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 CY3Surface 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 experimentsTo determine factors limiting heterotrophic bacterial production

Nitrate/Ammonium 2 µMphosphate 0.25 µM

glucose 10 µM C

Addition of all elementsunenriched

N P G

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 24-48 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 activityELF

fluorescent, insoluble

Methodolology : phosphatase activity

- Short-term stations : noon cast ?

9 layers 0-200 m bacterial production (total) ------------------ 50 mlSurface layer ------------------------------------------------------ 2.3 liters

phosphatase, aminopeptidase activities, size class BPbioassay experiment

- Long occupation stations (gyres, Marquises, Upwelling):

1) Focusing vertical variability of limiting factorson noon cast : ---------------------------------------------------- 2,3 liters

size class BP 0.2 and 2 µm phosphatase, aminopeptidase activitiesbioasssays 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 activitiesOn surface layers, four times a day---------------------------------- 2,3 litersstarting 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