Instituto de Recursos Naturales y Agrobiología de Sevilla Consejo Superior de Investigaciones Científicas José Julio Ortega ASSESSING THE MICROBIAL AVAILABILITY.

Instituto de Recursos Naturales y Agrobiología de SevillaConsejo Superior de Investigaciones Científicas

José Julio Ortega

ASSESSING THE MICROBIAL AVAILABILITY OF POLYCYCLIC AROMATIC HYDROCARBONS PRESENT IN

MARINE SPILLS

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 theirmodification by biological factors and spill management strategies

OBJ. 3 Bioavailability and metabolism of PAH in microcosms

OBJECTIVES

Dissolved in macropores

Sorbed to organic matter and clay

fractions1

Sorbed in micropores smaller than bacteria

2

Dissolved in non-aqueous phase

liquids

3

LOW BIOAVAILABILITY

(slow kinetics, residual fractions)

BIOSURFACTANT PRODUCTION

RE (g/ml)0 50 100 150

Sur

face

tens

ion

(mN

/m)

30

40

50

60

70

80

CMC

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

Minutes0 20 40 60

Phe

nan

thre

ne

(g/

ml)

0

1

2

3

4

60

01

25

156

Growth and biosurfactant production byPseudomonas aeruginosa 19SJ from solid phenanthrene (10 mg/ml)

Hours

0 200 400 600 800

log

CF

U

7,00

7,75

8,50

9,25

RE

(µ

g/m

l)

0

50

100

150

200

250

M. Garcia-Junco et al., Environ. Microbiol. 2001, 3, 561-569

Effect of biosurfactants from Pseudomonas aeruginosa 19SJ on partitioning of pyrene from a NAPL

Biosurf. Solids Part. rate CeqCeqsolids

(µg/mL) (mg/mL) (ng/mL/h) (ng/mL) (ng/mg)

0

10

100

0

100

0

0

0

1

1

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

CHEMOTAXIS

CHEMOTAXISCAPILLARY METHOD

slide

coverslip

Bacterial suspension

Chemoattractant or control

Capillary tube

1 L

Chemotaxis towards phenanthrene of Pseudomonas putida 10D

J.J. Ortega-Calvo et al., FEMS Microbiol. Ecol. 2003, 44, 373-381

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

control 0,32 0,65 1,29

Phen conc (µg/ml)

Sp

eed

(m

m/m

in)

Environmental sample

TENAX

SAMPLESUSPENSION

DESORPTION BIODEGRADATION

SAMPLESUSPENSION

NaOH TRAP

- TENAX EXTRACTION- HPLC ANALYSIS

- MC EXTRACTION- HPLC ANALYSIS (native PAH)- 14CO2 MEASUREMENTS (14C-PAH)

BIOAVAILABILITY ASSAY

250 ml erlenmeyer flask

B. HPLC analysis

40 g Sample

Teflon-lined stopper

Lateral body

A. 14CO2 production

Syringe sampling

Alkali trap40 g sample + radiolabelled PAH

Main body

Teflon-lined stoppers

Biometric flask

14CO2

St / S0 = Frap * exp (-Krap * t) + Fslow * exp (-Kslow * t)

Tiempo (h)

0 200 400 600 800 1000

Ln St /

S 0

-5

-4

-3

-2

-1

0

Tiempo (h)

0 200 400 600 800 1000

Ln St /

S0

-6

-5

-4

-3

-2

-1

0

Flu Fen

Tiempo (h)

0 200 400 600 800 1000

Ln St /

S0

-4

-3

-2

-1

0

Tiempo (h)

0 200 400 600 800 1000

Ln S

t / S

0

-6

-5

-4

-3

-2

-1

0

AntFlut

Tiempo (h)

0 200 400 600 800 1000

Ln S

t / S

0

-5

-4

-3

-2

-1

0

Tiempo (h)

0 200 400 600 800 1000

Ln S

t / S

0

-4

-3

-2

-1

0

Pyr Benz(a)pyr

DESORPTION OF PAHs WITH TENAX

CREOSOTE-POLLUTED SOIL

DESORPTION & BIODEGRADATION

Time (h)

0 200 400 600 800 1000

Ln St /

S0

-6

-5

-4

-3

-2

-1

0

Phen desorpPhen biodeg

Time (h)

0 200 400 600 800 1000

Ln S

t / S

0

-4

-3

-2

-1

0

BaP desorpBaP biodeg

Phenanthrene Benz(a)pyrene Tenax Biodegradation Tenax Biodegradation

Frap (%) 96,1 94,9 86,7 31,4

Fslow (%) 3,9 5,1 13,3 68,6

Krap (h-1) 0,38 0,15 0,11 0,03

Kslow (x10-3 h-1) 2,70 2,20 1,80 0,08

St / S0 = Frap * exp (-Krap * t) + Fslow * exp (-Kslow * t)

Tiempo (h)

0 200 400 600 800 1000

Ln St /

S 0

-0,5

-0,4

-0,3

-0,2

-0,1

0,0

Ant

Tiempo (h)

0 200 400 600 800 1000

Ln St /

S 0

-0,5

-0,4

-0,3

-0,2

-0,1

0,0

Fen

Tiempo (h)

0 200 400 600 800 1000

Ln St /

S 0

-0,5

-0,4

-0,3

-0,2

-0,1

0,0Fluor

Tiempo (h)0 200 400 600 800 1000

Ln St /

S 0

-0,5

-0,4

-0,3

-0,2

-0,1

0,0

Pyr

Tiempo (h)

0 200 400 600 800 1000

Ln St /

S 0

-0,5

-0,4

-0,3

-0,2

-0,1

0,0Benz(a)ant

Tiempo (h)

0 200 400 600 800 1000

Ln St /

S 0

-0,5

-0,4

-0,3

-0,2

-0,1

0,0Benz(a)pyr

DESORPTION OF PAHs WITH TENAX

BIOREMEDIATED SOIL

Time (days)

0 5 10 15 20 25 30 35 40 45

% 1

4 C m

ine

rali

zed

0

10

20

30

40

50

60

70

80

HA

P (

mg

/Kg

)

0

20

40

60

80

Fen Ftno Pir

31.49 %

23.80 %0.35 %

BIOAVAILABILITY OF PAH IN BIOREMEDIATED SOIL (SOILREM E6068)(0.2 g/Kg PAH)

Fen

Pir

Ftno

42

98 186

1366

1250

372

118

404

59111

85

346142

222

16612455

429

GIBRALTAR

SAMPLING POINTS

ROADS

URBAN ZONES

INDUSTRIAL ZONES

Background PAH pollution in Gibraltar Area (Cádiz)(µg/kg 16 EPA PAH)

Phenanthrene content in soils from Gibraltar Area

*Wild, S.R. & Jones, K.C., Environ. Pollut. 1995, 88, 91-108

% Organic matter

0 1 2 3 4 5 6 7 8 9 10 11

µg

/kg

ph

en

an

thre

ne

0

20

40

60

80

100

120

Average: 40.6 µg/kg

UK rural soils: 14 µg/kg*

Bioaccessibility of native phenanthrene to autochtonous microbial populations in soils with different % OM

Time (days)

0 20 40 60 80 100 120

% 14

C m

iner

aliz

ed

0

10

20

30

40

50

1.72

10.04 4.504.55

2.01

Conditions: • 40 g soil amended with 11.8 µg/kg 14C-phen dissolved in distilled water

14C

% OM

Ph

e (

g/k

g)

0

20

40

60

80

100

120

140

160

total concentrationnot bioaccessible

1.72 2.01 4.5 4.55 10.04

31.6 %17.4 %

24.6 %

native

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-RESISTANTCOMPOUNDS, WHERE THE PHYSICAL CONSTRAINTS FOR BIODEGRADATION ARE HARDLY MODIFIABLE BY PHYSIOLOGICAL MEANS

Instituto de Recursos Naturales y Agrobiología de SevillaC.S.I.C.

Grupo “Biodegradación y Biorremediación” (PAI-RNM312)

Marisa BuenoMarta García-Junco*César Gómez*Mohammed Lahlou*Jose Luis NiquiRosa Posada Patricia VelascoJosé Julio Ortega

M. Grifoll (Univ. Barcelona)Hauke Harms (UFZ, Leipzig)Anatoly Marchenko (R. Center Toxicol., Rusia)