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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
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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

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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)
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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
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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)

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Methodology bioassays
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
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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
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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.
• gt Ratio of both activities related to N vs P
  limitation of heterotrophic bacteria (inducible
  enzymes)
• gt functional diversity of phosphatase-positive
  cells

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Methodolology phosphatase activity
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
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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

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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)