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Bacterial production and factors limiting bacterial production BIOSOPE project France Van Wambeke

Bacterial production and factors limiting bacterial production BIOSOPE project France Van Wambeke LMGEM, Marseille. Villefranche-sur-Mer, presentation 27/01/2004. Specific objectives. Studying bacterial production in extreme oligotrophy

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Bacterial production and factors limiting bacterial production BIOSOPE project France Van Wambeke

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

  2. Specific objectives • 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

  3. Methodologies : bacterial production - 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)

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

  5. Methodologies • - 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)

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

  7. Methodology : bioassays Incubation 24-48 h under in situ – simulated conditions .... Running sea-water bath Then subsampling for : • - bacterial abundance • - bacterial production • ectoenzymatic activity • bacterial diversity Volume incubated varying according final parameters ; 60 to 500 ml

  8. Methodologies • - 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

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

  10. Sampling strategy • - Short-term stations : noon cast ? • 9 layers 0-200 m bacterial production (total) ------------------ 50 ml • Surface layer ------------------------------------------------------ 2.3 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 : ---------------------------------------------------- 2,3 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 -------------------------------- 500 ml • - Size class BP 0.2 and 2 µm • phosphatase activities • On surface layers, four times a day---------------------------------- 2,3 liters • starting a bioassay experiment

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

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