Laboratory and In Situ Experiments of
Limestone Colonization by Aerobic Bacteria
in Karst
and
Preliminary Study of Populations of Rotifers
and Crustaceans at the site of Lowest
Colonization
JCP Karst workshop AUSTIN February 2005
Part I
BACTERIAL COLONIZATION
IN KARST
JCP Karst workshop AUSTIN February 2005
In situ investigations
Objective:
To observe the evolution of the
biofilms developed on sterile stone
chips immersed in karst ground
water
JCP Karst workshop AUSTIN February 2005
Study sites
Borehole
environments
Low permeability
and
uncontaminated
zone
Fissured and
contaminated
zones
JCP Karst workshop AUSTIN February 2005
Methodology
„
Immersion of rock chips
Fissured sites : at the level where the borehole
crosses a main drain.
„ Low permeability site : 15 m below the average water
level.
„
„
„
Monthly sampling of rock chips
Enumeration and identification of the bacterial
biofilm fixed on the sampled rock chips
JCP Karst workshop AUSTIN February 2005
Rock types and immersion device
Stainless steel connection
Stopper
„
„
„
Portland limestone
Limestone marl
Granite
Holes
Rock
chips
JCP Karst workshop AUSTIN February 2005
Bacterial analysis
„
„
„
„
Estimation of the sampled rock chip surface
Rinsing in sterile water, sonication to release and
disaggregate the biofilm attached
Enumeration of the obtained suspension on
R2A medium
Identification of each colony type to the genus
level
JCP Karst workshop AUSTIN February 2005
Laboratory
experiments
Objective
To determine to what degree limestone is colonized
compared to colonization of PVC (known to be readily
colonized by bacteria)
2 bacteria
2 media
JCP Karst workshop AUSTIN February 2005
Materials
„
„
Supports: Portland limestone chips and PVC fragments
sterilized by autoclave
Media
„
„
„
Medium A is a synthetic medium designed to facilitate
biofilm formation on a variety of surfaces
Medium B consists of karst ground water from HV4,
sterilized by filtration (0.22µm)
Bacteria
„
„
E. coli ATCC 10536
P. fluorescens isolated from HV4
JCP Karst workshop AUSTIN February 2005
Methodology
„
„
„
In flasks: one type of support, medium (500ml), inoculum
of one bacterial strain resulting in initial concentration of
1.105 CFU/ml
Incubation 20°C, 3 weeks with 2 agitations daily
Every 3 days
„
„
„
replacement of media by new sterile media of the same type
Sampling of supports
Estimation of support surface, rinsing, sonication and
enumeration of the disaggregated biofilm as described
above
JCP Karst workshop AUSTIN February 2005
Results
In situ experiments
Bacterial colonization of rock chips
JCP Karst workshop AUSTIN February 2005
Statistical study
No significant
differences between
the different rock
types
Significant
differences between
the boreholes
Contaminated boreholes
JCP Karst workshop AUSTIN February 2005
Qualitative study
JCP Karst workshop AUSTIN February 2005
Results
Laboratory experiments
JCP Karst workshop AUSTIN February 2005
Conclusions
„
„
„
Limestone (and granite) surfaces can support
bacterial colonization
Karst water environments are hostile to biofilm
development
Enteric bacterial contamination seems unlikely
to be persistent once the contamination source
is removed
FLUSHING ?
PREDATION ?
JCP Karst workshop AUSTIN February 2005
Part II
Preliminary study to
investigate populations of
rotifers and crustaceans in
Borehole LB8
JCP Karst workshop AUSTIN February 2005
Materials and methods
„
„
„
„
„
Sampling of 1.5 L from the karst drain
intersected by LB8
Filtration of the sample (5 µm)
Agitation of the membrane in a fixative and
preservative solution
Coloring zooplankton by adding a vital dye
Microscopic identification of zooplankton
JCP Karst workshop AUSTIN February 2005
« SEBA TM « Sampler
Core of water
1.5 l
JCP Karst workshop AUSTIN February 2005
Qualitative study
Most of zooplankton were
Rotifers
Amphipods gammaridae (Niphargus)
Copepods and their larval step
JCP Karst workshop AUSTIN February 2005
Rotifer lepadella
40 µm
JCP Karst workshop AUSTIN February 2005
Amphipod Gammaridae
(Niphargus)
1 cm
JCP Karst workshop AUSTIN February 2005
Copepod
Kiefferiella delamarei
Nauplius
♂
♀
♂
100 µm
JCP Karst workshop AUSTIN February 2005
Quantitative observations
Organism
units in 1.5 L
Evolution of zooplankton from LB8
60
50
40
30
20
10
0
14/01/02
Rotifers
14/03/02
Amphipods
11/05/02
13/05/02
K. dellamarei
22/05/02
Nauplius
30/05/02
others
JCP Karst workshop AUSTIN February 2005
dates