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

Bacterial production and factors limiting bacterial production

BIOSOPE project

France Van Wambeke

LMGEM, Marseille

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


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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)


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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)


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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


Bacterial production and factors limiting bacterial production biosope project france van wambeke

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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