Spin-Off of Polytechnic University of Marche

1. Spin-Off of Polytechnic University of Marche ULIXES 1ST year meeting Mazara del Vallo, 22nd January 2012

2. OVERVIEW In the framework of ULIXES, EcoTechSystems is involved in the following Work Packages: WP2 - Sampling and characterization of the Mediterranean Sea sites WP3 - Microbial diversity exploration in the Mediterranean Sea sites WP4 - Establishment of collections of microbial isolates and metabolites WP7 - Establishing new product/processes for bioremediation WP8 - Testing new product/processes by pilot bioremediation treatments in the field

3. WP2 WP2 - Sampling and characterization of the Mediterranean Sea sites Anconaharbour U10 U9 U3 U1 U2 U7 U6 15 sampling stations investigated U1-U10 impacted areas U11-15 BLK stations U5 U8 U4

4. WP2 Sampling activities After collection, sediment samples were collected by means of Van Veen Grab and Box Corer. After equipment recovery, physico-chemical features of surface sediments were determined. A sub-sampling was performed by means of Plexiglas cores up to ca. 25 cm depth. Cores were then extruded and sliced in 5 cm thickness slices for pysico-chemical analyses and quantification of pollutants. Samples for lab experiments were transferred into anaerobic jars.

5. Granulometric determinations WP2 Impacted sites Control sites Prevalence of clay and silty sediments, with low percentages of sands and gravels in all the investigated stations (harbor area) and in control stations

6. Mineralogic features of sediments WP2 Since the different geochemical components can deeply influence the microbial heavy-metal sequestration, and provide important insights on the mobilization of each metal, in all the investigated stations a mineralogic analysis through X-ray diffraction was performed. Results revealed the presence of the following components:

7. WP2 Chemical Determinations Control sites Impacted sites

8. WP2 Chemical Determinations Control sites Impacted sites

9. WP2 Chemical Determinations Control sites Impacted sites

10. WP2 Chemical Determinations Control sites Impacted sites

11. WP2 Chemical Determinations Control sites Impacted sites

12. WP3 WP3 - Microbial diversity exploration in the Mediterranean Sea sites In each sampling stations, the autochthonous microbial assemblages is being characterized in terms of: • Total prokaryotic abundance and biomass • Physical-chemical cell detachment , staining and counts under epifluorescence microscopy (SYBR Green I staining) • Total abundance of Bacteria and Archaea • CARD-FISH/Real-time PCR analyses • Microbial biodiversity • ARISA analyses

13. WP3 Total prokaryotic abundance Control sites Impacted sites Total abundances fall in typical ranges of coastal sediments with high organic load

14. WP3 Total prokaryotic abundance Control sites Impacted sites Huge cells: up to 30 fg C cell-1

15. WP3 Bacterial biodiversity: preliminary results RFU DNA was extracted from water and sediment samples with the UltraClean soil DNA isolation kit. Extracted DNA was amplified using universal bacterial primers 16S-1392F and and 23S-125R (amplifying ITS1 region) which amplify the ITS1 region in the rRNA operon plus ca. 282 bases of the 16S and 23S rRNA.

16. WP3 H’ = 3.01 ± 0.207 J = 0.72 ± 0.050 Bacterial genotype richness expressed as the total number of peaks within each electropherogram. For the calculation of bacterial diversity indices (Shannon-Wiener index, Hand the evenness, J), it was assumed that the number of peaks of electropherograms represented the species number (genotype richness) and that the peak height represented the relative abundance of each bacterial species.

17. WP4 – WP7 WP4 - Establishment of collections of microbial isolates and metabolites WP7 - Establishing new product/processes for bioremediation Relationships between heavy metal partition and evolution of microbial community in contaminated sediments: a common basis for WP4 and WP7 Microcosm experiments, with the main aim of optimizing biodegradation yields of organic contaminants together with the development of suitable diagenetic models of metals and metalloids Dedicated anaerobic incubations to understand the effects of changes in prokaryotic growth and diversity on hydrocarbon degradation rates and heavy metal partitioning on contaminated anoxic harbor sediments

18. WP4 - WP7 Fate of metal/metalloids in sediments Selected sediment samples, collected and characterized in WP2 and WP3, were used for the preparation of specific microcosm dedicated for time-course experiments. In microcosms, the biodegradation yields of organic contaminants by the autochtonous microbial consortia will be tested and optimized through anaerobic incubations and different environmental conditions. Different ending points were selected for each experimental set, in order to monitor the changing of prokaryotic growth and diversity together with hydrocarbon degradation rates and heavy metal partitioning

19. WP4 - WP7 Fate of metal/metalloids in sediments Experimental microcosms Factorial experimental design: • Anaerobic incubations of 250 mL slurries prepared with 25% of homogenized sediment and 0.2 μm prefilteredseawater, on continuous rotary shacking. • Controlledtemperatures (25°C and 15°C) • Incubationtimes: 0, 28, 60 days • Presence/absence of nutrients (C:N:P=100:10:1 molar) • Presence/absence of substrate (Lactose/acetate, 20 mM of C)

20. WP4 - WP7 Experimental microcosms In each microcosms, the following parameters will be monitored at each incubation time: • pH • Eh • Total prokaryotic abundance, biomass and diversity • Heavy metal concentration (overlying water) • Heavy metal concentrations and partitioning in the diffrent geoghemical phases • Hydrocarbon concentrations

21. WP4 - WP7 Total prokaryotic abundance

22. WP4 - WP7 Total prokaryotic biomass µg C g-1 d.s.

23. WP4 - WP7 Hydrocarbon biodegradation yields %