Archives of Microbiology

Syngas fermentation by carbon monoxide (CO)-utilising acetogens offers a sustainable route for converting gasified waste materials into value-added chemicals. In this study, we isolated a novel thermophilic CO-utilising bacterium, strain AZ2, from marine hydrothermal sediment collected on the island of Sao Miguel, Azores, Portugal. Strain AZ2 is an obligately anaerobic, spore-forming bacterium. Average nucleotide identity (ANI; 78.4-86.7%) and digital DNA-DNA hybridization (dDDH; 23.4-32.5 %) analyses indicate that strain AZ2 represents a novel species within a previously uncharacterised lineage represented by the GTDB placeholder genus UBA2545 in the Neomoorellaceae family. Strain AZ2 was able to grow fermentatively on CO, producing acetate. We further demonstrated that its closest isolated relatives - Thermanaeromonas toyohensis, T. burensis, and Thermanaeromonas sp. strain 9S - are capable of growing on CO, producing either acetate or hydrogen gas (H2). Additionally, we unveiled the genomic potential for CO utilisation within other members of the GTDB placeholder class DSM-521 (previously Moorellia) to which our isolate belongs, expanding the list of possible thermophilic CO-utilising acetogens. We propose that strain AZ2T represents the type strain of a novel genus and species, named Thermobium azorense gen. nov., sp. nov. (= DSM 121889T = JCM 39698T).

From Brazilian limestone caves, we isolated 29 bacteria utilizing phenol (23 bacteria), toluene (all bacteria), and/or benzene (all bacteria) as sole carbon sources. One isolate showed phosphate solubilization, while lipase/esterase activity occurred in two isolates; no amylase activity was detected, but 16 isolates ([~]55%) exhibited protease activity. Among them, Nocardioides sp. SF1 was selected for whole-genome sequencing due to its aromatic compound tolerance and protease activity. Additionally, catechol cleavage assays yielded unexpected purple pigmentation, suggesting non-canonical aromatic metabolism. Its high-quality draft genome (4.25 Mbp, 16 contigs, N50 of 887 kb) lacks canonical phenol hydroxylase but encodes alternative oxidation systems, phenylacetyl-CoA pathway, besides, desferrioxamine siderophore, biosurfactants, and phosphate solubilization, key adaptations for oligotrophic caves and biotechnologically interesting activities. Whole-genome comparisons (TYGS/GGDC, OrthoANI and k-mer) suggest potential new species. Lacks acquired antimicrobial resistance genes (ResFinder) and pathogenicity potential (PathogenFinder). Nocardioides sp. SF1 emerges as a non-pathogenic candidate for aromatic bioremediation and plant growth promotion in contaminated, nutrient-poor environments, highlighting cave actinobacterias unexplored biotechnological potential.

Background: Human activities such as mining generally lead to increased heavy metal concentrations in the environment. While traditional remediation techniques are often costly, the use of fungi as bioremediators, known as mycoremediation, is increasingly gaining attention as a sustainable approach for removal of heavy metals. Here, we evaluated heavy metal levels inside the Kiirunavaara iron ore mine in Northern Sweden and analysed fungal responses to various metal concentrations by comparing growth and metal uptake in mine-derived isolates and closely related control isolates. Results: Sediments inside the mine were enriched in heavy metals compared to those from the outlet of the mine to natural lakes. Six Fusarium isolates were recovered from contaminated mining environments: five isolates from inside the mine were identified as Fusarium oxysporum, and one isolate from the outlet was identified as Fusarium tricinctum. Isolates from the mine and outlet showed overall higher survival and biomass production in presence of copper, iron, and zinc across a range of concentrations (up to 1000 mg/L) compared to control isolates. At the same time, these isolates often exhibited reduced relative metal uptake. As a result, mycoremediation potential, assessed as total uptake in the grown mycelium, was isolate-dependent. Conclusions: Based on these results, we conclude that Fusarium isolates from the Kiirunavaara mine show increased growth in media enriched with heavy metals compared to closely related control isolates. We additionally show that mycoremediation potential is not necessarily associated with environmental origin. Instead, mycoremediation potential should be evaluated on a case-by-case basis for each isolate and based on specific needs for mycoremediation.

This study investigated the genomic and biochemical characteristics of two amylolytic microbial strains, NATIVEDY160T (= JE7B6T = NRRL B-68523T) and NATIVEDY161T (= JL13D9T, = NRRL B-68524T) isolated from the rumen of healthy Holstein dairy cattle. Both strains are obligately anaerobic, non-motile, Gram positive, catalase-negative, and oxidase-negative. Morphologically, NATIVEDY160T grows in long coccoid chains while NATIVEDY161T grows in short chains or pairs. NATIVEDY160T can catabolize amygdalin, esculin/ferric citrate, and starch, compared to NATIVEDY161T which utilizes amygdalin, arbutin, esculin/ferric citrate, glycogen, and D-maltose as determined by API 50 CH carbon panels. Starch degradation ability was verified for both strains, but neither showed cellulolytic activity as confirmed by starch agar and Congo red agar assays, respectively. HPLC analysis revealed that lactate was the primary end product of both strains carbohydrate fermentation, while strain NATIVEDY161T also produced small amounts of acetate. 16S rRNA sequences from both strains cluster with the Oscillospiraceae (formerly Ruminococcaceae) lineage Ruminococcus species, but average nucleotide identity of either strain compared to closely related Ruminococcus members was under the species threshold (95%). Genomic, phylogenetic, and phenotypic interrogation support NATIVEDY160T and NATIVEDY161T as novel species. Each strain was isolated from the rumen of dairy cows located within the central valley of southern California, which has a rich history of Dutch and Basque dairy farm ownership and is still the case today in the region. In recognition of the contributions and heritage of the central and southern California dairy industry, the names Ruminococcus hollandia and Ruminococcus vasco are proposed with NATIVEDY160T and NATIVEDY161T as their respective type strains.

Compact poultry raising has turned poultry litter into an environmental problem, as it may all be packed with heavy metals and drug-resistant germs. Of all the metals, chromium contamination not only disturbs the general environment but is also a source of concern for public health. Poultry litters were taken from 14 farms in different places, and the bacteria characters from different places were tested for their capacity to tolerate Cr(VI). A total of 31 bacterial isolates were initially screened, and three of them (AH-2, AZ-1, and AMF-3) appeared to be very resistant to chromium. The isolates were able to survive at the highest concentration, 800 mg/L of the Cr(VI); however, AH-2 was the most resistant one (MIC: 900 mg/L; MBC: 1000 mg/L). Chromium reduction tests showed that AMF-3 at high concentration showed the maximum chromium reduction, while AH-2 achieved higher chromium reduction at medium concentration. Phenotypic and biochemical analysis showed that the isolates were Staphylococcus spp., which was confirmed by 16S rRNA gene sequencing as S. cohnii, S. saprophyticus, and S. gallinarum. Moreover, chromium was detected at higher levels in poultry litter compared to the feed, with the highest accumulation in AZ farm litter (4464.0 {micro}g/kg). The highlighting feature of our article is the presence of chromium-tolerant and reducing bacteria in poultry environments. Besides that, the level of chromium in poultry litter is really high, and it points to the need for better waste management.

Melanins are biological macromolecule with immense functionality synthesised by a wide spectrum of living organism. It is mainly synthesised by the oxidative polymerization of indolic and phenolic compounds through several enzymatic process. It has wide spread application in agriculture, cosmetic and therapeutic industry due to its various properties including antioxidation ability, UV protection efficiency and anticancer activity. Because of this wide range of application in different sectors, large scale production and commercialization attains enormous consideration. The present study deals with the effect of 12 different process parameters on melanin production viz., production media, incubation time, inoculum concentration, pH, temperature, agitation, carbon source, phosphate and magnesium source, CuSO4.5H2O, sodium chloride and L-tyrosine on melanin production by Pseudomonas stutzeri strain BTCZ 109 obtained from Arabian sea sediments was evaluated. After optimizing the important process parameters, the bacteria showed about ~4.65 fold increase in melanin production compared to unoptimized cultural conditions. The melanin optimized through this method was found to be nano sized. The Nano sized DOPA melanin in treating Skin cancer cell line SK ML28 which showed a dose-dependent activity with an IC50 value of 164 g/mL. All these results highlight the therapeutic efficiency of DOPA melanin Nano particle as promising bioactive molecule.

During mycological surveys in Yunnan Province, China, specimens of a fungus producing massive, upright stromata up to 50 cm high and individually 2.2 Kg in weight were sampled. Through an integrative taxonomic approach combining detailed morphology, multilocus phylogeny (ITS, LSU, RPB2, TUB2), and phylogenomic analyses, this fungus is proposed as the new species Dianjunus rex gen. et sp. nov., the type of the new family Dianjunaceae (Xylariales). Phylogenetic analyses robustly place Dianjunaceae as a distinct sister clade to Graphostromataceae. Divergence time estimation dates the origin of this family to the early Paleocene (~65 Mya), coinciding with the post-K-Pg extinction period, when an estimated 75% of all plant and animal species went extinct, and a significant ecological reorganization of life on earth happened. The stromata of D. rex represent the largest fructifications documented within the Ascomycota, significantly expanding the known morphological range of the Xylariales. The study provides a comprehensive description, including a nodulisporium-like anamorph with periconiella-like branching patterns, and discusses the taxon's phylogenetic placement, and distinctive morphology. This discovery highlights the unexplored fungal diversity in East Asian forests.

Oligella otitidis MSHR-50489EDL strain (ATCC: TSD462; DSMZ: DSM118617) is a new species of the genus Oligella that was isolated from a middle ear discharge swab from a child with chronic suppurative otitis media (CSOM). This Gram-negative coccobacillus produces small, circular, smooth, whitish-opaque and occasionally mucoid colonies. It grows in aerobic conditions at a temperature range from 25-42oC. Phylogenetic analysis demonstrates a relationship to other species of the genera Oligella and average nucleotide identity and digital DNA/DNA hybridization values indicate a distinct species in comparison to other Oligella species. Thus far, the majority of isolates exhibit resistance to ciprofloxacin, the first line treatment for CSOM.

Microorganisms are the key drivers of carbon cycling in coastal marine sediment ecosystems, significantly influencing carbon storage and release during Gracilariopsis lemaneiformis cultivation. This study employed 16S rRNA sequencing, a high-throughput qPCR chip, and carbon isotope labeling to assess the impact of G. lemaneiformis cultivation on carbon cycling processes in coastal sediments. A comparative analysis was conducted between cultivated zones (GZ) of G. lemaneiformis and adjacent control zones (CZ). The results indicated that macroalgae cultivation significantly modified sediment-seawater exchange dynamics and accelerated carbon cycling within coastal marine sediment ecosystems. Furthermore, G. lemaneiformis cultivation increased the abundance of genes linked to polysaccharide degradation and carbon fixation pathways, thereby enhancing carbon cycling efficiency. The ecosystem multifunctional index, calculated based on carbon fixation gene abundance, was significantly higher in GZ compared to CZ. Incubation experiments using 13C-NaHCO3 demonstrated that cultivation markedly elevated the carbon fixation rate of sediment, emphasizing a higher potential for carbon sequestration in sedimentary environments cultivated with macroalgae. Additionally, cultivation significantly altered sediment microbial communities, simplifying their structural complexity. Key microbial taxa identified via k-core species analysis--including Subgroup10 of Desulfobacterota and MBNT15, correlated strongly with carbon fixation rates, indicating their pivotal roles in sediment carbon cycling processes. This study provides critical insights into how large-scale macroalgae cultivation influences coastal carbon dynamics and informs strategies for optimizing carbon management in aquaculture ecosystems.

Caldimonas thermodepolymerans is a thermophilic polyhydroxyalkanoate (PHA)-producing bacterium with strong potential for sustainable bioplastic production. Besides serving as intracellular carbon and energy storage compounds, PHAs are increasingly associated with bacterial stress resistance and cellular robustness. This study aimed to investigate the physiological and transcriptomic response of C. thermodepolymerans to osmotic stress induced by elevated NaCl concentrations. Growth analysis demonstrated tolerance up to a supplementation of 2% NaCl, while moderate salt concentrations enhanced PHA accumulation, reaching 65% cell dry weight at 1.5% NaCl supplementation. To better understand the bacterial response to osmotic stress, RNA sequencing was performed under sublethal salt stress conditions. Differential expression analysis revealed major changes in genes related to osmoprotection, trehalose metabolism and type VI secretion systems, whereas motility and chemotaxis genes were strongly repressed. Phenotypic assays confirmed increased biofilm formation and reduced swarming motility under salt-induced osmotic stress. Although canonical PHA biosynthesis genes were not significantly differentially expressed, increased polymer accumulation suggests other underlying mechanisms linked to osmoadaptation. Together, these findings demonstrate that osmotic stress induces metabolic, physiological and regulatory responses in C. thermodepolymerans, highlighting the importance of PHA in stress adaptation besides its industrial applicability.

The present-day martian surface is generally considered inhospitable to life because of low atmospheric pressure, intense surface radiation, global desiccation, and oxidizing chemistry which has been increasing since the late Noachian. However, shallow martian subsurface regions where mineralogy has shown groundwater movement may include localized hypersaline environments capable of retaining liquid water and supporting microbial metabolism. Haloferax volcanii, a model halophilic archaeon, has previously been shown to survive under low-pressure martian conditions (24 mbar) and to grow anaerobically supported by the Mars-relevant oxyanions nitrate and perchlorate under high-salinity conditions. Here, we investigated whether H. volcanii could actively grow under a combination of environmental and chemical conditions relevant to potentially habitable shallow subsurface martian lacustrine settings. Cultures were incubated for 160 days under anoxic, CO2-enriched, low-pressure conditions (24 mbar) in hypersaline liquid media supplemented with nitrate or perchlorate. Growth was observed in all low-pressure treatments and was confirmed by increases in optical density and biological reduction of nitrate and perchlorate. Scanning electron microscopy revealed extensive biofilm formation in low-pressure cultures, and Raman spectroscopy demonstrated the persistence of carotenoid biosignatures after prolonged incubation under martian conditions. Water loss remained below 4% across all treatments, indicating long-term stability of hypersaline brines throughout the experiment. These results demonstrate for the first time that a halophilic archaeon is capable of active growth and metabolism under a Mars-relevant combination of low pressure, high salinity, anoxia, and oxidizing chemistry, providing experimental support for the potential habitability of localized shallow subsurface martian environments. ImportanceThe search for cellular life is a major objective of future Mars exploration. While many studies have examined whether microorganisms can survive under martian conditions, far fewer have demonstrated active growth and metabolism. Here, we document Haloferax volcanii as the first halophilic archaeon capable of active growth under a defined combination of Mars-relevant low atmospheric pressure, high salinity, anoxia, and oxidizing chemical conditions. These findings expand the current understanding of the environmental limits of microbial growth and provide experimental evidence that localized brine environments in the shallow martian subsurface could support active microbial metabolism, if suitable organics and liquid water are present. In addition, this study establishes a practical framework for cultivating halophilic microorganisms under low-pressure martian conditions and may help guide future efforts to detect, cultivate, and characterize potential extant life on Mars.

The aim of this study was to evaluate the impact of two sustainable dietary protein sources on the structure and composition of the gut microbiota in European sea bass (Dicentrarchus labrax) juveniles. These protein sources were incorporated to the aquafeeds containing (a) Lupinus albus meal, treated with either exogenous enzymes (Solid state hydrolysis-SSH) or fermented with Saccharomyces cerevisiae (Solid state fermentation, SSF) and (b) Lagocephalus sceleratus meal. In the first case (a), the control aquafeed simulated a standard commercial diet, containing soybean meal whereas in the rest of the diets soybean meal was partially or totally replaced by hydrolysed or fermented Lupin meal. In the second case (b) the fish were fed Lagocephalus sceleratus unprocessed fishmeal as well as treated at different temperatures to deactivate tetrodotoxin (TTX). A control diet with 30% commercial fish meal was also fed as a reference diet. Both diets in all inclusion levels did not cause any significant gut microbiota change, suggesting their neutral role in this aspect. However, the gut bacterial communities of the fish fed with 12.5% lupin meal inclusion, had increased amino acid biosynthetic pathways suggesting a beneficial effect.

Microbial fuel cells (MFCs) technology offers sustainable electricity production. Current research largely focuses on few select model organisms, therefore the true prevalence of exoelectrogenesis amongst bacteria remaining largely unknown. We present a broad-scale survey of monomicrobial electricity production among environmental bacterial isolates inoculated in MFCs, using model organism Shewanella oneidensis MR-1 as a benchmark. Of the assessed taxa, 11-22% displayed exoelectrogenic activity, exceeding current predictions and identifying a further three novel exoelectrogenic species. Phylogenetic analysis based on the 16S sequences enabled the evolutionary relationship between isolates to be visualised, revealing that exoelectrogenesis is non-randomly distributed and phylogenetically conserved. Polarisation studies were implemented, revealing that numerous electron transfer mechanism were being utilised to perform exoelectrogenesis. The results of this study imply that bacterial electricity production is more widespread amongst culturable bacteria than previously estimated, with implications for bioprospecting novel exoelectrogens and predicting electrogenic activity in diverse microbial communities.

Evidence from the Cassini mission confirmed that Saturn's moon Enceladus hosts a subsurface alkaline ocean where rock-water reactions may generate redox disequilibria capable of supporting microbial metabolisms. To investigate potential microbial survival under simulated Enceladus ocean conditions, we used thermodynamic modeling to develop a salt formulation consistent with one possible Enceladus ocean composition and supplemented it with putative microbial energy sources to create a growth medium. The medium was inoculated with samples from diverse ocean world analog environments on Earth to determine which microorganisms could persist under Enceladus-like conditions. The microorganisms persisting in this geochemically bounded medium were heterotrophic, metabolically versatile bacteria with low carbon requirements. Genomic and physiological analyses further showed the presence of multiple stress-response pathways, sodium- based bioenergetic systems, osmoregulation strategies, and other adaptations consistent with survival in alkaline, low-nutrient settings. These results suggest that some stress-tolerant heterotrophic bacteria may serve as useful model organisms for life in Enceladus' subsurface ocean. These findings demonstrate the value of geochemically modeled media as a framework for constraining habitability, identifying relevant biosignatures, and probing potential microbial survival strategies beyond Earth.

It was recently reported that Methylobacterium extorquens AM1 produces the citrate-hydroxamate siderophore N-deoxyschizokinen A, identified by LC-HRMS. Multiple properties were inconsistent with the assignment: the feature eluted far later than the other schizokinen derivatives (17 min versus 6-8 min), a reversed-phase shift larger than a single-hydroxyl difference in a molecule can explain, further its accurate mass deviated from the calculated one by 28 ppm, well outside the error on the co-analyzed standards and its diagnostic m/z 105 and 77 fragments suggest a molecule with an aromatic moiety. A replicate comparison of identical samples in plastic versus glass autosampler vials was decisive: the m/z 387 feature was reproducibly present with plastic vials and absent with glass. We therefore conclude that the reported detection of N-deoxyschizokinen A in M. extorquens AM1 is an artifact, and recommend glass-vial and solvent-blank controls, an explicit accurate-mass threshold, and narrow MS/MS isolation when assigning trace siderophore-like features from complex extracts.

Background: Biochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside Escherichia coli cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (E. coli MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of E. coli lacking the endogenous SodFMs. Here, we characterised this E. coli BL21 (DE3) {Delta}sodA{Delta}sodB strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for in vitro characterisation and use. Results: Genomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein. Conclusions: The E. coli strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.

Mangrove forests are major blue carbon ecosystems but are often characterized by low surface methane (CH4) emissions. Such low emissions, however, do not necessarily indicate weak methanogenesis, because CH4 production may be offset by internal CH4 consumption before reaching the atmosphere. Although previous community, genomic, and transcriptomic studies have implicated methylotrophic methanogenesis in mangrove sediments, direct taxon-resolved evidence linking methylated carbon assimilation to potentially active methanogens remains limited. Here, we combined methanogenic activity assays, DNA stable isotope probing (DNA-SIP), mcrA and 16S rRNA gene analyses, and phylogenetic comparisons to identify potentially active methanogens across saline-influenced mangrove soils. The results showed that CH4 production potentials were consistently dominated by methylotrophic pathways (1.86-2.78 g CH4 g-1 soil hr-1) across all sites. DNA-SIP, together with consistent community patterns in fresh soils, indicated the potential activity of methylotrophic and mixotrophic methanogens under saline conditions. Methanolobus-affiliated methanogens were associated with salinity, Na+, Cl-, and NH4+, whereas Methanosarcina and unclassified Methanosarcinaceae were linked to soil soluble organic carbon availability and water content, indicating niche differentiation among active methanogenic groups. Phylogenetic analyses incorporating reference sequences from diverse environments further showed that potentially active mangrove methanogens were dominated by saline-associated lineages. Together with our previous methanotrophic evidence from the same sites, these findings suggest that low CH4 emissions from mangrove blue carbon ecosystems can mask substantial internal CH4 cycling sustained by active methanogenesis and CH4 consumption.

Native ash trees are destined for functional extinction in North America due to the spread of the non-native emerald ash borer. Yet, the consequences of ash loss for soil fungi are unclear. To address this, we employed a factorial study of forest soil fungi in two hydropedological soil types beneath four canopy tree species -- including white ash (Fraxinus americana). Sporocarp surveys and community DNA metabarcoding from soil samples revealed patterns in fungal communities related to canopy tree species but not soil type. Ash trees supported a particularly rich soil fungal community that was distinguishable from communities beneath beech, birch, and maple. We identified over 100 fungal taxa (OTUs) that are at risk of decline or loss from the studied forest, due to their association with ash. Our results indicate that canopy tree species influence soil fungi much more broadly than just the species with which they have mycorrhizal associations.

Manual intervention for concrete repair and replacement comes at high environmental and economic costs. Bioconcrete, which can be formed by bacteria via microbially-induced carbonate precipitation (MICP), is a sustainable method for concrete repair. Bioconcrete-forming bacteria can be incorporated into the concrete at mixing and then heal cracks where and when they occur. Bioconcrete is not intentionally made by bacteria; rather, it is a byproduct of alterations to the local environment that occur during their normal metabolic activities. Bacteria thus make bioconcrete by different metabolic mechanisms, and the environment plays a substantial role in the yield and physical properties of the bioconcrete produced by a given bacterium. The ureolytic bacterium Sporosarcina pasteurii is the most commonly used model organism for MICP, but it requires urea supplementation, which is not feasible for all applications because of nitrogenous waste. In particular, the marine environment is understudied for bioconcrete applications, yet there is a need for self-healing structures in this environment, wherein urea and nitrogenous waste would be detrimental to native biota. Here, we assessed the ability of S. pasteurii to form bioconcrete under marine-like media conditions with urea and calcium supplementation. We found that S. pasteurii generated higher bioconcrete yields in these media conditions compared to standard growth media. We then designed an enrichment protocol to isolate and characterize non-urea-requiring bioconcrete-forming bacteria from Atlantic seawater. We identified three isolates, from the Sulflitobacter, Marinobacter, and Bacillus genera, two of which yielded higher bioconcrete yields in seawater-mimicking media compared to non-ureolytic bacteria utilized in prior literature. Moreover, scanning electron microscopy (SEM)/energy dispersive spectroscopy (EDS) and Fourier transform infrared (FTIR) spectroscopy revealed distinct chemical and structural features of the bioconcrete produced by bacteria in seawater-mimicking medium and between ureolytic and non-ureolytic cultures. Overall, our work establishes a pipeline for the isolation and characterization of novel bioconcrete-forming bacteria from marine samples, with potential for application to marine self-healing materials.

Pelagibacterales gMED is the dominant epipelagic genomospecies in the western Mediterranean Sea. We used the O-chain biosynthesis gene clusters, OBCs, as clonal barcodes to analyse strain-level population structure. In total, 385 OBC-defined clonal lineages were tracked across Mediterranean metagenomes spanning 14 years and depths from 5 to 90 m within the photic zone, with between 128 and 336 detected per metagenome. The relative conservation of dominant OBC types across years, seasons, and geographic locations indicated a persistently high and stable clonal diversity.