Laboratory and In Situ Experiments of Limestone Colonization by Aerobic Bacteria in Karst. and

Transcription

1 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

2 Part I BACTERIAL COLONIZATION IN KARST

3 In situ investigations Objective: To observe the evolution of the biofilms developed on sterile stone chips immersed in karst ground water

4 Study sites Borehole environments Low permeability and uncontaminated zone Fissured and contaminated zones

5 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

6 Rock types and immersion device Stainless steel connection Stopper Portland limestone Limestone marl Granite Holes Rock chips

7 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

8 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

9 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 P. fluorescens isolated from HV4

10 Methodology In flasks: one type of support, medium (500ml), inoculum of one bacterial strain resulting in initial concentration of 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

11 Results In situ experiments Bacterial colonization of rock chips

12 Statistical study No significant differences between the different rock types Significant differences between the boreholes Contaminated boreholes

13 Qualitative study

14 Results Laboratory experiments

15 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?

16 Part II Preliminary study to investigate populations of rotifers and crustaceans in Borehole LB8

17 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

18 «SEBA TM «Sampler Core of water 1.5 l

19 Qualitative study Most of zooplankton were Rotifers Amphipods gammaridae (Niphargus) Copepods and their larval step

20 Rotifer lepadella 40 µm

21 Amphipod Gammaridae (Niphargus) 1 cm

22 Copepod Kiefferiella delamarei Nauplius 100 µm

23 Quantitative observations Organism units in 1.5 L Evolution of zooplankton from LB /01/02 14/03/02 11/05/02 13/05/02 22/05/02 30/05/02 dates Rotifers Amphipods K. dellamarei Nauplius others