Bacterial production and factors limiting bacterial production
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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

LMGEM, Marseille

Villefranche-sur-Mer, presentation 27/01/2004


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


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)


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


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)


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


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


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


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


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


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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