CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare

1. Daniele Daffonchio Tullio Brusa Sara Borin Enrica Canzi Alessandro Favini Lorenzo Brusetti Claudia Sorlini Partner 1c CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare DISTAM Università degli Studi di Milano WP4: Determination of the distribution, taxonomy and diversity of microorganisms from DHABs, and isolation of strains with biotechnological potential WP5: understanding of ecological relations between the microbial communities and the functioning of DHABs environment WP6: genetic characterization of isolated microorganisms and of enzymes interesting for biotechnological application

2. Urania 2001 samples

3. 2001 isolates collection Strict anaerobes aerobes URANIA 25 BANNOCK 23 DISCOVERY 21 L’ATALANTE 23 URANIA BANNOCK DISCOVERY 4 L’ATALANTE 2 95 6 • Transferred to Proteus • Characterized for diversity • Species identification • Screened for activity • Under isolation procedure

4. Rep-PCR interspecies diversity characterisation ITS-PCR species/subspecies specific 16S rDNA sequencing identification Isolated strains Halobacillus Group 1 Halomonas Group 2 Group 3 Alteromonas Group 4 nnn Group 5 nnn Group n nnn Molecular typing of isolated strains

5. n° strains 7 17 20 25 ITS groups 1, 3, 4, 5, 6 1, 2, 14, 15, 22, 23, 24, 38, 39, 40, 41, 42 4, 18, 19, 20, 21, 25, 33, 34, 35, 36, 37 1, 4, 7, 9, 10, 11, 12, 13, 14, 16, 17, 26, 27, 28, 29, 30, 31, 32 L’Atalante Bannock Discovery Urania 46 ITS groups 3 groups found in different basins 31 Groups with only 1 isolate Molecular typing of isolates based on 16S-23S rDNA Intergenic Transcribed Spacer PCR

6. Species diversity among basins/samples n° of strains

7. Identification of ITS groups Most common isolates

8. Phylogenetic groups Bacillaceae and proteobacteria in all the basins proteobacteria in water/brine interface Bacillaceae in sediments

9. New species New species?

10. 5Bt WB 7B WB 8B WB 15B S 10B BS 23D S 14D S 15D S 9B WB 18B S 8D BS 30D WB 19D WB 19Db WB 2D WB 4D WB 5D WB 11Ub BS 10U BS 5A WB 2B WB 11A BS 14B BS 6D WB 22U BS 1D WB 24U WB 4A WB 6A WB 13D BS 13U BS 9A BS 17B S 16B S 11U BS 12D BS 11D BS 5Bbis WB 16Ubis S 15U S 16U S 6 29 53 76 100 % similarity Aerobic isolates intra-species diversity based on BOX-PCR fingerprinting • In each ITS group the strains display a different degree of genomic variability. • Strains belonging to the same ITS group, are not grouped according to the basin of origin, or according to the same sample type. • In some cases strains of the same origin show relatively high similarity

11. Cultivable/uncultivable microflora DGGE fingerprint seawater DWB DWB 10D 11D 17D 20D 21D 22D 30D 31D 2D 6D 9D Isolated strains do not seem to represent the total eubacterial community inhabiting the water/brine interface

12. Salt tolerance • Growth in rich solid media (Plate Count Agar) with NaCl

13. Catabolic genes characterization 19% of the isolates are able to grow on oil and catechol as unique carbon sources Isolated strains Research for xyl E-type genes (catechol 2,3 dioxigenase) gene via PCR All negatives Community DNA

14. Urania 2002 samples

15. Media inoculated on-board Brines Brine/sediment interface Sediments Mud pit SAMPLES: High salinity Anaerobiosis

16. Mixed cultures obtained No growth from mud pit Oil degrading bacteria from concentrated samples 29 enrichment cultures

17. SaRB 1.3: Medium for sulphate reducing bacteria (Procariota). E- acceptor: sulphate; Carbon source: succinate, fumatate, malate, pyruvate SaRB 1.4: Medium for sulphate reducing bacteria (Procariota). E- acceptor: sulphate; Carbon source: ethanol SaRB 1.5: Medium for sulphate reducing bacteria (Procariota). E- acceptor: sulphate; Carbon source: lactate SaRB 1.6: Medium for sulphate reducing bacteria (Procariota). E- acceptor: sulphate; Carbon source: phenol, catechol SuRB 2.1: Medium for Sulphor reducing bacteria ( Procariota). E- acceptor: S; Carbon source: acetate, formate SuRB 2.2: Medium for Sulphor reducing bacteria ( Procariota). E- acceptor: S; Carbon source: propionate, pyruvate, palmitate SuRB 2.3: Medium for Sulphor reducing bacteria ( Procariota). E- acceptor: S; Carbon source: succinate, fumarate, malate, pyruvate SuRB 2.4: Medium for Sulphor reducing bacteria ( Procariota). E- acceptor: S; Carbon source: ethanol SuRB 2.5: Medium for Sulphor reducing bacteria ( Procariota). E- acceptor: S; Carbon source: lactate SuRB 2.6: Medium for Sulphor reducing bacteria ( Procariota). E- acceptor: S; Carbon source: phenol, catechol MB: Medium for methanobacteria (Balch). E- donor: formate, acetate, H2; E- acceptor: CO2; Carbon source: yeast extract, tryptone MOB: Medium for methanotrophic bacteria (DSM 1 modified) Carbon source: meat extract, peptone, CH4 M 1.1: Carbon source: yeast extract, triptycase M 1.2: Carbon source: lactate M 1.3: Carbon source: propionate, palmitate, pyruvate M 2:Medium for Haloanaerobiaceae (DSM). E- acceptor: sulphate; Carbon source: yeast extract, glucose SOB: Medium sulphur-oxidizing bacteria (DSM 484 modified). E-donor: sulfide SRB2: Medium for sulphate-reducing bacteria (Procariota modified). Carbon source: CH4 ODB: Medium for crude-oil degrading bacteria. Carbon source: crude-oil

18. donor MATING host “Fishing” of functional genes in the metagenomic DNA Plasmids donor: cells recovered filtrating Discovery water/brines interface concentrated Plasmids exogenous isolation Plasmids hosts: Pseudomonas putida Screening for: Hg resistance NaCl tolerance Oil degradation Naphtalene degradation Search fortransconiugants

19. sample AMO: Anaerobic Methane Oxidation search samples Bromodeoxyuridine BrdU On board CH4 • Brines • Mud pit Fluxed with N2 to eliminate endogenous CH4 Incubation DNA extraction GC CH4 oxidation measurement Immunocapture BrdU-labelled DNA Identification of the actively growing bacteria = methane oxidizing

20. AMO: Anaerobic Methane Oxidation search CH4 consumption GC measurement 30 days of incubation: 15°C brines 45°C mud pit