CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare

1. Daniele Daffonchio Tullio Brusa Sara Borin Adriana Bona Enrica Canzi Claudia Sorlini Partner 1c CoNISMa, Consorzio Nazionale Interuniversitario Scienze del Mare DISTAM Università degli Studi di Milano WP5: understanding of ecological relations between the microbial communities and the functioning of DHABs environment

2. Screening of the 2001 isolates collection for selected metabolic activities Evaluation of the metabolic differences among the basins microflora • Oil utilisation as unique carbon source • Catechol utilization as unique carbon source • Esterase activity on long chain satured fatty acids (Tween60) • Esterase activity on long chain insatured fatty acids (tryoleine) • Production of polysaccarides • Salinity tolerance

3. Oil utilization • Inoculum preparation in M9 mineral broth with yeast extract as carbon source • Cells wash, and resuspension in mineral medium (NaCl concentration optimum for each strain) with 3‰ crude oil as unique carbon source • Incubation at 28°C and growth revealing with INT (iodoniltetrazolium violet) Almost alll the positive strains were able to grow on catechol as unique carbon source • Bannock shows the highest activity in oil degradation • The activity is prevalent in the sediments

4. Inoculum preparation on rich liquid medium (PCB) with 5% NaCl • Inoculation of minimal solid media with Tween60 and 3/5% NaCl • Incubation 6 days at 30°C • Screening of results (CaCl2 precipitation) Lipase-esterase activity on long chain satured fatty acids • Lipase-esterase activity is highly present among the isolated strains • L’Atalante shows the lowest activity for the idrolysis of long chain fatty acids • The activity is prevalent at the water/brine interface

5. Inoculum preparation on rich liquid medium (PCB) with 5% NaCl • Inoculation of minimal solid media with tryolein and 3/5% NaCl • Incubation 6 days at 30°C • Screening of results (CuSO4 precipitation) Lipase-esterase activity on long chain unsatured fatty acids • Lipase-esterase activity on unsatured fatty acids is very low in all the basins • Urania shows the lowest number of active strains • The activity is expressed mainly in the water/brine interface, at seawater salinity value

6. Polysaccarides production • Growth on rich liquid medium (PCB) with 5% NaCl • Precipitation with 1 volume of acetone • Half of the strains exhibit production of exopolysaccarides • Discovery has the lowest range of polysaccarides production

7. Antibacterial activity • Growth on rich solid medium (PCA) with 3% NaCl • Killing of cells with chloroform • Overlay with Sarcina lutea culture, incubation at 30°C • Screening of inhibition rings Tested all 94 collection strains: no antibacterial activity found

8. In the Bannock basin a large amount of the cultivable microflora has interesting metabolic capabilities • Discovery shows the lowest metabolic activity

9. Salt tolerance • Growth in solid media containing different concentrations of NaCl • All the strains grow at seawater salinity • Few strains do not tolerate absence of NaCl in the growing medium • Few strains isolated mostly from Urania and Discovery tolerate >15% NaCl • L’Atalante isolates have the lowest values of tolerance

10. 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

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