Unlocking marine treasures: isolation and mining strategies of natural products from sponge-associated bacteria

Contents

• 1. Introduction
  • 1.1. The importance of marine sponges and symbionts
  • 1.2. Major natural products and their biological activities found in sponge symbionts in the last 5 years
  • 1.3. Trends in sponge-associated NP research

• 2. Separation of bacteria from sponges
  • 2.1. Importance of separating sponge-associated bacteria
  • 2.2. Simple methods for isolation and its limitations
  • 2.3. Density gradient centrifugation method
  • 2.4. Targeted cell sorting by using flow cytometry
  • 2.5. Targeted cell sorting by using magnetic-activated cell sorting

• 3. Cell culture attempts for sponge-associated bacteria
  • 3.1. Fine control of medium composition (in vitro cultivation methods)
  • 3.2. In situ cultivation methods
  • 3.3. Microcapsule-based cultivation methods

• 4. Genome-based approach
  • 4.1. Metagenome-based NPs biosynthesis gene discovery
  • 4.2. Single cell-based NPs biosynthesis gene discovery
  • 4.3. Heterologous expression of NP BGCs

• 5. Chemical-based bacterial NPs mining method
  • 5.1. In situ chemical capture using artificial marine sponge devices
  • 5.2. Small molecule in situ resin capture
  • 5.3. Integration of metabolomics and genome mining
  • 5.4. AI-driven strategies for natural product discovery

• 6. Conclusion

• 7. Data availability

• 8. Conflicts of interest

• Acknowledgements

• References

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Abstract

Covering: 2019 to early 2025

Marine sponges form unique ecosystems through symbiosis with diverse microbial communities, producing natural products including bioactive compounds. This review comprehensively addresses the key steps in the discovery of natural products from sponge-associated microorganisms, encompassing microbial isolation and cultivation, compound identification, and characterisation. Various cultivation methods, such as floating filter cultivation, microcapsule-based cultivation, and in situ systems, are examined to highlight their applications and strategies for overcoming limitations of conventional approaches. Additionally, the integration of genome-based methodologies and compound screening is explored to enhance the discovery of novel bioactive substances and establish a sustainable platform for natural product research. This review provides insights into the latest trends in sponge-associated microbial research and offers practical perspectives for expanding the utilization of marine biological resources.

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Jeong-A Kim

Dr Jeong-A Kim is a postdoctoral researcher at the Korea Institute of Ocean Science & Technology (KIOST), Jeju Bio Center. She received her PhD in Life Sciences from Sogang University, South Korea, where she investigated microbial regulatory mechanisms and host–microbe interactions. Her research interests have expanded to marine microbial ecology, with a focus on sponge-associated bacteria and their potential as a source of bioactive natural products. She applies her expertise in bacterial physiology, genetic regulation, and microbial interactions to improve the selective isolation and cultivation of marine microorganisms, aiming to facilitate the discovery of novel secondary metabolites with pharmaceutical applications.

Si-sun Choi

Dr Si-sun Choi is a research Professor at the Inha University, South Korea. She received her bioengineering PhD at Inha University, focusing on isolation, genetic manipulation and characterization of the Tautomycetin biosynthetic gene cluster in Streptomyces sp. CK4412. She also carried out postdoctoral research at Inha University, focusing on bioactive compound production from metabolic engineered Streptomyces and Corynebacteria. The main research interests of her are (i) genetic engineering for production of nature compound in Streptomyces (ii) discovery of microbial fungicide using actinomycetes genomics-driven approaches.

Jae Kyu Lim

Dr Jae Kyu Lim obtained his PhD in Marine Biotechnology from the University of Science and Technology, South Korea, in 2013. He is currently a scientist at the Marine Biotechnology Center, Korea Institute of Ocean Science & Technology (KIOST). His research focuses on metagenomic exploration of sponge-associated symbiotic microorganisms that produce bioactive natural products. He investigates microbial genes involved in biosynthetic pathways, including PKS, NRPS, and terpene cyclases. His current work emphasizes developing cultivation techniques for previously unculturable bacteria and applying heterologous gene expression to enable the biotechnological production of novel marine-derived compounds.

Eung-Soo Kim

Dr Kim obtained his PhD in Microbiology from the University of Minnesota, USA, in 1994. He then joined Inha University, where he has been a professor at the department of biological engineering since 2001. His primary research focuses on biosynthesis and regulation of Streptomyces and rare actinomyces that produce valuable secondary metabolites. His work involves actinomycetes genome mining, natural product development, heterologous expression of large-sized biosynthetic gene cluster, and synthetic & chemical biology.

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1. Introduction

1.1. The importance of marine sponges and symbionts

Sponges (Phylum: Porifera) are one of the oldest existing multicellular organisms, with fossil records indicating their appearance at least 600 million years ago.^1,2 Approximately 15 000 species of sponges have been described, primarily categorized into Calcarea, Hexactinellida, Homoscleromorpha, and Demospongiae.^3,4 Among these, Demospongiae represents the largest group, comprising the majority of currently known species.^5,6 The histological structure of sponges is unique, lacking homologous organs found in other animal species. The outer epithelial layer (exopinacoderm) consists of flattened exopinacocytes, while the aquiferous system, a network of canals responsible for water circulation and filtration, is lined with endopinacocytes and choanocytes. Choanocytes, equipped with flagella and microvilli, facilitate water pumping, nutrient uptake, and capturing microorganisms. The mesohyl, a connective tissue located between the outer epithelial layer and the aquiferous system, contains an extracellular matrix and various mobile cells. Among these, archaeocytes serve as macrophage-like stem cells, while lophocytes secrete the extracellular matrix, and spongocytes and sclerocytes generate skeletal elements.⁷ Sponges are sessile filter feeders that efficiently capture suspended food particles from the water.⁸ This filtration introduces diverse microorganisms into their canals, allowing them to form mutualistic symbiotic relationships.⁹ Sponge-associated microbial communities include archaea, cyanobacteria, heterotrophic bacteria, fungi, and microalgae, establishing complex symbioses.^10,11 These relationships involve extracellular, intracellular, and even intranuclear symbiosis, constituting 50–60% of the host's biomass.¹² Depending on their persistence, symbionts are classified as either transient or resident. Upon establishing stability and functionality, transient microbes become resident symbionts, forming integrated relationships with the sponge host.^13–15

Sponges have evolved unique chemical defence systems in response to ecological pressures such as competition, overgrowth, infection, and predation. These defences involve secondary metabolites, including amino acids, nucleosides, polyethers, alkaloids, macrolides, porphyrins, terpenes, and steroids, many of which exhibit potent anticancer, anti-inflammatory, antifungal, and antiviral activities.¹⁶ Between 2010 and 2019, a total of 2659 compounds were identified from sponges, making them a “goldmine” of bioactive compounds.¹⁷ Interestingly, these bioactive compounds are primarily produced by sponge-associated microorganisms rather than the sponges. Among these, sponge-associated bacteria are the most extensively studied symbionts due to their ease of cultivation and the superior properties of their metabolites.¹¹ Structural similarities between natural products derived from sponges and bacterial metabolites suggest that many bioactive compounds likely originate from sponge-associated bacteria.^18–21

In this context, numerous sponge-derived compounds have served as lead structures for pharmaceutical development, including clinically approved drugs such as cytarabine and eribulin.²² However, translating these discoveries into therapeutics often faces the critical barrier of sustainable supply, as compound yields from natural sponge biomass are typically low, and large-scale harvesting raises ecological concerns.^23,24 Identifying the true producer, whether sponge or symbiont, is essential for enabling sustainable production strategies, such as microbial fermentation or synthetic biology. Moreover, the metabolite structure similarity between host sponges and their symbionts makes biosynthetic origin assignments increasingly complex, highlighting the need for integrated chemical and genomic approaches.^25–30

Given these challenges, there is a growing need for integrated knowledge that bridges natural product chemistry, microbial symbiosis, and modern biotechnological tools. This review aims to provide an integrated overview of progress in the discovery of natural products from sponges and examines strategies for isolating and cultivating sponge-associated bacteria. Additionally, it discusses genome-based approaches for identifying biosynthetic gene clusters (BGCs) and their heterologous expression, as well as chemical-based approaches for the discovery of natural products.

1.2. Major natural products and their biological activities found in sponge symbionts in the last 5 years

Research on natural products derived from marine sponges has been extensively documented in numerous studies. Therefore, this section focuses on natural products reported in the past five years, categorizing them based on their biological activities to examine recent research trends (Table 1 and Fig. S1–S5†). Through this, we aim to gain a clearer understanding of the biological activity patterns and structural diversity of these compounds while highlighting the latest advancements in the field. Additionally, this review seeks to contribute to future natural product discovery by providing insights into research directions targeting specific biological activities.

Table 1 List of natural products reported in the past five years

Activity classification	Compound	Origin	Activity	Ref.
Antimicrobial and antiparasitic activities	Secodinorspongin A 1	Spongia sp.	Antibacterial activity against S. aureus	Tai et al.^31
	Scalimides J 2 and K 3	Spongia sp.	Antibacterial activity against Bacillus subtilis and Micrococcus luteus	Shin et al.^32
	Phyllospongianes A 4, B 5, and D 6	Phyllospongia foliascens	Antibacterial activity against S. aureus, E. coli, B. subtilis, E. faecalis, V. vulnificus, V. parahaemolyticus, and P. aeruginosa	Yu et al.^33
	Sceptrin 7	Stylissa massa	Antibacterial activity against S. aureus and E. coli	Kosgahakumbura et al.^34
	Stonikacidin A 8	Lissodendoryx papillosa	Antibacterial activity against S. aureus and E. coli	Tabakmakher et al.^35
	3,5-Dibromo-6-(3′,5′-dibromo-2′-hydroxyphenoxy)phenol 9	Lamellodysidea herbacea	Antibacterial activity against S. aureus and K. pneumoniae	Hanif et al.^36
	Aspergetherins A 10 and C 11	Aspergillus terreus	Antibacterial activity against MRSA	Li et al.^37
	Bacillimidazole F 12	Bacillus sp. WMMC1349	Antibacterial activity against MRSA	Yan et al.^38
	Xanthoelegansin 13	Aspergillus elegans	Antibacterial activity against MRSA, VRE, and E. faecalis	Kumla et al.^39
	Xishaeleganin B 14	Dactylospongia elegans	Antibacterial activity against S. aureus, S. pyogenes, and E. faecium	Chen et al.^40
	(+)- and (−)-Tedanine 15	Tedania sp.	Antibacterial activity against E. faecalis	Guo et al.^41
	2-Ethylhexyl 1H-imidazole-4-carboxylate 16	Verrucosispora sp.	Antibacterial activity against H. pylori, S. aureus, and K. pneumoniae	Chen et al.^42
	3β-hydroxy-5α,6β-methoxyergosta-7,22-dien-15-one 17	Aspergillus asclerogenus	Antibacterial activity against S. aureus	Wen et al.^43
	Aspergilluone A 18	Aspergillus sp. LS57	Antibacterial activity against Mycobacterium tuberculosis H37Rv and S. aureus	Liu et al.^44
	Duryne 19	Petrosia sp.	Antimycobacterial activity against M. tuberculosis H37Rv	Chhetri et al.^45
	Odoripenoids A 20 and B 21	Streptomyces spp.	Antifungal activity against Candida albicans	Wen et al.^46
	Oceanalin B 22	Oceanapia sp.	Antifungal activity against Candida glabrata	Makarieva et al.^47
	7-Dechloronidulin 23	Aspergillus nidulans	Antibacterial and antifungal activities against Gram-positive bacteria and C. albicans	Thi Hoang Anh et al.^48
	Chlocarbazomycin C 24	Streptomyces diacarni LHW51701	Antibacterial and antifungal activity against MRSA, Mycobacterium smegmatis, Bacillus mycoides, and C. albicans	Cheng et al.^49
	Marmaricines A–C 25–27	Agelas sp. aff. marmarica	Antibacterial and antifungal activity against MRSA and C. albicans	Youssef et al.^50
	Bacillisporin A 28 and B 29	Talaromyces pinophilus KUFA 1767	Antibacterial activity against MRSA and S. aureus, antibiofilm activity	Machado et al.^51
	Spinolactone 30	Neosartorya spinosa KUFA 1047	Inhibition of biofilm formation by E. coli, S. aureus, and E. faecalis	de Sá et al.^52
	Tenellic acid C 31
	Neospinosic acid 32
	Citronamine A 33	Citronia astra	Actiparasitic activity against Plasmodium falciparum	Prebble et al.^53
	Saframycin Y3 34	Streptomyces sp.	Inhibited P. falciparum lysyl-tRNA synthetase, showing high binding affinity	Gamaleldin et al.^54
	Juglomycin E 35
	Plakortinic acids C 36	Plakortis symbiotica	Antiparasitic activity against Plasmodium berghei, with favourable ADMET profiles	Amador et al.^55
	Plakortinic acids D 37	Xestospongia deweerdtae
Ant and cytotoxic activities	Furospinulosin-1 38	Hippospongia fistulosa	Induces apoptosis in triple-negative breast cancer spheroids	Guzmán et al.^56
	Neviotin A 39	Haliclona fascigera	Induces oxidative stress and mitochondrial dysfunction, leading to cytotoxicity in MCF-7 breast cancer cells	Saquib et al.^57
	Phakefustatin A 40	Phakellia fusca	Affects the PI3K/Akt pathway; modulates RXRα receptor	Wu et al.^58
	(+)-Spondomine 41	Tedania anhelans	Inhibits Wnt and HIF1 signalling; cytotoxicity against K562 leukemia cells	Jin et al.^59
	Swinhopeptolides A 42 and B 43	Theonella swinhoei	Disrupt Ras–Raf interactions	Kim et al.^60
	2Z- and 6Z-onnamides A 44–45	Theonella conica	Potent activity against HeLa and P388 cells	Nakamura et al.^61
	Neopetrothiazide 46	Neopetrosia spp.	Selectively inhibits PAX3-FOXO1-mediated transcription	Wang et al.^62
	Scalarane-type sesterterpenoid (featuring C-24 pentenone E-ring) 47	Hyrtios erectus and Dysidea spp.	Cytotoxicity against MDA-MB-231 breast cancer cells	Shin et al.^63
	Neopetrosidines A–D 48–51	Neopetrosia chaliniformis	Inhibit proliferation in HeLa/Fucci2 cells	Hitora et al.^64
	Sarcotragusolides A1 52, A2 53, and B1 54	Sarcotragus sp.	Cytotoxicity against K562 leukemia and ASPC-1 pancreatic cancer cells	Xu et al.^65
	24R,25S-luffariellin A 55	Luffariella spp.	Cytotoxicity against leukemia cell lines	Lai et al.^66
	24R-O-methyl-25S-luffariellin A 56
	24R-O-methyl-25S-manoalide 57
	24R,25S-manoalide 58		Cytotoxicity against leukemia cell lines and induced mitochondria-dependent apoptosis and inhibited human topoisomerase I and II
	Phyllospongiane C 59	Phyllospongia foliascens	Cytotoxicity across multiple cancer cell lines	Yu et al.^33
	Phyllofolactone S 60	Phyllospongia foliascens	Selective activity against HCT116 and HN6 cells	Lu et al.^67
	Ascandinine D 61	Aspergillus candidus HDN15-152	Selectively inhibited HL-60 leukemia cells	Zhou et al.^68
	(3R)-(3′,5′-Dihydroxyphenyl)butan-2-one 62	Ascomycota sp. VK12	Cytotoxicity across various cancer cell lines; anti-inflammatory activity via NO inhibition in microglial cells	Quang et al.^69
	Unguisol B 63	Aspergillus unguis	Cytotoxicity in MDA-MB-231 cells; induced apoptosis with S-phase arrest, associated with down-regulation of BCL2L1 and AKT1	Bashari et al.^70
	19-Methoxy-dictyoceratin-A 64	Dactylospongia elegans	Cytotoxicity activity across several cancer cell lines	Yu et al.^71
	Halaminol E 65	Haliclona chrysa	Cytotoxicity in the NCI-60 cancer cell panel; notable selectivity toward melanoma and colon cancer cell lines	Grkovic et al.^72
	Batzelladines O 66 and P 67	Monanchora pulchra	Cytotoxicity against drug-resistant prostate cancer lines; induced both apoptosis and autophagy	Dyshlovoy et al.^73
	Pelorol 68	Dactylospongia elegans	Cytotoxic to melanoma cells; caused G1 arrest and apoptosis through modulation of apoptosis-related genes and miRNAs	Carpi et al.^74
	5-epi-ilimaquinone 69	Dactylospongia elegans
Immunomodulation and host defence-related activities	Secodinorspongin D 70	Spongia spp.	Inhibited neutrophil-derived superoxide and elastase release	Tai et al.^31
	Sponginolide 71
	Fistularin-1 72	Ecionemia acervus	Significant anti-inflammatory effects; reduced NO and prostaglandin E2 production	Ji et al.^75
	11,19-Dideoxyfistularin-3 73
	17-Dehydroxysponalactone 74	Spongia sp.	Inhibited superoxide anion generation and elastase release without cytotoxicity	Tai et al.^76
	(−)-Sponalisolide B 75	Spongia sp.	Inhibited superoxide anion generation	Tai et al.^76
	Spongianol 76	Spongia sp.	Suppressed superoxide and elastase release	Tai et al.^77
	3β,5α,9α-Trihydroxy-24S-ethylcholest-7-en-6-one 77	Spongia sp.	Exhibited superoxide inhibition	Tai et al.^77
	(22E,24S)-Ergosta-7,22-dien-3β,5α-diol-6,5-olide 78
	Thalysiaketide A 79 and B 80	Clathria (Thalysias) vulpina	Dual inhibition of COX-2 and 5-LOX	Chakraborty & Francis^78
	Stylimassalins A 81 and B 82	Stylissa massa	Reduced macrophage migration in a CuSO4-induced zebrafish inflammation model	Liu et al.^79
	(−)-Subertieslide B 83	Suberties sp.	Suppressed NO production in LPS-stimulated RAW 264.7 macrophages	Li et al.^80
	Ircinialactam J 84	Ircinia felix	Inhibited human adenovirus type 5 by interfering with the viral life cycle prior to nuclear genome entry	Ruiz-Molina et al.^81
	2-Bromoaldisine 85	Stylissa massa	Inhibitory activity against HIV-1 Vpr-mediated suppression of cell proliferation in the TREx-HeLa-Vpr model	Win et al.^82
	Ascandinine C 86	Aspergillus candidus HDN15-152	Displayed potent activity against influenza A virus	Zhou et al.^68
	Spiroetherone A 87	Dysidea etheria	Inhibited angiogenesis in a zebrafish model	Jiao et al.^83
	Cyclobutastellettolides A 88 and B 89	Stelletta sp.	Significantly increased ROS production in murine peritoneal macrophages	Kolesnikova et al.^84
	Assimiloside A 90	Hymeniacidon assimilis	Stimulated lysosomal activity and elevated intracellular ROS levels in RAW 264.7 macrophages	Kudryashova et al.^85
Neuroactive and neuroprotective activities	Veranamine 91	Verongula rigida	Selective binder to 5HT2B and sigma-1 receptors, exhibiting antidepressant activity in the forced-swim test without interacting with serotonin, norepinephrine, or dopamine transporters	Kochanowska-Karamyan et al.^86
	Aaptamine 92	Aaptos aaptos	Demonstrated analgesic effects as a delta-opioid receptor agonist in a neuropathic pain model	Sung et al.^87
	Lamellosterols A–C 93–95	Lamellodysidea cf. chlorea	Identified as anti-prion compounds, exhibiting inhibition of α-synuclein aggregation	Jennings et al.^88
	Frondoplysins A 96 and B 97	Dysidea frondosa	Inhibit protein tyrosine phosphatase 1B	Jiao et al.^89
	Erectcyanthins A–C 98–100	Hyrtios erectus	Inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase and 5-lipoxygenase, with erectcyanthin B exhibiting strong inhibition of HMGCR	Chakraborty & Francis^78
	Eutypoid F 101	Penicillium sp. SCSIO 41413	Inhibits PI3K, with potential as an anticancer lead compound	Ye et al.^90
Other functional activities	Aaptodines A–D 102–105	Aaptos suberitoides	Inhibit RANKL-induced osteoclastogenesis	Wang et al.^91
	Tedaniophorbasins A 106 and B 107	Tedaniophorbas ceratosis	Exhibited strong yellow colouration, UV absorbance, cyan fluorescence, and potential ecological functions such as UV shielding or bioluminescence	Hiranrat et al.^92
	9,11-Dihydrogracillinone A 108	Dendrilla antarctica	Antifouling activity	Prieto et al.^93

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1.2.1. Antimicrobial and antiparasitic activities. Marine sponge-derived natural products have yielded a wide array of antibacterial compounds, many of which show activity against clinically relevant pathogens, including multidrug-resistant strains. Several terpenoid compounds have demonstrated notable broad-spectrum or Gram-positive antibacterial activity. Secodinorspongin A 1, a 3,4-seco-3,19-dinorspongian diterpenoid lactone from Spongia sp., exhibited inhibitory activity against Staphylococcus aureus, suppressing bacterial growth by 43%, 54%, and 75% at concentrations of 50, 100, and 200 μM, respectively.³¹ Scalimides J 2 and K 3, scalarane sesterterpenes from the same genus, exhibited antimicrobial activity against Micrococcus luteus and Bacillus subtilis, respectively, with MIC values ranging from 4 to 16 μg mL⁻¹.³² In contrast, Phyllospongianes A 4, B 5, and D 6, isolated from Phyllospongia foliascens, exhibit broad-spectrum antibacterial activity with MIC values ranging from 1 to 8 μg mL⁻¹ against S. aureus, E. coli, B. subtilis, Enterococcus faecalis, Vibrio vulnificus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa.³³ Other compounds with unique structural features have also been reported. Sceptrin 7, a bromopyrrole alkaloid from Stylissa massa, and stonikacidin A 8, a 4-bromopyrrole alkaloid containing an L-idonic acid core from Lissodendoryx papillosa, both exhibit antibacterial activity against S. aureus and E. coli. The latter acts via sortase A inhibition, a novel antibacterial mechanism.^34,35 Additionally, 3,5-dibromo-6-(3′,5′-dibromo-2′-hydroxyphenoxy)phenol 9 from Lamellodysidea herbacea showed strong antibacterial activity with a minimum inhibitory dose (MID) of 0.078 μg per disk against S. aureus and Klebsiella pneumoniae.³⁶ A number of compounds target antibiotic-resistant bacteria, particularly MRSA and VRE. Aspergetherins A 10 and C 11, isolated from Aspergillus terreus, inhibited methicillin-resistant S. aureus (MRSA) with MICs of 64–128 μg mL⁻¹.³⁷ Bacillimidazole F 12, an imidazolium-containing compound from sponge-associated Bacillus sp. WMMC1349, also inhibited MRSA with a MIC of 38.3 μM.³⁸ Notably, xanthoelegansin 13, a viomellein derivative from Aspergillus elegans, showed potent bactericidal activity against MRSA, vancomycin-resistant Enterococcus (VRE), and E. faecalis, with MICs ranging from 0.5 to 2 μg mL⁻¹.³⁹ Xishaeleganin B 14, a sesquiterpene hydroquinone from Dactylospongia elegans, exhibited comparable potency to vancomycin, with MIC values of 1.5 μg mL⁻¹ against S. aureus and Streptococcus pyogenes, and 3.0 μg mL⁻¹ against E. faecium.⁴⁰ Several sponge-associated sources have yielded alkaloids and sterols with moderate to weak antibacterial activities. (+)- and (−)-Tedanine 15, enantiomeric indolone alkaloids from Tedania sp., inhibited E. faecalis with a minimum inhibitory concentration (MIC) of 64 μg mL⁻¹.⁴¹ 2-Ethylhexyl 1H-imidazole-4-carboxylate 16, isolated from Verrucosispora sp., exhibited antibacterial activity against Helicobacter pylori (MIC = 8 μg mL⁻¹), S. aureus (16 μg mL⁻¹), and K. pneumoniae (64 μg mL⁻¹).⁴² A novel ergostane-type steroid, 3β-hydroxy-5α,6β-methoxyergosta-7,22-dien-15-one 17, derived from Aspergillus asclerogenus, inhibited S. aureus with an MIC of 64 μg mL⁻¹.⁴³ Aspergilluone A 18, a chromone derivative from Aspergillus sp. LS57 isolated from the sponge Haliclona sp., exhibited antibacterial activity against Mycobacterium tuberculosis H37Rv (MIC = 32 μg mL⁻¹) and moderate activity against S. aureus (MIC = 64 μg mL⁻¹), while showing weak activity against B. subtilis and E. coli (both MICs = 128 μg mL⁻¹).⁴⁴

Several sponge-derived compounds have demonstrated activity against fungal pathogens, mycobacteria, and in some cases, also exhibited antibiofilm effects. Among compounds with antimycobacterial or antifungal activity, Duryne 19, a linear acetylene compound from Petrosia sp., was active against M. tuberculosis H37Rv, with a MIC of 1.4 μM.⁴⁵ Odoripenoids A 20 and B 21, geosmin-related metabolites from Streptomyces spp. isolated from mesophotic sponges, showed antifungal activity against Candida albicans with MIC values of 16 and 32 μg mL⁻¹, respectively.⁴⁶ Likewise, oceanalin B 22, a bipolar sphingoid tetrahydroisoquinoline β-glycoside from Oceanapia sp., inhibited Candida glabrata with a MIC of 25 μg mL⁻¹, and its activity was proposed to be linked to interference with the sphingolipid pathway.⁴⁷ In addition, 7-dechloronidulin 23, a chlorinated depsidone isolated from Aspergillus nidulans, exhibited strong activity against Gram-positive bacteria and C. albicans, with MIC values ranging from 2–8 μg mL⁻¹.⁴⁸ Chlocarbazomycin C 24, a chlorinated carbazole alkaloid from Streptomyces diacarni LHW51701, exhibited broad-spectrum antimicrobial activity against MRSA, Mycobacterium smegmatis, Bacillus mycoides, and C. albicans, with MICs of 32 μg mL⁻¹ for all strains, and showed no cytotoxicity against human lung cancer cells.⁴⁹ Similarly, marmaricines A–C 25–27, brominated pyrrole alkaloids from the Red Sea sponge Agelas sp. aff. marmarica, were active against MRSA and C. albicans. Specifically, marmaricines A and B inhibited MRSA with MIC values of 8 μg mL⁻¹, while B and C showed equivalent inhibition of C. albicans at the same concentration.⁵⁰

In addition to direct antimicrobial effects, several sponge-derived metabolites demonstrated antibiofilm activity. Bacillisporins A 28 and B 29, isolated from Talaromyces pinophilus KUFA 1767, exhibited antibacterial and antibiofilm activity, inhibiting MRSA and S. aureus with MIC values of 4–16 μg mL⁻¹, and effectively suppressing biofilm formation.⁵¹ Furthermore, Fungal metabolites such as spinolactone 30, tenellic acid C 31, and neospinosic acid 32, produced by Neosartorya spinosa KUFA 1047 isolated from a Thai sponge, significantly inhibited biofilm formation by E. coli, S. aureus, and E. faecalis, despite showing no direct growth inhibition (MIC > 64 μg mL⁻¹). At 64 μg mL⁻¹, tenellic acid C and neospinosic acid reduced biofilm biomass by up to 82.78 ± 0.51% for E. coli, 85.46 ± 0.25% for S. aureus, and 85.06 ± 0.42% for E. faecalis.⁵²

Marine sponges have also yielded compounds with notable antiparasitic activities, particularly against Plasmodium species. Citronamine A 33, an isoquinoline alkaloid isolated from Citronia astra, exhibited moderate antiplasmodial activity against both drug-sensitive (Plasmodium falciparum 3D7, IC₅₀ = 4.3 μM) and drug-resistant (P. falciparum Dd2, IC₅₀ = 5.8 μM) strains. It showed low cytotoxicity toward HEK293 cells (66% inhibition at 40 μM), indicating a selectivity index greater than nine. The compound features a novel pentacyclic spiro[indene-2,2′-pyrrolo[2,1-a]isoquinoline] scaffold, highlighting its potential as a lead for the development of antiplasmodial drugs.⁵³ From Callyspongia siphonella-associated Streptomyces, saframycin Y3 34 and juglomycin E 35 were identified as potential inhibitors of P. falciparum lysyl-tRNA synthetase, showing high binding affinity in docking studies.⁵⁴ Plakortinic acids C 36 and D 37, isolated from a two-sponge association of Plakortis symbiotica and Xestospongia deweerdtae, exhibited antiplasmodial activity against Plasmodium berghei (EC₅₀ = 5.3 μM), along with favourable ADMET properties including high intestinal absorption and hepatic metabolism.⁵⁵

1.2.2. Anticancer and cytotoxic activities. Marine sponge-derived natural products have yielded a wide array of cytotoxic compounds with significant anticancer potential. Several of these compounds demonstrate well-defined mechanisms of action. For example, furospinulosin-1 38, isolated from Hippospongia fistulosa, selectively induces apoptosis in triple-negative breast cancer spheroids, despite limited activity in 2D cultures.⁵⁶ Neviotin A 39, isolated from Haliclona fascigera, induces oxidative stress and mitochondrial dysfunction, leading to cytotoxicity in MCF-7 breast cancer cells. At 25 μg mL⁻¹, it caused a 23.5% increase in late apoptosis and triggered the activation of p53, BAX, and caspase-3.⁵⁷ Phakefustatin A 40, a kynurenine-bearing cycloheptapeptide from Phakellia fusca, acts as an RXRα modulator and down-regulates PI3K/Akt signalling, inducing G2/M arrest and apoptosis in cancer cells. It showed potent cytotoxicity against MCF-7 (IC₅₀ = 3.4 μM), HeLa (6.2 μM), and NCI-H460 (7.1 μM), with no effect on normal cells at 100 μM.⁵⁸ Similarly, (+)-Spondomine 41, isolated from Tedania anhelans, exhibits strong cytotoxicity against K562 leukemia cells (IC₅₀ = 2.2 μM) and dual inhibition of Wnt (72.4%) and HIF1 (70.6%) signalling pathways.⁵⁹ Swinhopeptolides A 42 and B 43, cyclic depsipeptides from the marine sponge Theonella swinhoei, inhibit Ras–Raf interactions with IC₅₀ values of 5.8 and 8.5 μM, respectively.⁶⁰ Similarly, 2Z- and 6Z-onnamides A (44 and 45), isolated from Theonella conica, exhibited strong cytotoxicity against HeLa cells with IC₅₀ values of 0.17 and 0.15 μM, and against P388 cells with IC₅₀ values of 1.8 and 4.8 μM, respectively.⁶¹ Neopetrothiazide 46, a structurally unique isoquinoline–quinone alkaloid containing a thiazide moiety, was isolated from Neopetrosia spp. It selectively inhibits PAX3–FOXO1-mediated transcription, a key oncogenic driver in rhabdomyosarcoma, with an IC₅₀ value of 1.4 μM, although nonspecific cytotoxicity was observed at concentrations ≥4.6 μM.⁶²

Many sponge-derived cytotoxic compounds fall into structurally distinct classes. Among scalarane sesterterpenes isolated from Hyrtios erectus and Dysidea spp., a compound featuring a C-24 pentenone ‘E-ring’ (compound 7) 47 exhibited potent cytotoxicity against MDA-MB-231 breast cancer cells (GI₅₀ = 4.21 μM), while other analogues with differing ‘E-ring’ modifications showed weaker activity.⁶³ Neopetrosidines A–D 48–51, pyridine alkaloids from Neopetrosia chaliniformis, inhibited the proliferation of HeLa/Fucci2 cells with IC₅₀ values of 1.2–2.6 μM. Among them, neopetrosidine A delayed the cell cycle from 16.9 to 36.2 hours by decreasing mitochondrial membrane potential and suppressing ATP production, suggesting a cytostatic mechanism via perturbation of cellular bioenergetics.⁶⁴ Sarcotragusolides A1 52, A2 53, and B 54, butenolide sesterterpenes from Sarcotragus sp., showed moderate cytotoxic activity. Notably, A1 and A2 inhibited K562 leukemia cells (IC₅₀ = 4.38 and 2.91 μM, respectively), while B showed activity against ASPC-1 pancreatic cancer cells (IC₅₀ = 4.71 μM).⁶⁵ Among the isolated stereoisomers from Luffariella spp., 24R,25S-luffariellin A 55, 24R-O-methyl-25S-luffariellin A 56, 24R,25S-manoalide 57, and 24R-O-methyl-25S-manoalide 58 showed the most potent anti-leukemic activity, with IC₅₀ values ranging from 0.50 to 7.67 μM across Molt 4, K562, Sup-T1, and U937 cell lines. Notably, 24R,25S-manoalide induced mitochondria-dependent apoptosis and inhibited human topoisomerase I and II (IC₅₀ = 1.80 and 1.18 μM, respectively), and significantly reduced Molt 4 tumor volume by 66.11% in a xenograft mouse model.⁶⁶ Of the phyllospongianes A–E isolated from P. foliascens, only phyllospongiane C 59 exhibited notable cytotoxicity across multiple cancer cell lines (IC₅₀ = 0.7–2.0 μM), while others, such as A, B, and D, were primarily reported for their antibacterial activity.³³ In addition, phyllofolactone S 60, a bishomoscalarane sesterterpenoid from the same sponge, demonstrated selective activity against HCT116 and HN6 cells.⁶⁷

Fungal symbionts associated with marine sponges have also been a source of notable cytotoxic compounds. Ascandinine D 61, an indole diterpenoid from Aspergillus candidus HDN15-152, selectively inhibited HL-60 leukemia cells (IC₅₀ = 7.8 μM).⁶⁸. (3R)-(3′,5′-Dihydroxyphenyl)butan-2-one 62, a phenolic compound isolated from Ascomycota sp. VK12, exhibited moderate cytotoxicity against HepG2, MCF-7, and SK-Mel2 carcinoma cells with IC₅₀ values ranging from 48.6 to 96.5 μM, and showed anti-inflammatory activity by inhibiting NO production in LPS-stimulated BV2 microglial cells (IC₅₀ = 24.2 μM).⁶⁹ From Aspergillus unguis, a sponge-associated fungus, Unguisol B 63 showed cytotoxicity in MDA-MB-231 cells and induced apoptosis with S-phase arrest, associated with down-regulation of BCL2L1 and AKT1.⁷⁰

Recent studies have also identified new compounds with selective or context-dependent cytotoxicity. A 19-methoxy-dictyoceratin-A 64 from Dactylospongia elegans showed moderate activity across several cancer cell lines (IC₅₀ = 17.4–37.8 μM).⁷¹ Halaminol E 65, isolated from Haliclona chrysa, exhibited moderate cytotoxicity in the NCI-60 cancer cell panel with a mean GI₅₀ value of 6.76 μM. Notably, it demonstrated selective activity against melanoma and colon cancer cell lines.⁷² Batzelladines O 66 and P 67, guanidine alkaloids from Monanchora pulchra, exhibited potent cytotoxicity against prostate cancer cell lines (PC3, PC3-DR, and 22Rv1) at low micromolar concentrations (IC₅₀ = 1.4–2.1 μM), and induced apoptosis via caspase-3 and PARP cleavage, as well as cytoprotective autophagy characterised by LC3B-II upregulation and mTOR suppression.⁷³ Additionally, Pelorol 68 and 5-epi-ilimaquinone 69, two meroterpenoids isolated from Dactylospongia elegans, exhibited cytotoxicity against 501Mel melanoma cells with IC₅₀ values of 3.02 μM and 1.72 μM, respectively (72 h). Both compounds induced G1 cell cycle arrest and triggered apoptosis via modulation of apoptosis-related genes (e.g., BAX, BCL2, BIRC5, MCL-1) and microRNAs (e.g., upregulation of miR-16-5p, miR-193a-3p, and down-regulation of miR-214-3p).⁷⁴

1.2.3. Immunomodulation and host defence-related activities. Recent studies have increasingly highlighted the potential of sponge-derived natural products as immune modulators and therapeutic agents for inflammatory conditions. Among these, secodinorspongin D 70 and sponginolide 71, isolated from Spongia spp., belong to the 3,4-seco-3,19-dinorspongian and classical spongian diterpenoid lactone classes, respectively. Both compounds were shown to inhibit neutrophil-derived superoxide (20.4% and 22.0% inhibition, respectively) and elastase release (30.8% and 22.5%, respectively).³¹ Similarly, a series of bromotyrosine alkaloids from Ecionemia acervus demonstrated significant anti-inflammatory effects in a Caco-2/THP-1 co-culture model. Notably, fistularin-1 72 and 11,19-dideoxyfistularin-3 73 markedly reduced nitric oxide (NO) and prostaglandin E₂ production, down-regulated iNOS and COX-2 expression, suppressed phosphorylation of MAPKs (p38, JNK, ERK1/2), and blocked nuclear translocation of NF-κB p65, leading to the overall reduction of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α.⁷⁵ Other sponge-derived compounds with neutrophil-targeted anti-inflammatory activity include 17-dehydroxysponalactone 74, which inhibited superoxide anion generation (IC₅₀ = 3.37 μM) and elastase release (IC₅₀ = 4.07 μM) in fMLF/CB-induced human neutrophils without cytotoxicity. Similarly, (−)-sponalisolide B 75 exhibited superoxide inhibition (IC₅₀ = 5.31 μM).⁷⁶ Spongianol 76, a chlorinated, polyoxygenated labdane-type diterpenoid from Spongia sp., significantly inhibited neutrophil-derived inflammatory responses, suppressing superoxide anion generation by 76.7% and elastase release by 85.6% at 10 μM without cytotoxicity. In addition, two newly identified steroidal compounds, 3β,5α,9α-trihydroxy-24S-ethylcholest-7-en-6-one 77 and (22E,24S)-ergosta-7,22-dien-3β,5α-diol-6,5-olide 78, exhibited moderate superoxide inhibition, showing 47.4% and 57.2% inhibition, respectively, at the same concentration.⁷⁷ Polyketides thalysiaketide A 79 and B 80, isolated from Clathria (Thalysias) vulpina, showed dual inhibition of COX-2 (IC₅₀ = 0.93 and 1.03 mM, respectively) and 5-lipoxygenase (5-LOX) (IC₅₀ = 0.87 and 0.98 mM), with thalysiaketide A exhibiting stronger COX-2 selectivity than ibuprofen.⁹⁴ Likewise, stylimassalin A 81 and B 82, two pyrrole-derived alkaloids from Stylissa massa, significantly reduced macrophage migration in a CuSO₄-induced zebrafish inflammation model at concentrations of 5 μM and 10 μM, respectively.⁷⁹ Another compound, (−)-subertieslide B 83, a butenolide alkaloid isolated from Suberties sp., suppressed NO production in LPS-stimulated RAW 264.7 macrophages with an IC₅₀ value of 40.8 μM.⁸⁰

In addition to anti-inflammatory effects, several sponge-derived compounds have exhibited notable antiviral activity. For example, ircinialactam J 84, isolated from Ircinia felix, potently inhibited human adenovirus type 5 by interfering with the viral life cycle prior to nuclear genome entry.⁸¹ 2-Bromoaldisine 85, a brominated pyrrolactam alkaloid isolated from Stylissa massa, showed inhibitory activity against HIV-1 Vpr-mediated suppression of cell proliferation in the TREx-HeLa-Vpr model at 10 μM, with a proliferation recovery comparable to that of the positive control damnacanthal.⁸² Furthermore, ascandinine C 86, isolated from Aspergillus candidus HDN15-152, displayed potent activity against influenza A virus, with an IC₅₀ value of 26 μM.⁶⁸

Sponge-derived compounds have also shown potential in vascular activity regulation. Spiroetherone A 87 (IC₅₀ = 2.4 μM) and B (IC₅₀ = 8.7 μM), two sesquiterpene naphthoquinones from Dysidea etheria, inhibited angiogenesis in a zebrafish model. Notably, spiroetherone A also showed selective cytotoxicity toward NCI–H929, HepG2, A549, and SK-OV-3 cells (IC₅₀ = 7.4–12.2 μM), but was non-cytotoxic to HEK293 cells at 32 μM.⁸³

Lastly, a number of compounds have demonstrated immunostimulatory or ROS-modulating activities. Cyclobutastellettolides A 88 and B 89, two C₁₉ norterpenoids from the sponge Stelletta sp., significantly increased ROS production in murine peritoneal macrophages at non-cytotoxic concentrations. At 10 μM, cyclobutastellettolide A and B enhanced ROS levels by 93% and 90%, respectively, suggesting their potential as immunostimulatory agents.⁸⁴ Similarly, assimiloside A 90, a glycolipid isolated from Hymeniacidon assimilis, stimulated lysosomal activity (25–57%) and elevated intracellular ROS levels (15–35%) in RAW 264.7 macrophages at non-cytotoxic concentrations ranging from 0.01 to 10 μM.⁸⁵

1.2.4. Neuroactive and neuroprotective activities. Certain marine sponge-derived natural products target the central nervous system and show promising potential for the treatment of neurological and psychiatric disorders. Veranamine 91, a compound with a unique heterocyclic structure derived from Verongula rigida, has been identified as a selective binder to 5HT2B and sigma-1 receptors. In vivo experiments exhibited antidepressant activity in the forced-swim test and displayed a distinct mechanism of action by not interacting with serotonin, norepinephrine, or dopamine transporters.⁸⁶ Aaptamine 92, an alkaloid isolated from Aaptos aaptos, demonstrated analgesic effects in a neuropathic pain model. Studies have shown that it functions as a delta-opioid receptor agonist within the spinal cord and was reported to reduce pain sensitivity in a chronic constriction injury model. Additionally, it was suggested that aaptamine modulates neuropathic pain by down-regulating vascular endothelial growth factor and lactate dehydrogenase A expression.⁸⁷ Meanwhile, Lamellosterols A–C 93–95, polyoxygenated sterol sulfates isolated from Lamellodysidea cf. chlorea, exhibited potent anti-prion activity against the [PSI⁺] yeast prion (EC₅₀ = 12.7, 13.8, and 9.8 μM, respectively). Among them, lamellosterol A additionally inhibited α-synuclein aggregation by 68.0% and directly bound to the protein in mass spectrometry assays. This suggests potential therapeutic relevance to prion and Parkinson's disease-related pathologies.⁸⁸

1.2.5. Enzyme inhibitory activities. Marine sponge-derived natural products have yielded structurally diverse enzyme inhibitors with therapeutic potential. Frondoplysins A 96 and B 97, terpene–alkaloid bioconjugates isolated from Dysidea frondosa, exhibit potent inhibitory activity against protein tyrosine phosphatase 1B, with IC₅₀ values of 0.39 μM and 0.65 μM, respectively. Notably, frondoplysin A exhibited in vivo antioxidative activity over five times stronger than vitamin C.⁸⁹ Three cyanthiwigin-type diterpenoids, erectcyanthins A–C 98–100, were obtained from Hyrtios erectus and evaluated for their activity against 3-hydroxy-3-methylglutaryl coenzyme A reductase. Erectcyanthin B showed the strongest inhibitory effect (IC₅₀ = 0.07 mM), comparable to atorvastatin (IC₅₀ = 0.08 mM), and also displayed moderate antioxidant (IC₅₀ = 0.4 mM) and 5-lipoxygenase inhibitory activity (IC₅₀ = 0.88 mM), indicating its potential as a multifunctional therapeutic candidate. Erectcyanthins A and C exhibited slightly weaker activity, but all three shared a common structural scaffold useful for structure–activity relationship analysis.⁷⁸ In addition, eutypoid F 101, a new butenolide derivative isolated from the sponge-derived fungus Penicillium sp. SCSIO 41413 collected from the Beibu Gulf, exhibited potent phosphatidylinositol 3-kinase (PI3K) inhibitory activity (IC₅₀ = 1.7 μM). Molecular docking studies supported its binding to the PI3K catalytic site, suggesting its mechanism of action and potential as an anticancer lead compound.⁹⁰

1.2.6. Other functional activities. Several marine sponge-derived natural products have shown unique biological activities beyond conventional categories such as antimicrobial, cytotoxic, or neuroactive effects. These include anti-osteoclastic, fluorescence, and antifouling properties, which expand the functional diversity of sponge-derived metabolites. Aaptodines A–D 102–105, hybrid alkaloids with a haptacyclic spiro naphthyridine-furooxazoloquinoline scaffold isolated from Aaptos suberitoides, were reported to inhibit RANKL-induced osteoclastogenesis. Among them, aaptodine D exhibited the strongest activity by significantly reducing TRAP-positive multinucleated osteoclasts and resorption pits in a dose-dependent manner (ED₅₀ = 2.3 μM) without cytotoxicity, suggesting its potential application in bone disease therapeutic.⁹¹

Tedaniophorbasins A 106 and B 107, isolated from the marine sponge Tedaniophorbas ceratosis, represent a structurally distinct class of pteridine alkaloids incorporating a rare thiomorpholine ring, a feature not previously observed in marine natural products.⁹² Tedaniophorbasin A possesses a 2-imino functional group, while B is its corresponding 2-oxo derivative, and both structures display substitution at the C-7 position, a modification unprecedented among known pteridines of natural origin. Although these compounds exhibited no detectable activity in antimalarial, cytotoxic, or antitrypanosomal assays at concentrations up to 40 μM, their strong yellow colouration and pronounced UV absorbance prompted further analysis of their photophysical behaviour. Both tedaniophorbasins showed striking cyan fluorescence with dual excitation wavelengths (λ_ex = 280 and 419 nm) and a shared emission maximum at 490 nm, accompanied by large Stokes shifts (∼14 000 cm⁻¹). These properties point to notable electronic transitions and potential excited-state rearrangements. The combination of broad UV absorption and intense visible fluorescence suggests that these metabolites may function in roles beyond direct antimicrobial defence. Specifically, their photophysical traits support potential ecological functions such as UV shielding, serving as biological sunscreens, or as donor luminophores involved in marine bioluminescence systems. These findings underscore the importance of investigating not only bioactivity but also physicochemical traits when evaluating the functional diversity of sponge-derived natural products.

9,11-Dihydrogracillinone A 108, a nor-diterpenoid from the Antarctic sponge Dendrilla antarctica, showed potent antifouling activity in a 90-day marine field assay. When incorporated into rosin-based paint at 25 mg%, it reduced macrofouling organism coverage on submerged panels to <10%, compared to ∼80% in the control group. This result demonstrates its potential as an eco-friendly antifouling agent for marine applications.⁹³

1.3. Trends in sponge-associated NP research

Over an extended period, natural product research from marine sponges has focused mainly on identifying novel compounds with biological activity, particularly through compound-first, bioactivity-guided isolation.^95,96 While this approach has yielded a remarkable array of structurally diverse metabolites, it presents several inherent limitations: the true producers of many compounds remain unknown, yields are often low and irreproducible, and many sponge-associated microorganisms remain uncultured.⁹⁵ These factors hinder sustainable supply, biosynthetic understanding, and the translation of promising metabolites into drug candidates.

In response to these challenges, the research focus is gradually shifting from compound-centred discovery toward microbe-centred and gene-guided strategies. Key developments include improved methods for separating and cultivating sponge symbionts, genome-based tools for predicting BGCs, and chemical-based approaches such as in situ detection and metabolomics.^20,97,98 These methods are increasingly used together to improve the reliability and scope of natural product discovery.^99–102

Recent reviews have provided valuable insights into the structural diversity and biological activities of sponge-associated microbial metabolites, with particular attention to antimicrobial compounds and the taxonomic classification of their microbial sources.^103,104 Some have also discussed cultivation strategies or genome-based discovery in specific contexts.^105,106 However, these aspects have typically been treated separately, and a comprehensive perspective that integrates the full range of methodologies, from microbial separation to chemical-based detection, remains lacking.

This review builds on previous efforts by outlining how evolving methodologies such as microbial separation, cultivation, genome-based discovery, and chemical approaches are being applied in a complementary way. Instead of focusing solely on compound inventories, it brings together these strategies to provide a broader understanding of how sponge-associated microbial natural products are explored today.

2. Separation of bacteria from sponges

2.1. Importance of separating sponge-associated bacteria

Marine sponges form complex ecological networks through symbiosis with diverse microbial communities. Symbiotic microorganisms play essential roles in the survival of sponges, contributing to key biological functions such as pathogen suppression, nutrient cycling, and the production of bioactive compounds.^8,107 To accurately evaluate the physiological roles and ecological contributions of symbiotic microorganisms, it is essential to separate sponge-associated microorganisms from sponge hosts. The separation of symbiotic microorganisms from sponges is crucial for several reasons. First, separating microbial communities from sponges is essential for accurately identifying the producers of natural products. While most sponge-derived natural products are known to be produced by symbiotic microorganisms, there is an ongoing debate about whether certain compounds originate from the sponge itself or its symbiotic microbes.¹⁰⁸ Clarifying the origin of these compounds is crucial for the sustainable development of sponge-derived drugs and the establishment of stable supply chains for future drug discovery. For instance, swinholide A, a compound with potent cytotoxic activity, was discovered in Theonella swinhoei. A study using density gradient centrifugation revealed that swinholide A was detected exclusively in fractions containing unicellular heterotrophic bacteria rather than in sponge tissue.¹⁰⁹ This suggests that swinholide A originates from symbiotic bacteria rather than the sponge itself. This demonstrates that separating microbial communities is crucial for accurately determining the true producers of bioactive compounds. Second, separating microbial communities from sponges is essential for accurately evaluating the ecological roles of sponge-associated microorganisms. Sponges derive 56–99% of their daily carbon intake from dissolved organic matter (DOM),^110–116 making them significant contributors to marine carbon cycling and important ecological components of aquatic ecosystems worldwide. Sponge-associated microorganisms also play essential roles in nutrient cycling and nitrogen retention, making them crucial subjects of study for understanding the ecological functions of sponges.^117–121 Various studies have attempted to evaluate the individual contributions of sponge host cells and microbial symbionts by measuring DOM assimilation using stable isotope probing of phospholipid fatty acids (PLFAs) without separating the sponge and its microbial symbionts.^122,123 However, this approach only measures the fraction of DOM incorporated into PLFAs and fails to account for the total DOM assimilation. Recently, a study employed physical separation of sponges and their microbial symbionts to quantify DOM uptake, revealing biomass. In contrast, microbial symbionts recycle nitrogenous waste produced by the host, playing a complementary role in nutrient cycling.¹²⁴ In conclusion, separating microbial communities enables researchers to accurately identify microbial taxa and their associated metabolic pathways, as well as to better understand the complexity of nutrient exchange that sponge host cells primarily assimilate DOM into cellular processes. This, in turn, facilitates a deeper understanding of the impact of sponge-microbe symbioses on marine biogeochemical cycles.

Finally, ensuring the reliability of metagenomic analysis of sponge-associated microbial communities requires minimizing contamination from host DNA and unrelated microbial DNA. Schmieder and Edwards (2011) pointed out that host-derived DNA acts as a major noise source in metagenomic data analysis, compromising the accuracy of similarity assessments and functional pathway interpretations.¹²⁵ Although they demonstrated that host DNA can be removed using the bioinformatics filtering tool DeconSeq, they indirectly suggested that the physical separation of host tissues and symbiotic microbes during sampling is essential for fundamentally improving data quality. Knights et al. (2011) further supported this view by showing that data refinement processes, such as host DNA removal, significantly enhance the accuracy of metabolic pathway analysis and similarity assessments of microbial communities.¹²⁶ They reported that datasets excluding host DNA more clearly revealed the structure and contributions of metabolic pathways. Pereira-Marques et al. (2019) reported that a high proportion of host DNA reduces the sensitivity of metagenomic sequencing, making it difficult to detect low-abundance microbial species.¹²⁷ Marotz et al. (2018) demonstrated that the chemical removal of host DNA increases the microbial DNA ratio, improving analytical accuracy.¹²⁸ These studies emphasize that combining physical separation with chemical host DNA removal during the sample preparation stage critically enhanced data quality. In conclusion, bioinformatics filtering and physical host DNA removal play complementary roles in metagenomic data analysis. By thoroughly excluding host DNA or combining physical and chemical separation methods during the initial sampling stage, researchers can reduce data noise and more clearly evaluate microbial communities' genetic contributions and metabolic pathways.

Separating microbial communities from host tissues in sponges is a critical step for the success of natural product discovery, ecological role research, and advanced genomic analysis. This separation improves data reliability, facilitates the interpretation of complex ecological networks, and maximizes the potential value of sponge-associated microorganisms. Fig. 1 illustrates an overview of the major microbial separation techniques used in recent studies, highlighting their key steps and methodologies.

Fig. 1 Methods for isolating sponge-associated bacteria. This figure illustrates various approaches used to isolate sponge-associated bacteria following the physical disruption of sponge tissue. Simple methods allow for the preliminary separation of microbial cells from sponge debris. Density gradient centrifugation methods utilize solutions like Ficoll, Percoll, and Urografin to achieve finer separation. Flow cytometry-based cell sorting enables the separation of microbial population on size, complexity, and fluorescence characteristics, making it particularly useful for isolating intracellular symbionts residing within sponge cells. Magnetic-based cell sorting further enhances specificity by using magnetic nanoparticle-conjugated antibodies or DNA probes to capture target bacteria selectively. Created with https://biorender.com/.

2.2. Simple methods for isolation and its limitations

Physical disruption, filtration, and centrifugation are widely used methods for isolating symbiotic microorganisms from sponge tissues. These methods are simple, cost-effective, and require no specialized skills, making them highly accessible and among the most commonly used techniques in sponge symbiont research. This approach typically involves cutting sponge tissues into small pieces and homogenizing them using a blender or a mortar and pestle. For example, in studies on Aplysina aerophoba and Dysidea avara, homogenized samples were filtered through a 100 μm nylon mesh to remove large tissue fragments. Low-speed centrifugation (770g or 1100g) was then used to pellet sponge cells, resulting in microbial cell fractions with over 99% purity.¹²⁹ Similarly, in a study on Cymbastela concentrica, sponge tissues were cut into ∼1 cm³ pieces, homogenized, and filtered through a 125 μm metal sieve to remove large debris. Subsequently, centrifugation at 100×g for 15 minutes eliminated the remaining sponge cells. Additional filtration through 11 μm and 3 μm filters captured microbial-sized particles, and high-speed centrifugation concentrated the bacterial cells into high-purity fractions.¹³⁰

Following physical disruption and filtration, stepwise centrifugation further enhances the purity and recovery of microbial communities. In a study on Halichondria okadai, stepwise centrifugation (500g, 1000g, 3000g, 8000g) progressively removed sponge cells and enriched microbial fractions, reducing host-derived DNA to 0.02%. Metagenomic analysis confirmed that the enriched pellet was predominantly composed of bacteria.¹³¹ Similarly, in research on Discodermia dissoluta, collagenase treatment (500 μg mL⁻¹) was employed to digest sponge tissues enzymatically, followed by blending and a combination of stepwise centrifugation and filtration, successfully increasing the purity of microbial communities.¹³²

However, these simple methods have several limitations. The heat generated during physical disruption may negatively affect certain sensitive microorganisms, potentially hindering their cultivation. Excessive pressure during filtration can also cause microbial cell damage or loss. Furthermore, while stepwise centrifugation provides high-purity fractions, it cannot completely remove sponge cells, leaving trace amounts of host contaminants (e.g., host DNA) that may introduce inaccuracies into analysis results. Despite these limitations, these methods remain a primary approach in sponge symbiont research due to their simplicity and ability to handle large sample volumes efficiently.

2.3. Density gradient centrifugation method

Sponges host diverse microorganisms, which can be categorized into intracellular symbionts residing within sponge cells and extracellular symbionts located outside the cells. Sponges consist of major cell types, such as archaeocytes and choanocytes, that harbor intracellular symbionts. Density gradient centrifugation is commonly employed to distinguish these microbial communities. This method isolates cells based on their physical properties, such as density and size, enabling effective separation of each cell type and facilitating independent studies of microbial communities associated with specific sponge cell types.^7,133

Density gradient media such as Ficoll, Percoll, and Urografin are widely used for sponge cell fractionation, as they create gradients with varying densities and viscosities, enabling the effective separation of sponge cells and symbiotic microorganisms. Among these, Ficoll is particularly favoured in sponge research due to its ability to form stable density gradients while minimizing cell damage.^133–138 A study on Euryspongia arenaria demonstrates the application of Ficoll-based density gradients.¹³³ In the experiment, differential centrifugation at 100×g was first performed to remove most extracellular microorganisms, followed by a gradual increase in centrifugal force to fractionate the microbial community. Subsequently, Ficoll density gradient centrifugation was used to isolate archaeocytes from the 20–30% Ficoll layer and choanocytes from the 10–20% Ficoll layer. Each cell type was fractionated with over 70% purity, and DNA sequencing analysis of these fractions revealed that archaeocytes and choanocytes harbored distinct microbial communities. Specifically, Chlamydiae, Spirochaetes, and Candidatus Tectomicrobia were detected in the archaeocyte fraction, suggesting that these bacteria are likely endosymbionts. In contrast, a distinct microbial community was also observed in the choanocyte fraction, but it did not include the specific bacterial taxa found in archaeocytes. These findings suggest that each sponge cell type maintains a unique symbiotic microbial community and may have functionally distinct roles.

Percoll consists of colloidal silica particles coated with polyvinylpyrrolidone and can maintain its density gradient even under high-speed centrifugation, making it useful for specific cell fractionation studies. Due to these properties, Percoll may be a better choice than Ficoll for fractionation based on cell size and density differences.^139,140 This approach was applied in a study on Haliclona sp., where Percoll density gradient centrifugation was used to effectively fractionate sponge cells and symbiotic microorganisms.¹³⁹ The research team applied Percoll density gradients to separate nine major cell layers (A1–A9) based on size and density differences. Transmission electron microscopy (TEM) analysis revealed that layers A1–A4 primarily contained sponge cells, including choanocytes, archaeocytes, and mesohyl cells, whereas layers A5–A9 were composed mainly of symbiotic dinoflagellates and nematocysts. Chemical analysis of each cell layer showed that the cytotoxic alkaloids haliclonacyclamines A and B were detected exclusively in layers A1–A4, while no traces were found in the symbiotic dinoflagellate-containing layers A5–A9. Based on these findings, the researchers concluded that the cytotoxic alkaloids of Haliclona sp. are more likely to originate from sponge cells rather than symbiotic dinoflagellates. These results suggest that density gradient centrifugation can serve as a crucial tool for accurately identifying the producer cells of specific secondary metabolites.

A study has applied an approach utilizing the sequential use of Ficoll and Percoll to perform both initial fractionation and fine-scale fractionation. In the study by Flowers et al. (1998), a two-step density gradient centrifugation method incorporating Ficoll and Percoll was employed to achieve a higher-purity separation of sponge cells and the symbiotic cyanobacterium Oscillatoria spongeliae in Dysidea herbacea.¹³⁸ They first applied a discontinuous Ficoll density gradient ranging from 15% to 46%, fractionating the sample into seven distinct cell layers (A1–A7). Subsequently, layers A4–A7, which were identified to contain cyanobacteria, were further purified using a Percoll density gradient (20–80%), yielding four additional major cell layers (B1–B4). Upon further fractionation with the Percoll gradient, the B4 layer still contained some sponge cells, whereas layers B5–B7 exhibited a higher purity of cyanobacteria. Notably, in layers B6 and B7, O. spongeliae was fractionated with over 99% purity. These findings demonstrated that an initial fractionation using Ficoll followed by additional purification with Percoll enables the separation of sponge cells and symbiotic microorganisms with higher precision.

Urografin is an iodinated contrast agent with high density and viscosity, which can enhance the stability of density gradients due to its high viscosity. It can also be used as a density gradient medium for the separation of cells and bioactive compounds. Song et al. (2011) utilized Urografin density gradient centrifugation to study the intracellular localization of the bioactive compounds debromohymenialdisine (DBH) and hymenialdisine (HD) in Axinella sp. The research team first performed an initial cell fractionation using differential centrifugation, followed by the application of a 10–30% Urografin density gradient to separate four major cell layers (C1–C4).¹⁴¹ TEM analysis revealed that the C1 layer was primarily composed of dark brown spherulous cells. High-performance liquid chromatography analysis showed that DBH (10.9%) and HD (6.1%) were detected at the highest concentrations in the C1 layer. Based on these findings, the researchers suggested that spherulous cells serve as the primary storage sites for DBH and HD. This demonstrates that Urografin can be used as an important tool for understanding the cellular localization and origin of metabolites.

Studies utilizing density gradient centrifugation have contributed to a more detailed understanding of sponge cells and their symbiotic microorganisms. As various density gradient media and methods continue to be applied, research on sponge-microbe interactions is expected to become more refined in the future.

2.4. Targeted cell sorting by using flow cytometry

Flow cytometry can be an effective technique for separating sponge cells and targeting symbiotic microorganisms. This method analyses and isolates individual cells based on differences in forward scatter (FSC) and side scatter (SSC) signals. These vary according to cell size, internal complexity, and fluorescence expression as cells pass through a laser beam in a fluid stream. This process is refined through fluorescence-activated cell sorting (FACS), which directly separates cells exhibiting specific fluorescence signals, enabling the high-purity isolation of target cell populations.¹⁴²

Mukherjee et al. (2015) utilized flow cytometry to classify cells from the sponge Eunapius carteri into three major cell populations (P1, P2, P3) based on cell size and internal complexity.¹⁴³ The analysis revealed that the P1 group consisted of small cells such as choanocytes, small amoebocytes, and blast-like cells, while the P2 group comprised large amoebocytes and archaeocytes characterised by their complex internal structures. The P3 group included granular cells and sclerocytes, distinguished by their highly complex internal structures and numerous vesicles. Notably, the researchers observed the production of superoxide anions (O₂⁻) and NO in the P2 group, indicating strong immune responses. This finding suggests that these cells play a critical role in maintaining symbiotic microbial populations while eliminating external pathogens.

In another study, sponge cells and the symbiotic bacterium O. spongeliae from D. herbacea were separated using flow cytometry.¹⁴⁴ The researchers obtained initial cell pellets from sponge tissues through centrifugation and successfully isolated O. spongeliae with over 95% purity using flow cytometry based on the natural fluorescence signal of phycoerythrin exhibited by the symbiotic cyanobacterium. Organic solvent extraction of the isolated symbiont fraction revealed that the major brominated metabolite, 2-(2′,4′-dibromophenyl)-4,6-dibromophenol, was detected exclusively in this fraction. These findings indicate that this metabolite is derived from the symbiotic cyanobacterium O. spongeliae.

In conclusion, these studies demonstrated that flow cytometry is a powerful tool for leveraging natural fluorescence signals to achieve high-purity separation of symbiotic microorganisms from marine sponges and to facilitate efficient research on their metabolites.

2.5. Targeted cell sorting by using magnetic-activated cell sorting

Magnetic-based cell separation techniques can separate and enrich a wide range of substances, from ions to cells. These techniques are relatively simple and cost-effective, making them an alternative to flow cytometry, which requires expensive equipment and highly skilled operators.^145–148 While direct applications of magnetic-based separation in sponge symbiotic microorganism research have not yet been reported, magnetic-based separation holds significant potential for high-efficiency selective cell sorting and is therefore introduced in this section as a promising approach.

Magnetic-activated cell sorting (MACS) is a technique that uses magnetic nanoparticles to bind to cell surface antigens and employs an external magnetic field to retain the labeled cells in a magnetic column (e.g., MACS® Column), allowing for the separation of target cells.^149,150 A study applied MACS with pipette tips to demonstrate its potential for bacterial separation.¹⁵¹ Researchers designed a highly magnetized 3D-printed pipette tip (MACS Tip) using commercial nickel mesh and neodymium magnets. They coated magnetic nanoparticles with mannose-binding lectin, which specifically binds to mannose residues on the lipopolysaccharides (LPS) of the E. coli outer membrane. By passing the bacterial mixture through the MACS Tip, selective separation of E. coli was achieved with a capture efficiency of approximately 90.5% ± 7.3%. These results demonstrate that magnetic-based cell separation techniques can exhibit excellent performance in removing non-target bacteria and enabling selective cell sorting. However, if target microorganisms lack specific antigens on their cell surface or possess extracellular capsules, antibody-based targeting can be challenging. Furthermore, non-specific binding and the presence of matrix contaminants may reduce the efficiency of MACS.

To overcome these limitations of MACS, magnetic probe-based cell fishing was developed.¹⁵² Unlike traditional MACS, this technique targets intracellular genetic sequences rather than cell surface antigens for selective bacterial separation. Researchers targeted the 23S rRNA sequences of E. coli DH10B and Acinetobacter sp. ADP1 to generate biotin-labeled RNA probes, which were hybridized in situ with intracellular ribosomal RNA. Streptavidin-coated paramagnetic beads were then added to bind with biotin, imparting magnetism to the cells. These magnetized cells were introduced into a magnetic cell sorting device containing NdFeB films. Through this process, E. coli DH10B, initially at a concentration of only 0.04%, was enriched to a high purity of 98.46%, with a cell recovery rate of approximately 50%. Similarly, Acinetobacter sp. ADP1 was enriched to a purity of 85%, with a recovery rate of around 40%. These results indicate that magnetic probe-based cell fishing significantly improves both cell recovery and selective separation efficiency compared to traditional MACS technologies. This study highlights that magnetic cell sorting, when applied using nucleic acid-targeted approaches, can achieve higher sensitivity and accuracy than antibody-based MACS. However, the requirement for cell fixation during in situ hybridization of RNA probes limits its applicability to live cell separation. Therefore, this technique is a useful tool for genome-based sponge symbiotic microorganism research and can be complementarily used with antibody-based MACS, depending on research objectives. The various microbial separation techniques discussed in Section 2 are summarized in Table 2.

Table 2 Comparison of microbial separation techniques from marine sponges

Method	Principle	Advantages	Limitations
Simple physical methods	Physical breakdown of tissue and stepwise separation by size/density	- Easy and cost-effective	- Possible microbial cell damage
		- No specialized equipment required	- Residual host contamination
		- Handles large sample volumes	- Limited separation precision
Density gradient centrifugation	Separation based on cell density in a stable gradient medium	- Enables high-resolution fractionation	- Requires gradient optimization
		- Reduces host DNA contamination	- Lower throughput
		- Allows cell-type-specific analysis	- Specialized reagents needed
Flow cytometry	Cell sorting using FSC/SSC and fluorescence signals	- High precision and purity	- Expensive instrumentation
		- Enables live cell separation	- Requires trained personnel
		- Useful for functionally distinct populations	- Often needs fluorescent labeling
Magnetic-activated cell sorting	Magnetic labeling of target cells using antibodies or RNA probes	- Selective and high-purity enrichment	- Requires target antigen or sequence
		- Relatively low-cost and scalable	- Cell fixation may be needed (for RNA probes)
		- Applicable to genome-level studies

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3. Cell culture attempts for sponge-associated bacteria

As previously mentioned, sponge-associated microorganisms are considered key producers of various bioactive compounds, making the cultivation of sponge-associated bacteria one of the most direct and essential approaches for obtaining natural products.^8,153–155 These symbionts depend on sponge-derived nutrients and chemical signals, allowing them to thrive in complex microenvironments. However, replicating their natural living conditions in the laboratory remains challenging. Due to these difficulties, current cultivation success rates are typically reported to range between 0.1% and 14%, significantly limiting the practical utilization of these valuable microbial strains.^156–158 To overcome these obstacles, researchers are actively developing innovative cultivation methods and tailored media formulations to improve the recovery and cultivation of new microbial strains. This section will introduce these cultivation methods and novel strategies to improve cultivation (Fig. 2).

Fig. 2 Various cultivation methods for sponge-associated microorganisms. In vitro cultivation included fine control of medium composition and floating filter cultivation using polycarbonate membranes to enhance microbial growth. In situ cultivation using diffusion growth chambers, I-tip for microbial enrichment from sponges, and chip-based systems (iChip, cChip) that enable selective microbial growth through controlled environmental exposure. Microcapsule-based cultivation, including culture columns and MD-Pod systems, encapsulates single microbial cells in microbeads, allowing stable interaction with external growth factors while minimizing competition with other microbes. Created with https://biorender.com/.

3.1. Fine control of medium composition (in vitro cultivation methods)

3.1.1. Effects of low-nutrient media. Several studies have demonstrated the effectiveness of low-nutrient media and sponge extract-based media in enhancing microbial diversity. The marine environments where sponges thrive are generally nutrient-poor, and this favours the dominance of slow-growing bacteria. For example, Sipkema et al. (2011) utilized low-nutrient media supplemented with sponge extracts and siderophores to isolate rare bacteria, such as Planctomycetes and Verrucomicrobia.¹⁵⁷

Similarly, Esteves et al. (2016) replicated the natural sponge environment using diluted marine broth and sponge extracts to create nutrient conditions that mirror the sponge habitat.¹⁵⁹ Building on earlier findings that anaerobic metabolism, such as denitrification, and ammonium oxidation are prevalent in sponges,¹⁶⁰ this study employed nitrate (NO₃⁻)-enriched media for anaerobic cultivation and ammonium (NH₄⁺)-added media for aerobic cultivation. As a result, microorganisms involved in nitrogen cycling, including bacteria known for nitrate reduction such as Photobacterium and Desulfovibrio, were successfully isolated.

Lavy et al. (2014) applied high-nutrient and low-nutrient media under various oxygen conditions to observe cultivation efficiency.¹⁶¹ Their results showed that 34% of OTUs grew in high-nutrient media, 49% in low-nutrient media, and 17% in both, indicating higher cultivation efficiency in low-nutrient media.

These findings indicate that low-nutrient media, those tailored to specific metabolic pathways, are crucial for enhancing the cultivation success of diverse sponge-associated bacteria.

3.1.2. Use of selective antibiotics and growth enhancers. The use of selective antibiotics and growth promoters during the cultivation process can suppress the growth of fast-growing dominant bacterial strains and effectively isolate rare, slow-growing microorganisms. In particular, inhibiting the rapid proliferation of Proteobacteria allows for the more effective isolation of Actinobacteria, a major microbial group involved in natural product production.

Selvin et al. (2009) incorporated sponge extracts, antibiotics, and signal molecules such as N-acyl-homoserine lactones (GMDs) into actinomycete-selective media to isolate Actinobacteria.¹⁶² Among these, AHLs function as quorum-sensing molecules, activating microbial metabolic processes, regulating growth, and providing a competitive advantage to slow-growing bacteria. The application of these cultivation conditions significantly increased the diversity of isolated strains, with Actinobacteria accounting for 46% of all isolates.

Versluis et al. (2017) applied 13 individual antibiotics or antibiotic combinations to inhibit the growth of certain Proteobacteria strains and successfully isolated 27 previously uncultured OTUs.¹⁶³ Among them, 14 belonged to the Actinobacteria group, suggesting that selective antibiotic pressure can facilitate the isolation of specific bacterial taxa.

Montalvo et al. (2014) utilized various culture media and antibiotics to isolate 434 pure bacterial strains from Xestospongia muta and Xestospongia testudinaria, identifying over 90 bacterial species.¹⁵⁸ To maximize the diversity of culturable microorganisms from sponge tissues, the researchers applied 18 different culture media, with some media supplemented with selective antibiotics (e.g., cycloheximide, nalidixic acid, nystatin) to regulate the growth of specific bacterial groups. Through these methods, a diverse microbial community, including Actinobacteria, Firmicutes, and Proteobacteria, was successfully cultivated, with Actinobacteria exhibiting the highest diversity.

3.1.3. Application of environmental factors and specialized media. Integrating environmental factors is a crucial strategy for selectively cultivating microorganisms with specific metabolic capabilities. For example, Keren et al. (2015) designed an arsenic-enriched medium (AsO₄³⁻ and AsO₃³⁻) to mimic the high arsenic accumulation characteristic of T. swinhoei.¹⁶⁴ In this study, the researchers first fractionated sponge cells and bacterial cells and found that arsenic was present in both fractions, though it accumulated at higher concentrations in the bacterial fraction. These results suggest that sponge-associated microorganisms may play a significant role in arsenic accumulation processes. To better replicate the natural sponge environment in the laboratory, the researchers formulated various culture media incorporating sponge skeletons and arsenic. By applying seven different culture media, they successfully isolated 54 bacterial strains, which were classified into 15 OTUs. Among these, 20 OTUs exclusively grew in media containing sponge skeletons, suggesting that certain microorganisms may possess physiological traits associated with sponge structures. This study highlights the importance of integrating environmental factors, such as sponge skeletons and chemical gradients, in the cultivation of sponge-associated microorganisms, demonstrating their effectiveness in isolating bacteria with unique physiological adaptations.

3.1.4. Insights from genome-based studies. Genome-based studies help improve the cultivation of sponge-associated microorganisms by revealing their metabolic needs and guiding the creation of suitable culture conditions. These approaches go beyond conventional methods, uncovering key metabolic pathways and environmental adaptation mechanisms of unculturable sponge symbionts, thereby supporting the development of more specialized and effective cultivation strategies.

For example, the major sponge symbiont Poribacteria possesses genes required for CO₂ fixation via the Wood–Ljungdahl pathway, suggesting its adaptation to carbon-limited environments.¹⁶⁵ Based on this, culture media with high CO₂ concentrations or low organic carbon content can be designed to promote its growth. Similarly, the sponge-associated archaeon Cenarchaeum symbiosum contains genes related to ammonia oxidation and CO₂ fixation.¹⁶⁶ This indicates that ammonia-supplemented media under low-oxygen conditions may facilitate its growth.

Furthermore, genomic analysis results of the sponge symbiotic cyanobacterium “Candidatus Synechococcus spongiarum” across three lineages revealed an abundance of genes related to DNA modification and recombination compared to free-living cyanobacteria, while genes associated with inorganic ion transport, metabolism, cell wall biosynthesis, and signal transduction were reduced.¹⁶⁷ Decreasing antioxidant enzymes and low-weight peptides in Photosystem II suggest unique physiological adaptations, such as siderophore-mediated iron transport and external methionine dependency. Consequently, culture conditions enriched with iron and supplemented with methionine may enhance their growth. Genomic analysis of sponge-associated Entotheonella spp. indicates that these microorganisms are likely mixotrophs, capable of utilizing various carbon sources but synthesizing only a limited set of proteinogenic amino acids (Cys, Met, Lys, Thr, Ser). Therefore, growth conditions may need to include diverse carbon sources and free amino acids to accommodate their metabolic characteristics. Additionally, their abilities for anaerobic respiration and sulfate reduction suggest that providing sulfate and maintaining controlled oxygen levels or anaerobic conditions could enhance their cultivation success.

Metagenomic studies of A. aerophoba revealed microbial groups enriched in genes associated with carnitine and sulfated polysaccharide metabolism, components abundant in the extracellular matrix of sponges.¹⁶⁸ Incorporating carnitine and sulfated polysaccharides into culture media could support the growth of sponge symbionts reliant on these resources.

To date, there have been few cases where genome-based research has directly led to the successful cultivation of sponge-associated microorganisms. However, genomic analysis provides crucial insights into their metabolic pathways, environmental adaptation mechanisms, and nutritional requirements. This information can help overcome the limitations of traditional cultivation methods and suggests the potential for designing tailored cultivation strategies based on specific metabolic traits. Future research should integrate experimental validation with genomic data to effectively enhance the cultivation success of sponge-associated microorganisms.

3.1.5. Floating-filter cultivation. The floating-filter cultivation method involves inoculating microbial cells onto a filter with a fine pore size and suspending the filter on the surface of a culture medium. The filter provides a growth surface for the cells while maintaining contact with the medium to ensure nutrient availability. Unlike conventional in vitro cultivation techniques conducted entirely under controlled laboratory conditions, this method partially simulates environmental exposure by allowing microbial cells to interact with diffusible factors across the medium–air interface.

Sipkema et al. (2011) applied the floating-filter cultivation method to culture sponge-associated microorganisms derived from Haliclona sp.¹⁵⁷ They used black polycarbonate filters (0.1 μm pore size) and observed that two types of colonies formed on the filter: macrocolonies and microcolonies. While microcolonies were barely visible to the naked eye, they accounted for approximately 80% of the total colonies, indicating a high frequency of occurrence. A total of 60 OTUs were isolated using the floating-filter method, among which 23 OTUs had not been cultured previously using conventional solid or liquid media.

Wiegand et al. (2018) successfully applied the floating-filter cultivation method to isolate microorganisms belonging to the Planctomycetes phylum from marine and freshwater environments.¹⁶⁹ The researchers placed culture media in six-well culture plates and used black polycarbonate filters with a 0.1 μm pore size to culture microorganisms in a floating state. The culture media contained various antibiotics, including cycloheximide, carbenicillin, and streptomycin, which were used to inhibit the growth of fast-growing bacteria and selectively isolate specific microorganisms. Using this approach, the researchers isolated four novel species of Planctomycetes, which were previously unculturable using conventional methods. 16S rRNA gene sequencing confirmed that these isolates represented previously unreported microbial species.

These studies suggest that the floating-filter cultivation method can serve as an alternative approach for isolating microorganisms that cannot be cultured using traditional methods. In particular, this method may be a valuable tool for studying the physiological characteristics of specific microbial groups, such as Planctomycetes.

3.2. In situ cultivation methods

Researchers have developed in situ cultivation techniques that utilize natural habitats to overcome the limitations of laboratory-based cultivation methods. This approach directly places cultivation devices in marine ecosystems, allowing essential factors such as microbial interactions, nutrients, and signalling molecules to be maintained, providing optimal growth conditions for previously uncultivable bacteria.

3.2.1. Diffusion growth chambers. Kaeberlein et al. (2002) designed a diffusion growth chamber using two polycarbonate membranes with a pore size of 0.03 μm to more effectively cultivate environmentally challenging microorganisms.¹⁷⁰ This chamber employed a structure where the membranes were secured using circular stainless steel, and the interior was filled with agar medium containing microorganisms. This setup allowed for free chemical exchange with the external environment (diffusion of nutrients and signalling molecules) while preventing the direct movement of microorganisms. In this study, they used the diffusion growth chamber to culture microorganisms derived from intertidal marine sediment. The chamber was placed on the sediment layer inside an aquarium filled with seawater and incubated for one week. As a result, 40 ± 13% of the inoculated cells formed colonies, achieving a maximum recovery rate up to 300 times higher than conventional Petri dish cultivation methods. Additionally, some of the microorganisms cultured within the chamber acquired the ability to sustain growth on petri dishes afterward. This suggests that the diffusion growth chamber provides a favourable environment for the proliferation and isolation of microorganisms that were previously uncultivable using conventional methods.

Bollmann et al. (2007) applied the diffusion growth chamber to culture microorganisms in a freshwater pond sediment rather than a marine sediment environment.¹⁷¹ The chamber used in this study consisted of a 0.03 μm pore-size polycarbonate membrane attached to a stainless-steel O-ring, with an interior filled with agar medium mixed with environmental samples. The chamber was then sealed and placed back into the natural environment for incubation. Over 16 weeks, four generations of diffusion growth chambers were used consecutively. Analysis of the distribution of cultured strains revealed that 59% of the newly isolated strains were detected exclusively through the diffusion growth chamber method. Specifically, 62 species of Alphaproteobacteria, 69 species of Betaproteobacteria, 7 species of Gammaproteobacteria, 1 species of Deltaproteobacteria, 12 species of Bacteroidetes, 5 species of Verrucomicrobia, 2 species of Acidobacteria, 4 species of Spirochaetes, 6 species of Firmicutes, and 6 species of Actinobacteria were successfully cultured. Among these, Verrucomicrobia and Acidobacteria required at least two generations of diffusion growth chambers before they could be successfully cultivated.

Steinert et al. (2014) were the first study to apply the diffusion growth chamber for the cultivation of symbiotic microorganisms within sponge tissue, utilizing a chamber with a structurally distinct design from previous studies.¹⁷² Instead of the stainless-steel ring-based design, this study employed a modified centrifuge microfilter section with an integrated 0.2 μm pore-size filter, allowing direct insertion into sponge tissue. The research team homogenized Rhabdastrella globostellata sponge tissue and introduced a suspension of microbial cells mixed with various concentrations of culture media into the diffusion growth chamber. The chamber was then inserted into the sponge tissue and incubated for four weeks. As a result, a total of 255 sequences of 16S rRNA genes were obtained. Analysis of these sequences revealed the presence of 83 sequences from Alphaproteobacteria, 96 from Gammaproteobacteria, 19 from Bacteroidetes, 14 from Actinobacteria, and 43 from Firmicutes. Additionally, 15 of the sequences represented previously uncultivated strains. This study marked the first case of inserting a diffusion growth chamber into sponge tissue for cultivating sponge-associated microorganisms, demonstrating a novel approach distinct from previous studies based on marine or freshwater sediment environments.

3.2.2. Isolation tip (I-tip). The I-tip method is an innovative in situ cultivation technique developed by Jung et al. (2014) and first applied to freshwater sponges in Lake Baikal.¹⁷³ I-tip uses standard micropipette tips as primary components, creating a simple yet efficient cultivation device that allows microbes to grow by absorbing nutrients and signalling molecules naturally diffused from their habitats. The narrow end of the pipette tip is filled with fine glass beads and agar medium to prevent large particles from entering, while the broader end is sealed with waterproof adhesive. The design enables the device to function as a solid support structure that absorbs environmental nutrients, promoting microbial growth when inserted into sponge tissue. The research team successfully isolated 103 bacterial strains from Baicalospongia sp. and Lubomirskia baicalensis sponges using the I-tip method. Major bacterial phyla identified included Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria. Actinobacteria and Alphaproteobacteria were notably absent in Petri dish cultures but were successfully isolated using the I-tip method. Additionally, another study showed that using I-tip cultivation on the sponge T. swinhoei resulted in higher microbial diversity and the isolation of new species from the Bacteroidetes, Alphaproteobacteria, and Gammaproteobacteria phyla, compared to standard direct cultivation methods. However, some microbial groups such as Cyanobacteria, Planctomycetes, and Verrucomicrobia were rarely isolated, indicating that the I-tip method may require complementary cultivation techniques.¹⁷⁴

3.2.3. Isolation chip (iChip). The iChip system, introduced by Nichols et al. (2010), leverages natural environments as cultivation chambers while physically isolating microbial cells to promote the formation of single-strain colonies.¹⁷⁵ The iChip consists of a central plate containing hundreds of small wells, each serving as a miniature microbial cultivation chamber. Environmental samples are diluted so that approximately one bacterial cell is introduced into each well. The plate is then covered with semipermeable membranes that allow nutrients and growth factors to diffuse from the environment while blocking microbial movement. The device is returned to the natural habitat for incubation.

Nichols et al. (2010) demonstrated that the iChip system enabled the cultivation of over 40% of bacterial cells in seawater samples and over 50% in soil samples.¹⁷⁵ This increase in cultivation efficiency represents a fivefold improvement in microbial recovery rates. Additionally, Berdy et al. (2017) reported that microbial recovery using iChip varied from 5- to 300-fold higher than traditional methods, depending on the specific environmental sample, further emphasizing its effectiveness in cultivating previously uncultivable species.^84,176

The iChip system also supports high-throughput screening by simultaneously cultivating thousands of micro-wells. This feature has proven useful for large-scale antimicrobial activity screening, including the first discovery of the antibiotic teixobactin by Ling et al. (2015).¹⁷⁷ They tested approximately 10 000 microbial isolates obtained from soil samples using the iChip system for activity against S. aureus. This led to the discovery of a new beta-proteobacterium species, Eleftheria terrae, which exhibited strong antimicrobial activity. MacIntyre et al. (2019) demonstrated the successful application of iChip technology to marine sponges, specifically Xestospongia muta.¹⁷⁸ Researchers implanted iChips into sponge tissues and isolated a novel bacterial strain, Alteromonas sp. RKMC-009, which produced a unique N-acyltyrosine derivative with potent antimicrobial properties against Gram-positive bacteria.

Recent advancements have explored combining iChip technology with co-culture techniques to further enhance microbial cultivation. Lodhi et al. (2018) hypothesized that while iChip is effective in isolating single microbial species, some bacteria may depend on metabolic interactions with neighboring species for growth. Accordingly, they proposed that integrating co-culture techniques with iChip could enhance the cultivation of previously unculturable microorganisms by facilitating metabolic interactions between different bacterial species. However, further experimental validation is required to confirm the effectiveness of this approach.¹⁷⁹

3.2.4. Culturing chip (cChip). Based on iChip, Lodhi et al. (2023) developed a cChip for the isolation of microorganisms from the aquatic environment.¹⁸⁰ The cChip was designed to enhance interaction with the environment by adjusting features such as the well diameter, chip thickness, number of membrane barriers, and operating protocol. These modifications create conditions that provide more stable growth in the aquatic environment for bacteria. This system demonstrated approximately five times higher microbial diversity than conventional Petri dish cultures, and a total of 45 new strains were isolated from the cChip. Notably, species such as Verrucomicrobia, rarely detected in standard cultures, were successfully isolated using cChip.

3.3. Microcapsule-based cultivation methods

3.3.1. Culture column with microbial encapsulated microbeads. Microcapsule-based cultivation is a single-cell cultivation system designed to encapsulate microbial cells within semipermeable gel materials such as agarose. This structure protects the cells while allowing the diffusion of nutrients and signal molecules from the external environment. This method addresses limitations of traditional cultivation, such as difficulties in isolating slow-growing strains in mixed cultures dominated by fast-growing microbes, and challenges in cultivating strains requiring essential environmental growth factors. In simple terms, microcapsules are produced using microfluidic devices and emulsion techniques, with each capsule serving as an independent cultivation chamber. Environmental cell samples are mixed with agarose, emulsified in a matrix through a microfluidic device, and formed gel microdroplets (GMDs). These GMDs are immobilized in columns that prevent external contamination while allowing the influx of nutrients and signal molecules for microbial growth.

Zengler et al. (2002) isolated microbial cells from marine samples and encapsulated them individually using agarose gel.¹⁸¹ These cells were cultivated under low-organic medium in a growth column designed to mimic a flow-through system. After cultivation, microcolony included GMDs were discriminated by using flow cytometry. As a result, previously uncultivable microbial lineages such as Planctomycetes and the Cytophaga-Flavobacterium-Bacteroides group were successfully cultivated.

Toledo et al. (2006) separated symbiotic microbial cells from the marine sponge Mycale armata and encapsulated them in agarose-based gel microcapsules.¹⁸² These capsules were cultured for five weeks in media containing sterile seawater diluted with sponge homogenates. Afterward, microcolonies formed within the capsules were isolated using flow cytometry, and Fourier-transform infrared (FT/IR) spectroscopy was employed for dereplication of redundant strains. Ultimately, the microcapsule-based cultivation method demonstrated approximately six times higher novel species isolation efficiency than traditional Petri dish methods, resulting in the discovery of five potential candidate strains which produce novel secondary metabolites.

3.3.2. Microbe domestication pod. Alkayyali et al. (2021) introduced the Microbe Domestication Pod (MD Pod), a system combining microbial encapsulation with in situ cultivation to address the challenges of cultivating hard-to-grow microorganisms.¹⁸³ The MD Pod encapsulates microbial cells in agarose microbeads through a microfluidic process, forming microbeads approximately 80–100 μm in size. These microbeads provide a protective barrier while promoting cell-to-cell interactions and maintaining microbial metabolic activity. To facilitate nutrient exchange and signal molecule diffusion while preventing microbial migration, the MD Pod is equipped with a polycarbonate track etch (PCTE) membrane with 30 nm pores. The semipermeable membrane connects the internal cultivation environment to the external habitat, enabling continuous nutrient supply and natural metabolic signals. Various MD Pod designs were tested, and researchers ultimately selected a flexible MD Pod design due to its quick assembly and durability, making it well-suited for long-term deployments in marine environments. The MD Pod demonstrated up to five times higher microbial diversity compared to traditional cultivation methods, leading to the successful isolation of 45 novel bacterial strains. Notably, previously undetectable microbial species such as Verrucomicrobia were successfully cultivated using the MD Pod, emphasizing its effectiveness in improving microbial recovery from marine environments.

The micron-scale Microbial Domestication Pod (μMD Pod) was developed as an enhanced version of the original MD Pod, enhancing its adaptability for in situ cultivation within small marine invertebrates while maintaining its core design principles.¹⁸⁴ The μMD Pod features porous wall structures with 10 μm pores, fabricated using two-photon polymerization technology. These structures facilitate efficient nutrient and signal molecule exchange, eliminating the need for fragile filter membranes used in the MD Pod. This improvement allows the μMD Pod to perform effectively in challenging environments, such as marine sediments and small invertebrates, where conventional cultivation devices face limitations. Another distinguishing feature of the μMD Pod is its replacement of semipermeable membranes with porous structures, simplifying the microbial encapsulation and nutrient delivery process while maintaining natural interactions with the surrounding environment. In experiments, microbial strains such as Sphingomonas phyllosphaerae and Streptomyces sp. were encapsulated in agarose beads and incubated. Fluorescence microscopy confirmed stable cell growth within the μMD Pod, confirming its viability as an in situ cultivation device. In conclusion, the μMD Pod builds on the MD Pod's foundational concept, enhancing structural durability and cultivation efficiency, thereby broadening the potential for microbial isolation and bioprospecting in previously inaccessible environments.

4. Genome-based approach

Marine sponges harbor diverse microbial communities that play essential roles in the biosynthesis of bioactive secondary metabolites. Advances in genomic technologies have significantly enhanced our ability to investigate the biosynthetic potential of these sponge-associated microorganisms. Due to the challenges associated with cultivating marine microorganisms, genome-based strategies such as metagenomics, single-cell genomics, and heterologous expression have been developed. These approaches enable the identification and functional characterisation of BGCs leading to the discovery of novel natural products with pharmaceutical and biotechnological significance. This chapter discusses these genome-based methodologies and their applications in natural product discovery from sponge-derived bacteria (Fig. 3).

Fig. 3 Genome-based approach for biosynthetic gene cluster (BGC) identification and isolation from sponge-associated bacteria. The process begins with metagenomic DNA extraction or single-cell genome amplification, followed by sequencing, assembly and binning to reconstruct microbial genomes. Identified BGCs are analysed using in silico tools. BGC isolation involves constructing a DNA library and employing in vivo (TAR cloning, DNA assembly, vector integration/excision in Chassis) of in vitro (CATCH, E. coli-based cloning, DiPac, long-amplicon PCR) cloning strategies. BGCs can be introduced into heterologous chassis for expression, facilitating the discovery of novel natural products from sponge-derived microbial communities. Created with https://biorender.com/.

4.1. Metagenome-based NPs biosynthesis gene discovery

Despite continuous advancements in microbial cultivation techniques, more than 99% of all microbial species, including marine microorganisms, remain uncultivable. To overcome this limitation, metagenomics has emerged as a powerful approach for exploring BGCs from marine microbes that are difficult or impossible to culture.¹⁸⁵ By analysing total environmental DNA extracted directly from natural habitats, metagenomics enables the reconstruction of individual microbial genomes and facilitates the identification of gene clusters associated with specific metabolic functions.

Marine environmental DNA can be sequenced using advanced technologies such as next-generation sequencing, PacBio, and nanopore sequencing, generating sequence data that can be assembled into contigs. These contigs are classified based on genomic characteristics such as GC content and codon usage patterns, allowing the reconstruction of microbial genomes. This process, known as binning, plays a crucial role in distinguishing genome fragments belonging to different microbial taxa. The identification of biosynthetic gene clusters through metagenomics is performed using bioinformatics tools such as antiSMASH, PRISM, MetaBGC, SMBP, and NaPDoS. These identified gene clusters can then be compared against large-scale databases such as antiSMASH, IMG-ABC, and MIBiG to analyse homology with known gene clusters and elucidate novel biosynthetic pathways.^186–194

Although the identification of biosynthetic gene clusters from marine microbes remains a challenging task, metagenomics has proven instrumental in the discovery of bioactive natural products derived from marine invertebrates such as sponges, tunicates, and bryozoans.¹⁹⁵ Comparative metagenomics, which involves comparing metagenomic datasets from different environments, is a useful approach for identifying unique biosynthetic gene clusters within specific microbial communities. Recent comparative metagenomic studies have identified 5082 BGCs in high-microbial-abundance sponges, with most of them classified as novel clusters showing no homology to previously known gene clusters.⁹⁷ This study demonstrated that BGCs found in sponges are markedly distinct from those in surrounding marine environments and that certain BGCs are consistently present in specific sponge species. Notably, non-ribosomal peptide synthetase (NRPS) and ribosomally synthesized and post-translationally modified peptides (RiPPs) were found to be predominant, suggesting that these biosynthetic pathways are evolutionarily conserved among sponge-associated microbes.⁹⁷

Targeted metagenomics is another approach that focuses on specific biosynthetic pathways or gene clusters associated with particular natural products, providing an advantage over conventional random metagenomic analysis in identifying functionally relevant biosynthetic gene clusters. This method selectively analyses target genes or pathways of interest, enhancing the identification of biosynthetic origins. It has played a critical role in determining the true microbial producers of marine-derived natural products with previously unclear origins. For example, the anticancer compound bryostatin, originally thought to be derived from the marine bryozoan Bugula neritina, was later confirmed to be produced by the symbiotic bacterium Candidatus Endobugula sertula through targeted metagenomic analysis.¹⁹⁶ Similarly, ET-743 (Yondelis®), a well-known anticancer drug initially attributed to the sea squirt Ecteinascidia turbinata, was later found to be biosynthesized by the symbiotic bacterium Candidatus Endoecteinascidia frumentensis.¹⁹⁷ By using metagenome-based biosynthetic pathway analysis, the complete biosynthetic route of ET-743 was identified, allowing its large-scale semi-synthetic production using an industrial microbial strain. This approach, adopted by the Spanish pharmaceutical company PharmaMar, enabled the commercialization of ET-743 as a novel anticancer drug, demonstrating the potential of metagenomics in natural product discovery. In addition to these examples, patellazole (antifungal), polytheonamide (anticancer), and calyculin A (phosphatase inhibitor) were also identified through targeted metagenomic analysis, significantly expanding our understanding of the complex biosynthetic pathways found in marine microorganisms.^198–200

Currently, metagenomic research on marine-derived natural products is primarily focused on elucidating biosynthetic pathways and analysing functional genes encoding biosynthetic enzymes, yet several major challenges remain. In particular, while metagenomic sequencing has identified numerous biosynthetic gene clusters, a significant proportion of these clusters remain uncharacterised and are classified as silent biosynthetic gene clusters, which are not naturally expressed under standard conditions. To overcome this issue, efforts have been made to optimize heterologous expression systems and apply synthetic biology techniques to activate gene expression.

4.2. Single cell-based NPs biosynthesis gene discovery

In natural environments, uncultivated microorganisms constitute the majority of microbial diversity, and traditional culture-based studies have limitations in fully elucidating their physiological characteristics and genomic information. While microbial communities harbor numerous BGCs, analysing these genes in uncultivable species remains challenging. Conventional metagenomic approaches allow for the broad exploration of genetic diversity within microbial communities but are limited in resolving the genomic context of individual microorganisms. To address this limitation, single-cell genomics has emerged as a powerful approach, enabling the isolation of individual cells via FACS, followed by whole-genome amplification (WGA) and sequencing for BGC analysis. This method provides a means to directly analyse the genomes of individual microbes, overcoming the constraints of traditional techniques.²⁰¹

Kvist et al. (2007) introduced a technique for targeting and isolating single cells from uncultivated microbial populations, followed by whole-genome amplification via multiple displacement amplification.¹⁰⁰ In this study, researchers employed fluorescence in situ hybridization (FISH) to label specific microbial cells, which were subsequently isolated using a micromanipulator, demonstrating the feasibility of genome analysis at the single-cell level even in non-cultivable microbes. While FISH-based cell sorting is effective for targeting specific microbes, it suffers from low throughput. To overcome this limitation, recent single-cell genomic studies have increasingly adopted FACS-based approaches to achieve rapid and high-throughput isolation of individual cells.²⁰²

In a study by Pachiadaki et al. (2019), this method was applied to isolate 12 715 single-amplified genomes (SAGs) from tropical and subtropical marine environments, enabling a systematic analysis of the biosynthetic potential of marine prokaryotes.²⁰³ The study revealed that many SAGs contained diverse BGCs encoding terpenes, bacteriocins, polyketides (PKS), and NRPS, suggesting that marine microorganisms possess significant genetic potential for bioactive compound production. Moreover, single-cell genome analysis allowed for the phylogenetic characterisation of NRPS and PKS clusters, uncovering these biosynthetic pathways in microbial lineages not previously represented in public databases. This finding expands the potential for discovering secondary metabolites from uncultivated marine microbes, reinforcing the value of single-cell genomics as a powerful tool for natural product discovery and biosynthetic pathway analysis.

A more recent study introduced MERMAID (Microfluidic Encapsulation, Raman Microscopy, and Integrated Digital Genomics), a novel method that integrates Raman microscopy with single-cell genomics for the targeted identification of secondary metabolite producers. MERMAID involves encapsulating individual cells within a microfluidic system, analysing their metabolic signatures using Raman microscopy, and selecting specific cells for single-cell genome sequencing. Kogawa et al. (2022) applied this technique to the symbiotic microbiome of the marine sponge T. swinhoei, successfully identifying an aurantoside producer that had previously remained undetected through metagenomic binning.²⁰⁴ This led to the discovery of a novel Chloroflexi symbiont, ‘Candidatus Poriflexus aureus,’ which harbors a 14 Mbp genome enriched with multiple BGCs. Among these, the researchers identified a PKS-NRPS hybrid BGC responsible for aurantoside biosynthesis, along with 19 additional BGCs, underscoring the greater-than-expected chemical diversity within sponge-associated microbiomes.

Compared to metagenomic approaches, single-cell sequencing offers cleaner and less contaminated genomic sequences, facilitating the identification of previously undetected BGCs and enhancing the potential for novel natural product discovery. However, the WGA process introduces risks of DNA loss and amplification errors, and successful cultivation of strains identified through single-cell genomics remains rare. Despite these challenges, the integration of FACS-based single-cell approaches with metagenomics is expected to play a crucial role in advancing NP BGC discovery.²⁰²

4.3. Heterologous expression of NP BGCs

The first step in heterologous expression of NP BGCs is capturing the target BGC. Traditionally, this has been done through cosmid-, fosmid-, or BAC-based genomic libraries, which allow for the isolation of multiple BGCs. More recently, Transformation-Associated Recombination cloning in Saccharomyces cerevisiae has become an attractive option due to yeast's robust recombination capabilities, enabling the cloning of large BGCs exceeding 200 kb. Another method involves integrating E. coli-host shuttle vectors and restriction enzyme sites adjacent to native BGC loci, allowing for precise excision and manipulation of target BGCs. Advances in genetic engineering techniques, including Large PCR, CRISPR/Cas9, Gibson Assembly, and In-Fusion cloning, have further improved the efficiency of direct in vitro cloning, although challenges remain in handling large BGCs.^205,206

Once isolated, BGCs can be expressed in their native form or undergo modifications to improve production yields and generate novel analogues. Engineering strategies involve modifying promoters, regulatory genes, post-modification genes, or even PKS domain structures to optimize BGC performance.^207–209

Actinobacteria, particularly Streptomyces species, are the primary chassis used for NP heterologous expression due to their established genetic tools and secondary metabolite production capabilities. S. coelicolor and S. lividans are widely used model strains, while S. albus and S. venezuelae have gained attention due to their rapid growth and abundant precursor supply.^209,210

To enhance chassis productivity, several modifications are commonly implemented, including the removal of native BGCs to reduce competition for precursors, mutation of ribosome binding sites to increase translation efficiency, elimination of negative global regulators, and introduction of additional attachment sites to amplify BGC copy numbers.

Marine-derived natural products pose unique challenges due to the specialized metabolic systems of their microbial producers. Recognizing this limitation, researchers have developed MGCEP 1.0 (Marine Gene Cluster Expression Platform 1.0), an optimized chassis strain derived from Streptomyces atratus SCSIO ZH16. Expression trials using MGCEP 1.0 successfully produced nineteen compounds, including the novel angucycline compound, demonstrating the effectiveness of marine-specific chassis systems for NP production.²¹¹

Despite these advancements, heterologous expression still faces limitations, especially for the expression of silent or cryptic BGCs from marine microorganisms. As a complementary strategy, BGC awakening approaches have emerged as an alternative to directly induce expression of these clusters. Traditional OSMAC (One Strain, Many Compounds) methods, such as altering salinity, temperature, pH, or co-culturing with other microbes, have shown success in activating silent gene clusters.²¹² Moreover, recent synthetic biology techniques, including promoter refactoring, overexpression of pathway-specific regulators, and chassis optimization, enable targeted activation of these silent BGCs.²¹² Integrating awakening strategies with marine-specific heterologous expression platforms is expected to greatly expand the accessible chemical space of marine-derived natural products, thereby accelerating novel compound discovery.

5. Chemical-based bacterial NPs mining method

5.1. In situ chemical capture using artificial marine sponge devices

An in situ capture method using artificial sponge devices was initially introduced as an alternative to traditional sponge sampling techniques.²¹³ This method allows for the direct collection of bioactive compounds from the environment without microbial cultivation.

La Clair et al. (2014) designed an artificial sponge device that mimics the physical structure and functional properties of natural marine sponges, and deployed it in marine environments for extended periods to successfully capture bioactive compounds.²¹³ The device is centred around a hollow-fiber bioreactor, which provides a surface for microbial colonization and includes a resin (Amberlite XAD-18) capable of adsorbing metabolites. It operates in three main stages: (1) inoculation through the introduction of seawater from sponge-rich environments, (2) incubation for several days in situ, and (3) recovery of the device followed by extraction of adsorbed metabolites from the internal resin. This approach enables the collection of microbial metabolites while preserving the native microbial community, minimizing ecological disturbance, and offering a sustainable strategy for natural product exploration.

A notable compound group collected using this device includes jasplakinolide-class depsipeptides, which were found to have identical structures to those previously isolated from sponges in the same region. This result suggests that device-based strategies can complement or even replace traditional sponge sampling techniques in certain contexts. Such device-based in situ compound capture strategies were later adapted into simpler resin-based systems, as exemplified by technologies like SMIRC.

5.2. Small molecule in situ resin capture

While artificial sponge devices offer a means to collect metabolites from marine environments, their application typically requires complex equipment and external deployment. In contrast, the Small Molecule in situ Resin Capture (SMIRC) method provides a simpler and more direct approach by inserting resin adsorbents into sponge tissue itself, allowing compound capture to occur within the native microenvironment.

SMIRC enables the direct capture of small molecules from marine environments using resin adsorbents, bypassing the need for microbial cultivation (Fig. 4).⁹⁸ This innovative method integrates high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) to identify novel compounds while leveraging genomic tools to explore their biosynthetic origins. In this study, the researchers deployed the resin adsorbent HP-20, known for its efficacy in capturing marine toxins across various polarities, in diverse marine environments. Among the identified compounds were cabrillostatin, which demonstrated selective bioactivity against cancer cell lines, and the newly discovered compounds cabrillospirals A and B, which exhibited no notable bioactivity. To explore potential links between these compounds and corresponding BGCs, the researchers extracted DNA from environmental samples and conducted the untargeted shotgun metagenomic analysis. This approach identified 1843 BGCs, grouped into 631 Gene Cluster Families based on sequence similarity. Many of these BGCs were classified as NRPS, terpenes, and RiPPs, with most representing novel pathways not listed in the MIBiG database. In their search for a PKS-NRPS hybrid BGC potentially responsible for producing cabrillostatin, the researchers examined 43 BGCs with similar characteristics but found no matches capable of synthesizing the same structure. Based on the chemical structure of cabrillospirals A and B, they predicted that its biosynthesis might involve a modular T1PKS, halogenases, a p-hydroxybenzoic acid starter unit, and methyltransferases responsible for the C-2 hydroxymethyl and C-8 methyl groups. Among the 68 T1PKS BGCs identified, two promising candidate genes were found in high-quality MAGs from the phylum Planctomycetes, though cultivation attempts for these Planctomycetes were unsuccessful. These results show the persistent challenges in linking chemical structures to BGCs within complex microbial communities. Although challenges in linking compounds to BGCs remain, SMIRC offers a cultivation-independent framework for discovering chemically diverse metabolites.

Fig. 4 A chemical-based strategy for bacterial NP discovery. This figure illustrates an integrated workflow combining in situ compound capture (artificial sponge devices and SMIRC), MS/MS- and NMR-based metabolomics, and genome mining to discover natural products from sponge-associated bacteria. AI-assisted tools such as SMART 2.0, CANOPUS, NPClassifier, and Chemprop are used to predict compound structures, biosynthetic origins, and bioactivities. Metabolite-BGC linkage is established through platforms including NPLinker, MetaMiner, and NPOmix, enabling the discovery of novel compounds without microbial cultivation. Created with https://biorender.com/.

5.3. Integration of metabolomics and genome mining

Recent studies have begun integrating metabolomics and genome mining to rapidly elucidate the structures of unknown natural products and trace their biosynthetic origins.^214,215 At the core of this integrated strategy lies tandem mass spectrometry (MS/MS)-based metabolomic analysis. Data generated through MS/MS are utilized by platforms such as GNPS (Global Natural Products Social Molecular Networking), MolNetEnhancer, and CANOPUS to cluster compounds, classify metabolic features, and predict structures.^216–218 However, structure prediction alone is insufficient to identify the corresponding BGCs, and therefore, integration with genomic information is essential.

Recently, Louwen et al. (2023) developed NPLinker, a platform designed to facilitate this integration, and NPClassScore, a complementary scoring system that prioritizes meaningful links between MS/MS spectra and BGCs.¹⁰² NPClassScore assigns likelihood scores to BGC-spectrum pairs based on chemical class ontology, thereby reducing false positives and enhancing link prioritization. Notably, NPClassScore quantitatively connects structure-based classifications (e.g., ClassyFire, NPClassifier) with genome-based predictions (e.g., antiSMASH), maintaining 96% of experimentally validated BGC-MS/MS links while reducing the number of candidate links by an average of over 63%, significantly alleviating the burden of manual inspection.

In cases such as RiPPs, where BGC architectures are often atypical and metabolic diversity is high, metabolomics-based information offers critical guidance to complement genome mining. For instance, MetaMiner enables structural-genomic linkage of RiPP compounds by comparing substructures derived from MS/MS spectra with predicted peptide sequences from BGCs.²¹⁹ This tool demonstrates high accuracy in predicting ribosomal core peptides and modification patterns, outperforming traditional spectrum-based comparison approaches. MetaMiner analysed approximately 10 million MS/MS spectra collected from eight diverse environmental sources, identifying 31 known RiPPs and 7 novel RiPPs. Among these, wewakazole was successfully isolated and structurally confirmed via NMR. Notably, in a sponge symbiont dataset derived from Theonella swinhoei, the genome of Candidatus Entotheonella sp. TSY1 was linked with ∼223 000 MS/MS spectra, allowing the identification of several BGCs (including four RiPP-type clusters) and suggesting structural relationships with the observed metabolites.

NPOmix is a k-nearest neighbor-based model that integrates chemical fingerprints and biosynthetic features to predict links between MS/MS spectra and BGCs.²²⁰ Utilizing 1040 paired omics datasets for training, NPOmix accurately predicted 13 MS/MS spectra corresponding to 11 experimentally validated BGC-spectrum links, including structural analogues. The model achieved a 92.9% top-3 accuracy, highlighting its high predictive power. Compounds such as albicidin, its analogues, and brasilicardin A were among those accurately linked, demonstrating the method's robustness.

These integrated approaches are especially effective in exploring unknown metabolites produced by complex microbial consortia such as sponge-associated microbiomes. By leveraging MS/MS data alongside genomic information, researchers can simultaneously infer structural characteristics and biosynthetic origins. Together, these tools enable metabolite-BGC linkage even in microbiomes where cultivation is not feasible.

5.4. AI-driven strategies for natural product discovery

Recent advances in artificial intelligence (AI) are reshaping multiple aspects of natural product-based drug discovery, particularly in chemically focused areas such as metabolomic analysis, structure elucidation, bioactivity prediction, and target identification. Among these domains, AI has demonstrated notable success in addressing the complexities of chemical data interpretation and in accelerating the identification of promising compound leads.

In metabolomics, AI has been instrumental in automating the analysis of large-scale MS/MS datasets and prioritizing compounds from complex mixtures. Tools such as CANOPUS and NPClassifier classify natural products into chemical classes and predict their biosynthetic origins directly from MS/MS spectra, even in the absence of complete structural information.^217,221 These classifiers have been successfully applied to the annotation of novel compounds such as rivulariapeptolide, offering insights into functional roles within structurally related families.²²²

AI has also advanced the field of structure elucidation. For example, SMART 2.0, a platform based on convolutional neural networks, enables rapid and accurate structural determination from NMR and MS data. This tool was used to resolve the structure of the complex macrolide symplocolide A with high efficiency.²²³

In addition, AI models are increasingly applied to predict the biological activity and potential molecular targets of natural products. A prominent example is Chemprop, a graph neural network-based framework that infers properties such as antimicrobial activity, toxicity, and protein target specificity from molecular structures.²²⁴ This approach facilitated the discovery of halicin and abaucin as antibiotic candidates, both of which were subsequently validated and shown to act through mechanisms distinct from traditional antibiotics.^225,226

These examples collectively illustrate AI's ability to enhance both the precision and scalability of chemically driven natural product discovery. When integrated with conventional methodologies, AI can produce synergistic gains across the pipeline, from dereplication and annotation to lead prioritization. Looking ahead, the convergence of AI with metabolomics, structure elucidation, and activity prediction is expected to yield unified platforms capable of accelerating early-stage discovery from natural sources while expanding the chemical and biological space accessible to researchers.

6. Conclusion

Sponge-associated microorganisms are key producers of bioactive compounds and have garnered attention as valuable resources for natural product discovery and understanding marine ecosystems. This review highlights the latest trends and technological advancements in sponge symbiont-based natural product research, showcasing the potential for continued progress in this field.

The precise isolation and cultivation of symbiotic microorganisms are necessary to discover natural products from the sponge. Recent advancements in physical and chemical separation techniques, along with fine-controlled cultivation strategies, have significantly improved cultivation rates and compound productivity by addressing the unique metabolic pathways and physiological requirements of these microorganisms. Cell separation techniques such as density gradient centrifugation and flow cytometry enhance the accuracy of microbial separation and clarify the origins of natural products.

The genome- and chemistry-based approaches are powerful tools for understanding the metabolic pathways for cultivation, exploring the BGCs and chemical properties of natural products from sponge-associated microorganisms. Genomic analysis enables the prediction of metabolic pathways and BGCs, providing critical insights into novel natural product structures and functions. Techniques like single-cell genomics and heterologous expression systems allow researchers to elucidate individual microbial gene functions and produce natural products under laboratory conditions. At the same time, chemical-based approaches bypass the need for cultivation by directly identifying natural products from environmental samples, facilitating the discovery of compounds that are otherwise challenging to access. Together, these approaches accelerate the research pipeline from sponge symbionts to drug development and expand the potential for discovering novel bioactive compounds.

Recent studies on sponge-associated microorganisms have transcended traditional limitations by incorporating techniques, offering a more systematic understanding of complex host–microbe interactions and natural product biosynthesis mechanisms. These multidisciplinary approaches provide a strong foundation for discovering new compounds and understanding the ecological roles of microorganisms.

Future research must integrate genomic analysis, chemical exploration, and advanced biological cultivation techniques to achieve a deeper understanding of the complex ecological interactions and metabolic pathways of sponge symbionts. This effort should prioritize developing novel technologies targeting unculturable microorganisms and creating sustainable strategies for natural product production and utilization. Such integrated approaches are expected to significantly contribute to the development of new therapeutics based on bioactive compounds from sponge-associated microorganisms while enhancing our understanding of microbial roles in marine ecosystems.

7. Data availability

No primary research results, software, or code have been included, and no new data were generated or analysed as part of this review.

8. Conflicts of interest

The authors declare no conflict of interest.

9. Acknowledgements

This work was supported by the Korea Institute of Ocean Science and Technology (KIOST) In-house Programme (no. PEA0176), and the Bio & Medical Technology Development Programme of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2024-00352229).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5np00013k

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