MetaboLights

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order to highlight specialised metabolites, MetaboScape software was first used to process monophasic protocol (MP) data. After recalibration of each sample analysis (< 1ppm, according to sodium formate internal standard), peaks with intensities greater than 5,000 counts in at least 10% of the set of samples and minimal RT-correlation coefficient of 0.7. Different states of charge (1+, 2+ and 3+ ) and classical adducts were grouped together, and the peak area was determined in order to generate a unique global data matrix containing semi-quantification results for each metabolite in all analysed samples. Feature annotations were attempted according to respective ion mass (< 2ppm) and isotopic pattern (< 20msigma) by automatic match with the CyanometDB 1.0 database [14] which contains over 2,100 chemical formulas of known specialised metabolite produced by cyanobacteria. Feature intensity was further normalised by the Total Ion Chromatogram (TIC) prior the use ofMS-cleanR [10] to filter the data according blank intensity, RSD, RMD, etc. For untargeted metabolomics processing, MSDial software was used to extract features from samples. Feature intensity was further normalised by the Total Ion Chromatogram (TIC) prior the use ofMS-cleanR [10] to filter the data according blank intensity, RSD, RMD, etc. Filtered data were finally export as peak lists (i.e. one per acquisition mode) for chemometrics analysis and as spectra (as individual .mat files) to implement SIRIUS (v6.1). SIRIUS software allowed to interrogate structural libraries (SIRIUS database and cyanoMetDB) and provide scoring of the formula (ZODIAC) and structure (CSI:FingerID). [8, 17, 26]. In has to be noted, that these filtered peak lists contained both ”only MS1” features (i.e. detected signals for which no fragmentation was performed) and ”MS2” features (i.e. signals for which a fragmentation was performed allowing to interrogate spectral or structural DBs). Output files from MS-DIAL and SIRIUS (Top 50 per feature) were further imported into MS-Net (developed in Knime Analytics Platform, v5.2) [11] in order to merge Pos and Neg acquisition for each gradient (hydrophilic-HSST3-pos/neg, hydrophilic-HILIC-pos/neg, lipophilic-pos/neg), suppress analytical redundancy, provide annotation confidence level and classification (NPclassifier, [16]), and highlight link between metabolites and identified bacterial genus and Microcystis sp. Briefly, among the top 50 in silico candidates per feature, those matching taxonomic criteria (genus: Gemmatimonas, Hydrogenophaga, Meiothermus, Neoroseomonas, Aminobacter, Limnobacter, Novosphingobium, Phenylobacterium, Nevskia, Rhabdaerophilum, Cnuella, Flavobacterium, Aquidulcibacter, Brevundimonas, Caulobacter, Silanimonas, Inhella, IdeonellaA, Pararheinheimera, Falsiroseomonas, PseudomonasE, Rubrivivax, LeptospiraA, Microcystis ; Famillies : Fimbriimonadaceae, Sphingomonadaceae, Gemmatimonadaceae, TH1-2, Burkholderiaceae, Thermaceae, Acetobacteraceae, Caulobacteraceae, Rhizobiaceae, Nevskiaceae, Beijerinckiaceae, Chitinophagaceae, Flavobacteriaceae, Tepidisphaeraceae, Xanthomonadaceae, Alteromonadaceae, Pseudomonadaceae, Usitatibacteraceae, Leptospiraceae, Microcystaceae) were elevated to Level 3a. Features were filtered using Ion Identity Network (IIN) results to remove redundant adducts and isotopes, followed by MS-CleanR-based RT clustering (∆RT ≤ 0.01 min). Within each cluster, the top 2 features by network degree and the top 2 by peak intensity were retained. The MS2 network was constrained to edges with cosine similarity ≥ 0.7 and ∆RT ≤ 8 min between connected nodes. High-confidence annotations (Levels 1, 2a, 3a) seeded the MS2 network for iterative annotation propagation. For each feature pair, candidate structures were ranked using a weighting parameter set to α = 0.3, prioritizing structural-spectral evidence (70%) over in silico ranking (30%). The top 5candidates by Link Score were retained per feature before looping through the entire MS2 network. Redundant annotations with identical InChIKey identifiers and Pearson correlation among samples > 0.7 were consolidated by selecting the candidate with the highest mean peak height. Positive and negative mode feature lists were merged using ∆RT ≤ 0.05 min, ∆m/z ≤ 0.005 Da, and a minimum Pearson correlation ≥ 0.6 across sample intensities. Final annotations were enriched with chemical ontology classifications from ClassyFire (kingdom, superclass, class, subclass) and NPClassifier (pathway, superclass, class). Database identifiers (PubChem CID, KEGG, HMDB, ChEBI) were retrieved using the Chemical Translation Service. Natural product-likeness scores were calculated using the NPlikeness calculator [13]. Structural similarity networks were constructed using Tanimoto coefficient ≥ 0.8, retaining the top 2 nearest neighbours per feature.In the Biphasic protocol method, about 2 mg of dried biomass was first extracted on ice by adding 1 mL of cold mixture of methyl-tert-butyl-ether and methanol (MTBE-MeOH, ratio 3:1, v:v) and homogenisation during 2 cycle of 15-second (6000 RPM, FastPrep-24 5G, MP-biomededical). Then, a 650 μL cold mixture of ultrapure water and methanol (UPW-MeOH, ratio 3:1, v:v) was added, followed by a third cycle of homogenization. Hydrophilic and lipophylic fractions were separated by centrifugation (12000 RPM, 4°C, 5 min) and further partly collected. A second similar extraction was performed to optimise metabolite recovery after the addition of 700 μL of MTBE-MeOH mixture and 455 μL of UPW-MeOH mixture. In total, lipophilic and hydrophilic fractions consisted in 1.1 mL and 1.3 mL extracts, respectively. Following the extraction, 500 μL of each fractions were evaported to dryness (EZ2-PLUS, Genevac) and resuspended in ACN:H20 (1:1, v/v) mixture and ISO:ACN (1:1, v/v) respectively for hydrophilic and lipophilic fractions. Both fractions were analysed on UHPLC Vanquish combined to Q-Exactive Plus (Qex+) HRMS equiped with a heated electropray ionisation probe (HESI-II) (ThermoFisherScientific, San Jose, CA).Biphasic extracts were separated on HSST3 RP-C18 column (150 × 2.1 mm × 1.7 μm, Waters) to analyse hydrophilic and lipophilic fractions and BEH-Amide (100 x 2.1 mm x 1.7 μm, Waters) to analyse only hydrophilic fraction.

Polar extracts from biphasic protocol were also separated on HSS T3 RP-C18 column (150 × 2.1 mm × 1.7 μm, Waters) with UHPLC Vanquish, as described below.Eluent A: Ultrapure water supplemented with 0.1% Formic acid (FA)Eluent B: Acetonitrile supplemented with 0.1% FA. The flow was 0.400 mL/min 0 min: 99% (Eluent A) 1% (Eluent B)4 min: 99% (Eluent A) 1% (Eluent B)20 min: 1% (Eluent A) 99% (Eluent B)22.1 min: 99% (Eluent A) 1% (Eluent B)25 min: 99% (Eluent A) 1% (Eluent B) For Biphasic protocol extraction, samples were also analysed in triplicates on QEx+ (ThermoFisher Scientific, San Jose, CA) in both positive and negative mode with a C18 and an HILIC columns. Each extract was analysed in triplicate in positive ionization mode with a mass range of 50–1500 m/z, and automatic fragmentation of the TOP 5 most intense ions.

MetaboLightsPublicIn the Monophasic protocol method, about 1 mg of lyophilised biomass or supernatant was extracted with 100 μL of solvent mix of methanol/water (3:1, v/v) acidified with 0.1 percent formic acid. Metabolite extraction was carried out on ice using an ultrasonic probe, performing four consecutive cycles of 30 seconds, each followed by a 30-second pause. The samples were then centrifuged (15,000 g, 4°C, 10 minutes). A volume of 50 μL of the supernatant was transferred into injection vials, and 1 μL was analysed by ultra-high-performance liquid chromatography (Elute, Bruker, Bremen, Germany) coupled with time of flight high-resolution tandem mass spectrometry (MS) (Maxis II-QTOF, Bruker, Bremen, Germany) (UHPLC-HRMS/MS).

Polar extracts from biphasic protocol were separated on BEH-Amide (100 x 2.1 mm x 1.7 μm,Waters) , as described below.Eluent A: Ultrapure water supplemented with and 5% of EtOHEluent B, Acetonitrile supplemented with 0.1% The flow was 0.400 mL/min 0 min: 30% (Eluent A) 70% (Eluent B)2 min: 30% (Eluent A) 70% (Eluent B)12 min: 70% (Eluent A) 30% (Eluent B)16 min: 70% (Eluent A) 30% (Eluent B)18 min: 30% (Eluent A) 70% (Eluent B)24 min: 30% (Eluent A) 70% (Eluent B) For Biphasic protocol extraction, samples were also analysed in triplicates on QEx+ (ThermoFisher Scientific, San Jose, CA) in both positive and negative mode with a C18 and an HILIC columns. Each extract was analysed in triplicate in positive ionization mode with a mass range of 50–1500 m/z, and automatic fragmentation of the TOP 5 most intense ions.

Liquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - hilicLiquid Chromatography MS - positive - reverse-phaseLiquid Chromatography MS - negative - hilicLC-MS Methods are described individually in sections below.Integrating metagenome-scale metabolic models and metabolomics to explore candidate biochemical interactions in cultivated Microcystis phycospheres. 10.1093/ismeco/ycag226.INRAE - UMR 1019 Human Nutrition Unit Metabolism Exploration Platform MetaboHUB – Clermont FdUniversite Clermont AuvergneBinta DIEMEFranck Giacomonisolventexometabolomeendometabolomemass spectrometry assayStudy samples were prepared for analysis by both monophasic and biphasic extraction methods.Extraction Methods are described individually in sections below. Monophasic extraction yielded a single extract, analysed by LC-MS methodology, in positive ion mode, using a C18 Polar Advances II chromatographic column (Reversed phase).Biphasic extraction yielded polar and apolar extracts, analysed by LC-MS methodlogy, in positive and negative ion modes, using:HSS T3 chromatographic column (Reversed phase).BEH-Amide chromatographic column (HILIC). Sample/Assay Combinations are as follows:Assay Sheet 1: all samples > monophasic extraction > extract > C18 Polar Advances II (RP) > positive ionisation modeAssay Sheet 2: all samples > biphasic extraction > polar extract > BEH-Amide (HILIC) > positive ionisation modeAssay Sheet 3: all samples > biphasic extraction > polar extract > BEH-Amide (HILIC) > negative ionisation modeAssay Sheet 4: all samples > biphasic extraction > polar extract > C18 HSS T3 (RP) > positive ionisation modeAssay Sheet 5: all samples > biphasic extraction > polar extract > C18 HSS T3 (RP) > negative ionisation modeAssay Sheet 6: all samples > biphasic extraction > apolar extract > C18 HSS T3 (RP) > positive ionisation modeAssay Sheet 7: all samples > biphasic extraction > apolar extract > C18 HSS T3 (RP) > negative ionisation modemicrobial communityblankPooled Samplehttps://www.ebi.ac.uk/metabolights/MTBLS13909MTH Clermont. MetaboHUB. mtbls-mth-clermont@inrae.fr.BINTA DIEME. Université Clermont Auvergne, INRAE, UNH, PFEM, MetaboHUB Clermont, 63000 Clermont-Ferrand, France. Centre Auvergne Rhône Alpes, Site de Theix, Route de Theix 63122 Saint Genès Champanelle. binta.dieme@uca.fr.MSconvert (Version: 3.0.24124-ba8a4fd (automated build)) was used to transforme raw data to open format mzML PhycosphereMicrocystisbinta.dieme@uca.frmtbls-mth-clermont@inrae.frWater samples were collected in September 2021, from a suburban freshwater pond near Paris, Ile-de-France (Cergy-Pontoise, GPS coordinates lat. 49.01343 long. 2.02575), experiencing a Microcystis bloom, (chlorophyl-content around 10 μg.L−1 )[33]. After prefiltration on 50 μm porosity, twelve monoclonal Microcystis strains and their associated bacterial consortia were isolated by repeated of single-cell or small-colony transfers on solid or liquid media under an inverted microscope (NIKON ECLIPSE TS100, Japan). Viable clones were then cultured in 25 cm2 culture flasks (CORNING-FALCON) containing 10 mL of Z8 medium. Cyanobacterial strains were maintained in the Paris Museum Collection (PMC, https://mcam.mnhn.fr/en/cyanobacteria-and-live-microalgae-470) at 18C, using white LED-powered lights providing an irradiance of 8-10 μm photons/m2/s, with a photoperiod of 13h light/11h dark. Isolated strains and cultures were all monoclonal and non-axenic. They were then cultured in BG11 medium, with a photoperiod of 16h light/8h dark and ∼14 μm photons/m2/s. After optimal growth, cultures were centrifuged to separately collect cell pellets and supernatants for genomic and metabolomic analyses. Two complementary extraction protocols were used for metabolomic analyses to maximize coverage.

Monophasic extract was separated on a C18 Polar Advances II column (2.1 x 100 mm, x 2.2 μm, ThermoScientific), as described below.Eluent A: Ultrapure water supplemented with 0.08% of Formic acid (FA)Eluent B, Acetonitrile supplemented with 0.08% of FA. The flow was 0.300 mL/min 0 min: 95% (Eluent A) 5% (Eluent B)2 min: 95% (Eluent A) 5% (Eluent B)16 min: 10% (Eluent A) 90% (Eluent B)18 min: 10% (Eluent A) 90% (Eluent B)19 min: 95% (Eluent A) 5% (Eluent B)21 min: 95% (Eluent A) 5% (Eluent B) Samples were acquired in positive ionisation mode for the monophasic extract with the Maxis II-QTOF, Bruker, Bremen, Germany. Each extract was analysed in triplicate in positive ionization mode with a mass range of 50–1500 m/z, and automatic fragmentation of the TOP 5 most intense ions. QC and Blank samples (injected every six samples and every triplicate, respectively) were examined to ensure the reproducibility and robustness of the whole data series.

MetabolomicsMetabolomicsblankuntargeted analysismetabolic modellingphycospherePooled SampleBruker Elute UHPLC systemMicrocystisSystems BiologyThermo Scientific Q Exactive PlusendometabolomeThermo Scientific Vanquish UHPLC SystemBruker maXis II UHR-ToFmicrobial communitysolventexperimental blankcyanospherefreshwater cyanobacteriaexometabolomeMetaboHUBMetabolomicsblankuntargeted analysismetabolic modellingphycospherePooled SampleBruker Elute UHPLC systemMicrocystisSystems BiologyThermo Scientific Q Exactive PlusendometabolomeThermo Scientific Vanquish UHPLC SystemBruker maXis II UHR-ToFmicrobial communitysolventexperimental blankcyanospherefreshwater cyanobacteriaexometabolomeMetaboHUBLC-MS Methods are described individually in sections below.glutamic acidThr-Pheergothioneine2,6-diaminopimelic acid(NAG)(NAM)-AqmAglutathione disulfideSerine7,8-dihydrobiopterinN-gamma-GlutamylmethionineAla-MetDiprotin AAla-LeuN-(Carboxymethyl)-3-(dodecanoylamino)-N,N-dimethylpropan-1-aminiumoctadecanamide2-(2,6-Diaminohexanoylamino)-3-methylbutanoic acidVal-ArgD-tryptophan5'-MethylthioadenosineThreonylleucinelys-leu(2R,3S)-2-[[3-(carboxymethylimino)-5-hydroxy-5-(hydroxymethyl)-2-methoxycyclohexen-1-yl]amino]-3-hydroxybutanoic acidalanylphenylalanineN-acetyl-?-D-mannosamineArg-IleN6,N6,N6-Trimethyl-L-lysineN-alpha-Acetyl-L-lysine2-Amino-6-propanoylpteridin-4(1H)-oneAla-GlnMicrocystin LYD-Ornithinespermidine9-Hydroxy-10,12,15-octadecatrienoic acid2-[4-(2-Hydroxyethyl)piperazin-4-ium-1-yl]ethanesulfonate1-hexadecanoyl-sn-glycerolMethionineagmatineAcetyl tributyl citrateDL-ThreonineAla-Thr[D-Asp3]MC-LRSer-LeuOleamide15-benzyl-18-[(1E,3E)-6-methoxy-3,5-dimethyl-7-phenylhepta-1,3-dienyl]-1,5,12,19-tetramethyl-2-methylidene-8-(2-methylpropyl)-3,6,9,13,16,20,25-heptaoxo-1,4,7,10,14,17,21-heptazacyclopentacosane-11,22-dicarboxylic acidS-adenosyl-L-methionineCoronaric acidmicrocystin-LRGlutaminefalseIntegrating metagenome-scale metabolic models and metabolomics to explore candidate biochemical interactions in cultivated Microcystis phycospheresFavoured by global changes, freshwater cyanobacterial harmful blooms generate major ecological, economical and public health challenges. Microcystis, one of the most widespread cyanobacterial genera, grows within a phycosphere where specialised interactions with its microbiome occur, and are suspected to influence bloom appearance and its potential toxicity. Using a combination of metagenomic, metabolomic and metabolic modelling, we characterised the culture-associated phycospheres of twelve Microcystis strains isolated from a French pond. The distribution of metabolic reactions within Microcystis was consistent with their genospecies, whereas the metabolic landscape at the community level diverged from cyanobacterial phylogeny indicating partial functional decoupling between cyanobacteria and their associated microbiomes. Bacteria associated with the simplified phycospheres substantially expand the metabolic repertoire of the system, while maintaining functional redundancy within and across communities. On the other hand, endometabolomic profiles were largely driven by cyanobacterial metabolic outputs whereas exometabolomic analysis did not reveal metabolites involved in exchange processes. Metabolic modelling, together with the identification of toxic specialised metabolites produced by specific biosynthetic gene clusters, further highlighted differences in metabolic potential among phycospheres. Together, these findings deepen the understanding of Microcystis’ phycosphere functioning and demonstrate the value of multi-omics systems biology approaches, while suggesting that metabolic complementarity between species and across phycospheres could play a role in bloom-associated microbiome structure.2026-08-142026-02-15MTBLS13909HMDB0030282MTBLC61553MTBLC17364MTBLC17295MTBLC73514MTBLC17361MTBLC21553MTBLC17115MTBLC17797MTBLC17345MTBLC35704MTBLC30753MTBLC166468MTBLC191000MTBLC34611MTBLC3362MTBLC170065MTBLC189140MTBLC73002MTBLC86092MTBLC15930MTBLC75542MTBLC63866MTBLC44920MTBLC6784MTBLC16365MTBLC28842MTBLC34687MTBLC44898MTBLC82965MTBLC16856MTBLC142245MTBLC15966MTBLC34876MTBLC48131MTBLC6650MTBLC177459MTBLC133298MTBLC22660MTBLC191135MTBLC16695MTBLC181836MTBLC75016MTBLC44992MTBLC18066MTBLC181445MTBLC84058MTBLC15741MTBLC30769MTBLC52742MTBLC16650MTBLC182838MTBLC74871MTBLC15918MTBLC29016MTBLC32398MTBLC17533MTBLC4828MTBLC181501MTBLC17596MTBLC32978MTBLC30832MTBLC181428MTBLC82966MTBLC16761MTBLC18397MTBLC28670MTBLC72816MTBLC73527MTBLC17964MTBLC9533MTBLC37023MTBLC141472MTBLC73770MTBLC182655MTBLC135063MTBLC16235MTBLC16238MTBLC18237MTBLC28300MTBLC16176MTBLC16610MTBLC74561MTBLC175415MTBLC38263MTBLC74860MTBLC73762MTBLC116314MTBLC34494MTBLC64277MTBLC16811MTBLC73806MTBLC23673MTBLC73788MTBLC73711MTBLC74862MTBLC46755MTBLC34900MTBLC15414MTBLC17822MTBLC165870MTBLC74815MTBLC73807MTBLC63154MTBLC16296MTBLC73814MTBLC17431MTBLC168067MTBLC157877MTBLC220610MTBLC17509MTBLC17858MTBLC173751MTBLC6925CHEBI:61553CHEBI:17364CHEBI:17295CHEBI:73514CHEBI:17361CHEBI:21553CHEBI:17115CHEBI:17797CHEBI:17345CHEBI:35704CHEBI:30753CHEBI:166468CHEBI:191000CHEBI:34611CHEBI:3362CHEBI:170065CHEBI:189140CHEBI:73002CHEBI:86092CHEBI:15930CHEBI:75542CHEBI:63866CHEBI:44920CHEBI:6784CHEBI:16365CHEBI:28842CHEBI:34687CHEBI:44898CHEBI:82965CHEBI:16856CHEBI:142245CHEBI:15966CHEBI:34876CHEBI:48131CHEBI:6650CHEBI:177459CHEBI:133298CHEBI:22660CHEBI:191135CHEBI:16695CHEBI:181836CHEBI:75016CHEBI:44992CHEBI:18066CHEBI:181445CHEBI:84058CHEBI:15741CHEBI:30769CHEBI:52742CHEBI:16650CHEBI:182838CHEBI:74871CHEBI:15918CHEBI:29016CHEBI:32398CHEBI:17533CHEBI:4828CHEBI:181501CHEBI:17596CHEBI:32978CHEBI:30832CHEBI:181428CHEBI:82966CHEBI:16761CHEBI:18397CHEBI:28670CHEBI:72816CHEBI:73527CHEBI:17964CHEBI:9533CHEBI:37023CHEBI:141472CHEBI:73770CHEBI:182655CHEBI:135063CHEBI:16235CHEBI:16238CHEBI:18237CHEBI:28300CHEBI:16176CHEBI:16610CHEBI:74561CHEBI:175415CHEBI:38263CHEBI:74860CHEBI:73762CHEBI:116314CHEBI:34494CHEBI:64277CHEBI:16811CHEBI:73806CHEBI:23673CHEBI:73788CHEBI:73711CHEBI:74862CHEBI:46755CHEBI:34900CHEBI:15414CHEBI:17822CHEBI:165870CHEBI:74815CHEBI:73807CHEBI:63154CHEBI:16296CHEBI:73814CHEBI:17431CHEBI:168067CHEBI:157877CHEBI:220610CHEBI:17509CHEBI:17858CHEBI:173751CHEBI:6925