M. Tedetti, B. Charrière, F. Joux*, M. Abboudi and R. Sempéré
LMGEM, UMR 6117, CNRS/INSU, COM, Université de la Méditerranée
*OOBanyuls, UMR CNRS/INSU, 7621 sempere@com.univ-mrs.fr

- 100 m aerosols
UVA
UVB
Atmosphere
CO
2
. : 750 Gt C
CO
2
7.5 Gt C yr -1
Gas exchanges
Ocean
CO
2
Photosynthesis
Primary production
50 Gt C yr -1
UVA
UVB phytoplankton photochemistry bacteria
Respiration
DOC
700 Gt C nutrients
Mineralization organic carbon fluxes
CO
2
~ 30 Gt C yr -1
Tedetti et al., 2003
- 4000 m
Sediment

air-water interface
Effects of UVR on bacterial mineralization of DOM
(adapted from Obernosterer, 2000) sunlight
UVR
UVR
DOM photolysis nitrates photolysis radicals biologically available photoproducts
OH . radicals refractory sugars
-
-
Bacteria
+ diacids
-
Bacterial production,
Bacterial growth efficiency ?
CO
2 production by respiration

Production of dicarboxylic acids and ketoacids in the atmosphere
OH .
h n
O3
CHO
R
Monaldehyde
+
R
Monoacide
COOH
+
HOOC
HOOC
Azelaic
COOH
CHO
R
O
4-oxoacid
COOH HOOC
COOH
Succinic acid (C4)
HOOC
COOH
OH
Malic acid (hC4)
Unsaturated fatty acid
HOOC COOH
Oxalic acid (C2)
HOOC COOH
Malonic acid (C3)
(Kawamura et al., 1996; Sempéré and Kawamura, 2003)

TOMS-derived map of surface UV irradiance weighted by the erythemal action spectra (January 1, 2004) in kJ m -2
(http://jwocky.gsfc.nasa.gov/)
BIOSOPE
10 % UV-B irradiance depth ~ 17 m (Vasilkov et al., 2001)

To study the importance and the variability of UVR (A and B) and PAR intensity at the ocean surface and in the water column
(0-200 m) in the South East Pacific Gyre
To study the effects of UVR (A and B) and PAR on the molecular transformations of DOM (sugars, amino acids, carbonyl compounds including dicaboxylic acids, keto-acids and dicarbonyls) and on bacterial DNA damages in the oceanic surface layer
To study the effects of UVR on the DOM bacterial cycling in term of bacterial production and bacterial growth efficiency (BGE)

1-Measurements of UVR and PAR intensity at the ocean surface using surface sensors (Satlantic OCR-504 I ) and in the water column
(0-200 m) using a MICRO-PRO Profiler (Satlantic OCR-504 I/R).
Relationships with TOMS products (collaboration with A. Vassilkov)
2-Water column profiles of dissolved organic compounds (sugars, amino acids, diacids) and bacterial DNA damages (ELISA protocols) at
4 depths (5, 80, 200, and 1000 m)
3-Irradiation experiments of freshly collected seawater: sunlight exposure of DOM (photoproduction of sugars, amino acids, diacids) and bacteria (photoproduction of bacterial DNA damages) followed by dark microbial degradation of DOM (bacterial production and bacterial respiration)

1-Measurements of UVR and PAR intensity at the ocean surface using surface sensors (Satlantic OCR-504 I ) and in the water column
(0-200 m) using a MICRO-PRO Profiler (Satlantic OCR-504 I/R).
Relationships with Seawifs, TOMS products if possible (collaboration with A. Vassilkov and Biosope scientists)

Air
UV: 305, 325, 340, 380 nm (bandwidth: 2 and 10 nm)
PAR: 412, 443, 490, 565 nm (bandwidth: 20 nm)
Surface sensors (OCR-504 I) (on the ship deck)
Real time surface reference for in-water measurements
Surface irradiance (E s
( λ ) in W m -2 )
Dose (in kJ m -2 )
1 m underwater
Simultaneously
Underwater : MICRO-PRO Profiler (OCR-504 I/R)
(Free Fall Profiling Vehicle)
Real time profiling deployments (200 m operating depth)
Measurements around solar noon
Downwelling irradiance (E d
(
λ
, z), Upwelling radiance (L u
(
λ
, z ),

®
7 cm
11 cm
OCR-504 UV (-B and -A)
λ = 305, 325, 340, 380 nm width = 2 et 10 nm
OCR-504 PAR
λ = 412, 443, 490, 565 nm width = 20 nm

Atmospheric UVR doses for August 2003, Marseille France
Roof top (25 m) of the Faculty of Science
Days

Sugars :Waters HPLC, Dionex column
HPAEC-PAD at 4 depths (5, 80, 200, and 1000 m)
Amino-acids, OH .
:Waters HPLC,
Fluo.

High Performance Anion-Exchange Chromatography and Pulsed Amperometry
Detection (HPAEC-PAD) (Panagiotopoulos et al., 2001; 2003; Panagiotopoulos and Sempéré, 2004)
Desalting of seawater samples using Bio-Rad ion-exchange resins (Mopper et al.,
1992)
Recoveries of desalting : 70–90 % at 20 nM ( Tedetti and Sempéré, in prep.)
Volume of seawater sample : 4 x 8 ml = 32 ml
Detection level : 5 nM
Total analysis time : 3h

HPAEC-PAD chromatogram of dissolved sugars after desalting
Recoveries of desalting : 70-90 %
Tedetti and Sempéré, 2004

OPA derivatisation technique followed by HPLC and spectrofluorimetry
(Lindroth et Mopper,1979)
- detection level : 2 -5 nM .
-Reprod. < 5%
Phe
Slight improvements : decrease coelution by using new column and new elution programme
Collaboration with C. Lee (Stony Brooks Univ., USA)

.
Derivatization by benzoic acid (Qian et al., 2001)
Salicylic acid :
Highly fluorescent (300)
COOH COOH
+
OH .

OH
+ no fluorescent compounds
BA OHBA
OH .
photoproduction = (OHAB photoproduction )
×
6,45
Volume of seawater sample : 2 x 100 ml = 200 ml
Detection level : 0,5 nM
Run : 25 min

0,00
-0,50
-1,00
1,00
0,50
HPLC Chromatogram of acid salicylic solution (1,5 nM)
Salicylic acid
1,00 2,00 3,00 4,00 5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00
Minutes

A new gas chromatography (GC) protocol (Tedetti et al., in prep.)
Dicarboxylic acids, ketoacids and dicarbonyls
Extraction by activated charcoal after intensive cleaning
Elution by NH
4
OH-MeOH, CH
2
Cl
2
, Milli-Q
Derivatisation by BF
3
/butanol (Kawamura, 1993). On board
GC injection, FID detection
Volume of seawater sample : 150 ml
Detection level : ~ 10 nM
Oxalic acid (C2) COOH
Recoveries
50-57 %
Malonic acid (C3) HOOC COOH
20 %
Succinic acid (C4) HOOC
COOH
90-95 %
Glutaric acid (C5) HOOC COOH
90-95 %
Adipic acid (C6) HOOC COOH
90-95 %
Azelaic acid (C9) HOOC COOH
85-95 %
Collaboration with K. Kawamura (Sapporo Univ. Japan) d
C13 for individual diacids

Detection of UV damages on bacterial DNA
UV-B
UV-C UV-B
ADN
Direct damages on DNA
STOP of DNA transcription and replication
Cellular death
Reparation
Mutation
Joux, 2003

UV-B
DNA damages
90%
2 bases pyrimidiques adjacentes
10%
Different reparation processes Dimères cyclobutane pyrimidine
300 times more efficient
6-4 photoproduits in DNA blocking replication pyrimidone
Detection: Seawater filtration (0.2 m m). Samples might be kept frozen.
immunodetection : (ELISA test: addition of antobodies, spectrophotometry detection)
Volume of seawater samples : 1-3 Liters

Niskin bottle
Seawater
Full Sun
Dark
0.8 µm
ELISA
BP bacterial inoculum
100 ml Quartz flasks
ELISA
BP
20% 80%
1 l shot bottles
0.2 µm sugars amino acids diacids
DOM solution
1 l Quartz flasks
Sunlight exposure
(8 h, around solar noon, on the ship deck) sugars amino acids diacids
Dark incubation (2 days)
BP, BR

Dissolved sugars (HPAEC - PAD)
Desalination using ion-exchange resins
Dissolved amino acids (HPLC) OPA derivatization (Lindroth and Mopper, 1979;
Lee et al., 2000)
OH .
Radical production (HPLC)
Diacids (GC - GC/MS)
BF
3
/butanol derivatization Extraction of diacids using activated charcoal
DNA damages
ELISA experiment
Bacterial Production
Measurement of 3 H-Leucine incorporation
Bacterial Respiration
Measurement of dissolved O
2
, concentrations (????)

Measurements of UVR and PAR intensity
Around solar noon, light measurements :
- 3 for each short stations (st 1-21)
- 6 for each long stations (G1, G2, G3)
- 6 for each station of specific sites (MARQ1-7, UPW1-7)
Profiles of dissolved organic compounds and DNA damages
Around solar noon, sampling:
- 1 at 5 m (surface ?) for all stations (short, long, and specific sites)
- 1 at 80, 200, and 1000 m for 7 stations (G1, G2, G3, UPW1, 7, MARQ1, 7)
Irradiation experiments
Around solar noon, at :
- 1 at MARQ1
- 1 at G3
- between (MARQ1 and G3)

Volume of Seawater
- All stations at the surface (5 m) : 3 Liters
- MARQ and G3 in surface water : 16 Liters
- G1, G2, G3, UPW1, 7, MARQ1, 7 at 80, 200, 1000 m : 3 Liters
Volume of sample storage
- Freezer: 35 Liters
- 4 °C: 410 Liters
Material : Problem with contamination for organics
-Oven: 70 l :
-Niskin bottles (Silicon rubbers and Viton o-rings)
-Access for underwater radiometer

Space :
Space on ship deck : 5 m 2 for surface sensors and irradiation experiments
Space for computer connected to the surface sensors : 1m
Space for storage of the underwater radiometer : 2 m2
Chemistry lab. for organics : Regular airbench and laminar flow airbench. Lab: 10 m 2
Access to isotope containers
Chemicals, isotopes :
Use of organic solvents, acids
Isotopes : 3H-leucine
Oxygen (??)

Aerosols : High volume air sampler for organics (K. Kawamura,
Sapporo, Japan)
Sediment trap particles (sugars, amino-acids)
Surface samples

PIs: R. Sempéré and F. Joux www.com.univ-mrs.fr/LMGEM/uveco/
U
V
ECO
May 2003-end May 2006
This projects involves 7 French laboratories and 30 scientists specialized in marine biogeochemistry other foreign scientists (Australia, Canada, Japan, and USA)

Stratospheric ozone depletion

UV-B
Increase of aerosols, ozone
(troposphere) Increase of winds frequency

UV-A and UV-B  vertical mixing
Increase of greenhouse gases
Variation of nebulosity

UV-A and UV-B
+

UV-B
+
+
Variation of UVR at the Earth’s surface
Variation of UV penetration in seawater
(UNEP/WMO, 2002; McKenzie et al ., 2003; Häder et al ., 2003).
Adapted from Joux, 2003

Objectives
To study cellular and molecular responses of marine microbial community to UV stress
To better understand the molecular and physiological bases of the capacity of marine picocyanobacteria and heterotrophic bacteria to resist high fluxes of visible and ultraviolet light occurring in the top layer of oceans.
To study degradation of DOM including polysaccharides, proteins, carbonyls and dimethylsulfide (DMS) as well as on subsequent effects on bacterial cycling.
Atmospheric (at ground level at the Oceanographic Centre of Banyuls/mer and at the University of Marseille-Luminy) as well as submersible irradiances
(in coastal areas of Banyuls/Mer and Marseille cities) will be monitored
Experiments will be conducted in north-western Mediterranean coastal waters (Banyuls/Mer and Marseille), likely in open Mediterranean waters
(cruise not defined yet) and in Pacific Ocean (Biosope).

NAME
JOUX Fabien
CONAN Pascal
PUJO-PAY Mireille
LANTOINE François
LEBARON Philippe
GHIGLIONE Jean-François
CATALA Philippe
ZUDAIRE Laurent
SEMPÉRÉ Richard
TEDETTI Marc
ABBOUDI Maher
VANWAMBEKE France
LEFEVRE Dominique
CHARRIERE Bruno
MONZIKOFF André
GOYET Catherine
TOURATIER Franck
BELVISO Sauveur
PARTENSKY Frédéric
MARIE Dominique
GARCZAREK Laurence
SIX Christophe
DUFRESNE Anne
CHAMI Malik
UVECO
LABORATORY
LOBB
LOBB
LOBB
LOBB
LOBB
LOBB
LOBB
LOBB,
LMGEM-COM
LMGEM-COM
LMGEM-COM
LMGEM-COM
LMGEM-COM
LMGEM-COM
LBCM
Univ. Perpi.
CEFREM
LSCE
SBR
SBR
SBR
SBR
SBR
LOV
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Marseille/FRA
Marseille/FRA
Marseille/FRA
Marseille/FRA
Marseille/FRA
Marseille/FRA
Paris/FRA
Perpignan/FRA
Perpignan/FRA
Gif/FRA
Roscoff/FRA
Roscoff/FRA
Roscoff/FRA
Roscoff/FRA
Roscoff/FRA
Villefranche/mer/FRA

COURBES DE CALIBRATION DO / DOMMAGES ADN (dosés par HPLC)
2,5
2 y = 0,1214x + 0,0935
R
2
= 0,9931
1,5
CPD
1
15 ng/puits
0,5
0
0 15 5 10
Lésions/10 4 b
6- 4 photoproduits
50 ng/puits
0,8
0,6 y = 2,1798x + 0,0087
R
2
= 0,9939
0,4
0,2
0
0
Jeffrey et al. 1996. Photochem. Photobiol. 64:419-427.
Joux et al. 1999. Appl. Envrion. Microbiol. 65:3820-3827.
0,1 0,2
Lésions/10 4 b
0,3 0,4

UV-B
DNA damages
90%
2 bases pyrimidiques adjacentes
10%
Different reparation processes Dimères cyclobutane pyrimidine
Photoreactivation
Excision of nucleotides
Recombination
Yes
Yes
Yes
6-4 photoproduits pyrimidone
NO
Yes
Yes
300 times more efficient in DNA blocking replication

DNA damage detection by immunodetection (ELISA Test)
1. Non specific absorbtion of the antigene for the detection of the DNA damages
3. Specific fixation of the antobodies on the antigene
4. Second specific antigene fixation associated to an enzyme against the first antibody
5. Colorimetric detection (spectrophotometry)