Aquatic Biomaterial Repositories: Comprehensive Guidelines, Recommendations, and Best Practices for Their Development, Establishment, and Sustainable Operation.

The alarming pace of species extinction severely threatens terrestrial and aquatic ecosystems, undermining the crucial ecological services vital for environmental sustainability and human well-being. Anthropogenic activities, such as urbanization, agriculture, industrialization, and those inducing climate change, intensify these risks, further imperiling biodiversity. Of particular importance are aquatic organisms, pivotal in biodiscovery and biotechnology. They contribute significantly to natural product chemistry, drug development, and various biotechnological applications. To safeguard these invaluable resources, establishing and maintaining aquatic biomaterial repositories (ABRs) is imperative. This review explores the complex landscape of ABRs, emphasizing the need for standardized procedures from collection to distribution. It identifies key legislative and regulatory frameworks, such as the Nagoya Protocol and EU directives, essential for ensuring responsible and equitable biorepository operations. Drawing on extensive literature and database searches, this study compiles existing recommendations and practices into a cohesive framework with which to guide the establishment and sustainable management of ABRs. Through collaborative efforts and adherence to best practices, ABRs can play a transformative role in the future of marine biotechnology and environmental conservation.

Extraction and Purification of Biopolymers from Marine Origin Sources Envisaging Their Use for Biotechnological Applications.

Biopolymers are a versatile and diverse class of materials that has won high interest due to their potential application in several sectors of the economy, such as cosmetics, medical materials/devices, and food additives. In the last years, the search for these compounds has explored a wider range of marine organisms that have proven to be a great alternative to mammal sources for these applications and benefit from their biological properties, such as low antigenicity, biocompatibility, and biodegradability, among others. Furthermore, to ensure the sustainable exploitation of natural marine resources and address the challenges of 3R's policies, there is a current necessity to valorize the residues and by-products obtained from food processing to benefit both economic and environmental interests. Many extraction methodologies have received significant attention for the obtention of diverse polysaccharides, proteins, and glycosaminoglycans to accomplish the increasing demands for these products. The present review gives emphasis to the ones that can be obtained from marine biological resources, as agar/agarose, alginate and sulfated polysaccharides from seaweeds, chitin/chitosan from crustaceans from crustaceans, collagen, and some glycosaminoglycans such as chondroitin sulfate and hyaluronic acids from fish. It is offered, in a summarized and easy-to-interpret arrangement, the most well-established extraction and purification methodologies used for obtaining the referred marine biopolymers, their chemical structure, as well as the characterization tools that are required to validate the extracted material and respective features. As supplementary material, a practical guide with the step-by-step isolation protocol, together with the various materials, reagents, and equipment, needed for each extraction is also delivered is also delivered. Finally, some remarks are made on the needs still observed, despite all the past efforts, to improve the current extraction and purification procedures to achieve more efficient and green methodologies with higher yields, less time-consuming, and decreased batch-to-batch variability.

Novel metabolite madeirone and neomarinone extracted from Streptomyces aculeoletus as marine antibiofilm and antifouling agents.

Introduction: Biofouling poses a significant economic threat to various marine industries, leading to financial losses that can reach billions of euros annually. This study highlights the urgent need for effective alternatives to traditional antifouling agents, particularly following the global ban on organotin compounds. Material and methods: Streptomyces aculeolatus PTM-346 was isolated from sediment samples on the shores of the Madeira Archipelago, Portugal. The crude extract was fractionated using silica flash chromatography and preparative HPLC, resulting in two isolated marinone compounds: madeirone (1), a novel marinone derivative discovered in this study, and neomarinone (2). The antifouling activities of these compounds were tested against five marine bacterial species and the larvae of the mussel Mytilus galloprovincialis. Additionally, in silico and in vivo environmental toxicity evaluations of madeirone (1) and neomarinone (2) were conducted. Results: Madeirone (1) demonstrated significant antibiofilm efficacy, inhibiting Phaeobacter inhibens by up to 66%, Marinobacter hydrocarbonoclasticus by up to 60%, and Cobetia marina by up to 40%. Neomarinone (2) also exhibited substantial antibiofilm activity, with inhibition rates of up to 41% against P. inhibens, 40% against Pseudo-oceanicola batsensis, 56% against M. hydrocarbonoclasticus, 46% against C. marina, and 40% against Micrococcus luteus. The growth inhibition activity at the same concentrations of these compounds remained below 20% for the respective bacteria, highlighting their effectiveness as potent antibiofilm agents without significantly affecting bacterial viability. Additionally, both compounds showed potent effects against the settlement of Mytilus galloprovincialis larvae, with EC50 values of 1.76 µg/mL and 0.12 µg/mL for compounds (1) and (2), respectively, without impairing the viability of the targeted macrofouling species. In silico toxicity predictions and in vivo toxicity assays both support their potential for further development as antifouling agents. Conclusion: The newly discovered metabolite madeirone (1) and neomarinone (2) effectively inhibit both micro- and macrofouling. This distinct capability sets them apart from existing commercial antifouling agents and positions them as promising candidates for biofouling prevention. Consequently, these compounds represent a viable and environmentally friendly alternative for incorporation into paints, primers, varnishes, and sealants, offering significant advantages over traditional copper-based compounds.

Iron(III) cross-linked hydrogels based on Alteromonas macleodii Mo 169 exopolysaccharide.

Recently, polysaccharide-based hydrogels crosslinked with the trivalent iron cation have attracted interest due to their remarkable properties that include high mechanical stability, stimuli-responsiveness, and enhanced absorptivity. In this study, a Fe3+ crosslinked hydrogel was prepared using the biocompatible extracellular polysaccharide (EPS) secreted by the marine bacterium Alteromonas macleodii Mo169. Hydrogels with mechanical strengths (G') ranging from 0.3 kPa to 44.5 kPa were obtained as a result of the combination of different Fe3+ (0.05-9.95 g L-1) and EPS (0.3-1.7 %) concentrations. All the hydrogels had a water content above 98 %. Three different hydrogels, named HA, HB, and HC, were chosen for further characterization. With strength values (G') of 3.2, 28.9, and 44.5 kPa, respectively, these hydrogels might meet the strength requirements for several specific applications. Their mechanical resistance increased as higher Fe3+ and polymer concentrations were used in their preparation (the compressive hardness increased from 8.7 to 192.1 kPa for hydrogel HA and HC, respectively). In addition, a tighter mesh was noticed for HC, which was correlated to its lower swelling ratio value compared to HA and HB. Overall, this preliminary study highlighted the potential of these hydrogels for tissue engineering, drug delivery, or wound healing applications.

Biocatalytic Conversion of Carrageenans for the Production of 3,6-Anhydro-D-galactose.

Marine biomass stands out as a sustainable resource for generating value-added chemicals. In particular, anhydrosugars derived from carrageenans exhibit a variety of biological functions, rendering them highly promising for utilization and cascading in food, cosmetic, and biotechnological applications. However, the limitation of available sulfatases to break down the complex sulfation patterns of carrageenans poses a significant limitation for the sustainable production of valuable bioproducts from red algae. In this study, we screened several carrageenolytic polysaccharide utilization loci for novel sulfatase activities to assist the efficient conversion of a variety of sulfated galactans into the target product 3,6-anhydro-D-galactose. Inspired by the carrageenolytic pathways in marine heterotrophic bacteria, we systematically combined these novel sulfatases with other carrageenolytic enzymes, facilitating the development of the first enzymatic one-pot biotransformation of ι- and κ-carrageenan to 3,6-anhdyro-D-galactose. We further showed the applicability of this enzymatic bioconversion to a broad series of hybrid carrageenans, rendering this process a promising and sustainable approach for the production of value-added biomolecules from red-algal feedstocks.

Bioprospection of the Antarctic Diatoms Craspedostauros ineffabilis IMA082A and Craspedostauros zucchelli IMA088A.

In extreme environments such as Antarctica, a diverse range of organisms, including diatoms, serve as essential reservoirs of distinctive bioactive compounds with significant implications in pharmaceutical, cosmeceutical, nutraceutical, and biotechnological fields. This is the case of the new species Craspedostauros ineffabilis IMA082A and Craspedostauros zucchellii IMA088A Trentin, Moschin, Lopes, Custódio and Moro (Bacillariophyta) that are here explored for the first time for possible biotechnological applications. For this purpose, a bioprospection approach was applied by preparing organic extracts (acetone and methanol) from freeze-dried biomass followed by the evaluation of their in vitro antioxidant properties and inhibitory activities on enzymes related with Alzheimer's disease (acetylcholinesterase: AChE, butyrylcholinesterase: BChE), Type 2 diabetes mellitus (T2DM, α-glucosidase, α-amylase), obesity (lipase) and hyperpigmentation (tyrosinase). Extracts were then profiled by ultra-high-performance liquid chromatography-mass spectrometry (UPLC-HR-MS/MS), while the fatty acid methyl ester (FAME) profiles were established by gas chromatography-mass spectrometry (GC-MS). Our results highlighted strong copper chelating activity of the acetone extract from C. ineffabilis and moderate to high inhibitory activities on AChE, BChE, α-amylase and lipase for extracts from both species. The results of the chemical analysis indicated polyunsaturated fatty acids (PUFA) and their derivatives as the possible compounds responsible for the observed activities. The FAME profile showed saturated fatty acids (SFA) as the main group and methyl palmitoleate (C16:1) as the predominant FAME in both species. Overall, our results suggest both Antarctic strains as potential sources of interesting molecules with industrial applications. Further studies aiming to investigate unidentified metabolites and to maximize growth yield and natural compound production are required.

Proteomic analysis of the mucus of the photosynthetic sea slug Elysia crispata.

Elysia crispata is a tropical sea slug that can retain intracellular functional chloroplasts from its algae prey, a mechanism termed kleptoplasty. This sea slug, like other gastropods, secretes mucus, a viscous secretion with multiple functions, including lubrication, protection, and locomotion. This study presents the first comprehensive analysis of the mucus proteome of the sea slug E. crispata using gel electrophoresis and HPLC-MS/MS. We identified 306 proteins in the mucus secretions of this animal, despite the limited entries for E. crispata in the Uniprot database. The functional annotation of the mucus proteome using Gene Ontology identified proteins involved in different functions such as hydrolase activity (molecular function), carbohydrate-derived metabolic processes (biological processes) and cytoskeletal organization (cell component). Moreover, a high proportion of proteins with enzymatic activity in the mucus of E. crispata suggests potential biotechnological applications including antimicrobial and antitumor activities. Putative antimicrobial properties are reinforced by the high abundance of hydrolases. This study also identified proteins common in mucus samples from various species, supporting a common mechanism of mucus in protecting cells and tissues while facilitating animal movement. SIGNIFICANCE: Marine species are increasingly drawing the interest of researchers for their role in discovering new bioactive compounds. The study "Proteomic Analysis of the Mucus of the Photosynthetic Sea Slug Elysia crispata" is a pioneering effort that uncovers the complex protein content in this fascinating sea slug's mucus. This detailed proteomic study has revealed proteins with potential use in biotechnology, particularly for antimicrobial and antitumor purposes. This research is a first step in exploring the possibilities within the mucus of Elysia crispata, suggesting the potential for new drug discoveries. These findings could be crucial in developing treatments for severe diseases, especially those caused by multidrug-resistant bacteria, and may lead to significant advances in medical research.

A guide to the use of bioassays in exploration of natural resources.

Bioassays are the main tool to decipher bioactivities from natural resources thus their selection and quality are critical for optimal bioprospecting. They are used both in the early stages of compounds isolation/purification/identification, and in later stages to evaluate their safety and efficacy. In this review, we provide a comprehensive overview of the most common bioassays used in the discovery and development of new bioactive compounds with a focus on marine bioresources. We present a comprehensive list of practical considerations for selecting appropriate bioassays and discuss in detail the bioassays typically used to explore antimicrobial, antibiofilm, cytotoxic, antiviral, antioxidant, and anti-ageing potential. The concept of quality control and bioassay validation are introduced, followed by safety considerations, which are critical to advancing bioactive compounds to a higher stage of development. We conclude by providing an application-oriented view focused on the development of pharmaceuticals, food supplements, and cosmetics, the industrial pipelines where currently known marine natural products hold most potential. We highlight the importance of gaining reliable bioassay results, as these serve as a starting point for application-based development and further testing, as well as for consideration by regulatory authorities.

A Novel Nanoporous Adsorbent for Pesticides Obtained from Biogenic Calcium Carbonate Derived from Waste Crab Shells.

A novel nanoporous adsorbent was obtained through the thermal treatment and chemical wash of the wasted crab shells (BC1) and characterized by various techniques. The structure of BC1 at the end of the treatments comprised a mixture of calcite and amorphous CaCO3, as evidenced by X-ray diffraction and Fourier transform infrared absorption. The BET surface area, BET pore volume, and pore diameter were 250.33 m2 g-1, 0.4 cm3 g-1, and <70 nm, respectively. The point of zero charge of BC1 was determined to be around pH 9. The prepared adsorbent was tested for its adsorption efficacy towards the neonicotinoid pesticide acetamiprid. The influence of pH (2-10), temperature (20-45 °C), adsorbent dose (0.2-1.2 g L-1), contact time (5-60 min), and initial pesticide concentration (10-60 mg L-1) on the adsorption process of acetamiprid on BC1 was studied. The adsorption capacity of BC1 was 17.8 mg g-1 under optimum conditions (i.e., 20 mg L-1 initial acetamiprid concentration, pH 8, 1 g L-1 adsorbent dose, 25 °C, and 15 min contact time). The equilibrium data obtained from the adsorption experiment fitted well with the Langmuir isotherm model. We developed an effective nanoporous adsorbent for the recycling of crab shells which can be applied on site with minimal laboratory infrastructure according to local needs.

Exploring the Potential of Using Marine-Derived Ingredients: From the Extraction to Cutting-Edge Cosmetics.

The growing understanding and knowledge of the potential of marine species, as well as the application of "blue biotechnology" have been motivating new innovative solutions in cosmetics. It is widely noted that that marine species are important sources of compounds with several biological activities that are yet to be discovered. This review explores various biological properties of marine-derived molecules and briefly outlines the main extraction methods. Alongside these, it is well known the legislative and normative framework of cosmetics is increasingly being developed. In this research segment, there is a growing concern with sustainability. In this sense, "blue biotechnology", together with the use of invasive species or marine waste products to obtain new active ingredients, haven been emerging as innovative and sustainable solutions for the future's cosmetics industry. This review also examines the regulatory framework and focus on the recent advancements in "blue biotechnology" and its relevance to the sustainable development of innovative cosmetics.

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.

Exopolysaccharide Production from Marine-Derived Brevundimonas huaxiensis Obtained from Estremadura Spur Pockmarks Sediments Revealing Potential for Circular Economy.

Marine environments represent an enormous biodiversity reservoir due to their numerous different habitats, being abundant in microorganisms capable of producing biomolecules, namely exopolysaccharides (EPS), with unique physical characteristics and applications in a broad range of industrial sectors. From a total of 67 marine-derived bacteria obtained from marine sediments collected at depths of 200 to 350 m from the Estremadura Spur pockmarks field, off the coast of Continental Portugal, the Brevundimonas huaxiensis strain SPUR-41 was selected to be cultivated in a bioreactor with saline culture media and glucose as a carbon source. The bacterium exhibited the capacity to produce 1.83 g/L of EPS under saline conditions. SPUR-41 EPS was a heteropolysaccharide composed of mannose (62.55% mol), glucose (9.19% mol), rhamnose (19.41% mol), glucuronic acid (4.43% mol), galactose (2.53% mol), and galacturonic acid (1.89% mol). Moreover, SPUR-41 EPS also revealed acyl groups in its composition, namely acetyl, succinyl, and pyruvyl. This study revealed the importance of research on marine environments for the discovery of bacteria that produce new value-added biopolymers for pharmaceutical and other biotechnological applications, enabling us to potentially address saline effluent pollution via a sustainable circular economy.

Improving awareness, understanding, and enforcement of responsibilities and regulations in Blue Biotechnology.

Blue Biotechnology is developing rapidly worldwide. However, the Nagoya Protocol (NP), Responsible Research and Innovation (RRI) and other regulatory requirements in this field are falling behind. This article identifies the main RRI, NP, and regulatory gaps and provides key recommendations to mitigate these challenges.

Advanced Methods for Natural Products Discovery: Bioactivity Screening, Dereplication, Metabolomics Profiling, Genomic Sequencing, Databases and Informatic Tools, and Structure Elucidation.

Natural Products (NP) are essential for the discovery of novel drugs and products for numerous biotechnological applications. The NP discovery process is expensive and time-consuming, having as major hurdles dereplication (early identification of known compounds) and structure elucidation, particularly the determination of the absolute configuration of metabolites with stereogenic centers. This review comprehensively focuses on recent technological and instrumental advances, highlighting the development of methods that alleviate these obstacles, paving the way for accelerating NP discovery towards biotechnological applications. Herein, we emphasize the most innovative high-throughput tools and methods for advancing bioactivity screening, NP chemical analysis, dereplication, metabolite profiling, metabolomics, genome sequencing and/or genomics approaches, databases, bioinformatics, chemoinformatics, and three-dimensional NP structure elucidation.

Selenium Bio-Nanocomposite Based on Alteromonas macleodii Mo169 Exopolysaccharide: Synthesis, Characterization, and In Vitro Antioxidant Activity.

In this study, the novel exopolysaccharide (EPS) produced by the marine bacterium Alteromonas macleodii Mo 169 was used as a stabilizer and capping agent in the preparation of selenium nanoparticles (SeNPs). The synthesized nanoparticles were well dispersed and spherical with an average particle size of 32 nm. The cytotoxicity of the EPS and the EPS/SeNPs bio-nanocomposite was investigated on human keratinocyte (HaCaT) and fibroblast (CCD-1079Sk) cell lines. No cytotoxicity was found for the EPS alone for concentrations up to 1 g L-1. A cytotoxic effect was only noticed for the bio-nanocomposite at the highest concentrations tested (0.5 and 1 g L-1). In vitro experiments demonstrated that non-cytotoxic concentrations of the EPS/SeNPs bio-nanocomposite had a significant cellular antioxidant effect on the HaCaT cell line by reducing ROS levels up to 33.8%. These findings demonstrated that the A. macleodii Mo 169 EPS can be efficiently used as a stabilizer and surface coating to produce a SeNP-based bio-nanocomposite with improved antioxidant activity.

Enhanced production of biobased, biodegradable, Poly(3-hydroxybutyrate) using an unexplored marine bacterium Pseudohalocynthiibacter aestuariivivens, isolated from highly polluted coastal environment.

The production and disposal of plastics from limited fossil reserves, has prompted research for greener and sustainable alternatives. Polyhydroxyalkanoates (PHAs) are biocompatible, biodegradable, and thermoprocessable polyester produced by microbes. PHAs found several applications but their use is limited due to high production cost and low yields. Herein, for the first time, the isolation and characterization of Pseudohalocynthiibacter aestuariivivens P96, a marine bacterium able to produce surprising amount of PHAs is reported. In the best growth condition P96 was able to reach a maximum production of 4.73 g/L, corresponding to the 87 % of total cell dry-weight. Using scanning and transmission microscopy, lab-scale fermentation, spectroscopic techniques, and genome analysis, the production of thermoprocessable polymer Polyhydroxybutyrate P(3HB), a PHAs class, endowed with mechanical and thermal properties comparable to that of petroleum-based plastics was confirmed. This study represents a milestone toward the use of this unexplored marine bacterium for P(3HB) production.

Marine-Derived Actinomycetes: Biodegradation of Plastics and Formation of PHA Bioplastics-A Circular Bioeconomy Approach.

Plastics are present in the majority of daily-use products worldwide. Due to society's production and consumption patterns, plastics are accumulating in the environment, causing global pollution issues and intergenerational impacts. Our work aims to contribute to the development of solutions and sustainable methods to mitigate this pressing problem, focusing on the ability of marine-derived actinomycetes to accelerate plastics biodegradation and produce polyhydroxyalkanoates (PHAs), which are biodegradable bioplastics. The thin plastic films' biodegradation was monitored by weight loss, changes in the surface chemical structure (Infra-Red spectroscopy FTIR-ATR), and by mechanical properties (tensile strength tests). Thirty-six marine-derived actinomycete strains were screened for their plastic biodegradability potential. Among these, Streptomyces gougerotti, Micromonospora matsumotoense, and Nocardiopsis prasina revealed ability to degrade plastic films-low-density polyethylene (LDPE), polystyrene (PS) and polylactic acid (PLA) in varying conditions, namely upon the addition of yeast extract to the culture media and the use of UV pre-treated thin plastic films. Enhanced biodegradation by these bacteria was observed in both cases. S. gougerotti degraded 0.56% of LDPE films treated with UV radiation and 0.67% of PS films when inoculated with yeast extract. Additionally, N. prasina degraded 1.27% of PLA films when these were treated with UV radiation, and yeast extract was added to the culture medium. The main and most frequent differences observed in FTIR-ATR spectra during biodegradation occurred at 1740 cm-1, indicating the formation of carbonyl groups and an increase in the intensity of the bands, which indicates oxidation. Young Modulus decreased by 30% on average. In addition, S. gougerotti and M. matsumotoense, besides biodegrading conventional plastics (LDPE and PS), were also able to use these as a carbon source to produce degradable PHA bioplastics in a circular economy concept.

Green approaches for extraction, chemical modification and processing of marine polysaccharides for biomedical applications.

Over the past few decades, natural-origin polysaccharides have received increasing attention across different fields of application, including biomedicine and biotechnology, because of their specific physicochemical and biological properties that have afforded the fabrication of a plethora of multifunctional devices for healthcare applications. More recently, marine raw materials from fisheries and aquaculture have emerged as a highly sustainable approach to convert marine biomass into added-value polysaccharides for human benefit. Nowadays, significant efforts have been made to combine such circular bio-based approach with cost-effective and environmentally-friendly technologies that enable the isolation of marine-origin polysaccharides up to the final construction of a biomedical device, thus developing an entirely sustainable pipeline. In this regard, the present review intends to provide an up-to-date outlook on the current green extraction methodologies of marine-origin polysaccharides and their molecular engineering toolbox for designing a multitude of biomaterial platforms for healthcare. Furthermore, we discuss how to foster circular bio-based approaches to pursue the further development of added-value biomedical devices, while preserving the marine ecosystem.

Assessing the genomic composition, putative ecological relevance and biotechnological potential of plasmids from sponge bacterial symbionts.

Plasmid-mediated transfer of genes can have direct consequences in several biological processes within sponge microbial communities. However, very few studies have attempted genomic and functional characterization of plasmids from marine host-associated microbial communities in general and those of sponges in particular. In the present study, we used an endogenous plasmid isolation method to obtain plasmids from bacterial symbionts of the marine sponges Stylissa carteri and Paratetilla sp. and investigated the genomic composition, putative ecological relevance and biotechnological potential of these plasmids. In total, we isolated and characterized three complete plasmids, three plasmid prophages and one incomplete plasmid. Our results highlight the importance of plasmids to transfer relevant genetic traits putatively involved in microbial symbiont adaptation and host-microbe and microbe-microbe interactions. For example, putative genes involved in bacterial response to chemical stress, competition, metabolic versatility and mediation of bacterial colonization and pathogenicity were detected. Genes coding for enzymes and toxins of biotechnological potential were also detected. Most plasmid prophage coding sequences were, however, hypothetical proteins with unknown functions. Overall, this study highlights the ecological relevance of plasmids in the marine sponge microbiome and provides evidence that plasmids of sponge bacterial symbionts may represent an untapped resource of genes of biotechnological interest.