To date, exploration of Gracilaria and Gelidiella’s bioactive compounds has been conducted using conventional methods that require a long time, high costs, and significant effort. Currently, metabolomic profiling and molecular networking have emerged as methods of exploring bioactive compounds. This study aimed to perform bioactive compound profiling through a metabolomic LC-HRMS-based and molecular networking approach in Gelidiella acerosa, Gracilaria arcuata, and Gracilaria verrucosa. All chromatograms and MS/MS spectra obtained for three crude extracts were digitally converted into an mzXML file using MSConvert, submitted to the Global Natural Product Social (GNPS), and visualized in Cytoscape 3.9.1. In total, nine dereplicated compounds were identified: 11-Deoxyprostaglandin (m/z 324.214), Diacylglyceryl trimethylhomoserines (DGTS) (m/z 684.575), Glycochenodeoxy acid (m/z 448.369), Lysophosphatidylcholine (m/z 522.350), Diacylglyceryl trimethylhomoserines (DGTS) (m/z 656.557), Pheophorbide A (m/z 593.266), Pyropheophorbide A (m/z 593.266), (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dihydro-4H-chromen-4-one (m/z 303.15), and Polyporic acid (m/z 293.156). These compounds are typically classified as fatty acids, lipids, terpenoids, alkaloids, shikimates, and phenylpropanoids. The molecular networking and metabolite clustering showed an interesting pattern where some compounds were produced only by one species, some by two species, and some by all three. These compounds may have pharmaceutical potential based on their chemical properties and reported activities.

Gracilaria edulis, a red seaweed that is widely known as an agar-producing seaweed, has a unique biosynthetic pathway for producing bioactive compounds. However, most of the bioactive compounds of this species have not explored yet. The environmental conditions as well as the extraction method might influence the bioactive compound production. Hence, this study aimed to explore untargeted liquid chromatography–high resolution mass spectrometry (LCHRMS)-based metabolomics profiling of G. edulis (Gracilariales, Rhodophyta) from three different beaches along the southern part of the Special Region of Yogyakarta (Java Island, Indonesia), which has different environmental characteristics. We also observed the effect of extraction solvent on metabolomic profiling of G. edulis extract using three different solvents with the different polarity. Metabolomic profiling was performed using an LC-HRMS instrument and analyzed using the Global Natural Product Social Molecular Networking (GNPS) database. The results revealed differences in the metabolomic profiles of G. edulis’s extracts based on coastal location and solvent. G. edulis from Kukup Beach exhibited the highest metabolomic diversity (277 nodes), followed by G. edulis from Sepanjang Beach (268) and G. edulis from Krakal Beach (204). Among the solvents, n-hexane was the most effective, extracting 311 nodes, followed by methanol (293) and ethyl acetate (197). Nine tentatively dereplicated compounds were found, i.e., pumilacidine C, pumilacidine E, lichenysin, cholesterol, AC1L1X1Z, sarmentoside B, 7-dehydrocholesterol, pheophytin A and porphyra-334. Some dereplicated compounds were found in a specific area. For example, lichenysin and pumilacidin compounds were produced by G. edulis from Sepanjang Beach, while cholesterol and AC1L1X1Z were found in from G. edulis extract, which were collected from Sepanjang and Kukup Beaches, and 7-dehydrocholesterol and sarmentoside B compounds from all beach locations, while pheophytin A and porphyra-334 from Krakal Beach. Further research is needed to obtain pure compounds that have the potential to be antibacterial, as well as antioxidant, anti-inflammatory, and photoprotective compounds.

Graphical Abstract Highlight Research Compounds produced by Roseivirga PAP.19 bacteria have potential as antimicrobials. The OSMAC approach was used successfully to activate BGCs of Roseivirga PAP.19. The use of medium in bacterial culture affects the secondary metabolites produced by Roseivirga PAP.19 bacteria. Bacteria cultured in A1Bfe+C medium produced more secondary metabolites compared to the other medium. The different incubation times during culture affect the amount of secondary metabolite compounds produced. Abstract Marine sponge-associated bacteria are a rich source of bioactive metabolites. The biochemical interaction between bacteria and sponges enables symbiotic bacteria to produce metabolites similar to those of their host. Metabolite extracts from symbiotic bacteria contain bioactive compounds with cytotoxic, antiviral, and antimicrobial properties. There is an urgent need for new antimicrobial agents due to the increasing resistance to existing drugs. This study aims to optimize the medium and incubation time of secondary metabolites produced by the symbiotic bacteria of the sponge Roseivirga sp. PAP.19. To stimulate biosynthesis of silent gene clusters, the One Strain Many Compounds (OSMAC) approach was used. Bacteria were cultured in various media and at different incubation times. This has the potential to increase the production of secondary metabolites. Secondary metabolites were analyzed using LC-HRMS. The potential of these metabolites as antimicrobial agents was subsequently evaluated. Results showed six compounds with antimicrobial potential. Notably, the A1BFe+C medium produced more bioactive compounds than the other medium, and the highest compound production occurred on day 4. These findings suggest that optimizing culture conditions using the OSMAC approach can enhance the yield of potentially valuable antimicrobial secondary metabolites from symbiotic bacteria, which may contribute to the discovery of new antimicrobial agents.

One of the most promising sources for the discovery and development of new antibiotics is marine microorganisms from the phylum Bacteroidetes. However, the output of exploration campaigns exploiting the Bacteroidetes phylum still needs to be improved. Co-culture represents an approach for discovering new metabolites that increases metabolite diversity and avoids the rediscovery of known metabolites. This research aims to determine the antimicrobial potential of the extracts derived from the co-culture of the bacteria Roseivirga sp. PAP.19 and Sinomicrobium sp. PAP.21. In this study, Roseivirga sp. PAP.19 and Sinomicrobium sp. PAP.21 were co-cultured under different culture conditions, following the principle of the One Strain Many Compounds (OSMAC) approach, to enhance metabolite diversity. The resulting extracts were analysed in a time-based fashion by molecular networking of the LC-HRMS profile. The OSMAC principle was applied to activate the production of secondary metabolite biosynthetic gene clusters (BGCs) and thereby hopefully trigger the production of new antimicrobial compounds. Extracts of the co-culture produced various secondary metabolites. In our study, we detected surfactin C13 (m/z 1008.65 [M+H]), surfactin C14 (m/z 1022.67 [M+H]), and surfactin C15 (m/z 1036.69 [M+H]+). These metabolites are known to possess antimicrobial activity with potential applications against pathogenic bacteria.

The mudskipper Boleophthalmus boddarti is known for secreting epidermal mucus that likely serves as a protective antibacterial barrier against microbial threats. This research aimed to investigate the effectiveness of mucus extracts from B. boddarti in inhibiting the growth of two pathogenic bacteria, Aeromonas hydrophila and Vibrio harveyi. Extraction of the mucus was conducted using a sterile physiological saline solution (0.85% NaCl), followed by centrifugation and freeze-drying to preserve the material. Two forms of the mucus extracts fresh and frozen were prepared in six different concentrations: 62.5, 125, 250, 500, 1000, 2000 µg/mL. The experimental setup included both positive (Ampicillin) and negative (saline) controls to benchmark antibacterial activity. Antimicrobial efficacy was evaluated using the disc diffusion method, where inhibition zones around discs impregnated with the extracts were measured. Results showed that the diameter of the inhibition zones indicated a mild antibacterial effect. Specifically, for A. hydrophila, the fresh mucus extract produced inhibition zones ranging from 1.17 to 3.50 mm, while the frozen extract ranged from 1.25 to 3.33 mm. Against V. harveyi, the fresh extract yielded zones between 0.92 and 3.67 mm, and the frozen extract showed zones between 1.67 and 3.67 mm. The inhibition value of mudskipper mucus extract included in low category. These findings suggest that B. boddarti mucus extract possesses promising antibacterial properties against the tested bacterial species