Aragno, Michel

1994, Rossi, Pierre, Aragno, Michel, Muller, Imre

This work was undertaken in the Laboratoire de Microbiologie of the University of Neuchâtel, in close collaboration with the Center of Hydrogeology (CHYN). This multi-disciplinary research project was instigated because of the growing need both for environmentally harmless hydrogeological investigation tools, and for new tracers that can be used in multitracing experiments. This work treats of the use of bacteriophages as biological tracers in underground and surface waters. The size of bacteriophages is of the order of a few hundred nm. These viruses only attack specific bacteria. They are absolutely harmless for any other living organism. Previous experiments have shown the value of bacteriophages as tracers in fissured environments. The aims of the present work were: to select several bacteriophages particularly well adapted to the use as hydrogeological tracers ; to optimize the production of these bacteriophages, and the analytical methods for detecting them; to study the behavior of these bacteriophages experimentally, under different physico-chemical conditions; to test the phages in karstic fissured environments and to extend their use to saturated porous environments and surface hydrology; to compare the migration of these phages to that of the best conventional hydrological tracers. The first part of this work treats of the isolation and characterization of new bacteriophages. Over twenty bacteriophage/host bacterium (BHB) systems were studied. Seven bacteriophages were selected for the second part of the project. The selection was based on the physiological properties of the host bacteria and the physico-chemical characteristics of the bacteriophages. These seven phages exhibit an interesting variety of shapes and physical characteristics. They include three phages of strictly marine bacteria. None of these BHB systems are naturally found in aquifer waters. The rest of this work includes the development of an analytical and enumeration technique. We chose to work with the double agar-layer technique, using Petri dishes. The growth media and the different steps of this technique were optimized for each BHB system. This increased the reliability and reproducibility of the technique, without adding to the cost or workload. The behavior of the phages was studied in laboratory experiments. As this behavior is mostly determined by the inactivation and adsorption of the phages, we investigated the influence on these two phenomena of various parameters, either physical (temperature, pH, agitation), or chemical (ionic concentrations, presence of proteins, of sand or colloidal clay particles). Our results show that in water, phages react very rapidly and massively to the presence of colloidal particles, even to very low concentrations. Agitation causes the viruses to be rapidly inactivated. Raising the temperature increases this inactivation. Colloidal clay particles (Montmorillonite and Attapulgite) as well as organic macromolecules efficiently protect the phages from inactivation. Only one phage was inactivated faster in the presence of mineral colloids. Each phage reacted individually to the presence of these mineral colloids. However, no correlation could be established with the physico-chemical characteristics of the bacteriophages. The reactions can be classified into three distinct types, described by Grant et al. [1993]. According to the type of reaction of a phage, it is possible to qualitatively predict its behavior during a tracing experiment. The tracing experiments performed in karstic fissured environments showed that the migration of bacteriophages is similar to that of the best fluorescent tracers (Uranine, Sulphorhodamine). The restitutions are generally high. The phages always reappear faster, which shows that they can be diluted more, before reaching their inferior detection limit. The trials performed in a porous saturated environment, on the Wilerwald (CH) test site, showed that bacteriophages are also adapted to this type of conditions. Their speed of migration is higher than that of dissolved chemical compounds (Uranine, Naphtionate). Only one trial was performed in a river. It nevertheless showed that phages are also perfectly suitable to these types of tracings. By the simultaneous injection of several bacteriophages at different points along the river, it was possible to scan its course and discover an infiltration zone. This work confirms that phages are of undeniable value to hydrology as biological tracers. Their impact on the environment is practically nil and they can therefore be used also in delicate situations (springs used for drinking water, tracings in inhabited areas). This method is not limited to karstic aquifers anymore. Our modifications and adaptations of the technique make it suitable also for saturated porous environments and rivers. It advantages are numerous: The phages are non-pathogenic, non-toxic and invisible. So this method can also be used for studying drinking water (springs, reservoirs, etc). Each phage generally attacks only one bacterial species. By a careful selection of the BHB systems, any effect on the aquifers microflora can be avoided. No background noise exists in the aquifers, since they phages are not found there naturally. None will be generated or will persist over time, because of their short life span. The analysis of the samples is fast and cheap. Only a few milliliters of water are necessary for the enumeration of the phages. The detection level of the routine-analysis technique is about 1 phage per 2 ml of water. This sensitivity is comparable, and most often superior, to that of the best fluorescent tracers. If need be, the sensitivity can be lowered to 1 phage per 10 ml. It is also possible to differentiate and count a mixture of phages in a single sample. Theoretically, bacteriophages offer unlimited possibilities of multitracings. Ten to twenty liters of phage culture are necessary for one tracing experiment. Such an amount is easily transportable, even to inaccessible injection sites. It will contain 1014 - 1015 phages, which represents in all about 1 gram of protein and a few grams of mineral salts and of various organic substances (amino acids, growth medium components). The influence on the aquifer will be negligible, even if the flow is small.