RESULTS AND DISCUSSION Metabolomics profile of Aspergillus niger through comprehensive two dimensional gas chromatography Carina Costa1,*, Sílvia M. Rocha2, Adelaide Almeida3 1 Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal, *carina.pedrosa@ua.pt 2 Departamento de Química & QOPNA, Universidade de Aveiro, 3810-193 Aveiro, Portugal 3 Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal RESULTS AND DISCUSSION INTRODUCTION Fungal infections have greatly increased in risk populations, namely in immunocompromised patients, because many of them became resistant to antifungals, as Aspergillus species. Survival of infected patients depends on early diagnosis, but conventional methods are unable to diagnose infections on their early stages (ca. 1 week). Microbial metabolomics arises as a powerful feature screening the metabolites produced by microorganisms. It provides information regarding the state of biological organisms which can be used as a diagnostic tool for diseases through fungal specific metabolites pattern. A methodology based on headspace-solid phase microextraction combined with comprehensive two-dimensional gas chromatography coupled to mass spectrometry with a high resolution time of flight analyser (HS-SPME/GC×GC-ToFMS) was developed in order to establish a specific metabolomic profile of Aspergillus niger, that can be further exploited to fungal diagnosis. Acids Alcohols Aldehydes Esters Hydrocarbons Ketones S-compounds compounds Terpenic PCA scores plot applied to GC×GC peak areas of a set of 314 metabolites identified for A. niger exo-metabolome in all growth conditions. PC1 and PC2 loadings plot explaining the separation observed in scores map. The variables are organized according the chemical families. METHODOLOGY Nutrients of the culture medium are important factors for the growth of A. niger After 5 days of growth, oxidative stress occurs Increased the amount of carotenoids (precursors of C13 norisoprenoids) Solid medium Liquid medium 1. Growth of A. niger Heatmap representation of the A. niger exo-metabolome (around 500 metabolites) in different growth conditions, such as culture medium (solid and liquid YGC), temperature of 25 and 37 ⁰C and incubation time of 3 and 5 days. Different intensities correspond to the normalized GC peak of each compound. 2. Centrifugation Metabolism of A. niger dependent on the growth condition Heatmap shows these differences in the metabolomic profile Temperature: 25 and 37 ºC Growth media: Yeast Glucose Chloramphenicol Agar (YGC) Incubation time: 3 and 5 days Culture medium: Solid and liquid YGC 10.000 rpm during 15 min. at 4 ºC and filtered for glass vial 3. Exo-metabolome determination: 3.1 Headspace-Solid phase microextraction (HS-SPME) Heatmap shows different intensities of each compound in different growth conditions. The chemical families reported in literature for A. niger were selected and a multivariate analysis unsupervised was applied – Principal Component Analysis (PCA) shows a separation between the tested conditions, revealing that PC1 and PC2 explained 32% and 25%, respectively. Due to the complexity of the results, in order to reduce analysis time, a sub-set of 30 metabolites produced in all growth conditions was selected. The multivariate analysis unsupervised was also applied for this sub-set of 30 metabolites and shows a separation between the tested conditions. Accordingly to the GC×GC peak area for this sub-set, the best growth conditions are 3 or 5 days at 25 ⁰C, using solid medium or 3 days at 37 ⁰C, using liquid medium. SPME Conditions: 20 mL of sample 4 g of NaCl Extraction Time: 30 min Agitation: 350 rpm Extraction Temperature: 50 ºC Fibre: 50/30 μm, DVB/CAR/PDMS PCA scores plot applied to GC×GC peak areas of a sub-set of 30 metabolites identified for A. niger exo-metabolome in all growth conditions. PC1 and PC2 loadings plot explaining the separation observed in scores map. The variables are organized according sub-set of 30 metabolites. The numbers represent the 30 metabolites: 1- 1-Butanol; 2- 3-Methyl-1-butanol; 3- 1-Hexanol; 4- 1-Heptanol; 5- 1-Octen-3-ol; 6- 3-Octanol; 7- 2-Ethyl- 1-hexanol; 8- 2,6-Dimethyl-7-octen-2-ol; 9- 1-Octanol; 10- 3-Methylbutanal; 11- Hexanal; 12- Heptanal; 13- Nonanal; 14-Decanal; 15- 2-Undecenal; 16- Dodecanal; 17- Benzaldehyde; 18- Benzeneacetaldehyde; 19- Methyl 2-methylpropenoate; 20- 3-hydroxy-2,4,4-trimethylpentyl 2-methyl-propanoate; 21- Hexadecane; 22- Heptadecane; 23- 2-Propanone; 24- 3-Heptanone; 25- 6-Methyl-5-hepten-2-one; 26- 3-Nonen-2-one; 27- 6,10-Dimethyl-,5,9-undecadien-2-one; 28- Endobornyl acetate; 29- Torreyol; 30- α-iso-methyl ionone. 3.2 HS-SPME/GC×GC-ToFMS a CONCLUDING REMARKS HS-SPME/GC×GC-ToFMS showed that the exo-metabolome of A. niger includes several hundreds of metabolites, distributed by several chemical families, mainly alcohols, aldehydes, esters, hydrocarbons, ketones and terpenic compounds; The results indicated the high complexity of A. niger exo-metabolome and allowed to characterize a specific metabolomic pattern; The specific metabolic pattern of A. niger can be explored in the clinical context in order to discriminate fungal infections through a more sensitive method when compared to conventional ones. ACKNOWLEDGMENTS GC peak area normalized by CFU's of a sub-set of 30 metabolites identified for A. niger. Each column represents the average calculated for three replicas and error bars represent the standard deviation. a - represent no significant differences (p > 0.05) among conditions. Funding is acknowledged from the European Regional Development Fund (FEDER) through the Competitive Factors Thematic Operational Program (COMPETE) and from the Foundation for Science and Technology (FCT), Portugal, for funding the Research Units QOPNA (Research Unit 62/94 QOPNA, under projects PEst-C/QUI/UI0062/2013 and FCOMP-01-0124-FEDER-037296), and CESAM (project PEst-C/MAR/LA0017/2013).