laboratory and in situ experiments of limestone ...rinsing in sterile water, sonication to release...

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

Fissured and contaminated

zones

Low permeability and

uncontaminated zone

Borehole environments

JCP Karst workshop AUSTIN February 2005

MethodologyImmersion 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 chipsEnumeration and identification of the bacterial biofilm fixed on the sampled rock chips

JCP Karst workshop AUSTIN February 2005

Rock types and immersion device

Portland limestoneLimestone marlGranite

Holes

Rock chips

Stainless steel connection

Stopper

JCP Karst workshop AUSTIN February 2005

Bacterial analysis

Estimation of the sampled rock chip surfaceRinsing in sterile water, sonication to release and disaggregate the biofilm attachedEnumeration of the obtained suspension on R2A mediumIdentification of each colony type to the genus level

JCP Karst workshop AUSTIN February 2005

Laboratory experiments

ObjectiveTo 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 autoclaveMedia

Medium A is a synthetic medium designed to facilitate biofilm formation on a variety of surfacesMedium B consists of karst ground water from HV4, sterilized by filtration (0.22µm)

BacteriaE. coli ATCC 10536P. 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/mlIncubation 20°C, 3 weeks with 2 agitations dailyEvery 3 days

replacement of media by new sterile media of the same typeSampling of supports

Estimation of support surface, rinsing, sonication and enumeration of the disaggregated biofilm as described above

JCP Karst workshop AUSTIN February 2005

ResultsIn 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 boreholesJCP Karst workshop AUSTIN February 2005

Qualitative study

JCP Karst workshop AUSTIN February 2005

ResultsLaboratory experiments

JCP Karst workshop AUSTIN February 2005

Conclusions

Limestone (and granite) surfaces can support bacterial colonizationKarst water environments are hostile to biofilm developmentEnteric 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 LB8Filtration of the sample (5 µm) Agitation of the membrane in a fixative and preservative solutionColoring zooplankton by adding a vital dyeMicroscopic 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

RotifersAmphipods gammaridae (Niphargus)

Copepods and their larval step

JCP Karst workshop AUSTIN February 2005

Rotifer lepadella

JCP Karst workshop AUSTIN February 2005

40 µm

Amphipod Gammaridae(Niphargus)

1 cm

JCP Karst workshop AUSTIN February 2005

CopepodKiefferiella delamarei

♂

♂♀100 µm

Nauplius

JCP Karst workshop AUSTIN February 2005

Quantitative observations

Evolution of zooplankton from LB8

0

10

20

30

40

50

60

14/01/02 14/03/02 11/05/02 13/05/02 22/05/02 30/05/02 dates

Organismunits in 1.5 L

Rotifers Amphipods K. dellamarei Nauplius others

JCP Karst workshop AUSTIN February 2005