1. Instituto de Recursos Naturales y Agrobiología de Sevilla Consejo Superior de Investigaciones Científicas José Julio Ortega ASSESSING THE MICROBIAL AVAILABILITY OF POLYCYCLIC AROMATIC HYDROCARBONS PRESENT IN MARINE SPILLS

2. Proyect VEM 2004-08556: “Microbial availability and metabolism of polycyclic aromatic hydrocarbons present in marine oil spills. Implications for their natural attenuation and bioremediation” Examine PAH bioavailability and metabolism in different scenarios relevant to marine oil spills: water column (dispersants), shoreline (bioremediation) and sediments (nat. attenuation) OBJ. 1 Microbial physiology and metabolism of PAH biodegradation in marine microorganisms OBJ. 2 Study of physicochemical processes involved in PAH bioavailability and their modification by biological factors and spill management strategies OBJ. 3 Bioavailability and metabolism of PAH in microcosms OBJECTIVES

4. Dissolved in macropores Sorbed to organic matter and clay fractions 1 Sorbed in micropores smaller than bacteria 2 Dissolved in non- aqueous phase liquids 3 LOW BIOAVAILABILITY (slow kinetics, residual fractions)

5. BIOSURFACTANT PRODUCTION

6. CMC Effect of Pseudomonas aeruginosa 19SJ biosurfactants on surface tension: critical micelle concentration

7. Growth and biosurfactant production by Pseudomonas aeruginosa 19SJ from solid phenanthrene (10 mg/ml) M. Garcia-Junco et al., Environ. Microbiol. 2001, 3, 561-569

8. Effect of biosurfactants from Pseudomonas aeruginosa 19SJ on partitioning of pyrene from a NAPL Biosurf.Solids Part. rate C eq C eqsolids (µg/mL)(mg/mL) (ng/mL/h) (ng/mL)(ng/mg) 0 10 100 0 100 0001100011 0,5 1,6 11,6 0,7 27,4 18 85 547 27 650 - 16 40 M. Garcia-Junco et al., Environ. Sci. Technol. 2003, 37, 2988-2996

9. CHEMOTAXIS

10. CHEMOTAXIS CAPILLARY METHOD slide coverslip Bacterial suspension Chemoattractant or control Capillary tube 1 L1 L

12. Chemotaxis towards phenanthrene of Pseudomonas putida 10D J.J. Ortega-Calvo et al., FEMS Microbiol. Ecol. 2003, 44, 373-381

13. Environmental sample TENAX SAMPLE SUSPENSION DESORPTIONBIODEGRADATION SAMPLE SUSPENSION NaOH TRAP - TENAX EXTRACTION - HPLC ANALYSIS - MC EXTRACTION - HPLC ANALYSIS (native PAH) - 14 CO 2 MEASUREMENTS ( 14 C-PAH)

14. BIOAVAILABILITY ASSAY 250 ml erlenmeyer flask B. HPLC analysis 40 g Sample Teflon-lined stopper Lateral body A. 14 CO 2 production Syringe sampling Alkali trap 40 g sample + radiolabelled PAH Main body Teflon-lined stoppers Biometric flask 14 CO 2

15. S t / S 0 = F rap * exp (-K rap * t) + F slow * exp (-K slow * t) Flu Fen Ant Flut PyrBenz(a)pyr DESORPTION OF PAHs WITH TENAX CREOSOTE-POLLUTED SOIL

16. DESORPTION & BIODEGRADATION

17. S t / S 0 = F rap * exp (-K rap * t) + F slow * exp (-K slow * t) Ant Fen Fluor Pyr Benz(a)ant Benz(a)pyr DESORPTION OF PAHs WITH TENAX BIOREMEDIATED SOIL

18. BIOAVAILABILITY OF PAH IN BIOREMEDIATED SOIL (SOILREM E6068) (0.2 g/Kg  PAH) Fen Pir Ftno

19. 42 98 186 1366 1250 372 118 404 59 111 85 346 142 222 166 124 55 429 GIBRALTAR SAMPLING POINTS ROADS URBAN ZONES INDUSTRIAL ZONES Background PAH pollution in Gibraltar Area (Cádiz) (µg/kg  16 EPA PAH)

20. Phenanthrene content in soils from Gibraltar Area *Wild, S.R. & Jones, K.C., Environ. Pollut. 1995, 88, 91-108

21. Bioaccessibility of native phenanthrene to autochtonous microbial populations in soils with different % OM Conditions: 40 g soil amended with 11.8 µg/kg 14 C-phen dissolved in distilled water 14 Cnative

22. CONCLUSIONS 1. FAR FROM BEHAVING AS PASSIVE CATALISTS, MICROORGANISMS INHABITING PAH-POLLUTED SOILS AND SEDIMENTS CAN SOLVE A RANGE OF LOW-BIOAVAILABILITY SITUATIONS BY IMPROVING THEIR MODES OF POLLUTANT ACQUISITION (BIOSURFACTANTS, CHEMOTAXIS, ETC.) 2. HOWEVER, THERE ARE A NUMBER OF ENVIRONMENTAL SITUATIONS, FOR EXAMPLE INVOLVING DESORPTION-RESISTANT COMPOUNDS, WHERE THE PHYSICAL CONSTRAINTS FOR BIODEGRADATION ARE HARDLY MODIFIABLE BY PHYSIOLOGICAL MEANS

23. Instituto de Recursos Naturales y Agrobiología de Sevilla C.S.I.C. Grupo “Biodegradación y Biorremediación” (PAI-RNM312) Marisa Bueno Marta García-Junco* César Gómez* Mohammed Lahlou* Jose Luis Niqui Rosa Posada Patricia Velasco José Julio Ortega M. Grifoll (Univ. Barcelona) Hauke Harms (UFZ, Leipzig) Anatoly Marchenko (R. Center Toxicol., Rusia)