Partner 1c CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare DISTAM Università degli Studi di Milano Daniele Daffonchio Tullio Brusa Sara Borin Adriana Bona Enrica Canzi Claudia Sorlini WP4: Determination of the distribution, taxonomy and diversity of microorganisms from DHABs, and isolation of strains with biotechnological potential

Urania 2001 samples

Species identification 2001 isolates collection Strict anaerobes aerobes URANIA 25 BANNOCK 23 DISCOVERY 21 L’ATALANTE 23 URANIA BANNOCK DISCOVERY 1 L’ATALANTE 2 95 3 Transferred to Proteus Characterized for diversity Species identification Screened for activity Species identification

interspecies diversity characterization Molecular typing of isolated strains Rep-PCR interspecies diversity characterization 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 Isolated strains

Molecular typing of isolates based on 16S-23S rDNA Intergenic Transcribed Spacer PCR 46 different ITS groups Only 3 groups found in different basins 31 Groups with only 1 isolate Very high subspecies diversity

Identification of ITS groups Most abundant groups Isolates, all aerobic/facultative anerobic, have a wide subspecies diversity but belong to few phylogenetic groups: -proteobacteria Bacillus -proteobacteria Enterococcaceae

Bacillaceae and proteobacteria in all the basins Phylogenetic groups Bacillaceae and proteobacteria in all the basins

Phylogenetic groups proteobacteria in water/brine interface Bacillaceae in sediments

New species: 99% similarity with unclassified sediment -proteobacterium 95% similarity with Ruegeria algicola 97% similarity with unclassified PAH-degrading -proteobacterium 96% similarity with Rhodospirillum

intra-species diversity based on BOX-PCR fingerprinting 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 6 29 53 76 100 % 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

Qualitative data on the environmental significance of cultivable microflora: DGGE fingerprint DWB 2D 6D 9D 10D 11D 17D 20D 21D 22D 30D Isolated strains do not seem to represent the total eubacterial community inhabiting the water/brine interface

Quantitative data on eubacteria/arhcaea prevalence in Urania and Bannock basins: Real Time PCR Water-brine interface is an enrichment phase for bacteria Eubacteria are 1-3 magnitude orders higher than archaea in the 2 basins. Urania Bannock

Kinetics of survival in the brines Aerobic strains isolated from water/brine interface: are adapted to survive in the brines? Kinetics of survival in the brines -proteobacteria Brines have bactericidal activity Cells in 10 minutes decrease of 9 magnitude orders A fraction of the cells remains vital for variable range of time (2 weeks or more than 30 days) Bacillus L’Atalante brines

Kinetics of cells survival in the brines Aerobic strains isolated from water/brine interface: are adapted to survive in the brines? Kinetics of cells survival in the brines -proteobacteria Bacillus L’Atalante brines Bannock brines Discovery brines Urania brines Discovery has the most aggressive brines: death of all the inoculated cells in 1 day Urania has the less aggressive brines: surviving of a fraction of the cells over 1 month

Urania 2002 samples

Mixed cultures obtained SAMPLES: Brines Brine/sediment interface Sediments Mud pit High salinity Anaerobiosis Sulfur red bac suphate red bac Sulfur red bac 47 mixed cultures obtained Methane bac Brine+substrate Geobacter Methane ox bac Sulphur ox bac

Isolated strains 23 isolated strains