Paul Adrian Iturbe Espinoza October 10, Monthly Oil Spill Incidents - 2015 Volume of oil spills / month (bbl) http://www.shell.com.ng http://www.bbc.com The once-pristine Niger Delta has been polluted for decades Musat, 2005 Study microbial community structure and functioning during oil degradation in soils. Isolation and characterization of dominant oil-degrading strains from Nigerian soils under different culture conditions. SOIL MEDIA pH Rich Poor Rich Contaminated Poor Uncontaminated Rich Poor 5 7 Plating on Nutrient agar 28 days of incubation Shaking at 120 rpm /30°C No soil 5 7 5 7 5 7 5 7 5 7 47.5 mL of medium 5 g of soil 2.5 mL of oil Purify DNA from precultures (MoBio Power Soil DNA Isolation kit) Primers 8f and 1512r were used to amplify almost complete 16S rDNA Sequence PCR products by using 23 ABI 3730XLs (Macrogen) Best hit after the BLAST search OD (600 nm) 4,0 R5-014Ly R7UO24Cw(bc) R7-O14Lc NCDO712 P5CO16Lw KF147 3,0 2,0 1,0 0,0 0 OD600=0.1 4 8 Sampling 12 Time (h) 16 20 24 Sampling Add 2 mL of the hydrocarbon measure OD600 vortex for 2 min 5 mL of cells (OD600=1) in buffer PUM Rosenberg et al, 1980 Wait 15 min Pseudomonas aeruginosa Sphingobium fuliginis Rhizobium sp E. coli R7CO16Bb R7-O14Sy R7-O26Lw P7UO26So P7-O24Lb P7CO26B P5-O26Sy P5UO15Bbc R7CO15Sy R5-O14Ly Marker R7-O14Lw R7-O14Lc P5CO16G P7-O16Woc R7UO24Clwbc P7UO26Lg SSOSw Marker R5CO24Gc P5CO16Lw R7UO16Vwc P7UO26Sw NENY SSOSy SSOLw SSOIc Marker Cont. soil R7CO16Bb Gram stain Hydrophobicity 84.68% Cont soil P7CO26B 86,16% Uncont. Soil P5UO15Bbc 47,94% R7C016Bb P7C026B P5U015B(bc) % Hydrophobicity 100 80 Octane 60 R7C016Bb P7C026B P5U015B(bc) 40 20 0 R7C016Bb P7C026 B P5U015B(bc) Hexane Octane Hexane No soil R7-O14Sy Cont. soil R7CO15Sy Cont. Soil P5CO16G Gram stain Hydrophobicity 6h 24 53.5% 83.5% 55.1 % 89 % 57 % 81.3 % 1 mL KPi 3 mL MES + 2.5 mL MgCl2 buffer of oil 100 L of the oil (top) layer were plated on Nutritive agar Shaking at 120 rpm/30°C Over night 50 mL of distilled water Streak spin oil SSOIc SSOLw Gram stain Hydrophobicity 6.7 % 11 % Rhodococcus sp. Patulibacter sp. Pseudomonas aeruginosa Kocuria rhizophila Micrococcus yunnanensis Pannonibacter phragmitetus 94,9% 88.0 % 86,2% 93.5 % 91.6% 71.1% Three strains change their hydrophobicity in stationary phase: Dyella sp. Pandoraea vervacti Sphingobium fuliginis 6h 18.9 % 7.1% 55.1 % 24h 57,28 % 31.23 % 89.05 % No soil P7-O24Lb Hydrophobicity 24,98% Uncont. soil R7UO16Vwc 88.02 % Poor media/pH 7/Uncont. soil P7UO26SW Hydrophobicity 94,86% Rich media/pH 5/No Soil R5-O14Ly 91.55% Pseudoxanthomonas sp. 9.2 % Sphingopyxis soli 15.1 % Dyella sp. 18.9 % Labrys portucalensis 10.1 % Pandoraea vervacti 7.1% Swaminathania salitolerans 9.7% Pseudomonas guguanensis 25% Rhizobium sp. 6.7 % Ralstonia sp. 27.8 % Rhizobium sp. 11 % Georgenia sp. 11 % Niabella thaonhiensis Pseudomonas aeruginosa Sphingobium fuliginis Achromobacter xylosoxidans Novosphingobium capsulatum 44.0 % 47.9 % 57.0 % 42.6 % 49.2 % Day 1 Ralstonia sp. Rhizobium sp. Georgenia sp. Novosphingobium capsulatum Day 28 Sphingobium fuliginis Swaminathania salitolerans Pseudomonas guguanensis Kocuria rhizophila Micrococcus yunnanensis Achromobacter xylosoxidans Pandoraea vervacti Pannonibacter phragmitetus After 28 days of incubation dominant strains are different as a result of a adaptive response to the change of conditions contaminated soils + oil Labrys portucalensis Dyella sp. Pseudomonas aeruginosa Sphingobium fuliginis Niabella thaonhiensis Sphingopyxis soli Pseudoxanthomonas sp. Patulibacter sp. Rhodococcus sp. uncontaminated soils + oil After long incubation (28 day) dominant strains from contaminated soils take over strains from uncontaminated soils. Both group at the same time take over dominant strains from only oil. 1. 25 strains associated with oil degradation were identify by partial 16S rDNA gen sequencing and were characterized by DGGE fingerprinting, Gram stain and cell surface hydrophobicity test. 1. Pseudomonas aeroginosa and Sphingobium fuliginis are the dominant isolated, both strains have high hydrophobicity and are potential hydrocarbon degraders. - Rob van Spanning - Martin Braster - Herwig Bachmann - David Brown - Juami van Gils - Madalina Maria Vita