflected in the composition of the soil TB-EPS fraction, indicating the role in soil stabilization of both the released and the cell attached EPS. Our results indicate that the features of the polysaccharidic matrix produced by different cyanobacteria can influence their growth success in soil. These results are of great relevance when selecting suitable candidates for large-scale cyanobacteria applications in soil restoration.

Protection of Wine from Protein Haze Using Schizosaccharomyces japonicus Polysaccharides.

Nowadays commercial preparations of yeast polysaccharides (PSs), in particular mannoproteins, are widely used for wine colloidal and tartrate salt stabilization. In this context, the industry has developed different processes for the isolation and purification of PSs from the cell wall of Saccharomyces cerevisiae. This yeast releases limited amounts of mannoproteins in the growth medium, thus making their direct isolation from the culture broth not economically feasible. On the contrary, Schizosaccharomyces japonicus, a non-Saccharomyces yeast isolated from wine, releases significant amounts of PSs during the alcoholic fermentation. In the present work, PSs released by Sch. japonicus were recovered from the growth medium by ultrafiltration and their impact on the wine colloidal stability was evaluated. Interestingly, these PSs contribute positively to the wine protein stability. The visible haziness of the heat-treated wine decreases as the concentration of added PSs increases. SDS-PAGE Gel electrophoresis results of the haze and of the supernatant after the heat stability test are consistent with the turbidity measurements. Moreover, particle size distributions of the heat-treated wines, as obtained by Dynamic Light Scattering (DLS), show a reduction in the average dimension of the protein aggregates as the concentration of added PSs increases.

Anti-Inflammatory Activity of Exopolysaccharides from Phormidium sp. ETS05, the Most Abundant Cyanobacterium of the Therapeutic Euganean Thermal Muds, Using the Zebrafish Model.

The Euganean Thermal District (Italy) represents the oldest and largest thermal center in Europe, and its therapeutic mud is considered a unique product whose beneficial effects have been documented since Ancient Roman times. Mud properties depend on the heat and electrolytes of the thermal water, as well as on the bioactive molecules produced by its biotic component, mainly represented by cyanobacteria. The investigation of the healing effects of compounds produced by the Euganean cyanobacteria represents an important goal for scientific validation of Euganean mud therapies and for the discovering of new health beneficial biomolecules. In this work, we evaluated the therapeutic potential of exopolysaccharides (EPS) produced by Phormidium sp. ETS05, the most abundant cyanobacterium of the Euganean mud. Specifically, Phormidium EPS resulted in exerting anti-inflammatory and pro-resolution activities in chemical and injury-induced zebrafish inflammation models as demonstrated using specific transgenic zebrafish lines and morphometric and expression analyses. Moreover, in vivo and in vitro tests showed no toxicity at all for the EPS concentrations tested. The results suggest that these EPS, with their combined anti-inflammatory and pro-resolution activities, could be one of the most important therapeutic molecules present in the Euganean mud and confirm the potential of these treatments for chronic inflammatory disease recovery.