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CNR IAMC - Istituto per l’Ambiente Marino Costiero Messina, Italy Università degli Studi di Messina. WP4-WP5. State of the Art April 2003. Isolates analysis. 0. Anaerobic chamber. SOLID Brine based media. O 2. O 2. Reisolation by liquid and solid cultures Strain collection

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  1. CNR IAMC - Istituto per l’Ambiente Marino Costiero Messina, Italy Università degli Studi di Messina WP4-WP5 State of the Art April 2003

  2. Isolates analysis 0 Anaerobic chamber SOLID Brine based media O2 O2 Reisolation by liquid and solid cultures Strain collection Phylogentical and physiological charachterisation ONR Saline Medium (3%Salinity) No Isolates from BodyBrine

  3. Isolates analysis WP4-WP5 Phylogenetical Analysis REP Fingerprinting Aerobic/Anaerobic growth curves Taxonomic Diversity Physiological Methabolic analysis “Bottle Neck” and others Application Conclusions

  4. Isolates analysis Bulleidia e. Methanococcus jannacii Desulfococcus biaticus Arcobacter butzleril Alteromonas macleodi Idiomarina loihensis Halomonas variabilis 339-27-530 364 365 367-27 369-27 369-1492 370 Marinobacter aqueoli 357 351g 348g-530 345 340-27 Isolated strains 350 Marinobacter aqueoli 374b 351b Alteromonas sp. 354 374l 363 Marinobacter sp. 348b Marinobacter hydrocarbonoclasticus 369 Pseudomonas sp. Pseudomonas sp. 328 Pseudomonas pseudoalcaligenes Pseudomonas sp. 353 359 Alcanivorax sp. 358 Alcanivorax Venustit 368 Fundibacter jadensi Alcanivorax borkumensis Sulfitobacter pontiacus 0.1 Roseobaccter sp.

  5. Isolates analysis Phylogenetical Analysis REP Fingerprinting Aerobic/Anaerobic growth curves Taxonomic Diversity Physiological Methabolic analysis “Bottle Neck” and others Application Conclusions

  6. Isolates analysis TAACGGCGCTCCACA ………………….TGTGGAGCGCCGTTA GENE ATTGCCGCGAGGTGT…………………..ACACCTCGCGGCAAT M S 5 3 2 6 1 4 4 2 1 5 6 3 REP Fingerprinting Repetitive Extragenic Palyndromic Analysis

  7. Isolates analysis REP Fingerprinting

  8. Isolates analysis REP Fingerprinting

  9. Isolates analysis Phylogenetical Analysis REP Fingerprinting Aerobic/Anaerobic growth curves Taxonomic Diversity Physiological Methabolic analysis “Bottle Neck” and others Application Conclusions

  10. Isolates analysis Aerobic/Anaerobic growth curves Bioscreen analysis Reading at 640nm every 1.30h For 10days 4 replicate Mineral oil Aerobic Anaerobic ONR + Pyruvate ONR + Pyruvate

  11. Isolates analysis Aerobic/Anaerobic growth curves Aerobic Anaerobic 345 350 363 114h 142h Marinobacter h. Marinobacter a. Marinobacter h. 340 348 134h 178h 42h 102h Alteromonas sp. Idiomarina l. 351 354 356 122h 138h 34h 82h 62h 64h 88h 128h Marinobacter h. Marinobacter a. Idiomarina l. Ten days, four replicate

  12. Isolates analysis Aerobic/Anaerobic growth curves Aerobic Anaerobic 50h 85h 339 353 369 72h 92h Halomonas v. Alteromonas sp. Halomonas v. 358 359 108h 116h Alcanivorax v. Alcanivorax v. 364 365 367 88h 92h 72h 104h 94h 78h Halomonas v. Halomonas v. Halomonas V. Ten days, four replicate

  13. Isolates analysis Aerobic/Anaerobic growth curves Aerobic Anaerobic 327 124h 182h Marinobacter h. 320 323 326 328 184h 200h 54h 80h Idiomarina l. Marinobacter a. Marinobacter h. Pseudomonas p. 330 333 116h 166h Pseudomonas p. Marinobacter a. Ten days, four replicate

  14. Isolates analysis 345 114h 142h 50h 85h Marinobacter h. 327 339 369 124h 182h 72h 92h Marinobacter h. Halomonas v. Halomonas v. REP Fingerprinting

  15. Isolates analysis Aerobic/Anaerobic growth curves • Problems: • Preliminary data (not shown) seem to suggest that the isolates are not able to grow on pure brine. • Future: • To try the same experiment in aerobic and anaerobic condition using a salt gradient from SeaWater to pure Brine • To try different substrates

  16. Isolates analysis Phylogenetical Analysis REP Fingerprinting Aerobic/Anaerobic growth curves Taxonomic Diversity Physiological Methabolic analysis “Bottle Neck” and others Application Conclusions

  17. Isolates analysis Methabolic analysis Why: • The study of the degradation of aromatic compounds has a very applicative meaning in order to evaluate the possible employ in bioremediation • The major part of dangerous or difficoult to remove compounds are aromatics • A great number of polluted areas is associated with hypersaline environment

  18. Isolates analysis Multiple culture on ONR + Aromatic compound Catechol Gentisate The samples were incubated in anaerobic chamber to avoid spontaneous oxydation of substrates Oxygen Oxygen Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) Tested by turbidimetry of growing cells after 4 days

  19. Isolates analysis Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) Catechol Gentisate

  20. Isolates analysis Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) Gentisate Catechol

  21. Isolates analysis Catechol Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) Gentisate

  22. Isolates analysis Methabolic analysis (Naphtalene,Fenantrene, Hexadecane) Hexadecane Direct cultivation on ONR Agar Blank in ONR without carbon source

  23. Isolates analysis Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) Preliminar data

  24. Isolates analysis

  25. Isolates analysis Methabolic analysis • Future: • To repeat the experiments in salinity gradient • To check and charachterize the genes coding for the enzymes involved in the HC pathways by PCR and sequencing • To purify and charachterize the obtained enzymes

  26. Isolates analysis Conclusions • Phylogenetical Analysis • The cultivatable components of bacterial communities from DHABs are not corresponding at all with the active components within the basins (Allochthnous/contaminant bacteria?) • REP Fingerprinting • Aerobic/Anaerobic growth curves • Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) • Conclusions

  27. Isolates analysis Conclusions • Phylogenetical Analysis • REP Fingerprinting • Isolates with identical 16S rDNA sequences show different REP fingerprint. • The REP analysis shows an higher and variegate diversity in respect to the 16S rDNA sequence analysis that henances the low efficiency of 16S rDNA analysis. • Aerobic/Anaerobic growth curves • Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) • Conclusions

  28. Isolates analysis Conclusions • Phylogenetical Analysis • REP Fingerprinting • Aerobic/Anaerobic growth curves • Every isolate shows peculiar growth curves and is able to grow both in aerobic and anaerobic conditions • Each isolate grows faster in aerobic than in anaerobic conditions but in general the maximum density for both conditions is comparable • The clustering of the isolates based on the obtained growth curves seems to correspond to the REP-16S rDNA grouping • Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) • Conclusions

  29. Isolates analysis Conclusions • Phylogenetical Analysis • REP Fingerprinting • Aerobic/Anaerobic growth curves • Methabolic analysis (Catechol, Gentisate, Protocatechuic acid) • Some isolates are able to degrade different aromatic compounds at the marine salt conditions. The question on the range of salinity at which this capability is maintained is still opened. • Further analyses on the aromatic compound spectra are planned (included the halogenated compounds) as well as the molecular study of the bacterial enzymes involved in the HC pathways. • Conclusions

  30. Isolates analysis Conclusions Phylogenetical Analysis REP Fingerprinting Aerobic/Anaerobic growth curves Methabolic analysis (aromatic compounds) Other substrates Conclusion We have a lot of work to do! Special Thank’s to the CNR staff

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