CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare Partner 1c DISTAM Università degli Studi di Milano Daniele Daffonchio Tullio Brusa Sara Borin Enrica Canzi Alessandro Favini Lorenzo Brusetti Claudia Sorlini 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

Urania 2001 samples

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

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

Molecular typing of isolates based on 16S-23S rDNA Intergenic Transcribed Spacer PCR 46 ITS groups 3 groups found in different basins 31 Groups with only 1 isolate L’Atalante Bannock Discovery Urania n° strains 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

Species diversity among basins/samples n° of strains

Identification of ITS groups Most common isolates

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

New species New species?

Aerobic isolates intra-species diversity based on BOX-PCR fingerprinting 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 % similarity 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

Cultivable/uncultivable microflora DGGE fingerprint DWB 2D6D9D 10D11D17D20D21D22D30D31D Isolated strains do not seem to represent the total eubacterial community inhabiting the water/brine interface seawater

Salt tolerance  Growth in rich solid media (Plate Count Agar) with NaCl

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

Urania 2002 samples

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

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

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, H 2 ; E - acceptor: CO 2 ; Carbon source: yeast extract, tryptone MOB: Medium for methanotrophic bacteria (DSM 1 modified) Carbon source: meat extract, peptone, CH 4 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: CH 4 ODB: Medium for crude-oil degrading bacteria. Carbon source: crude-oil

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

AMO: Anaerobic Methane Oxidation search  Brines  Mud pit samples sample CH 4 Bromodeoxyuridine BrdU On board Immunocapture BrdU-labelled DNA Identification of the actively growing bacteria = methane oxidizing Incubation GC CH 4 oxidation measurement Fluxed with N2 to eliminate endogenous CH 4 DNA extraction

AMO: Anaerobic Methane Oxidation search 30 days of incubation:15°C brines 45°C mud pit CH 4 consumption GC measurement