Bacterial production and factors limiting bacterial production BIOSOPE project France Van Wambeke LMGEM, Marseille Villefranche-sur-Mer, presentation 27/01/2004

- Studying bacterial production in extreme oligotrophy - Looking for factors controlling heterotrophic bacterial growth along : - surface gradients - vertical gradients - diel cycle - Studying one functional diversity aspect in heterotrophic bacteria : phosphatase alkaline activity in relation to P cycle Specific objectives

- 3H leucine incorporation into proteins : - total (microcentrifuge technique) - size class (0,2 and 0,6 µm), relation P cycle (coll T Moutin) - microautoradiography - FISH, relation bacterial diversity (coll P Lebaron) Methodologies : bacterial production

DAPI CY3 Transmitted light micro-fish probe eub338 CY3 Surface water DYFAMED, mars 2003 Coll D kirchman, M Cottrell, Lewes, July 2003 Experience with MICRO-FISH Expected results : Percentage of active cells Identification of specific active groups

- 3H leucine incorporation into proteins : - total (microcentrifuge technique) - size class (0,2 and 0,6 µm), relation P cycle (coll T Moutin) - microautoradiography - FISH, relation bacterial diversity (coll P Lebaron) - Enrichment experiments (bioassays) Methodologies

Enrichment experiments To determine factors limiting heterotrophic bacterial production Nitrate/Ammonium 2 µM phosphate 0.25 µM glucose 10 µM C Addition of all elements unenriched NPG NPG Surface sea water, pre-filtered through 60 µm Fe In areas of potential Fe limitation (coll S. Blain) Fe NPG Methodology : bioassays

Running sea-water bath Incubation h under in situ – simulated conditions bacterial abundance - bacterial production - ectoenzymatic activity - bacterial diversity Then subsampling for : Volume incubated varying according final parameters ; 60 to 500 ml Methodology : bioassays

- 3H leucine incorporation into proteins : - total (microcentrifuge technique) - size class (0,2 and 2 µm), relation P cycle (coll T Moutin) - microautoradiography - FISH, relation bacterial diversity (coll P Lebaron) - Enrichment experiments (bioassays) - Ectoenzyme activities : phosphatase and aminopeptidase activities with fluorogenic substrates. => Ratio of both activities related to N vs P limitation of heterotrophic bacteria (inducible enzymes) => functional diversity of phosphatase-positive cells Methodologies

Cell membrane Alkaline phosphatase MUF-PO4 MUF fluorescent, soluble and diffusible Classical method (spectrofluorimetry) - quantitative - global flux - kinetic approach (Vm, Km) - do not allow detection of the origin of activity Use of fluorogenic substrate. Looking for bacteria expressing phosphatase activity, a proxy for phosphorus limitation Alkaline phosphatase ELF-PO4 New method (epifluorescence microscopy): - qualitative - allows detection of the origin of the activity ELF fluorescent, insoluble Methodolology : phosphatase activity

- Short-term stations : noon cast ? 9 layers m bacterial production (total) ml Surface layer liters phosphatase, aminopeptidase activities, size class BP bioassay experiment - Long occupation stations (gyres, Marquises, Upwelling): 1) Focusing vertical variability of limiting factors on noon cast : ,3 liters size class BP 0.2 and 2 µm phosphatase, aminopeptidase activities bioasssays along vertical profiles 2) Focusing diel variability of limiting factors (Marquises) On surface layers, every 3 hours ml - Size class BP 0.2 and 2 µm - phosphatase activities On surface layers, four times a day ,3 liters starting a bioassay experiment Sampling strategy

- Bacterial production during UV biodegradation experiments (coll M Tedetti, R Sempéré) - Bacterial production on surface – microlayer - Bacterial diversity (coll P. Lebaron, I Obernosterer) Other collaborations