Identification of Marine Biotechnology Value Chains with High Potential in the Northern Mediterranean Region.

Marine (blue) biotechnology is an emerging field enabling the valorization of new products and processes with massive potential for innovation and economic growth. In the Mediterranean region, this innovation potential is not exploited as well as in other European regions due to a lack of a clear identification of the different value chains and the high fragmentation of business innovation initiatives. As a result, several opportunities to create an innovative society are being missed. To address this problem, eight Northern Mediterranean countries (Croatia, France, Greece, Italy, Montenegro, Portugal, Slovenia and Spain) established five national blue biotechnology hubs to identify and address the bottlenecks that prevent the development of marine biotechnology in the region. Following a three-step approach (1. Analysis: setting the scene; 2. Transfer: identification of promising value chains; 3. Capitalization: community creation), we identified the three value chains that are most promising for the Northern Mediterranean region: algae production for added-value compounds, integrated multi-trophic aquaculture (IMTA) and valorization aquaculture/fisheries/processing by-products, unavoidable/unwanted catches and discards. The potential for the development and the technical and non-technical skills that are necessary to advance in this exciting field were identified through several stakeholder events which provided valuable insight and feedback that should be addressed for marine biotechnology in the Northern Mediterranean region to reach its full potential.

Photosynthesis Monitoring in Microalgae Cultures Grown on Municipal Wastewater as a Nutrient Source in Large-Scale Outdoor Bioreactors.

Microalgae cultures were used for a WW treatment to remediate nutrients while producing biomass and recycling water. In these trials, raceway ponds (RWPs; 1 and 0.5 ha) were located next to a municipal (WW) treatment plant in Mérida, Spain. The ponds were used for continuous, all-year-round microalgae production using WW as a source of nutrients. Neither CO2 nor air was supplied to cultures. The objective was to validate photosynthesis monitoring techniques in large-scale bioreactors. Various in-situ/ex-situ methods based on chlorophyll fluorescence and oxygen evolution measurements were used to follow culture performance. Photosynthesis variables gathered with these techniques were compared to the physiological behavior and growth of cultures. Good photosynthetic activity was indicated by the build-up of dissolved oxygen concentration up to 380% saturation, high photochemical yield (Fv/Fm = 0.62-0.71), and relative electron transport rate rETR between 200 and 450 µmol e- m-2 s-1 at midday, which resulted in biomass productivity of about 15-25 g DW m-2 day-1. The variables represent reliable markers reflecting the physiological status of microalgae cultures. Using waste nutrients, the biomass production cost can be significantly decreased for abundant biomass production in large-scale bioreactors, which can be exploited for agricultural purposes.

Centrate as a sustainable growth medium: Impact on microalgal inocula and bacterial communities in tubular photobioreactor cultivation systems.

Centrate is a low-cost alternative to synthetic fertilizers for microalgal cultivation, reducing environmental burdens and remediation costs. Adapted microalgae need to be selected and characterised to maximise biomass production and depuration efficiency. Here, the performance and composition of six microalgal communities cultivated both on synthetic media and centrate within semi-open tubular photobioreactors were investigated through Illumina sequencing. Biomass grown on centrate, exposed to a high concentration of ammonium, showed a higher quantity of nitrogen (5.6% dry weight) than the biomass grown on the synthetic media nitrate (3.9% dry weight). Eukaryotic inocula were replaced by other microalgae while cyanobacterial inocula were maintained. Communities were generally similar for the same inoculum between media, however, inoculation with cyanobacteria led to variability within the eukaryotic community. Where communities differed, centrate resulted in a higher richness and diversity. The higher nitrogen of centrate possibly led to higher abundance of genes coding for N metabolism enzymes.

Extracellular Polymeric Substances Produced by the Thermophilic Cyanobacterium Gloeocapsa gelatinosa: Characterization and Assessment of Their Antioxidant and Metal-Chelating Activities.

Cyanobacteria, particularly thermophilic strains, represent an important potential source of EPSs, harboring structural complexity that predicts diverse and specific bioactive potential. The thermophilic cyanobacteria Gloeocapsa gelatinosa, isolated from a natural hot source in Ain Echfa (Tunisia), was cultivated in a cylindrical reactor, and the production of biomass and EPSs was investigated. Results revealed that the strain is amongst the most efficient EPSs producers (0.89 g L-1) and that EPSs production was not correlated with the growth phase. EPSs were sulfated heteropolysaccharides containing carbohydrates (70%) based on nine different monosaccharides, mainly mannose (22%), and with the presence of two uronic acids. EPSs were formed by two polymers moieties with a molecular weight of 598.3 ± 7.2 and 67.2 ± 4.4 kDa. They are thermostable in temperatures exceeding 100 °C and have an anionic nature (zeta potential of -40 ± 2 mV). Atomic force microscopy showed that EPSs formed multimodal lumps with 88 nm maximum height. EPSs presented high water holding capacity (70.29 ± 2.36%) and solubility index (97.43 ± 1.24%), and a strong bivalent metal sorption capacity especially for Cu2+ (91.20 ± 1.25%) and Fe2+ (75.51 ± 0.71%). The antioxidant activity of G. gelatinosa EPSs was investigated using four methods: the ß-carotene-bleaching activity, DPPH assays, iron-reducing activity, and metal-chelating activity. EPS has shown high potential as free radicals' scavenger, with an IC50 on DPPH (0.2 g L-1) three-fold lower than ascorbic acid (0.6 g L -1) and as a metal chelating activity (IC50 = 0.4 g L-1) significantly lower than EDTA. The obtained results allow further exploration of the thermophilic G. gelatinosa for several biotechnological and industrial applications.

Characterization of Novel Selected Microalgae for Antioxidant Activity and Polyphenols, Amino Acids, and Carbohydrates.

The biochemical composition of three novel selected microalgae strains (Chlorophyta) was evaluated to confirm their potential possibilities as new sustainably produced biomass with nutritional, functional, and/or biomedical properties. Extracts from cultured Pseudopediastrum boryanum, Chloromonas cf. reticulata, and Chloroidium saccharophilum exhibited higher radical scavenging activity of DPPH (1,1-diphenyl-2-picrylhydrazyl) when compared to butylated hydroxytoluene (BHT), but lower than butylated hydroxyanisole (BHA). Total phenolic compounds and amino acids were determined by newly developed RP-HPLC methods. Total phenolic contents, as µg g-1 of dry biomass, reached 27.1 for C. cf. reticulata, 26.4 for P. boryanum, and 55.8 for C. saccharophilum. Percentages of total analysed amino acids were 24.3, 32.1, and 18.5% of dry biomass, respectively, presenting high values for essential amino acids reaching 54.1, 72.6, and 61.2%, respectively. Glutamic acid was the most abundant free amino acid in all microalgae samples, followed by proline and lysine in C. saccharophilum and P. boryanum, and methionine and lysine in C. reticulata. Soluble carbohydrates in aqueous extracts ranged from 39.6 for C. saccharophilum to 49.3% for C. reticulata, increasing values to 45.1 for C. saccharophilum and 52.7% for P. boryanum in acid hydrolysates of dried biomass. Results confirmed the potential possibilities of these microalgae strains.

Cyanobacteria and Red Macroalgae as Potential Sources of Antioxidants and UV Radiation-Absorbing Compounds for Cosmeceutical Applications.

In recent years, research on natural products has gained considerable attention, particularly in the cosmetic industry, which is looking for new bio-active and biodegradable molecules. In this study, cosmetic properties of cyanobacteria and red macroalgae were analyzed. The extractions were conducted in different solvents (water, ethanol and two combinations of water:ethanol). The main molecules with antioxidant and photoprotective capacity were mycosporine-like amino acids (MAAs), scytonemin and phenolic compounds. The highest contents of scytonemin (only present in cyanobacteria) were observed in Scytonema sp. (BEA 1603B) and Lyngbya sp. (BEA 1328B). The highest concentrations of MAAs were found in the red macroalgae Porphyra umbilicalis, Gelidium corneum and Osmundea pinnatifida and in the cyanobacterium Lyngbya sp. Scytonema sp. was the unique species that presented an MAA with maximum absorption in the UV-B band, being identified as mycosporine-glutaminol for the first time in this species. The highest content of polyphenols was observed in Scytonema sp. and P. umbilicalis. Water was the best extraction solvent for MAAs and phenols, whereas scytonemin was better extracted in a less polar solvent such as ethanol:dH2O (4:1). Cyanobacterium extracts presented higher antioxidant activity than those of red macroalgae. Positive correlations of antioxidant activity with different molecules, especially polyphenols, biliproteins and MAAs, were observed. Hydroethanolic extracts of some species incorporated in creams showed an increase in the photoprotection capacity in comparison with the base cream. Extracts of these organisms could be used as natural photoprotectors improving the diversity of sunscreens. The combination of different extracts enriched in scytonemin and MAAs could be useful to design broad-band natural UV-screen cosmeceutical products.

Natural Pigments and Biogas Recovery from Microalgae Grown in Wastewater.

This study assessed the recovery of natural pigments (phycobiliproteins) and bioenergy (biogas) from microalgae grown in wastewater. A consortium of microalgae, mainly composed by Nostoc, Phormidium, and Geitlerinema, known to have high phycobiliproteins content, was grown in photobioreactors. The growth medium was composed by secondary effluent from a high rate algal pond (HRAP) along with the anaerobic digestion centrate, which aimed to enhance the N/P ratio, given the lack of nutrients in the secondary effluent. Additionally, the centrate is still a challenging anaerobic digestion residue since the high nitrogen concentrations have to be removed before disposal. Removal efficiencies up to 52% of COD, 86% of NH4 +-N, and 100% of phosphorus were observed. The biomass composition was monitored over the experimental period in order to ensure stable cyanobacterial dominance in the mixed culture. Phycocyanin and phycoerythrin were extracted from harvested biomass, achieving maximum concentrations of 20.1 and 8.1 mg/g dry weight, respectively. The residual biomass from phycobiliproteins extraction was then used to produce biogas, with final methane yields ranging from 159 to 199 mL CH4/g VS. According to the results, by combining the extraction of pigments and the production of biogas from residual biomass, we would not only obtain high-value compounds, but also more energy (around 5-10% higher), as compared to the single recovery of biogas. The proposed process poses an example of resource recovery from biomass grown in wastewater, moving toward a circular bioeconomy.