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BIOSOPE
Effects of UV radiation (UVR) on the molecular
structure of DOM and its subsequent utilization by
marine bacterioplankton in the South East Pacific
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
[email protected]
Atmosphere
UVA
UVB
aerosols
CO2
7.5 Gt C yr-1
CO2. : 750 Gt C
Ocean
Gas
exchanges
CO2
UVA
UVB
Photosynthesis
Primary
production
50 Gt C yr-1
phytoplankton
photochemistry
CO2
bacteria
Respiration
DOC
700 Gt C
nutrients
- 100 m
Mineralization
organic carbon fluxes
Tedetti et al., 2003
- 4000 m
Sediment
~ 30 Gt C yr-1
Effects of UVR on bacterial mineralization of DOM
(adapted from Obernosterer, 2000)
sunlight
UVR
UVR
air-water interface
nitrates
DOM
photolysis
photolysis
biologically available
photoproducts
radicals
refractory
photoproducts
-
-
sugars
amino acids
diacids
+
Bacteria
OH. radicals
-
Bacterial production,
Bacterial growth efficiency ?
CO2 production by respiration
Production of dicarboxylic acids and ketoacids in the atmosphere
CHO
R
HOOC
COOH
Monaldehyde
COOH
R
+
Monoacide
Azelaic
+
HOOC
CHO
Oxononanoic(
COOH
COOH
O
4- oxoacid
Succinic acid (C4)
HOOC
COOH
OH
Malic acid (hC4)
HOOC
R
hn
O3
COOH
R
OH.
HOOC
COOH
Oxalic acid (C2)
HOOC
COOH
Malonic acid (C3)
Unsaturated fatty acid
(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)
Our objectives during Biosope
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)
Scientific approach
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)
Methods
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)
Satlantic's OCR-504 UV/PAR radiometers
UV: 305, 325, 340, 380 nm (bandwidth: 2 and 10 nm)
PAR: 412, 443, 490, 565 nm (bandwidth: 20 nm)
Air
Surface sensors (OCR-504 I) (on the ship deck)
Real time surface reference for in-water measurements
Surface irradiance (Es(λ) in W m-2)
Dose (in kJ m-2)
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
1m
underwater
Downwelling irradiance (Ed(λ, z), Upwelling radiance (Lu(λ, z),
Surface radiometres UV and PAR (Satlantic®)
7 cm
11 cm
4.6 cm
17 cm
OCR-504 UV (-B and -A)
OCR-504 PAR
λ = 305, 325, 340, 380 nm
λ = 412, 443, 490, 565 nm
width = 20 nm
width = 2 et 10 nm
Atmospheric UVR doses for August 2003, Marseille France
Roof top (25 m) of the Faculty of Science
Days
Analysis of dissolved organic
compounds
Sugars :Waters HPLC, Dionex column
HPAEC-PAD
at 4 depths (5, 80, 200, and 1000 m)
Amino-acids, OH.:Waters HPLC,
Fluo.
Dissolved (free and combined)
individual sugars by HPAEC-PAD
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
Amino-acid analysis by HPLC
Phe
Ileu
Val
Leu
Lys
OPA derivatisation technique followed by HPLC and spectrofluorimetry
(Lindroth et Mopper,1979)
- detection level : 2 -5 nM .
-Reprod. < 5%
Gaba
Ala
Thr
Gly
Ser
Glu
His
Asp
Tyr
Phe
Slight improvements : decrease coelution by using new column and new elution programme
Collaboration with C. Lee (Stony Brooks Univ., USA)
OH. radical production by HPLC
Derivatization by benzoic acid (Qian et al., 2001)
Salicylic acid :
Highly fluorescent (300)
COOH
COOH
OH
+
OH.
BA

+
no fluorescent compounds
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
HPLC Chromatogram of acid salicylic solution (1,5 nM)
1,00
Salicylic acid
mV
0,50
0,00
-0,50
-1,00
1,00
2,00
3,00
4,00
5,00
6,00
7,00
8,00
Minutes
9,00
10,00
11,00
12,00
13,00
14,00
15,00
Dicarboxylic acids in seawater : 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 NH4OH-MeOH, CH2Cl2, Milli-Q
Derivatisation by BF3/butanol (Kawamura, 1993). On board
GC injection, FID detection
Volume of seawater sample : 150 ml
Detection level : ~ 10 nM
Oxalic acid (C2)
HOOC
Malonic acid (C3)
Succinic acid (C4)
Glutaric acid (C5)
Adipic acid (C6)
Azelaic acid (C9)
HOOC
HOOC
HOOC
HOOC
HOOC
Recoveries
50-57 %
COOH
COOH
COOH
20 %
90-95 %
COOH
90-95 %
COOH
90-95 %
COOH
85-95 %
Collaboration with K. Kawamura (Sapporo Univ. Japan)dC13 for individual diacids
Detection of UV damages on bacterial DNA
UV-B
UV-C
UV-B
ADN
Direct damages on DNA
Cellular death
Reparation
STOP of DNA transcription and replication
Mutation
Joux, 2003
DNA damages
2 bases
pyrimidiques
adjacentes
UV-B
90%
Different reparation processes
Dimères cyclobutane
pyrimidine
10%
300 times more efficient
6-4 photoproduits
in DNA blocking replication
pyrimidone
Detection: Seawater filtration (0.2 mm). Samples might be kept frozen.
immunodetection : (ELISA test: addition of antobodies, spectrophotometry
detection)
Volume of seawater samples : 1-3 Liters
Irradiation/incubation protocol
Niskin bottle
Seawater
0.8 µm
ELISA
BP
0.2 µm
16 l
8l
8l
bacterial inoculum
sugars
amino acids
diacids
DOM solution
100 ml Quartz flasks
1 l Quartz flasks
Sunlight exposure
(8 h, around solar noon,
on the ship deck)
Full Sun
Dark
20%
80%
ELISA
BP
1 l shot bottles
sugars
amino acids
diacids
Dark incubation (2 days)
BP, BR
Analytical protocol: summary
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)
BF3/butanol derivatization Extraction of diacids using activated charcoal
DNA damages
ELISA experiment
Bacterial Production
Measurement of 3H-Leucine incorporation
Bacterial Respiration
Measurement of dissolved O2, concentrations (????)
Strategy, types of samples
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)
Needs
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
Needs
Space :
Space on ship deck : 5 m2 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 m2
Access to isotope containers
Chemicals, isotopes :
Use of organic solvents, acids
Isotopes : 3H-leucine
Oxygen (??)
Other types of samples
Aerosols : High volume air sampler for organics (K. Kawamura,
Sapporo, Japan)
Sediment trap particles (sugars, amino-acids)
Surface samples
UVECO-program (CNRS PROOF / SOLAS)
PIs: R. Sempéré and F. Joux
www.com.univ-mrs.fr/LMGEM/uveco/
Induction of microbial community responses and dissolved
organic matter transformations by UltraViolet radiation in
marine ECOsystems
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)
UVR and global change
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).
UVECO
NAME
LABORATORY
JOUX Fabien
CONAN Pascal
PUJO-PAY Mireille
LANTOINE François
LEBARON Philippe
GHIGLIONE Jean-François
CATALA Philippe
ZUDAIRE Laurent
SEMPÉRÉ Richard
TEDETTI Marc
LOBB
LOBB
LOBB
LOBB
LOBB
LOBB
LOBB
LOBB,
LMGEM-COM
LMGEM-COM
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Banyuls/FRA
Marseille/FRA
Marseille/FRA
ABBOUDI Maher
VANWAMBEKE France
LEFEVRE Dominique
CHARRIERE Bruno
MONZIKOFF André
GOYET Catherine
TOURATIER Franck
BELVISO Sauveur
PARTENSKY Frédéric
LMGEM-COM
LMGEM-COM
LMGEM-COM
LMGEM-COM
LBCM
Univ. Perpi.
CEFREM
LSCE
SBR
SBR
SBR
SBR
Marseille/FRA
Marseille/FRA
Marseille/FRA
Marseille/FRA
Paris/FRA
Perpignan/FRA
Perpignan/FRA
Gif/FRA
Roscoff/FRA
MARIE Dominique
GARCZAREK Laurence
SIX Christophe
DUFRESNE Anne
CHAMI Malik
SBR
LOV
Roscoff/FRA
Roscoff/FRA
Roscoff/FRA
Roscoff/FRA
Villefranche/mer/FRA
COURBES DE CALIBRATION DO / DOMMAGES ADN (dosés
par HPLC)
2,5
y = 0,1214x + 0,0935
R2 = 0,9931
CPD
15 ng/puits
DO 490 nm
2
1,5
1
0,5
0
0
5
10
15
Lésions/104 b
0,8
50 ng/puits
0,6
DO 490 nm
6- 4 photoproduits
y = 2,1798x + 0,0087
R2 = 0,9939
0,4
0,2
0
0
0,1
Jeffrey et al. 1996. Photochem. Photobiol. 64:419-427.
Joux et al. 1999. Appl. Envrion. Microbiol. 65:3820-3827.
0,2
Lésions/104 b
0,3
0,4
DNA damages
2 bases
pyrimidiques
adjacentes
UV-B
90%
Different reparation processes
Photoreactivation
10%
Dimères cyclobutane
pyrimidine
6-4 photoproduits
pyrimidone
Yes
NO
Excision of nucleotides
Yes
Yes
Recombination
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)